WO2017156423A2 - Broadly protective antibody cocktails for treatment of filovirus hemorrhagic fever - Google Patents
Broadly protective antibody cocktails for treatment of filovirus hemorrhagic fever Download PDFInfo
- Publication number
- WO2017156423A2 WO2017156423A2 PCT/US2017/021842 US2017021842W WO2017156423A2 WO 2017156423 A2 WO2017156423 A2 WO 2017156423A2 US 2017021842 W US2017021842 W US 2017021842W WO 2017156423 A2 WO2017156423 A2 WO 2017156423A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- antibody
- fragment
- amino acid
- filovirus
- Prior art date
Links
- 241000711950 Filoviridae Species 0.000 title claims abstract description 104
- 206010061192 Haemorrhagic fever Diseases 0.000 title claims description 9
- 238000011282 treatment Methods 0.000 title description 25
- 230000001681 protective effect Effects 0.000 title description 8
- 239000012634 fragment Substances 0.000 claims abstract description 263
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 100
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 100
- 238000000034 method Methods 0.000 claims abstract description 80
- 239000000427 antigen Substances 0.000 claims abstract description 76
- 108091007433 antigens Proteins 0.000 claims abstract description 76
- 102000036639 antigens Human genes 0.000 claims abstract description 76
- 208000007136 Filoviridae Infections Diseases 0.000 claims abstract description 23
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 121
- 241001115402 Ebolavirus Species 0.000 claims description 109
- 241000282414 Homo sapiens Species 0.000 claims description 54
- 108090000623 proteins and genes Proteins 0.000 claims description 36
- 238000006467 substitution reaction Methods 0.000 claims description 34
- 150000004676 glycans Chemical class 0.000 claims description 31
- 238000012217 deletion Methods 0.000 claims description 30
- 230000037430 deletion Effects 0.000 claims description 30
- 208000015181 infectious disease Diseases 0.000 claims description 30
- 238000003780 insertion Methods 0.000 claims description 28
- 230000037431 insertion Effects 0.000 claims description 28
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 claims description 26
- 241000884921 Bundibugyo ebolavirus Species 0.000 claims description 25
- 241001115401 Marburgvirus Species 0.000 claims description 23
- 241000700605 Viruses Species 0.000 claims description 22
- 102000004169 proteins and genes Human genes 0.000 claims description 19
- 241000700198 Cavia Species 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 17
- 241000283984 Rodentia Species 0.000 claims description 15
- 241001529936 Murinae Species 0.000 claims description 12
- 241001115374 Tai Forest ebolavirus Species 0.000 claims description 12
- 230000004580 weight loss Effects 0.000 claims description 12
- 238000010172 mouse model Methods 0.000 claims description 10
- 108020003175 receptors Proteins 0.000 claims description 10
- 102000005962 receptors Human genes 0.000 claims description 10
- 230000004083 survival effect Effects 0.000 claims description 10
- 241000894007 species Species 0.000 claims description 8
- 230000004927 fusion Effects 0.000 claims description 7
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 241000282553 Macaca Species 0.000 claims description 6
- 241000288906 Primates Species 0.000 claims description 6
- 230000003247 decreasing effect Effects 0.000 claims description 4
- 238000011554 guinea pig model Methods 0.000 claims description 4
- 150000002632 lipids Chemical class 0.000 claims description 4
- 229920000642 polymer Polymers 0.000 claims description 4
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 3
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 3
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims description 3
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims description 3
- 239000003443 antiviral agent Substances 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 238000011808 rodent model Methods 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 61
- 108090000765 processed proteins & peptides Proteins 0.000 description 61
- 229920001184 polypeptide Polymers 0.000 description 59
- 235000001014 amino acid Nutrition 0.000 description 43
- 241001465754 Metazoa Species 0.000 description 38
- 210000004027 cell Anatomy 0.000 description 38
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 32
- 150000001413 amino acids Chemical class 0.000 description 31
- 229940024606 amino acid Drugs 0.000 description 30
- 201000010099 disease Diseases 0.000 description 29
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 27
- 150000007523 nucleic acids Chemical group 0.000 description 23
- 108091033319 polynucleotide Proteins 0.000 description 23
- 102000040430 polynucleotide Human genes 0.000 description 23
- 239000002157 polynucleotide Substances 0.000 description 23
- 108060003951 Immunoglobulin Proteins 0.000 description 21
- 238000003556 assay Methods 0.000 description 21
- 102000018358 immunoglobulin Human genes 0.000 description 21
- 102000039446 nucleic acids Human genes 0.000 description 21
- 108020004707 nucleic acids Proteins 0.000 description 21
- 241000699670 Mus sp. Species 0.000 description 20
- 241000711975 Vesicular stomatitis virus Species 0.000 description 20
- 235000018102 proteins Nutrition 0.000 description 17
- 239000013598 vector Substances 0.000 description 17
- 238000006386 neutralization reaction Methods 0.000 description 15
- 230000003472 neutralizing effect Effects 0.000 description 15
- 102100023448 GTP-binding protein 1 Human genes 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 12
- 239000000047 product Substances 0.000 description 10
- 108091026890 Coding region Proteins 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 102100024952 Protein CBFA2T1 Human genes 0.000 description 8
- 230000036961 partial effect Effects 0.000 description 8
- 238000013518 transcription Methods 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- 241000589158 Agrobacterium Species 0.000 description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 230000037396 body weight Effects 0.000 description 7
- 238000002703 mutagenesis Methods 0.000 description 7
- 231100000350 mutagenesis Toxicity 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 238000002560 therapeutic procedure Methods 0.000 description 7
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 6
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 6
- 239000004480 active ingredient Substances 0.000 description 6
- 239000011230 binding agent Substances 0.000 description 6
- 230000036541 health Effects 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 5
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 229940072221 immunoglobulins Drugs 0.000 description 5
- 238000007912 intraperitoneal administration Methods 0.000 description 5
- 230000002195 synergetic effect Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241000700199 Cavia porcellus Species 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000013595 glycosylation Effects 0.000 description 4
- 238000006206 glycosylation reaction Methods 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 231100000518 lethal Toxicity 0.000 description 4
- 230000001665 lethal effect Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000207746 Nicotiana benthamiana Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 241000709992 Potato virus X Species 0.000 description 3
- 102000028391 RNA cap binding Human genes 0.000 description 3
- 108091000106 RNA cap binding Proteins 0.000 description 3
- 241000723873 Tobacco mosaic virus Species 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000004797 therapeutic response Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- -1 tripeptides Proteins 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 235000005749 Anthriscus sylvestris Nutrition 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 2
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 description 2
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 241001112090 Pseudovirus Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 206010058874 Viraemia Diseases 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000012867 alanine scanning Methods 0.000 description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 238000001212 derivatisation Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- BRMWTNUJHUMWMS-UHFFFAOYSA-N 3-Methylhistidine Natural products CN1C=NC(CC(N)C(O)=O)=C1 BRMWTNUJHUMWMS-UHFFFAOYSA-N 0.000 description 1
- 229940117976 5-hydroxylysine Drugs 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 244000303258 Annona diversifolia Species 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 108010088468 Ebola virus envelope glycoprotein Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 206010016334 Feeling hot Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 102000006471 Fucosyltransferases Human genes 0.000 description 1
- 108010019236 Fucosyltransferases Proteins 0.000 description 1
- 108090001126 Furin Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101710114810 Glycoprotein Proteins 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 101000998953 Homo sapiens Immunoglobulin heavy variable 1-2 Proteins 0.000 description 1
- 101001008255 Homo sapiens Immunoglobulin kappa variable 1D-8 Proteins 0.000 description 1
- 101001047628 Homo sapiens Immunoglobulin kappa variable 2-29 Proteins 0.000 description 1
- 101001008321 Homo sapiens Immunoglobulin kappa variable 2D-26 Proteins 0.000 description 1
- 101001047619 Homo sapiens Immunoglobulin kappa variable 3-20 Proteins 0.000 description 1
- 101001008263 Homo sapiens Immunoglobulin kappa variable 3D-15 Proteins 0.000 description 1
- 101000997654 Homo sapiens N-acetylmannosamine kinase Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 102100036887 Immunoglobulin heavy variable 1-2 Human genes 0.000 description 1
- 102100022949 Immunoglobulin kappa variable 2-29 Human genes 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 1
- 241000711937 Marburg marburgvirus Species 0.000 description 1
- 101000933447 Mus musculus Beta-glucuronidase Proteins 0.000 description 1
- JDHILDINMRGULE-LURJTMIESA-N N(pros)-methyl-L-histidine Chemical compound CN1C=NC=C1C[C@H](N)C(O)=O JDHILDINMRGULE-LURJTMIESA-N 0.000 description 1
- 102100033341 N-acetylmannosamine kinase Human genes 0.000 description 1
- 108010081778 N-acylneuraminate cytidylyltransferase Proteins 0.000 description 1
- 108010069465 N-acylneuraminate-9-phosphate synthase Proteins 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000577979 Peromyscus spicilegus Species 0.000 description 1
- ZPHBZEQOLSRPAK-UHFFFAOYSA-N Phosphoramidon Natural products C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O ZPHBZEQOLSRPAK-UHFFFAOYSA-N 0.000 description 1
- 101710194807 Protective antigen Proteins 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 102100029954 Sialic acid synthase Human genes 0.000 description 1
- 101710167605 Spike glycoprotein Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 108090001109 Thermolysin Proteins 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 241000282458 Ursus sp. Species 0.000 description 1
- 230000010530 Virus Neutralization Effects 0.000 description 1
- 102000010199 Xylosyltransferases Human genes 0.000 description 1
- 108050001741 Xylosyltransferases Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- WYUKJASPBYYQRJ-VSJOFRJTSA-N beta-D-GlcpNAc-(1->2)-alpha-D-Manp-(1->3)-[beta-D-GlcpNAc-(1->2)-alpha-D-Manp-(1->6)]-beta-D-Manp-(1->4)-beta-GlcpNAc-(1->4)-beta-D-GlcpNAc Chemical compound O[C@@H]1[C@@H](NC(=O)C)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O[C@@H]3[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)NC(C)=O)[C@H](O)[C@@H](CO[C@@H]3[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)NC(C)=O)O2)O)[C@@H](CO)O1 WYUKJASPBYYQRJ-VSJOFRJTSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940126587 biotherapeutics Drugs 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-UKLRSMCWSA-N dextrose-2-13c Chemical compound OC[C@H]1OC(O)[13C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-UKLRSMCWSA-N 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 125000002446 fucosyl group Chemical group C1([C@@H](O)[C@H](O)[C@H](O)[C@@H](O1)C)* 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 244000144993 groups of animals Species 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 229940065638 intron a Drugs 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 238000002439 negative-stain electron microscopy Methods 0.000 description 1
- 229940060155 neuac Drugs 0.000 description 1
- 238000004305 normal phase HPLC Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000012510 peptide mapping method Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- BWSDNRQVTFZQQD-AYVHNPTNSA-N phosphoramidon Chemical compound O([P@@](O)(=O)N[C@H](CC(C)C)C(=O)N[C@H](CC=1[C]2C=CC=CC2=NC=1)C(O)=O)[C@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@@H]1O BWSDNRQVTFZQQD-AYVHNPTNSA-N 0.000 description 1
- 108010072906 phosphoramidon Proteins 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 229950008882 polysorbate Drugs 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 210000003935 rough endoplasmic reticulum Anatomy 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000001296 transplacental effect Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- A61K2039/507—Comprising a combination of two or more separate antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Definitions
- Filoviruses ebolavirus and marburgvirus, cause severe hemorrhagic fevers in humans, with high mortality rates as well as epizootic diseases in nonhuman primates and probably other mammals.
- the main filovirus species causing outbreaks in humans are ebolaviruses Zaire (EBOV) and Sudan (SUDV), as well as the Lake Victoria Marburg virus (MARV).
- Filoviruses are enveloped, single-stranded, negative sense RNA filamentous viruses and encode seven proteins, of which the spike glycoprotein (GP) is considered the main protective antigen.
- EBOV and MARV GP is proteolytically cleaved by furin protease into two subunits linked by a disulfide linkage: GP1 (-140 kDa) and GP2 (-38 kDa) (Manicassamy, et al, 2005, J Virol, 79 (8):4793-4805).
- Three GP1-GP2 units form the trimeric GP envelope spike (-550 kDa) on the viral surface (Feldmann, et al, 1993, Arch Virol Suppl, 7:81-100; Feldmann, et al, 1991, Virology, 182 (l):353-356; Geisbert and Jahrling, 1995, Virus Res, 39 (2-3): 129-150; Kiley, et al, 1988a, J Gen Virol, 69 (Pt 8): 1957-1967).
- GP1 mediates cellular attachment (Kiley, et al, 1988b, J Gen Virol, 69 (Pt 8):1957-1967; Kuhn, et al, 2006, J Biol Chem, 281 (23): 15951-15958), and contains a mucin-like domain (MLD) which is heavily glycosylated and variable and has little or no predicted secondary structure (Sanchez, et al, 1998, J Virol, 72 (8):6442- 6447).
- Other filoviruses include Ravn virus (RAW), Tai Forest virus (TAFV), Reston virus (RESTV), and Bundibugyo virus (BDBV).
- This disclosure provides a method for preventing, treating, or managing a filovirus infection in a subject, where the method includes administering to a subject in need thereof an effective amount of an antibody cocktail comprising at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus glycoprotein (filovirus GP).
- an antibody cocktail comprising at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus glycoprotein (filovirus GP).
- the cocktail can include a first anti- filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof, where the first antibody or fragment thereof specifically binds to a filovirus GP1/GP2 base epitope, where the second antibody or fragment thereof specifically binds to a filovirus GP receptor binding site (RBS) epitope, a filovirus GP glycan cap epitope, a filovirus GP internal fusion loop (IFL) epitope, or any combination thereof.
- RBS filovirus GP receptor binding site
- IFL filovirus GP internal fusion loop
- at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains.
- administration of the antibody cocktail can be effective against two or more filovirus species or strains.
- the antibody cocktail can further include additional anti-filovirus GP antibodies, e.g., a third anti-filovirus GP antibody or antigen-binding fragment thereof that can specifically bind to a filovirus glycan cap epitope.
- additional anti-filovirus GP antibodies e.g., a third anti-filovirus GP antibody or antigen-binding fragment thereof that can specifically bind to a filovirus glycan cap epitope.
- the disclosure further provides a method for preventing, treating, or managing a filovirus infection in a subject, where the method includes administering to a subject in need thereof an effective amount of an antibody cocktail that includes at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus GP.
- the cocktail can include a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof, where the first antibody or fragment thereof specifically binds to a filovirus RBS epitope, where the second antibody or fragment thereof specifically binds to a filovirus IFL epitope.
- at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains.
- administration of the antibody cocktail can be effective against two or more filovirus species or strains.
- the filovirus infection is hemorrhagic fever.
- the subject is a nonhuman primate or a human.
- the two or more filovirus species can be, e.g., two or more of Marburg virus (MARV), Ravn virus (RAW), Tai Forest virus (TAFV), Reston virus
- the ability of the antibody cocktail to prevent, treat, or manage a filovirus infection can be measured in a model comprising administering the antibody cocktail to a group of rodents, e.g., mice or guinea pigs, and challenging rodents with a wild-type or rodent-adapted filovirus before, at the same time as, or after administering the antibody cocktail to the rodents.
- rodents e.g., mice or guinea pigs
- challenging rodents with a wild-type or rodent-adapted filovirus before, at the same time as, or after administering the antibody cocktail to the rodents.
- Efficacy in the model systems can be determined by monitoring the test rodents for, e.g., increased survival time, decreased weight loss, or a combination thereof as compared to control rodents.
- Antibody cocktails suitable for the provided methods are also provided.
- FIG. 1 Trimeric structure of EBOV glycoprotein (GP) lacking the mucin like domain (MLD).
- MLD mucin like domain
- the MLD is modeled on the structure as large gray balls.
- the different regions of the GP, the glycan cap, the core GPl in the head domain, the base consisting of the GP2 and the N-terminus of GP are shown.
- the stalk, which was not part of the solved structure is shown as a box.
- the mapped epitope regions of several monoclonal antibodies described herein are specified.
- Figure 2 The position of m8C4 binding sites Q251, F252, and R136 shown on EBOV GP monomelic structure.
- FIG. 3A-C Post-exposure efficacy of FVM04 in mouse model of EBOV and SUDV infection.
- A&B Groups of ten BALB/c mice (6-8 weeks old) were infected by intraperitoneal (i.p.) with 100 PFU of mouse adapted (MA)-EBOV and treated i.p. with the doses and at time points indicated in the figure or left untreated.
- Figure 4A-E Efficacy of FVM04 treatment and an FVM04 containing cocktail in guinea pig models of SUDV and EBOV infection.
- A&B Efficacy of a single dose of 5 mg/animal FVM04 (-15 mg/kg) injected i.p. at 1 dpi compared to control group receiving DPBS in animals challenged with guinea pig-adapted (GPA)-SUDV (A) or GPA-EBOV (B), with 6 animals per group.
- FIG. 5 FVM09 displays neutralizing activity in presence of 2G4.
- FIG. 6A-B FVM02 enhances the neutralization of filoviruses by RBS binding antibodies.
- a or “an” entity refers to one or more of that entity; for example, “polypeptide subunit” is understood to represent one or more polypeptide subunits.
- polypeptide subunit is understood to represent one or more polypeptide subunits.
- the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
- non-naturally occurring substance, composition, entity, and/or any combination of substances, compositions, or entities, or any grammatical variants thereof is a conditional term that explicitly excludes, but only excludes, those forms of the substance, composition, entity, and/or any combination of substances, compositions, or entities that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or could be at any time, determined or interpreted by a judge or an administrative or judicial body to be, "naturally-occurring.”
- polypeptide is intended to encompass a singular "polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
- polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
- polypeptides peptides, dipeptides, tripeptides, oligopeptides, "protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
- polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids.
- a polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
- a "protein” as used herein can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein.
- an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
- an isolated polypeptide can be removed from its native or natural environment.
- Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.
- non-naturally occurring polypeptide is a conditional term that explicitly excludes, but only excludes, those forms of the polypeptide that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or could be at any time, determined or interpreted by a judge or an administrative or judicial body to be, "naturally-occurring.”
- polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof.
- fragment can include any polypeptide or protein that retain at least some of the activities of the complete polypeptide or protein, but which is structurally different. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments.
- variants include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur spontaneously or be intentionally constructed.
- variants can be produced using art-known mutagenesis techniques.
- Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions.
- Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins.
- Variant polypeptides can also be referred to herein as "polypeptide analogs.”
- a "derivative” refers to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as “derivatives" are those peptides that contain one or more standard or synthetic amino acid derivatives of the twenty standard amino acids.
- 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.
- a "conservative amino acid substitution” is one in which one amino acid is replaced with another amino acid having a similar side chain.
- Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
- basic side chains e.g.,
- substitution of a phenylalanine for a tyrosine is a conservative substitution.
- Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate protein activity are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al, Protein Eng. 12(10):879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).
- antibody encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally-occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.
- an antibody or fragment thereof can comprise one or more "binding domains.”
- a "binding domain” or "antigen binding domain” is a two- or three-dimensional structure, e.g., a polypeptide structure that cans specifically bind a given antigenic determinant, e.g., the region formed by the heavy and light chain variable regions of an antibody or fragment thereof.
- the terms "antibody” and “immunoglobulin” can be used interchangeably herein.
- An antibody (or a fragment, variant, or derivative thereof as disclosed herein comprises at least the variable domain of a heavy chain and at least the variable domains of a heavy chain and a light chain.
- Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et ah, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
- immunoglobulin comprises various broad classes of polypeptides that can be distinguished biochemically.
- heavy chains are classified as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ 1- ⁇ 4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
- immunoglobulin subclasses e.g., IgGj, IgG 2 , IgG 3 , IgG 4 , IgA I , etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.
- Light chains are classified as either kappa or lambda ( ⁇ , ⁇ ). Each heavy chain class can be bound with either a kappa or lambda light chain.
- the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
- the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
- Both the light and heavy chains are divided into regions of structural and functional homology.
- the terms "constant” and “variable” are used functionally.
- the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
- the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
- the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
- variable region allows an antibody or fragment thereof to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody or fragment thereof combine to form the variable region that defines a three dimensional antigen binding site.
- This quaternary antibody structure forms the antigen- binding site present at the end of each arm of the Y. More specifically, the antigen- binding site is defined by three CDRs on each of the VH and VL chains.
- each antigen binding domain is short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
- the remainder of the amino acids in the antigen binding domains referred to as "framework” regions, show less inter-molecular variability.
- the framework regions largely adopt a ⁇ - sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure.
- framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
- the antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
- the amino acids comprising the CDRs and the framework regions, respectively can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined ⁇ see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J Mol. Biol., 796:901-917 (1987), which are incorporated herein by reference in their entireties).
- CDR complementarity determining region
- Immunoglobulin variable domains can also be analyzed using the IMGT information system (www://imgt.cines.fr/) (IMGT®/V-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. 5 ⁇ 5:W503-508 (2008).
- Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
- One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself.
- Kabat numbering refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).
- Antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library.
- ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
- Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type ⁇ e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class ⁇ e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
- an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope.
- an antibody or fragment thereof is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
- the term “specificity” is used herein to qualify the relative affinity by which a certain antibody or fragment thereof binds to a certain epitope.
- antibody or fragment thereof "A” can be deemed to have a higher specificity for a given epitope than antibody or fragment thereof "B,” or antibody or fragment thereof “A” can be said to bind to epitope "C” with a higher specificity than it has for related epitope "D.”
- the methods and compositions provided in this disclosure combine various human-, murine-, and/or non-human primate (NHP)-derived antibodies or fragments, variants, or derivatives thereof in antibody cocktails.
- the antibodies or fragments thereof disclosed herein can be said to bind a target antigen, e.g., a filovirus glycoprotein subunit or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5 X 10 '2 sec '1 , 1(T sec "1 , 5 X 10 "J sec “ or 10 " sec “ .
- a human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to bind a target antigen, e.g., a filovirus glycoprotein subunit, with an off rate (k(off)) less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 "5 sec “1 , or 10 "5 sec “1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X 10 "7 sec '1 or 10 '7 sec “1 .
- a target antigen e.g., a filovirus glycoprotein subunit
- a human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to bind a target antigen, e.g., a filovirus glycoprotein subunit with an on rate (k(on)) of greater than or equal to 10 3 M "1 sec "1 , 5 X 10 3 M “1 sec '1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
- a target antigen e.g., a filovirus glycoprotein subunit with an on rate (k(on)) of greater than or equal to 10 3 M "1 sec "1 , 5 X 10 3 M "1 sec '1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
- a human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to bind a target antigen, e.g., a filovirus glycoprotein subunit with an on rate (k(on)) greater than or equal to 10 s M "1 sec “1 , 5 X 10 s M “1 sec “1 , 10 6 M “1 sec “1 , or 5 X 10 6 M “1 sec “1 or 10 7 M " ' sec “1 .
- a target antigen e.g., a filovirus glycoprotein subunit with an on rate (k(on)) greater than or equal to 10 s M "1 sec “1 , 5 X 10 s M “1 sec “1 , 10 6 M “1 sec “1 , or 5 X 10 6 M “1 sec “1 or 10 7 M " ' sec “1 .
- a human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to competitively inhibit binding of a reference antibody or antigen binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen binding fragment to the epitope.
- Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays.
- a human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
- the term "affinity” refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28.
- the term “avidity” refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g., Harlow at pages 29-34.
- Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. An interaction between a between a bivalent monoclonal antibody with a receptor present at a high density on a cell surface would also be of high avidity.
- Antibodies or antigen-binding fragments, variants or derivatives thereof as disclosed herein can also be described or specified in terms of their cross-reactivity.
- cross-reactivity refers, e.g., to the ability of a human-, murine-, and/or NHP- derived antibody or fragment thereof specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances.
- an antibody or fragment thereof is cross-reactive if it binds to an epitope other than the one that induced its formation, e.g., various different filovirus receptor binding regions.
- the cross-reactive epitope contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.
- a Human-, murine- and/or NHP-derived antibody or fragment, variant, or derivative thereof for use in the antibody cocktails provided herein can also be described or specified in terms of their binding affinity to an antigen.
- an antibody can bind to an antigen with a dissociation constant or 3 ⁇ 4 no greater than 5 10 " M, 10 " M, 5 x 10 "3 M, 10 "3 M, 5 x lO ⁇ M, lO ⁇ M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 " 7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 " M, 10 “9 M, 5 x 10 " '° M, 10 " '° M, 5 x 10 " “ M, 10 “n M, 5 x 10 "I2 M, 10 “I2 M, 5 x 10 "13 M, 10 "I3 M, 5 x 10 "I M, 10 “I4 M, 5
- Antibody fragments including single-chain antibodies can comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included are antigen-binding fragments that comprise any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains. Antibodies, or antigen-binding fragments thereof disclosed herein can be from any animal origin including birds and mammals.
- the antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
- the variable region can be condricthoid in origin (e.g., from sharks).
- human antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
- the term "heavy chain portion” includes amino acid sequences derived from an immunoglobulin heavy chain, an antibody comprising a heavy chain portion comprises at least one of: a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
- a Human-, murine- and/or NHP-derived antibody or fragment, variant, or derivative thereof for use in the antibody cocktails provided herein can comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain.
- a human-, murine- and/or NHP-derived antibody or fragment, variant, or derivative thereof for use in the antibody cocktails provided herein comprises a polypeptide chain comprising a CH3 domain.
- a human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails provided herein can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
- these domains e.g., the heavy chain portions
- these domains can be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
- the heavy chain portions of a human-, murine-, and/or NHP-derived antibody as disclosed herein can be derived from different immunoglobulin molecules.
- a heavy chain portion of a polypeptide can comprise a CHI domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule.
- a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule.
- a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
- the term "light chain portion” includes amino acid sequences derived from an immunoglobulin light chain.
- the light chain portion comprises at least one of a VL or CL domain.
- Human-, murine-, and/or NHP-derived antibodies or fragments thereof for use in the antibody cocktails disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target a filovirus glycoprotein subunit that they recognize or specifically bind.
- the portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope," or an "antigenic determinant.”
- a target antigen, e.g., a filovirus glycoprotein subunit can comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.
- orthologous epitope refers to versions of an epitope found in related organisms, e.g., different filovirus species or strains. Orthologous epitopes can be similar in structure, but can vary in one or more amino acids.
- VH domain includes the amino terminal variable domain of an immunoglobulin heavy chain
- CHI domain includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain.
- the CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
- CH2 domain includes the portion of a heavy chain molecule that extends, e.g., from about amino acid 244 to amino acid 360 of an antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat EA et al. op. cit.
- the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N- linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 amino acids.
- Hinge region includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 amino acids and is flexible, thus allowing the two N-terminal antigen- binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al, J. Immunol. 7 «57:4083 (1998)).
- disulfide bond includes the covalent bond formed between two sulfur atoms.
- the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
- the CHI and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
- chimeric antibody will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial or modified) is obtained from a second species.
- the target binding region or site will be from a non-human source ⁇ e.g. mouse or primate) and the constant region is human.
- bispecific antibody refers to an antibody that has binding sites for two different antigens within a single antibody molecule. It will be appreciated that other molecules in addition to the canonical antibody structure can be constructed with two binding specificities. It will further be appreciated that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol. tf:1387-94 (2010); Mabry and Snavely, IDrugs. 75:543-9 (2010)). A bispecific antibody can also be a diabody.
- the term “engineered antibody” refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, by partial framework region replacement and sequence changing.
- the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class, e.g., from an antibody from a different species.
- an engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody.”
- a humanized antibody In some instances, not all of the CDRs are replaced with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another, instead, minimal amino acids that maintain the activity of the target-binding site are transferred.
- U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370 it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional engineered or humanized antibody.
- Human-, murine-, and/or NHP-derived antibodies or fragments thereof for use in the antibody cocktails disclosed herein can be glycoengineered to contain predominantly a single glycoform.
- the engineered glycan can be, e.g., GnGn (GlcNAc 2 -Man 3 -GlcNAc 2 ), mono- or di-galactosylated (Gal ( i/ 2) -GlcNAc 2 -Man 3 - GlcNAc 2 ), mono- or di-sialylated (NaNa ( i i2) -Gal ( i /2) -GlcNAc 2 -Man 3 -GlcNAc 2 ) containing little or no fucose, or xylose.
- a predominantly single glycoform is any glycoform that represents more than half (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) of all glycoform
- the RAMP system has been used for glycoengineering of antibodies, antibody fragments, idiotype vaccines, enzymes, and cytokines. Dozens of antibodies have been produced in the RAMP system (see, e.g., Hiatt A, et al., (2013) Clinical & Developmental Immunology 2013:632893; Whaley KJ, et al, (2014) Current topics in Microbiology and Immunology 375: 107-26; Bendandi M, et al. (2010) Annals of Oncology 21 :2420-2427; Castilho A & Steinkellner H (2012) Biotechnology J. 7:1088-1098).
- IgGs IgM
- IgM isotypes
- IgM Lios A, et al., (2014) Proc. Natl. Acad. Sci. U.S.A. 7/7:6263-8; Hiatt, A., (2014) Proc. Natl. Acad. Set U.S.A 777:6124-5
- IgM Lios A, et al., (2014) Proc. Natl. Acad. Sci. U.S.A. 7/7:6263-8; Hiatt, A., (2014) Proc. Natl. Acad. Set U.S.A 777:6124-5
- cDNAs encoding a mAb of interest can be expressed in suitable Nicotiana benthamiana expression hosts and/or in combination with Agrobacterium vectors encoding one or more appropriate glycosylation enzymes to produce the desired glycan profile in the expressed mAb.
- the suitable Nicotiana benthamiana expression host can be simultaneously exposed to a mixed population of recombinant Agrobacterium where some of the Agrobacteria contain a first T-DNA vector comprising a cDNA encoding the heavy chain of the mAb in a first non-competing viral vector and the remainder of the Agrobacteria contain a second T-DNA comprising a cDNA encoding the light chain of the mAb in a second non-competing viral vector.
- one of the non-competing viral vectors is a Tobacco Mosaic Virus (TMV) vector and one of the non-competing vectors is a Potato Virus X (PVX) vector (Giritch et al, (2006) Proc. Natl. Acad. Sci. U.S.A. 103: 14701-14706).
- TMV Tobacco Mosaic Virus
- PVX Potato Virus X
- wild-type glycans ⁇ i.e. native, plant-produced glycosylation
- wild-type N. benthamiana can be inoculated with only the Agrobacterium containing the antibody cDNA(s).
- the same Agrobacterium can be used to inoculate plants that contain little or no fucosyl or xylosyl transferases (AXF plants; Strasser et al. Plant Biotechnol J. 26:392- 402).
- AXF plants can be inoculated with the Agrobacterium containing the antibody cDNA(s) as well as an Agrobacterium containing the cDNA for p-l,4-galactosyl transferase expression contained on a binary Agrobacterium vector to avoid recombination with the TMV and PVX vectors (Castilho A, el al, (2010) J Biol.
- sialylated glycans six additional genes can be introduced in binary vectors to reconstitute the mammalian sialic acid biosynthetic pathway.
- the genes are UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N- acetylneuraminic acid phosphate synthase, CMP-N-acetylneuraminic acid synthetase, CMP-NeuAc transporter, p-l,4-galactosylatransferase, and a2,6-sialyltransferase ⁇ Id.).
- Glycanalysis of glycoengineered mAbs can be achieved by release of N-linked glycans by digestion with N-glycosidase F (PNGase F). Subsequent derivatization of the free glycan can be achieved with anthranilic acid (2-AA). The 2-AA-derivatized oligosaccharide is separated from any excess reagent via normal-phase HPLC. The column is calibrated with 2-AA-labeled glucose homopolymers and glycan standards. The test samples and 2-AA-labeled glycan standards are detected fluorometrically.
- PNGase F N-glycosidase F
- 2-AA anthranilic acid
- Glycoforms are assigned either by comparing their glucose unit (GU) values with those of the 2-AA-labeled glycan standards or by comparing with the theoretical GU values (Guile GR, et al., (1996) Analytical Biochem. 240:210-226). Confirmation of glycan structure can be accomplished with LC/MS.
- GU glucose unit
- RAMP system is an effective method of producing various glycoengineered and wild-type mABs
- other expression systems can be used to accomplish the same result.
- mammalian cell lines such as CHO or NSO cells (Davies, J., et al, Biotechnol Bioeng 74:288-294), yeast cells such as Pichia pastoris (Gerngross T. Adv Exp Med Biol. (2005); 564:139) and bacterial cells such as E. coli have been used produce such mAbs.
- polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).
- mRNA messenger RNA
- pDNA plasmid DNA
- a polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond ⁇ e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
- PNA peptide nucleic acids
- nucleic acid refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
- isolated nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
- a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein.
- Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
- Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically.
- polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
- a "non-naturally occurring" polynucleotide is a conditional definition that explicitly excludes, but only excludes, those forms of the polynucleotide that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or that could be at any time, determined or interpreted by a judge or an administrative or judicial body to be, "naturally- occurring.”
- a "coding region” is a portion of nucleic acid comprising codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
- any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region.
- a vector, polynucleotide, or nucleic acid can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a polypeptide subunit or fusion protein as provided herein.
- Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
- the polynucleotide or nucleic acid is DNA.
- a polynucleotide comprising a nucleic acid that encodes a polypeptide normally can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions.
- An operable association or linkage can be when a coding region for a gene product, e.g., a polypeptide, can be associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
- Two DNA fragments can be "operably associated” or “operably linked” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
- a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
- the promoter can be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
- transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
- Suitable promoters and other transcription control regions are disclosed herein.
- transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
- Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit ⁇ -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
- translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
- a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA).
- mRNA messenger RNA
- Polynucleotide and nucleic acid coding regions can be associated with additional coding regions that encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein, e.g., a polynucleotide encoding a polypeptide subunit provided herein.
- proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
- polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature” form of the polypeptide.
- the native signal peptide e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
- a heterologous mammalian signal peptide, or a functional derivative thereof can be used.
- the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse ⁇ - glucuronidase.
- a "vector” is nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
- a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
- a vector can also include one or more selectable marker gene and other genetic elements known in the art.
- a "transformed” cell, or a "host” cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques.
- transformation encompasses those techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
- a transformed cell or a host cell can be a bacterial cell or a eukaryotic cell.
- expression refers to a process by which a gene produces a biochemical, for example, a polypeptide.
- the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.
- mRNA messenger RNA
- a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript.
- Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
- treat refers to reducing the potential for disease pathology, reducing the occurrence of disease symptoms, e.g., to an extent that the subject has a longer survival rate or reduced discomfort.
- treating can refer to the ability of a therapy when administered to a subject, to reduce disease symptoms, signs, or causes. Treating also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.
- protection when used in the context of the ability of a therapeutic agent to affect the course of an infectious disease refers to any protective effect observed in comparison to a control agent. For example if two groups of animals are challenged with an infectious agent, e.g., a lethal dose of EBOV, and one group of animals is administered the therapeutic agent while the other group is administered a control, if a statistically significant number of animals in the therapeutic group survive relative to the number of survivors in the control group, a protective effect is observed. "Protection" can be, but does not have to be, 100%.
- subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
- Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals, including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.
- composition refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.
- Such composition can be sterile.
- an “effective amount” of an antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose.
- An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
- Certain therapies can provide "synergy” and prove “synergistic", i.e., an effect can be achieved when the active ingredients used together that is greater than the sum of the effects that results from using the compounds separately.
- a synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
- alternation therapy a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes.
- an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
- a synergistic effect can be measured by potency assays, e.g., mouse our guinea pig challenge assays or neutralization assays as described elsewhere herein.
- cocktails of two or more anti-filovirus glycoprotein antibodies can provide life-sustaining benefit to subjects, e.g., patients and/or healthcare workers exposed to or susceptible to exposure to, filovirus infection, e.g., EBOV, SUDV, or MARV.
- filovirus infection e.g., EBOV, SUDV, or MARV.
- the exact species or strain of filovirus might not be immediately determined, or the outbreak could be caused by more than one filovirus species or strain.
- this disclosure provides methods for preventing, treating, and/or managing filovirus infections or outbreaks using cocktails of antibodies that can be effective against more than one filovirus species or strain.
- this disclosure provides a method for preventing, treating, or managing a filovirus infection in a subject where the method entails administering to a subject in need thereof an effective amount of an antibody cocktail that includes, or comprises, at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus glycoprotein (filovirus GP).
- an antibody cocktail that includes, or comprises, at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus glycoprotein (filovirus GP).
- at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains.
- the antibody in other words, if the antibody or fragment thereof was raised against the EBOV glycoprotein and is found to bind to the EBOV glycan cap, the antibody "can specifically bind to its orthologous epitope on two or more filovirus species or strains" if the antibody or fragment thereof also binds to, e.g., the glycan cap of other strains of EBOV and/or the MARV, RAW, TAFV, RESTV, SUDV, and/or BDBV GP glycan cap, or any strain thereof.
- administration of the antibody cocktail can be effective against two or more filovirus species or strains, e.g., the antibody cocktail can neutralize two or more filovirus species or strains and/or protect against disease caused by two or more filovirus species or strains, e.g., in an animal challenge model.
- the two or more filovirus species can be two or more of Marburg virus (MARV), Ravn virus (RAW), Tai Forest virus (TAFV), Reston virus (RESTV), Sudan virus (SUDV) Ebola virus (EBOV), Bundibugyo virus (BDBV), or any strain thereof.
- the filovirus infection is hemorrhagic fever.
- the subject is a nonhuman primate or a human.
- the antibodies or fragments thereof for use in the methods provided herein can each independently be, e.g., a mouse antibody, a non-human primate (NHP) antibody, a humanized antibody, a chimeric antibody, or a fragment thereof.
- the antibody or fragment thereof can be a monoclonal antibody, a component of a polyclonal antibody mixture, a recombinant antibody, a multispecific antibody, or any combination thereof.
- the ability of the antibody cocktail to prevent, treat, or manage a filovirus infection, and or the potency relative to individual antibodies can be measured in a model comprising administering the antibody cocktail to a group of rodents and challenging rodents with a rodent-adapted filovirus before, at the same time as, or after administering the antibody cocktail to the rodents.
- the rodent model can be, for example, a guinea pig model or a mouse model. In either model, the challenged rodents can be monitored for increased survival time, decreased weight loss, or a combination thereof as compared to control rodents.
- the ability of the antibody cocktail to prevent, treat, or manage a filovirus infection, and or the potency relative to individual antibodies can be measured in a neutralization assay as described elsewhere herein.
- the subject is administered an effective amount of an antibody cocktail as described above.
- the antibody cocktail can prevent, treat, or manage filovirus infection in the subject with a potency that is greater than the additive potency of the antibodies or fragments thereof when administered individually.
- administration of the provided antibody cocktail to the subject is effective against two or more filovirus species or strains.
- the method includes administering the antibody cocktail to, e.g., improve efficacy, reduce the number of treatments, to allow efficacy when administered at a later time from the inception of infection in the subject, and/or to allow dose sparing.
- administration of the antibody cocktail can result in synergistic efficacy, e.g., efficacy that is more potent than would be expected based on the efficacy of the antibodies administered individually.
- the antibody cocktails provided herein can be useful for treatment of a filovirus infection without it being necessary to know the exact filovirus species or strain. Any antibody cocktail described herein can include, in addition to the recited antibodies, other antibodies that bind to the same or different filovirus glycoprotein epitopes.
- each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently comprise a heavy chain constant region or fragment thereof.
- the heavy chain can be, e.g., a murine constant region or fragment thereof, a human constant region or fragment thereof, e.g., an IgM, IgG, IgA, IgE, IgD, or IgY constant region or fragment thereof.
- a human IgG constant region subtypes or fragments thereof can also be included, e.g., a human IgGl, IgG2, IgG3, or IgG4 constant region or fragment thereof.
- each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently further comprise a light chain constant region or fragment thereof.
- the light chain constant region or fragment thereof can be a murine constant region or fragment thereof, e.g., a human light chain constant region or fragment thereof, e.g., a human kappa or lambda constant region or fragment thereof.
- each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently comprise a full-size antibody comprising two heavy chains and two light chains.
- the binding domain(s) of each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently be an Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv fragment, an scFab fragment, an sc(Fv)2 fragment, or any combination thereof.
- each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can, either independently or collectively, fully or partially neutralize infectivity of a filovirus upon binding of the binding domain to one or more orthologous epitopes on the filovirus.
- each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently be conjugated to an antiviral agent, a protein, a lipid, a detectable label, a polymer, or any combination thereof.
- the antibody cocktail includes at least a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof.
- the first antibody or fragment thereof can specifically bind to a filovirus GP1/GP2 base epitope
- the second antibody or fragment thereof can specifically bind to a filovirus GP receptor binding site (RBS) epitope, a filovirus GP glycan cap epitope, a filovirus GP internal fusion loop (IFL) epitope, or any combination thereof.
- Antibodies binding to the base epitope include, without limitation, 2G4, 4G7, and/or ADI- 15734, described elsewhere herein.
- Antibodies that bind to the RBS epitope include, without limitation, FVM04, and MR191.
- Antibodies that bind to the glycan cap epitope include, without limitation, 13C6FR1, FVM09, ADI- 15731, m8C4 (in part), and ADI-15750 (in part).
- Antibodies binding to the IFL epitope include, without limitation, FVM02, ADI-15742, ADI-15878, and ADI-15946.
- the first antibody or fragment thereof of the antibody cocktail can be, or can be related to the base-binding antibody 2G4 and the second antibody or fragment thereof of the antibody cocktail can be, or can be related to the RBS-binding antibody FVM04.
- the first antibody or fragment thereof of the antibody cocktail can comprise a heavy chain variable region (VH) and a light chain variable region (VL) collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 , respectively (2G4)
- the second antibody or fragment thereof can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-C
- the first antibody or fragment thereof can comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 29, respectively (2G4)
- the second antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 5, respectively (FVM04).
- the antibody cocktail can further comprise a third anti- filovirus GP antibody or antigen-binding fragment thereof, wherein the third antibody or fragment thereof can specifically bind to a filovirus glycan cap epitope.
- the third antibody or fragment thereof can be, or can be related to the glycan cap binding Mab 13C6 (See U.S. Patent No. 7,335,356) or the related glycan cap binding Mab 13C6FR1, which contains various amino acid substitutions in the VH framework- 1 (FR1) and the VL FR1 regions relative to the original 13C6. 13C6FR1 is described in more detail elsewhere herein.
- the third antibody or fragment thereof in the antibody cocktail can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, or a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 9 and SEQ ID NO: 13, respectively (13C6FR1).
- an effective amount of an antibody cocktail comprising 2G4 or an antigen-binding fragment thereof, FVM04 or an antigen binding fragment thereof, and 13C6FR1 or an antigen binding fragment thereof can protect guinea pigs from challenge with guinea pig-adapted EBOV (GPA-EBOV) and can also protect guinea pigs from challenge with guinea pig-adapted SUDV (GPA-SUDV).
- GPA-EBOV guinea pig-adapted EBOV
- GPA-SUDV guinea pig-adapted SUDV
- the first antibody or fragment thereof of the antibody cocktail can be, or can be related to the base-binding antibody 2G4 and the second antibody or fragment thereof of the antibody cocktail can be, or can be related to the glycan cap binding antibody FVM09.
- the first antibody or fragment thereof of the antibody cocktail can comprise a heavy chain variable region (VH) and a light chain variable region (VL) collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL- CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 , respectively (2G4)
- the second antibody or fragment thereof can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to S
- the first antibody or fragment thereof can comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 29, respectively (2G4)
- the second antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 17 and SEQ ID NO: 21, respectively (FVM09).
- the antibody cocktail includes at least a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof.
- the first antibody or fragment thereof can specifically bind to a filovirus RBS epitope
- the second antibody or fragment thereof can specifically bind to a filovirus IFL epitope.
- the method provides that at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains.
- the method provides that administration of the antibody cocktail is effective against two or more filovirus species or strains , e.g., the antibody cocktail can neutralize two or more filovirus species or strains and/or protect against disease caused by two or more filovirus species or strains, e.g., in an animal challenge model.
- the first antibody or fragment thereof of the antibody cocktail can be, or can be related to the RBS-binding antibody FVM04 and the second antibody or fragment thereof of the antibody cocktail can be, or can be related to the IFL-binding antibody FVM02.
- the first antibody or fragment thereof of the antibody cocktail can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively (FVM04), and the second antibody or fragment thereof can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH- CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 34, SEQ ID NO: 35,
- the first antibody or fragment thereof can comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 5, respectively (FVM04), and the second antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 33 and SEQ ID NO: 37, respectively (FVM02).
- the first antibody or fragment thereof of the antibody cocktail can be, or can be related to the RBS-binding antibody MR191 and the second antibody or fragment thereof of the antibody cocktail can be, or can be related to the IFL-binding antibody FVM02.
- the first antibody or fragment thereof of the antibody cocktail can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively (MR191)
- the second antibody or fragment thereof can comprise a VH and a VL collectively comprising VH-CDR1, VH- CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 34, S
- the first antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 41 and SEQ ID NO: 45, respectively (MR191)
- the second antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 33 and SEQ ID NO: 37, respectively (FVM02).
- treatment includes the application or administration of an antibody cocktail as provided herein, to a subject or patient, where the subject or patient has been exposed to a filovirus, infected with a filovirus, has a filovirus disease, a symptom of a filovirus disease, or a predisposition toward contracting a filovirus disease.
- treatment can also include the application or administration of a pharmaceutical composition comprising an antibody cocktail as provided herein, to a subject or patient, so as to target the pharmaceutical composition to an environment where the antibody cocktail can be most effective, e.g., the endosomal region of a virus- infected cell.
- an antibody cocktail as provided herein can be used to promote a positive therapeutic response.
- positive therapeutic response is intended any improvement in the disease conditions associated with the activity of the antibody cocktail, and/or an improvement in the symptoms associated with the disease.
- an improvement in the disease can be characterized as a complete response.
- complete response is intended an absence of clinically detectable disease with normalization of any previously test results. Such a response can in some cases persist, e.g., for at least one month following treatment according to the methods of the disclosure.
- an improvement in the disease can be categorized as being a partial response.
- the disclosure further provides antibody cocktails as described above, for use in the provided treatment methods.
- Such antibody cocktails can be formulated as pharmaceutical compositions.
- Suitable and administering antibody cocktails as provided herein, to a subject in need thereof are well known to or are readily determined by those skilled in the art.
- the route of administration of an antibody cocktail for use in the methods provided herein can be, for example, oral, parenteral, by inhalation or topical.
- parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are clearly contemplated as suitable forms, another example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip.
- a suitable pharmaceutical composition can comprise a buffer (e.g.
- an antibody cocktail as provided herein can be delivered directly to a site where the antibody or fragment thereof can be effective in virus neutralization.
- an antibody cocktail as provided herein can be administered in a pharmaceutically effective amount for the in vivo treatment of diseases or disorders associated with filovirus infection.
- the disclosed antibody cocktails can be formulated so as to facilitate administration and promote stability of the active agent(s).
- compositions accordingly can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, nontoxic buffers, preservatives and the like.
- a pharmaceutically effective amount of an antibody cocktail means an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or condition or to detect a substance or a cell.
- the components of a given antibody cocktail can be provided in equimolar amounts or can be provided in different ratios to produce the best clinical result. Methods to determine the best ratios of active components can be readily determined. Suitable formulations for use in the therapeutic methods disclosed herein can be described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).
- the relative amounts of the antibodies or fragments thereof in the antibody cocktail that can be combined with carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration.
- the composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide an optimum response (e.g., a therapeutic or prophylactic response).
- an antibody cocktail for use in the methods provided herein can be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic effect.
- An antibody cocktail provided herein can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody or antigen-binding fragment, variant, or derivative thereof of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques.
- the form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
- therapeutically effective dose or amount or "effective amount” is intended an amount of an antibody cocktail that when administered brings about a positive therapeutic response with respect to treatment of a patient with a disease or condition to be treated.
- compositions disclosed herein for treatment of diseases or disorders associated with filovirus infection, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
- the patient is a human, but non- human mammals including non-human primates can also be treated.
- Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
- the amount of an antibody cocktail to be administered can be readily determined by one of ordinary skill in the art without undue experimentation given this disclosure. Factors influencing the mode of administration and the respective amount of an antibody cocktail include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Similarly, the amount of an antibody cocktail to be administered will be dependent upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent.
- This disclosure also provides for the use of an antibody cocktail in the manufacture of a medicament for treating, preventing, or managing a disease or disorder associated with filovirus infection, e.g., hemorrhagic fever.
- a disease or disorder associated with filovirus infection e.g., hemorrhagic fever.
- VSV vesicular stomatitis virus
- filovirus GP a replication defective (single round infection) rVSV-GP-Luc expressing firefly luciferase
- rVSV-GFP replication competent expressing green fluorescence protein
- rVSV-GP Luciferase pseudotype assay Pseudotyped viruses were generated based on a modification of previously published method (Whitt, . A. 2010. J Virol Methods 169:2)65-1 ).
- HEK 293T cells were grown to 80% confluency, and transfected with plasmids encoding EBOV-GP, MARV-GP or SUDV GP using Fugene HD (Promega) according to the manufacture's protocol. The next day, these cells were infected with rVSV-AGP pseudotype (Kerafast) at an MOI of 3 and the virus was washed off after 1 h with DPBS.
- rVSV GP-GFP assay Recombinant Indiana VSV (rVSV) expressing eGFP, as well as EBOV or MARV GP in place of VSV G were described previously (Miller, E.H., et al., (2012) EMBO J. 31 :1947-1960; Ng, M., et al., (2014) Virology 468-470:637-646; Wong., A.C. et al., (2010) J. Virol. 84:163-175.). rVSVs bearing TAFV GP, SUDV GP, or BDBV GPAMuc were generated by the same method.
- VSV particles containing cleaved GP were generated by incubating rVSV-GPs with thermolysin (200 ⁇ g mL) for lh at 37 °C, followed by treatment with phosphoramidon (1 mM), and reaction mixtures were used immediately. Infectivity of rVSVs was measured by counting eGFP-positive cells at 12-14 h post-infection using a Celllnsight CX5 automated microscope and onboard software (Thermo Scientific). For neutralization experiments, serial dilutions of mAbs were incubated with the rVSV-GP for lh at room temperature.
- FVM04 is a macaque-human chimeric monoclonal antibody that binds to an exposed epitope within the RBS of multiple ebolaviruses including the virulent strains Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). See PCT Application No. PCT/US 15/57627.
- EBOV Ebola virus
- SUDV Sudan virus
- BDBV Bundibugyo virus
- Human HEK-293T cells were transfected with the entire mutation library in a 384-well array format (one clone per well) and assessed for reactivity to FVM04 using high-throughput flow cytometry.
- the method for shotgun mutagenesis is described in patent application 61/938,894 and (Davidson, E., and Doranz, B.J., 2014, Immunology, 143, 13-20).
- the shotgun mutagenesis revealed that FVM04 contact sites included Kl 15, Dl 17, and Gl 18. This is located within a hydrophilic region of the RBS known as the Crest ((Hashiguchi, et al, 2015, Cell, 160, 904-912; Wang et al, 2016, Cell, 164(l-2):258-68).
- FVM04 is also weakly reactive to marburgvirus glycoprotein (PCT/US 15/57627; Keck, et al., 2015, J Virol, 90:279 -291 ("Keck et al.”)).
- FVM04 neutralizes EBOV and SUDV (PCT/US 15/57627; Keck et al.), as well as BDBV and protects mice and guinea pigs against lethal EBOV and SUDV infection (see below).
- Antibodies that bind to the glycan cap :
- 13C6FR1 Monoclonal antibody 13C6 was developed using mouse hybridoma technology and was shown to protect mice from lethal challenge with Ebola virus (Wilson et al, 2000, Science, 287(5458): 1664-6). 13C6 binds on the top of EBOV GP glycan cap (Murin, et al., 2014, Proc Natl Acad Sci U S A, 111(48): 17182-7) with key contact residues being T270 and K272 (Davidson et al, 2015, J Virol, 89(21):10982-9). 13C6FR1, a variant of 13C6 (U.S. Patent No.
- FVM09 binds with high affinity to a linear epitope within the disordered loop connecting the ⁇ strands 17 and 18 in the glycan cap region of EBOV GP (PCT/US 15/57627; Keck et al.).
- Using overlapping peptide mapping we mapped the epitope for FVM09 to amino acids 286-290 (GEWAF) of EBOV GP, and this epitope is 100% conserved among all ebolaviruses (PCT US 15/57627; Keck et al.).
- FVM09 alone does not neutralize or provide protection in vivo against EBOV, but in combination with several other antibodies it enhances their neutralizing and protective potency as described in (Keck et al.) and below.
- ADI-15731 The ADI- 15731 mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt, et al., 2016, Science, pii:aad5788. [Epub ahead of print] "Bornholdt et al "). Negative stain electron microscopy reconstructions of the ADI-15731 fab bound to EBOV GP showed that ADI-15731 bound directly to the GP1 glycan cap structure in a manner reflective of 13C6 (Murin, et al, 2014, Proc Natl Acad Sci U S A, l l l(48):17182-7 and Bornholdt et al.).
- ADI- 15731 binds to EBOV GP, BDBV GP and SUDV GP. ADI-15731 only effectively neutralizes vesicular stomatitis virus (VSV) pseudovirions displaying either EBOV GP or BDBV GP (as determined by the rVSV GP-GFP assay described in Example 1).
- VSV vesicular stomatitis virus
- m8C4 Mouse monoclonal antibody m8C4 cross neutralizes EBOV and SUDV and provides partial protection against both viruses in mice (PCT Publication No. WO2015/200522; Holtsberg, et al., 2015, J Virol, 90:266 -278). Efficacy of m8C4 was enhanced when used in combination with FVM09 (PCT Publication No. WO2015/200522; PCT US 15/57627; Keck et al).
- Shotgun mutagenesis epitope mapping identified EBOV GP residues R136, Q251, and F252 as critical for m8C4 binding. Of these residues, Q251 and F252 are located within the glycan cap, while R136 is located within the core GP1 head domain ( Figure 2). m8C4 bridges the core GP1 region with the glycan cap, and the epitope is conserved across ebolaviruses.
- ADI-15750 was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al.). ADI-15750 can compete for binding with 13C6 and binds with high affinity to a quaternary epitope in the EBOV GP1 structure also present on EBOV soluble GP (sGP). ADI-15750 demonstrated neutralization activity against VSV pseudoviruses displaying either EBOV GP or SUDV GP with IC50 values of 8.80 nM and 32.30 nM, respectively (as determined by the rVSV GP-GFP assay described in Example 1).
- ADI-15968 was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al.). ADI-15968 can compete for binding with 13C6 and binds with high affinity to a quaternary epitope in the EBOV GP1 structure also present on EBOV sGP. ADI-15968 demonstrated neutralization activity against VSV pseudoviruses displaying either EBOV GP or SUDV GP with IC50 values of 11.74 nM and 47.30 nM respectively (as determined by the rVSV GP-GFP assay described in Example 1).
- 2G4 and 4G7 The most studied EBOV neutralizing epitope is in a region at the base of the trimeric GP that involves contact sites within GP1 and GP2. Antibodies such as KZ52 (Maruyama, et al., J. Virol. 1999;73:6024-6030), as well as two of ZMapp components 2G4 and 4G7 bind to this region (Lee, et al, 2008, Nature, 454 (7201): 177- 182; Murin, et al, 2014, Proc Natl Acad Sci U S A, l l l(48):17182-7). The epitopes for 2G4 and 4G7 are largely overlapping Davidson et al, 2015, J Virol., 89(21):10982-9) but the angle of binding for these two antibodies is different.
- 2G4 and 4G7 were shown to provide significant protection in mice and guinea pig models of EBOV infection (Qiu, et al, 2012, PLoS Negl Trop Dis, 6: 1575). Both of these antibodies, along with 13C6FR1, are components of the ZMappTM antibody cocktail (Qiu, et al, 2014, Nature, 514(7520):47-53). 2G4 and 4G7 are specific to EBOV and do not cross react with other filovirus glycoproteins.
- ADI-15734 was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al). ADI-15734 binds specifically to the EBOV GP and directly competes with KZ52. ADI-15734 neutralizes EBOV (as determined by the rVSV GP-GFP assay described in Example 1) and provides significant levels of protection in the EBOV murine model.
- FVM02 (also called FVM02p): FVM02 is a macaque-derived panfilovirus antibody that binds to the tip of the internal fusion loop (IFL) of all ebolaviruses and marburgvirus (IBT PCT/US 15/57627; Keck et al). FVM02 provides partial protection against EBOV and MARV and potentiates the efficacy of FVM09 against EBOV in mouse models (IBT PCT/US 15/57627; Keck et al.).
- IFL internal fusion loop
- FVM02 provides partial protection against EBOV and MARV and potentiates the efficacy of FVM09 against EBOV in mouse models (IBT PCT/US 15/57627; Keck et al.).
- ADI-15742 The ADI- 15742 mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al.). ADI-15742 is an ultra-potent pan- ebolavirus neutralizing antibody demonstrating sub-nanomolar IC50 values against VSV pseudovirions displaying GP from the following species: EBOV, BDBV, SUDV, RESTV and TAFV (as determined by the rVSV GP-GFP assay described in Example 1). ADI- 15742 also provides complete protection against either EBOV or SUDV in their respective murine models.
- ADI-15878 The ADI-15878 mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak and is a clonal relative of ADI-15742 (Bornholdt et al.). ADI-15878 is also an ultra-potent pan-ebolavirus neutralizing antibody demonstrating sub-nanomolar IC50 values against VSV pseudovirions displaying GP from the following species: EBOV, BDBV, SUDV, RESTV and TAFV (as determined by the rVSV GP-GFP assay described in Example 1). ADI-15878 showed significant levels of protection against EBOV and complete protection against SUDV in their respective murine models.
- ADI-15946 The ADI-15946 mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al.). ADI-15946 is a potent pan-ebolavirus neutralizing antibody demonstrating sub-nanomolar IC50 values against VSV pseudovirions displaying GP from the following species: EBOV, BDBV, and SUDV (as determined by the rVSV GP-GFP assay described in Example 1).
- Antibodies that bind to the viral membrane proximal (stalk) region of filovirus glycoprotein are described in detail below.
- ADI-16061 is a human mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bomholdt et al). ADI-16061 binds to the heptad repeat 2 helices in the stalk region of EBOV GP, BDBV GP, and SUDV GP. However, ADI-16061 only effectively neutralizes VSV pseudovirions displaying GP from EBOV and BDBV with IC50 values 0.21 nM and 0.59 nM, respectively (as determined by the rVSV GP-GFP assay described in Example 1). Further ADI-16061 provided significant levels of protection from EBOV in the murine infection model post infection.
- ADI-15974, ADI-15956, and ADI-15758 are clonally related human mAbs derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bomholdt et al.). These mAbs bind to the heptad repeat 2 helices in the stalk region of EBOV GP and BDBV GP. All three mAb effectively neutralize VSV pseudovirions displaying GP from EBOV and BDBV with sub-nanomolar IC50 values.
- ADI-15974 ADI-15956, and ADI-15758 potently neutralize EBOV (as determined by the rVSV GP-GFP assay described in Example 1) and provided significant levels of protection from EBOV in the murine infection model post infection.
- ADI-15848 is a human mAb derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bomholdt et al). ADI-15848 binds to the heptad repeat 2 helices in the stalk region of EBOV GP and BDBV GP. ADI-15848 effectively neutralizes VSV pseudovirions displaying GP from EBOV and BDBV with IC50 values 0.50 nM and 0.77 nM, respectively (as determined by the rVSV GP-GFP assay described in Example 1). In plaque reduction neutralization (PRNT) assays ADI-15848 potently neutralized EBOV and provided significant levels of protection from EBOV in the murine infection model post infection.
- PRNT plaque reduction neutralization
- ADI-16021 is a human mAb derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bomholdt et al). ADI-16021 binds to the heptad repeat 2 helices in the stalk region of EBOV GP, BDBV GP and SUDV GP.
- Example 3 Combination of pan-ebolavirus antibody with strong activity towards Sudan virus with EBOV specific antibodies included in ZMappTM cocktail
- FVM04 binds to all ebolaviruses and neutralizes EBOV and SUDV; moreover, FVM04 was effective in mouse challenge models of EBOV infection when administered at two doses starting immediately after infection (PCT/US 15/57627; Keck, et al, 2015, J Virol, 90:279 -291).
- PCT/US 15/57627 Keck, et al, 2015, J Virol, 90:279 -291
- FVM04 binds to all ebolaviruses and neutralizes EBOV and SUDV; moreover, FVM04 was effective in mouse challenge models of EBOV infection when administered at two doses starting immediately after infection (PCT/US 15/57627; Keck, et al, 2015
- mice were infected with 100 pfu of mouse-adapted EBOV (MA-EBOV) (Bray, et al, 1999, J Infect Dis., 179 Suppl 1, S248-258) and treated with a single injection of 10 mg/kg (200 ⁇ g/mouse) of FVM04 either 1, 2, or 3 days post infection (dpi).
- MA-EBOV mouse-adapted EBOV
- FVM04 either 1, 2, or 3 days post infection
- dpi days post infection
- mice treated at 1 dpi showed no weight loss or sign of disease ⁇ Figure 3A).
- a second experiment we evaluated the dose response by treating the mice 2 dpi with 10, 5, or 2.5 mg/kg (200, 100, or 50 ⁇ g/mouse) of FVM04 or PBS as control.
- Efficacy of FVM04 was also examined in guinea pigs using guinea pig adapted EBOV and SUDV (GPA-EBOV and GPA-SUDV) (Wong, et al, 2015, J Virol, 90(l):392-9).
- Four groups of 6 guinea pigs were challenged either with 1000 X LD50 of GPA-SUDV or GPA-EBOV followed by a single intraperitoneal injection of 5 mg FVM04 ( ⁇ 15 mg/kg) or DPBS as vehicle control at 1 dpi. Animals were monitored for 16 days for weight change and 28 days for survival.
- ZMAPPTM consisting of the two base binders c2G4 and c4G7 and the glycan cap binder cl3C6, was selected for testing in NHPs based on significant, but partial, protection in guinea pigs (4 out of 6) when administered once at 3 dpi (Qiu, et al., 2014, Nature, 514(7520):47-53).
- the cocktail consisting of FVM04, cl3C6 and c2G4 was also tested in the GPA- EBOV model.
- Four out of 6 animals treated with a single dose of 5 mg cocktail ( ⁇ 1.6 mg of each component) at 3 dpi survived the challenge while all control animals succumbed to infection within 7-9 days (Figure 4D) (P 0.0061).
- a survival rate of 67% in guinea pigs for the cocktail of FVM04/cl3C6/c2G4 is well within the range of protection afforded by ZMAPPTM as shown here and reported previously (Qiu, et al, 2014, Nature, 514(7520):47-53).
- FIG. 5 shows an experiment in which the neutralizing activity of FVM09 towards Ebola virus GP pseudotyped vesicular stomatitis virus (VSV) was tested in presence or absence of a low concentration of 2G4, using the rVSV GP luciferase pseudotype assay described in Example 1.
- VSV Ebola virus GP pseudotyped vesicular stomatitis virus
- Example 5 The IFL-binder FVM02 enhances the neutralizing potency of
- MR191 is a monoclonal antibody that binds to the RBS of the marburgvirus and neutralizes marburgvirus (Flyak, et al, 2015, Cell, 160(5):893-903; sequences of MR191 are published in PCT Pub. WO2016/179511 and US Pub. No. 2016/0326234, both of which are incorporated by reference herein in their entireties).
- FVM02 also enhanced the neutralizing potency of MR191 towards marburgvirus while FVM02 by itself did not have any neutralizing activity.
Abstract
This disclosure provides a method for preventing, treating, or managing a filovirus infection in a subject, where the method includes administering to a subject in need thereof an effective amount of at least two anti-filovirus glycoprotein antibodies or antigen-binding fragments thereof where at least one of the antibodies or fragments binds to its orthologous filovirus glycoprotein epitope on two or more filovirus species or strains.
Description
BROADLY PROTECTIVE ANTIBODY COCKTAILS FOR TREATMENT OF
FILO VIRUS HEMORRHAGIC FEVER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 62/307,118, filed March 11, 2016 and U.S. Provisional Application No. 62/368,688, filed July 29, 2016, both of which are incorporated by reference herein in their entireties.
BACKGROUND
[0002] Filoviruses, ebolavirus and marburgvirus, cause severe hemorrhagic fevers in humans, with high mortality rates as well as epizootic diseases in nonhuman primates and probably other mammals. The main filovirus species causing outbreaks in humans are ebolaviruses Zaire (EBOV) and Sudan (SUDV), as well as the Lake Victoria Marburg virus (MARV). Filoviruses are enveloped, single-stranded, negative sense RNA filamentous viruses and encode seven proteins, of which the spike glycoprotein (GP) is considered the main protective antigen. EBOV and MARV GP is proteolytically cleaved by furin protease into two subunits linked by a disulfide linkage: GP1 (-140 kDa) and GP2 (-38 kDa) (Manicassamy, et al, 2005, J Virol, 79 (8):4793-4805). Three GP1-GP2 units form the trimeric GP envelope spike (-550 kDa) on the viral surface (Feldmann, et al, 1993, Arch Virol Suppl, 7:81-100; Feldmann, et al, 1991, Virology, 182 (l):353-356; Geisbert and Jahrling, 1995, Virus Res, 39 (2-3): 129-150; Kiley, et al, 1988a, J Gen Virol, 69 (Pt 8): 1957-1967). GP1 mediates cellular attachment (Kiley, et al, 1988b, J Gen Virol, 69 (Pt 8):1957-1967; Kuhn, et al, 2006, J Biol Chem, 281 (23): 15951-15958), and contains a mucin-like domain (MLD) which is heavily glycosylated and variable and has little or no predicted secondary structure (Sanchez, et al, 1998, J Virol, 72 (8):6442- 6447). Other filoviruses include Ravn virus (RAW), Tai Forest virus (TAFV), Reston virus (RESTV), and Bundibugyo virus (BDBV).
[0003] It is well established that the filovirus GPs represent the primary protective antigens (Feldmann, et al, 2003, Nat Rev Immunol, 3 (8):677-685; Feldmann, et al, 2005, Curr Opin Investig Drugs, 6 (8):823-830; Geisbert, et al, 2010, Rev Med Virol, 20(6):344-57).
A specific region of the MARV and EBOV GP1, consisting of -150 amino acids binds filovirus receptor-positive cells, but not receptor-negative cells (Kuhn, et al, 2006, J Biol Chem, 281 (23): 15951-15958). This region of GP is referred to here as receptor binding region (RBR).
[0004] Role of antibodies in protection against filovirus hemorrhagic fever: While both T and B cell responses are reported to play a role in protective immune responses to filoviruses (Warfield, et al, 2005, J Immunol, 175 (2):1184-1191), a series of recent reports indicate that antibody alone can provide significant protection. Dye et al. showed that purified convalescent IgG from macaques can protect NHPs against challenge with MARV and EBOV when administered as late as 48h post exposure (Dye, et al., 2012, Proc Natl Acad Sci USA, 109(13):5034-9). Olinger et al. reported significant protection from EBOV challenge in NHPs treated with a cocktail of three monoclonal antibodies (mAbs) to GP administered 24h and 48h post exposure (Olinger, et al., 2012, Proc Natl Acad Sci U S A, 109 (44): 18030-18035). Similar results were also reported in two other studies (Qiu, et al, 2013, Sci Transl Med, 5 (207):207ral43; Qiu, et al, 2013, J Virol, 87 (13):7754-7757). A recent study shows that a combination of three monoclonal antibodies called ZMapp can protect monkeys when administered five days after exposure to EBOV, at a time when the disease is fully manifest and the viremia is at its peak (Qiu, et al, 2014, Nature, 514:47-53). Collectively these data demonstrate the ability of the humoral response to control filovirus infection.
[0005] A number of antibodies have been developed and/or evaluated by Mapp Biopharmaceuticals and Integrated Biotherapeutics that target shared epitopes within the glycoprotein of multiple filovirus species. This disclosure describes combinations of these antibodies for effective treatment of hemorrhagic fever caused by ebolaviruses and marburgvirus. Figure 1 shows an overview of the location of these antibodies.
SUMMARY
[0006] This disclosure provides a method for preventing, treating, or managing a filovirus infection in a subject, where the method includes administering to a subject in need thereof an effective amount of an antibody cocktail comprising at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus
glycoprotein (filovirus GP). In certain aspects, the cocktail can include a first anti- filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof, where the first antibody or fragment thereof specifically binds to a filovirus GP1/GP2 base epitope, where the second antibody or fragment thereof specifically binds to a filovirus GP receptor binding site (RBS) epitope, a filovirus GP glycan cap epitope, a filovirus GP internal fusion loop (IFL) epitope, or any combination thereof. In certain aspects, at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains. In certain aspects, administration of the antibody cocktail can be effective against two or more filovirus species or strains.
[0007] In certain aspects, the antibody cocktail can further include additional anti-filovirus GP antibodies, e.g., a third anti-filovirus GP antibody or antigen-binding fragment thereof that can specifically bind to a filovirus glycan cap epitope.
[0008] The disclosure further provides a method for preventing, treating, or managing a filovirus infection in a subject, where the method includes administering to a subject in need thereof an effective amount of an antibody cocktail that includes at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus GP. In certain aspects, the cocktail can include a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof, where the first antibody or fragment thereof specifically binds to a filovirus RBS epitope, where the second antibody or fragment thereof specifically binds to a filovirus IFL epitope. In certain aspects, at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains. In certain aspects, administration of the antibody cocktail can be effective against two or more filovirus species or strains.
[0009] In certain aspects, the filovirus infection is hemorrhagic fever. In certain aspects, the subject is a nonhuman primate or a human.
[0010] In the provided methods the two or more filovirus species can be, e.g., two or more of Marburg virus (MARV), Ravn virus (RAW), Tai Forest virus (TAFV), Reston virus
(RESTV), Sudan virus (SUDV) Ebola virus (EBOV), Bundibugyo virus (BDBV), or any strain thereof.
[0011] In certain aspects, the ability of the antibody cocktail to prevent, treat, or manage a filovirus infection can be measured in a model comprising administering the antibody cocktail to a group of rodents, e.g., mice or guinea pigs, and challenging rodents with a wild-type or rodent-adapted filovirus before, at the same time as, or after administering the antibody cocktail to the rodents. Efficacy in the model systems can be determined by monitoring the test rodents for, e.g., increased survival time, decreased weight loss, or a combination thereof as compared to control rodents.
[0012] Antibody cocktails suitable for the provided methods are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0013] Figure 1: Trimeric structure of EBOV glycoprotein (GP) lacking the mucin like domain (MLD). The MLD is modeled on the structure as large gray balls. The different regions of the GP, the glycan cap, the core GPl in the head domain, the base consisting of the GP2 and the N-terminus of GP are shown. The stalk, which was not part of the solved structure is shown as a box. The mapped epitope regions of several monoclonal antibodies described herein are specified.
[0014] Figure 2: The position of m8C4 binding sites Q251, F252, and R136 shown on EBOV GP monomelic structure.
[0015] Figure 3A-C: Post-exposure efficacy of FVM04 in mouse model of EBOV and SUDV infection. (A&B) Groups of ten BALB/c mice (6-8 weeks old) were infected by intraperitoneal (i.p.) with 100 PFU of mouse adapted (MA)-EBOV and treated i.p. with the doses and at time points indicated in the figure or left untreated. (C) Three groups of IFNapR"7' mice were infected with 1000 PFU of SUDV; one group (n=7) received two intraperitoneal injections of mAb 16F6 at 1 and 3 dpi, a second group received FVM04 once at 1 dpi, and a third group was left untreated. Mice were monitored for 21 days for survival, weight change, and signs of disease.
[0016] Figure 4A-E: Efficacy of FVM04 treatment and an FVM04 containing cocktail in guinea pig models of SUDV and EBOV infection. (A&B) Efficacy of a single dose of 5 mg/animal FVM04 (-15 mg/kg) injected i.p. at 1 dpi compared to control group receiving DPBS in animals challenged with guinea pig-adapted (GPA)-SUDV (A) or GPA-EBOV
(B), with 6 animals per group. (C) Groups of 6 guinea pigs were challenged with GPA- SUDV and treated with either DPBS, 5 mg/animal of FVM04, or 1.6 mg/animal each of FVM04, c2G4, and cl3C6, 3 dpi. (D) Guinea pigs (6 animals per group) were infected with GPA-EBOV and treated with 1.6 mg/animal each of FVM04, c2G4, and cl3C6 at 3 dpi. (E) Compiled data of three experiments with ZMapp™. Guinea pigs were infected with GPA-EBOV and treated with DPBS (n=19) or 5 mg/animal of ZMAPP™ (n=20), 3dpi. Challenge was performed with either 1000X LD50 of GPA-SUDV or GPA-EBOV as indicated. Survival was monitored for 21 dpi and weights for 16 days dpi.
[0017] Figure 5: FVM09 displays neutralizing activity in presence of 2G4. FVM09 mediated neutralization by was determined using vesicular stomatitis virus pseudotyped with EBOV GP in presence or absence of 0.2 μg/ml of 2G4. Note that 2G4 at this concentration only mediates about 20% neutralization.
[0018] Figure 6A-B: FVM02 enhances the neutralization of filoviruses by RBS binding antibodies. A) FVM04 mediated neutralization by FVM02 determined using vesicular stomatitis virus pseudotyped with EBOV GP. B) MR191 mediated neutralization by FVM02 determined using vesicular stomatitis virus pseudotyped with MARV GP.
DETAILED DESCRIPTION
[0019] The term "a" or "an" entity refers to one or more of that entity; for example, "polypeptide subunit" is understood to represent one or more polypeptide subunits. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
[0020] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
[0021] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1 99, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.
[0022] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
[0023] As used herein, the term "non-naturally occurring" substance, composition, entity, and/or any combination of substances, compositions, or entities, or any grammatical variants thereof, is a conditional term that explicitly excludes, but only excludes, those forms of the substance, composition, entity, and/or any combination of substances, compositions, or entities that are well-understood by persons of ordinary skill in the art as being "naturally-occurring," or that are, or could be at any time, determined or interpreted by a judge or an administrative or judicial body to be, "naturally-occurring."
[0024] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is
not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
[0025] A "protein" as used herein can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein.
[0026] By an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.
[0027] As used herein, the term "non-naturally occurring" polypeptide, or any grammatical variants thereof, is a conditional term that explicitly excludes, but only excludes, those forms of the polypeptide that are well-understood by persons of ordinary skill in the art as being "naturally-occurring," or that are, or could be at any time, determined or interpreted by a judge or an administrative or judicial body to be, "naturally-occurring."
[0028] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment," "variant," "derivative" and "analog" when referring to polypeptide subunit or multimeric protein as disclosed herein can include any polypeptide or protein that retain at least some of the activities of the complete polypeptide or protein, but which is structurally different. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments. Variants include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur spontaneously or be intentionally constructed. Intentionally constructed variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides can also be referred to herein as "polypeptide analogs." As used
herein a "derivative" refers to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides that contain one or more standard or synthetic amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.
[0029] A "conservative amino acid substitution" is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate protein activity are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al, Protein Eng. 12(10):879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).
[0030] Disclosed herein are certain antibodies, or antigen-binding fragments, variants, or derivatives thereof. Unless specifically referring to full-sized antibodies such as naturally- occurring antibodies, the term "antibody" encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally-occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.
[0031] As described further herein, an antibody or fragment thereof can comprise one or more "binding domains." As used herein, a "binding domain" or "antigen binding domain" is a two- or three-dimensional structure, e.g., a polypeptide structure that cans specifically bind a given antigenic determinant, e.g., the region formed by the heavy and light chain variable regions of an antibody or fragment thereof.
[0032] The terms "antibody" and "immunoglobulin" can be used interchangeably herein. An antibody (or a fragment, variant, or derivative thereof as disclosed herein comprises at least the variable domain of a heavy chain and at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et ah, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
[0033] As will be discussed in more detail below, the term "immunoglobulin" comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGj, IgG2, IgG3, IgG4, IgA I , etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.
[0034] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
[0035] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is
a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
[0036] As indicated above, the variable region allows an antibody or fragment thereof to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody or fragment thereof combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen- binding site present at the end of each arm of the Y. More specifically, the antigen- binding site is defined by three CDRs on each of the VH and VL chains.
[0037] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen binding domains, referred to as "framework" regions, show less inter-molecular variability. The framework regions largely adopt a β- sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined {see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J Mol. Biol., 796:901-917 (1987), which are incorporated herein by reference in their entireties).
[0038] In the case where there are two or more definitions of a term that is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain
polypeptides. This particular region has been described by Kabat et al, U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al, J. Mol Biol. 196:901-917 (1987), which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acids when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acids that encompass the CDRs as defined by each of the above-cited references are set forth below in Table 1 as a comparison. The exact amino acid numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which amino acids comprise a particular CDR given the variable region amino acid sequence of the antibody.
Table 1
*Numbering of CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below).
[0039] Immunoglobulin variable domains can also be analyzed using the IMGT information system (www://imgt.cines.fr/) (IMGT®/V-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. 5<5:W503-508 (2008).
[0040] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).
[0041] Antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019. Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type {e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class {e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
[0042] By "specifically binds," it is meant that an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody or fragment thereof is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen-binding domain more readily
than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain antibody or fragment thereof binds to a certain epitope. For example, antibody or fragment thereof "A" can be deemed to have a higher specificity for a given epitope than antibody or fragment thereof "B," or antibody or fragment thereof "A" can be said to bind to epitope "C" with a higher specificity than it has for related epitope "D."
[0043] The methods and compositions provided in this disclosure combine various human-, murine-, and/or non-human primate (NHP)-derived antibodies or fragments, variants, or derivatives thereof in antibody cocktails. The antibodies or fragments thereof disclosed herein can be said to bind a target antigen, e.g., a filovirus glycoprotein subunit or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5 X 10'2 sec'1, 1(T sec"1, 5 X 10"J sec" or 10" sec" . A human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to bind a target antigen, e.g., a filovirus glycoprotein subunit, with an off rate (k(off)) less than or equal to 5 X 10"4 sec"1, 10"4 sec"1, 5 X 10"5 sec"1, or 10"5 sec"1 5 X 10"6 sec"1, 10"6 sec"1, 5 X 10"7 sec'1 or 10'7 sec"1.
[0044] A human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to bind a target antigen, e.g., a filovirus glycoprotein subunit with an on rate (k(on)) of greater than or equal to 103 M"1 sec"1, 5 X 103 M"1 sec'1, 104 M"1 sec"1 or 5 X 104 M"1 sec"1. A human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to bind a target antigen, e.g., a filovirus glycoprotein subunit with an on rate (k(on)) greater than or equal to 10s M"1 sec"1, 5 X 10s M"1 sec"1, 106 M"1 sec"1, or 5 X 106 M"1 sec"1 or 107 M"' sec"1.
[0045] A human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to competitively inhibit binding of a reference antibody or antigen binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. A human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails disclosed herein can be said to competitively inhibit binding of the
reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
[0046] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. An interaction between a between a bivalent monoclonal antibody with a receptor present at a high density on a cell surface would also be of high avidity.
[0047] Antibodies or antigen-binding fragments, variants or derivatives thereof as disclosed herein can also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers, e.g., to the ability of a human-, murine-, and/or NHP- derived antibody or fragment thereof specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody or fragment thereof is cross-reactive if it binds to an epitope other than the one that induced its formation, e.g., various different filovirus receptor binding regions. The cross-reactive epitope contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.
[0048] A Human-, murine- and/or NHP-derived antibody or fragment, variant, or derivative thereof for use in the antibody cocktails provided herein can also be described or specified in terms of their binding affinity to an antigen. For example, an antibody can bind to an antigen with a dissociation constant or ¾ no greater than 5 10" M, 10" M, 5 x 10"3 M, 10"3 M, 5 x lO^ M, lO^ M, 5 x 10"5 M, 10"5 M, 5 x 10"6 M, 10"6M, 5 x 10"7 M, 10" 7 M, 5 x 10"8 M, 10"8 M, 5 x 10" M, 10"9 M, 5 x 10"'° M, 10"'° M, 5 x 10"" M, 10"n M, 5 x 10"I2 M, 10"I2M, 5 x 10"13 M, 10"I3 M, 5 x 10"I M, 10"I4M, 5 x 10"I5 M, or 10"15 M.
[0049] Antibody fragments including single-chain antibodies can comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included are antigen-binding fragments that comprise any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains. Antibodies, or antigen-binding fragments thereof disclosed herein can be from any animal origin including birds and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g., from sharks). As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0050] As used herein, the term "heavy chain portion" includes amino acid sequences derived from an immunoglobulin heavy chain, an antibody comprising a heavy chain portion comprises at least one of: a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a Human-, murine- and/or NHP-derived antibody or fragment, variant, or derivative thereof for use in the antibody cocktails provided herein can comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a human-, murine- and/or NHP-derived antibody or fragment, variant, or derivative thereof for use in the antibody cocktails provided herein comprises a polypeptide chain comprising a CH3 domain. Further, a human-, murine-, and/or NHP-derived antibody or fragment thereof for use in the antibody cocktails provided herein can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) can be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
[0051] The heavy chain portions of a human-, murine-, and/or NHP-derived antibody as disclosed herein can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CHI domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
[0052] As used herein, the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain. The light chain portion comprises at least one of a VL or CL domain.
[0053] Human-, murine-, and/or NHP-derived antibodies or fragments thereof for use in the antibody cocktails disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target a filovirus glycoprotein subunit that they recognize or specifically bind. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope," or an "antigenic determinant." A target antigen, e.g., a filovirus glycoprotein subunit can comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen. As used herein, an "orthologous epitope" refers to versions of an epitope found in related organisms, e.g., different filovirus species or strains. Orthologous epitopes can be similar in structure, but can vary in one or more amino acids.
[0054] As previously indicated, the subunit structures and three-dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the term "CHI domain" includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
[0055] As used herein the term "CH2 domain" includes the portion of a heavy chain molecule that extends, e.g., from about amino acid 244 to amino acid 360 of an antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering
system; and amino acids 231-340, EU numbering system; see Kabat EA et al. op. cit. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N- linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 amino acids.
[0056] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 amino acids and is flexible, thus allowing the two N-terminal antigen- binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al, J. Immunol. 7 «57:4083 (1998)).
[0057] As used herein the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CHI and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
[0058] As used herein, the term "chimeric antibody" will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial or modified) is obtained from a second species. In some embodiments the target binding region or site will be from a non-human source {e.g. mouse or primate) and the constant region is human.
[0059] The term "bispecific antibody" as used herein refers to an antibody that has binding sites for two different antigens within a single antibody molecule. It will be appreciated that other molecules in addition to the canonical antibody structure can be constructed with two binding specificities. It will further be appreciated that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol. tf:1387-94 (2010); Mabry and Snavely, IDrugs. 75:543-9 (2010)). A bispecific antibody can also be a diabody.
[0060] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, by partial framework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class, e.g., from an antibody from a different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody." In some instances, not all of the CDRs are replaced with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another, instead, minimal amino acids that maintain the activity of the target-binding site are transferred. Given the explanations set forth in, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional engineered or humanized antibody.
[0061] In certain aspects, Human-, murine-, and/or NHP-derived antibodies or fragments thereof for use in the antibody cocktails disclosed herein can be glycoengineered to contain predominantly a single glycoform. The engineered glycan can be, e.g., GnGn (GlcNAc2-Man3-GlcNAc2), mono- or di-galactosylated (Gal(i/2)-GlcNAc2-Man3- GlcNAc2), mono- or di-sialylated (NaNa(ii2)-Gal(i/2)-GlcNAc2-Man3-GlcNAc2) containing little or no fucose, or xylose. A predominantly single glycoform is any glycoform that represents more than half (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) of all glycoforms present in the antibody solution.
[0062] The RAMP system has been used for glycoengineering of antibodies, antibody fragments, idiotype vaccines, enzymes, and cytokines. Dozens of antibodies have been produced in the RAMP system (see, e.g., Hiatt A, et al., (2013) Clinical & Developmental Immunology 2013:632893; Whaley KJ, et al, (2014) Current topics in Microbiology and Immunology 375: 107-26; Bendandi M, et al. (2010) Annals of Oncology 21 :2420-2427; Castilho A & Steinkellner H (2012) Biotechnology J. 7:1088-1098). These have predominantly been IgGs but other isotypes, including IgM (Loos A, et al., (2014) Proc.
Natl. Acad. Sci. U.S.A. 7/7:6263-8; Hiatt, A., (2014) Proc. Natl. Acad. Set U.S.A 777:6124-5), have been glycoengineered.
[0063] In certain aspects, cDNAs encoding a mAb of interest can be expressed in suitable Nicotiana benthamiana expression hosts and/or in combination with Agrobacterium vectors encoding one or more appropriate glycosylation enzymes to produce the desired glycan profile in the expressed mAb. In certain embodiments, the suitable Nicotiana benthamiana expression host can be simultaneously exposed to a mixed population of recombinant Agrobacterium where some of the Agrobacteria contain a first T-DNA vector comprising a cDNA encoding the heavy chain of the mAb in a first non-competing viral vector and the remainder of the Agrobacteria contain a second T-DNA comprising a cDNA encoding the light chain of the mAb in a second non-competing viral vector. In certain embodiments, one of the non-competing viral vectors is a Tobacco Mosaic Virus (TMV) vector and one of the non-competing vectors is a Potato Virus X (PVX) vector (Giritch et al, (2006) Proc. Natl. Acad. Sci. U.S.A. 103: 14701-14706). For wild-type glycans {i.e. native, plant-produced glycosylation), wild-type N. benthamiana can be inoculated with only the Agrobacterium containing the antibody cDNA(s). For the GnGn glycan, the same Agrobacterium can be used to inoculate plants that contain little or no fucosyl or xylosyl transferases (AXF plants; Strasser et al. Plant Biotechnol J. 26:392- 402). For galactosylated glycans, AXF plants can be inoculated with the Agrobacterium containing the antibody cDNA(s) as well as an Agrobacterium containing the cDNA for p-l,4-galactosyl transferase expression contained on a binary Agrobacterium vector to avoid recombination with the TMV and PVX vectors (Castilho A, el al, (2010) J Biol. Chem. 285:15923-15930). For sialylated glycans, six additional genes can be introduced in binary vectors to reconstitute the mammalian sialic acid biosynthetic pathway. The genes are UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N- acetylneuraminic acid phosphate synthase, CMP-N-acetylneuraminic acid synthetase, CMP-NeuAc transporter, p-l,4-galactosylatransferase, and a2,6-sialyltransferase {Id.).
[0064] Glycanalysis of glycoengineered mAbs can be achieved by release of N-linked glycans by digestion with N-glycosidase F (PNGase F). Subsequent derivatization of the free glycan can be achieved with anthranilic acid (2-AA). The 2-AA-derivatized oligosaccharide is separated from any excess reagent via normal-phase HPLC. The column is calibrated with 2-AA-labeled glucose homopolymers and glycan standards.
The test samples and 2-AA-labeled glycan standards are detected fluorometrically. Glycoforms are assigned either by comparing their glucose unit (GU) values with those of the 2-AA-labeled glycan standards or by comparing with the theoretical GU values (Guile GR, et al., (1996) Analytical Biochem. 240:210-226). Confirmation of glycan structure can be accomplished with LC/MS.
[0065] While the RAMP system is an effective method of producing various glycoengineered and wild-type mABs, it will be recognized that other expression systems can be used to accomplish the same result. For example, mammalian cell lines (such as CHO or NSO cells (Davies, J., et al, Biotechnol Bioeng 74:288-294), yeast cells such as Pichia pastoris (Gerngross T. Adv Exp Med Biol. (2005); 564:139) and bacterial cells such as E. coli have been used produce such mAbs.
[0066] The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond {e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
[0067] As used herein, a "non-naturally occurring" polynucleotide, or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the polynucleotide that are well-understood by persons of ordinary skill in the art as being "naturally-occurring," or that are, or that could be at any time, determined
or interpreted by a judge or an administrative or judicial body to be, "naturally- occurring."
[0068] As used herein, a "coding region" is a portion of nucleic acid comprising codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a polypeptide subunit or fusion protein as provided herein. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
[0069] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid that encodes a polypeptide normally can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association or linkage can be when a coding region for a gene product, e.g., a polypeptide, can be associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) can be "operably associated" or "operably linked" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other
transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.
[0070] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
[0071] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
[0072] In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA).
[0073] Polynucleotide and nucleic acid coding regions can be associated with additional coding regions that encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein, e.g., a polynucleotide encoding a polypeptide subunit provided herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the
ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse β- glucuronidase.
[0074] A "vector" is nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker gene and other genetic elements known in the art.
[0075] A "transformed" cell, or a "host" cell, is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses those techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transformed cell or a host cell can be a bacterial cell or a eukaryotic cell.
[0076] The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
[0077] As used herein the terms "treat," "treatment," or "treatment of (e.g., in the phrase "treating a subject") refers to reducing the potential for disease pathology, reducing the occurrence of disease symptoms, e.g., to an extent that the subject has a longer survival
rate or reduced discomfort. For example, treating can refer to the ability of a therapy when administered to a subject, to reduce disease symptoms, signs, or causes. Treating also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness. The term "protection" and related grammatical terms, when used in the context of the ability of a therapeutic agent to affect the course of an infectious disease refers to any protective effect observed in comparison to a control agent. For example if two groups of animals are challenged with an infectious agent, e.g., a lethal dose of EBOV, and one group of animals is administered the therapeutic agent while the other group is administered a control, if a statistically significant number of animals in the therapeutic group survive relative to the number of survivors in the control group, a protective effect is observed. "Protection" can be, but does not have to be, 100%.
[0078] By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals, including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.
[0079] The term "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.
[0080] An "effective amount" of an antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.
[0081] Certain therapies can provide "synergy" and prove "synergistic", i.e., an effect can be achieved when the active ingredients used together that is greater than the sum of the effects that results from using the compounds separately. A synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. In
general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. A synergistic effect can be measured by potency assays, e.g., mouse our guinea pig challenge assays or neutralization assays as described elsewhere herein.
Treatment Methods using Cocktails of Cross-Reactive Anti-Filovirus Antibodies
[0082] Mounting evidence suggests that cocktails of two or more anti-filovirus glycoprotein antibodies, e.g., anti-EBOV, SUDV, or MARV GP antibodies, can provide life-sustaining benefit to subjects, e.g., patients and/or healthcare workers exposed to or susceptible to exposure to, filovirus infection, e.g., EBOV, SUDV, or MARV. At the initiation of an outbreak or infection, the exact species or strain of filovirus might not be immediately determined, or the outbreak could be caused by more than one filovirus species or strain. Accordingly, this disclosure provides methods for preventing, treating, and/or managing filovirus infections or outbreaks using cocktails of antibodies that can be effective against more than one filovirus species or strain.
[0083] In certain aspects, this disclosure provides a method for preventing, treating, or managing a filovirus infection in a subject where the method entails administering to a subject in need thereof an effective amount of an antibody cocktail that includes, or comprises, at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus glycoprotein (filovirus GP). In certain aspects, at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains. In other words, if the antibody or fragment thereof was raised against the EBOV glycoprotein and is found to bind to the EBOV glycan cap, the antibody "can specifically bind to its orthologous epitope on two or more filovirus species or strains" if the antibody or fragment thereof also binds to, e.g., the glycan cap of other strains of EBOV and/or the MARV, RAW, TAFV, RESTV, SUDV, and/or BDBV GP glycan cap, or any strain thereof. Moreover, in certain aspects, administration of the antibody cocktail can be effective against two or more filovirus species or strains, e.g., the antibody cocktail can neutralize two or more filovirus species or strains and/or protect against disease caused by two or more filovirus species or strains, e.g., in an animal challenge model. The two or more filovirus species can be two or more of Marburg virus (MARV), Ravn virus (RAW), Tai Forest virus
(TAFV), Reston virus (RESTV), Sudan virus (SUDV) Ebola virus (EBOV), Bundibugyo virus (BDBV), or any strain thereof. In certain aspects, the filovirus infection is hemorrhagic fever. In certain aspects, the subject is a nonhuman primate or a human.
[0084] In certain aspects the antibodies or fragments thereof for use in the methods provided herein can each independently be, e.g., a mouse antibody, a non-human primate (NHP) antibody, a humanized antibody, a chimeric antibody, or a fragment thereof. Moreover, the antibody or fragment thereof can be a monoclonal antibody, a component of a polyclonal antibody mixture, a recombinant antibody, a multispecific antibody, or any combination thereof.
[0085] In certain aspects, the ability of the antibody cocktail to prevent, treat, or manage a filovirus infection, and or the potency relative to individual antibodies, can be measured in a model comprising administering the antibody cocktail to a group of rodents and challenging rodents with a rodent-adapted filovirus before, at the same time as, or after administering the antibody cocktail to the rodents. The rodent model can be, for example, a guinea pig model or a mouse model. In either model, the challenged rodents can be monitored for increased survival time, decreased weight loss, or a combination thereof as compared to control rodents. In other aspects the ability of the antibody cocktail to prevent, treat, or manage a filovirus infection, and or the potency relative to individual antibodies, can be measured in a neutralization assay as described elsewhere herein.
[0086] In certain aspects of the provided method, the subject is administered an effective amount of an antibody cocktail as described above. In certain aspects, the antibody cocktail can prevent, treat, or manage filovirus infection in the subject with a potency that is greater than the additive potency of the antibodies or fragments thereof when administered individually.
[0087] In certain aspects, administration of the provided antibody cocktail to the subject is effective against two or more filovirus species or strains. In certain aspects the method includes administering the antibody cocktail to, e.g., improve efficacy, reduce the number of treatments, to allow efficacy when administered at a later time from the inception of infection in the subject, and/or to allow dose sparing. In certain aspects administration of the antibody cocktail can result in synergistic efficacy, e.g., efficacy that is more potent than would be expected based on the efficacy of the antibodies administered individually. Moreover, the antibody cocktails provided herein can be useful for treatment of a
filovirus infection without it being necessary to know the exact filovirus species or strain. Any antibody cocktail described herein can include, in addition to the recited antibodies, other antibodies that bind to the same or different filovirus glycoprotein epitopes.
[0088] In certain aspects each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently comprise a heavy chain constant region or fragment thereof. The heavy chain can be, e.g., a murine constant region or fragment thereof, a human constant region or fragment thereof, e.g., an IgM, IgG, IgA, IgE, IgD, or IgY constant region or fragment thereof. Various human IgG constant region subtypes or fragments thereof can also be included, e.g., a human IgGl, IgG2, IgG3, or IgG4 constant region or fragment thereof.
[0089] In certain aspects each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently further comprise a light chain constant region or fragment thereof. For example, the light chain constant region or fragment thereof can be a murine constant region or fragment thereof, e.g., a human light chain constant region or fragment thereof, e.g., a human kappa or lambda constant region or fragment thereof.
[0090] In certain aspects each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently comprise a full-size antibody comprising two heavy chains and two light chains. In other aspects, the binding domain(s) of each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently be an Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv fragment, an scFab fragment, an sc(Fv)2 fragment, or any combination thereof.
[0091] In certain aspects each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can, either independently or collectively, fully or partially neutralize infectivity of a filovirus upon binding of the binding domain to one or more orthologous epitopes on the filovirus.
[0092] In certain aspects, each antibody or fragment thereof of an antibody cocktail for use in the methods provided herein can independently be conjugated to an antiviral agent, a protein, a lipid, a detectable label, a polymer, or any combination thereof.
[0093] In certain aspects the antibody cocktail includes at least a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or
antigen-binding fragment thereof. According to one aspect the first antibody or fragment thereof can specifically bind to a filovirus GP1/GP2 base epitope, and the second antibody or fragment thereof can specifically bind to a filovirus GP receptor binding site (RBS) epitope, a filovirus GP glycan cap epitope, a filovirus GP internal fusion loop (IFL) epitope, or any combination thereof. Antibodies binding to the base epitope include, without limitation, 2G4, 4G7, and/or ADI- 15734, described elsewhere herein. Antibodies that bind to the RBS epitope include, without limitation, FVM04, and MR191. Antibodies that bind to the glycan cap epitope include, without limitation, 13C6FR1, FVM09, ADI- 15731, m8C4 (in part), and ADI-15750 (in part). Antibodies binding to the IFL epitope include, without limitation, FVM02, ADI-15742, ADI-15878, and ADI-15946.
In one aspect, the first antibody or fragment thereof of the antibody cocktail can be, or can be related to the base-binding antibody 2G4 and the second antibody or fragment thereof of the antibody cocktail can be, or can be related to the RBS-binding antibody FVM04. Thus the first antibody or fragment thereof of the antibody cocktail can comprise a heavy chain variable region (VH) and a light chain variable region (VL) collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 , respectively (2G4), and the second antibody or fragment thereof can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively (FVM04). Similarly, the first antibody or fragment thereof can comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 29, respectively (2G4), and the second antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 5, respectively (FVM04).
[0095] According to this aspect, the antibody cocktail can further comprise a third anti- filovirus GP antibody or antigen-binding fragment thereof, wherein the third antibody or fragment thereof can specifically bind to a filovirus glycan cap epitope. For example, the third antibody or fragment thereof can be, or can be related to the glycan cap binding Mab 13C6 (See U.S. Patent No. 7,335,356) or the related glycan cap binding Mab 13C6FR1, which contains various amino acid substitutions in the VH framework- 1 (FR1) and the VL FR1 regions relative to the original 13C6. 13C6FR1 is described in more detail elsewhere herein. Thus the third antibody or fragment thereof in the antibody cocktail can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, or a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 9 and SEQ ID NO: 13, respectively (13C6FR1).
[0096] In a related aspect, an effective amount of an antibody cocktail comprising 2G4 or an antigen-binding fragment thereof, FVM04 or an antigen binding fragment thereof, and 13C6FR1 or an antigen binding fragment thereof can protect guinea pigs from challenge with guinea pig-adapted EBOV (GPA-EBOV) and can also protect guinea pigs from challenge with guinea pig-adapted SUDV (GPA-SUDV).
[0097] In another aspect, the first antibody or fragment thereof of the antibody cocktail can be, or can be related to the base-binding antibody 2G4 and the second antibody or fragment thereof of the antibody cocktail can be, or can be related to the glycan cap binding antibody FVM09. Thus the first antibody or fragment thereof of the antibody cocktail can comprise a heavy chain variable region (VH) and a light chain variable region (VL) collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL- CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 , respectively (2G4), and the second antibody or fragment thereof can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except
for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 , respectively (FVM09). Similarly, the first antibody or fragment thereof can comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 29, respectively (2G4), and the second antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 17 and SEQ ID NO: 21, respectively (FVM09).
[0098] In certain aspects the antibody cocktail includes at least a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof. According to one aspect the first antibody or fragment thereof can specifically bind to a filovirus RBS epitope, and the second antibody or fragment thereof can specifically bind to a filovirus IFL epitope. As noted above, the method provides that at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains. Moreover, the method provides that administration of the antibody cocktail is effective against two or more filovirus species or strains , e.g., the antibody cocktail can neutralize two or more filovirus species or strains and/or protect against disease caused by two or more filovirus species or strains, e.g., in an animal challenge model.
[0099] In one aspect, the first antibody or fragment thereof of the antibody cocktail can be, or can be related to the RBS-binding antibody FVM04 and the second antibody or fragment thereof of the antibody cocktail can be, or can be related to the IFL-binding antibody FVM02. Thus the first antibody or fragment thereof of the antibody cocktail can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively (FVM04), and the second antibody or fragment thereof can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH- CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions
in one or more CDRs to SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively (FVM02). Similarly, the first antibody or fragment thereof can comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 5, respectively (FVM04), and the second antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 33 and SEQ ID NO: 37, respectively (FVM02).
[0100] In another aspect, the first antibody or fragment thereof of the antibody cocktail can be, or can be related to the RBS-binding antibody MR191 and the second antibody or fragment thereof of the antibody cocktail can be, or can be related to the IFL-binding antibody FVM02. Thus the first antibody or fragment thereof of the antibody cocktail can comprise a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively (MR191), and the second antibody or fragment thereof can comprise a VH and a VL collectively comprising VH-CDR1, VH- CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively (FVM02). Similarly, the first antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 41 and SEQ ID NO: 45, respectively (MR191), and the second antibody or fragment thereof can comprise a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 33 and SEQ ID NO: 37, respectively (FVM02).
[0101] In one embodiment, treatment includes the application or administration of an antibody cocktail as provided herein, to a subject or patient, where the subject or patient has been exposed to a filovirus, infected with a filovirus, has a filovirus disease, a symptom of a filovirus disease, or a predisposition toward contracting a filovirus disease.
In another embodiment, treatment can also include the application or administration of a pharmaceutical composition comprising an antibody cocktail as provided herein, to a subject or patient, so as to target the pharmaceutical composition to an environment where the antibody cocktail can be most effective, e.g., the endosomal region of a virus- infected cell.
[0102] In accordance with the methods of the present disclosure, an antibody cocktail as provided herein can be used to promote a positive therapeutic response. By "positive therapeutic response" is intended any improvement in the disease conditions associated with the activity of the antibody cocktail, and/or an improvement in the symptoms associated with the disease. Thus, for example, an improvement in the disease can be characterized as a complete response. By "complete response" is intended an absence of clinically detectable disease with normalization of any previously test results. Such a response can in some cases persist, e.g., for at least one month following treatment according to the methods of the disclosure. Alternatively, an improvement in the disease can be categorized as being a partial response.
Pharmaceutical Compositions and Administration Methods
[0103] The disclosure further provides antibody cocktails as described above, for use in the provided treatment methods. Such antibody cocktails can be formulated as pharmaceutical compositions.
[0104] Methods of preparing and administering antibody cocktails as provided herein, to a subject in need thereof are well known to or are readily determined by those skilled in the art. The route of administration of an antibody cocktail for use in the methods provided herein can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are clearly contemplated as suitable forms, another example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. In some cases a suitable pharmaceutical composition can comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. In other methods compatible with the teachings herein, an antibody cocktail as provided herein can be delivered directly to a site where the antibody or fragment thereof can be effective in virus neutralization.
[0105] As discussed herein, an antibody cocktail as provided herein can be administered in a pharmaceutically effective amount for the in vivo treatment of diseases or disorders associated with filovirus infection. In this regard, it will be appreciated that the disclosed antibody cocktails can be formulated so as to facilitate administration and promote stability of the active agent(s). Pharmaceutical compositions accordingly can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, nontoxic buffers, preservatives and the like. A pharmaceutically effective amount of an antibody cocktail means an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or condition or to detect a substance or a cell. Persons or ordinary skill in the art will appreciate that the components of a given antibody cocktail can be provided in equimolar amounts or can be provided in different ratios to produce the best clinical result. Methods to determine the best ratios of active components can be readily determined. Suitable formulations for use in the therapeutic methods disclosed herein can be described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).
[0106] The relative amounts of the antibodies or fragments thereof in the antibody cocktail that can be combined with carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide an optimum response (e.g., a therapeutic or prophylactic response).
[0107] In keeping with the scope of the present disclosure, an antibody cocktail for use in the methods provided herein can be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic effect. An antibody cocktail provided herein can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody or antigen-binding fragment, variant, or derivative thereof of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
[0108] By "therapeutically effective dose or amount" or "effective amount" is intended an amount of an antibody cocktail that when administered brings about a positive therapeutic response with respect to treatment of a patient with a disease or condition to be treated.
[0109] Therapeutically effective doses of the compositions disclosed herein, for treatment of diseases or disorders associated with filovirus infection, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non- human mammals including non-human primates can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
[0110] The amount of an antibody cocktail to be administered can be readily determined by one of ordinary skill in the art without undue experimentation given this disclosure. Factors influencing the mode of administration and the respective amount of an antibody cocktail include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Similarly, the amount of an antibody cocktail to be administered will be dependent upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent.
[0111] This disclosure also provides for the use of an antibody cocktail in the manufacture of a medicament for treating, preventing, or managing a disease or disorder associated with filovirus infection, e.g., hemorrhagic fever.
Examples
Example 1 : Methods
[0112] Two different neutralization assays based on pseudotyped recombinant vesicular stomatitis virus (VSV) expressing filovirus GP were used: a replication defective (single round infection) rVSV-GP-Luc expressing firefly luciferase, and a replication competent expressing green fluorescence protein (rVSV-GFP).
[0113] rVSV-GP Luciferase pseudotype assay. Pseudotyped viruses were generated based on a modification of previously published method (Whitt, . A. 2010. J Virol Methods
169:2)65-1 ). HEK 293T cells were grown to 80% confluency, and transfected with plasmids encoding EBOV-GP, MARV-GP or SUDV GP using Fugene HD (Promega) according to the manufacture's protocol. The next day, these cells were infected with rVSV-AGP pseudotype (Kerafast) at an MOI of 3 and the virus was washed off after 1 h with DPBS. The next day, the supernatant was collected and clarified by centrifugation. To titer the pseudotyped virus, BHK-21 cells were transfected in 6 well plates with pCAGGS VSV-G (Kerafast), and after 48h, serial dilution of VSV-EBOV-GP-Luc or VSV-SUDV GP-Luc pseudotype was added to each well for lh before the addition of 0.9% agar in DMEM. The next day, wells were fixed with 500 μΐ. of 5% glutaraldehyde for 30 min before removing the agar and staining with crystal violet to count the plaques. The details of the luciferase assay for determination of VSV-GP-Luc infectivity was previously described (Keck, Z., et al, (2015) J. Virol. 90:279-291). Data was fit to a 4PL curve using GraphPad Prism 6. Percent neutralization was calculated based on wells containing virus only.
rVSV GP-GFP assay. Recombinant Indiana VSV (rVSV) expressing eGFP, as well as EBOV or MARV GP in place of VSV G were described previously (Miller, E.H., et al., (2012) EMBO J. 31 :1947-1960; Ng, M., et al., (2014) Virology 468-470:637-646; Wong., A.C. et al., (2010) J. Virol. 84:163-175.). rVSVs bearing TAFV GP, SUDV GP, or BDBV GPAMuc were generated by the same method. VSV particles containing cleaved GP (GPCL) were generated by incubating rVSV-GPs with thermolysin (200 μg mL) for lh at 37 °C, followed by treatment with phosphoramidon (1 mM), and reaction mixtures were used immediately. Infectivity of rVSVs was measured by counting eGFP-positive cells at 12-14 h post-infection using a Celllnsight CX5 automated microscope and onboard software (Thermo Scientific). For neutralization experiments, serial dilutions of mAbs were incubated with the rVSV-GP for lh at room temperature. Monolayers of Vero cells seeded in 96-well plates were inoculated with the mAb-virus mixture in triplicate and then incubated at 37°C overnight. Infection was scored 12 to 16 hours post-infection by enumeration of eGFP-positive cells. Infection levels were normalized to no-antibody control taken to represent 100%.
Example 2: Exemplary Anti-Filovirus Glycoprotein Antibodies
[0115] This Example lists examples of anti-filo virus glycoprotein antibodies that can be evaluated for efficacy in the antibody cocktails provided herein. The heavy and light chain variable domain sequences and CDR sequences of selected antibodies are provided in Table 2.
Antibodies binding to the receptor binding site (RBS) of ebolavirus:
[0116] FVM04: FVM04 is a macaque-human chimeric monoclonal antibody that binds to an exposed epitope within the RBS of multiple ebolaviruses including the virulent strains Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). See PCT Application No. PCT/US 15/57627. In order to define the epitope recognized by FVM04 we employed a comprehensive alanine scanning approach, where FVM04 binding was evaluated against a 'shotgun mutagenesis' mutation library of EBOV GP with 641 of 644 target residues individually mutated. Human HEK-293T cells were transfected with the entire mutation library in a 384-well array format (one clone per well) and assessed for reactivity to FVM04 using high-throughput flow cytometry. The method for shotgun mutagenesis is described in patent application 61/938,894 and (Davidson, E., and Doranz, B.J., 2014, Immunology, 143, 13-20). The shotgun mutagenesis revealed that FVM04 contact sites included Kl 15, Dl 17, and Gl 18. This is located within a hydrophilic region of the RBS known as the Crest ((Hashiguchi, et al, 2015, Cell, 160, 904-912; Wang et al, 2016, Cell, 164(l-2):258-68). This epitope is conserved across all ebolaviruses. FVM04 is also weakly reactive to marburgvirus glycoprotein (PCT/US 15/57627; Keck, et al., 2015, J Virol, 90:279 -291 ("Keck et al.")).
[0117] FVM04 neutralizes EBOV and SUDV (PCT/US 15/57627; Keck et al.), as well as BDBV and protects mice and guinea pigs against lethal EBOV and SUDV infection (see below).
Antibodies that bind to the glycan cap:
[0118] 13C6FR1: Monoclonal antibody 13C6 was developed using mouse hybridoma technology and was shown to protect mice from lethal challenge with Ebola virus (Wilson et al, 2000, Science, 287(5458): 1664-6). 13C6 binds on the top of EBOV GP glycan cap (Murin, et al., 2014, Proc Natl Acad Sci U S A, 111(48): 17182-7) with key
contact residues being T270 and K272 (Davidson et al, 2015, J Virol, 89(21):10982-9). 13C6FR1, a variant of 13C6 (U.S. Patent No. 7,335,356), is a component of the ZMapp therapeutic cocktail (Qiu, et al, 2014, Nature, 514(7520):47-53). Effective neutralization of EBOV by 13C6 requires the presence of complement (Wilson et al, 2000, Science, 287(5458): 1664-6).
[0119] FVM09: FVM09 binds with high affinity to a linear epitope within the disordered loop connecting the β strands 17 and 18 in the glycan cap region of EBOV GP (PCT/US 15/57627; Keck et al.). Using overlapping peptide mapping we mapped the epitope for FVM09 to amino acids 286-290 (GEWAF) of EBOV GP, and this epitope is 100% conserved among all ebolaviruses (PCT US 15/57627; Keck et al.).
[0120] FVM09 alone does not neutralize or provide protection in vivo against EBOV, but in combination with several other antibodies it enhances their neutralizing and protective potency as described in (Keck et al.) and below.
[0121] ADI-15731: The ADI- 15731 mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt, et al., 2016, Science, pii:aad5788. [Epub ahead of print] "Bornholdt et al "). Negative stain electron microscopy reconstructions of the ADI-15731 fab bound to EBOV GP showed that ADI-15731 bound directly to the GP1 glycan cap structure in a manner reflective of 13C6 (Murin, et al, 2014, Proc Natl Acad Sci U S A, l l l(48):17182-7 and Bornholdt et al.). However unlike 13C6, ADI- 15731 binds to EBOV GP, BDBV GP and SUDV GP. ADI-15731 only effectively neutralizes vesicular stomatitis virus (VSV) pseudovirions displaying either EBOV GP or BDBV GP (as determined by the rVSV GP-GFP assay described in Example 1).
Antibodies that simultaneously bind to glycan cap and core of GP1
[0122] m8C4: Mouse monoclonal antibody m8C4 cross neutralizes EBOV and SUDV and provides partial protection against both viruses in mice (PCT Publication No. WO2015/200522; Holtsberg, et al., 2015, J Virol, 90:266 -278). Efficacy of m8C4 was enhanced when used in combination with FVM09 (PCT Publication No. WO2015/200522; PCT US 15/57627; Keck et al). In order to define the epitope recognized by m8C4 we employed a comprehensive alanine scanning approach, where m8C4 binding was evaluated against a 'shotgun mutagenesis' mutation library of EBOV GP with 641 of 644 target residues individually mutated. Human HEK-293T cells were transfected with the entire mutation library in a 384-well array format (one clone per
well) and assessed for reactivity to m8C4 using high-throughput flow cytometry. The method for shotgun mutagenesis is described in patent application 61/938,894 and (Davidson, E., and Doranz, B.J., 2014, Immunology, 143, 13-20). Shotgun mutagenesis epitope mapping identified EBOV GP residues R136, Q251, and F252 as critical for m8C4 binding. Of these residues, Q251 and F252 are located within the glycan cap, while R136 is located within the core GP1 head domain (Figure 2). m8C4 bridges the core GP1 region with the glycan cap, and the epitope is conserved across ebolaviruses.
[0123] ADI-15750: ADI-15750 was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al.). ADI-15750 can compete for binding with 13C6 and binds with high affinity to a quaternary epitope in the EBOV GP1 structure also present on EBOV soluble GP (sGP). ADI-15750 demonstrated neutralization activity against VSV pseudoviruses displaying either EBOV GP or SUDV GP with IC50 values of 8.80 nM and 32.30 nM, respectively (as determined by the rVSV GP-GFP assay described in Example 1).
[0124] ADI-15968: ADI-15968 was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al.). ADI-15968 can compete for binding with 13C6 and binds with high affinity to a quaternary epitope in the EBOV GP1 structure also present on EBOV sGP. ADI-15968 demonstrated neutralization activity against VSV pseudoviruses displaying either EBOV GP or SUDV GP with IC50 values of 11.74 nM and 47.30 nM respectively (as determined by the rVSV GP-GFP assay described in Example 1).
Antibodies that bind to GP1/GP2 base epitope (Base binders)
[0125] 2G4 and 4G7: The most studied EBOV neutralizing epitope is in a region at the base of the trimeric GP that involves contact sites within GP1 and GP2. Antibodies such as KZ52 (Maruyama, et al., J. Virol. 1999;73:6024-6030), as well as two of ZMapp components 2G4 and 4G7 bind to this region (Lee, et al, 2008, Nature, 454 (7201): 177- 182; Murin, et al, 2014, Proc Natl Acad Sci U S A, l l l(48):17182-7). The epitopes for 2G4 and 4G7 are largely overlapping Davidson et al, 2015, J Virol., 89(21):10982-9) but the angle of binding for these two antibodies is different.
[0126] 2G4 and 4G7 were shown to provide significant protection in mice and guinea pig models of EBOV infection (Qiu, et al, 2012, PLoS Negl Trop Dis, 6: 1575). Both of these antibodies, along with 13C6FR1, are components of the ZMapp™ antibody cocktail
(Qiu, et al, 2014, Nature, 514(7520):47-53). 2G4 and 4G7 are specific to EBOV and do not cross react with other filovirus glycoproteins.
[0127] ADI-15734: ADI-15734 was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al). ADI-15734 binds specifically to the EBOV GP and directly competes with KZ52. ADI-15734 neutralizes EBOV (as determined by the rVSV GP-GFP assay described in Example 1) and provides significant levels of protection in the EBOV murine model.
Antibodies that bind the fusion loop
[0128] FVM02 (also called FVM02p): FVM02 is a macaque-derived panfilovirus antibody that binds to the tip of the internal fusion loop (IFL) of all ebolaviruses and marburgvirus (IBT PCT/US 15/57627; Keck et al). FVM02 provides partial protection against EBOV and MARV and potentiates the efficacy of FVM09 against EBOV in mouse models (IBT PCT/US 15/57627; Keck et al.).
Antibodies that bind between the tip of the fusion loop and the base GP1/GP2 epitope:
[0129] ADI-15742: The ADI- 15742 mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al.). ADI-15742 is an ultra-potent pan- ebolavirus neutralizing antibody demonstrating sub-nanomolar IC50 values against VSV pseudovirions displaying GP from the following species: EBOV, BDBV, SUDV, RESTV and TAFV (as determined by the rVSV GP-GFP assay described in Example 1). ADI- 15742 also provides complete protection against either EBOV or SUDV in their respective murine models.
[0130] ADI-15878: The ADI-15878 mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak and is a clonal relative of ADI-15742 (Bornholdt et al.). ADI-15878 is also an ultra-potent pan-ebolavirus neutralizing antibody demonstrating sub-nanomolar IC50 values against VSV pseudovirions displaying GP from the following species: EBOV, BDBV, SUDV, RESTV and TAFV (as determined by the rVSV GP-GFP assay described in Example 1). ADI-15878 showed significant levels of protection against EBOV and complete protection against SUDV in their respective murine models.
[0131] ADI-15946: The ADI-15946 mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bornholdt et al.). ADI-15946 is a potent pan-ebolavirus neutralizing antibody demonstrating sub-nanomolar IC50 values against VSV
pseudovirions displaying GP from the following species: EBOV, BDBV, and SUDV (as determined by the rVSV GP-GFP assay described in Example 1).
Antibodies that bind to the viral membrane proximal (stalk) region of filovirus glycoprotein:
[0132] ADI-16061: ADI-16061 is a human mAb was derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bomholdt et al). ADI-16061 binds to the heptad repeat 2 helices in the stalk region of EBOV GP, BDBV GP, and SUDV GP. However, ADI-16061 only effectively neutralizes VSV pseudovirions displaying GP from EBOV and BDBV with IC50 values 0.21 nM and 0.59 nM, respectively (as determined by the rVSV GP-GFP assay described in Example 1). Further ADI-16061 provided significant levels of protection from EBOV in the murine infection model post infection.
[0133] ADI-15974, ADI-15956, and ADI-15758: ADI-15974, ADI-15956, and ADI-15758 are clonally related human mAbs derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bomholdt et al.). These mAbs bind to the heptad repeat 2 helices in the stalk region of EBOV GP and BDBV GP. All three mAb effectively neutralize VSV pseudovirions displaying GP from EBOV and BDBV with sub-nanomolar IC50 values. In PRNT assays ADI-15974 ADI-15956, and ADI-15758 potently neutralize EBOV (as determined by the rVSV GP-GFP assay described in Example 1) and provided significant levels of protection from EBOV in the murine infection model post infection.
[0134] ADI-15848: ADI-15848 is a human mAb derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bomholdt et al). ADI-15848 binds to the heptad repeat 2 helices in the stalk region of EBOV GP and BDBV GP. ADI-15848 effectively neutralizes VSV pseudovirions displaying GP from EBOV and BDBV with IC50 values 0.50 nM and 0.77 nM, respectively (as determined by the rVSV GP-GFP assay described in Example 1). In plaque reduction neutralization (PRNT) assays ADI-15848 potently neutralized EBOV and provided significant levels of protection from EBOV in the murine infection model post infection.
[0135] ADI-16021: ADI-16021 is a human mAb derived from a human survivor (subject 45) of the 2014 Ebola virus outbreak (Bomholdt et al). ADI-16021 binds to the heptad repeat 2 helices in the stalk region of EBOV GP, BDBV GP and SUDV GP.
Example 3: Combination of pan-ebolavirus antibody with strong activity towards Sudan virus with EBOV specific antibodies included in ZMapp™ cocktail
Mouse Efficacy of FVM04
[0136] FVM04 binds to all ebolaviruses and neutralizes EBOV and SUDV; moreover, FVM04 was effective in mouse challenge models of EBOV infection when administered at two doses starting immediately after infection (PCT/US 15/57627; Keck, et al, 2015, J Virol, 90:279 -291). In this example we demonstrate efficacy in post exposure treatment in both the EBOV mouse model as well as a recently developed mouse model for SUDV (Brannan et al, 2015, J Infect Dis., 212 Suppl 2:S282-94). First, we evaluated the delayed administration of a single dose of FVM04. Groups of 10 mice were infected with 100 pfu of mouse-adapted EBOV (MA-EBOV) (Bray, et al, 1999, J Infect Dis., 179 Suppl 1, S248-258) and treated with a single injection of 10 mg/kg (200 μg/mouse) of FVM04 either 1, 2, or 3 days post infection (dpi). As shown in Figure 3 A, a single injection of FVM04 at 1 dpi led to full protection from lethal challenge (P<0.0001; determined by Manel-Cox method), while delayed treatment on day 2 or day 3 (peak of viremia) resulted in 80% (P=0.0012) and 30% (P=0.108) protection, respectively. Consistent with survival data, mice treated at 1 dpi showed no weight loss or sign of disease {Figure 3A). Mice treated on day 2 or 3 dpi lost a maximum of 8 and 10% body weight, respectively, compared to 18% weight loss in the control group, and milder clinical signs of disease were observed {Figure 3A). In a second experiment we evaluated the dose response by treating the mice 2 dpi with 10, 5, or 2.5 mg/kg (200, 100, or 50 μg/mouse) of FVM04 or PBS as control. In this study both 10 and 5 mg/kg FVM04 provided full protection (P<0.0001) while 70% of the mice receiving 2.5 mg/kg survived the challenge (P=0.0004) {Figure 3B). PBS treated mice lost about 13% of their body weight before succumbing to infection, while mice treated with FVM04 showed less weight loss and less severe disease as determined by health scores {Figure 3B).
[0137] Efficacy of FVM04 was further tested in mice in which the genes for IFNa/IFNP receptor are knocked out (IFNa R'/_) (Brannan et al, 2015, J Infect Dis., 212 Suppl 2:S282-94). Groups of 7 four week old IFNcxpR' " mice were infected with 1000 pfu of SUDV followed by intraperitoneal injection of 10 mg/kg of anti-SUDV GP mAb 16F6 at 1 and 3 dpi, or FVM04 at 1 dpi. A control group of 6 mice received no treatment after the infection. The SUDV specific mAb 16F6 fully protected mice with minimal weight loss
or signs of disease (Figure 3C). Five out of seven mice treated with FVM04 were protected from lethal challenge, while the effect on average weight loss and health scores was not apparent (Figure 3C).
Guinea pig Efficacy of FV 04
[0138] Efficacy of FVM04 was also examined in guinea pigs using guinea pig adapted EBOV and SUDV (GPA-EBOV and GPA-SUDV) (Wong, et al, 2015, J Virol, 90(l):392-9). Four groups of 6 guinea pigs were challenged either with 1000 X LD50 of GPA-SUDV or GPA-EBOV followed by a single intraperitoneal injection of 5 mg FVM04 (~15 mg/kg) or DPBS as vehicle control at 1 dpi. Animals were monitored for 16 days for weight change and 28 days for survival. As shown in Figure 4 A, a single injection of FVM04 protected all guinea pigs from GPA-SUDV challenge, while the controls succumbed to infection within 10-13 days (P=0.0004). While the controls lost up to 40% body weight after GPA-SUDV challenge, no weight loss or sign of disease was observed among FVM04 treated animals (Figure 4A). All DPBS-treated guinea pigs infected with GPA-EBOV succumbed to infection within 6-7 days, while 2 out of 6 FVM04 treated animals survived the challenge and the remaining died between days 9 and 11 post infection (P=0.0012) (Figure 4B).
Efficacy of a cocktail of FVM04 with two ZMAPP™ components against EBOV and SUDV
[0139] The partial protection against EBOV is consistent with previous reports indicating that an antibody cocktail is required for effective post-exposure protection against EBOV in guinea pigs and nonhuman primates (Qiu, et al., 2014, Nature, 514(7520):47-53). ZMAPP™, consisting of the two base binders c2G4 and c4G7 and the glycan cap binder cl3C6, was selected for testing in NHPs based on significant, but partial, protection in guinea pigs (4 out of 6) when administered once at 3 dpi (Qiu, et al., 2014, Nature, 514(7520):47-53). Based on the above study we hypothesized that replacing one of the components of ZMAPP™ with FVM04 would lead to an effective EBOV cocktail that is also protective against Sudan virus. We selected the mAb c4G7 to be replaced since it binds to an epitope closely overlapping the c2G4 epitope (Murin, et al, 2014, Proc Natl AcadSci USA, 1 11(48): 17182-7). We first tested if FVM04 alone (5 mg) or a cocktail of c2G4/cl3C6/cFVM04 (1.6 mg each) would protect against GPA-SUDV when
administered at 3 dpi. While all GPA-SUDV infected control animals died within 10-14 days, all FVM04 treated animals and 5 out of 6 animals treated with the cocktail survived the challenge (Figure 4C). The protection was highly significant with P=0.0008 for both treatment groups compared with the control group. Animals treated with FVM04 exhibited no weight loss, while control animals lost an average of 25% body weight (Figure 4C). While the animals treated with the cocktail also showed no weight loss on average (Figure 4C), the only fatal case in this group lost 17% body weight before dying on day 14 post infection.
[0140] The cocktail consisting of FVM04, cl3C6 and c2G4 was also tested in the GPA- EBOV model. Four out of 6 animals treated with a single dose of 5 mg cocktail (~1.6 mg of each component) at 3 dpi survived the challenge while all control animals succumbed to infection within 7-9 days (Figure 4D) (P=0.0061). The control animals lost an average of 20% weight, while the average weight within the cocktail treated group showed a steady increase over 16 days post infection (Figure 4D). Of the two cocktail treated animals that died, one animal lost about 9% body weight by the day of death (7 dpi) and the second animal actually gained 12% body weight before dying on day 8. As a comparison, Figure 4E shows compiled survival and weight loss data from three studies that we have performed with ZMAPP™ (5 mg/animal, n=20). A survival rate of 67% in guinea pigs for the cocktail of FVM04/cl3C6/c2G4 is well within the range of protection afforded by ZMAPP™ as shown here and reported previously (Qiu, et al, 2014, Nature, 514(7520):47-53).
Example 4: FVM09 displays neutralizing activity in presence of base binder 2G4
[0141] In presence of a subneutralizing concentration of 2G4, FVM09 behaved like a neutralizing antibody. Figure 5 shows an experiment in which the neutralizing activity of FVM09 towards Ebola virus GP pseudotyped vesicular stomatitis virus (VSV) was tested in presence or absence of a low concentration of 2G4, using the rVSV GP luciferase pseudotype assay described in Example 1. In the absence of 2G4, FVM09 did not show any significant neutralizing activity while when 2G4 was added at 0.2 μβ/πιΐ FVM09 showed a clear dose dependent neutralization.
Example 5: The IFL-binder FVM02 enhances the neutralizing potency of
neutralizing antibodies that target the RBS
[0142] We made the observation that the neutralizing potency of the FV 04 that binds to the RBS of various Ebolavirus species is significantly enhanced by FVM02, using the rVSV GP luciferase pseudotype assay described in Example 1, while FV 02 alone does not neutralize by itself {Figure 6Λ)
[0143] MR191 is a monoclonal antibody that binds to the RBS of the marburgvirus and neutralizes marburgvirus (Flyak, et al, 2015, Cell, 160(5):893-903; sequences of MR191 are published in PCT Pub. WO2016/179511 and US Pub. No. 2016/0326234, both of which are incorporated by reference herein in their entireties). As shown in Figure 6B, FVM02 also enhanced the neutralizing potency of MR191 towards marburgvirus while FVM02 by itself did not have any neutralizing activity. These data provide evidence that a combination of FVM02 with an RBS binder can lead to effective therapeutic cocktail for pan-ebolavirus and pan-filovirus treatment.
Table 2: Antibody Sequences
Claims
1. A method for preventing, treating, or managing a filovirus infection in a subject, comprising administering to a subject in need thereof an effective amount of an antibody cocktail comprising at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus glycoprotein (filovirus GP), wherein the cocktail comprises a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof, wherein the first antibody or fragment thereof specifically binds to a filovirus GP1/GP2 base epitope, wherein the second antibody or fragment thereof specifically binds to a filovirus GP receptor binding site ( BS) epitope, a filovirus GP glycan cap epitope, a filovirus GP internal fusion loop (IFL) epitope, or any combination thereof, wherein at least one antibody or fragment thereof in the antibody cocktail can specifically bind to the orthologous epitope on two or more filovirus species or strains, and wherein administration of the antibody cocktail is effective against two or more filovirus species or strains.
2. The method of claim 1, wherein the first antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 , respectively (2G4), and the second antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH- CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively (FVM04).
3. The method of claim 1, wherein the first antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 29, respectively (2G4), and the second antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 5, respectively (FVM04).
4. The method of claim 2 or claim 3, wherein the antibody cocktail further comprises a third anti-filovirus GP antibody or antigen-binding fragment thereof, wherein the third antibody or fragment thereof can specifically bind to a filovirus glycan cap epitope.
5. The method of claim 4, wherein the third antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL- CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, or a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 9 and SEQ ID NO: 13, respectively (13 C6FR1).
6. The method of claim 5, wherein the antibody cocktail can protect guinea pigs from challenge with guinea pig-adapted EBOV (GPA-EBOV) and guinea pig-adapted SUDV (GPA-SUDV).
7. The method of claim 1, wherein the first antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 , respectively (2G4), and the second antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL- CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 , respectively (FVM09).
8. The method of claim 1, wherein the first antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 29, respectively (2G4), and the second antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 17 and SEQ ID NO: 21, respectively (FVM09).
9. A method for preventing, treating, or managing a filovirus infection in a subject, comprising administering to a subject in need thereof an effective amount of an antibody cocktail comprising at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus GP, wherein the cocktail comprises a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen- binding fragment thereof, wherein the first antibody or fragment thereof specifically binds to a filovirus RBS epitope, wherein the second antibody or fragment thereof specifically binds to a filovirus IFL epitope, wherein at least one antibody or fragment thereof in the antibody cocktail can specifically bind to the orthologous epitope on two or more filovirus species or strains, and wherein administration of the antibody cocktail is effective against two or more filovirus species or strains.
10. The method of claim 9, wherein the first antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDRl, VH-CDR2, VH-CDR3, VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively (FVM04), and the second antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDRl, VH-CDR2, VH-CDR3, VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively (FVM02).
11. The method of claim 9, wherein the first antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 5, respectively (FVM04), and the second antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 33 and SEQ ID NO: 37, respectively (FVM02).
12. The method of claim 9, wherein the first antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDRl, VH-CDR2, VH-CDR3, VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48,
respectively (MR1 1), and the second antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL- CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively (FVM02).
13. The method of claim 9, wherein the first antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 41 and SEQ ID NO: 45, respectively (MR191), and the second antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 33 and SEQ ID NO: 37, respectively (FVM02).
14. The method of any one of claims 1 to 13, wherein the two or more fllovirus species can be two or more of Marburg virus (MARV), Ravn virus (RAW), Tai Forest virus (TAFV), Reston virus (RESTV), Sudan virus (SUDV) Ebola virus (EBOV), Bundibugyo virus (BDBV), or any strain thereof.
15. The method of any one of claims 1 to 14, wherein the ability of the antibody cocktail to prevent, treat, or manage a fllovirus infection can be measured in a model comprising administering the antibody cocktail to a group of rodents and challenging rodents with a rodent- adapted fllovirus before, at the same time as, or after administering the antibody cocktail to the rodents.
16. The method of claim 15, wherein the rodent model is a guinea pig model or a mouse model.
17. The method of claim 15 or claim 16, wherein the rodents are monitored for increased survival time, decreased weight loss, or a combination thereof as compared to control rodents.
18. The method of any one of claims 1 to 17, wherein the antibody cocktail can prevent, treat, or manage filovirus infection in the subject with a potency that is greater than the additive potency of the antibodies or fragments thereof when administered individually.
19. The method of any one of claims 1 to 18, wherein each antibody or fragment thereof included in the antibody cocktail is, independently, a murine antibody or fragment thereof, a non-human primate (NHP) antibody or fragment thereof, a humanized antibody or fragment thereof, or a chimeric antibody or fragment thereof.
20. The method of any one of claims 1 to 19, wherein each antibody or fragment thereof included in the antibody cocktail is, independently, a monoclonal antibody or fragment thereof, a component of a polyclonal antibody mixture, a recombinant antibody or fragment thereof, a multispecific antibody or fragment thereof, or any combination thereof.
21. The method of any one of claims 1 to 20, wherein one or more antibodies or fragments thereof included in the antibody cocktail comprises a heavy chain constant region or fragment thereof.
22. The method of claim 21, wherein the heavy chain constant region or fragment thereof is a murine constant region or fragment thereof, a macaque constant region or fragment thereof, or a human constant region or fragment thereof.
23. The method of claim 21 or claim 22, wherein the heavy chain constant region or fragment thereof is an IgM, IgG, IgA, IgE, IgD, or IgY constant region or fragment thereof.
24. The method of any one of claims 21 to 23, wherein one or more antibodies included in the antibody cocktail comprises a light chain constant region or fragment thereof.
25. The method of claim 24, wherein the light chain constant region or fragment thereof is a murine constant region or fragment thereof, a macaque constant region or fragment thereof, or a human constant region or fragment thereof.
26. The method of any one of claims 1 to 25, wherein one or more antibodies or fragments thereof included in the antibody cocktail comprises an Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv fragment, an scFab fragment, an sc(Fv)2 fragment, or any combination thereof.
27. The method of any one of claims 1 to 26, wherein one or more antibodies or fragments thereof included in the antibody cocktail is conjugated to an antiviral agent, a protein, a lipid, a detectable label, a polymer, or any combination thereof.
28. The method of any one of claims 1 to 27, wherein the filovirus infection is hemorrhagic fever.
29. The method of any one of claims 1 to 28 wherein the subject is a nonhuman primate or a human.
30. An antibody cocktail comprising at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus glycoprotein (filovirus GP), wherein the cocktail comprises a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof, wherein the first antibody or fragment thereof specifically binds to a filovirus GP1/GP2 base epitope, wherein the second antibody or fragment thereof specifically binds to a filovirus GP receptor binding site (RBS) epitope, a filovirus GP glycan cap epitope, a filovirus GP internal fusion loop (IFL) epitope, or any combination thereof, wherein at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains, wherein administration of an effective amount of the antibody cocktail can prevent, treat, or manage a filovirus infection in a subject, and wherein the antibody cocktail is effective against two or more filovirus species or strains.
31. The antibody cocktail of claim 30, wherein the first antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 , respectively (2G4), and the second antibody or fragment thereof comprises a VH and a VL collectively comprising VH- CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively (FVM04).
32. The antibody cocktail of claim 30, wherein the first antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 29, respectively (2G4), and the second antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 5, respectively (FVM04).
33. The antibody cocktail of claim 31 or claim 32, which further comprises a third anti-filovirus GP antibody or antigen-binding fragment thereof, wherein the third antibody or fragment thereof can specifically bind to a filovirus glycan cap epitope.
34. The antibody cocktail of claim 33, wherein the third antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, or a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 9 and SEQ ID NO: 13, respectively (13 C6FR1).
35. The antibody cocktail of claim 34, which can protect guinea pigs from challenge with guinea pig-adapted EBOV (GPA-EBOV) and/or guinea pig-adapted SUDV (GPA-SUDV).
36. The antibody cocktail of claim 30, wherein the first antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 , respectively (2G4), and the second antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 , respectively (FVM09).
37. The antibody cocktail of claim 30, wherein the first antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 29, respectively (2G4), and the second antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100%) identical to reference amino acid sequences SEQ ID NO: 17 and SEQ ID NO: 21, respectively (FVM09).
38. An antibody cocktail comprising at least two antibodies or antigen-binding fragment thereof that specifically bind to different epitopes on a filovirus GP, wherein the cocktail comprises a first anti-filovirus GP antibody or antigen-binding fragment thereof and a second anti-filovirus GP antibody or antigen-binding fragment thereof, wherein the first antibody or fragment thereof specifically binds to a filovirus RBS epitope, wherein the second antibody or fragment thereof specifically binds to a filovirus IFL epitope, wherein at least one antibody or fragment thereof in the antibody cocktail can specifically bind to its orthologous epitope on two or more filovirus species or strains, wherein administration of an effective amount of the antibody cocktail can prevent, treat, or manage a filovirus infection in a subject, and wherein the antibody cocktail is effective against two or more filovirus species or strains.
39. The antibody cocktail of claim 38, wherein the first antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively (FVM04), and the second antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL- CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively (FVM02).
40. The antibody cocktail of claim 38, wherein the first antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 5, respectively (FVM04), and the second antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 33 and SEQ ID NO: 37, respectively (FVM02).
41. The antibody cocktail of claim 38, wherein the first antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ
ID NO: 48, respectively (MR191), and the second antibody or fragment thereof comprises a VH and a VL collectively comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively (FV 02).
42. The antibody cocktail of claim 38, wherein the first antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 41 and SEQ ID NO: 45, respectively ( R191), and the second antibody or fragment thereof comprises a VH and a VL comprising amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 33 and SEQ ID NO: 37, respectively (FVM02).
43. The antibody cocktail of any one of claims 30 to 42, wherein each antibody or fragment thereof included in the antibody cocktail is, independently, a murine antibody or fragment thereof, a non-human primate (NHP) antibody or fragment thereof, a humanized antibody or fragment thereof, or a chimeric antibody or fragment thereof.
44. The antibody cocktail of any one of claims 30 to 43, wherein each antibody or fragment thereof included in the antibody cocktail is, independently, a monoclonal antibody or fragment thereof, a component of a polyclonal antibody mixture, a recombinant antibody or fragment thereof, a multispecific antibody or fragment thereof, or any combination thereof.
45. The antibody cocktail of any one of claims 30 to 44, wherein one or more antibodies or fragments thereof included in the antibody cocktail comprises a heavy chain constant region or fragment thereof.
46. The antibody cocktail of claim 45, wherein the heavy chain constant region or fragment thereof is a murine constant region or fragment thereof, a macaque constant region or fragment thereof, or a human constant region or fragment thereof.
47. The antibody cocktail of claim 44 or claim 45, wherein the heavy chain constant region or fragment thereof is an IgM, IgG, IgA, IgE, IgD, or IgY constant region or fragment thereof.
48. The antibody cocktail of any one of claims 45 to 47, wherein one or more antibodies included in the antibody cocktail comprises a light chain constant region or fragment thereof.
49. The antibody cocktail of claim 48, wherein the light chain constant region or fragment thereof is a murine constant region or fragment thereof, a macaque constant region or fragment thereof, or a human constant region or fragment thereof.
50. The antibody cocktail of any one of claims 30 to 49, wherein one or more antibodies or fragments thereof included in the antibody cocktail comprises an Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv fragment, an scFab fragment, an sc(Fv)2 fragment, or any combination thereof.
51. The antibody cocktail of any one of claims 30 to 50, wherein one or more antibodies or fragments thereof included in the antibody cocktail is conjugated to an antiviral agent, a protein, a lipid, a detectable label, a polymer, or any combination thereof.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662307118P | 2016-03-11 | 2016-03-11 | |
US62/307,118 | 2016-03-11 | ||
US201662368688P | 2016-07-29 | 2016-07-29 | |
US62/368,688 | 2016-07-29 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2017156423A2 true WO2017156423A2 (en) | 2017-09-14 |
WO2017156423A3 WO2017156423A3 (en) | 2017-10-19 |
Family
ID=59789746
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2017/021842 WO2017156423A2 (en) | 2016-03-11 | 2017-03-10 | Broadly protective antibody cocktails for treatment of filovirus hemorrhagic fever |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2017156423A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190240328A1 (en) * | 2016-09-24 | 2019-08-08 | The Trustees Of The University Of Pennsylvania | Novel humanized anti-ebola antibodies useful in preventing ebola infections |
JP2020508990A (en) * | 2017-02-17 | 2020-03-26 | マップ バイオファーマシューティカル、インコーポレイテッド | Monoclonal antibodies and cocktails for the treatment of Ebola infection |
EP3583122A4 (en) * | 2017-02-17 | 2020-11-11 | Mapp Biopharmaceutical, Inc. | Monoclonal antibodies and cocktails for treatment of ebola infections |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110372792A (en) * | 2008-06-25 | 2019-10-25 | 艾斯巴技术-诺华有限责任公司 | Inhibit the stabilization and soluble antibodies of VEGF |
JP6507097B2 (en) * | 2012-09-19 | 2019-04-24 | イナート・ファルマ・ソシエテ・アノニムInnate Pharma Pharma S.A. | KIR3DL2 binding agent |
US9346875B2 (en) * | 2013-06-03 | 2016-05-24 | Albert Einstein College Of Medicine, Inc. | Therapy for filovirus infection |
WO2015200522A2 (en) * | 2014-06-25 | 2015-12-30 | Integrated Biotherapeutics, Inc. | Monoclonal antibodies directed against envelope glycoproteins from multiple filovirus species |
US10611827B2 (en) * | 2014-10-28 | 2020-04-07 | Integrated Biotherapeutics, Inc. | Non-human primate-derived pan-ebola and pan-filovirus monoclonal antibodies directed against envelope glycoproteins |
WO2016145385A2 (en) * | 2015-03-11 | 2016-09-15 | Integrated Biotherapeutics, Inc. | Pan-ebola and pan-filovirus protective epitopes, antibodies, and antibody cocktails |
US20160326234A1 (en) * | 2015-05-07 | 2016-11-10 | Andrew Hiatt | Monoclonal antibody cocktails for treatment of ebola infections |
-
2017
- 2017-03-10 WO PCT/US2017/021842 patent/WO2017156423A2/en active Application Filing
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190240328A1 (en) * | 2016-09-24 | 2019-08-08 | The Trustees Of The University Of Pennsylvania | Novel humanized anti-ebola antibodies useful in preventing ebola infections |
JP2020508990A (en) * | 2017-02-17 | 2020-03-26 | マップ バイオファーマシューティカル、インコーポレイテッド | Monoclonal antibodies and cocktails for the treatment of Ebola infection |
EP3583122A4 (en) * | 2017-02-17 | 2020-11-11 | Mapp Biopharmaceutical, Inc. | Monoclonal antibodies and cocktails for treatment of ebola infections |
US11407816B2 (en) | 2017-02-17 | 2022-08-09 | Mapp Biopharmaceutical, Inc. | Monoclonal antibodies and cocktails for treatment of Ebola infections |
US11407817B2 (en) | 2017-02-17 | 2022-08-09 | Mapp Biopharmaceutical, Inc. | Monoclonal antibodies and cocktails for treatment of Ebola infections |
JP7128829B2 (en) | 2017-02-17 | 2022-08-31 | マップ バイオファーマシューティカル、インコーポレイテッド | Monoclonal antibodies and cocktails for the treatment of Ebola infection |
Also Published As
Publication number | Publication date |
---|---|
WO2017156423A3 (en) | 2017-10-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107847591B (en) | Multivalent human immunodeficiency virus antigen binding molecules and uses thereof | |
CN107532188B (en) | CD20 binding molecules and uses thereof | |
CN107921285B (en) | Multivalent hepatitis b virus antigen binding molecules and uses thereof | |
US20180237502A1 (en) | Pan-ebola and pan-filovirus protective epitopes, antibodies, and antibody cocktails | |
US9499608B2 (en) | Bispecific monoclonal antibody therapeutics against West Nile virus with improved CNS penetration | |
US20220403009A1 (en) | Multimeric coronavirus binding molecules and uses thereof | |
US8663950B2 (en) | Production of a monoclonal antibody therapeutic against west nile virus in plants | |
US20170158753A1 (en) | Monoclonal antibodies directed against envelope glycoproteins from multiple filovirus species | |
US20190263894A1 (en) | Monoclonal antibody cocktails for treatment of ebola infections | |
US20220267415A1 (en) | Multimeric sars-cov-2 binding molecules and uses thereof | |
KR20220143768A (en) | Use of anti-family with sequence similarity 19, member a5 antibodies for the treatment of glaucoma | |
WO2017156423A2 (en) | Broadly protective antibody cocktails for treatment of filovirus hemorrhagic fever | |
US10611827B2 (en) | Non-human primate-derived pan-ebola and pan-filovirus monoclonal antibodies directed against envelope glycoproteins | |
US10982221B2 (en) | Plant-derived antibodies and derivatives that reduce risk of antibody-dependent enhancement (ADE) of infection | |
JP2018537117A (en) | Antibodies with improved stability against intestinal digestion | |
EP3525813B1 (en) | Broadly neutralizing antibody targeting the ebolavirus glycoprotein internal fusion loop | |
WO2022122788A1 (en) | Multispecific antibodies against severe acute respiratory syndrome coronavirus 2 | |
KR20230006456A (en) | Humanized Anti-C5a Antibody | |
US20220372114A1 (en) | Sars-cov-2 spike protein antibodies | |
US20240109954A1 (en) | Fc-enhanced antibodies for prevention and treatment of ebola virus infection | |
CN116601169A (en) | Broadly neutralizing binding molecules against marburg virus | |
WO2017192483A1 (en) | Monoclonal antibody cocktail for treatment of ebola infections | |
CN116621975A (en) | Humanized monoclonal antibody aiming at Nipah virus G protein and application thereof | |
He et al. | Generation and Analysis of Novel Plant-Derived Antibody-Based Therapeutic Molecules against |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17764204 Country of ref document: EP Kind code of ref document: A2 |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 17764204 Country of ref document: EP Kind code of ref document: A2 |