US20210347906A1 - Fusion proteins for immunotherapy against cancer and infectious diseases - Google Patents
Fusion proteins for immunotherapy against cancer and infectious diseases Download PDFInfo
- Publication number
- US20210347906A1 US20210347906A1 US17/240,160 US202117240160A US2021347906A1 US 20210347906 A1 US20210347906 A1 US 20210347906A1 US 202117240160 A US202117240160 A US 202117240160A US 2021347906 A1 US2021347906 A1 US 2021347906A1
- Authority
- US
- United States
- Prior art keywords
- fusion protein
- antigen
- virus
- seq
- amino acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 162
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 162
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 64
- 201000011510 cancer Diseases 0.000 title abstract description 10
- 208000035473 Communicable disease Diseases 0.000 title abstract description 5
- 238000009169 immunotherapy Methods 0.000 title abstract 2
- 239000000427 antigen Substances 0.000 claims abstract description 158
- 108091007433 antigens Proteins 0.000 claims abstract description 158
- 102000036639 antigens Human genes 0.000 claims abstract description 158
- 230000005945 translocation Effects 0.000 claims abstract description 81
- 102000004961 Furin Human genes 0.000 claims abstract description 36
- 108090001126 Furin Proteins 0.000 claims abstract description 36
- 102000004172 Cathepsin L Human genes 0.000 claims abstract description 34
- 108090000624 Cathepsin L Proteins 0.000 claims abstract description 34
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 30
- 230000007017 scission Effects 0.000 claims abstract description 30
- 244000052769 pathogen Species 0.000 claims abstract description 22
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 21
- 201000010099 disease Diseases 0.000 claims abstract description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 10
- 230000021633 leukocyte mediated immunity Effects 0.000 claims abstract description 9
- 101150013553 CD40 gene Proteins 0.000 claims abstract 19
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims abstract 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 84
- 239000012634 fragment Substances 0.000 claims description 46
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 41
- 108010079723 Shiga Toxin Proteins 0.000 claims description 40
- 102100032937 CD40 ligand Human genes 0.000 claims description 38
- 108010029697 CD40 Ligand Proteins 0.000 claims description 37
- 108700033844 Pseudomonas aeruginosa toxA Proteins 0.000 claims description 37
- 125000000539 amino acid group Chemical group 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 241000700721 Hepatitis B virus Species 0.000 claims description 9
- 241001678559 COVID-19 virus Species 0.000 claims description 6
- 241000711549 Hepacivirus C Species 0.000 claims description 6
- 241000701806 Human papillomavirus Species 0.000 claims description 6
- 208000031261 Acute myeloid leukaemia Diseases 0.000 claims description 5
- 241000709721 Hepatovirus A Species 0.000 claims description 5
- 241000713772 Human immunodeficiency virus 1 Species 0.000 claims description 5
- 241000606768 Haemophilus influenzae Species 0.000 claims description 4
- 241000724675 Hepatitis E virus Species 0.000 claims description 4
- 208000037262 Hepatitis delta Diseases 0.000 claims description 4
- 241000724709 Hepatitis delta virus Species 0.000 claims description 4
- 241000701085 Human alphaherpesvirus 3 Species 0.000 claims description 4
- 241000701044 Human gammaherpesvirus 4 Species 0.000 claims description 4
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 claims description 4
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 claims description 4
- 241000710772 Yellow fever virus Species 0.000 claims description 4
- 229940045808 haemophilus influenzae type b Drugs 0.000 claims description 4
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 4
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 4
- 229940051021 yellow-fever virus Drugs 0.000 claims description 4
- 241000725619 Dengue virus Species 0.000 claims description 3
- 241000712461 unidentified influenza virus Species 0.000 claims description 3
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 claims description 2
- 241000193738 Bacillus anthracis Species 0.000 claims description 2
- 206010004593 Bile duct cancer Diseases 0.000 claims description 2
- 206010005003 Bladder cancer Diseases 0.000 claims description 2
- 241000588807 Bordetella Species 0.000 claims description 2
- 241000589969 Borreliella burgdorferi Species 0.000 claims description 2
- 206010006187 Breast cancer Diseases 0.000 claims description 2
- 208000026310 Breast neoplasm Diseases 0.000 claims description 2
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 2
- 241001502567 Chikungunya virus Species 0.000 claims description 2
- 241000193163 Clostridioides difficile Species 0.000 claims description 2
- 241000193155 Clostridium botulinum Species 0.000 claims description 2
- 206010009944 Colon cancer Diseases 0.000 claims description 2
- 241000711573 Coronaviridae Species 0.000 claims description 2
- 241000709687 Coxsackievirus Species 0.000 claims description 2
- 241001115402 Ebolavirus Species 0.000 claims description 2
- 241001466953 Echovirus Species 0.000 claims description 2
- 206010014733 Endometrial cancer Diseases 0.000 claims description 2
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 2
- 241000224432 Entamoeba histolytica Species 0.000 claims description 2
- 241000709661 Enterovirus Species 0.000 claims description 2
- 241000991587 Enterovirus C Species 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000710842 Japanese encephalitis virus Species 0.000 claims description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 2
- 241000712902 Lassa mammarenavirus Species 0.000 claims description 2
- 241000186779 Listeria monocytogenes Species 0.000 claims description 2
- 241001115401 Marburgvirus Species 0.000 claims description 2
- 241000712079 Measles morbillivirus Species 0.000 claims description 2
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 claims description 2
- 241000711386 Mumps virus Species 0.000 claims description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims description 2
- 241000588650 Neisseria meningitidis Species 0.000 claims description 2
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 claims description 2
- 241001263478 Norovirus Species 0.000 claims description 2
- 241000606693 Orientia tsutsugamushi Species 0.000 claims description 2
- 241000150452 Orthohantavirus Species 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 241000223960 Plasmodium falciparum Species 0.000 claims description 2
- 241000223821 Plasmodium malariae Species 0.000 claims description 2
- 241001505293 Plasmodium ovale Species 0.000 claims description 2
- 241000223810 Plasmodium vivax Species 0.000 claims description 2
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 2
- 241000711798 Rabies lyssavirus Species 0.000 claims description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 2
- 206010038389 Renal cancer Diseases 0.000 claims description 2
- 241000606697 Rickettsia prowazekii Species 0.000 claims description 2
- 241000606726 Rickettsia typhi Species 0.000 claims description 2
- 241000713124 Rift Valley fever virus Species 0.000 claims description 2
- 241000702670 Rotavirus Species 0.000 claims description 2
- 241000710799 Rubella virus Species 0.000 claims description 2
- 241001138501 Salmonella enterica Species 0.000 claims description 2
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 claims description 2
- 241000369757 Sapovirus Species 0.000 claims description 2
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 claims description 2
- 241000607766 Shigella boydii Species 0.000 claims description 2
- 241000607764 Shigella dysenteriae Species 0.000 claims description 2
- 241000607762 Shigella flexneri Species 0.000 claims description 2
- 206010041067 Small cell lung cancer Diseases 0.000 claims description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 2
- 241000193998 Streptococcus pneumoniae Species 0.000 claims description 2
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 2
- 241000700647 Variola virus Species 0.000 claims description 2
- 241000607626 Vibrio cholerae Species 0.000 claims description 2
- 241000710886 West Nile virus Species 0.000 claims description 2
- 241000607479 Yersinia pestis Species 0.000 claims description 2
- 241000907316 Zika virus Species 0.000 claims description 2
- 244000309743 astrovirus Species 0.000 claims description 2
- 229940065181 bacillus anthracis Drugs 0.000 claims description 2
- 208000026900 bile duct neoplasm Diseases 0.000 claims description 2
- 201000010881 cervical cancer Diseases 0.000 claims description 2
- 208000006990 cholangiocarcinoma Diseases 0.000 claims description 2
- 208000029742 colonic neoplasm Diseases 0.000 claims description 2
- 229940007078 entamoeba histolytica Drugs 0.000 claims description 2
- 206010017758 gastric cancer Diseases 0.000 claims description 2
- 208000005017 glioblastoma Diseases 0.000 claims description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 claims description 2
- 201000010982 kidney cancer Diseases 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 2
- 229940118768 plasmodium malariae Drugs 0.000 claims description 2
- 206010038038 rectal cancer Diseases 0.000 claims description 2
- 201000001275 rectum cancer Diseases 0.000 claims description 2
- 229940046939 rickettsia prowazekii Drugs 0.000 claims description 2
- 229940007046 shigella dysenteriae Drugs 0.000 claims description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 claims description 2
- 201000011549 stomach cancer Diseases 0.000 claims description 2
- 229940031000 streptococcus pneumoniae Drugs 0.000 claims description 2
- 201000002510 thyroid cancer Diseases 0.000 claims description 2
- 241000701161 unidentified adenovirus Species 0.000 claims description 2
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 2
- 229940118696 vibrio cholerae Drugs 0.000 claims description 2
- 241000193403 Clostridium Species 0.000 claims 1
- 241000607760 Shigella sonnei Species 0.000 claims 1
- 229940115939 shigella sonnei Drugs 0.000 claims 1
- 239000013604 expression vector Substances 0.000 abstract description 14
- 239000008194 pharmaceutical composition Substances 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 description 42
- 102000004169 proteins and genes Human genes 0.000 description 41
- 108090000623 proteins and genes Proteins 0.000 description 41
- 108010074328 Interferon-gamma Proteins 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 23
- 102100037850 Interferon gamma Human genes 0.000 description 22
- 210000004988 splenocyte Anatomy 0.000 description 22
- 210000001744 T-lymphocyte Anatomy 0.000 description 17
- 210000002966 serum Anatomy 0.000 description 17
- 239000000902 placebo Substances 0.000 description 16
- 229940068196 placebo Drugs 0.000 description 16
- 108700025184 hepatitis B virus X Proteins 0.000 description 15
- 210000000612 antigen-presenting cell Anatomy 0.000 description 14
- 230000004927 fusion Effects 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 12
- 239000002953 phosphate buffered saline Substances 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 102000004127 Cytokines Human genes 0.000 description 11
- 108090000695 Cytokines Proteins 0.000 description 11
- 239000002671 adjuvant Substances 0.000 description 11
- 230000003053 immunization Effects 0.000 description 11
- 238000002649 immunization Methods 0.000 description 11
- 230000006698 induction Effects 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 230000014759 maintenance of location Effects 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 230000003248 secreting effect Effects 0.000 description 8
- 238000007920 subcutaneous administration Methods 0.000 description 8
- 102100034540 Adenomatous polyposis coli protein Human genes 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000004071 biological effect Effects 0.000 description 7
- 230000028993 immune response Effects 0.000 description 7
- 230000001939 inductive effect Effects 0.000 description 7
- 238000002255 vaccination Methods 0.000 description 7
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 6
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 6
- 230000030741 antigen processing and presentation Effects 0.000 description 6
- 230000000890 antigenic effect Effects 0.000 description 6
- 210000000172 cytosol Anatomy 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 210000003071 memory t lymphocyte Anatomy 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 108010002350 Interleukin-2 Proteins 0.000 description 5
- 102000000588 Interleukin-2 Human genes 0.000 description 5
- 108091054437 MHC class I family Proteins 0.000 description 5
- 108010091769 Shiga Toxin 1 Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000004443 dendritic cell Anatomy 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- SSOORFWOBGFTHL-OTEJMHTDSA-N (4S)-5-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[(2S)-2-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-5-carbamimidamido-1-[[(2S)-5-carbamimidamido-1-[[(1S)-4-carbamimidamido-1-carboxybutyl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-[[(2S)-2-[[(2S)-2-[[(2S)-2,6-diaminohexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-5-oxopentanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SSOORFWOBGFTHL-OTEJMHTDSA-N 0.000 description 4
- -1 CEA-NE3 Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 210000001163 endosome Anatomy 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 230000003389 potentiating effect Effects 0.000 description 4
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 4
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 3
- 102100026245 E3 ubiquitin-protein ligase RNF43 Human genes 0.000 description 3
- 108700039791 Hepatitis C virus nucleocapsid Proteins 0.000 description 3
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 3
- 101000692702 Homo sapiens E3 ubiquitin-protein ligase RNF43 Proteins 0.000 description 3
- 101000880770 Homo sapiens Protein SSX2 Proteins 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 102000043129 MHC class I family Human genes 0.000 description 3
- 102100037686 Protein SSX2 Human genes 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 230000010534 mechanism of action Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 102100024423 Carbonic anhydrase 9 Human genes 0.000 description 2
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 2
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 2
- 102000043131 MHC class II family Human genes 0.000 description 2
- 108091054438 MHC class II family Proteins 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 2
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000006044 T cell activation Effects 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DRHZYJAUECRAJM-DWSYSWFDSA-N (2s,3s,4s,5r,6r)-6-[[(3s,4s,4ar,6ar,6bs,8r,8ar,12as,14ar,14br)-8a-[(2s,3r,4s,5r,6r)-3-[(2s,3r,4s,5r,6s)-5-[(2s,3r,4s,5r)-4-[(2s,3r,4r)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxy-3,5-dihydroxyoxan-2-yl]oxy-3,4-dihydroxy-6-methyloxan-2-yl]oxy-5-[(3s,5s, Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O1)O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@H]5CC(C)(C)CC[C@@]5([C@@H](C[C@@]4(C)[C@]3(C)CC[C@H]2[C@@]1(C=O)C)O)C(=O)O[C@@H]1O[C@H](C)[C@@H]([C@@H]([C@H]1O[C@H]1[C@@H]([C@H](O)[C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@](O)(CO)CO3)O)[C@H](O)CO2)O)[C@H](C)O1)O)O)OC(=O)C[C@@H](O)C[C@H](OC(=O)C[C@@H](O)C[C@@H]([C@@H](C)CC)O[C@H]1[C@@H]([C@@H](O)[C@H](CO)O1)O)[C@@H](C)CC)C(O)=O)[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O DRHZYJAUECRAJM-DWSYSWFDSA-N 0.000 description 1
- KKVYYGGCHJGEFJ-UHFFFAOYSA-N 1-n-(4-chlorophenyl)-6-methyl-5-n-[3-(7h-purin-6-yl)pyridin-2-yl]isoquinoline-1,5-diamine Chemical compound N=1C=CC2=C(NC=3C(=CC=CN=3)C=3C=4N=CNC=4N=CN=3)C(C)=CC=C2C=1NC1=CC=C(Cl)C=C1 KKVYYGGCHJGEFJ-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- LKKMLIBUAXYLOY-UHFFFAOYSA-N 3-Amino-1-methyl-5H-pyrido[4,3-b]indole Chemical compound N1C2=CC=CC=C2C2=C1C=C(N)N=C2C LKKMLIBUAXYLOY-UHFFFAOYSA-N 0.000 description 1
- 101800000504 3C-like protease Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 description 1
- 101710163881 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 description 1
- 102100036464 Activated RNA polymerase II transcriptional coactivator p15 Human genes 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 102100024003 Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 Human genes 0.000 description 1
- 102100035526 B melanoma antigen 1 Human genes 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 102000036365 BRCA1 Human genes 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 102100021663 Baculoviral IAP repeat-containing protein 5 Human genes 0.000 description 1
- 102100027522 Baculoviral IAP repeat-containing protein 7 Human genes 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108700012439 CA9 Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101800001318 Capsid protein VP4 Proteins 0.000 description 1
- 108090000538 Caspase-8 Proteins 0.000 description 1
- 102100026548 Caspase-8 Human genes 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 241000193449 Clostridium tetani Species 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 241000186227 Corynebacterium diphtheriae Species 0.000 description 1
- 108010060385 Cyclin B1 Proteins 0.000 description 1
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 1
- 102000013701 Cyclin-Dependent Kinase 4 Human genes 0.000 description 1
- 102100027417 Cytochrome P450 1B1 Human genes 0.000 description 1
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 1
- 102100022874 Dexamethasone-induced Ras-related protein 1 Human genes 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 238000011510 Elispot assay Methods 0.000 description 1
- 108010055196 EphA2 Receptor Proteins 0.000 description 1
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 102100035139 Folate receptor alpha Human genes 0.000 description 1
- 102000003817 Fos-related antigen 1 Human genes 0.000 description 1
- 108090000123 Fos-related antigen 1 Proteins 0.000 description 1
- 102100039717 G antigen 1 Human genes 0.000 description 1
- 102100032340 G2/mitotic-specific cyclin-B1 Human genes 0.000 description 1
- 102100039554 Galectin-8 Human genes 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 108090000369 Glutamate Carboxypeptidase II Proteins 0.000 description 1
- 102000010956 Glypican Human genes 0.000 description 1
- 108050001154 Glypican Proteins 0.000 description 1
- 108050007237 Glypican-3 Proteins 0.000 description 1
- 102100036242 HLA class II histocompatibility antigen, DQ alpha 2 chain Human genes 0.000 description 1
- 101000874316 Homo sapiens B melanoma antigen 1 Proteins 0.000 description 1
- 101000936083 Homo sapiens Baculoviral IAP repeat-containing protein 7 Proteins 0.000 description 1
- 101000910338 Homo sapiens Carbonic anhydrase 9 Proteins 0.000 description 1
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 1
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 1
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 1
- 101000725164 Homo sapiens Cytochrome P450 1B1 Proteins 0.000 description 1
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 1
- 101000620808 Homo sapiens Dexamethasone-induced Ras-related protein 1 Proteins 0.000 description 1
- 101001023230 Homo sapiens Folate receptor alpha Proteins 0.000 description 1
- 101000886137 Homo sapiens G antigen 1 Proteins 0.000 description 1
- 101000608769 Homo sapiens Galectin-8 Proteins 0.000 description 1
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 1
- 101000930801 Homo sapiens HLA class II histocompatibility antigen, DQ alpha 2 chain Proteins 0.000 description 1
- 101001011441 Homo sapiens Interferon regulatory factor 4 Proteins 0.000 description 1
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 1
- 101001131670 Homo sapiens PWWP domain-containing DNA repair factor 3A Proteins 0.000 description 1
- 101000613490 Homo sapiens Paired box protein Pax-3 Proteins 0.000 description 1
- 101000601724 Homo sapiens Paired box protein Pax-5 Proteins 0.000 description 1
- 101000691463 Homo sapiens Placenta-specific protein 1 Proteins 0.000 description 1
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 description 1
- 101001123448 Homo sapiens Prolactin receptor Proteins 0.000 description 1
- 101001136981 Homo sapiens Proteasome subunit beta type-9 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 101000894428 Homo sapiens Transcriptional repressor CTCFL Proteins 0.000 description 1
- 101000638154 Homo sapiens Transmembrane protease serine 2 Proteins 0.000 description 1
- 102100030126 Interferon regulatory factor 4 Human genes 0.000 description 1
- 102100031413 L-dopachrome tautomerase Human genes 0.000 description 1
- 101710093778 L-dopachrome tautomerase Proteins 0.000 description 1
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 1
- 102000000440 Melanoma-associated antigen Human genes 0.000 description 1
- 108050008953 Melanoma-associated antigen Proteins 0.000 description 1
- 102000003735 Mesothelin Human genes 0.000 description 1
- 108090000015 Mesothelin Proteins 0.000 description 1
- 229930191564 Monensin Natural products 0.000 description 1
- GAOZTHIDHYLHMS-UHFFFAOYSA-N Monensin A Natural products O1C(CC)(C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CCC1C(O1)(C)CCC21CC(O)C(C)C(C(C)C(OC)C(C)C(O)=O)O2 GAOZTHIDHYLHMS-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100381978 Mus musculus Braf gene Proteins 0.000 description 1
- 101100346932 Mus musculus Muc1 gene Proteins 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 108060006580 PRAME Proteins 0.000 description 1
- 102000036673 PRAME Human genes 0.000 description 1
- 102100040891 Paired box protein Pax-3 Human genes 0.000 description 1
- 102100037504 Paired box protein Pax-5 Human genes 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 102100026181 Placenta-specific protein 1 Human genes 0.000 description 1
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 description 1
- 102100029000 Prolactin receptor Human genes 0.000 description 1
- 102100035764 Proteasome subunit beta type-9 Human genes 0.000 description 1
- 102100022851 Rab5 GDP/GTP exchange factor Human genes 0.000 description 1
- 102100037421 Regulator of G-protein signaling 5 Human genes 0.000 description 1
- 101710140403 Regulator of G-protein signaling 5 Proteins 0.000 description 1
- 101710203837 Replication-associated protein Proteins 0.000 description 1
- 102100027609 Rho-related GTP-binding protein RhoD Human genes 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 101710173693 Short transient receptor potential channel 1 Proteins 0.000 description 1
- 101710173694 Short transient receptor potential channel 2 Proteins 0.000 description 1
- 102100035748 Squamous cell carcinoma antigen recognized by T-cells 3 Human genes 0.000 description 1
- 101710185775 Squamous cell carcinoma antigen recognized by T-cells 3 Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 108010002687 Survivin Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 230000037453 T cell priming Effects 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 102000002689 Toll-like receptor Human genes 0.000 description 1
- 108020000411 Toll-like receptor Proteins 0.000 description 1
- 229940123384 Toll-like receptor (TLR) agonist Drugs 0.000 description 1
- 102100021393 Transcriptional repressor CTCFL Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 102100031989 Transmembrane protease serine 2 Human genes 0.000 description 1
- LVTKHGUGBGNBPL-UHFFFAOYSA-N Trp-P-1 Chemical compound N1C2=CC=CC=C2C2=C1C(C)=C(N)N=C2C LVTKHGUGBGNBPL-UHFFFAOYSA-N 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 102100027244 U4/U6.U5 tri-snRNP-associated protein 1 Human genes 0.000 description 1
- 101710155955 U4/U6.U5 tri-snRNP-associated protein 1 Proteins 0.000 description 1
- 102000015979 Uroplakin-3 Human genes 0.000 description 1
- 108050004262 Uroplakin-3 Proteins 0.000 description 1
- NKVLDFAVEWLOCX-GUSKIFEASA-N [(2s,3r,4s,5r,6r)-3-[(2s,3r,4s,5r,6s)-5-[(2s,3r,4s,5r)-4-[(2s,3r,4r)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxy-3,5-dihydroxyoxan-2-yl]oxy-3,4-dihydroxy-6-methyloxan-2-yl]oxy-4,5-dihydroxy-6-methyloxan-2-yl] (4ar,5r,6as,6br,9s,10s,12ar)-10-[(2r,3r,4s, Chemical compound O([C@H]1[C@H](O)CO[C@H]([C@@H]1O)O[C@H]1[C@H](C)O[C@H]([C@@H]([C@@H]1O)O)O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](C)O[C@H]1OC(=O)[C@]12CCC(C)(C)CC1C1=CCC3[C@@]([C@@]1(C[C@H]2O)C)(C)CCC1[C@]3(C)CC[C@@H]([C@@]1(C)C=O)O[C@@H]1O[C@@H]([C@H]([C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)CO2)O)[C@H]1O[C@H]1[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O1)O)O)C(=O)NCCCCCCCCCCCC)[C@@H]1OC[C@](O)(CO)[C@H]1O NKVLDFAVEWLOCX-GUSKIFEASA-N 0.000 description 1
- UZQJVUCHXGYFLQ-AYDHOLPZSA-N [(2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-4-[(2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-6-(hy Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)CC(O)[C@]1(CCC(CC14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O[C@H]4[C@@H]([C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)[C@H](O)[C@@H](CO)O4)O)[C@H](O)[C@@H](CO)O3)O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UZQJVUCHXGYFLQ-AYDHOLPZSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 230000008350 antigen-specific antibody response Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 108010087914 epidermal growth factor receptor VIII Proteins 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229960005358 monensin Drugs 0.000 description 1
- GAOZTHIDHYLHMS-KEOBGNEYSA-N monensin A Chemical compound C([C@@](O1)(C)[C@H]2CC[C@@](O2)(CC)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C[C@@]21C[C@H](O)[C@@H](C)[C@@H]([C@@H](C)[C@@H](OC)[C@H](C)C(O)=O)O2 GAOZTHIDHYLHMS-KEOBGNEYSA-N 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 231100000065 noncytotoxic Toxicity 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 101800000607 p15 Proteins 0.000 description 1
- 239000006201 parenteral dosage form Substances 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 description 1
- 108040000983 polyphosphate:AMP phosphotransferase activity proteins Proteins 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 108010056119 protease So Proteins 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 108010014186 ras Proteins Proteins 0.000 description 1
- 102000016914 ras Proteins Human genes 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000011537 solubilization buffer Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 101150047061 tag-72 gene Proteins 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 229940021747 therapeutic vaccine Drugs 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/25—Shigella (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- 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.
- 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.
- 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.
- the invention in another aspect, 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.
- the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutical acceptable carrier and/or an adjuvant.
- 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.
- 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.
- 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.
- FIG. 1 is a vector map.
- FIG. 2 is a vector map. MCS, multiple cloning sites.
- FIG. 3 is a vector map.
- FIG. 4 is a vector map.
- FIGS. 5A-E are schematic drawings illustrating various embodiments of the invention.
- FIG. 6 is a graph showing relative cytokine inductions in each animal group.
- FIG. 7 is a graph showing IFN- ⁇ + immunospots in the splenocytes from each animal group.
- FIG. 8 is a graph showing serum HPV 16 E7-specific antibody level in each animal group.
- FIG. 9 is a graph showing serum HPV 18 E7-specific antibody level in each animal group.
- FIG. 10 shows an immunization schedule and animal groups treated and untreated with the indicated fusion proteins, respectively.
- FIG. 11 is a graph showing tumor size in each animal group treated or untreated with the fusion protein indicated.
- FIG. 12 is a graph showing survival rate in each animal group treated or untreated with the fusion protein indicated.
- FIG. 13 is a graph showing tumor free rate in each animal group treated or untreated with the fusion protein indicated.
- FIG. 14 is a graph showing tumor size in each animal group treated or untreated with the fusion protein 18sCD40L-T PE -E7 at various doses indicated.
- FIG. 15 is a graph showing tumor size in each animal group treated or untreated with the fusion protein E7-T Stx -18sCD4L at various doses indicated.
- FIG. 16 shows an immunization scheme (upper panel), animal groups and respective dosing schedules (lower panel) of the fusion protein HBx-preS1-T Stx -18sCD40L.
- FIG. 17 is a graph showing IFN- ⁇ + immunospots in the splenocytes from each animal group in FIG. 16 .
- FIG. 18 is a graph showing serum HBx-specific antibody level in each animal group in FIG. 16 .
- 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.
- MHCs major histocompatibility complexes
- T cells may recognize these complexes using their T cell receptors (TCRs).
- APCs process antigens and present them to T cells.
- 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.
- 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.
- MHC class I a peptide that originates 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.
- 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.
- 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.
- 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.”
- 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.
- anti-CD40 antibody and “CD40-specific antibody” are interchangeable.
- 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.
- the starting amino acid “M” could be omitted or kept.
- 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.
- MHC-1 major histocompatibility complex
- a Pseudomonas Exotoxin A (PE) translocation peptide refers to a PE domain II peptide or a functional fragment thereof that has the biological activity in translocation.
- Shiga toxin (Stx) translocation peptide refers to a Stx translocating domain or a functional fragment thereof that has the biological activity in translocation.
- 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.
- 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).
- tumor and cancer are interchangeable.
- an antigen of a cancer cell and “a tumor antigen” are interchangeable.
- 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.
- CD28 Cluster of Differentiation 28
- 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.
- 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.
- treating 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.
- a subject can be identified by a health care professional based on results from any suitable diagnostic method.
- 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.
- 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 PE -E7 as an example, the mechanism of action is illustrated below:
- the fusion protein binds to a CD40-expressing cell (e.g., dendritic cell or macrophage) and is internalized via a CD40-mediated endocytosis;
- a CD40-expressing cell e.g., dendritic cell or macrophage
- 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 PE -E7 fragment;
- T PE -E7 fragment is digested by cytosol proteasome to generate small E7 antigens with epitopes
- 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.
- the above mechanism of action is applicable to the fusion protein E7-T Stx -18sCD40L, in which case the furin and/or cathepsin L protease cleavage removes the E7-T Stx fragment away from the 18sCD40L fragment.
- the E7-T 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.
- no furin and/or cathepsin L cleavage site is present in the fusion protein between the antigen and the translocation domain.
- 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.
- 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.
- the translocation domain is derived from a Pseudomonas Exotoxin A (PE). In another embodiment, the translocation domain is derived from a Shiga toxin (Stx).
- PE Pseudomonas Exotoxin A
- Stx Shiga toxin
- the translocation domain comprises or is a Pseudomonas Exotoxin A (PE) translocation peptide (T PE ), with the proviso that the CD40-binding domain is located at the N-terminal of the fusion protein.
- PE Pseudomonas Exotoxin A
- the translocation domain comprises or is a Shiga toxin (Stx) translocation peptide (T Stx ), with the proviso that the antigen is located at the N-terminal of the fusion protein.
- Stx Shiga toxin
- 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 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.
- T PE PE translocation peptide
- the translocation domain is a functional moiety of T PE and the furin and/or cathepsin L cleavage site is an intrinsic furin cleavage site from PE.
- 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 PE ); and (iv) an antigen of a pathogen or a tumor antigen.
- 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 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.
- the translocation domain is a functional moiety of T Stx
- said furin and/or cathepsin L cleavage site is an intrinsic furin cleavage site from Stx.
- 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 Stx ); (iii) a cleavable linker comprising a furin and/or cathepsin L cleavage site; and (iv) a CD40-binding domain.
- 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.
- a PE translocation peptide 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).
- a PE translocation peptide comprises the minimal functional fragment GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO: 5).
- a PE translocation peptide (T 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).
- a PE translocation peptide comprises an amino acid sequence that is at least 90%, 95% or 99% identical to SEQ ID NO: 5, 6, 7, 8 or 9.
- a PE translocation peptide is selected from the group consisting of PE 280-305 (SEQ ID NO: 5), PE 280-313 (SEQ ID NO: NO: 6), PE 268-313 (SEQ ID NO: 7), PE 253-313 (SEQ ID NO: 8), and PE 253-364 (SEQ ID NO: 9; full-length PE domain II).
- a Stx translocation peptide 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).
- 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.
- a Stx translocation peptide (T Stx ) consists of 8-84 amino acid residues in length.
- a Stx translocation peptide comprises a minimal functional fragment of LNCHHHAS (SEQ ID NO: 12).
- a Stx translocation peptide (T Stx ) consists of 8-84 amino acid residues in length, said T Stx comprising a minimal fragment of LNCHHHAS (SEQ ID NO: 12).
- a Stx translocation peptide 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.
- a Stx translocation peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15 and 16.
- a Stx translocation peptide is seleted from the group consisting of Stx 240-247 (SEQ ID NO: 12), Stx 240-251 (SEQ ID NO: 13), Stx 211-247 (SEQ ID NO: 14), Stx 211-251 (SEQ ID NO: 15) and Stx 168-251 (SEQ ID NO: 16) of Stx subunit A.
- 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).
- 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.
- 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.
- a functional fragment of CD40L is a truncated CD40L with biological activity, substantially lacking transmembrane and cytoplasmic regions of the full-length CD40L 1-261 protein (SEQ ID NO: 17).
- a CD40L or a functional fragment thereof consists of 154-261 amino acid residues in length.
- a truncated CD40L with functional activity is selected from the group consisting of CD40L 47-261 (SEQ ID NO: 18) and CD40L 108-261 (named 18sCD40L; SEQ ID NO: 19).
- a CD40 ligand (CD40L) or a functional fragment thereof consists of 154-261 amino acid residues in length, said functional fragment thereof comprises CD40L 108-261 (SEQ ID NO: 19).
- 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.
- a CD40 ligand is selected from the group consisting of CD40L 47-261 (SEQ ID NO: 18), CD40L 108-261 (SEQ ID NO: 19; referred to as 18sCD40L) and CD40L 1-261 (SEQ ID NO: 17).
- 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.
- the CD40-specific antibody comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the Vu comprises the amino acid sequence of SEQ ID NO: 22; and the V L comprises the amino acid sequence of SEQ ID NO: 23.
- 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 2 , a minibody and a fully antibody.
- scFv single chain variable fragment
- dscFv diabody
- dscFv diabody
- a triabody a tetrabody
- a bispecific-scFv a scFv-Fc
- scFab single chain antigen-binding fragment
- Fab antigen-binding fragment
- a CD40-binding domain according to the invention is a CD40-specific scFv (anti-CD40 scFv) comprising a heavy chain variable domain (V H ), a light chain variable domain (V L ) and a flexible linker (L) connecting the V H and the V L .
- V H heavy chain variable domain
- V L light chain variable domain
- L flexible linker
- a CD40-specific scFv comprises the amino acid sequence of SEQ ID NO: 20 or 21.
- 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 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.
- the CD40-specific antibody or CD40-specific scFv comprises 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.
- the CD40-binding domain is a CD40-specific 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.
- 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 PE ).
- ER endoplasmic reticulum
- the ER retention sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 31, 32, 33 and 34.
- 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.
- the CD28-activating peptide consisting of 28-53 amino acid residues in length.
- 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.
- 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.
- the CD28-activating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36 and 37.
- 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.
- An antigen in the fusion protein of the invention is an antigen of a pathogen or a tumor antigen.
- 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), Cercop
- 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.
- 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.
- the antigen is a pathogenic antigen selected from the group consisting of HPV 16 E7 protein, HPV 18 E7 protein, HBV X protein (HBx), HBV preS1 protein, HCV core protein (HCVcore) and SARS-CoV-2 spike protein (CoV2S).
- HBx HBV X protein
- HBV preS1 protein HBV preS1 protein
- HCV core protein HCV core protein
- CoV2S SARS-CoV-2 spike protein
- 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.
- 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.
- 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.
- the antigen comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 38, 39, 40, 41, 42 and 43.
- the antigen is a tumor antigen.
- a tumor antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).
- 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.
- AML acute myelogenous leukemia
- CML chronic myelogenous leukemia
- non-Hodgkin's lymphoma and thyroid cancer.
- 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, ⁇ -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.
- 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.
- 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.
- 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.
- An antigen may be a single antigen or an antigenic fragment thereof, or a fusion antigen comprising at least two antigens fused together.
- an antigen may be a single antigen of HPV 16 E7 protein or a fusion antigen comprising HPV 16 E7 and HPV 18 E7 proteins.
- a fusion antigen may or may not have a linker connecting different antigens.
- the antigen is a fusion antigen having at least one linker connecting different antigens.
- the antigen is a fusion antigen having a rigid linker, (EAAAAK) n , connecting different antigens, wherein n is an integer from 0-12, preferably from 2-6, more preferably from 3-4.
- the rigid linker comprises 0 to 12 repeats, 2 to 6 repeats or 3-4 repeats of the sequence EAAAAK (SEQ ID NO: 56).
- 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).
- the rigid linker may be (EAAAAK) n , or (SEQ ID NO: 56) n , wherein n is an integer from 0-12, preferably from 2-6, more preferably from 3-4.
- the rigid linker comprises 2 to 6 repeats or 3-4 repeats of SEQ ID NO: 56.
- 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.
- 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.
- the invention in another aspect, 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.
- 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.
- 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).
- the adjuvant is a CpG oligonucleotide.
- the pharmaceutical composition may also be administered orally, e.g., in the form of tablets, coated tablets, drages, hard and soft gelatine capsules.
- 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.
- 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.
- 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.
- Rap1, Ras-proximate-1 or Ras-related protein 1 Rap1, Ras-proximate-1 or Ras-related protein 1
- CD40 Cluster of differentiation 40
- CDR Complementarity-determining region.
- HPV 16 E6- and E7-expressing tumor cell line from lung epithelial cells of C57BL/6 mice was used to establish a mouse HPV 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° C., 5% CO2.
- Table 1 shows SEQ ID NOs. and corresponding peptides, polypeptides and fusion proteins.
- Splenocytes were stimulated with an antigenic stimulator for 2 hours at 37° C., followed by treating with 50 ⁇ g/mL of Brefeldin A and Monensin at 37° 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.
- the cells were permeabilized, fixed and intracellularly stained with PE-conjugated anti-IFN- ⁇ antibody, PE/Cy7-conjugated anti-IL-2 antibody and eFluor450-conjugated anti-TNF- ⁇ antibody simultaneously.
- the intracellular cytokine characterization (IFN- ⁇ , IL-2 or TNF- ⁇ ) of splenocytes with CD8+ or CD4+ memory T cell phenotypes (CD3 + CD44 hi CD62L lo ) were further analyzed by Gallios flow cytometer and Kaluza software.
- Enzyme-linked immunospot (ELISpot) assay Splenocytes were seeded in triplicate in a pretreated murine IFN- ⁇ capturing 96-well plate (CTL IMMUNOSPOT®) at a cell density of 2 ⁇ 10 5 cells/well in the presence or absence of an antigenic stimulator. The cells were discarded after 24 hours of incubation at 37° C. After wash, the captured IFN- ⁇ was detected by biotin-conjugated anti-murine IFN- ⁇ antibody at room temperature for 2 hours and the IFN- ⁇ -immunospots were developed according to the manufacturer's instructions. The scanning and counting of IFN- ⁇ -immunospots was performed by IMMUNOSPOT® S5 Micro analyzer (CTL).
- CTL IMMUNOSPOT® S5 Micro analyzer
- Indirect enzyme-linked immunosorbent assay Collected whole blood samples were left undisturbed at 4° 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° C.
- the purified coating protein for antigen-specific antibody binding was diluted in guanidine coating buffer (2 M guanidine hydrochloride, 500 mM Na 2 HPO 4 , 25 mM citrate, pH 4.0-4.4) and distributed into 96-well plate at 1 ⁇ g/well. After overnight incubation at 4° C., the 96-well plate was blocked with 1% BSA in PBS at 37° 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 ⁇ l of 1000-fold diluted serum sample at 37° C. for 2 hours. After 4 times washing with phosphate buffered saline TWEEN®-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° C. for 30 minutes.
- HRP horseradish peroxidase
- the HRP-mediated color development was catalyzed in the presence of 100 ⁇ L of TMB substrate and quenched by 100 ⁇ L of 1 N HCl.
- the relative titers of antigen-specific antibody in the serum samples were determined by the absorbance at 450 nm.
- FIGS. 5A-E illustrates various embodiments of the fusion protein according to the invention.
- the fusion protein CD40L 47-261 -T PE -E7 (SEQ ID NO: 51; FIG. 5A ) comprises (a) a truncated CD40 ligand CD40L 47-261 (SEQ ID NO: 18); (b) a cleavable linker comprising (EAAAAK) 3 (SEQ ID NO: 3) and RX 1 RX 2 X 3 R (SEQ ID NO: 2) (wherein X 1 is A, X 2 is Y, X 3 is K); (c) a PE translocation peptide of SEQ ID NO: 5 (PE 280-305 ); and (d) a fusion antigen HPV 16/18 E7, comprising a HPV 16 E7 protein of SEQ ID NO: 38 and a HPV 18 E7 protein of SEQ ID NO: 39.
- An expression vector for CD40L 47-261 -T PE -E7 ( FIG. 1 ) is constructed as follows: A DNA fragment encoding HindIII CD40L-Linker-PE Ncol, XhoI, SalI , comprising the CD40L 47-261, the cleavable linker and the PE translocation peptide (PE 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 ).
- a cleavable linker allows furin and/or cathepsin L protease to cut the fusion protein for releasing the T PE -E7 fragment from the fusion protein.
- 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.
- An expression vector for the fusion protein 18sCD40L-T PE -E7 (SEQ ID NO: 52; FIG. 5B ) was constructed using a similar method described above, in which the truncated CD40 ligand: CD40L 47-261 (SEQ ID NO: 18) was replaced by 18sCD40L, another truncated CD40 ligand: CD40L 108-261 (SEQ ID NO: 19).
- the fusion protein E7-T Stx -CD40L 47-261 (SEQ ID NO: 53; FIG. 5C ) comprises (a) a fusion antigen HPV 16/18 E7 (comprising HPV 16 E7 protein (SEQ ID NO: 38) and HPV 18 E7 protein (SEQ ID NO: 39)), (b) a Stx translocation peptide of SEQ ID NO: 14 (Stx 211-247 ), (c) a cleavable linker comprising RX 1 X 2 R of SEQ ID NO: 1 (wherein X 1 is V, X 2 is A) and (EAAAAK) 3 of SEQ ID NO: 3, and (d) a truncated CD40 ligand of SEQ ID NO: 18 (CD40L 47-261 ).
- An expression vector for E7-T Stx -CD40L 47-261 ( FIG. 3 ) is constructed as follows:
- 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 Stx fragment from the fusion protein. For example, see FIG. 5C .
- 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.
- an expression vector for the fusion protein E7-T Stx -18sCD40L (SEQ ID NO: 54; FIG. 5D ) was constructed, in which the truncated CD40 ligand: CD40 47-261 (SEQ ID NO: 18) was replaced by 18sCD40L, another truncated CD40 ligand: CD40L 108-261 (SEQ ID NO: 19).
- RAP1-CD28 conv PE 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 conv PE 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 268-313 ), an antigen E7 protein and an endoplasmic reticulum retention sequence.
- the antigen E7 protein used here is a fusion antigen HPV 16/18 E7, which comprises a HPV 16 E7 protein (SEQ ID NO: 38) and HPV 18 E7 protein (SEQ ID NO: 39), while the antigen E7 protein used in the prior art is HPV 16 E7 protein.
- the fusion protein HBx-preS1-T 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 211-247 ), (c) a cleavable linker comprising RX 1 X 2 R of SEQ ID NO: 1 (wherein X 1 is V, X 2 is A) and (EAAAAK) 3 of SEQ ID NO: 3, and (d) a truncated CD40 ligand of SEQ ID NO: 19 (18sCD40L).
- an expression vector for the fusion protein HBx-preS1-T 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.
- E. coli BL21 cells harboring the protein expression vector CD40L 47-261 -T 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° C.
- ZY media 10 g/L tryptone and 5 g/L yeast extract
- IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
- 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.
- solubilisation buffer 6 M guanidine hydrochloride, 20 mM potassium phosphate, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, pH 7.4
- the refolding of the fusion protein was performed by dialysis against 20- to 50-fold volume of dialysis buffer (10 mM PBS) at 4° 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.
- the following fusion proteins were also expressed and refolded by using a similar method as described above: (1) 18sCD40L-T PE -E7; (2) E7-T Stx -CD40L 47-261 ; (3) E7-T Stx -18sCD40L; (4) RAP1-E7; (5) CD40L 47-261 -T PE -HBx-preS1; (6) 18sCD40L-T PE -HBx-preS1; (7) HBx-preS1-T Stx -CD40L 47-261 ; (8) HBx-preS1-T Stx -18sCD40L; (9) CD40L 47-261 -T PE -HCVcore; (10) 18sCD40L-T PE -HCVcore; (11) HCVcore-T Stx -CD40L 47-261; (12) HCVcore-T Stx -18sCD40L; (13) CD40L 47-261 -T PE -CoV2S; (14) 18sCD40
- the fusion proteins CD40L 47-261 -T PE -E7, 18sCD40L-T PE -E7, E7-T stx -18sCD40L, RAP1-E7 and HBx-preS1-T Stx -18sCD40L were further subjected to an immunogenicity analysis or an efficacy analysis in the following experiments.
- 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.
- the splenocytes were used to analyze intracellular cytokine induction (IFN- ⁇ , IL-2 and TNF- ⁇ ) in the CD8 + and CD4 + 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 ⁇ g/mL of HPV 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 + /CD8 + and cytokine + /CD4 + splenocytes in the presence of the stimulating antigen E7 to that of the unstimulated (untreated) control.
- the splenocytes were also used to analyze the frequency of IFN- ⁇ -secreting splenocytes in the presence or absence of antigen stimulation (2 ⁇ g/mL of HPV 16 E7 peptide pool) by using Enzyme-linked immunospot (ELISpot) assay. The results were presented as IFN- ⁇ + immunospots per million splenocytes.
- the blood samples were used to analyze the level of serum HPV 16 E7-specific and HPV 18 E7-specific antibody by using ELISA, in which the purified HPV 16 E7 and HPV 18 E7 recombinant proteins were used as coating proteins, respectively.
- FIG. 6 shows cytokine induction results after antigen stimulation of the splenocytes with HPV 16 E7 peptide pool.
- the fusion protein of the invention is superior to the prior art fusion protein in inducing the expression of IFN- ⁇ and TNF- ⁇ in CD8 + memory T cells in response to the stimulation of the antigen HPV 16 E7.
- FIG. 7 shows IFN- ⁇ + immunospots in the splenocytes stimulated with the HPV 16 E7 peptide pool in vitro.
- FIG. 8 shows the serum HPV 16 E7-specific antibody levels in the animals immunized with various fusion proteins on day 0, 7 and 14.
- the HPV 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 47-261 -T PE -E7, 18sCD40L-T PE -E7 or E7-T Stx 18sCD40L (Groups B-D respectively).
- the serum HPV 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).
- the fusion protein RAP1-E7 failed to elicit HPV 16 E7-specific antibody level after two vaccinations (on day 0 and 7). It started to induce HPV 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 16 E7-specific antibody level after two shots of the vaccine on day 0 and 7.
- RAP1-HBx RAP1-CD28convPEt-HBx-K3
- 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).
- FIG. 9 shows the serum HPV 18 E7-specific antibody level in the animals immunized with various fusion proteins on day 0, 7 and 14.
- the fusion proteins CD40L 47-261 -T PE -E7, 18sCD40L-T PE -E7 and E7-T Stx -18sCD40L significantly increased the serum HPV 18 E7-specific antibody level as compared to the placebo group.
- the fusion protein of the invention is effective in inducing antigen-specific antibodies and the antibody induction occurs after twice vaccinations.
- the fusion protein of the invention can induce antigen-specific T cell response, increase the expression of proinflammatory cytokines, e.g., IFN- ⁇ and TNF- ⁇ , and generate antigen-specific antibody response.
- proinflammatory cytokines e.g., IFN- ⁇ and TNF- ⁇
- the fusion proteins were dissolved in PBS and CpG1826 (50 ⁇ 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.
- tumor cells (1 ⁇ 10 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 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 PE -E7 and E7-T Stx -18 s CD40L, 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 47-261 -T PE -E7 and RAP1-E7, respectively) were initially well controlled, however, gradually grew after ceasing immunization.
- the fusion protein of the invention particularly the fusion proteins 18sCD40L-T PE -E7 and E7-T Stx -18sCD40L, can effectively suppress tumor growth.
- the survival rate in the animal groups B-E (immunized with CD40L 47-261 -T PE -E7, 18sCD40L-T PE -E7, E7-T 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 ).
- the results indicate that the fusion protein of the invention can effectively maintain the survival rate in the animal tumor model.
- 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.
- the tumor volume was determined. AU the dosages (25 ⁇ g, 50 ⁇ g or 100 ⁇ g) of the fusion protein 18sCD40L-T 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 PE , both lacking the antigen E7, had no effect in suppressing tumor growth ( FIG. 14 ).
- 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 Stx -18sCD40L (100 ⁇ g; without the fusion antigen E7); (C) E7-T Stx -18sCD40L (100 ⁇ g); (D) E7-T Stx -18sCD40L (50 ⁇ g); and (E) E7-T Stx -18sCD40L (25 ⁇ g), respectively.
- the fusion protein of the invention has potent effects in suppressing tumor growth with outstanding therapeutic efficacy.
- FIG. 16 shows each animal group's dosing schedule.
- the placebo group received PBS via s.c. on Day 0, 7 and 14.
- Mice in other groups received HBx-preS1-T Stx -18sCD40L (100 ⁇ g) adjuvanted with CpG1826 ODN (50 ⁇ g) via s.c. according to the dosing schedule in FIG. 16 .
- an antigenic stimulator a HBx-specific peptide pool, i.e., HBV 32aa overlap 9 peptide
- FIG. 17 shows the IFN- ⁇ + 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- ⁇ -secreting splenocytes as compared to the placebo.
- the frequency of IFN- ⁇ -secreting splenocytes was positively correlated with the number of immunizations.
- the group D0-D7-D14 (vaccinated three times) showed the best induction of IFN- ⁇ -secreting splenocytes.
- a single priming dose (D14 group) of HBx-preS1-T Stx -18sCD40L did not apparently induce HBx-specific antibody response.
- 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- ⁇ production in a dosing number dependent manner ( FIG. 17 ).
- the fusion protein HBx-preS1-T 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.
Abstract
Description
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
-
FIG. 1 is a vector map. -
FIG. 2 is a vector map. MCS, multiple cloning sites. -
FIG. 3 is a vector map. -
FIG. 4 is a vector map. -
FIGS. 5A-E are schematic drawings illustrating various embodiments of the invention. -
FIG. 6 is a graph showing relative cytokine inductions in each animal group. -
FIG. 7 is a graph showing IFN-γ+ immunospots in the splenocytes from each animal group. -
FIG. 8 is a graph showing serum HPV16 E7-specific antibody level in each animal group. -
FIG. 9 is a graph showing serum HPV18 E7-specific antibody level in each animal group. -
FIG. 10 shows an immunization schedule and animal groups treated and untreated with the indicated fusion proteins, respectively. -
FIG. 11 is a graph showing tumor size in each animal group treated or untreated with the fusion protein indicated. -
FIG. 12 is a graph showing survival rate in each animal group treated or untreated with the fusion protein indicated. -
FIG. 13 is a graph showing tumor free rate in each animal group treated or untreated with the fusion protein indicated. -
FIG. 14 is a graph showing tumor size in each animal group treated or untreated with the fusion protein 18sCD40L-TPE-E7 at various doses indicated. -
FIG. 15 is a graph showing tumor size in each animal group treated or untreated with the fusion protein E7-TStx-18sCD4L at various doses indicated. -
FIG. 16 shows an immunization scheme (upper panel), animal groups and respective dosing schedules (lower panel) of the fusion protein HBx-preS1-TStx-18sCD40L. -
FIG. 17 is a graph showing IFN-γ+ immunospots in the splenocytes from each animal group inFIG. 16 . -
FIG. 18 is a graph showing serum HBx-specific antibody level in each animal group inFIG. 16 . - 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.”
- 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.
- The terms “anti-CD40 antibody” and “CD40-specific antibody” are interchangeable.
- 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 HPV18 E7 protein (SEQ ID NO: 39) fused to another polypeptide (e.g., another antigen), the starting amino acid “M” could be omitted or kept.
- 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.
- The term “a Pseudomonas Exotoxin A (PE) translocation peptide (TPE)” refers to a PE domain II peptide or a functional fragment thereof that has the biological activity in translocation.
- The term “a Shiga toxin (Stx) translocation peptide (TStx)” refers to a Stx translocating domain or a functional fragment thereof that has the biological activity in translocation.
- 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.
- 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).
- The terms “tumor” and “cancer” are interchangeable.
- The terms “an antigen of a cancer cell” and “a tumor antigen” are interchangeable.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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-TPE-E7 as an example, the mechanism of action is illustrated below:
- (1) the fusion protein binds to a CD40-expressing cell (e.g., dendritic cell or macrophage) and is internalized via a CD40-mediated endocytosis;
- (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 TPE-E7 fragment;
- (3) the TPE-E7 fragment is translocated across the endosomal membrane of the endosome into the cytosol;
- (4) the TPE-E7 fragment is digested by cytosol proteasome to generate small E7 antigens with epitopes;
- (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.
- The above mechanism of action is applicable to the fusion protein E7-TStx-18sCD40L, in which case the furin and/or cathepsin L protease cleavage removes the E7-TStx fragment away from the 18sCD40L fragment. Thus, the E7-TStx 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- In one embodiment, the translocation domain comprises or is a Pseudomonas Exotoxin A (PE) translocation peptide (TPE), with the proviso that the CD40-binding domain is located at the N-terminal of the fusion protein.
- In another embodiment, the translocation domain comprises or is a Shiga toxin (Stx) translocation peptide (TStx), with the proviso that the antigen is located at the N-terminal of the fusion protein.
- 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 (TPE); 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.
- In another embodiment, the translocation domain is a functional moiety of TPE and the furin and/or cathepsin L cleavage site is an intrinsic furin cleavage site from PE.
- 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 (TPE); and (iv) an antigen of a pathogen or a tumor antigen.
- 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 (TStx); 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.
- In one embodiment, the translocation domain is a functional moiety of TStx, and said furin and/or cathepsin L cleavage site is an intrinsic furin cleavage site from Stx.
- 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 (TStx); (iii) a cleavable linker comprising a furin and/or cathepsin L cleavage site; and (iv) a CD40-binding domain.
- 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.
- 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).
- In another embodiment, a PE translocation peptide (TPE) comprises the minimal functional fragment GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO: 5).
- In another embodiment, a PE translocation peptide (TPE) 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).
- In another embodiment, a PE translocation peptide (TPE) comprises an amino acid sequence that is at least 90%, 95% or 99% identical to SEQ ID NO: 5, 6, 7, 8 or 9.
- In another embodiment, a PE translocation peptide (TPF) is selected from the group consisting of PE280-305 (SEQ ID NO: 5), PE280-313 (SEQ ID NO: NO: 6), PE268-313 (SEQ ID NO: NO: 7), PE253-313 (SEQ ID NO: 8), and PE253-364 (SEQ ID NO: 9; full-length PE domain II).
- In one embodiment, a Stx translocation peptide (TStx) 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.
- In another embodiment, a Stx translocation peptide (TStx) consists of 8-84 amino acid residues in length.
- In another embodiment, a Stx translocation peptide (TStx) comprises a minimal functional fragment of LNCHHHAS (SEQ ID NO: 12).
- In another embodiment, a Stx translocation peptide (TStx) consists of 8-84 amino acid residues in length, said TStx comprising a minimal fragment of LNCHHHAS (SEQ ID NO: 12).
- In another embodiment, a Stx translocation peptide (TStx) 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.
- 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.
- In another embodiment, a Stx translocation peptide (TStx) is seleted from the group consisting of Stx240-247 (SEQ ID NO: 12), Stx240-251 (SEQ ID NO: 13), Stx211-247 (SEQ ID NO: 14), Stx211-251 (SEQ ID NO: 15) and Stx168-251 (SEQ ID NO: 16) of Stx subunit A.
- 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).
- 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.
- 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.
- A functional fragment of CD40L is a truncated CD40L with biological activity, substantially lacking transmembrane and cytoplasmic regions of the full-length CD40L1-261 protein (SEQ ID NO: 17).
- In another embodiment, a CD40L or a functional fragment thereof consists of 154-261 amino acid residues in length.
- In another embodiment, a truncated CD40L with functional activity is selected from the group consisting of CD40L47-261 (SEQ ID NO: 18) and CD40L108-261 (named 18sCD40L; SEQ ID NO: 19).
- 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 CD40L108-261 (SEQ ID NO: 19).
- 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.
- In another embodiment, a CD40 ligand (CD40L) is selected from the group consisting of CD40L47-261 (SEQ ID NO: 18), CD40L108-261 (SEQ ID NO: 19; referred to as 18sCD40L) and CD40L1-261 (SEQ ID NO: 17).
- 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.
- In one embodiment, the CD40-specific antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the Vu comprises the amino acid sequence of SEQ ID NO: 22; and the VL comprises the amino acid sequence of SEQ ID NO: 23.
- 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), Fab2, a minibody and a fully antibody.
- 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 (VH), a light chain variable domain (VL) and a flexible linker (L) connecting the VH and the VL.
- In one embodiment, a CD40-specific scFv comprises the amino acid sequence of SEQ ID NO: 20 or 21.
- 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 VH and a VL, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 22; and (b) the VL comprises the amino acid sequence of SEQ ID NO: 23.
- In another embodiment, the CD40-specific antibody or CD40-specific scFv comprises a VH and a VL, the VH comprising VH CDR1, VH CDR2 and VH CDR3; and the VL comprising VL CDR1, VL CDR2 and VL CDR3, wherein: (i) the VH CDR1, VH CDR2 and VH CDR3 comprises the amino acid sequence of SEQ ID NOs: 24, 25 and 26, respectively; and (ii) the VL CDR1, VL CDR2 and VL CDR3 comprises the amino acid sequence of SEQ ID NOs: 27, 28 and 29, respectively.
- In another embodiment, the CD40-binding domain is a CD40-specific scFv comprising a VH and a VL, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 22; and (b) the VL comprises the amino acid sequence of SEQ ID NO: 23.
- 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 (TPE).
- 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.
- 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.
- In another embodiment, the CD28-activating peptide consisting of 28-53 amino acid residues in length.
- 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.
- 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.
- 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.
- 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.
- An antigen in the fusion protein of the invention is an antigen of a pathogen or a tumor antigen.
- 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.
- 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.
- In another embodiment, the antigen is a pathogenic antigen selected from the group consisting of HPV16 E7 protein, HPV18 E7 protein, HBV X protein (HBx), HBV preS1 protein, HCV core protein (HCVcore) and SARS-CoV-2 spike protein (CoV2S).
- 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.
- 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.
- 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.
- 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.
- In another embodiment, the antigen is a tumor antigen. A tumor antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).
- 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.
- 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, β-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-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3.
- 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.
- 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.
- 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.
- 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 HPV16 E7 protein or a fusion antigen comprising HPV16 E7 and HPV18 E7 proteins. A fusion antigen may or may not have a linker connecting different antigens.
- In another embodiment, the antigen is a fusion antigen having at least one linker connecting different antigens.
- In another embodiment, the antigen is a fusion antigen having a rigid linker, (EAAAAK)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).
- 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).
- The rigid linker may be (EAAAAK)n, or (SEQ ID NO: 56)n, wherein n is an integer from 0-12, preferably from 2-6, more preferably from 3-4.
- In another embodiment, the rigid linker comprises 2 to 6 repeats or 3-4 repeats of SEQ ID NO: 56.
- 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.
- 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.
- 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.
- 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.
- 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.
- The pharmaceutical composition may also be administered orally, e.g., in the form of tablets, coated tablets, drages, hard and soft gelatine capsules.
- 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.
- 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.
- 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.
- Abbreviations: Rap1, Ras-proximate-1 or Ras-related
protein 1; CD40, Cluster ofdifferentiation 40; CDR, Complementarity-determining region. - An HPV16 E6- and E7-expressing tumor cell line from lung epithelial cells of C57BL/6 mice was used to establish a mouse HPV16 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° C., 5% CO2.
- Table 1 shows SEQ ID NOs. and corresponding peptides, polypeptides and fusion proteins.
-
TABLE 1 SEQ ID No. Component name or sequence (N→C) Length (aa) 1 Cleavable linker 1 4 RX1X2R, wherein X1 and X2 are any amino acid residue. 2 Cleavable linker 2 6 RX1RX2X3R, wherein X1 and X2 are any amino acid residue, and X3 is K, F or R. 3 Rigid linker 1 (EAAAAK)3 18 4 Full length PE 613 5 PE translocation peptide (PE280-305, minimal) 26 6 PE translocation peptide (PE280-313) 34 7 PE translocation peptide (PE268-313) 46 8 PE translocation peptide (PE253-313) 61 9 PE translocation peptide (PE253-364) 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 (Stx240-247, minimal) 8 13 Stx translocationpsptide (Stx240-251) 12 14 Stx translocation peptide (Stx211-247) 37 15 Stx translocation peptide (Stx211-251) 41 16 Stx translocation peptide (Stx168-251) 84 17 Full length CD40 ligand (CD40L1-261) 261 18 Truncated CD40 ligand (CD40L47-261) 215 19 Truncated CD40 ligand (CD40L108-261, also referred to as 154 18sCD40L) 20 Anti-CD40 scFv (VH-L-VL) 246 21 Anti-CD40 scFv (VL-L-VH) 246 22 VH of the anti-CD40 scFv 119 23 VL of the anti-CD40 scFv 112 24 VH CDR1 GFTFSTYGMH 10 25 VH CDR2 GKGLEWLSYISGGSSYIFYADSVRGR 26 26 VH CDR3 CARILRGGSGMDL 13 27 VL CDR1 CTGSSSNIGAGYNVY 15 28 VL CDR2 GNINRPS 7 29 VL 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 T1D2I3Y4F5C6K7X8E9X10X11Y12P13P14P15Y16X17D18N19 E20K21S22N23G24T25I26I27H28, wherein X8 is I or L, X10 is V, F or A, X11 is M or L, X17 is L or I. 36 CD28-activating peptide (minimal) 28 37 CD28-activating peptide 53 38 Antigen HPV16 E7 protein 98 39 Antigen HPV18 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 CD40L47-261-TPE-E7 528 52 Fusion protein 18sCD40L-TPE-E7 467 53 Fusion protein E7-TStx-CD40L47-261 535 54 Fusion protein E7-TStx-18sCD40L 474 55 Fusion protein HBx-preS1-TStx-18sCD40L 541 56 Rigid linker EAAAAK 6 - Flow cytometry. Splenocytes were stimulated with an antigenic stimulator for 2 hours at 37° C., followed by treating with 50 μg/mL of Brefeldin A and Monensin at 37° 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-γ antibody, PE/Cy7-conjugated anti-IL-2 antibody and eFluor450-conjugated anti-TNF-α antibody simultaneously. The intracellular cytokine characterization (IFN-γ, IL-2 or TNF-α) of splenocytes with CD8+ or CD4+ memory T cell phenotypes (CD3+CD44hiCD62Llo) were further analyzed by Gallios flow cytometer and Kaluza software.
- Enzyme-linked immunospot (ELISpot) assay. Splenocytes were seeded in triplicate in a pretreated murine IFN-γ capturing 96-well plate (CTL IMMUNOSPOT®) at a cell density of 2×105 cells/well in the presence or absence of an antigenic stimulator. The cells were discarded after 24 hours of incubation at 37° C. After wash, the captured IFN-γ was detected by biotin-conjugated anti-murine IFN-γ antibody at room temperature for 2 hours and the IFN-γ-immunospots were developed according to the manufacturer's instructions. The scanning and counting of IFN-γ-immunospots was performed by IMMUNOSPOT® S5 Micro analyzer (CTL).
- Indirect enzyme-linked immunosorbent assay (ELISA). Collected whole blood samples were left undisturbed at 4° 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° C. The purified coating protein for antigen-specific antibody binding was diluted in guanidine coating buffer (2 M guanidine hydrochloride, 500 mM Na2HPO4, 25 mM citrate, pH 4.0-4.4) and distributed into 96-well plate at 1 μg/well. After overnight incubation at 4° C., the 96-well plate was blocked with 1% BSA in PBS at 37° 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 μl of 1000-fold diluted serum sample at 37° C. for 2 hours. After 4 times washing with phosphate buffered saline TWEEN®-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° C. for 30 minutes. Following 4 times of washing with PBST, the HRP-mediated color development was catalyzed in the presence of 100 μL of TMB substrate and quenched by 100 μL of 1 N HCl. The relative titers of antigen-specific antibody in the serum samples were determined by the absorbance at 450 nm.
- Statistical analysis. The significance of all comparisons was calculated by using t-test, and results considered significant when p<0.05.
-
FIGS. 5A-E illustrates various embodiments of the fusion protein according to the invention. - The fusion protein CD40L47-261-TPE-E7 (SEQ ID NO: 51;
FIG. 5A ) comprises (a) a truncated CD40 ligand CD40L47-261 (SEQ ID NO: 18); (b) a cleavable linker comprising (EAAAAK)3 (SEQ ID NO: 3) and RX1RX2X3R (SEQ ID NO: 2) (wherein X1 is A, X2 is Y, X3 is K); (c) a PE translocation peptide of SEQ ID NO: 5 (PE280-305); and (d) a fusion antigen HPV16/18 E7, comprising a HPV16 E7 protein of SEQ ID NO: 38 and a HPV18 E7 protein of SEQ ID NO: 39. - An expression vector for CD40L47-261-TPE-E7 (
FIG. 1 ) is constructed as follows: A DNA fragment encoding HindIIICD40L-Linker-PENcol, XhoI, SalI, comprising the CD40L47-261, the cleavable linker and the PE translocation peptide (PE280-305), 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 HPV16/18 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 CD40L47-261-TPE-E7 (FIG. 1 ). - A cleavable linker allows furin and/or cathepsin L protease to cut the fusion protein for releasing the TPE-E7 fragment from the fusion protein.
- 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 ofFIG. 1 for a fusion protein comprising the antigen of interest according to the invention. - An expression vector for the fusion protein 18sCD40L-TPE-E7 (SEQ ID NO: 52;
FIG. 5B ) was constructed using a similar method described above, in which the truncated CD40 ligand: CD40L47-261 (SEQ ID NO: 18) was replaced by 18sCD40L, another truncated CD40 ligand: CD40L108-261 (SEQ ID NO: 19). - The fusion protein E7-TStx-CD40L47-261 (SEQ ID NO: 53;
FIG. 5C ) comprises (a) a fusion antigen HPV16/18 E7 (comprising HPV16 E7 protein (SEQ ID NO: 38) and HPV18 E7 protein (SEQ ID NO: 39)), (b) a Stx translocation peptide of SEQ ID NO: 14 (Stx211-247), (c) a cleavable linker comprising RX1X2R of SEQ ID NO: 1 (wherein X1 is V, X2 is A) and (EAAAAK)3 of SEQ ID NO: 3, and (d) a truncated CD40 ligand of SEQ ID NO: 18 (CD40L47-261). - An expression vector for E7-TStx-CD40L47-261 (
FIG. 3 ) is constructed as follows: - A DNA fragment encoding HindIII, XhoIStx-Linker-CD40LSalI, comprising the Stx translocation peptide (Stx211-247), the cleavable linker and the CD40L47-261, 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 HPV16/18 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-Tstx-CD40L47-261 (FIG. 3 ). - 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-TStx fragment from the fusion protein. For example, see
FIG. 5C . - 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 ofFIG. 3 for a fusion protein comprising the antigen of interest according to the invention. - Using a similar method described above, an expression vector for the fusion protein E7-TStx-18sCD40L (SEQ ID NO: 54;
FIG. 5D ) was constructed, in which the truncated CD40 ligand: CD4047-261 (SEQ ID NO: 18) was replaced by 18sCD40L, another truncated CD40 ligand: CD40L108-261 (SEQ ID NO: 19). - For a comparison purpose, we have constructed the fusion protein RAP1-CD28convPEt-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-CD28convPEt-E7-K3 (referred as “RAP1-E7” in the application) comprises a RAP1 domain III, a CD28 sequence, a linker, a PE translocation domain II (PE268-313), an antigen E7 protein and an endoplasmic reticulum retention sequence. The antigen E7 protein used here is a fusion antigen HPV16/18 E7, which comprises a HPV16 E7 protein (SEQ ID NO: 38) and HPV18 E7 protein (SEQ ID NO: 39), while the antigen E7 protein used in the prior art is HPV16 E7 protein.
- The fusion protein HBx-preS1-TStx-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 (Stx211-247), (c) a cleavable linker comprising RX1X2R of SEQ ID NO: 1 (wherein X1 is V, X2 is A) and (EAAAAK)3 of SEQ ID NO: 3, and (d) a truncated CD40 ligand of SEQ ID NO: 19 (18sCD40L). - Using a similar method described in Example 2, an expression vector for the fusion protein HBx-preS1-TStx-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.
- E. coli BL21 cells harboring the protein expression vector CD40L47-261-TPE-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° C. When the culture reached an early log phase, (OD600=2 to 5), the expression of fusion protein was induced by isopropyl-1-thio-β-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° 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.
- The following fusion proteins were also expressed and refolded by using a similar method as described above: (1) 18sCD40L-TPE-E7; (2) E7-TStx-CD40L47-261; (3) E7-TStx-18sCD40L; (4) RAP1-E7; (5) CD40L47-261-TPE-HBx-preS1; (6) 18sCD40L-TPE-HBx-preS1; (7) HBx-preS1-TStx-CD40L47-261; (8) HBx-preS1-TStx-18sCD40L; (9) CD40L47-261-TPE-HCVcore; (10) 18sCD40L-TPE-HCVcore; (11) HCVcore-TStx-CD40L47-261; (12) HCVcore-TStx-18sCD40L; (13) CD40L47-261-TPE-CoV2S; (14) 18sCD40L-TPE-CoV2S; (15) CoV2S-TStx-CD40L47-261; (16) CoV2S-TStx-18sCD40L; (17) CD40L47-261-TPE-SSX2; (18) 18sCD40L-TPE-SSX2; (19) SSX2-TStx-CD40L47-261; (20) SSX2-TStx-18sCD40L. The fusion proteins CD40L47-261-TPE-E7, 18sCD40L-TPE-E7, E7-Tstx-18sCD40L, RAP1-E7 and HBx-preS1-TStx-18sCD40L were further subjected to an immunogenicity analysis or an efficacy analysis in the following experiments.
- 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 CD40L47-261-TPE-E7 (100 μg); (C) fusion protein 18sCD40L-TPE-E7 (100 μg); (D) fusion protein E7-TStx-18sCD40L (100 μg); and (E) fusion protein RAP1-E7 (100 μg). The fusion proteins were dialyzed into PBS. CpG1826 (50 μg) 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 onday day 21, the blood samples were harvested and the splenocytes were resuspended in RPMI 1640 medium containing FBS (10%) and PSA. - The splenocytes were used to analyze intracellular cytokine induction (IFN-γ, IL-2 and TNF-α) in the CD8+ and CD4+ 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 μg/mL of HPV16 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+/CD8+ and cytokine+/CD4+ splenocytes in the presence of the stimulating antigen E7 to that of the unstimulated (untreated) control.
- The splenocytes were also used to analyze the frequency of IFN-γ-secreting splenocytes in the presence or absence of antigen stimulation (2 μg/mL of HPV16 E7 peptide pool) by using Enzyme-linked immunospot (ELISpot) assay. The results were presented as IFN-γ+ immunospots per million splenocytes.
- The blood samples were used to analyze the level of serum HPV16 E7-specific and HPV18 E7-specific antibody by using ELISA, in which the purified HPV16 E7 and HPV18 E7 recombinant proteins were used as coating proteins, respectively.
-
FIG. 6 shows cytokine induction results after antigen stimulation of the splenocytes with HPV16 E7 peptide pool. The relative cytokine induction of IFN-γ and TNF-α, but not IL2, in CD8+ memory T cells from the animals immunized with the fusion protein CD40L47-261-TPE-E7, 18sCD40L-TPE-E7 or E7-TStx-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+ memory T cells, and the cytokines IFN-γ, IL-2 or TNF-α in CD4+ memory T cells in the animal groups B-E slightly increased, however, showed no significant difference as compared to placebo group (Group A). - 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-γ and TNF-α in CD8+ memory T cells in response to the stimulation of the antigen HPV16 E7.
-
FIG. 7 shows IFN-γ+ immunospots in the splenocytes stimulated with the HPV16 E7 peptide pool in vitro. The frequency of IFN-γ-secreting splenocytes from the animal groups immunized with CD40L47-261-TPE-E7, 18sCD40L-TPE-E7, E7-TStx-18sCD40L and RAP1-E7 (Groups B-E), respectively, significantly increased as compared to the placebo group. Particularly, E7-TStx-18sCD40L induced significantly higher frequency of IFN-γ-secreting cells than CD40L47-261-TPE-E7 (p=0.035). - The results indicate that the fusion protein of the invention can significantly increase IFN-γ-secreting T cell population upon or after stimulation with the antigenic HPV16 E7 peptide pool.
-
FIG. 8 shows the serum HPV16 E7-specific antibody levels in the animals immunized with various fusion proteins onday day 7, and further rose after the third vaccination onday 14 in animals vaccinated with CD40L47-261-TPE-E7, 18sCD40L-TPE-E7 or E7-TStx18sCD40L (Groups B-D respectively). Onday 21, the serum HPV16 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). - The fusion protein RAP1-E7 (RAP1-CD28convPEt-E7-K3) failed to elicit HPV16 E7-specific antibody level after two vaccinations (on
day 0 and 7). It started to induce HPV16 E7-specific antibody after the third vaccination onday 14, and the serum antibody level was only modest onday 21 as compared to Groups B-D. In contrast, the fusion protein of the invention elicited serum HPV16 E7-specific antibody level after two shots of the vaccine onday - 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 onday 21 was only modest as compared to animals vaccinated with the fusion protein of the invention (data not shown). -
FIG. 9 shows the serum HPV18 E7-specific antibody level in the animals immunized with various fusion proteins onday - Thus, the fusion protein of the invention is effective in inducing antigen-specific antibodies and the antibody induction occurs after twice vaccinations.
- In summary, the fusion protein of the invention can induce antigen-specific T cell response, increase the expression of proinflammatory cytokines, e.g., IFN-γ and TNF-α, and generate antigen-specific antibody response.
- 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, CD40L47-261-TPE-E7 (25 μg; n=5); Group C, 18sCD40L-TPE-E7 (25 μg; n=4); Group D, E7-TStx-18sCD40L (25 μg; n=5); and Group E, RAP1-E7 (25 μg; n=5). The fusion proteins were dissolved in PBS and CpG1826 (50 μg) was used as an adjuvant in vaccinating animals in Groups B to E.
FIG. 10 shows an immunization schedule, fusion proteins and the dosages. - To challenge mice, tumor cells (1×105 in 0.1 mL) were injected s.c. into the left flank of each mouse on
day 0. Three immunizations were s.c. given onday - 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-TPE-E7 and E7-TStx-18sCD40L, 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 CD40L47-261-TPE-E7 and RAP1-E7, respectively) were initially well controlled, however, gradually grew after ceasing immunization. - The results indicate that the fusion protein of the invention, particularly the fusion proteins 18sCD40L-TPE-E7 and E7-TStx-18sCD40L, can effectively suppress tumor growth.
- The survival rate in the animal groups B-E (immunized with CD40L47-261-TPE-E7, 18sCD40L-TPE-E7, E7-TStx-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 ). - The results indicate that the fusion protein of the invention can effectively maintain the survival rate in the animal tumor model.
- 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-TPE-E7 and E7-TStx-18sCD40L, 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-TPE-E7 or E7-TStx-18sCD40L. - 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.
- 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-TPE (100 μg; without the fusion antigen E7); (C) 18sCD40L-TPE-E7 (100 μg); (D) 18sCD40L-TPE-E7 (50 lig); (E) 18sCD40L-T″-E7 (25 μg). The fusion proteins were dissolved in PBS and CpG1826 (50 μg) used as an adjuvant in Groups B to E. Tumor cells (1×106in 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. onday - The tumor volume was determined. AU the dosages (25 μg, 50 μg or 100 μg) of the fusion protein 18sCD40L-TPE-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-TPE, both lacking the antigen E7, had no effect in suppressing tumor growth (FIG. 14 ). - Mice were grouped, challenged with tumor cells, and dosed on
day 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 TStx-18sCD40L, both lacking the antigen E7, had no effect in suppressing tumor growth (FIG. 15 ). - Thus, the fusion protein of the invention has potent effects in suppressing tumor growth with outstanding therapeutic efficacy.
-
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 ondays days 7, 14); and (4) D14 group (one dose, vaccinated on day 14). The placebo group received PBS via s.c. onDay FIG. 16 . Blood samples were collected onday day 21, the animals were sacrificed, splenocytes harvested and cultured. The frequency of IFN-γ-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. -
FIG. 17 shows the IFN-γ+ immunospots in the splenocytes stimulated with theHBV 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-γ-secreting splenocytes as compared to the placebo. The frequency of IFN-γ-secreting splenocytes was positively correlated with the number of immunizations. The group D0-D7-D14 (vaccinated three times) showed the best induction of IFN-γ-secreting splenocytes. - In contrast, a single priming dose (D14 group) of HBx-preS1-TStx-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-γ production in a dosing number dependent manner (FIG. 17 ). - Thus, the fusion protein HBx-preS1-TStx-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.
- 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.
Claims (20)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/240,160 US20210347906A1 (en) | 2020-05-06 | 2021-04-26 | Fusion proteins for immunotherapy against cancer and infectious diseases |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202063020545P | 2020-05-06 | 2020-05-06 | |
US17/240,160 US20210347906A1 (en) | 2020-05-06 | 2021-04-26 | Fusion proteins for immunotherapy against cancer and infectious diseases |
Publications (1)
Publication Number | Publication Date |
---|---|
US20210347906A1 true US20210347906A1 (en) | 2021-11-11 |
Family
ID=78412282
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/240,160 Pending US20210347906A1 (en) | 2020-05-06 | 2021-04-26 | Fusion proteins for immunotherapy against cancer and infectious diseases |
Country Status (12)
Country | Link |
---|---|
US (1) | US20210347906A1 (en) |
EP (1) | EP4146701A1 (en) |
JP (1) | JP2023524814A (en) |
KR (1) | KR20230008162A (en) |
CN (1) | CN115956086A (en) |
AU (1) | AU2021268885A1 (en) |
BR (1) | BR112022021797A2 (en) |
CA (1) | CA3172293A1 (en) |
IL (1) | IL296880A (en) |
MX (1) | MX2022013809A (en) |
TW (1) | TWI817113B (en) |
WO (1) | WO2021225820A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023081595A1 (en) * | 2021-11-05 | 2023-05-11 | Navicure Biopharmaceuticals Limited | Immunogenic fusion proteins against infectious animal diseases |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8119117B2 (en) * | 2002-11-12 | 2012-02-21 | Vaxum, Llc | Adenoviral expression vector comprising a CD40L fusion protein adapted to elicit cellular immunity |
US9340599B2 (en) * | 2008-07-21 | 2016-05-17 | Apogenix Ag | Single chain CD40L fusion polypeptides |
US20170101636A1 (en) * | 2014-06-11 | 2017-04-13 | Molecular Templates, Inc. | Protease-cleavage resistant, shiga toxin a subunit effector polypeptides and cell-targeted molecules comprising the same |
US10293058B2 (en) * | 2015-04-22 | 2019-05-21 | Curevac Ag | RNA containing composition for treatment of tumor diseases |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2951209A4 (en) * | 2013-01-31 | 2016-06-22 | Univ Jefferson | Agonist fusion protein for cd40 ox40 and uses thereof |
CN109715651A (en) * | 2016-09-19 | 2019-05-03 | 生控基因疫苗股份有限公司 | Treating hepatitis B type vaccine |
-
2021
- 2021-04-26 CA CA3172293A patent/CA3172293A1/en active Pending
- 2021-04-26 EP EP21799774.1A patent/EP4146701A1/en active Pending
- 2021-04-26 MX MX2022013809A patent/MX2022013809A/en unknown
- 2021-04-26 WO PCT/US2021/029203 patent/WO2021225820A1/en unknown
- 2021-04-26 US US17/240,160 patent/US20210347906A1/en active Pending
- 2021-04-26 IL IL296880A patent/IL296880A/en unknown
- 2021-04-26 AU AU2021268885A patent/AU2021268885A1/en active Pending
- 2021-04-26 JP JP2022567573A patent/JP2023524814A/en active Pending
- 2021-04-26 KR KR1020227042378A patent/KR20230008162A/en unknown
- 2021-04-26 BR BR112022021797A patent/BR112022021797A2/en unknown
- 2021-04-26 CN CN202180029571.6A patent/CN115956086A/en active Pending
- 2021-05-04 TW TW110116026A patent/TWI817113B/en active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8119117B2 (en) * | 2002-11-12 | 2012-02-21 | Vaxum, Llc | Adenoviral expression vector comprising a CD40L fusion protein adapted to elicit cellular immunity |
US9340599B2 (en) * | 2008-07-21 | 2016-05-17 | Apogenix Ag | Single chain CD40L fusion polypeptides |
US20170101636A1 (en) * | 2014-06-11 | 2017-04-13 | Molecular Templates, Inc. | Protease-cleavage resistant, shiga toxin a subunit effector polypeptides and cell-targeted molecules comprising the same |
US10293058B2 (en) * | 2015-04-22 | 2019-05-21 | Curevac Ag | RNA containing composition for treatment of tumor diseases |
Also Published As
Publication number | Publication date |
---|---|
JP2023524814A (en) | 2023-06-13 |
MX2022013809A (en) | 2022-11-30 |
EP4146701A1 (en) | 2023-03-15 |
WO2021225820A1 (en) | 2021-11-11 |
CN115956086A (en) | 2023-04-11 |
CA3172293A1 (en) | 2021-11-11 |
KR20230008162A (en) | 2023-01-13 |
BR112022021797A2 (en) | 2022-12-13 |
TWI817113B (en) | 2023-10-01 |
TW202200625A (en) | 2022-01-01 |
AU2021268885A1 (en) | 2022-10-27 |
IL296880A (en) | 2022-12-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
ES2432357T3 (en) | Entirely human antibodies against human 4-1BB (CD137) | |
US20170080064A1 (en) | Methods and compositions for increasing a t-effector cell to regulatory t cell ratio | |
CA2786940C (en) | Tumor vaccination involving a humoral immune response, and proteins therefor including all or a portion of a hepatitis b virus core antigen protein and an epitope of claudin 18.2 | |
TW201639594A (en) | Combination therapies with recombinant listeria strains | |
Chua et al. | Soluble proteins induce strong CD8+ T cell and antibody responses through electrostatic association with simple cationic or anionic lipopeptides that target TLR2 | |
TW201100096A (en) | Antigen presenting cell targeted anti-viral vaccines | |
Jemon et al. | An enhanced heterologous virus-like particle for human papillomavirus type 16 tumour immunotherapy | |
Sadraeian et al. | Induction of antitumor immunity against cervical cancer by protein HPV-16 E7 in fusion with ricin B chain in tumor-bearing mice | |
Neukirch et al. | Adenovirus based virus-like-vaccines targeting endogenous retroviruses can eliminate growing colorectal cancers in mice | |
Mohit et al. | The contribution of NT‐gp96 as an adjuvant for increasing HPV16 E7‐specific immunity in C57BL/6 mouse model | |
US20060051373A1 (en) | Particle-bound human immunodeficiency virus envelope glycoproteins and related compositions and methods | |
JP6698541B2 (en) | Medicament for use in a method of inducing or prolonging a cellular cytotoxic immune response | |
RU2556128C2 (en) | Immunoprophylactic cancer vaccine | |
US20210347906A1 (en) | Fusion proteins for immunotherapy against cancer and infectious diseases | |
Silva et al. | Antigen delivery to DEC205+ dendritic cells induces immunological memory and protective therapeutic effects against HPV-associated tumors at different anatomical sites | |
EP2366709A1 (en) | Tumor vaccination involving a humoral immune response against self-proteins | |
Biragyn et al. | Tumor-associated embryonic antigen-expressing vaccines that target CCR6 elicit potent CD8+ T cell-mediated protective and therapeutic antitumor immunity | |
Chu et al. | Th2-dominated antitumor immunity induced by DNA immunization with the genes coding for a basal core peptide PDTRP and GM-CSF | |
WO2023076768A1 (en) | Combination therapies against cancer and infectious diseases | |
WO2008107641A1 (en) | Liposome preparation | |
WO2023081595A1 (en) | Immunogenic fusion proteins against infectious animal diseases | |
AU2022380700A1 (en) | Immunogenic fusion proteins against infectious animal diseases | |
WO2023076820A1 (en) | Immunogenic fusion proteins against coronavirus | |
Johansson et al. | Responses of mice immunized with a DNA vaccine encoding carcinoembryonic antigen (CEA) | |
Zeng et al. | Soluble Proteins Induce Strong CD8+ T Cell |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: NAVICURE BIOPHARMACEUTICALS LIMITED, SAMOA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:WU, CHIA-MAO;REEL/FRAME:056044/0152 Effective date: 20210423 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE AFTER FINAL ACTION FORWARDED TO EXAMINER |