US20230338465A1 - Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases - Google Patents
Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases Download PDFInfo
- Publication number
- US20230338465A1 US20230338465A1 US18/022,096 US202118022096A US2023338465A1 US 20230338465 A1 US20230338465 A1 US 20230338465A1 US 202118022096 A US202118022096 A US 202118022096A US 2023338465 A1 US2023338465 A1 US 2023338465A1
- Authority
- US
- United States
- Prior art keywords
- sequence
- seq
- sequence identity
- fusion construct
- sequences
- 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
- 238000011282 treatment Methods 0.000 title claims abstract description 18
- 108020001507 fusion proteins Proteins 0.000 title claims description 17
- 102000037865 fusion proteins Human genes 0.000 title claims description 17
- 208000035473 Communicable disease Diseases 0.000 title 1
- 238000003745 diagnosis Methods 0.000 title 1
- 238000011321 prophylaxis Methods 0.000 title 1
- 230000004927 fusion Effects 0.000 claims abstract description 280
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 71
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 66
- 208000015181 infectious disease Diseases 0.000 claims abstract description 31
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 claims abstract description 30
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 claims abstract description 29
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 23
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims abstract description 17
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 11
- 241000197306 H1N1 subtype Species 0.000 claims abstract description 8
- 102000009027 Albumins Human genes 0.000 claims abstract description 7
- 108010088751 Albumins Proteins 0.000 claims abstract description 7
- 101800000795 Proadrenomedullin N-20 terminal peptide Proteins 0.000 claims abstract description 6
- 201000004792 malaria Diseases 0.000 claims abstract description 6
- PIRWNASAJNPKHT-SHZATDIYSA-N pamp Chemical compound C([C@@H](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](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)N)C(C)C)C1=CC=CC=C1 PIRWNASAJNPKHT-SHZATDIYSA-N 0.000 claims abstract description 6
- 241000711573 Coronaviridae Species 0.000 claims abstract description 5
- 241000709661 Enterovirus Species 0.000 claims abstract description 5
- 208000004554 Leishmaniasis Diseases 0.000 claims abstract description 4
- 102100032814 ATP-dependent zinc metalloprotease YME1L1 Human genes 0.000 claims abstract 2
- 239000012634 fragment Substances 0.000 claims description 72
- 230000035772 mutation Effects 0.000 claims description 46
- 125000000539 amino acid group Chemical group 0.000 claims description 41
- 235000018102 proteins Nutrition 0.000 claims description 36
- 210000004027 cell Anatomy 0.000 claims description 29
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical group SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 27
- 241000907316 Zika virus Species 0.000 claims description 27
- 108010037897 DC-specific ICAM-3 grabbing nonintegrin Proteins 0.000 claims description 24
- 101001068136 Homo sapiens Hepatitis A virus cellular receptor 1 Proteins 0.000 claims description 24
- 101000831286 Homo sapiens Protein timeless homolog Proteins 0.000 claims description 23
- 101000752245 Homo sapiens Rho guanine nucleotide exchange factor 5 Proteins 0.000 claims description 23
- 229960005486 vaccine Drugs 0.000 claims description 22
- 230000003612 virological effect Effects 0.000 claims description 21
- 101100284386 Homo sapiens HAVCR1 gene Proteins 0.000 claims description 20
- 101100369806 Homo sapiens TIMELESS gene Proteins 0.000 claims description 20
- 241000700605 Viruses Species 0.000 claims description 18
- 239000000833 heterodimer Substances 0.000 claims description 18
- 238000005734 heterodimerization reaction Methods 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 16
- 229940082999 Furin inhibitor Drugs 0.000 claims description 15
- 108090000342 C-Type Lectins Proteins 0.000 claims description 14
- 102000003930 C-Type Lectins Human genes 0.000 claims description 14
- 210000004369 blood Anatomy 0.000 claims description 12
- 239000008280 blood Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 229920000057 Mannan Polymers 0.000 claims description 11
- 230000021615 conjugation Effects 0.000 claims description 11
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 claims description 10
- 230000005888 antibody-dependent cellular phagocytosis Effects 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 9
- 239000000710 homodimer Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 201000010099 disease Diseases 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- 229940079593 drug Drugs 0.000 claims description 8
- 239000013598 vector Substances 0.000 claims description 8
- 230000003993 interaction Effects 0.000 claims description 7
- 241000233866 Fungi Species 0.000 claims description 6
- 241000710886 West Nile virus Species 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 150000007523 nucleic acids Chemical class 0.000 claims description 6
- 244000045947 parasite Species 0.000 claims description 6
- 150000003904 phospholipids Chemical class 0.000 claims description 6
- 238000007920 subcutaneous administration Methods 0.000 claims description 6
- 241001115402 Ebolavirus Species 0.000 claims description 5
- 238000007918 intramuscular administration Methods 0.000 claims description 5
- 241001502567 Chikungunya virus Species 0.000 claims description 4
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 4
- 241000725619 Dengue virus Species 0.000 claims description 4
- 241000186362 Mycobacterium leprae Species 0.000 claims description 4
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims description 4
- 208000036142 Viral infection Diseases 0.000 claims description 4
- 239000000969 carrier Substances 0.000 claims description 4
- 238000012544 monitoring process Methods 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 241000712461 unidentified influenza virus Species 0.000 claims description 4
- 208000035143 Bacterial infection Diseases 0.000 claims description 3
- 208000010362 Protozoan Infections Diseases 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 208000022362 bacterial infectious disease Diseases 0.000 claims description 3
- 239000011230 binding agent Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 230000003292 diminished effect Effects 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 claims description 2
- 229920002498 Beta-glucan Polymers 0.000 claims description 2
- 210000002865 immune cell Anatomy 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 230000004936 stimulating effect Effects 0.000 claims description 2
- 102100021688 Rho guanine nucleotide exchange factor 5 Human genes 0.000 claims 5
- 241000004176 Alphacoronavirus Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 206010012310 Dengue fever Diseases 0.000 abstract description 13
- 208000020329 Zika virus infectious disease Diseases 0.000 abstract description 12
- 208000001490 Dengue Diseases 0.000 abstract description 11
- 208000025729 dengue disease Diseases 0.000 abstract description 11
- 201000011001 Ebola Hemorrhagic Fever Diseases 0.000 abstract description 9
- 230000001717 pathogenic effect Effects 0.000 abstract description 9
- 235000000346 sugar Nutrition 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 239000000427 antigen Substances 0.000 description 23
- 102000036639 antigens Human genes 0.000 description 22
- 108091007433 antigens Proteins 0.000 description 22
- 102100034459 Hepatitis A virus cellular receptor 1 Human genes 0.000 description 18
- 108090001126 Furin Proteins 0.000 description 16
- 102000004961 Furin Human genes 0.000 description 14
- 241000282414 Homo sapiens Species 0.000 description 14
- 230000006870 function Effects 0.000 description 14
- 210000001744 T-lymphocyte Anatomy 0.000 description 13
- 238000003556 assay Methods 0.000 description 10
- BMLTZEAFQLJTIZ-TZNXUKFXSA-N (2r)-2-amino-n-[(2r)-1-[[(2r)-1-[[(2r)-1-[[(2r)-1-[[(2r)-1-amino-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxop Chemical compound NC(N)=NCCC[C@@H](N)C(=O)N[C@H](CCCN=C(N)N)C(=O)N[C@H](CCCN=C(N)N)C(=O)N[C@H](CCCN=C(N)N)C(=O)N[C@H](CCCN=C(N)N)C(=O)N[C@H](CCCN=C(N)N)C(N)=O BMLTZEAFQLJTIZ-TZNXUKFXSA-N 0.000 description 9
- 239000008186 active pharmaceutical agent Substances 0.000 description 9
- NHBJTTGFHCHQHS-VZTVMPNDSA-N decanoyl-L-Arg-L-Val-L-Lys-L-Arg-chloromethylketone Chemical compound CCCCCCCCCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)CCl NHBJTTGFHCHQHS-VZTVMPNDSA-N 0.000 description 9
- 239000012636 effector Substances 0.000 description 9
- 108010085290 hexa-D-arginine Proteins 0.000 description 8
- 244000052769 pathogen Species 0.000 description 8
- 125000006850 spacer group Chemical group 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 102000003886 Glycoproteins Human genes 0.000 description 7
- 108090000288 Glycoproteins Proteins 0.000 description 7
- 108060003951 Immunoglobulin Proteins 0.000 description 7
- 235000018417 cysteine Nutrition 0.000 description 7
- 102000018358 immunoglobulin Human genes 0.000 description 7
- 238000006386 neutralization reaction Methods 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 7
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 6
- -1 cysteine amino acids Chemical class 0.000 description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 5
- 208000025721 COVID-19 Diseases 0.000 description 5
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 5
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 5
- 108010067390 Viral Proteins Proteins 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 208000037797 influenza A Diseases 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 241000711920 Human orthopneumovirus Species 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 102400001018 Proadrenomedullin N-20 terminal peptide Human genes 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 208000009714 Severe Dengue Diseases 0.000 description 4
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 4
- 108010046722 Thrombospondin 1 Proteins 0.000 description 4
- 102100036034 Thrombospondin-1 Human genes 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000000562 conjugate Substances 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 229910001868 water Inorganic materials 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 3
- 239000012515 MabSelect SuRe Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108020000999 Viral RNA Proteins 0.000 description 3
- HYSPJPGXSALJRR-DHIFEGFHSA-N [4-[[(2s)-5-(carbamoylamino)-2-[[(2s)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate Chemical compound N([C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=O)C(=O)NC=1C=CC(COC(=O)OC=2C=CC(=CC=2)[N+]([O-])=O)=CC=1)C(=O)CCCCCN1C(=O)C=CC1=O HYSPJPGXSALJRR-DHIFEGFHSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 230000000840 anti-viral effect Effects 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 150000001945 cysteines Chemical class 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000000834 fixative Substances 0.000 description 3
- 238000005755 formation reaction Methods 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 230000002132 lysosomal effect Effects 0.000 description 3
- 230000034217 membrane fusion Effects 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000012562 protein A resin Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 101100369802 Caenorhabditis elegans tim-1 gene Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 150000008555 D-arginines Chemical class 0.000 description 2
- 108010016626 Dipeptides Proteins 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 208000032163 Emerging Communicable disease Diseases 0.000 description 2
- 101710121417 Envelope glycoprotein Proteins 0.000 description 2
- 241000710781 Flaviviridae Species 0.000 description 2
- 241000710831 Flavivirus Species 0.000 description 2
- 208000035895 Guillain-Barré syndrome Diseases 0.000 description 2
- 241000700721 Hepatitis B virus Species 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 206010049567 Miller Fisher syndrome Diseases 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 102100029565 NPC intracellular cholesterol transporter 1 Human genes 0.000 description 2
- 101710187018 NPC intracellular cholesterol transporter 1 Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 102000006437 Proprotein Convertases Human genes 0.000 description 2
- 108010044159 Proprotein Convertases Proteins 0.000 description 2
- 108010008038 Synthetic Vaccines Proteins 0.000 description 2
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 241001115400 Zaire ebolavirus Species 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229940049595 antibody-drug conjugate Drugs 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- BULLHNJGPPOUOX-UHFFFAOYSA-N chloroacetone Chemical compound CC(=O)CCl BULLHNJGPPOUOX-UHFFFAOYSA-N 0.000 description 2
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 2
- 201000002950 dengue hemorrhagic fever Diseases 0.000 description 2
- 201000009892 dengue shock syndrome Diseases 0.000 description 2
- 206010014599 encephalitis Diseases 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000003818 flash chromatography Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000002998 immunogenetic effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 210000005007 innate immune system Anatomy 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 210000003563 lymphoid tissue Anatomy 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 230000000269 nucleophilic effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229940124551 recombinant vaccine Drugs 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 150000003573 thiols Chemical group 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- PVBORIXVWRTHOZ-UHFFFAOYSA-N (2,5-dioxopyrrol-1-yl)methyl cyclohexanecarboxylate Chemical compound C1CCCCC1C(=O)OCN1C(=O)C=CC1=O PVBORIXVWRTHOZ-UHFFFAOYSA-N 0.000 description 1
- ALBODLTZUXKBGZ-JUUVMNCLSA-N (2s)-2-amino-3-phenylpropanoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound NCCCC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CC1=CC=CC=C1 ALBODLTZUXKBGZ-JUUVMNCLSA-N 0.000 description 1
- 102100027518 1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial Human genes 0.000 description 1
- FUHCFUVCWLZEDQ-UHFFFAOYSA-N 1-(2,5-dioxopyrrolidin-1-yl)oxy-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonic acid Chemical compound O=C1CCC(=O)N1OC(=O)C(S(=O)(=O)O)CCSSC1=CC=CC=N1 FUHCFUVCWLZEDQ-UHFFFAOYSA-N 0.000 description 1
- PJUPKRYGDFTMTM-UHFFFAOYSA-N 1-hydroxybenzotriazole;hydrate Chemical compound O.C1=CC=C2N(O)N=NC2=C1 PJUPKRYGDFTMTM-UHFFFAOYSA-N 0.000 description 1
- 241000256111 Aedes <genus> Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 description 1
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000031504 Asymptomatic Infections Diseases 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 101000849787 Brucella melitensis biotype 1 (strain 16M / ATCC 23456 / NCTC 10094) Ribosome-recycling factor Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000004225 Cathepsin B Human genes 0.000 description 1
- 108090000712 Cathepsin B Proteins 0.000 description 1
- 102000005600 Cathepsins Human genes 0.000 description 1
- 108010084457 Cathepsins Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 201000009182 Chikungunya Diseases 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- 102000005853 Clathrin Human genes 0.000 description 1
- 108010019874 Clathrin Proteins 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 241000710844 Dengue virus 4 Species 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000032274 Encephalopathy Diseases 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 101710204837 Envelope small membrane protein Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000001860 Eye Infections Diseases 0.000 description 1
- 208000007136 Filoviridae Infections Diseases 0.000 description 1
- 102100035233 Furin Human genes 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010007712 Hepatitis A Virus Cellular Receptor 1 Proteins 0.000 description 1
- 102000007343 Hepatitis A Virus Cellular Receptor 1 Human genes 0.000 description 1
- 101000861278 Homo sapiens 1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial Proteins 0.000 description 1
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 1
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical group C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 101710145006 Lysis protein Proteins 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 208000003926 Myelitis Diseases 0.000 description 1
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 101800001030 Non-structural protein 2A Proteins 0.000 description 1
- 101710144111 Non-structural protein 3 Proteins 0.000 description 1
- 101800001020 Non-structural protein 4A Proteins 0.000 description 1
- 101800001019 Non-structural protein 4B Proteins 0.000 description 1
- 101710144121 Non-structural protein 5 Proteins 0.000 description 1
- 102000008212 P-Selectin Human genes 0.000 description 1
- 108010035766 P-Selectin Proteins 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 206010034620 Peripheral sensory neuropathy Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 206010036105 Polyneuropathy Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 208000035977 Rare disease Diseases 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241001479493 Sousa Species 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 108091077436 Tam family Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 101150098329 Tyro3 gene Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 208000011312 Vector Borne disease Diseases 0.000 description 1
- 230000010530 Virus Neutralization Effects 0.000 description 1
- 208000001455 Zika Virus Infection Diseases 0.000 description 1
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 208000002552 acute disseminated encephalomyelitis Diseases 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000003441 anti-flavivirus Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229960003008 blinatumomab Drugs 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 229960000455 brentuximab vedotin Drugs 0.000 description 1
- 239000000872 buffer Chemical group 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001201 calcium disodium ethylene diamine tetra-acetate Substances 0.000 description 1
- 239000001427 calcium tartrate Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229960000419 catumaxomab Drugs 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 229930193282 clathrin Natural products 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000010402 computational modelling Methods 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 244000096108 cunha Species 0.000 description 1
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000000368 destabilizing effect Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 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
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000013504 emergency use authorization Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000004651 endocytosis pathway Effects 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 208000011323 eye infectious disease Diseases 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 229940125777 fusion inhibitor Drugs 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229960003297 gemtuzumab ozogamicin Drugs 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 244000000013 helminth Species 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 208000005252 hepatitis A Diseases 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 102000051539 human HAVCR1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 229950004101 inotuzumab ozogamicin Drugs 0.000 description 1
- 229940030980 inova Drugs 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- LUEWUZLMQUOBSB-GFVSVBBRSA-N mannan Chemical class O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-GFVSVBBRSA-N 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 201000011475 meningoencephalitis Diseases 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 208000004141 microcephaly Diseases 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000003007 myelin sheath Anatomy 0.000 description 1
- 210000001178 neural stem cell Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000065 noncytotoxic Toxicity 0.000 description 1
- 230000002020 noncytotoxic effect Effects 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000007824 polyneuropathy Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000012910 preclinical development Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- RWWYLEGWBNMMLJ-MEUHYHILSA-N remdesivir Drugs C([C@@H]1[C@H]([C@@H](O)[C@@](C#N)(O1)C=1N2N=CN=C(N)C2=CC=1)O)OP(=O)(N[C@@H](C)C(=O)OCC(CC)CC)OC1=CC=CC=C1 RWWYLEGWBNMMLJ-MEUHYHILSA-N 0.000 description 1
- RWWYLEGWBNMMLJ-YSOARWBDSA-N remdesivir Chemical compound NC1=NC=NN2C1=CC=C2[C@]1([C@@H]([C@@H]([C@H](O1)CO[P@](=O)(OC1=CC=CC=C1)N[C@H](C(=O)OCC(CC)CC)C)O)O)C#N RWWYLEGWBNMMLJ-YSOARWBDSA-N 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 210000003660 reticulum Anatomy 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 201000005572 sensory peripheral neuropathy Diseases 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 229950001694 vadastuximab talirine Drugs 0.000 description 1
- BNJNAEJASPUJTO-DUOHOMBCSA-N vadastuximab talirine Chemical compound COc1ccc(cc1)C2=CN3[C@@H](C2)C=Nc4cc(OCCCOc5cc6N=C[C@@H]7CC(=CN7C(=O)c6cc5OC)c8ccc(NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CCCCCN9C(=O)C[C@@H](SC[C@H](N)C(=O)O)C9=O)C(C)C)cc8)c(OC)cc4C3=O BNJNAEJASPUJTO-DUOHOMBCSA-N 0.000 description 1
- 230000033041 viral attachment to host cell Effects 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 244000052613 viral pathogen Species 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
- A61K39/0002—Fungal antigens, e.g. Trichophyton, Aspergillus, Candida
-
- 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
- A61K39/002—Protozoa antigens
-
- 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
- A61K39/02—Bacterial antigens
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
-
- 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
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
-
- 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
- C07K14/76—Albumins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- 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 to the use of phosphotidylserine or pathogenic sugar targeted therapeutics for the management and treatment of microbial infections, including Zika, Dengue, Respiratory Syncytial Virus, West Nile, Ebola, H1N1, Mycobacterium Leprae , Mycobacterium tuberculosis , Enteroviruses , Leishmaniasis , Malaria and Coronaviruses SARS-CoV.
- microbial infections including Zika, Dengue, Respiratory Syncytial Virus, West Nile, Ebola, H1N1, Mycobacterium Leprae , Mycobacterium tuberculosis , Enteroviruses , Leishmaniasis , Malaria and Coronaviruses SARS-CoV.
- compositions related to novel, therapeutic proteins including pathogen neutralizing proteins that may be conjugated to furin protease inhibitors, T cell engagers, platforms with cytotoxic functions, mono and multivalent molecules, drug conjugates and adjuvants, carriers and methods of administration, in particular subcutaneous, oral or nasal administration.
- This invention further relates to a companion diagnostic as a method of selection of subjects that may benefit from such therapies and a blood biomarker for rapid and easy monitoring of response of treatment.
- a live, recombinant vaccine consisting of the envelope glycoprotein of one of the Ebola strains, Zaire Ebolavirus (Ervebo, 2019) was recently approved by the FDA for adult use only, although the duration of protection is still not entirely known.
- Another recombinant vaccine consisting of the pre-M and E proteins for all 4 strains of Dengue (Dengvaxia, 2019) has also been approved however Lim et al 2019 reported new mutations in DENV with different antigenic properties.
- ZIKV, WNV and DENV are flaviviruses (family Flaviviridae) primarily transmitted by mosquito vectors (i.e. arboviruses).
- Zika is a positive single stranded RNA flaviviridae virus mainly transmitted by the Aedes mosquito and an increasing number of strains in two phylogenetic lineages (Asian and African) have been identified since its first isolation in Kenya in 1947 (Ramos da Silva 2016).
- Zika is also spread through sexual contact (Musso 2015) and according to Rasmussen 2016, Zika is also spread from maternal to fetal blood (Rasmussen 2016).
- Rasmussen 2016 a particularly serious co-morbidity of Zika infection in pregnant women is severe congenital microcephaly to their progeny (Rasmussen 2016).
- Barbi 2018 in adults, Guillain-Barré syndrome (GBS), an auto-immune disease that destroys the myelin sheath and causes progressive ascending paralysis has been estimated to affect 1.23% of patients (Barbi 2018).
- GRS Guillain-Barré syndrome
- Other reported ZIKV neurological complications include encephalitis/meningoencephalitis, acute disseminated encephalomyelitis, myelitis, cerebrovascular complications, seizures and encephalopathy, sensory polyneuropathy and sensory neuropathy.
- Primary hosts of ZIKV include human, monkey, and mosquito. According to Hou 2017 neural stem cells, fibroblasts, epithelial and blood cells are permissive to ZIKV infection (Hou 2017).
- Dengue virus is a negative RNA strand flavivirus that causes the most prevalent arthropod-born viral disease in the world (1 million cases/year).
- DENV infection causes human diseases with a wide spectrum of clinical symptoms, ranging from asymptomatic infection or self-limited febrile illness named Dengue fever (DF) to life-threatening diseases including Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS).
- DF Dengue fever
- DHF Dengue hemorrhagic fever
- DSS Dengue shock syndrome
- Dengvaxia is a vaccine approved for individuals 9 through 16 years of age with laboratory-confirmed previous dengue infection and living in endemic areas.
- IgG Fc-binding mortif-conjugated HIV-1 fusion inhibitor exhibits improved potency and in vivo half-life: Potential application in combination with broad neutralizing antibodies, PLOS Pathogens, Dec. 5, 2019.
- a strategy have been developed to extend the in vivo half-life of a short HIV-1 fusion inhibitory peptide, CP24, by fusing it with the human IgG Fc-binding peptide (IBP).
- Glycans are essential structural and functional components of microbes. Among these, glucans, polysaccharide moieties derived from D-glucose, are prominent constituents of the cell walls of fungi, plants, and mycobacteria. High mannose containing structures (mannans) are expressed by many viruses, fungi, and bacteria, and fucose structures (fucans) are found on the surface of helminths and some bacteria (Geijtenbeek and Gringhuis, 2009; Robinson et al., 2006).
- the human immune system has evolved innate pattern-recognition receptors that discern self from non-self-glycans. Binding of C-type lectins to pathogen sugars triggers both innate and adaptive immune events that lead to pathogen clearance however this interaction may also be exploited to enhance pathogenicity.
- the myeloid, dendritic and macrophage cell specific C-type lectin receptor CD209 (also known as DC-SIGN), (Zelensky and Gready, 2005) is an important host cell receptor for entry of ZIKV (Perera Lecoin, 2013, Osorio and Sousa 2011), Influenza (Gillespie 2016), DENV (Cruz-Oliveira, 2015), WNV (Davis 2006), Ebola (Alvarez 2002), enterovirus (REN 2014), mycobacterium tuberculosis (Tailleux 2003) and mycobacterium Leprae (Barreiro 2006) and SARS-COV2/COVID19 (Amraei 2020, Cai 2020, Jeffers 2004).
- CD209 binds to both mannan (high-mannose N-linked oligosaccharides) and fucan moieties that comprise viral signatures or “pathogen associated molecular patterns (PAMPs). The binding occurs within a compact protein region with a unique structural fold that became known as the “C-type carbohydrate recognition domain” or “C-type lectin domain (CTLD)” (Weis and Drick- Amer, 1996).
- the outer virus membrane layer of several viruses is rich in phospholipid phosphatidylserine (PS) whereas in the host cell membrane, PS is normally restricted to the inner membrane layer.
- PS phospholipid phosphatidylserine
- T-cell immunoglobulin and mucin domain 1 human membrane receptor functions as a potent co-stimulatory molecule for T-cell activation.
- TIM1 (also known as HAVCR1) is a type I transmembrane glycoprotein that contains an extracellular domain composed of an N-terminal immunoglobulin variable (IgV)-like domain followed by a glycosylated mucin domain, a single transmembrane domain, and a short cytoplasmic tail with tyrosine phosphorylation motifs.
- IgV immunoglobulin variable
- the Ig V domain of TIM1 is predicted to contain a conserved PS binding site (Santiago et al., 2007).
- TIM-1 is expressed preferentially on T-helper 2 (Th2) cells in the brain, gastrointestinal tract, liver and gallbladder, kidney, testis and lymphoid tissue.
- Th2 T-helper 2
- TIM-1 recognizes and attaches to exposed PS with high specificity in dying, apoptotic cells and triggers their phagocytosis by the immune system (Freeman 2010).
- TIM-1 promotes apoptotic clearance by binding to PS through its metal ion-dependent ligand binding site (MILIBS) within the IgV domain.
- MILIBS metal ion-dependent ligand binding site
- TIM1 is an entry factor for highly divergent viruses (Jemielity, 2013), including Zika (Lee 2018), Ebola (Brunton 2019), Dengue (Chu 2019, Amara 2015), West Nile (Richard 2015), Hepatitis A and possibly Malaria (Nuchnoi 2020).
- TIM-1 functions as a common attachment factor for a range of enveloped viruses through direct interaction with PS of the viral envelope independent of glycoproteins.
- TIM1 is the most well-known PS receptor although other PS receptors have been described to a lesser extent such as Tyro3, Axl and Mer of the TAM family of proteins.
- Tim-1 as a Receptor for Viral Proteins
- TIM-1 plays a role in autoimmune and inflammatory disease development by controlling T cell adhesion through binding to P-selectin proteins via the TIM-1 mucin and IgV domains (Angiari 2014).
- TIM-1 may also participate in viral membrane fusion.
- human TIM-1 directly binds to EBOV glycoprotein (GP) and the authors determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4 as well as the binding region.
- GP EBOV glycoprotein
- TIM-1 on host epithelial cells of the trachea, cornea, and conjunctiva, binds directly to the receptor binding domain of the Zaire Ebola virus (EBOV) glycoprotein and enhances airborne and hand-to-eye infection. Blockage of this interaction with antibodies inhibited binding and Ebola infection.
- EBOV Zaire Ebola virus
- TIM-1 may have both protein dependent and independent functions in viral infection.
- Furin cleavage sites are present in entry proteins of Zika, Dengue, COVID-19 (Coutard 2020), Ebola, HIV, and Hepatitis B viruses among others (Braun 2019).
- Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) comprising SEQ ID NO. 81 and hexa-D-arginine (D6R) are small synthetic furin inhibitors that have been used to show reduction of viral infectivity in vitro (Owczarek, 2019, Imran 2019, Remacle 2010, Couture 2015).
- CMK is more effective than D6R in the reduction of Hepatitis replication by inhibiting furin-mediated processing of the hepatitis B e-antigen (HBeAg) precursor into mature HBeAg.
- Dec-RVKR-cmk is a small, synthetic, irreversible, and cell-permeable competitive inhibitor of all proprotein convertases (PC1, PC2, PC4, PACE 4, PC5, PC7, and furin).
- CMK is reported to inhibit furin-mediated cleavage and fusion activity of viral glycoproteins, and acts as an antiviral agent against different viruses, including human immunodeficiency virus, Chikungunya virus, chronic hepatitis B virus, influenza A, Ebola virus infection and papilloma virus. Smith et al. and Steinmetzer et al.
- furin knockout mice Wide-range furin/proprotein inhibitors are thought to have minimal off-pathogen, on-target effects in the host given that proprotein convertases are highly redundant, as shown by furin knockout mice.
- CMK has been shown to have anti-flavivirus activity at non-cytotoxic concentration (Imran 2019).
- Zika virus contains 3 structural (capsid-pC, envelope-pE and membrane-prM) and 7 non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5).
- Zika virus replication occurs in the permissive host cell after internalization via clathrin-mediated pH dependent endocytosis and maturation of viral proteins in the lysosomal compartment (Owczarek, 2019).
- furin or furin-like proteases cleave viral surface glycoprotein prM into its active form destabilizing the viral membrane and promoting release of the viral RNA for replication in mitochondria and endoplasmatic reticulum.
- furin inhibition causes the immature virion to be transported to late compartments where it undergoes proteolytic degradation.
- the degradation products are ejected from the cell via slow recycling vesicles (Owczarek, 2019).
- DENV binding of viral protein E with cellular receptors allows viral particles to internalize into the permissible cell via the chlathrin mediated endocytosis pathway.
- DENV virions undergo an acid-induced conformational change and membrane fusion.
- Newly synthesized viral proteins generated near the endoplasmic reticulum (ER) promote replication of the viral RNA genome, induction of membrane rearrangement, and assembly of new viral particles.
- ER endoplasmic reticulum
- DENV To facilitate the process of DENV replication, DENV not only interacts with various cellular components, but also triggers various host responses, such as autophagy.
- CD3 is a protein complex and T cell co-receptor that is involved in activating T cells. CD3 is selectively expressed on T cells in blood, bone marrow and lymphoid tissues, but not on other normal tissues and with no cross reactivity to other animals except for chimpanzee. Recently, human CD3 transgenic mice have been engineered, facilitating the study of anti-CD3 immunotherapies.
- Anti-CD3 based therapies such as muromomab-CD3 (Janssen, Orthoclone, OKT3) have been extensively studied in humans both systemically and orally to block reactive T cells and ameliorate ulcerative cholitis and metabolic syndrome (da Cunha 2011, Ilan 2010-NCT01287195, NCT01205087).
- Anti-CD3 bispecific antibody platforms that bridge tumors and engage T cells such as blinatumomab and catumaxomab have been approved for the treatment of cancer and several others CD3 bispecifics are in clinical development (Suurs, 2019).
- the invention concerns a fusion construct comprising an Ig-Fc domain or other protein scaffold, such as albumin, and
- PAMP refers to Pathogen-associated molecular pattern: conserved molecular structures produced by microbial pathogens, but not by the host organism that are recognized by the host innate immune system.
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- ADCC may be defined as Antibody-Dependent Cellular Cytotoxicity.
- ADCP may be defined as Antibody-Dependent Cellular Phagocytosis.
- CDC may be defined as Complement-dependent cytotoxicity.
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- the Furin inhibitor is selected among chloromethylketone and D-arginine derivatives such as hexa-D-arginine and dec-RVKR-cmk (comprising SEQ ID NO. 81).
- linker and spacers may be conjugated to furin, see Table 7.
- the invention concerns a fusion construct, wherein said fusion construct is an IgG3 construct, and wherein said IgG3 construct comprises a hinge region, wherein said hinge region has been modified.
- the invention concerns a fusion construct, a fusion protein or an antibody comprising the constant region(s) of IgG3 and a hinge, wherein said hinge preferably is selected among an IgG1 or IgG4 hinge.
- the invention concerns IgG3 homodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
- the invention concerns IgG3 heterodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
- the invention concerns IgG3, wherein said IgG3 comprises a mutation at position 405 and/or position 409.
- the invention concerns IgM heterodimers obtainable by changing the charge pairs of the CH2 and/or CH4 domains.
- the invention concerns IgM heterodimers, comprising one or more of the mutations of Table 8.
- the invention concerns a fusion construct, wherein said fusion construct comprises an IgG3 homodimer, an IgG3 heterodimer and/or an IgM heterodimer according to the invention.
- the invention concerns use of a fusion construct according to the invention for the treatment of an infection.
- the invention concerns use, wherein said infections are selected among viral, bacterial, and protozoan infections.
- the invention concerns use, wherein the treatment comprising administration of the fusion construct with an administration form selected among subcutaneous, intradermal, intramuscular, oral and nasal.
- the invention concerns use of IgG4 or a part of IgG4 for payload delivery, wherein said IgG4 has been modified to comprise no Fc or wherein the activity of the Fc of said IgG4 has been nullified or diminished by one or more mutations.
- the invention concerns a vaccine comprising a fusion construct according to the invention.
- the invention concerns a vaccine comprising a mannan, a high mannose containing structure, a fucan and/or a phospholipid phosphatidylserine (PS).
- a vaccine comprising a mannan, a high mannose containing structure, a fucan and/or a phospholipid phosphatidylserine (PS).
- the invention concerns a composition
- a composition comprising a fusion construct according to the invention, optionally comprising one or more excipients such as diluents, binders or carriers.
- the invention concerns a method of treating and/or preventing an infection in a subject, comprising a step of administration of a fusion construct and/or a vaccine and/or a composition according to the invention.
- the invention concerns a method of screening and/or monitoring progression of a disease in a subject, wherein said method comprises the following steps:
- the invention concerns an isolated nucleic acid molecule encoding a fusion construct according to the invention.
- the invention concerns a recombinant vector comprising the nucleic acid molecule of the invention.
- the invention concerns a host cell comprising the recombinant vector of the invention.
- the invention concerns a method to produce a fusion construct according to the invention comprising a step of culturing the host cell according to the invention in a culture medium under conditions allowing the expression of the fusion construct and separating the fusion construct from the culture medium.
- the invention concerns a fusion construct comprising an Ig-Fc domain or other protein scaffold, such as albumin, and
- PAMP refers to Pathogen-associated molecular pattern: conserved molecular structures produced by microbial pathogens, but not by the host organism that are recognized by the host innate immune system.
- protein scaffold refers to a protein structure on which the active elements defined in a. and b. above can be bound.
- the protein scaffold should preferably be soluble in plasma and preferably have a high residence time in plasma, which typically can be provided if the complete fusion protein has a size above the renal clearance limit, such as above 60 kDa, or by selecting a protein scaffold that is subject to an active retention system e.g. proteins binding and recycled to the plasma via the FcRn receptor.
- suitable protein scaffolds include plasma proteins or fragments thereof, such as constant regions of immunoglobulins, albumin, albumin domain I, II, or III, transferrin and lactoferrin.
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- the invention concerns a fusion construct comprising
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- ADCC may be defined as Antibody-Dependent Cellular Cytotoxicity.
- ADCP may be defined as Antibody-Dependent Cellular Phagocytosis.
- CDC may be defined as Complement-dependent cytotoxicity.
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- the Furin inhibitor is selected among chloromethylketone and D-arginine derivatives such as hexa-D-arginine and dec-RVKR-cmk (comprising SEQ ID NO. 81).
- linker and spacers may be conjugated to furin, see Table 7.
- the invention concerns the fusion construct, wherein said peptide, protein or antibody fragment is capable of binding to and/or stimulating an immune cell.
- the invention concerns the fusion construct, wherein said TIM1 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 50-180 amino acid residues, 60-160 amino acid residues, 70-140 amino acid residues, 80-130 amino acid residues, 90-120 amino acid residues, 100-120 amino acid residues and 100-110 amino acid residues.
- the invention concerns the fusion construct, wherein said CD209 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 40-190 amino acid residues, 50-180 amino acid residues, 60-170 amino acid residues, 70-160 amino acid residues, 80-150 amino acid residues, 90-150 amino acid residues, 100-150 amino acid residues, 110-150 amino acid residues, 120-150 amino acid residues and 130-140 amino acid residues.
- the invention concerns the fusion construct, wherein said TIM1 and/or CD209 fragment has a sequence homology of at least 70%, alternatively 75%, alternatively 80%, alternatively 85%, alternatively 90%, alternatively 95% to wildtype TIM1 or CD209.
- the invention concerns the fusion construct, wherein said TIM1 and/or CD209 fragment has intact TIM1 and/or CD209 function.
- the invention concerns the fusion construct, wherein said IgG-Fc domain is an IgG3-Fc domain.
- the invention concerns the fusion construct, comprising additionally at least one of the following:
- the invention concerns the fusion construct, wherein said fusion construct comprises a sequence according to SEQ ID No.: 1 and/or SEQ ID No.: 2.
- the invention concerns the fusion construct, wherein said fusion construct comprises a sequence according to SEQ ID No.: 3 and/or SEQ ID No.: 4.
- the invention concerns the fusion construct, wherein said fusion construct comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or preferably at least 8 disulfide bonds.
- the invention concerns the fusion construct, wherein said fusion construct is capable of binding to a target, and wherein said target is a mannan, a high-mannose containing structure, a fucan, a phospholipid phosphatidylserine and/or CD3.
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises
- the invention concerns the fusion construct, wherein said fusion construct comprises a linker.
- the invention concerns the fusion construct, wherein said linker is selected among a (GGGGS)3 linker (SEQ ID NO. 41), a (GGGGS)4 linker (SEQ ID NO. 70), a (GGGGS)5 linker (SEQ ID NO. 71) and a (GGGGS)6 linker (SEQ ID NO. 72).
- said linker is selected among a (GGGGS)3 linker (SEQ ID NO. 41), a (GGGGS)4 linker (SEQ ID NO. 70), a (GGGGS)5 linker (SEQ ID NO. 71) and a (GGGGS)6 linker (SEQ ID NO. 72).
- a (GGGGS) linker may be defined as a Gly-Gly-Gly-Gly-Ser linker (SEQ ID NO. 69).
- the invention concerns the fusion construct, wherein said fusion construct comprises at least one free cysteine residue, at least two free cysteine residues, at least three free cysteine residues, at least four free cysteine residues, at least five free cysteine residues or preferably at least six free cysteine residues.
- the invention concerns the fusion construct, wherein said free cysteine allows interaction with a drug and/or a payload.
- the invention concerns the fusion construct, wherein said payload is a furin inhibitor.
- the invention concerns the fusion construct, wherein said fusion construct comprises a A339C mutation, a S337C mutation and/or a K340C mutation.
- the invention concerns the fusion construct, wherein said fusion construct comprises a sequence selected among any of the sequences SEQ ID No.: 36, 37, SEQ ID No.: 38, 39, 40, 42, 44 or 46.
- the invention concerns the fusion construct, wherein said fusion construct is an IgG1, IgG2, IgG3 or an IgG4.
- the invention concerns the fusion construct, wherein said fusion construct is an IgG, IgM, IgA, IgD or an IgE.
- the invention concerns the fusion construct, wherein said fusion construct comprises a null fc.
- the invention concerns the fusion construct, wherein said null fc comprises an Ala substitution at position 234 and/or Ala substitution at 235, and/or N297A, and/or a K322A mutation.
- the invention concerns the fusion construct, wherein said fusion construct comprises a heterodimerization domain.
- the invention concerns the fusion construct, wherein said heterodimerization domain comprises a sequence according to SEQ ID No.: 48, 49 or 50.
- the invention concerns the fusion construct, wherein said fusion construct comprises a heterodimerization mutation.
- the invention concerns the fusion construct, wherein said heterodimerization mutation is an F405L and/or K409R mutation.
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein said fusion construct comprises:
- the invention concerns the fusion construct, wherein the ratio of fusion construct to said drug and/or payload is selected among 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
- the invention concerns the fusion construct, wherein said fusion construct comprises a kappa light chain according to SEQ ID No.: 51 or a lambda light chain according to SEQ ID No.: 52 or 53.
- the invention concerns a fusion construct, wherein said fusion construct is an IgG3 construct, and wherein said IgG3 construct comprises a hinge region, wherein said hinge region has been modified.
- the invention concerns the fusion construct, wherein said hinge region comprises a sequence having a total of at least 10% identity, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the sequence according to SEQ ID No.: 6 or SEQ ID No.: 8.
- the invention concerns the fusion construct, wherein said fusion construct comprises the sequence according to SEQ ID No.: 5, 7, 9, 10, 11, 12 and/or 13.
- the invention concerns the fusion construct, wherein said hinge region comprises at least one free cysteine residue, at least two free cysteine residues or preferably at least three free cysteine residues.
- the invention concerns the fusion construct, wherein said hinge region comprises a S228P mutation.
- the invention concerns the fusion construct, wherein said hinge region comprises a sequence according to SEQ ID No.: 6 and/or SEQ ID No.: 8 and/or SEQ ID No.: 68.
- the invention concerns the fusion construct, wherein said fusion construct is used to detect phosphatidylserine.
- the invention concerns the fusion construct, wherein said fusion construct is used to detect phosphatidylserine in the blood of a subject.
- the invention concerns the fusion construct, wherein said fusion construct comprises a sequence according to SEQ ID No.: 1, and/or a sequence according to SEQ ID No.: 2.
- the invention concerns the fusion construct, wherein said fusion construct is used to detect C-type lectin binding mannan or fucan moieties.
- the invention concerns the fusion construct, wherein said fusion construct is used to detect C-type lectin binding mannan or fucan moieties in the blood of a subject.
- the invention concerns the fusion construct, wherein said fusion construct comprises a sequence according to SEQ ID No.: 3 and/or a sequence according to SEQ ID No.: 4.
- the invention concerns a fusion construct, a fusion protein or an antibody comprising the constant region(s) of IgG3 and a hinge, wherein said hinge preferably is selected among an IgG1 or IgG4 hinge.
- the invention concerns the fusion construct, fusion protein or antibody, comprising one or more heterodimerization mutations.
- the invention concerns the fusion construct, fusion protein or antibody, comprising heterodimerization mutations involve positions 405 and/or 409 (EU numbering).
- the invention concerns IgG3 homodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
- the invention concerns IgG3 heterodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
- the invention concerns IgG3, wherein said IgG3 comprises a mutation at position 405 and/or position 409.
- the invention concerns IgM heterodimers obtainable by changing the charge pairs of the CH2 and/or CH4 domains.
- the invention concerns IgM heterodimers, comprising one or more of the mutations of Table 8.
- the invention concerns the IgM, wherein said IgM comprises a sequence according to SEQ ID No.: 64 and/or 65.
- the invention concerns a fusion construct, wherein said fusion construct comprises an IgG3 homodimer, an IgG3 heterodimer and/or an IgM heterodimer according to the invention.
- the invention concerns the fusion construct, wherein said fusion construct is for use in the treatment of an infection.
- the invention concerns the fusion construct, wherein said infection is an infection caused by a virus, a parasite, a bacterium, a fungi or a protozoan.
- the invention concerns the fusion construct, wherein said virus is selected among an arborvirus, Zika virus, Dengue virus, West Nile virus, Ebola virus, influenza virus, influenza virus H1N1, Chikungunya virus, Enterovirus and Coronaviruses SARS-COV.
- said virus is selected among an arborvirus, Zika virus, Dengue virus, West Nile virus, Ebola virus, influenza virus, influenza virus H1N1, Chikungunya virus, Enterovirus and Coronaviruses SARS-COV.
- the invention concerns the fusion construct, wherein said bacteria is selected among mycobacterium tuberculosis and mycobacterium leprae .
- the invention concerns the fusion construct, wherein said parasite is selected among Leishmaniasis and Malaria.
- the invention concerns use of a fusion construct according to the invention for the treatment of an infection.
- the invention concerns use, wherein said infections are selected among viral, bacterial and protozoan infections.
- the invention concerns use, wherein the treatment comprising administration of the fusion construct with an administration form selected among subcutaneous, intradermal, intramuscular, oral and nasal.
- the invention concerns use of IgG4 or a part of IgG4 for payload delivery, wherein said IgG4 has been modified to comprise no Fc or wherein the activity of the Fc of said IgG4 has been nullified or diminished by one or more mutations.
- the invention concerns the use, wherein said IgG4 comprises one or more heterodimerization mutations.
- the invention concerns the use, wherein said IgG4 comprises one or more Cys mutations, preferably thereby allowing site specific conjugation.
- the invention concerns the use, wherein said IgG4 comprises a Cys at position 339 (EU numbering).
- the invention concerns a vaccine comprising a fusion construct according to the invention.
- the invention concerns a vaccine comprising a mannan, a high mannose containing structure, a fucan and/or a phospholipid phosphatidylserine (PS).
- a vaccine comprising a mannan, a high mannose containing structure, a fucan and/or a phospholipid phosphatidylserine (PS).
- the invention concerns the vaccine further comprising a ⁇ -glucan adjuvant to potentiate immune response.
- the invention concerns the vaccine, for the prevention and/or treatment of an infection.
- the invention concerns the vaccine, wherein said infection is caused by a virus, a parasite, a bacterium, a fungus or a protozoan.
- the invention concerns the fusion construct and/or vaccine, wherein said fusion construct and/or vaccine allows administration through a route selected among subcutaneous administration, intradermal administration, intramuscular administration, oral administration and/or nasal administration.
- the invention concerns a composition
- a composition comprising a fusion construct according to the invention, optionally comprising one or more excipients such as diluents, binders or carriers.
- the invention concerns a method of treating and/or preventing an infection in a subject, comprising a step of administration of a fusion construct and/or a vaccine and/or a composition to the invention.
- the invention concerns a method of screening and/or monitoring progression of a disease in a subject, wherein said method comprises the following steps:
- the invention concerns an isolated nucleic acid molecule encoding a fusion construct according to the invention.
- the invention concerns a recombinant vector comprising the nucleic acid molecule according to the invention.
- the invention concerns a host cell comprising the recombinant vector according to the invention.
- the invention concerns a method to produce a fusion construct according to the invention comprising a step of culturing the host cell according to the invention in a culture medium under conditions allowing the expression of the fusion construct and separating the fusion construct from the culture medium.
- the invention concerns a fusion construct, wherein said fusion construct comprises a hinge region, wherein said hinge region comprises any of the sequences as described below:
- the invention concerns a fusion construct, wherein said fusion construct comprises an Fc heterodimerization sequence at residue 405-409, wherein said Fc heterodimerization sequence comprises a sequence according to SEQ ID No.: 48, 49 or 50.
- IgG Fc heterodimerization sequences (residues 405-409) Construct Sequence IgG2 FLYSK SEQ ID No.: 48 V-IGG2/-C FLYSK SEQ ID No.: 48 V-IGG2-A/-D FLYSR SEQ ID No.: 49 V-IGG2-B/-E LLYSK SEQ ID No.: 50 IgG3 FLYSK SEQ ID No.: 48 V-IGG3/-C FLYSK SEQ ID No.: 48 V-IGG3-A/-D FLYSR SEQ ID No.: 49 V-IGG3-B/-E LLYSK SEQ ID No.: 50 IgG4 FLYSR SEQ ID No.: 49 V-IGG4-A FLYSR SEQ ID No.: 49 V-IGG4-B LLYSK SEQ ID No.: 50
- Immunoglobulins are glycoproteins composed of one or more units, each containing four polypeptide chains: two identical heavy chains (HCs) and two identical light chains (LCs).
- the amino terminal ends of the polypeptide chains show considerable variation in amino acid composition and are referred to as the variable (V) regions to distinguish them from the relatively constant (C) regions.
- Each light chain consists of one variable domain, VL, and one constant domain, CL.
- the heavy chains consist of a variable domain, VH, and three constant domains CH1, CH2 and CH3. Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure.
- the V regions of H and L chains comprise the antigen-binding sites of the immunoglobulin (Ig) molecules. Each Ig monomer contains two antigen-binding sites and is said to be bivalent.
- the Fab contains one complete L chain in its entirety and the V and CH1 portion of one H chain.
- the Fab can be further divided into a variable fragment (Fv) composed of the VH and VL domains, and a constant fragment (Fb) composed of the CL and CH1 domains.
- the H chain constant domain is generally defined as CH1-CH2-CH3 (IgG, IgA, IgD) with an additional domain (CH4) for IgM and IgE.
- CH1 domain is located within the F(ab) region whereas the remaining CH domains (CH2—CH3 or CH2—CH4) comprise the Fc fragment.
- This Fc fragment defines the isotype and subclass of the immunoglobulin.
- CH3 domain The terms CH3 domain and CH3 region are used interchangeable herein.
- CH1 domain The terms CH1 domain and CH1 region are used interchangeable herein.
- Hinge region The hinge region is the area of the heavy chains between the first and second C region domains and is held together by disulfide bonds.
- a hinge region typically comprises between 10 and 30 amino acid residues.
- a linker might be a peptide linker or a non-peptide linker.
- An example of a peptide linker is a Gly/Ser peptide linker comprising a five amino acid residue unit, GGGGS (SEQ ID NO:71), that can be repeated a suitable amount of times.
- a linker might be a naturally occurring linker or a synthetically produced linker. A linker might occur naturally in a molecule or might be synthetically added to a molecule.
- an “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody.
- antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments.
- antibody fragments include isolated fragments, “Fv” fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker (“ScFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
- an antibody fragment contains sufficient sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some embodiments, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen.
- antigen binding fragments of an antibody include, but are not limited to, Fab fragment, Fab′ fragment, F(ab′)2 fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd′ fragment, Fd fragment, and an isolated complementarity determining region (CDR) region.
- An antigen-binding fragment of an antibody may be produced by any means.
- an antigen-binding fragment of an antibody may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence.
- antigen-binding fragment of an antibody may be wholly or partially synthetically produced.
- An antigen-binding fragment of an antibody may optionally comprise a single chain antibody fragment.
- an antigen-binding fragment of an antibody may comprise multiple chains that are linked together, for example, by disulfide linkages.
- An antigen-binding fragment of an antibody may optionally comprise a multi-molecular complex.
- a functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.
- Antibody or fragment thereof refers to an antibody or antibody fragment as defined above.
- Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.
- IMGT the international ImMunoGeneTics information system, is an international reference in immunogenetics and immunoinformatics.
- Single-chain Fv Single-chain Fv
- scFvs Single-chain Fvs
- a single-chain Fv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, often connected by a short linker peptide (see, e.g., see, e.g., Benny K. C. Lo (ed.), Antibody Engineering - Methods and Protocols, Humana Press 2004, and references cited therein).
- FIG. 1 shows TIM1 and CTLD constructs with enhanced ADCC, ADCP and CDC.
- FIG. 2 shows TIM1 and CTLD constructs with T cell engager activity.
- FIG. 3 shows TIM1 and CTLD constructs with furin inhibitor payload.
- FIG. 4 shows TIM1 and CTLD constructs with IgM effector function.
- FIGS. 5 a and 5 b shows SEC-HPLC analysis of VP019 and VP020 respectively.
- FIG. 6 a shows that VP025 consists of 4 distinct peaks.
- FIG. 6 b intact mass spectrometry Fraction 3 from peak 3 (VP025-F3).
- FIG. 6 c intact mass spectrometry: Fraction 4 from peak 4 (VP025-F4).
- FIGS. 7 a and 7 b show the schematics and purity of VP300 and VP301 by SEC-HPLC.
- FIG. 8 shows binding of SARS-CoV-2 S protein (D614G)
- FIG. 9 shows binding of VP025-CT to viral proteins
- FIGS. 10 a and 10 b show binding curves of VP019, VP020, VP025-CT (heterodimer mixture) and VP025-F4 (78% pure heterodimer) to a biotin-phosphatidyl serine and a select group of viral antigens.
- FIG. 11 shows the structure of Hexa-D-arginine linker-compound and Decanoyl-RVKR-CMK linker compound
- FIG. 12 shows the reaction scheme for the Decanoyl-RVKR-CMK linker compound
- FIGS. 13 a and b shows a mass-spectrogram demonstrating that conjugation of furin inhibitor payload to VP020 has been accomplished.
- FIG. 14 a shows a neutralization assay for selected fusion proteins of the invention. For more details see example 16.
- FIG. 14 b shows a neutralization assay for selected fusion protein of the invention. For more details see example 17.
- V-TIM1-1 was selected as residues 21-125 of the full length TIM-1 sequence (https://www.uniprot.org/uniprot/Q96D42), and V-TIM1-2 was selected as residues 21-127.
- V-TIM1-2 contains an extra two Pro residues at the C-terminal domain boundary.
- V-CTLD-1 was selected as residues 250-385 of the full length DC-SIGN sequence (https://www.uniprot.org/uniprot/Q9NNX6), and V-CTLD-2 was selected as residues 254-383.
- V-CTLD-1 contains 4 internal disulfide bonds
- V-CTLD-2 contains 3 internal disulfide bonds.
- IgG3 has the highest effector functions in terms of ADCC, ADCP and CDC (https://www.frontiersin.org/articles/10.3389/fimmu.2014.00520/full). IgG3 has not typically been used for therapeutics because of the short serum half-life due to proteolytic cleavage of the prolonged hinge region between the CH1 and CH2 domains.
- the V-IGG3 construct was designed where the IgG3 hinge (LKTPLGDTTHTPEPKSCDTPPPCPRCPAP) (SEQ ID NO.
- V-IGG3 constructs were designed based on including K409R (on one half-antibody) and F405L (on second antibody) mutation in the CH3 domains (https://www.nature.com/articles/nprot.2014.169). Each half antibody is first generated as a single homodimer, then mixed together and allowed to recombine as heterodimers under reducing and oxidizing conditions. The resulting sequences are noted as V-IGG3-A and V-IGG3-B and pair together, or V-IGG3-D and V-IGG3-E that pair together. Sequences are found in Table 3, including truncated version that include a (GGGGS)3 linker (SEQ ID NO. 41) to replace the CH1 domains.
- GGGGS GGGGS
- TIM1 and CTLD fusion proteins were designed with the modified IgG3-Fc domains and are depicted in FIG. 1 and Table 4.
- VIT-303 V-TIM1-1/V-TIM1-2)-(V-IGG4-A-Fc/ V-IGG2-A-Fc/V-IGG2-D-Fc) (V-TIM1-1/V-TIM1-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) VIT-304 (V-CTLD-1/V-CTLD-2)-(V-IGG4-A-Fc/ V-IGG2-A-Fc /V-IGG2-D-Fc) (V-CTLD-1/V-CTLD-2)-(V-IGG4-B-Fc/ V-IGG2-E-Fc)-GGGGSGGSG
- TIM1 and CTLD fusion proteins with Fc domain with payload conjugation sites were designed and are shown in FIG. 3 and Table 8.
- Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) (SEQ ID NO. 81) or hexa-D-arginine (D6R) were linked to TIM-1 and CTLD constructs using cleavable linkers such as acid sensitive N-acyl-hydrazone or enzyme sensitive malemeide-conjugated dipeptides, valine-alanine, valine-citrulline, or phenylalanine-Lysine.
- cleavable linkers such as acid sensitive N-acyl-hydrazone or enzyme sensitive malemeide-conjugated dipeptides, valine-alanine, valine-citrulline, or phenylalanine-Lysine.
- Acid sensitive linkers are cleaved in the lysosome acidic environment after internalization of the construct. This strategy has been used in two approved ADCs, Gemtuzumab ozogamicin and Inotuzumab ozogamicin. Lysosomal protease sensitive dipeptides release the drug after cleavage by proteases such as cathepsin B- lysosomal protease. This type of linker chemistry has been used for FDA approved Brentuximab vedotin.
- Linkage to the polypeptide of antibodies is done through the nucleophilic groups of lysine or cysteine by random conjugation, generating a heterogeneous mixture of conjugates, or by site-directed conjugation to engineered cysteines, reducing the heterogeneity of the product to an antibody-drug ratio (ADR) of 1 or 2.
- ADR antibody-drug ratio
- the nucleophilic reactivity of the thiol functionality of a Cys residue to a maleimide group is about 1000 times higher compared to any other amino acid functionality in a protein, such as amino group of lysine residues or the N-terminal amino group.
- Thiol specific functionality in maleimide reagents may react with amine groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).
- the first FDA approved site-directed ADC through engineered cysteines was vadastuximab talirine (Seattle Genetics).
- Spacer 1 The purpose of the mc spacer is to provide enough room so that the vc group can be recognized by cathepsin B, which cleaves the citrulline-PABC amide bond.
- Spacer 2 Self-immolative spacer
- IgM molecules have robust Fc effector functions, particularly with CDC. IgM molecules naturally homodimerize and then covalently associate into pentamers or hexamers. IgM do not contain hinge regions like IgG molecules and instead contain an extra CH domain (CH1-CH2-CH3-CH4). The homodimeric heavy chains come together at the CH2 and CH4 domains.
- IgM CH2-CH3-CH4 which can used for fusing to antibody fragments (Fab, scFv, VHH, etc) or targeting proteins (TIM-1, CTLD/DC-SIGN) for adding IgM effector functions (residues 105-453): V-IGM
- TIM1 and CTLD fusion proteins with IgM effector functions were designed and shown in FIG. 4 and Table 11.
- VP011 100 mL
- VP012 100 mL
- VP013 100 mL
- VP014 100 mL
- VP019 1L
- VP020 250 mL
- VP011, VP019 and VP020 were purified by MabSelect SuRe protein A resin column chromatography.
- VP012, VP013 and VP014 were purified by HiTrap Protein G resin column chromatography. Expression yields and % monomeric purity are shown in Table 13.
- FIG. 5 a SEC-HPLC analysis of VP019 shows that VP019 form the expected homodimer (Fraction F6) and multimer ⁇ 600 kDa (Fraction F2). The fractions were purified out and assessed separately for functional activity (VP019-F6 and VP019-F2, respectively named).
- FIG. 5 b SEC-HPLC analysis of VP020.
- the resulting co-transfected sample product was named VP025-CT.
- FIG. 6 a shows that VP025-CT contains 4 distinct peaks which were purified out and assessed separately by intact mass spectrometry. The fractions from Peak 1 and Peak 2 showed a number of species.
- Fraction 3 from peak 3 (VP025-F3) showed a mass of 86,721 Da, corresponding to a VP020 homodimer ( FIG. 6 b ).
- Fraction 4 from peak 4 (VP025-F4) showed a mass of 83,881 Da, corresponding to the properly formed VP019/VP020 heterodimer, at an approximate purity of 78% ( FIG. 6
- Example 10 Expression and Purification of VP300 and VP301
- Example 11 Binding of VP019, VP020 and VP025 to SARS-COV2 S Protein
- VP019-F2, VP020 and VP025-CT were investigated by ELISA.
- This protein is representative of the dominant SARS-COV-2 strain in early 2020. All ELISA assays in this and subsequent examples were done in the presence of 2.5 mM CaCl 2 since DC-SIGN is known to use calcium at the binding site. Binding curves are shown in FIG. 8 and EC50 values are shown in Table 16. VP019-F2 had unexpectedly high binding to SARS-COV2 S Protein, while VP020 had modest binding.
- Example 12 Binding of VP025-CT to Wide Range of Viral Antigens
- Example 13 Binding of VP019, VP020, VP025-CT and VP025-F4 to Phosphatidyl Serine And viral antigens
- VP019, VP020, VP025-CT (heterodimer mixture) and VP025-F4 (78% pure heterodimer) to a biotin-phosphatidyl serine and a select group of viral antigens (Influenza A H1N1 HA, Human RSV Glycoprotein G, Zika Virus Envelope Protein and SARS-Cov-2 S D614G) was investigated by ELISA. Binding curves are shown in FIGS. 10 a and 10 b and EC50 values are shown in Table 18 and Table 19. VP025-F4 had stronger binding to all of the antigens than VP025-CT.
- Solvents and reagents were purchased from Sigma-Aldrich, VWR, or Fisher Scientific, and used without further purification. Reactions were monitored either by thin-layer chromatography (TLC) or by analytical liquid chromatography-mass spectrometry (LC-MS) employing a Waters Acquity Ultra Performance LC system and a Synapt high-definition mass spectrometer. 1 H NMR spectra were recorded on a Varian Unity INOVA spectrometer (500 MHz). All chemical shifts are reported in ppm and coupling constants, J, are reported in hertz (Hz).
- TLC thin-layer chromatography
- LC-MS analytical liquid chromatography-mass spectrometry
- NMR solvent peaks were referenced as follows: ( 1 H NMR) CDCl 3 : 7.27 ppm, DMSO-d 6 : 2.50 ppm.
- Compounds were purified by flash column chromatography on a Teledyne ISCO Combi-Flash system using normal phase silica gel (SiliCycle Inc.) or reverse phase (Teledyne Gold- C18 or C18-Aq) pre-packed columns. The purity of compounds was determined by analytical HPLC (Waters Acquity Ultra Performance) using an Acquity UPLC CSH C18 1.7 ⁇ m (50 mm x 2.1 mm) column and flow rate of 0.3 mL/min.
- the reaction scheme is shown in FIG. 12 .
- N-methyl morpholine (13.8 ⁇ lL, 0.126 mmol) was added to a solution of hexa-D-Arg (D-Argininamide D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-alanine; Ambeed, cat# A333458) (30 mg, 0.0314 mmol), MC-Val-Cit-PAB-PNP (BroadPharm Cat#: BP-23292, CAS: 159857-81-5) (46.4 mg, 0.0629 mmol) and HOBt.H 2 O (1-hydroxybenzotriazole monohydrate; 5.3 mg, 0.0345) in anhydrous DMF (1 mL) under argon atmosphere.
- the structure is shown in FIG. 11 .
- a test conjugation of Hexa-D-arginine linker-compound to VP020 was performed by reacting VP025 with 4 equivalents of TCEP and incubating at 37° C. for 1 hour to reduce the free cysteines.
- the sample was run through a Zeba column to remove TCEP and buffer exchanged into 1x PBS containing 1 mM DTPA pH 6.5.
- the sample was then reacted with 2.5 equivalents of the payload (Mc-VC-PAB-(D-Arg6)-CONH2 at Room temperature for 1 hour.
- the final product was analyzed by mass spectrometry (see FIG. 13 ) and shown to have the mass of 88,136 Da, near the expected mass of 88,138 Da.
- Example 16 RSV Neutralization Assay for VP019, VP020 and VP025
- a microneutralization assay was done to determine the antiviral properties of three compounds (VP019-F2, VP020 and VP025-F4) against RSV. Each virus was incubated with each antibody for 1 hour, after which the mix was added to A549 cells (human lung cancer cell line). Antiviral activity was determined 24 h later using an immunofluorescence-based assay. After 24 h, the infection plates were washed with PBS, fixed for 30 mins with 4% formaldehyde, washed again with PBS, and stored in PBS at 4° C. until staining.
- test articles were used in concentrations of 0.5 ⁇ M and samples were tested in triplicate. The resulting data are shown in FIG. 14 .
- VP019-F2 had 97% viral inhibition, compared to 55% inhibition for VP025-F5 and 28% inhibition for VP020.
- Example 17 ZIKV Neutralization Assay for VP025
- a second neutralization assay was done for Zika virus using VP025-F4 using the following procedures.
- Vero E6 cells were maintained with DMEM supplemented with 2% FBS and (1% pen-strep -need to confirm with Allen) and stored at 37° C. with 5% CO 2 . Cells were seeded onto 48-well plates at a concentration of 8.0*10 4 cells per well and allowed to adhere overnight. On the morning of infection cell monolayers were examined to ensure 90-95% confluency.
- VP025-F4 was serially diluted in triplicate using infection media at a ratio of 1:3 for a total of eight dilutions (220 to 0.1 ⁇ g/mL).).
- Zika virus (ZIKV), strain MEX-I-44, at a MOI of 1.0 (8.0*10 4 FFU) was added to each dilution, mixed, and incubated at 37° C. and 5% CO 2 for one hour.
- Mouse ⁇ -ZIKV MIAF mouse immune ascitic fluid antibody
- Vero E6 well plate was removed from the incubator. Media was aspirated from the cells and the test and positive control samples were transferred to the Vero E6 plate and returned to the incubator to allow non-neutralized virus to infect cells for one hour.
- ZIKV was serially diluted (10 -2 to 10 -5 ) and samples were allowed to infect Vero E6 cells in triplicate.
- Virus was inactivated with a 1:1 fixative mixture of methanol and acetone which was allowed to fix plates for 30 minutes. Following inactivation, fixative was removed, and plates were allowed to air dry until no fixative remained.
- Antibody was incubated on wells for one hour and then wells were washed with PBST with BSA.
- Vector labs ImmPACT AMEC developing solution was prepared according to kit instructions and added to each well. Plate was incubated in the dark but checked regularly for staining. After foci were clearly developed ( ⁇ 15 minutes), wells were rinsed with deionized water and the plate was allowed to dry.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention inter alia relates to the use of phosphotidylserine or pathogenic sugar targeted therapeutics for the management and treatment of microbial infections, including Zika, Dengue, West Nile, Ebola, H1N1, enteroviruses, Leishmaniasis, Malaria and Coronaviruses SARS-COV. In an aspect, the invention concerns a fusion construct comprising an Ig-Fc domain or other protein scaffold, such as albumin, and a peptide, protein, or antibody fragment binding to phosphatidylserine and/or a peptide or protein binding to and/or recognizing a PAMP expressed by a microbe. Other aspects are described.
Description
- The present specification comprises a sequence listing in computer readable format, submitted together with the application. The sequence listing forms part of the disclosure and is incorporated in the specification in its entirety.
- The present invention relates to the use of phosphotidylserine or pathogenic sugar targeted therapeutics for the management and treatment of microbial infections, including Zika, Dengue, Respiratory Syncytial Virus, West Nile, Ebola, H1N1, Mycobacterium Leprae, Mycobacterium tuberculosis, Enteroviruses, Leishmaniasis, Malaria and Coronaviruses SARS-CoV.
- Provided are compositions related to novel, therapeutic proteins including pathogen neutralizing proteins that may be conjugated to furin protease inhibitors, T cell engagers, platforms with cytotoxic functions, mono and multivalent molecules, drug conjugates and adjuvants, carriers and methods of administration, in particular subcutaneous, oral or nasal administration. This invention further relates to a companion diagnostic as a method of selection of subjects that may benefit from such therapies and a blood biomarker for rapid and easy monitoring of response of treatment.
- According to WHO, the Arborviruses Zika Virus (ZIKV), Chikungunya (CHIKV), Dengue (DENV), West Nile (WNV), as well as Ebola (EBLV) and SARS are relatively recent, life-threatening, rare diseases prone to pandemic spread that pose a high global public health risk (WHO report 2020).
- There are no FDA approved therapies for these diseases. A live, recombinant vaccine consisting of the envelope glycoprotein of one of the Ebola strains, Zaire Ebolavirus (Ervebo, 2019) was recently approved by the FDA for adult use only, although the duration of protection is still not entirely known. Another recombinant vaccine consisting of the pre-M and E proteins for all 4 strains of Dengue (Dengvaxia, 2019) has also been approved however Lim et al 2019 reported new mutations in DENV with different antigenic properties.
- ZIKV, WNV and DENV are flaviviruses (family Flaviviridae) primarily transmitted by mosquito vectors (i.e. arboviruses). Zika is a positive single stranded RNA flaviviridae virus mainly transmitted by the Aedes mosquito and an increasing number of strains in two phylogenetic lineages (Asian and African) have been identified since its first isolation in Uganda in 1947 (Ramos da Silva 2016). According to WHO outbreaks in 2015-2017 resulted in more than 30,000 cases worldwide (Worlds Health Organization Zika Epidemiology update July 2019, Website: www_who.int/emergencies/diseases/zika/zika-epidemiology-update-july-2019.pdf?ua=1, accessed 5 Aug. 2020).
- According to Musso 2015, Zika is also spread through sexual contact (Musso 2015) and according to Rasmussen 2016, Zika is also spread from maternal to fetal blood (Rasmussen 2016).
- An expanding spectrum of neurological sequelae has been reported. According to Rasmussen 2016 a particularly serious co-morbidity of Zika infection in pregnant women is severe congenital microcephaly to their progeny (Rasmussen 2016). According to Barbi 2018, in adults, Guillain-Barré syndrome (GBS), an auto-immune disease that destroys the myelin sheath and causes progressive ascending paralysis has been estimated to affect 1.23% of patients (Barbi 2018). Other reported ZIKV neurological complications include encephalitis/meningoencephalitis, acute disseminated encephalomyelitis, myelitis, cerebrovascular complications, seizures and encephalopathy, sensory polyneuropathy and sensory neuropathy.
- Primary hosts of ZIKV include human, monkey, and mosquito. According to Hou 2017 neural stem cells, fibroblasts, epithelial and blood cells are permissive to ZIKV infection (Hou 2017).
- Dengue virus (DEGV) is a negative RNA strand flavivirus that causes the most prevalent arthropod-born viral disease in the world (1 million cases/year). DENV infection causes human diseases with a wide spectrum of clinical symptoms, ranging from asymptomatic infection or self-limited febrile illness named Dengue fever (DF) to life-threatening diseases including Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS). There are currently no therapies for Dengue nor a vaccine for individuals not previously infected by Dengue or travelling from non-endemic areas. Dengvaxia is a vaccine approved for individuals 9 through 16 years of age with laboratory-confirmed previous dengue infection and living in endemic areas.
- The Pat. Application US2009175865A1 describes antibodies that are engineered by replacing one or more amino acids of a parent antibody with non-cross-linked, highly reactive cysteine amino acids. Among other mutations, the patent application mentions A339C and S337C.
- The patent application WO2015157595 describes conjugate compounds comprising antibodies and fragments thereof engineered with one or more reactive cysteine residues. Among other mutations, the patent application mentions K340C.
- According to Wenwen Bi et al. (IgG Fc-binding mortif-conjugated HIV-1 fusion inhibitor exhibits improved potency and in vivo half-life: Potential application in combination with broad neutralizing antibodies, PLOS Pathogens, Dec. 5, 2019.) a strategy have been developed to extend the in vivo half-life of a short HIV-1 fusion inhibitory peptide, CP24, by fusing it with the human IgG Fc-binding peptide (IBP).
- There is an unmet need for new technologies to manage emerging as well as re-emerging infectious diseases prone to genetic variability.
- Similarities in the way viruses bind to permissive human cells, are activated in the endosomal-lysosomal compartments and become infectious, offer insights towards a potential pan-therapeutic approach to their treatment.
- Glycans are essential structural and functional components of microbes. Among these, glucans, polysaccharide moieties derived from D-glucose, are prominent constituents of the cell walls of fungi, plants, and mycobacteria. High mannose containing structures (mannans) are expressed by many viruses, fungi, and bacteria, and fucose structures (fucans) are found on the surface of helminths and some bacteria (Geijtenbeek and Gringhuis, 2009; Robinson et al., 2006).
- The human immune system has evolved innate pattern-recognition receptors that discern self from non-self-glycans. Binding of C-type lectins to pathogen sugars triggers both innate and adaptive immune events that lead to pathogen clearance however this interaction may also be exploited to enhance pathogenicity.
- The myeloid, dendritic and macrophage cell specific C-type lectin receptor CD209 (also known as DC-SIGN), (Zelensky and Gready, 2005) is an important host cell receptor for entry of ZIKV (Perera Lecoin, 2013, Osorio and Sousa 2011), Influenza (Gillespie 2016), DENV (Cruz-Oliveira, 2015), WNV (Davis 2006), Ebola (Alvarez 2002), enterovirus (REN 2014), mycobacterium tuberculosis (Tailleux 2003) and mycobacterium Leprae (Barreiro 2006) and SARS-COV2/COVID19 (Amraei 2020, Cai 2020, Jeffers 2004). The protozoan vector borne disease Leshmaniasis and Malaria are non-viral pathogens that may exploit CD209 for host entry (Colmenares 2002, Morenikeji 2020). CD209 binds to both mannan (high-mannose N-linked oligosaccharides) and fucan moieties that comprise viral signatures or “pathogen associated molecular patterns (PAMPs). The binding occurs within a compact protein region with a unique structural fold that became known as the “C-type carbohydrate recognition domain” or “C-type lectin domain (CTLD)” (Weis and Drick- Amer, 1996).
- There is no cure for COVID-19, currently a worldwide pandemic, however the FDA has granted emergency use authorization for the antiviral Remdesivir, although their effectiveness against Covid-19 has yet to be demonstrated in large-scale, randomized clinical trials. Among approaches in early preclinical development are ACE2 decoy proteins to block viral attachment to host cells, and off-label use of dexamethasone to reduce inflammation in patients on ventilators but not patients with early stage symptoms. Thus, there is an urgent need for effective and safe means for treating and alleviating COVID-19 and related symptoms. Thus, there is an urgent need for a diagnostic that can precisely select patients that may benefit from a particular treatment.
- The outer virus membrane layer of several viruses is rich in phospholipid phosphatidylserine (PS) whereas in the host cell membrane, PS is normally restricted to the inner membrane layer.
- The T-cell immunoglobulin and mucin domain 1 (TIM-1) human membrane receptor functions as a potent co-stimulatory molecule for T-cell activation.
- TIM1 (also known as HAVCR1) is a type I transmembrane glycoprotein that contains an extracellular domain composed of an N-terminal immunoglobulin variable (IgV)-like domain followed by a glycosylated mucin domain, a single transmembrane domain, and a short cytoplasmic tail with tyrosine phosphorylation motifs. The Ig V domain of TIM1 is predicted to contain a conserved PS binding site (Santiago et al., 2007).
- TIM-1 is expressed preferentially on T-helper 2 (Th2) cells in the brain, gastrointestinal tract, liver and gallbladder, kidney, testis and lymphoid tissue. According to Freeman 2010 TIM-1 recognizes and attaches to exposed PS with high specificity in dying, apoptotic cells and triggers their phagocytosis by the immune system (Freeman 2010). TIM-1 promotes apoptotic clearance by binding to PS through its metal ion-dependent ligand binding site (MILIBS) within the IgV domain.
- It has also been shown that TIM1 is an entry factor for highly divergent viruses (Jemielity, 2013), including Zika (Lee 2018), Ebola (Brunton 2019), Dengue (Chu 2019, Amara 2015), West Nile (Richard 2015), Hepatitis A and possibly Malaria (Nuchnoi 2020).
- These studies indicate that TIM-1, functions as a common attachment factor for a range of enveloped viruses through direct interaction with PS of the viral envelope independent of glycoproteins.
- TIM1 is the most well-known PS receptor although other PS receptors have been described to a lesser extent such as Tyro3, Axl and Mer of the TAM family of proteins.
- According to Angiari et al. 2014, TIM-1 plays a role in autoimmune and inflammatory disease development by controlling T cell adhesion through binding to P-selectin proteins via the TIM-1 mucin and IgV domains (Angiari 2014).
- Kuroda et al. 2015 reported that filovirus infection and GP-mediated membrane fusion were significantly suppressed by treatment with a TIM-1-specific monoclonal antibody that interfered with the interaction between TIM-1 and Niemann-Pick C1 Protein (NPC1). This study suggested that TIM-1 may also participate in viral membrane fusion.
- According to Yuan et al. 2015, human TIM-1 directly binds to EBOV glycoprotein (GP) and the authors determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4 as well as the binding region.
- According to Kondratowicza et al. 2010, TIM-1 on host epithelial cells of the trachea, cornea, and conjunctiva, binds directly to the receptor binding domain of the Zaire Ebola virus (EBOV) glycoprotein and enhances airborne and hand-to-eye infection. Blockage of this interaction with antibodies inhibited binding and Ebola infection.
- Thus, TIM-1 may have both protein dependent and independent functions in viral infection.
- Furin cleavage sites are present in entry proteins of Zika, Dengue, COVID-19 (Coutard 2020), Ebola, HIV, and Hepatitis B viruses among others (Braun 2019).
- Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) comprising SEQ ID NO. 81 and hexa-D-arginine (D6R) are small synthetic furin inhibitors that have been used to show reduction of viral infectivity in vitro (Owczarek, 2019, Imran 2019, Remacle 2010, Couture 2015). CMK is more effective than D6R in the reduction of Hepatitis replication by inhibiting furin-mediated processing of the hepatitis B e-antigen (HBeAg) precursor into mature HBeAg. Dec-RVKR-cmk is a small, synthetic, irreversible, and cell-permeable competitive inhibitor of all proprotein convertases (PC1, PC2, PC4,
PACE 4, PC5, PC7, and furin). CMK is reported to inhibit furin-mediated cleavage and fusion activity of viral glycoproteins, and acts as an antiviral agent against different viruses, including human immunodeficiency virus, Chikungunya virus, chronic hepatitis B virus, influenza A, Ebola virus infection and papilloma virus. Smith et al. and Steinmetzer et al. also patented a peptidomimetic furin inhibitor by modifying the C-terminal of dec-RVKR-cmk with decarboxylated arginine mimetics, resulting in highly potent furin inhibitors (Couture 2015). - Wide-range furin/proprotein inhibitors are thought to have minimal off-pathogen, on-target effects in the host given that proprotein convertases are highly redundant, as shown by furin knockout mice.
- CMK has been shown to have anti-flavivirus activity at non-cytotoxic concentration (Imran 2019).
- Zika virus contains 3 structural (capsid-pC, envelope-pE and membrane-prM) and 7 non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Zika virus replication occurs in the permissive host cell after internalization via clathrin-mediated pH dependent endocytosis and maturation of viral proteins in the lysosomal compartment (Owczarek, 2019). In the lysosome, furin or furin-like proteases cleave viral surface glycoprotein prM into its active form destabilizing the viral membrane and promoting release of the viral RNA for replication in mitochondria and endoplasmatic reticulum.
- There is no vaccine for ZIKV although there are investigational agents in clinical development.
- It has been shown that furin inhibition causes the immature virion to be transported to late compartments where it undergoes proteolytic degradation. The degradation products are ejected from the cell via slow recycling vesicles (Owczarek, 2019).
- Like ZIKV, DENV binding of viral protein E with cellular receptors allows viral particles to internalize into the permissible cell via the chlathrin mediated endocytosis pathway. To release the viral RNA genome, DENV virions undergo an acid-induced conformational change and membrane fusion. Newly synthesized viral proteins generated near the endoplasmic reticulum (ER) promote replication of the viral RNA genome, induction of membrane rearrangement, and assembly of new viral particles. To facilitate the process of DENV replication, DENV not only interacts with various cellular components, but also triggers various host responses, such as autophagy.
- CD3 is a protein complex and T cell co-receptor that is involved in activating T cells. CD3 is selectively expressed on T cells in blood, bone marrow and lymphoid tissues, but not on other normal tissues and with no cross reactivity to other animals except for chimpanzee. Recently, human CD3 transgenic mice have been engineered, facilitating the study of anti-CD3 immunotherapies.
- Anti-CD3 based therapies such as muromomab-CD3 (Janssen, Orthoclone, OKT3) have been extensively studied in humans both systemically and orally to block reactive T cells and ameliorate ulcerative cholitis and metabolic syndrome (da Cunha 2011, Ilan 2010-NCT01287195, NCT01205087). Anti-CD3 bispecific antibody platforms that bridge tumors and engage T cells such as blinatumomab and catumaxomab have been approved for the treatment of cancer and several others CD3 bispecifics are in clinical development (Suurs, 2019).
- According to an aspect, the invention concerns a fusion construct comprising an Ig-Fc domain or other protein scaffold, such as albumin, and
- a. a peptide, protein, or antibody fragment binding to phosphatidylserine and/or
- b. a peptide or protein binding to and/or recognizing a PAMP expressed by a microbe.
- PAMP refers to Pathogen-associated molecular pattern: conserved molecular structures produced by microbial pathogens, but not by the host organism that are recognized by the host innate immune system.
- According to another aspect, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant human TIM1 fragment and/or
- b. a recombinant human CD209 fragment.
- According to another aspect, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant human TIM1 fragment and/or
- b. a recombinant human CD209 fragment
- ADCC may be defined as Antibody-Dependent Cellular Cytotoxicity. ADCP may be defined as Antibody-Dependent Cellular Phagocytosis. CDC may be defined as Complement-dependent cytotoxicity.
- According to another aspect, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant human TIM1 fragment and/or
- b. a recombinant human CD209 fragment
- According to another aspect, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant human TIM1 fragment and/or
- b. a recombinant human CD209 fragment
- Preferably the Furin inhibitor is selected among chloromethylketone and D-arginine derivatives such as hexa-D-arginine and dec-RVKR-cmk (comprising SEQ ID NO. 81).
- The linker and spacers may be conjugated to furin, see Table 7.
- According to another aspect, the invention concerns a fusion construct, wherein said fusion construct is an IgG3 construct, and wherein said IgG3 construct comprises a hinge region, wherein said hinge region has been modified.
- According to another aspect, the invention concerns a fusion construct, a fusion protein or an antibody comprising the constant region(s) of IgG3 and a hinge, wherein said hinge preferably is selected among an IgG1 or IgG4 hinge.
- According to another aspect, the invention concerns IgG3 homodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
- According to another aspect, the invention concerns IgG3 heterodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
- According to another aspect, the invention concerns IgG3, wherein said IgG3 comprises a mutation at position 405 and/or position 409. According to another aspect, the invention concerns IgM heterodimers obtainable by changing the charge pairs of the CH2 and/or CH4 domains.
- According to another aspect, the invention concerns IgM heterodimers, comprising one or more of the mutations of Table 8.
- According to another aspect, the invention concerns a fusion construct, wherein said fusion construct comprises an IgG3 homodimer, an IgG3 heterodimer and/or an IgM heterodimer according to the invention.
- According to another aspect, the invention concerns use of a fusion construct according to the invention for the treatment of an infection.
- According to another aspect, the invention concerns use, wherein said infections are selected among viral, bacterial, and protozoan infections.
- According to another aspect, the invention concerns use, wherein the treatment comprising administration of the fusion construct with an administration form selected among subcutaneous, intradermal, intramuscular, oral and nasal.
- According to another aspect, the invention concerns use of IgG4 or a part of IgG4 for payload delivery, wherein said IgG4 has been modified to comprise no Fc or wherein the activity of the Fc of said IgG4 has been nullified or diminished by one or more mutations.
- According to another aspect, the invention concerns a vaccine comprising a fusion construct according to the invention.
- According to another aspect, the invention concerns a vaccine comprising a mannan, a high mannose containing structure, a fucan and/or a phospholipid phosphatidylserine (PS).
- According to another aspect, the invention concerns a composition comprising a fusion construct according to the invention, optionally comprising one or more excipients such as diluents, binders or carriers.
- According to another aspect, the invention concerns a method of treating and/or preventing an infection in a subject, comprising a step of administration of a fusion construct and/or a vaccine and/or a composition according to the invention.
- According to another aspect, the invention concerns a method of screening and/or monitoring progression of a disease in a subject, wherein said method comprises the following steps:
- i. Providing a blood sample from said subject.
- ii. Contacting said blood sample with a fusion construct according to the invention.
- According to another aspect, the invention concerns an isolated nucleic acid molecule encoding a fusion construct according to the invention.
- According to another aspect, the invention concerns a recombinant vector comprising the nucleic acid molecule of the invention.
- According to another aspect, the invention concerns a host cell comprising the recombinant vector of the invention.
- According to another aspect, the invention concerns a method to produce a fusion construct according to the invention comprising a step of culturing the host cell according to the invention in a culture medium under conditions allowing the expression of the fusion construct and separating the fusion construct from the culture medium.
- According to an embodiment, the invention concerns a fusion construct comprising an Ig-Fc domain or other protein scaffold, such as albumin, and
- a. a peptide, protein, or antibody fragment binding to phosphatidylserine and/or
- b. a peptide or protein binding to and/or recognizing a PAMP expressed by a microbe.
- PAMP refers to Pathogen-associated molecular pattern: conserved molecular structures produced by microbial pathogens, but not by the host organism that are recognized by the host innate immune system.
- The term “protein scaffold” refers to a protein structure on which the active elements defined in a. and b. above can be bound. The protein scaffold should preferably be soluble in plasma and preferably have a high residence time in plasma, which typically can be provided if the complete fusion protein has a size above the renal clearance limit, such as above 60 kDa, or by selecting a protein scaffold that is subject to an active retention system e.g. proteins binding and recycled to the plasma via the FcRn receptor. Examples of suitable protein scaffolds include plasma proteins or fragments thereof, such as constant regions of immunoglobulins, albumin, albumin domain I, II, or III, transferrin and lactoferrin.
- According to an embodiment, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant human TIM1 fragment and/or
- b. a recombinant human CD209 fragment.
- According to an embodiment, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant Ig-like V-type domain of a human TIM1 fragment and/or
- b. a recombinant C-type lectin domain of a human CD209 fragment.
- According to an embodiment, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. two or more recombinant Ig-like V-type domains from one or more human TIM1 fragment(s) and/or
- b. two or more recombinant C-type lectin domains from one or more human CD209 fragment(s).
- According to an embodiment, the invention concerns a fusion construct comprising
- a. a recombinant Ig-like V-type domain of a human TIM1 fragment and
- b. a recombinant C-type lectin domain of a human CD209 fragment.
- According to an embodiment, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant human TIM1 fragment and/or
- b. a recombinant human CD209 fragment
- ADCC may be defined as Antibody-Dependent Cellular Cytotoxicity. ADCP may be defined as Antibody-Dependent Cellular Phagocytosis. CDC may be defined as Complement-dependent cytotoxicity.
- According to an embodiment, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant human TIM1 fragment and/or
- b. a recombinant human CD209 fragment
- According to an embodiment, the invention concerns a fusion construct comprising an IgG-Fc domain or other protein scaffold and
- a. a recombinant human TIM1 fragment and/or
- b. a recombinant human CD209 fragment
- Preferably the Furin inhibitor is selected among chloromethylketone and D-arginine derivatives such as hexa-D-arginine and dec-RVKR-cmk (comprising SEQ ID NO. 81).
- The linker and spacers may be conjugated to furin, see Table 7.
- According to an embodiment, the invention concerns the fusion construct, wherein said peptide, protein or antibody fragment is capable of binding to and/or stimulating an immune cell.
- According to an embodiment, the invention concerns the fusion construct, wherein said TIM1 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 50-180 amino acid residues, 60-160 amino acid residues, 70-140 amino acid residues, 80-130 amino acid residues, 90-120 amino acid residues, 100-120 amino acid residues and 100-110 amino acid residues.
- According to an embodiment, the invention concerns the fusion construct, wherein said CD209 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 40-190 amino acid residues, 50-180 amino acid residues, 60-170 amino acid residues, 70-160 amino acid residues, 80-150 amino acid residues, 90-150 amino acid residues, 100-150 amino acid residues, 110-150 amino acid residues, 120-150 amino acid residues and 130-140 amino acid residues.
- According to an embodiment, the invention concerns the fusion construct, wherein said TIM1 and/or CD209 fragment has a sequence homology of at least 70%, alternatively 75%, alternatively 80%, alternatively 85%, alternatively 90%, alternatively 95% to wildtype TIM1 or CD209.
- According to an embodiment, the invention concerns the fusion construct, wherein said TIM1 and/or CD209 fragment has intact TIM1 and/or CD209 function.
- According to an embodiment, the invention concerns the fusion construct, wherein said IgG-Fc domain is an IgG3-Fc domain.
- According to an embodiment, the invention concerns the fusion construct, comprising additionally at least one of the following:
- a) An IgG3, wherein the hinge sequence has been replaced, preferably with an IgG4 hinge sequence;
- b) CDR regions according to SEQ ID No.: 54 - 59 and/or
- c) A furin inhibitor.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a sequence according to SEQ ID No.: 1 and/or SEQ ID No.: 2.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a sequence according to SEQ ID No.: 3 and/or SEQ ID No.: 4.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or preferably at least 8 disulfide bonds.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct is capable of binding to a target, and wherein said target is a mannan, a high-mannose containing structure, a fucan, a phospholipid phosphatidylserine and/or CD3.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A protein fragment comprising or consisting of a sequence according to SEQ ID No.: 1, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence, and
- b. a protein fragment comprising or consisting of a sequence according to SEQ ID No.: 3 or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 9, SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 9, SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 9, SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 9, SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 11, SEQ ID No.: 45, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 13, SEQ ID No.: 47, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 14, 15, 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 16, 17, 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- v. a linker sequence, preferably according to SEQ ID No.: 41, and
- vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 14, 15, 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID No.: 3 and/or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 16, 17, 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- v. a linker sequence preferably according to SEQ ID No.: 41, and
- vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 14, 15, 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 16, 17, 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- v. a linker sequence preferably according to SEQ ID No.: 41, and
- vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 16, 17, 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iii. a linker sequence preferably according to SEQ ID No.: 41, and
- iv. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- b. A second chain comprising
- v. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences,
- vi. a linker sequence preferably according to SEQ ID No.: 41, and
- vii. a sequence according SEQ ID No.: 14, 15, 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a linker.
- According to an embodiment, the invention concerns the fusion construct, wherein said linker is selected among a (GGGGS)3 linker (SEQ ID NO. 41), a (GGGGS)4 linker (SEQ ID NO. 70), a (GGGGS)5 linker (SEQ ID NO. 71) and a (GGGGS)6 linker (SEQ ID NO. 72).
- A (GGGGS) linker may be defined as a Gly-Gly-Gly-Gly-Ser linker (SEQ ID NO. 69).
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises at least one free cysteine residue, at least two free cysteine residues, at least three free cysteine residues, at least four free cysteine residues, at least five free cysteine residues or preferably at least six free cysteine residues.
- According to an embodiment, the invention concerns the fusion construct, wherein said free cysteine allows interaction with a drug and/or a payload.
- According to an embodiment, the invention concerns the fusion construct, wherein said payload is a furin inhibitor.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a A339C mutation, a S337C mutation and/or a K340C mutation.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a sequence selected among any of the sequences SEQ ID No.: 36, 37, SEQ ID No.: 38, 39, 40, 42, 44 or 46.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct is an IgG1, IgG2, IgG3 or an IgG4.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct is an IgG, IgM, IgA, IgD or an IgE.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a null fc.
- According to an embodiment, the invention concerns the fusion construct, wherein said null fc comprises an Ala substitution at position 234 and/or Ala substitution at 235, and/or N297A, and/or a K322A mutation.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a heterodimerization domain.
- According to an embodiment, the invention concerns the fusion construct, wherein said heterodimerization domain comprises a sequence according to SEQ ID No.: 48, 49 or 50.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a heterodimerization mutation.
- According to an embodiment, the invention concerns the fusion construct, wherein said heterodimerization mutation is an F405L and/or K409R mutation.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence, and
- b. A second chain comprising
- iii. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- iv. a sequence according to SEQ ID No.: 40, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a linker sequence according to SEQ ID No.: 41, and
- iii. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence.
- b. A second chain comprising
- v. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- vi. a linker sequence according to SEQ ID No.: 41, and
- vii. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a linker sequence according to SEQ ID No.: 41, and
- iii. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence;
- b. A second chain comprising
- iv. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- v. a linker sequence according to SEQ ID No.: 41, and
- vi. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of this sequence.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises:
- a. A first chain comprising
- i. a sequence according to SEQ ID No.: 1, SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- ii. a linker sequence according to SEQ ID No.: 41, and
- iii. a sequence according to SEQ ID No.: 65, wherein said sequence ID No.: 65 comprises one or more of the mutations of table 8
- b. A second chain comprising
- iv. a sequence according to SEQ ID No.: 3, SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
- v. a linker sequence according to SEQ ID No.: 41, and
- vi. a sequence according to SEQ ID No.: 65, wherein said sequence ID No.: 65 comprises one or more of the mutations of table 8.
- According to an embodiment, the invention concerns the fusion construct, wherein the ratio of fusion construct to said drug and/or payload is selected among 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a kappa light chain according to SEQ ID No.: 51 or a lambda light chain according to SEQ ID No.: 52 or 53.
- According to an embodiment, the invention concerns a fusion construct, wherein said fusion construct is an IgG3 construct, and wherein said IgG3 construct comprises a hinge region, wherein said hinge region has been modified.
- According to an embodiment, the invention concerns the fusion construct, wherein said hinge region comprises a sequence having a total of at least 10% identity, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the sequence according to SEQ ID No.: 6 or SEQ ID No.: 8.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises the sequence according to SEQ ID No.: 5, 7, 9, 10, 11, 12 and/or 13.
- According to an embodiment, the invention concerns the fusion construct, wherein said hinge region comprises at least one free cysteine residue, at least two free cysteine residues or preferably at least three free cysteine residues.
- According to an embodiment, the invention concerns the fusion construct, wherein said hinge region comprises a S228P mutation.
- According to an embodiment, the invention concerns the fusion construct, wherein said hinge region comprises a sequence according to SEQ ID No.: 6 and/or SEQ ID No.: 8 and/or SEQ ID No.: 68.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct is used to detect phosphatidylserine.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct is used to detect phosphatidylserine in the blood of a subject.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a sequence according to SEQ ID No.: 1, and/or a sequence according to SEQ ID No.: 2.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct is used to detect C-type lectin binding mannan or fucan moieties.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct is used to detect C-type lectin binding mannan or fucan moieties in the blood of a subject.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct comprises a sequence according to SEQ ID No.: 3 and/or a sequence according to SEQ ID No.: 4.
- According to an embodiment, the invention concerns a fusion construct, a fusion protein or an antibody comprising the constant region(s) of IgG3 and a hinge, wherein said hinge preferably is selected among an IgG1 or IgG4 hinge.
- According to an embodiment, the invention concerns the fusion construct, fusion protein or antibody, comprising one or more heterodimerization mutations.
- According to an embodiment, the invention concerns the fusion construct, fusion protein or antibody, comprising heterodimerization mutations involve positions 405 and/or 409 (EU numbering).
- According to an embodiment, the invention concerns IgG3 homodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
- According to an embodiment, the invention concerns IgG3 heterodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
- According to an embodiment, the invention concerns IgG3, wherein said IgG3 comprises a mutation at position 405 and/or position 409.
- According to an embodiment, the invention concerns IgM heterodimers obtainable by changing the charge pairs of the CH2 and/or CH4 domains.
- According to an embodiment, the invention concerns IgM heterodimers, comprising one or more of the mutations of Table 8.
- According to an embodiment, the invention concerns the IgM, wherein said IgM comprises a sequence according to SEQ ID No.: 64 and/or 65.
- According to an embodiment, the invention concerns a fusion construct, wherein said fusion construct comprises an IgG3 homodimer, an IgG3 heterodimer and/or an IgM heterodimer according to the invention.
- According to an embodiment, the invention concerns the fusion construct, wherein said fusion construct is for use in the treatment of an infection.
- According to an embodiment, the invention concerns the fusion construct, wherein said infection is an infection caused by a virus, a parasite, a bacterium, a fungi or a protozoan.
- According to an embodiment, the invention concerns the fusion construct, wherein said virus is selected among an arborvirus, Zika virus, Dengue virus, West Nile virus, Ebola virus, influenza virus, influenza virus H1N1, Chikungunya virus, Enterovirus and Coronaviruses SARS-COV.
- According to an embodiment, the invention concerns the fusion construct, wherein said bacteria is selected among mycobacterium tuberculosis and mycobacterium leprae.
- According to an embodiment, the invention concerns the fusion construct, wherein said parasite is selected among Leishmaniasis and Malaria.
- According to an embodiment, the invention concerns use of a fusion construct according to the invention for the treatment of an infection.
- According to an embodiment, the invention concerns use, wherein said infections are selected among viral, bacterial and protozoan infections.
- According to an embodiment, the invention concerns use, wherein the treatment comprising administration of the fusion construct with an administration form selected among subcutaneous, intradermal, intramuscular, oral and nasal.
- According to an embodiment, the invention concerns use of IgG4 or a part of IgG4 for payload delivery, wherein said IgG4 has been modified to comprise no Fc or wherein the activity of the Fc of said IgG4 has been nullified or diminished by one or more mutations.
- According to an embodiment, the invention concerns the use, wherein said IgG4 comprises one or more heterodimerization mutations.
- According to an embodiment, the invention concerns the use, wherein said IgG4 comprises one or more Cys mutations, preferably thereby allowing site specific conjugation.
- According to an embodiment, the invention concerns the use, wherein said IgG4 comprises a Cys at position 339 (EU numbering).
- According to an embodiment, the invention concerns a vaccine comprising a fusion construct according to the invention.
- According to an embodiment, the invention concerns a vaccine comprising a mannan, a high mannose containing structure, a fucan and/or a phospholipid phosphatidylserine (PS).
- According to an embodiment, the invention concerns the vaccine further comprising a β -glucan adjuvant to potentiate immune response.
- According to an embodiment, the invention concerns the vaccine, for the prevention and/or treatment of an infection.
- According to an embodiment, the invention concerns the vaccine, wherein said infection is caused by a virus, a parasite, a bacterium, a fungus or a protozoan.
- According to an embodiment, the invention concerns the fusion construct and/or vaccine, wherein said fusion construct and/or vaccine allows administration through a route selected among subcutaneous administration, intradermal administration, intramuscular administration, oral administration and/or nasal administration.
- According to an embodiment, the invention concerns a composition comprising a fusion construct according to the invention, optionally comprising one or more excipients such as diluents, binders or carriers.
- According to an embodiment, the invention concerns a method of treating and/or preventing an infection in a subject, comprising a step of administration of a fusion construct and/or a vaccine and/or a composition to the invention.
- According to an embodiment, the invention concerns a method of screening and/or monitoring progression of a disease in a subject, wherein said method comprises the following steps:
- i. Providing a blood sample from said subject.
- ii. Contacting said blood sample with a fusion construct according to the invention.
- According to an embodiment, the invention concerns an isolated nucleic acid molecule encoding a fusion construct according to the invention.
- According to an embodiment, the invention concerns a recombinant vector comprising the nucleic acid molecule according to the invention.
- According to an embodiment, the invention concerns a host cell comprising the recombinant vector according to the invention.
- According to an embodiment, the invention concerns a method to produce a fusion construct according to the invention comprising a step of culturing the host cell according to the invention in a culture medium under conditions allowing the expression of the fusion construct and separating the fusion construct from the culture medium.
- Additional embodiments of the invention are described below.
- According to an embodiment, the invention concerns a fusion construct, wherein said fusion construct comprises a hinge region, wherein said hinge region comprises any of the sequences as described below:
-
Construct Hinge region sequence IgG1 -KS--CDKTHT-----------CPPCPAP (SEQ ID NO. 73) IgG2 -K----------------CCVECPPCPAP (SEQ ID NO. 74) IgG3 LKTPLGDTTHTPEPKSCDTPPPCPRCPAP (SEQ ID NO. 6) IgG4 SKY--G--------------PPCPSCPAP (SEQ ID NO. 75) V-IGG2, -A, -B -KD---------------KTHTCPPCPAP (SEQ ID NO. 76) V-IGG2-C, -D, -E -K----------------YGPPCPPCPAP (SEQ ID NO. 77) V-IGG3, -A, -B SKY--G--------------PPCPPCPAP (SEQ ID NO. 78) V-IGG3-C, -D, -E LKT--GDTTHT-----------CPRCPAP (SEQ ID NO. 79) V-IGG4-A, -B SKY--G--------------PPCPPCPAP (SEQ ID NO. 80) - According to an embodiment, the invention concerns a fusion construct, wherein said fusion construct comprises an Fc heterodimerization sequence at residue 405-409, wherein said Fc heterodimerization sequence comprises a sequence according to SEQ ID No.: 48, 49 or 50.
-
IgG Fc heterodimerization sequences (residues 405-409) Construct Sequence IgG2 FLYSK SEQ ID No.: 48 V-IGG2/-C FLYSK SEQ ID No.: 48 V-IGG2-A/-D FLYSR SEQ ID No.: 49 V-IGG2-B/-E LLYSK SEQ ID No.: 50 IgG3 FLYSK SEQ ID No.: 48 V-IGG3/-C FLYSK SEQ ID No.: 48 V-IGG3-A/-D FLYSR SEQ ID No.: 49 V-IGG3-B/-E LLYSK SEQ ID No.: 50 IgG4 FLYSR SEQ ID No.: 49 V-IGG4-A FLYSR SEQ ID No.: 49 V-IGG4-B LLYSK SEQ ID No.: 50 - Immunoglobulins are glycoproteins composed of one or more units, each containing four polypeptide chains: two identical heavy chains (HCs) and two identical light chains (LCs). The amino terminal ends of the polypeptide chains show considerable variation in amino acid composition and are referred to as the variable (V) regions to distinguish them from the relatively constant (C) regions. Each light chain consists of one variable domain, VL, and one constant domain, CL. The heavy chains consist of a variable domain, VH, and three constant domains CH1, CH2 and CH3. Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure. The V regions of H and L chains comprise the antigen-binding sites of the immunoglobulin (Ig) molecules. Each Ig monomer contains two antigen-binding sites and is said to be bivalent.
- The Fab contains one complete L chain in its entirety and the V and CH1 portion of one H chain. The Fab can be further divided into a variable fragment (Fv) composed of the VH and VL domains, and a constant fragment (Fb) composed of the CL and CH1 domains.
- The H chain constant domain is generally defined as CH1-CH2-CH3 (IgG, IgA, IgD) with an additional domain (CH4) for IgM and IgE. As described above, the CH1 domain is located within the F(ab) region whereas the remaining CH domains (CH2—CH3 or CH2—CH4) comprise the Fc fragment. This Fc fragment defines the isotype and subclass of the immunoglobulin.
- CH3 domain: The terms CH3 domain and CH3 region are used interchangeable herein.
- CH1 domain: The terms CH1 domain and CH1 region are used interchangeable herein.
- Hinge region: The hinge region is the area of the heavy chains between the first and second C region domains and is held together by disulfide bonds. A hinge region typically comprises between 10 and 30 amino acid residues.
- Linker: A linker might be a peptide linker or a non-peptide linker. An example of a peptide linker is a Gly/Ser peptide linker comprising a five amino acid residue unit, GGGGS (SEQ ID NO:71), that can be repeated a suitable amount of times. A linker might be a naturally occurring linker or a synthetically produced linker. A linker might occur naturally in a molecule or might be synthetically added to a molecule.
- Antibody fragment: As used herein, an “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments, “Fv” fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker (“ScFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region. In many embodiments, an antibody fragment contains sufficient sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some embodiments, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen. Examples of antigen binding fragments of an antibody include, but are not limited to, Fab fragment, Fab′ fragment, F(ab′)2 fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd′ fragment, Fd fragment, and an isolated complementarity determining region (CDR) region. An antigen-binding fragment of an antibody may be produced by any means. For example, an antigen-binding fragment of an antibody may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, or additionally, antigen-binding fragment of an antibody may be wholly or partially synthetically produced. An antigen-binding fragment of an antibody may optionally comprise a single chain antibody fragment. Alternatively, or additionally, an antigen-binding fragment of an antibody may comprise multiple chains that are linked together, for example, by disulfide linkages. An antigen-binding fragment of an antibody may optionally comprise a multi-molecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.
- Antibody or fragment thereof: As used herein, an “antibody or fragment thereof” refers to an antibody or antibody fragment as defined above.
- Humanized antibodies: Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.
- IMGT: the international ImMunoGeneTics information system, is an international reference in immunogenetics and immunoinformatics.
- Single-chain Fv (scFv): Single-chain Fvs (scFvs) are widely known and used in the art. A single-chain Fv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, often connected by a short linker peptide (see, e.g., see, e.g., Benny K. C. Lo (ed.), Antibody Engineering - Methods and Protocols, Humana Press 2004, and references cited therein).
-
FIG. 1 shows TIM1 and CTLD constructs with enhanced ADCC, ADCP and CDC. -
FIG. 2 shows TIM1 and CTLD constructs with T cell engager activity. -
FIG. 3 shows TIM1 and CTLD constructs with furin inhibitor payload. -
FIG. 4 shows TIM1 and CTLD constructs with IgM effector function. -
FIGS. 5 a and 5 b shows SEC-HPLC analysis of VP019 and VP020 respectively. -
FIG. 6 a shows that VP025 consists of 4 distinct peaks.FIG. 6 b : intactmass spectrometry Fraction 3 from peak 3 (VP025-F3).FIG. 6 c : intact mass spectrometry:Fraction 4 from peak 4 (VP025-F4). -
FIGS. 7 a and 7 b show the schematics and purity of VP300 and VP301 by SEC-HPLC. -
FIG. 8 shows binding of SARS-CoV-2 S protein (D614G) -
FIG. 9 shows binding of VP025-CT to viral proteins -
FIGS. 10 a and 10 b show binding curves of VP019, VP020, VP025-CT (heterodimer mixture) and VP025-F4 (78% pure heterodimer) to a biotin-phosphatidyl serine and a select group of viral antigens. -
FIG. 11 shows the structure of Hexa-D-arginine linker-compound and Decanoyl-RVKR-CMK linker compound -
FIG. 12 shows the reaction scheme for the Decanoyl-RVKR-CMK linker compound -
FIGS. 13 a and b shows a mass-spectrogram demonstrating that conjugation of furin inhibitor payload to VP020 has been accomplished. -
FIG. 14 a shows a neutralization assay for selected fusion proteins of the invention. For more details see example 16. -
FIG. 14 b shows a neutralization assay for selected fusion protein of the invention. For more details see example 17. - All cited references are incorporated by reference.
- The accompanying Figures and Examples are provided to explain rather than limit the present invention. It will be clear to the person skilled in the art that aspects, embodiments, claims and any items of the present invention may be combined.
- Unless otherwise mentioned, all percentages are in weight/weight. Unless otherwise mentioned, all measurements are conducted under standard conditions (ambient temperature and pressure). Unless otherwise mentioned, test conditions are according to European Pharmacopoeia 8.0.
- Construct V-TIM1-1 was selected as residues 21-125 of the full length TIM-1 sequence (https://www.uniprot.org/uniprot/Q96D42), and V-TIM1-2 was selected as residues 21-127. V-TIM1-2 contains an extra two Pro residues at the C-terminal domain boundary.
-
TABLE 1 Sequences of Recombinant human TIM1 fragment Construct Sequence Mw Predicted pl V-TIM1-1 SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTC QNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENT AVSDSGVYCCRVEHRGWFNDMKITVSLEIV SEQ ID No.: 1 11.6 KDa 8.26 V-TIM1-2 SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTC QNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENT AVSDSGVYCCRVEHRGWFNDMKITVSLEIVPP SEQ ID No.: 2 11.8 KDa 8.26 - Construct V-CTLD-1 was selected as residues 250-385 of the full length DC-SIGN sequence (https://www.uniprot.org/uniprot/Q9NNX6), and V-CTLD-2 was selected as residues 254-383. V-CTLD-1 contains 4 internal disulfide bonds, whereas V-CTLD-2 contains 3 internal disulfide bonds.
-
TABLE 2 Sequences of Recombinant human CTLD fragment of DC-SIGN Construct Sequence MW Predicted pl V-CTLD-1 ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACK EVGAQLVVIKSAEEQNFLQLQSSRSNRFTWMGLSDLN QEGTWQWVDGSPLLPSFKQYWNRGEPNNVGEEDCA EFSGNGWNDDKCNLAKFWICKKSAASCS SEQ ID No.: 3 15.7 KDa 5.12 V-CTLD-2 HPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVG AQLVVI KSAEEQN FLQLQSSRSN RFTWMG LSDLNQEG TWQWVDGSPLLPSFKQYWN RGEPN NVG EEDCAEFSG NGWNDDKCNLAKFWICKKSAAS SEQ ID No.: 4 15.0 KDa 5.08 - Among all human IgG subclasses, IgG3 has the highest effector functions in terms of ADCC, ADCP and CDC (https://www.frontiersin.org/articles/10.3389/fimmu.2014.00520/full). IgG3 has not typically been used for therapeutics because of the short serum half-life due to proteolytic cleavage of the prolonged hinge region between the CH1 and CH2 domains. To utilize the strong effector functions of the IgG3 subclass, the V-IGG3 construct was designed where the IgG3 hinge (LKTPLGDTTHTPEPKSCDTPPPCPRCPAP) (SEQ ID NO. 6) was replaced with an IgG4 hinge sequence containing an IgG4 hinge S228P mutation to prevent Fab arm exchange (SKYGPPCPPCPAP) (SEQ ID NO. 8) or an IgG1-like hinge (KTGDTTHTCPRCPAP) (SEQ ID NO. 68).
- Heterodimeric V-IGG3 constructs were designed based on including K409R (on one half-antibody) and F405L (on second antibody) mutation in the CH3 domains (https://www.nature.com/articles/nprot.2014.169). Each half antibody is first generated as a single homodimer, then mixed together and allowed to recombine as heterodimers under reducing and oxidizing conditions. The resulting sequences are noted as V-IGG3-A and V-IGG3-B and pair together, or V-IGG3-D and V-IGG3-E that pair together. Sequences are found in Table 3, including truncated version that include a (GGGGS)3 linker (SEQ ID NO. 41) to replace the CH1 domains.
-
TABLE 3 Modified IgG3 domains Construct Sequence Notes WT huIgG3 CH1-CH2-CH3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVN H KPSNTKVDKRVE LKTPLG DTTHTPEPKSCDTPPPCPR CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPEN NYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVM HEALHNRFTQKSLSLSPGK SEQ ID No.: 5 Hinge region underlined as SEQ ID No.: 6 V-IGG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K SEQ ID No.: 7 Hinge region underlined (SEQ ID No.: 8) with IgG4 hinge S228P mutation in bold V-lGG3-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSKL TVDKSRWQQGNI FSCSVM HEALH NRFTQKSLSLSPG K SEQ ID No.: 9 Same as above, but with linker and fc only V-IGG3-A ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K SEQ ID No.: 10 K409R V-lGG3-A-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL Same as above, but with linker and fc only YSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K SEQ ID No.: 11 V-IGG3-B ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLL YSKL TVDKSRWQQGNI FSCSVM HEALH NRFTQKSLSLSPG K SEQ ID No.: 12 F405L V-IGG3-B-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLL YSKL TVDKSRWQQGNI FSCSVM HEALH NRFTQKSLSLSPG K SEQ ID No.: 13 Same as above, but with linker and fc only V-IGG3-C ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK SEQ ID No.: 42 Utilizes an IgG1-like hinge V-IGG3-C-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK SEQ ID No.: 43 Same as above, but with linker and fc only V-IGG3-D ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVH NAKTKPREEQYNSTFRVVSVL TVLHQDWLNG KEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPEN NYNTTPPM LDSD K409R GSFFLYSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK SEQ ID No.: 44 V-IGG3-D-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFFLYSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK SEQ ID No.: 45 Same as above, but with linker and fc only V-IGG3-E ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVH NAKTKPREEQYNSTFRVVSVL TVLHQDWLNG KEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFLLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK SEQ ID No.: 46 F405L V-IGG3-E-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFLLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK SEQ ID No.: 47 Same as above, but with linker and fc only - TIM1 and CTLD fusion proteins were designed with the modified IgG3-Fc domains and are depicted in
FIG. 1 and Table 4. -
TABLE 4 Sequences of TIM-1 and CTLD constructs with enhanced ADCC, ADCP and CDC Construct Chain 1Chain 2VIT-300 (V-TIM1-1/V-TIM1-2) -V-IGG3-Fc/V-IGG3-C-Fc Same as Chain 1VIT-301 (V-CTLD-1/V-CTLD-2) -V-IGG3-Fc/ V-IGG3-C-Fc Same as Chain 1VIT-302 (V-TIM1-1/V-TIM1-2) -V-IGG3-A-Fc/ V-IGG3-D-Fc (V-CTLD-1/V-CTLD-2) -V-IGG3-B-Fc/ V-IGG3-E-Fc - Additional constructs were designed to engage T cell effector functions by fusing the TIM-1 and CTLD with a single anti-CD3 scFv. The designs are shown in
FIG. 2 and Table 5. -
TABLE 5 Sequences of TIM-1 and CTLD constructs with T cell engager activity Construct Chain 1 Chain 2 VIT-303 (V-TIM1-1/V-TIM1-2)-(V-IGG4-A-Fc/ V-IGG2-A-Fc/V-IGG2-D-Fc) (V-TIM1-1/V-TIM1-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) VIT-304 (V-CTLD-1/V-CTLD-2)-(V-IGG4-A-Fc/ V-IGG2-A-Fc /V-IGG2-D-Fc) (V-CTLD-1/V-CTLD-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) VIT-305 (V-TIM1-1/V-TIM1-2)-(V-IGG4-A-Fc/ V-IGG2-A-Fc /V-IGG2-D-Fc) (V-CTLD-1/V-CTLD-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/ V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) VIT-306 (V-TIM1-1/V-TIM1-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/ V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) (V-CTLD-1/V-CTLD-2)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(V-IGG4-A-Fc/ V-IGG2-A-Fc /V-IGG2-D-Fc) -
TABLE 6 Anti-CD3 scFv sequences are described below Construct Sequence VCD3-H1L1 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 18) VCD3-H1L1-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 19) VCD3-H1L2 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 20) VCD3-H1L2-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 21) VCD3-H1L3 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 22) VCD3-H1L3-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 23) VCD3-H2L1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYD DHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 24) VCD3-H2L1-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 25) VCD3-H2L2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQG RVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 26) VCD3-H2L2-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 27) VCD3-H2L3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 28) VCD3-H2L3-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 29) VCD3-H3L1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 30) VCD3-H3L1-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 31) VCD3-H3L2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 32) VCD3-H3L2-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 33) VCD3-H3L3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 34) VCD3-H3L3-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 35) - Site specific addition of drug payloads to the antibody Fc region was devised by analysis of the co-crystal structure of a human IgG1 Fc with the 3-helix bundle of bacterial protein A (PDB structure 5U4Y https://www.rcsb.org/sequence/5U4Y). Computational modelling revealed that A339C would have a stabilizing effect to the structure and S337C or K340C would have a neutral effect to the stability of the Fc domain. A339C was chosen as the site for site specific conjugation.
-
TABLE 7 IgG4 sequences with engineered free cys for site specific payload conjugation Construct Sequence Notes V-IGG4-ADC-A ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCA VSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK SEQ ID No.: 37 S228P FALA (F234A, L235A) K322A Naturally contains F405 and R409 A339C V-IGG4-ADC-A-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCAVSNKGLPSSIEKTISK CKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF Same as above, but with linker and fc only V-IGG4-ADC-B ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCA VSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK SEQ ID No.: 39 S228P FALA (F234A, L235A) K322A F405L, R409K A339C V-IGG4-ADC-B-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCAVSNKGLPSSIEKTISK CKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFL LYSKLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK SEQ ID No.: 40 Same as above, but with linker and fc only - TIM1 and CTLD fusion proteins with Fc domain with payload conjugation sites were designed and are shown in
FIG. 3 and Table 8. -
TABLE 8 Sequences of TIM1 and CTLD therapeutic proteins with free cys for payload conjugation Construct Chain 1 Chain 2VIT-307 (V-TIM1-1/V-TIM1-2) -V-IGG4-ADC-A-Fc Same as Chain 1VIT-308 (V-CTLD-1/V-CTLD-2) -V-IGG4-ADC-A-Fc Same as Chain 1VIT-309 (V-TIM1-1/V-TIM1-2) -V-IGG4-ADC-A-Fc (V-CTLD-1/V-CTLD-2) -V-IGG4-ADC-B-Fc - Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) (SEQ ID NO. 81) or hexa-D-arginine (D6R) were linked to TIM-1 and CTLD constructs using cleavable linkers such as acid sensitive N-acyl-hydrazone or enzyme sensitive malemeide-conjugated dipeptides, valine-alanine, valine-citrulline, or phenylalanine-Lysine.
- Acid sensitive linkers are cleaved in the lysosome acidic environment after internalization of the construct. This strategy has been used in two approved ADCs, Gemtuzumab ozogamicin and Inotuzumab ozogamicin. Lysosomal protease sensitive dipeptides release the drug after cleavage by proteases such as cathepsin B- lysosomal protease. This type of linker chemistry has been used for FDA approved Brentuximab vedotin.
- Linkage to the polypeptide of antibodies is done through the nucleophilic groups of lysine or cysteine by random conjugation, generating a heterogeneous mixture of conjugates, or by site-directed conjugation to engineered cysteines, reducing the heterogeneity of the product to an antibody-drug ratio (ADR) of 1 or 2.
- The nucleophilic reactivity of the thiol functionality of a Cys residue to a maleimide group is about 1000 times higher compared to any other amino acid functionality in a protein, such as amino group of lysine residues or the N-terminal amino group. Thiol specific functionality in maleimide reagents may react with amine groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).
- The first FDA approved site-directed ADC through engineered cysteines was vadastuximab talirine (Seattle Genetics).
-
TABLE 9 Linker Chemistry Residue Spacer 1 Linker Spacer 2 Drug Cysteine, engineered Cysteine Maleimidocaproyl (mc), maleimidomethyl VC, VA, PL para-amino benzyloxycarbonyl (PABC) hexa-D-arginine, dec-RVKR-cmk cyclohexane- 1-carboxylate (comprising SEQ ID NO. 81) Lysine NA N-acyl-hydrozone, N-succinimidyl-4-(2-pyridyldithio) Butanoate-disulfide (SPDB-disulfide), maleimidomethyl cyclohexane-1-carboxylate, sulfo-SPDB NA hexa-D-arginine, dec-RVKR-cmk (comprising SEQ ID NO. 81) Spacer 1: The purpose of the mc spacer is to provide enough room so that the vc group can be recognized by cathepsin B, which cleaves the citrulline-PABC amide bond. Spacer 2: Self-immolative spacer - IgM molecules have robust Fc effector functions, particularly with CDC. IgM molecules naturally homodimerize and then covalently associate into pentamers or hexamers. IgM do not contain hinge regions like IgG molecules and instead contain an extra CH domain (CH1-CH2-CH3-CH4). The homodimeric heavy chains come together at the CH2 and CH4 domains. Based on visual analyses of the crystal structure of the murine IgM CH2 domain (pdb 4JVU), the crystal structure of the murine IgM CH4 domain (pdb 4JVW), and a sequence alignment of the human IgM CH2 and CH4 sequences with the homologous mouse sequences, mutations were designed to induce IgM heavy chain heterodimerization by inducing charge differences at the homodimerization interfaces.
- Sequence of human IgM constant region, numbered residues 1-453 by uniprot (www_uniprot.org/uniprot/P01871):
-
GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDI SSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKN VPLPVIAELPPKVSVFVPPRDGFFGNPRKS K LIC Q ATGFSPRQIQVSWLR EGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVD HRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLT TYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATIT CLV T GFSPADVFVQWMQRGQPLSPEKYVTSAPMP E PQAPGRYFAHSILTV SEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAG TCY SEQ ID No.: 64 - Sequence of IgM CH2-CH3-CH4 which can used for fusing to antibody fragments (Fab, scFv, VHH, etc) or targeting proteins (TIM-1, CTLD/DC-SIGN) for adding IgM effector functions (residues 105-453): V-IGM
-
VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGL TFQQONASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLYVTDLTTY DSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFT CTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCL VTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSE EEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTC Y SEQID No.: 65 - Based on the structural analysis, the underlined residues K131 and Q135 were found to be in close proximity in the CH2:CH2 interface, and residues T354 and E385 were found to be in close proximity in the CH4:CH4 interface. The following mutations were made to alter the charge pattern in V-IGM-A and V-IGM-B to induce heterodimer formation of A:B and repel the formations of A:A or B:B.
-
TABLE 10 IgM constant region mutations to induce heavy chain heterodimer formation Position Wildtype residue in V-IGM V-IGM-A V-IGM-B 131 K K/R/H D/E 135 Q K/R/H D/E 354 T D/E K/R/H 385 E D/E K/R/H - TIM1 and CTLD fusion proteins with IgM effector functions were designed and shown in
FIG. 4 and Table 11. -
TABLE 11 Sequences of TIM1 and CTLD therapeutic proteins with IgM effector functions Construct Chain 1 Chain 2VIT-310 (V-TIM1-1/V-TIM1-2) -GGGGSGGGGSGGGGS (SEQ ID No.: 41)-V-IGM Same as Chain 1VIT-311 (V-CTLD-1/V-CTLD-2) -GGGGSGGGGSGGGGS (SEQ ID No.: 41) -V-IGM Same as Chain 1VIT-312 (V-TIM1-1/V-TIM1-2) -GGGGSGGGGSGGGGS (SEQ ID No.: 41) -V-IGM-A (V-CTLD-1/V-CTLD-2) -GGGGSGGGGSGGGGS (SEQ ID No.: 41)-V-IGM-B - The 10 proteins from [Table 1 and
FIGS. 1-3 ] were expressed and batch purified form 2.5 mL ExpiCHO cultures using Protein A/G magnetic agarose beads. Expression yields and % monomeric purity are shown in Table 12. -
TABLE 12 Small scale expression of protein constructs Protein Name Gene Name Description Fc domain Format Yield (µg) %Purity by SEC-HPLC VP011 TM-G1 V-TIM1-1 with V-IGG1-Fc IgG1 VIT-300-IgG1 143 28.6 VP012 TM-G3 V-TIM1-1 with V-IGG3-D-Fc IgG3 VIT-300-IgG3 150 34 VP013 CT-G1 V-CTLD-1 with IgG1-Fc IgG1 VIT-301-IgG1 209 56.5 VP014 CT-G3 V-CTLD-1 with V-IGG3-E-Fc IgG3 VIT-301-IgG3 225 73.4 VP015 TM-G4-A V-TIM1-1 with V-IgG4-A-Fc IgG4 VIT-300-IgG4 113 11 VP016 TM-G4-A-F V-TIM1-1 with V-IgG4-A-Fc-CD3 (H3L3) IgG4 73 16.8 VP017 CT-G4-B V-CTLD-1 with V-IgG4-B-Fc IgG4 VIT-301-IgG4 386 73.1 VP018 CT-G4-B-F V-CTLD-1 with V-IgG4-B-Fc-CD3 (H3L3) IgG4 290 74.4 VP019 TM-G4-A-DC V-TIM1-1 with V-IGG4-ADC-A-Fc IgG4 VIT-307 77 11.5 VP020 CT-G4-B-DC V-CTLD-1 with V-IGG4-ADC-B-Fc IgG4 VIT-308 645 60.9 - Larger scale preps were done in ExpiCHO cells for VP011 (100 mL), VP012 (100 mL), VP013 (100 mL), VP014 (100 mL), VP019 (1L) and VP020 (250 mL). VP011, VP019 and VP020 were purified by MabSelect SuRe protein A resin column chromatography. VP012, VP013 and VP014 were purified by HiTrap Protein G resin column chromatography. Expression yields and % monomeric purity are shown in Table 13.
-
TABLE 13 Larger scale expression of protein constructs Protein Name Gene Name Description Fc domain Format Yield (mg) %Purity by SEC-HPLC VP011 TM-G1 V-TIM1-1 with V-IGG1-Fc IgG1 VIT-300-IgG1 0.78 78.5∗ VP012 TM-G3 V-TIM1-1 with V-IGG3-D-Fc IgG3 VIT-300-IgG3 9.18 68.0∗ VP013 CT-G1 V-CTLD-1 with IgG1-Fc IgG1 VIT-301-IgG1 15.6 59.3 VP014 CT-G3 V-CTLD-1 with V-IGG3-E-Fc IgG3 VIT-301-IgG3 17.03 49.0∗ VP019 TM-G4-A-DC V-TIM1-1 with V-IGG4-ADC-A-Fc IgG4 VIT-307 12.73 60.7∗ VP020 CT-G4-B-DC V-CTLD-1 with V-IGG4-ADC-B-Fc IgG4 VIT-308 19.18 91.9 ∗multimer peak - The sequences of the expressed recombinant proteins are shown in table 14.
-
TABLE 14 Sequences of expressed recombinant proteins. Protein Name Description Sequence VP011 V-TIM1-1 with V-IGG1-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG NIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 82) VP012 V-TIM1-1 with V-IGG3-D-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSRLTVDKSRW QQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 83) VP013 V-CTLD-1 with IgG1-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG NIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 84) VP014 V-CTLD-1 with V-IGG3-E-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLLYSKLTVDKSRW QQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 85) VP015 V-TIM1-1 with V-IgG4-A-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 86) VP016 V-TIM1-1 with V-IgG4-A-Fc-CD3 (H3L3) SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHW VRQSPGQCLEWMGYINPSRGYTNYNQKFQGRVTMTTDKSTSTAYMELSSLRSE DTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSElQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPR RWIYDTSKLATGIPARFSGSGSGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGC GTKLEIK (SEQ ID NO: 87) VP017 V-CTLD-1 with V-IgG4-B-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 88) VP018 V-CTLD-1 with V-IgG4-B-Fc-CD3 (H3L3) ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHW VRQSPGQCLEWMGYINPSRGYTNYNQKFQGRVTMTTDKSTSTAYMELSSLRSE DTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPR RWIYDTSKLATGIPARFSGSGSGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGC GTKLEIK (SEQ ID NO: 89) VP019 V-TIM1-1 with V-IGG4-ADC-A-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 90) VP020 V-CTLD-1 with V-IGG4-ADC-B-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 91) VP300 V-TIM1-1 and V-CTLD-1 with V-IGG4-ADC-A-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIVGG GGSGGGGSGGGGSERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEV GAQLVVIKSAEEQNFLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSF KQYWNRGEPNNVGEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 92) VP301 V-CTLD-1 and V-TIM1-1 with V-IGG4-ADC-A-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCSGGGGSGGGGSGGGGSSV KVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYRKD TRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 93) - SEC-HPLC analysis of VP019 (
FIG. 5 a ) shows that VP019 form the expected homodimer (Fraction F6) andmultimer ~600 kDa (Fraction F2). The fractions were purified out and assessed separately for functional activity (VP019-F6 and VP019-F2, respectively named). -
FIG. 5 b : SEC-HPLC analysis of VP020. - The protein product VP025, which is a heterodimer of VP019 and VP020, was generated by co-expressing Genes TM-G4-A-DC and CT-G4-B-DC in ExpiCHO cells and purified by MabSelect SuRe protein A resin column chromatography. The resulting co-transfected sample product was named VP025-CT.
FIG. 6 a shows that VP025-CT contains 4 distinct peaks which were purified out and assessed separately by intact mass spectrometry. The fractions fromPeak 1 andPeak 2 showed a number of species.Fraction 3 from peak 3 (VP025-F3) showed a mass of 86,721 Da, corresponding to a VP020 homodimer (FIG. 6 b ).Fraction 4 from peak 4 (VP025-F4) showed a mass of 83,881 Da, corresponding to the properly formed VP019/VP020 heterodimer, at an approximate purity of 78% (FIG. 6 c ). - Two additional bispecific molecules containing the TIM-1 and CTLD domains on a single polypeptide chain (Table 15) were generated in 100 mL ExpiCHO cells and purified by MabSelect SuRe protein A resin column chromatography. The schematics and purity by SEC-HPLC are shown in
FIGS. 7 a and 7 b . - The sequences of the expressed recombinant proteins are shown in table 14.
-
TABLE 15 Larger scale expression of protein constructs Protein Name Gene Name Description Fc domain Yield (mg) VP300 TM-CT-G4-A-DC V-TIM1-1 and V-CTLD-1 with V-IGG4-ADC- A-Fc IgG4 2 VP301 CT-TM-G4-A-DC V-CTLD-1 and V-TIM1-1 with V-IGG4-ADC- A-Fc IgG4 2 - The binding of VP019-F2, VP020 and VP025-CT to SARS-Cov-2 S D614G was investigated by ELISA. This protein is representative of the dominant SARS-COV-2 strain in early 2020. All ELISA assays in this and subsequent examples were done in the presence of 2.5 mM CaCl2 since DC-SIGN is known to use calcium at the binding site. Binding curves are shown in
FIG. 8 and EC50 values are shown in Table 16. VP019-F2 had unexpectedly high binding to SARS-COV2 S Protein, while VP020 had modest binding. -
TABLE 16 EC50 for Binding of VP019, VP020 and VP025 to SARS-COV2 S Protein EC50 (mg/mL) EC50 (nM) VP019-F2 0.0009 10.6 VP020 0.0556 641.0 VP025-CT 0.0069 82.5 - The binding of VP025-CT to many diverse viral surface protein antigens was investigated by ELISA. Binding curves are shown in
FIG. 9 and EC50 values are shown in Table 17. -
TABLE 17 EC50 for binding of VP025-CT to several viral antigens EC50 (mg/mL) EC50 (nM) West Nile Virus Envelope Protein 0.0049 57.9 Chikungunya Virus E1 0.0102 121.5 Human RSV (A) Glycoprotein G 0.0696 829.6 Influenza A H1N1 HA 0.0062 73.9 SARS-Cov-2 S D614G 0.0040 47.6 Zika Virus Envelope Protein 0.0069 82.6 Dengue Virus 4 Envelope Protein0.0041 49.2 HIV-1 Clade C GP120 0.0055 65.9 EBOV Envelope Glycoprotein 0.0106 126.1 - The binding of VP019, VP020, VP025-CT (heterodimer mixture) and VP025-F4 (78% pure heterodimer) to a biotin-phosphatidyl serine and a select group of viral antigens (Influenza A H1N1 HA, Human RSV Glycoprotein G, Zika Virus Envelope Protein and SARS-Cov-2 S D614G) was investigated by ELISA. Binding curves are shown in
FIGS. 10 a and 10 b and EC50 values are shown in Table 18 and Table 19. VP025-F4 had stronger binding to all of the antigens than VP025-CT. -
TABLE 18 EC50 in mg/mL for binding of VP019, VP020, VP025-CT and VP025-F4 to phosphatidyl serine and viral antigens VP019-F2 VP020 VP025-CT VP025-F4 Biotin-PS 0.0098 0.0296 0.0056 0.0018 Influenza A H1N1 HA 0.0021 0.0320 0.0034 0.0014 HRSV (A) Glycoprotein G 0.0073 0.0069 0.0043 0.0020 Zika Virus Envelope Protein 0.0010 0.0074 0.0020 0.0008 SARS-Cov-2 S D614G 0.0017 0.0045 0.0012 0.0008 -
TABLE 19 EC50 in nM for binding of VP019, VP020, VP025-CT and VP025-F4 to phosphatidyl serine and viral antigens VP019-F2 VP020 VP025-CT VP025-F4 Biotin-PS 121.1 340.7 66.6 21.9 Influenza A H1N1 HA 25.8 368.8 40.6 17.0 HRSV (A) Glycoprotein G 89.9 79.6 51.4 23.6 Zika Virus Envelope Protein 12.2 85.1 24.0 10.0 SARS-Cov-2 S D614G 21.1 52.5 13.8 10.0 - Solvents and reagents were purchased from Sigma-Aldrich, VWR, or Fisher Scientific, and used without further purification. Reactions were monitored either by thin-layer chromatography (TLC) or by analytical liquid chromatography-mass spectrometry (LC-MS) employing a Waters Acquity Ultra Performance LC system and a Synapt high-definition mass spectrometer. 1H NMR spectra were recorded on a Varian Unity INOVA spectrometer (500 MHz). All chemical shifts are reported in ppm and coupling constants, J, are reported in hertz (Hz). NMR solvent peaks were referenced as follows: (1H NMR) CDCl3: 7.27 ppm, DMSO-d6: 2.50 ppm. Compounds were purified by flash column chromatography on a Teledyne ISCO Combi-Flash system using normal phase silica gel (SiliCycle Inc.) or reverse phase (Teledyne Gold- C18 or C18-Aq) pre-packed columns. The purity of compounds was determined by analytical HPLC (Waters Acquity Ultra Performance) using an Acquity UPLC CSH C18 1.7 µm (50 mm x 2.1 mm) column and flow rate of 0.3 mL/min. Gradient conditions: solvent A (0.05% formic acid in water) and solvent B (0.05% formic acid in acetonitrile): 0-0.1 min 95% A, 0.1-4.0 min 5-95% B (linear gradient), 4.0-5.0 min 95% B, UV detection at 254 nm and 220 nm.
- The reaction scheme is shown in
FIG. 12 . - N-methyl morpholine (13.8 µlL, 0.126 mmol) was added to a solution of hexa-D-Arg (D-Argininamide D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-alanine; Ambeed, cat# A333458) (30 mg, 0.0314 mmol), MC-Val-Cit-PAB-PNP (BroadPharm Cat#: BP-23292, CAS: 159857-81-5) (46.4 mg, 0.0629 mmol) and HOBt.H2O (1-hydroxybenzotriazole monohydrate; 5.3 mg, 0.0345) in anhydrous DMF (1 mL) under argon atmosphere. The solution was stirred at r.t. for 18 hours. The reaction was diluted with 1:1 ACN/water (0.05% HCO2H) (10 mL) and purified by reverse phase C18-Aq flash chromatography (gradient elution; 100% water -100% ACN with 0.05% HCO2H as mobile phase additive) to afford MC-VC-PAB-(D-Arg)6-NH2 (12 mg, 0.00773 mmol, 25%) as a white solid after lyophilization. 1H NMR (500 MHz, DMSO-d6) d 10.09 (s, 1H), 8.65 (br. s, 6H), 8.47 (s, 6H), 8.32 - 8.45 (m, 4H), 8.12 - 8.20 (m, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.50 - 7.80 (m, 18H), 7.29 (d, J = 7.8 Hz, 2H), 7.17 (s, 1H), 7.01 (s, 2H), 6.08 - 6.10 (m, 1H), 5.43 - 5.50 (s, 2H), 4.89 - 5.01 (m, 2H), 4.36 - 4.41 (m, 1H), 4.12 - 4.30 (m, 6H), 4.01 - 4.09 (m, 1H), 3.35 - 3.40 (m, 2H), 2.91- 3.12 (m, 12H), 2.09 - 2.21 (m, 2H), 1.93 - 2.00 (m, 1H), 1.65 -1.75 (m, 6H), 1.40 -1.62 (m, 28H), 1.30 -1.40 (m, 1H), 1.15 - 1.25 (m, 2H), 0.80 - 0.89 (m, 6H); MS (ESI) m/z: 1552.4 [M+H]+.
- The structure is shown in
FIG. 11 . - A test conjugation of Hexa-D-arginine linker-compound to VP020 was performed by reacting VP025 with 4 equivalents of TCEP and incubating at 37° C. for 1 hour to reduce the free cysteines. The sample was run through a Zeba column to remove TCEP and buffer exchanged into 1x PBS containing 1 mM DTPA pH 6.5. The sample was then reacted with 2.5 equivalents of the payload (Mc-VC-PAB-(D-Arg6)-CONH2 at Room temperature for 1 hour. The final product was analyzed by mass spectrometry (see
FIG. 13 ) and shown to have the mass of 88,136 Da, near the expected mass of 88,138 Da. - A microneutralization assay was done to determine the antiviral properties of three compounds (VP019-F2, VP020 and VP025-F4) against RSV. Each virus was incubated with each antibody for 1 hour, after which the mix was added to A549 cells (human lung cancer cell line). Antiviral activity was determined 24 h later using an immunofluorescence-based assay. After 24 h, the infection plates were washed with PBS, fixed for 30 mins with 4% formaldehyde, washed again with PBS, and stored in PBS at 4° C. until staining. Any residual formaldehyde was quenched with 50 mM ammonium chloride, after which cells were permeabilized (0.1% Triton X100) and stained with an antibody recognizing RSV fusion protein (GeneTex GTX40697). The primary antibody was detected with an Alexa-488 conjugate secondary antibody (Life Technologies, A21244 and A11001), and nuclei were stained with Hoechst. Images were acquired on an Celllnsight CX5 high content platform (Thermo Scientific), and percentage infection calculated using CellInsight CX5 software (infected cells/total cells x 100).
- The test articles were used in concentrations of 0.5 µM and samples were tested in triplicate. The resulting data are shown in
FIG. 14 . For RSV, VP019-F2 had 97% viral inhibition, compared to 55% inhibition for VP025-F5 and 28% inhibition for VP020. - A second neutralization assay was done for Zika virus using VP025-F4 using the following procedures.
- Vero E6 cells were maintained with DMEM supplemented with 2% FBS and (1% pen-strep -need to confirm with Allen) and stored at 37° C. with 5% CO2. Cells were seeded onto 48-well plates at a concentration of 8.0*104 cells per well and allowed to adhere overnight. On the morning of infection cell monolayers were examined to ensure 90-95% confluency.
- VP025-F4 was serially diluted in triplicate using infection media at a ratio of 1:3 for a total of eight dilutions (220 to 0.1 µg/mL).). Zika virus (ZIKV), strain MEX-I-44, at a MOI of 1.0 (8.0*104 FFU) was added to each dilution, mixed, and incubated at 37° C. and 5% CO2 for one hour.
- Simultaneous to the VP025:ZIKV incubation, positive control samples were also incubated. Mouse α-ZIKV MIAF (mouse immune ascitic fluid antibody) was diluted 1:500, 1:1000, and 1:1500 and combined with ZIKV, in triplicate, using the same concentration of virus as the test wells.
- Following incubation of VP025-F4:ZIKV and α-ZIKV MIAF:ZIKV, the Vero E6 well plate was removed from the incubator. Media was aspirated from the cells and the test and positive control samples were transferred to the Vero E6 plate and returned to the incubator to allow non-neutralized virus to infect cells for one hour.
- Simultaneous to the Plate Infection incubation, ZIKV was serially diluted (10-2 to 10-5) and samples were allowed to infect Vero E6 cells in triplicate.
- Following the one hour incubation on Vero E6 cells an overlay of 0.8% methylcellulose was added to all wells and they were maintained at 37° C. and 5% CO2 for approximately 60 hours.
- Plates were removed from the incubator, overlay was aspirated, and cells were gently washed twice with phosphate buffered saline. Virus was inactivated with a 1:1 fixative mixture of methanol and acetone which was allowed to fix plates for 30 minutes. Following inactivation, fixative was removed, and plates were allowed to air dry until no fixative remained.
- All incubations and washes were performed at room temperature and plate was placed on a plate rocker. Cells were permeabilized with 0.5% Triton in PBS and washed with 0.02
% Tween 20 in PBS (PBST). Blocking solution of PBST with BSA and normal goat serum was prepared and incubated on all wells for one hour. Primary antibody, mouse α-ZIKV MIAF, was diluted in PBST with BSA and stored on ice until used. Blocking solution was removed and 1° antibody was incubated for one hour. Antibody was removed and plates were washed with PBST. Secondary antibody, goat α-mouse IgG (high and low chain) HRP conjugated, was diluted in PBST with BSA. Antibody was incubated on wells for one hour and then wells were washed with PBST with BSA. Vector labs ImmPACT AMEC developing solution was prepared according to kit instructions and added to each well. Plate was incubated in the dark but checked regularly for staining. After foci were clearly developed (~15 minutes), wells were rinsed with deionized water and the plate was allowed to dry. - No foci were observed in the VP025-F4 test wells or the positive control wells (see
FIG. 14 b ). Foci developed as expected in the negative control focus-forming assay wells demonstrating decreasing foci with each successive virus dilution. This indicates that the VP025 test article was able to neutralize ZIKV at dilutions from (220 to 0.1 µg/mL).
Claims (97)
1. A fusion construct comprising an Ig-Fc domain or other protein scaffold, such as albumin or an antibody fragment binding to albumin, and
a. a peptide, protein or antibody fragment binding to phosphatidylserine and/or
b. a peptide or protein binding to and/or recognizing a PAMP expressed by a microbe.
2. A fusion construct, preferably according to claim 1 , comprising an IgG-Fc domain or other protein scaffold and
a. a recombinant human TIM1 fragment and/or
b. a recombinant human CD209 fragment.
3. A fusion construct according to any of claims 1-2 , comprising an IgG-Fc domain or other protein scaffold, and
a. A recombinant Ig-like V-type domain of a human TIM1 and/or
b. A recombinant C-type lectin domain of a human CD209 fragment.
4. A fusion construct according to claim 3 , comprising an IgG-Fc domain or other protein scaffold, and
a. Two or more recombinant Ig-like V-type domains from one or more human TIM1 and/or
b. Two or more recombinant C-type lectin domains from one or more human CD209 fragment(s).
5. A fusion construct comprising
a. A recombinant Ig-like V-type domain of a human TIM1 and
b. A recombinant C-type lectin domain of a human CD209 fragment.
6. A fusion construct, preferably according to any of the preceding claims comprising an IgG-Fc domain or other protein scaffold and
a. a recombinant human TIM1 fragment and/or
b. a recombinant human CD209 fragment
and wherein said fusion construct provides enhanced ADCC, ADCP and/or CDC. 7. A fusion construct, preferably according to any of the preceding claims comprising an IgG-Fc domain or other protein scaffold and
a. a recombinant human TIM1 fragment and/or
b. a recombinant human CD209 fragment
and wherein said fusion construct additionally comprises the CDR regions according to SEQ ID No.: 54 - 59. 8. A fusion construct, preferably according to any of the preceding claims comprising an IgG-Fc domain or other protein scaffold and
a. a recombinant human TIM1 fragment and/or
b. a recombinant human CD209 fragment
and wherein said fusion construct further comprises a Furin inhibitor. 9. The fusion construct according to any of the preceding claims , wherein said peptide, protein or antibody fragment is capable of binding to and/or stimulating an immune cell.
10. The fusion construct according to any of the preceding claims , wherein said TIM1 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 50-180 amino acid residues, 60-160 amino acid residues, 70-140 amino acid residues, 80-130 amino acid residues, 90-120 amino acid residues, 100-120 amino acid residues and 100-110 amino acid residues.
11. The fusion construct according to any of the preceding claims , wherein said CD209 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 40-190 amino acid residues, 50-180 amino acid residues, 60-170 amino acid residues, 70-160 amino acid residues, 80-150 amino acid residues, 90-150 amino acid residues, 100-150 amino acid residues, 110-150 amino acid residues, 120-150 amino acid residues and 130-140 amino acid residues.
12. The fusion construct according to any of the preceding claims , wherein said TIM1 and/or CD209 fragment has a sequence homology of at least 70%, alternatively 75%, alternatively 80%, alternatively 85%, alternatively 90%, alternatively 95% to wildtype TIM1 or CD209.
13. The fusion construct according to any of the preceding claims , wherein said TIM1 and/or CD209 fragment has intact TIM1 and/or CD209 function.
14. The fusion construct according to any of the preceding claims , wherein said IgG-Fc domain is an IgG3-Fc domain.
15. The fusion construct according to any of the preceding claims , comprising additionally at least one of the following:
a) An IgG3, wherein the hinge sequence has been replaced, preferably with an IgG4 or IgG1 hinge sequence;
b) CDR regions according to SEQ ID No.: 54 - 59; and/or
c) A furin inhibitor.
16. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a sequence according to SEQ ID No.: 1 and/or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
17. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a sequence according to SEQ ID No.: 3 and/or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
18. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or preferably at least 8 disulfide bonds.
19. The fusion construct according to any of the preceding claims , wherein said fusion construct is capable of binding to a target, and wherein said target is a mannan, a high-mannose containing structure, a fucan, a phospholipid phosphatidylserine and/or CD3.
20. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A protein fragment comprising or consisting of a sequence according to SEQ ID No.: 1, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to a sequence according to SEQ ID No.: 1, and
b. a protein fragment comprising or consisting of a sequence according to SEQ ID No.: 3 or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to a sequence according to SEQ ID No.: 3.
21. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences and
ii. a sequence according to SEQ ID No.: 9 or a sequence according to SEQ ID No.: 43,, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences and
b. A second chain comprising
iii. a sequence according to SEQ ID No.: 1or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 9 or a sequence according to SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
22. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 9 or a sequence according to SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
b. A second chain comprising
iii. a sequence according to SEQ ID SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 9 or a sequence according to SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
23. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 11 or a sequence according to SEQ ID No.: 45, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
b. A second chain comprising
iii. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 13 or a sequence according to SEQ ID No.: 47, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
24. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 14 or 15, or SEQ ID No.: 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
b. A second chain comprising
iii. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 16 or 17, or SEQ ID No.: 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences and
v. a linker sequence, preferably according to SEQ ID No.: 41, and
vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
25. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 14 or 15, or SEQ ID No.: 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
b. A second chain comprising
iii. a sequence according to SEQ ID No.: 3 and/or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 16 or 17, or SEQ ID No.: 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences and
v. a linker sequence preferably according to SEQ ID No.: 41, and
vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
26. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 14 or 15, or SEQ ID No.: 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
b. A second chain comprising
iii. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 16 or 17, or SEQ ID No.: 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
v. a linker sequence preferably according to SEQ ID No.: 41, and
vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
27. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 16 or 17, or SEQ ID No.: 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iii. a linker sequence preferably according to SEQ ID No.: 41, and
iv. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
b. A second chain comprising
v. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences,
vi. a linker sequence preferably according to SEQ ID No.: 41, and
vii. a sequence according SEQ ID No.: 14 or 15, or SEQ ID No.: 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
28. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a linker.
29. The fusion construct according to any of the preceding claims , wherein said linker is selected among a (GGGGS)3 linker (SEQ ID NO. 41), a (GGGGS)4 linker (SEQ ID NO. 70), a (GGGGS)5 linker (SEQ ID NO. 71) and a (GGGGS)6 linker (SEQ ID NO. 72).
30. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises at least one free cysteine residue, at least two free cysteine residues, at least three free cysteine residues, at least four free cysteine residues, at least five free cysteine residues or preferably at least six free cysteine residues.
31. The fusion construct according to any of the preceding claims , wherein said free cysteine allows interaction with a drug and/or a payload.
32. The fusion construct according to any of the preceding claims , wherein said payload is a furin inhibitor.
33. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a A339C mutation, a S337C mutation and/or a K340C mutation.
34. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a sequence selected among any of the sequences SEQ ID No.: 36, 37, SEQ ID No.: 38, 39, 40, 42, 44 or 46.
35. The fusion construct according to any of the preceding claims , wherein said fusion construct is an IgG1, IgG2, IgG3 or an IgG4.
36. The fusion construct according to any of the preceding claims , wherein said fusion construct is an IgG, IgM, IgA, IgD or an IgE.
37. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a null fc.
38. The fusion construct according to any of the preceding claims , wherein said null fc comprises an Ala substitution at position 234 and/or Ala substitution at 235, and/or N297A, and/or a K322A mutation.
39. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a heterodimerization domain.
40. The fusion construct according to any of the preceding claims , wherein said heterodimerization domain comprises a sequence according to SEQ ID No.: 48, 49 or 50.
41. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a heterodimerization mutation.
42. The fusion construct according to any of the preceding claims , wherein said heterodimerization mutation is an F405L, R409K and/or K409R mutation.
43. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence, and
b. A second chain comprising
iii. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence.
44. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
b. A second chain comprising
iii. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
45. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
b. A second chain comprising
iii. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
iv. a sequence according to SEQ ID No.: 40, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
46. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a linker sequence according to SEQ ID No.: 41, and
iii. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences
b. A second chain comprising
iv. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
v. a linker sequence according to SEQ ID No.: 41, and
vi. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
47. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 3 or SEQ ID No.:4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a linker sequence according to SEQ ID No.: 41, and
iii. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences
b. A second chain comprising
iv. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
v. a linker sequence according to SEQ ID No.: 41, and
vi. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.
48. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises:
a. A first chain comprising
i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
ii. a linker sequence according to SEQ ID No.: 41, and
iii. a sequence according to SEQ ID No.: 65, wherein said sequence ID No.: 65 comprises one or more of the mutations of table 8
b. A second chain comprising
iv. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and
v. a linker sequence according to SEQ ID No.: 41, and
vi. a sequence according to SEQ ID No.: 65, wherein said sequence ID No.: 65 comprises one or more of the mutations of table 8.
49. The fusion construct according to any of the preceding claims , wherein the ratio of fusion construct to said drug and/or payload is selected among 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
50. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises a kappa light chain according to SEQ ID No.: 51 or a lambda light chain according to SEQ ID No.: 52 or 53.
51. A fusion construct, preferably according to any of the preceding claims , wherein said fusion construct is an IgG3 construct, and wherein said IgG3 construct comprises a hinge region, wherein said hinge region has been modified.
52. The fusion construct according to claim 51 , wherein said hinge region comprises a sequence having a total of at least 10% identity, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the sequence according to SEQ ID No.: 6 or SEQ ID No.: 8.
53. The fusion construct according to any of the preceding claims , wherein said fusion construct comprises the sequence according to SEQ ID No.: 5, 7, 9, 10, 11, 12 and/or 13.
54. The fusion construct according to any of the preceding claims , wherein said hinge region comprises at least one free cysteine residue, at least two free cysteine residues or preferably at least three free cysteine residues.
55. The fusion construct according to any of the preceding claims , wherein said hinge region comprises a S228P mutation.
56. The fusion construct according to any of the preceding claims , wherein said hinge region comprises a sequence according to SEQ ID No.: 6 and/or SEQ ID No.: 8 and/or SEQ ID No.: 68.
57. The fusion construct according to any of the preceding claims , wherein said fusion construct is used to detect phosphatidylserine.
58. The fusion construct according to any of the preceding claims , wherein said fusion construct is used to detect phosphatidylserine in the blood of a subject.
59. The fusion construct according to claim 57 , wherein said fusion construct comprises a sequence according to SEQ ID No.: 1, and/or a sequence according to SEQ ID No.: 2.
60. The fusion construct according to any of the preceding claims , wherein said fusion construct is used to detect C-type lectin binding mannan or fucan moieties.
61. The fusion construct according to any of the preceding claims , wherein said fusion construct is used to detect C-type lectin binding mannan or fucan moieties in the blood of a subject.
62. The fusion construct according to claim 60 , wherein said fusion construct comprises a sequence according to SEQ ID No.: 3 and/or a sequence according to SEQ ID No.: 4.
63. A fusion construct, a fusion protein or an antibody comprising the constant region(s) of IgG3 and a hinge, wherein said hinge preferably is selected among an IgG1 or IgG4 hinge.
64. The fusion construct, fusion protein or antibody according to any claim 63 , comprising one or more heterodimerization mutations.
65. The fusion construct, fusion protein or antibody according to claim 64 , comprising heterodimerization mutations involving or including positions 405 and/or 409 (EU numbering).
66. IgG3 homodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
67. IgG3 heterodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.
68. IgG3 according to any of claims 66-67 , wherein said IgG3 comprises a mutation at position 405 and/or position 409.
69. IgM heterodimers obtainable by changing the charge pairs of the CH2 and/or CH4 domains.
70. IgM heterodimers according to claim 69 , comprising one or more of the mutations of Table 8.
71. The IgM according to any claims 69-70 , wherein said IgM comprises a sequence according to SEQ ID No.: 64 and/or 65.
72. A fusion construct according to any of claims 1-65 , wherein said fusion construct comprises an IgG3 homodimer, an IgG3 heterodimer and/or an IgM heterodimer according to any of claims 66-71 .
73. The fusion construct according to any of the preceding claims , wherein said fusion construct is for use in the treatment of an infection.
74. The fusion construct according to claim 73 , wherein said infection is an infection caused by a virus, such as Coronaviruses SARS-COV, a parasite, a bacteria, a fungi or a protozoan.
75. The fusion construct according to claims 74 , wherein said virus is selected among an arborvirus, Zika virus, Dengue virus, West Nile virus, Ebola virus, influenza virus, influenza virus H1N1, Chikungunya virus, enterovirus, Coronavirus SARS-COV-2 and Coronaviruses SARS-COV.
76. The fusion construct according to claim 74 , wherein said bacteria is selected among mycobacterium tuberculosis and mycobacterium leprae.
77. The fusion construct according to claim 74 , wherein said parasite is selected among Leishmaniasis and Malaria.
78. Use of a fusion construct according to any of claims 1-71 , for the treatment of an infection.
79. Use according to claim 78 , wherein said infections are selected among viral, bacterial and protozoan infections.
80. Use according to any claims 78-79 , wherein the treatment comprising administration of the fusion construct with an administration form selected among subcutaneous, intradermal, intramuscular, oral and nasal.
81. Use of IgG4 or a part of IgG4 for payload delivery, wherein said IgG4 has been modified to comprise no Fc or wherein the activity of the Fc of said IgG4 has been nullified or diminished by one or more mutations.
82. The use according to any of claims 78-81 , wherein said IgG4 comprises one or more heterodimerization mutations.
83. The use according to any of claims 81-82 , wherein said IgG4 comprises one or more Cys mutations, preferably thereby allowing site specific conjugation.
84. The use according to any of claims 81-83 , wherein said IgG4 comprises a Cys at position 339 (EU numbering).
85. A vaccine comprising a fusion construct according to any of claims 1-65 .
86. A vaccine comprising a mannan, a high mannose containing structure, a fucan and/or a phospholipid phosphatidylserine (PS).
87. The vaccine according to claim 85 or 86 , further comprising β-glucan.
88. The vaccine according to any of claims 85-87 , for the prevention and/or treatment of an infection.
89. The vaccine according to claim 88 , wherein said infection is caused by a virus, preferably according to claim 75 , and/or Coronaviruses SARS-COV, a parasite, preferably according to claim 77 , a bacteria, preferably according to claim 76 , a fungi or a protozoan.
90. The fusion construct according to any of claims 1-65 and/or vaccine according to any of claims 85-89 , wherein said fusion construct and/or vaccine allows administration through a route selected among subcutaneous administration, intradermal administration, intramuscular administration, oral administration and/or nasal administration.
91. A composition comprising a fusion construct according to any of claims 1-65 , optionally comprising one or more excipients such as diluents, binders or carriers.
92. A method of treating and/or preventing an infection in a subject, comprising a step of administration of a fusion construct according to any of claims 1-65 and/or a vaccine according to any of claims 85-89 and/or a composition according to any of claims 90-91 .
93. A method of screening and/or monitoring progression of a disease in a subject, wherein said method comprises the following steps:
i. Providing a blood sample from said subject.
ii. Contacting said blood sample with a fusion construct according to any of claims 1-65 .
94. An isolated nucleic acid molecule encoding a fusion construct according to any of claims 1-65 .
95. A recombinant vector comprising the nucleic acid molecule of claim 94 .
96. A host cell comprising the recombinant vector of claim 95 .
97. A method for the production of a fusion construct according to any of the precedent claims comprising a step of culturing the host cell according to claim 96 in a culture medium under conditions allowing the expression of the fusion construct and separating the fusion construct from the culture medium.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/022,096 US20230338465A1 (en) | 2020-08-19 | 2021-08-19 | Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/997,639 US11197910B1 (en) | 2020-08-19 | 2020-08-19 | Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases |
PCT/US2021/046713 WO2022040436A2 (en) | 2020-08-19 | 2021-08-19 | Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases |
US18/022,096 US20230338465A1 (en) | 2020-08-19 | 2021-08-19 | Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases |
Publications (1)
Publication Number | Publication Date |
---|---|
US20230338465A1 true US20230338465A1 (en) | 2023-10-26 |
Family
ID=78828615
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/997,639 Active US11197910B1 (en) | 2020-08-19 | 2020-08-19 | Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases |
US18/022,096 Pending US20230338465A1 (en) | 2020-08-19 | 2021-08-19 | Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/997,639 Active US11197910B1 (en) | 2020-08-19 | 2020-08-19 | Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases |
Country Status (6)
Country | Link |
---|---|
US (2) | US11197910B1 (en) |
EP (1) | EP4200415A2 (en) |
JP (1) | JP2023538119A (en) |
CA (1) | CA3189711A1 (en) |
MX (1) | MX2023001988A (en) |
WO (1) | WO2022040436A2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023150672A1 (en) * | 2022-02-04 | 2023-08-10 | Duke University | Compositions for and methods of treating hematological cancers |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2356401A1 (en) * | 1999-01-07 | 2000-07-13 | Lexigen Pharmaceuticals, Corp. | Expression and export of anti-obesity proteins as fc fusion proteins |
EP1417965A1 (en) * | 2002-11-07 | 2004-05-12 | Vereniging Voor Christelijk Wetenschappelijk Onderwijs | C-type lectin binding molecules, identification and uses thereof |
AU2005231685A1 (en) * | 2004-03-24 | 2005-10-20 | Telos Pharmaceuticals Llc | Compositions as adjuvants to improve immune responses to vaccines and methods of use |
GB0426481D0 (en) * | 2004-12-02 | 2005-01-05 | Vaccine Technology Ltd | Composition |
WO2008064031A2 (en) * | 2006-11-13 | 2008-05-29 | Government Of The United States, As Represented Bythe Secretary Of The Department Of Health And Human Services | Potent activation of antigen presenting cells by the hepatitis a virus cellular receptor 1 and its role in the regulation of immune responses |
CN101636168B (en) | 2007-01-09 | 2013-05-29 | 比奥根艾迪克Ma公司 | Sp35 antibodies and uses thereof |
MX2010011598A (en) * | 2008-04-22 | 2010-12-15 | Univ Rockefeller | Methods of identifying anti-inflammatory compounds. |
EP2403531A4 (en) | 2009-03-05 | 2013-02-27 | Abbott Lab | Il-17 binding proteins |
RU2578420C2 (en) * | 2010-05-10 | 2016-03-27 | Эсенд Байофармасьютикалз Пти Лтд | Immune stimulating and vaccine compositions |
WO2013096948A1 (en) * | 2011-12-23 | 2013-06-27 | Lydon Nicholas B | Immunoglobulins and variants directed against pathogenic microbes |
BR112014021068A8 (en) * | 2012-02-21 | 2018-01-23 | Centre National De La Recherce Scient | inhibitor of an interaction between phosphatidylserine and a thymic receptor, pharmaceutical composition and use of an inhibitor |
US9605084B2 (en) | 2013-03-15 | 2017-03-28 | Xencor, Inc. | Heterodimeric proteins |
TWI682941B (en) * | 2013-02-01 | 2020-01-21 | 美商再生元醫藥公司 | Antibodies comprising chimeric constant domains |
EP3041865A2 (en) * | 2013-09-03 | 2016-07-13 | NovImmune S.A. | Readily isolated bispecific binding molecules with native format having mutated constant regions |
US9493563B2 (en) | 2013-11-04 | 2016-11-15 | Glenmark Pharmaceuticals S.A. | Production of T cell retargeting hetero-dimeric immunoglobulins |
WO2015134671A1 (en) * | 2014-03-04 | 2015-09-11 | Targeson, Inc. | Molecular imaging contrast agents and uses thereof |
CA2991799A1 (en) | 2015-07-15 | 2017-01-19 | Zymeworks Inc. | Drug-conjugated bi-specific antigen-binding constructs |
MX2018012469A (en) | 2016-04-15 | 2019-07-04 | Immunext Inc | Anti-human vista antibodies and use thereof. |
IL263542B1 (en) | 2016-06-14 | 2024-06-01 | Xencor Inc | Bispecific checkpoint inhibitor antibodies |
WO2018031419A1 (en) * | 2016-08-12 | 2018-02-15 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions and methods for enhancing immunogenic cross-presentation of tumor antigens |
CN111201242A (en) | 2017-06-22 | 2020-05-26 | 财团法人生物技术开发中心 | Asymmetric heterodimeric FC-SCFV fusion anti-GLOBO H and anti-CD 3 bispecific antibodies and uses thereof in cancer therapy |
-
2020
- 2020-08-19 US US16/997,639 patent/US11197910B1/en active Active
-
2021
- 2021-08-19 CA CA3189711A patent/CA3189711A1/en active Pending
- 2021-08-19 EP EP21859140.2A patent/EP4200415A2/en active Pending
- 2021-08-19 US US18/022,096 patent/US20230338465A1/en active Pending
- 2021-08-19 WO PCT/US2021/046713 patent/WO2022040436A2/en unknown
- 2021-08-19 JP JP2023512390A patent/JP2023538119A/en active Pending
- 2021-08-19 MX MX2023001988A patent/MX2023001988A/en unknown
Also Published As
Publication number | Publication date |
---|---|
MX2023001988A (en) | 2023-03-23 |
US11197910B1 (en) | 2021-12-14 |
WO2022040436A3 (en) | 2022-03-31 |
EP4200415A2 (en) | 2023-06-28 |
JP2023538119A (en) | 2023-09-06 |
CA3189711A1 (en) | 2022-02-24 |
WO2022040436A2 (en) | 2022-02-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3390441B1 (en) | Human immunodeficiency virus neutralizing antibodies | |
EP3271390B1 (en) | A serotype cross-reactive, dengue neutralizing antibody and uses thereof | |
US20150344546A1 (en) | Monoclonal Antibodies for Ebola and Marburg Viruses | |
JP2023520468A (en) | Binding proteins useful against ACE2-targeted viruses | |
US11370830B2 (en) | Neutralizing antibodies that bind to the zika virus domain III envelope region | |
CN107074879A (en) | Novel cytotoxic molecule applied to cell-binding molecules drug conjugates | |
Baykov et al. | A protective chimeric antibody to tick-borne encephalitis virus | |
US20230338465A1 (en) | Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases | |
CN116785427B (en) | Use of respiratory syncytial virus specific binding molecules | |
JP2023534922A (en) | Antigen-binding molecule targeting SARS-CoV-2 | |
KR20220020228A (en) | Stable pharmaceutical formulation | |
CA3096901A1 (en) | Methods and compositions for treating yellow fever | |
US11028150B1 (en) | Anti-SARS-CoV-2 antibodies derived from 2DD8 | |
Bouayad | Multifaceted roles of Fcγ receptors in COVID-19 and vaccine responses | |
US20230212271A1 (en) | Compositions and methods for linear and conformational site-specific antibodies and methods of making the same | |
US20240083982A1 (en) | Therapeutic neutralizing antibodies for sars-cov-2 | |
US20230374153A1 (en) | METHODS OF PREVENTING OR TREATING INFECTION BY RESPIRATORY VIRUSES INCLUDING SARS-CoV-2 | |
WO2016139941A1 (en) | Antibody, fragment, molecule, and anti-hcv treatment agent | |
US20240150441A1 (en) | Monoclonal antibodies against sars-cov-2 and variants | |
WO2023201306A1 (en) | Compositions for preventing or treating influenza infections | |
CN116601291A (en) | Modified angiotensin converting enzyme 2 (ACE 2) and uses thereof | |
JP2023521667A (en) | Plasma kallikrein inhibitors and their use to treat acute respiratory distress syndrome | |
TW202241497A (en) | Antibody cocktail against sars-cov-2 spike protein | |
CN116848242A (en) | ACE2 fusion proteins and uses thereof | |
WO2021194886A1 (en) | Anti-sars-cov-2 antibodies derived from 3bgf |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: APPLICATION UNDERGOING PREEXAM PROCESSING |
|
AS | Assignment |
Owner name: VITRUVIAE LLC, NEW JERSEY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:AHMED, MAHIUDDIN;SEQUEIRA, SONIA;REEL/FRAME:062764/0382 Effective date: 20200825 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |