CA2704119C - Humanized anti-venezuelan equine encephalitis virus recombinant antibodies - Google Patents
Humanized anti-venezuelan equine encephalitis virus recombinant antibodies Download PDFInfo
- Publication number
- CA2704119C CA2704119C CA2704119A CA2704119A CA2704119C CA 2704119 C CA2704119 C CA 2704119C CA 2704119 A CA2704119 A CA 2704119A CA 2704119 A CA2704119 A CA 2704119A CA 2704119 C CA2704119 C CA 2704119C
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- veev
- human
- seq
- furin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000700605 Viruses Species 0.000 title description 10
- 208000002687 Venezuelan Equine Encephalomyelitis Diseases 0.000 title description 6
- 206010014611 Encephalitis venezuelan equine Diseases 0.000 title description 5
- 201000009145 Venezuelan equine encephalitis Diseases 0.000 title description 5
- 241000710959 Venezuelan equine encephalitis virus Species 0.000 claims abstract description 77
- 241001529936 Murinae Species 0.000 claims abstract description 59
- 208000015181 infectious disease Diseases 0.000 claims abstract description 26
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 16
- 239000013604 expression vector Substances 0.000 claims abstract description 14
- 238000011321 prophylaxis Methods 0.000 claims abstract description 13
- 238000011282 treatment Methods 0.000 claims abstract description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 23
- 108090000623 proteins and genes Proteins 0.000 claims description 23
- 150000007523 nucleic acids Chemical class 0.000 claims description 19
- 108020004707 nucleic acids Proteins 0.000 claims description 12
- 102000039446 nucleic acids Human genes 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 4
- 241000124008 Mammalia Species 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims 2
- 230000036039 immunity Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 23
- 239000008194 pharmaceutical composition Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 34
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 32
- 239000013598 vector Substances 0.000 description 26
- 241000699670 Mus sp. Species 0.000 description 25
- 101710117290 Aldo-keto reductase family 1 member C4 Proteins 0.000 description 24
- 108090001126 Furin Proteins 0.000 description 15
- 102000004961 Furin Human genes 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 14
- 230000003472 neutralizing effect Effects 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 230000027455 binding Effects 0.000 description 11
- 229960005486 vaccine Drugs 0.000 description 11
- 241000282412 Homo Species 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 238000002649 immunization Methods 0.000 description 10
- 230000003053 immunization Effects 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 239000000427 antigen Substances 0.000 description 9
- 210000004602 germ cell Anatomy 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- 238000013459 approach Methods 0.000 description 7
- 206010014599 encephalitis Diseases 0.000 description 7
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000006386 neutralization reaction Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 102100021966 Coiled-coil domain-containing protein 34 Human genes 0.000 description 4
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 4
- 241000283073 Equus caballus Species 0.000 description 4
- 101150008942 J gene Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 101710120037 Toxin CcdB Proteins 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 231100000636 lethal dose Toxicity 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000003259 recombinant expression Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 101150117115 V gene Proteins 0.000 description 3
- 101150063325 ab gene Proteins 0.000 description 3
- 239000000443 aerosol Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000002238 attenuated effect Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 102000008482 12E7 Antigen Human genes 0.000 description 2
- 108010020567 12E7 Antigen Proteins 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 241000710929 Alphavirus Species 0.000 description 2
- 101710121417 Envelope glycoprotein Proteins 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000283074 Equus asinus Species 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 101000690301 Homo sapiens Aldo-keto reductase family 1 member C4 Proteins 0.000 description 2
- 101001116548 Homo sapiens Protein CBFA2T1 Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 108020000999 Viral RNA Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 229940000406 drug candidate Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 102000054751 human RUNX1T1 Human genes 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000002962 plaque-reduction assay Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 210000003501 vero cell Anatomy 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 101150076401 16 gene Proteins 0.000 description 1
- 101150092328 22 gene Proteins 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 101150117081 51 gene Proteins 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 101710125507 Integrase/recombinase Proteins 0.000 description 1
- 101150045458 KEX2 gene Proteins 0.000 description 1
- 231100000111 LD50 Toxicity 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 102000006437 Proprotein Convertases Human genes 0.000 description 1
- 108010044159 Proprotein Convertases Proteins 0.000 description 1
- 102100038946 Proprotein convertase subtilisin/kexin type 6 Human genes 0.000 description 1
- 101710132593 Protein E2 Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 101800001473 Spike glycoprotein E2 Proteins 0.000 description 1
- 101710135785 Subtilisin-like protease Proteins 0.000 description 1
- 241000710924 Togaviridae Species 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 208000011312 Vector Borne disease Diseases 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000002009 allergenic effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000001130 anti-lysozyme effect Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000000305 astragalus gummifer gum Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 239000011545 carbonate/bicarbonate buffer Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 101150046339 fur gene Proteins 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 101150030901 mab-21 gene Proteins 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002625 monoclonal antibody therapy Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 235000020030 perry Nutrition 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000011808 rodent model Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 108010018381 streptavidin-binding peptide Proteins 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000007501 viral attachment Effects 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
-
- 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
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
- C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Mycology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A CDR grafted humanized recombinant antibody against infection from Venezuelan equine encephalitis virus (VEEV) comprises a human Ig framework having CDRs from murine mAb 1A4A1 VH and VL. DNA sequences, expression vectors incorporating such sequences and transformed host cells are also provided. Also provided are pharmaceutical compositions and methods of prophylaxis and treatment against VEEV infection using the humanized recombinant antibodies of the invention.
Description
CA 2,704,119 Blakes Ref: 67853/00022 4 [0001] The present invention relates to a humanized antibodies (Abs) and, more specifically, to humanized recombinant antibodies (rAbs) against infection by the Venezuelan equine encephalitis 6 virus (VEEV). The invention provides methods of prophylaxis and treatment against VEEV using 7 such antibodies.
9 [0002] Venezuelan equine encephalitis virus (VEEV), a member of the alphavirus genus of the family Togaviridae, is an important mosquito-borne pathogen in humans and equides [1]. VEEV
11 infections mainly target the central nervous system and lymphoid tissues causing severe 12 encephalitis in equines and a spectrum of human diseases ranging from unapparent or sub-clinical 13 infection to acute encephalitis. Neurological disease appears in 4-14%
of cases. The incidence of 14 human infection during equine epizootics could be up to 30%. Mortality associated with the encephalitis in children is as high as 35%. Recent outbreaks in Venezuela and Colombia in 1995 16 resulted in around 100,000 human cases with more than 300 fatal encephalitis cases [2].
17 Furthermore, VEEV is highly infectious by aerosol inhalation in humans and other animals.
18 However, there are no antiviral drugs available that are effective against VEEV although currently 19 there are two forms of IND (investigational new drug) VEEV vaccines available for human and veterinary use: TC-83, a live-attenuated Trinidad donkey strain and C-84, a formalin-inactivated TC-21 83 [3,4]. However, for various reasons, these vaccines are far from satisfactory. For example, 22 approximately 20% of recipients that receive the TC-83 vaccine fail to develop neutralizing Abs, 23 while another 20% exhibit reactogenicity. In addition, the TC-83 vaccine could revert to wild-type 24 form. The vaccine C-84 is well tolerated, but requires multiple immunizations, periodic boosts, and fails to provide protection against aerosol challenge in some rodent models.
26 [0003] Like the other alphaviruses, VEEV is an enveloped virus, consisting of three structural 27 proteins: a capsid encapsidating the viral RNA genome, and two envelope glycoproteins, El and 28 E2. El and E2 form heterodimers, which project from the virus envelope as trimer spikes. Epitopes 29 on the spikes are the targets of neutralizing Abs. Studies have shown that the viral neutralizing epitopes are mainly located on the E2 protein, and that the E2C epitope appears to be the hub of the 31 neutralization epitopes [5,6]. The murine monoclonal Ab (mAb) 1A4A1 [14]
is specific for E2C. This 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 mAb has been shown to be efficient in protecting animals from a lethal peripheral challenge with 2 virulent VEEV [7].
3 [0004] Murine mAbs, however, have serious disadvantages as therapeutic agents in humans 4 [8]. For example, one of the problems associated with using murine mAbs in humans is that they may induce an anti-mouse Ab response. Further, repeat administration of murine mAbs may result 6 in rapid clearance of the murine mAbs and anaphylaxis, which can sometimes be fatal. To 7 overcome this hurdle, the humanization of murine mAbs has been proposed, by which process 8 murine Ab frameworks are replaced by human Ab ones in order to reduce immunogenicity of Abs in 9 humans [9,10].
9 [0002] Venezuelan equine encephalitis virus (VEEV), a member of the alphavirus genus of the family Togaviridae, is an important mosquito-borne pathogen in humans and equides [1]. VEEV
11 infections mainly target the central nervous system and lymphoid tissues causing severe 12 encephalitis in equines and a spectrum of human diseases ranging from unapparent or sub-clinical 13 infection to acute encephalitis. Neurological disease appears in 4-14%
of cases. The incidence of 14 human infection during equine epizootics could be up to 30%. Mortality associated with the encephalitis in children is as high as 35%. Recent outbreaks in Venezuela and Colombia in 1995 16 resulted in around 100,000 human cases with more than 300 fatal encephalitis cases [2].
17 Furthermore, VEEV is highly infectious by aerosol inhalation in humans and other animals.
18 However, there are no antiviral drugs available that are effective against VEEV although currently 19 there are two forms of IND (investigational new drug) VEEV vaccines available for human and veterinary use: TC-83, a live-attenuated Trinidad donkey strain and C-84, a formalin-inactivated TC-21 83 [3,4]. However, for various reasons, these vaccines are far from satisfactory. For example, 22 approximately 20% of recipients that receive the TC-83 vaccine fail to develop neutralizing Abs, 23 while another 20% exhibit reactogenicity. In addition, the TC-83 vaccine could revert to wild-type 24 form. The vaccine C-84 is well tolerated, but requires multiple immunizations, periodic boosts, and fails to provide protection against aerosol challenge in some rodent models.
26 [0003] Like the other alphaviruses, VEEV is an enveloped virus, consisting of three structural 27 proteins: a capsid encapsidating the viral RNA genome, and two envelope glycoproteins, El and 28 E2. El and E2 form heterodimers, which project from the virus envelope as trimer spikes. Epitopes 29 on the spikes are the targets of neutralizing Abs. Studies have shown that the viral neutralizing epitopes are mainly located on the E2 protein, and that the E2C epitope appears to be the hub of the 31 neutralization epitopes [5,6]. The murine monoclonal Ab (mAb) 1A4A1 [14]
is specific for E2C. This 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 mAb has been shown to be efficient in protecting animals from a lethal peripheral challenge with 2 virulent VEEV [7].
3 [0004] Murine mAbs, however, have serious disadvantages as therapeutic agents in humans 4 [8]. For example, one of the problems associated with using murine mAbs in humans is that they may induce an anti-mouse Ab response. Further, repeat administration of murine mAbs may result 6 in rapid clearance of the murine mAbs and anaphylaxis, which can sometimes be fatal. To 7 overcome this hurdle, the humanization of murine mAbs has been proposed, by which process 8 murine Ab frameworks are replaced by human Ab ones in order to reduce immunogenicity of Abs in 9 humans [9,10].
[0005] An effective means of immunization against VEEV is needed. In particular, a means of 11 prophylaxis against VEEV and/or a therapy for VEEV infection is desired.
13 [0006] In one aspect, the present invention provides prophylaxis and post-exposure therapy 14 against VEEV infection.
[0007] In one aspect, the invention provides a humanized rAb comprising a human 16 immunoglobulin (Ig) framework and having grafted thereon complementarity determining regions 17 (CDRs) from the murine mAb 1A4A1. In a preferred embodiment, the human Ig framework is 18 obtained from IgG1.
19 [0008] In another aspect, the invention provides a humanized rAb having specificity to the E2 envelope protein of VEEV. More specifically, the rAb has specificity to the E2c epitope of the E2 21 protein.
22 [0009] In another aspect, the invention provides a humanized rAb wherein the complementarity 23 determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) have the 24 following amino acid sequences:
CDR1: SEQ ID NO: 1 26 CDR2: SEQ ID NO: 2 27 CDR3: SEQ ID NO: 3.
28 [0010] In another aspect, the invention provides a humanized rAb wherein the complementarity 29 determining regions CDR1, CDR2 and CDR3 of the light chain variable region (VL) have the following amino acid sequences:
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 CDR1: SEQ ID NO: 4 2 CDR2: SEQ ID NO: 5 3 CDR3: SEQ ID NO: 6.
4 [0011] In a further aspect, the invention provides a humanized rAb having a VH comprising the amino acid sequence of SEQ ID NO: 7.
13 [0006] In one aspect, the present invention provides prophylaxis and post-exposure therapy 14 against VEEV infection.
[0007] In one aspect, the invention provides a humanized rAb comprising a human 16 immunoglobulin (Ig) framework and having grafted thereon complementarity determining regions 17 (CDRs) from the murine mAb 1A4A1. In a preferred embodiment, the human Ig framework is 18 obtained from IgG1.
19 [0008] In another aspect, the invention provides a humanized rAb having specificity to the E2 envelope protein of VEEV. More specifically, the rAb has specificity to the E2c epitope of the E2 21 protein.
22 [0009] In another aspect, the invention provides a humanized rAb wherein the complementarity 23 determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) have the 24 following amino acid sequences:
CDR1: SEQ ID NO: 1 26 CDR2: SEQ ID NO: 2 27 CDR3: SEQ ID NO: 3.
28 [0010] In another aspect, the invention provides a humanized rAb wherein the complementarity 29 determining regions CDR1, CDR2 and CDR3 of the light chain variable region (VL) have the following amino acid sequences:
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 CDR1: SEQ ID NO: 4 2 CDR2: SEQ ID NO: 5 3 CDR3: SEQ ID NO: 6.
4 [0011] In a further aspect, the invention provides a humanized rAb having a VH comprising the amino acid sequence of SEQ ID NO: 7.
6 [0012] In a further aspect, the invention provides a humanized rAb having a VL comprising the 7 amino acid sequence of SEQ ID NO: 8.
8 [0013] In another aspect, the invention provides a DNA sequence which encodes a polypeptide 9 corresponding to a CDR grafted VH having the amino acid sequence according to SEQ ID NO: 7.
[0014] In another aspect, the invention provides a DNA sequence which encodes a polypeptide 11 corresponding to a CDR grafted VL having the amino acid sequence according to SEQ ID NO: 8.
12 [0015] In a further aspect, the invention provides a DNA construct having a nucleic acid 13 sequence according to SEQ ID NO:11 or SEQ ID NO:13.
14 [0016] In another aspect, the invention provides an expressed protein comprising a humanized rAb having an amino acid sequence according to SEQ ID NO: 12 or SEQ ID NO: 14.
16 [0017] The invention provides vectors containing such DNA
sequences and host cells 17 transformed thereby.
18 [0018] In other aspects, the invention provides methods and uses for treatment and/or 19 prophylaxis against VEEV infection comprising the antibodies described herein. The invention also provides pharmaceutical preparations for such treatment or prophylaxis.
22 [0019] These and other features of the invention will become more apparent in the following 23 detailed description in which reference is made to the appended drawings wherein:
24 [0020] Figure 1 is a representation of the external structure of the VEEV.
[0021] Figures 2a to 2d schematically illustrate murine, human, chimeric and humanized Abs, 26 respectively.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0022] Figures 3a to 3c schematically illustrate the humanization of the murine Ab variable 2 region.
3 [0023] Figure 4 schematically illustrates the cloning of the murine Ab VH and VL.
4 [0024] Figure 5 schematically illustrates the humanization of the Ab VH and shows its amino acid sequence (SEQ ID NO: 7).
6 [0025] Figure 6 schematically illustrates the humanization of the Ab VL and shows its amino 7 acid sequence (SEQ ID NO: 8).
8 [0026] Figure 7 schematically illustrates the design of a full Hu1A4A1IgG1 rAb gene in a single 9 open reading frame with two versions, Hu1A4A1IgG1-furin (SEQ ID NO: 11) and Hu1A4A1IgG1-2A
(SEQ ID NO: 13).
11 [0027] Figure 8 schematically illustrates the cloning of the Hu1A4A1IgG1-furin and 12 Hu1A4A1IgG1-2A genes into an adenoviral vector respectively.
13 [0028] Figure 9 schematically illustrates expression and purification of the Hu1A4A1IgG1-furin 14 and Hu1A4A1IgG1-2A rAbs.
[0029] Figures 10 and 11 illustrate the results from the SDS-PAGE
separation of the produced 16 Hu1A4A1IgG1-furin rAb.
17 [0030] Figure 12 illustrates the results from the sodium dodecyl sulfate-polyacrylamide gel 18 electrophoresis (SDS-PAGE) separation of the produced Hu1A4A1IgG1-2A
rAb.
19 [0031] Figure 13 illustrates the results of the enzyme-linked immunosorbent assays (ELISA) for the reactivity of the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A rAbs.
21 [0032] Figure 14 schematically illustrates Hu1A4A1IgG1-2A was cleaved between the heavy 22 and light chains as expected, whereas Hu1A4A1IgG1-furin was not cleaved.
23 [0033] Figure 15 schematically illustrates the neutralization assay used in assessing the 24 neutralizing activity of the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A rAbs against VEEV.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 2 [0034] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein 3 refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody 4 composition displays a single binding specificity and affinity for a particular epitope.
[0035] The term "recombinant antibody", as used herein, refers to antibodies that are prepared, 6 expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an 7 animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes or a 8 hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the 9 antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means 11 that involve splicing of immunoglobulin gene sequences to other DNA
sequences.
12 [0036] As used herein the terms "expression vector" or "cloning vector" include vectors which 13 are designed to provide transcription of the nucleic acid sequence. The transcribed nucleic acid may 14 be translated into a polypeptide or protein product. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One 16 type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which 17 additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional 18 DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous 19 replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors are integrated into the genome of a host cell upon introduction 21 into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors 22 are capable of directing the expression of genes to which they are operatively-linked. Such vectors 23 are referred to herein as "expression vectors" or "cloning vectors". In general, expression vectors of 24 utility in recombinant DNA techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form 26 of vector. However, the invention is intended to include such other forms of expression vectors, 27 such as viral vectors or plant transformation vectors, binary or otherwise, which serve equivalent 28 functions.
29 [0037] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the 31 recombinant expression vectors include one or more regulatory sequences, selected on the basis of 32 the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 expressed. Within a recombinant expression vector, "operatively-linked"
or "operably-linked" is 2 intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a 3 manner that allows for expression of the nucleotide sequence (e.g., in an in vitro 4 transcription/translation system or in a host cell when the vector is introduced into the host cell).
[0038] The term "regulatory sequence" is intended to include promoters, enhancers and other 6 expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well 7 known in the art such as, for example, in Goeddel, Gene Expression Technology: Methods in 8 Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those 9 that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific 11 regulatory sequences) or inducible promoters (e.g., induced in response to abiotic factors such as 12 environmental conditions, heat, drought, nutrient status or physiological status of the cell or biotic 13 such as pathogen responsive). Examples of suitable promoters include for example constitutive 14 promoters, ABA inducible promoters, tissue specific promoters and abiotic or biotic inducible promoters. It will be appreciated by those skilled in the art that the design of the expression vector 16 can depend on such factors as the choice of the host cell to be transformed, the level of expression 17 of protein desired as well as timing and location of expression, etc.
The expression vectors of the 18 invention can be introduced into host cells to thereby produce proteins or peptides, including fusion 19 proteins or peptides, encoded by nucleic acids as described herein.
[0039] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is 21 understood that such terms refer not only to the particular subject cell but also to the progeny or 22 potential progeny of such a cell. Because certain modifications may occur in succeeding 23 generations due to either mutation or environmental influences, such progeny may not, in fact, be 24 identical to the parent cell, but are still included within the scope of the term as used herein.
[0040] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional 26 transformation or transfection techniques. As used herein, the terms "transformation" and 27 "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign 28 nucleic acid (e.g., DNA) into a host cell.
29 [0041] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention encoded in an open reading frame 31 of a polynucleotide of the invention. Accordingly, the invention further provides methods for 32 producing a polypeptide using the host cells of the invention. In one embodiment, the method 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 comprises culturing the host cell of invention (into which a recombinant expression vector encoding 2 a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is 3 produced. In another embodiment, the method further comprises isolating the polypeptide from the 4 medium or the host cell.
[0042] Figure 1 illustrates the external structure of the VEEV. As shown, the virus 10 includes a 6 nucleocapsid 12 enveloping the viral RNA genome. The envelope comprises glycoproteins El and 7 E2, arranged in the form of heterodimers 14. Protein E2, which is responsible for viral attachment to 8 the host cell, contains neutralizing epitopes.
9 [0043] As has been described in the prior art, the murine mAb 1A4A1 has been found to be specific to the VEEV E2 envelope protein and, further, has been found to have a strong neutralizing 11 function against VEEV. The murine mAb, however, causes a sometimes fatal allergenic reaction in 12 humans, resulting in the formation of human anti-mouse Abs (HAMA). It is for this reason that the 13 present inventors have sought to humanize the 1A4A1 mAb so as to provide an effective agent to 14 counter VEEV infection in humans. In the course of this research, humanized recombinant anti-VEEV monoclonal antibodies have recently been designed and developed [18].
Such recombinant 16 antibodies are described further herein and are the subject of Canadian patent application number 17 2,607,771 and U.S. patent application number 11/933,948, both filed on November 1, 2007. The 18 present invention provides methods and uses involving such antibodies for the prevention 19 (prophylaxis) and treatment against VEEV infection in mammals.
[0044] In vivo efficacy studies in mice have demonstrated that treatment with murine mAb 21 1A4A1 leads to protection of animals from a lethal peripheral challenge with virulent VEEV. Thus, 22 the present invention builds upon these findings by providing a humanized mAb 1A4A1 to reduce the 23 foreignness of murine mAb in humans. For doing this, the majority of the non-human protein 24 sequence (in one embodiment, more than 90%) of mAb 1A4A1 is replaced with a human Ab sequence and the resultant whole humanized mAb gene is then synthesized and cloned to an 26 expression vector such as an adenoviral vector. The recombinant adenoviral vector can be 27 delivered as a therapeutic agent for prophylaxis or treatment of VEEV
infection in humans. One 28 advantage of this method is that the vector can express the humanized Ab in the human body for a 29 long period of time. The humanized Ab can also be produced in cell culture and delivered directly as a therapeutic.
31 [0045] The humanization of the present anti-VEEV mAb 1A4A1 has not been done previously 32 and particularly not for the prophylaxis or treatment of VEEV infection.
The present invention 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 provides in one embodiment a humanized Ab, referred to herein as Hu1A4A1IgG1, that retains the 2 VEEV-binding specificity and neutralizing activity of murine 1A4A1 while not eliciting a HAMA
3 response. As described further below, the humanized Ab comprises an Ig framework of human 4 IgG1 and CDRs obtained from murine mAb 1A4A1. The rAb of the present invention is specific to an epitope of the E2 envelope glycoprotein of VEEV and, more specifically, to the E2c epitope 6 thereon.
7 [0046] The construction of the humanized Ab of the invention is schematically illustrated in 8 Figures 2a to 2d. Figure 2a illustrates schematically the structure of a murine Ab 16 containing 9 murine CDRs 18 on the respective variable regions. Figure 2b shows a human Ab 20 containing human CDRs 22. As shown in Figure 2c, a chimeric Ab 26 would comprise the murine variable 11 regions 24, containing the murine CDRs 18, joined to the constant regions of the human Ab. On the 12 other hand, Figure 2d illustrates a humanized Ab 28 according to an embodiment of the invention, 13 wherein only the murine CDRs 18 are grafted to the variable regions of the human Ab 20.
14 [0047] The substitution of the murine CDRs into the human Ig framework is illustrated also in Figures 3a to 3c. As shown, the humanized Ab variable region comprises the grafted CDRs, 18, 16 from the murine Ab.
17 [0048] The protein sequences of the rAbs of the invention include linker sequences. The 18 expressed rAbs of the invention have amino acid sequences as shown in SEQ ID NO:12 and SEQ
19 ID NO:14. The nucleic acid constructs used in transforming cells to express the above rAbs are shown in SEQ ID NO:11 and SEQ ID NO:13.
21 [0049] As illustrated further below, the humanized recombinant antibodies of the present 22 invention have been found to be effective as both a prophylaxis and a treatment against VEEV
23 infection.
24 [0050] Examples [0051] The following examples are provided to illustrate embodiments of the present invention.
26 The examples are not intended to limit the scope of the invention in any way.
27 [0052] Example 1: Construction of HulA4A11pG1 and in vitro studies 28 [0053] In the study described below, murine mAb 1A4A1 CDRs of VH, VL were grafted onto the 29 frameworks of germline variable and joining (V, J) gene segments of human Ig heavy and light chains, respectively, which were chosen based on the CDR similarities between human Igs and 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 murine mAb 1A4A1. Furthermore, the humanized VH and VL were, respectively, grafted onto 2 human gamma 1 heavy chain constant regions (CHs) and kappa 1 light chain constant region (CL) 3 to assemble the whole humanized Ab gene. The resultant whole humanized mAb gene was 4 synthesized and cloned to an adenoviral vector. After the humanized Ab was expressed in HEK 293 cells and purified with protein L column, the Ab was demonstrated to retain antigen-binding 6 specificity and neutralizing activity.
7 [0054] Materials and Methods 8 [0055] Humanization of murine mAb 1A4A1 9 [0056] Murine mAb 1A4A1 was provided by Dr. J.T. Roehrig (Division of Vector-borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Colins, CO, USA).
The VH and VL of 11 mAb 1A4A1 were cloned in a single chain variable fragment (ScFv) format, mA116 previously [7], 12 which showed to retain the same binding specificity as mAb 1A4A1 [11].
The humanization of VH
13 and VL of murine mAb 1A4A1 was done by Absalus Inc. (Mountain View, CA, USA). Briefly, in order 14 to select human VH and VL frameworks 1-3, the VH and VL amino acid sequences of murine 1A4A1 were separately subjected to IgBlast and IMGT searches against the entire human Ig germline V
16 gene segments and then human heavy and light chain germline V gene segments were selected 17 based on their highest CDR 1 and 2 similarities with those of murine 1A4A1 VH and VL without 18 consideration of framework similarity. Both human VH and VL framework 4 were selected, 19 respectively, from human heavy and light chain J gene segments based on the highest similarities between human J gene segments and murine 1A4A1 VH and VL CDR3. Finally, CDRs of murine 21 1A4A1 VH and VL were, respectively, grafted onto the frameworks of selected germline V and J
22 gene segments of human Ab heavy and light chains, resulting in humanized 1A4A1 (Hu1A4A1).
23 Furthermore, the Hu1A4A1 VH and VL were, respectively, grafted onto human gamma 1 heavy 24 chain CHs and kappa 1 light chain CL to assemble the whole humanized Ab gene, resulting in humanized 1A4A1IgG1 (Hu1A4A1IgG1). This process is illustrated in Figures 3 to 6.
26 [0057] Construction, expression and purification of Hu1A4A1IpG1 (Hu1A4A1loG1-furin and 27 Hu1A4A1IoG1-2A) 28 [0058] The Hu1A4A1IgG1 DNA sequence (-2 kb) is schematically illustrated in Figure 7. The 29 nucleic acid sequence of the Hu1A4A1IgG1-furin rAb is provided in SEQ ID
NO:11 and the nucleic acid sequence of the Hu1A4A1IgG1-2A rAb is provided in SEQ ID NO:13.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0059] The Hu1A4A1IgG1 DNA sequences were synthesized as follows.
As shown in Figure 7, 2 a light chain leader sequence was provided upstream from the light chain, followed by a furin or 2A
3 linker (discussed further below) before the heavy chain. The whole DNA
sequence flanked by Kpn I
4 and Hind III was synthesized by GenScript Corporation (Scotch Plaines, NJ, USA) and cloned into pUC57 vector, resulting in pUC57-Hu1A4A1IgG1-furin or pUC57-Hu1A4A1IgG1-2A.
6 [0060] Recombinant adenovirus vectors expressing either Hu1A4A1IgG1-furin or 7 Hu1A4A1IgG1-2A were constructed using AdEasy TM system (Qbiogene, Carlsbad, CA, USA) 8 according to the manufacturer's protocol. Briefly, the Kpn I¨Hind III
fragment of Hu1A4A1IgG1-furin 9 or Hu1A4A1IgG1-2A was ligated to a Kpn I¨Hind III-digested pShuttle-CMV
vector. The resulting pShuttle construct was co-transformed with the pAdEasy-1 vector into Escherichia coli BJ5183 cells 11 to produce recombinant adenoviral genomic constructs for Hu1A4A1IgG1-furin or Hu1A4AlIgG1-2A
12 proteins. The recombinant adenoviral constructs, pAd-Hu1A4A1IgG1-furin and pAd-Hu1A4A1IgG1-13 2A were linearized with Pac I and transfected into HEK 293 cells (American Type Culture Collection, 14 Manassas, VA, USA) cultured in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum (FBS) for amplification and then the amplified adenovirus was purified by a 16 chromatographic method. This procedure is illustrated in Figure 8.
17 [0061] As illustrated in Figure 9, the expression of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was 18 achieved by first infecting HEK 293 cells with the recombinant adenovirus pAd-Hu1A4A1IgG1-furin 19 or pAd-Hu1A4A1IgG1-2A at a multiplicity of infection (M01) of 1. The infected cells were cultured for one week and the culture supernatant was harvested. The expressed Hu1A4A1IgG1-furin or 21 Hu1A4A1IgG1-2A was purified using protein L agarose gel from Pierce (Brockville, Ont., Canada).
22 Briefly, culture supernatant was dialyzed against phosphate buffer saline (PBS) (Sigma¨Aldrich, 23 Oakville, Ont., Canada) for 12h and then concentrated using PEG
(Sigma¨Aldrich) to less than 50 24 ml. The concentrated sample was incubated with 2m1 protein L agarose gel at 4 C for 1 h. The gel and supernatant mixture was then loaded to an empty column, which was subsequently washed with 26 binding buffer. Bound Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was eluted with elution buffer. The 27 eluted Ab was further desalted using an excellulose column (Pierce) and then concentrated by a 28 Centracon TM YM-30 (Millipore Corp., Bedford, MA, USA).
29 [0062] The amino acid sequence of the expressed Hu1A4A1IgG1-furin is shown in SEQ ID
NO:12 and the amino acid sequence of the expressed Hu1A4A1IgG1-2A is shown in SEQ ID NO:14.
31 [0063] Cells that were transformed to express the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A
32 humanized antibodies have been deposited at the International Depositary Authority of Canada 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 (IDAC) (National Microbiology Laboratory, Winnipeg, Manitoba, Canada) under accession numbers 2 141107-01 and 141107-02, respectively.
3 [0064] SDS-PAGE
4 [0065] Abs were separated by 10% SDS-PAGE gels using a Mini-PROTEANTm II apparatus (Bio-Rad Laboratories, Mississauga, Ont., Canada). The bands were visualized by SimplyBlue TM
6 safestain staining (Invitrogen, Burlington, Ont., Canada). The molecular weights of the samples 7 were estimated by comparison to the relative mobility values of standards of known molecular 8 weights. The SDS-PAGE analyses of the purified Hu1A4A1IgG1-furin are illustrated in Figures 10 9 and 11. Figure 12 illustrates the SDS-PAGE analysis of the purified Hu1A4A1IgG1-2A. As shown, lanes 1 and 3 correspond to purified Hu1A4A1IgG1 and control human IgG1 in a non-reducing 11 condition and lanes 2 and 4 correspond to purified Hu1A4A1IgG1 and control human IgG1 in a 12 reducing condition.
13 [0066] ELISA
14 [0067] The reactivity of purified Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to VEEV E2 antigen was determined by ELISA. Nunc Maxisorp7m flat bottomed 96-well plates (Canadian Life 16 Technologies, Burlington, Ont., Canada) were coated overnight at 4 C
with recombinant VEEV E2 17 antigen at a concentration of 10pg/m1 in carbonate bicarbonate buffer, pH 9.6. The plates were 18 washed five times with PBS containing 0.1% TweenTm-20 (PBST) and then blocked in 2% bovine 19 serum albumin for 2h at room temperature. After five washes with PBST, the plates were incubated for 2h at room temperature with various concentrations of Hu1A4A1IgG1-furin, Hu1A4A1IgG1-2A or 21 1A4A1 Abs diluted in PBST. Following five washes with PBST, the plates were incubated for 2h at 22 room temperature with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG fragment 23 crystallizable portion or HRP-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch 24 Laboratories Inc., West Grove, PA, USA) diluted 1:5000 in PBST. Finally, the plates were washed five times with PBST and developed for 10 min at room temperature with a 3,3',5,5'-26 tetramethylbenzidine substrate (Kirkegaard and Perry Laboratories). The reactions were read at an 27 absorbance of 650 nm by a microplate autoreader (Molecular Devices, Sunnyvale, CA, USA). The 28 results of the ELISA Hu1A4A1IgG1-antigen binding assay are illustrated in Figure 13.
29 [0068] Neutralization assay in vitro [0069] Neutralizing activity of each of Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A against VEEV
31 (strain TC-83) was analyzed by a plaque reduction assay. Briefly, each Ab was serially two-fold 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 diluted (1:32, 1:64, 1:128, etc.) and mixed with an equal volume containing 50 plaque-forming units 2 of virus per 100 pl. Afterwards, the mixtures were incubated for 1 h at room temperature, 200 pl of 3 the mixture was inoculated in duplicate into wells of six-well plates containing confluent Vero cell 4 monolayers and incubated at 37 C for 1 h. At the end of the incubation, the virus/Ab mixtures were removed from the wells before the wells were overlaid by tragacanth gum and then incubated for 2 6 days. The wells were stained with 0.3% crystal violet and plaques were counted. Neutralization titre 7 was expressed as the highest Ab dilution that inhibited 50% of virus plaques. This procedure is 8 illustrated in Figure 15.
9 [0070] Results and Discussion [0071] Different approaches have been developed to humanize murine Abs in order to reduce 11 the antigenicity of murine Abs in humans [9,10]. One widely used approach is CDR-grafting, which 12 involves the grafting of all murine CDRs onto a human Ab frameworks. The human Ab frameworks 13 are chosen based on their similarities to the frameworks of the murine Ab to be humanized. The 14 CDR-grafting approach has been proven successful in some cases. However, in many more instances, this humanization process could result in CDR conformation changes, which affect the 16 antigen-binding affinity. To restore the affinity, additional work for back-mutation of several murine 17 framework amino acids, which are deemed to be critical for CDR loop conformation, have to be 18 done.
19 [0072] Recently, Hwang et al. [12] employed an approach which consisted of grafting CDRs onto human germline Ab frameworks based on the CDR sequence similarities between the murine 21 and human Abs while basically ignoring the frameworks. Because the selection of the human 22 frameworks is driven by the sequence of the CDRs, this strategy minimizes the differences between 23 the murine and human CDRs. This approach has the potential to generate humanized Abs that 24 retain their binding affinity to their cognate antigen. Further, since all residues in frameworks are from human Ab germline sequences, the potential immunogenicity of non-human Abs is highly 26 reduced.
27 [0073] Using the above approach, and as disclosed herein, the present inventors humanized an 28 anti-VEEV murine mAb 1A4A1. The amino acid sequences of VH and VL from murine 1A4A1 were 29 first aligned with human Ig germline V and J genes. As shown in Figure 5, the human heavy chain V
gene segment H5-51 and J gene segment JH4 were selected to provide the frameworks for the 31 murine 1A4A1 VH. Similarly, as shown in Figure 6, for the murine 1A4A1 VL, the human light chain 32 V gene segment L15 and J gene segment Jk3 were selected.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0074] The identities of the CDR1 and CDR2 amino acid sequences between murine 1A4A1 VH
2 and the human H5-51 gene segment were 20% and 47%, respectively, while the identity of the 3 CDR3 between murine 1A4A1 VH and the JH4 gene segment was 33%. For the light chain, the 4 identities of the CDR1 and CDR2 between murine 1A4A1 VL and the human L15 gene segment were 27% and 14%, respectively, while the identity of the CDR3 between murine 1A4A1 VL and 6 human Jk3 gene segment was 22%. The CDRs of murine 1A4A1 VH were then grafted onto the 7 frameworks of selected human Ig germline H5-51 and JH4 gene segments, while the CDRs of 8 murine 1A4A1 VL were grafted onto human L15 and Jk3 gene segments. The hu1A4A1 VH was 9 further grafted onto the human gamma 1 heavy chain CHs to form a complete heavy chain, while the VL was grafted onto the human kappa 1 light chain CL to form a whole humanized light chain. This 11 procedure is schematically illustrated in Figures 5 and 6 with the end structure being illustrated in 12 Figure 7.
13 [0075] As shown in Figure 5, the murine 1A4A1 VH CDRs grafted onto the human framework 14 comprised the following amino acid sequences:
VH CDR1: DYHVH (SEQ ID NO: 1) 16 VH CDR2: MTYPGFDNTNYSETFKG (SEQ ID NO: 2) 17 VH CDR3: GVGLDY (SEQ ID NO: 3) 18 [0076] As shown in Figure 6, the murine 1A4A1 VL CDRs grafted onto the human framework 19 comprised the following amino acid sequences:
VL CDR1: KASQDVDTAVG (SEQ ID NO: 4) 21 VL CDR2: WSSTRHT (SEQ ID NO: 5) 22 VL CDR3: HQYSSYPFT (SEQ ID NO: 6) 23 [0077] As shown in Figure 5, the VH of the humanized Ab according to the present invention 24 comprises the following amino acid sequence:
Hu-VH:
27 KGQVTISADKSISTAYLQWSSLKASDTAMYYCARGVGLDYWGQGTLVTVSS (SEQ ID NO: 7).
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0078] Thus, as shown in Figure 6, the VL of the humanized Ab according to the present 2 invention comprises the following amino acid sequence:
3 Hu-VL:
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPFTFGPGTKVDIKR (SEQ ID NO: 8).
6 [0079] In order to express heavy and light chains in a monocistronic construct, a six-residue 7 peptide, RGRKRR (SEQ ID NO: 9) containing the recognition site for the protease furin, designated 8 as "furin linker", or a twenty-four-residue peptide of the foot-and-mouth-disease virus (FMDV)-9 derived 2A self-processing sequence, APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:
[0014] In another aspect, the invention provides a DNA sequence which encodes a polypeptide 11 corresponding to a CDR grafted VL having the amino acid sequence according to SEQ ID NO: 8.
12 [0015] In a further aspect, the invention provides a DNA construct having a nucleic acid 13 sequence according to SEQ ID NO:11 or SEQ ID NO:13.
14 [0016] In another aspect, the invention provides an expressed protein comprising a humanized rAb having an amino acid sequence according to SEQ ID NO: 12 or SEQ ID NO: 14.
16 [0017] The invention provides vectors containing such DNA
sequences and host cells 17 transformed thereby.
18 [0018] In other aspects, the invention provides methods and uses for treatment and/or 19 prophylaxis against VEEV infection comprising the antibodies described herein. The invention also provides pharmaceutical preparations for such treatment or prophylaxis.
22 [0019] These and other features of the invention will become more apparent in the following 23 detailed description in which reference is made to the appended drawings wherein:
24 [0020] Figure 1 is a representation of the external structure of the VEEV.
[0021] Figures 2a to 2d schematically illustrate murine, human, chimeric and humanized Abs, 26 respectively.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0022] Figures 3a to 3c schematically illustrate the humanization of the murine Ab variable 2 region.
3 [0023] Figure 4 schematically illustrates the cloning of the murine Ab VH and VL.
4 [0024] Figure 5 schematically illustrates the humanization of the Ab VH and shows its amino acid sequence (SEQ ID NO: 7).
6 [0025] Figure 6 schematically illustrates the humanization of the Ab VL and shows its amino 7 acid sequence (SEQ ID NO: 8).
8 [0026] Figure 7 schematically illustrates the design of a full Hu1A4A1IgG1 rAb gene in a single 9 open reading frame with two versions, Hu1A4A1IgG1-furin (SEQ ID NO: 11) and Hu1A4A1IgG1-2A
(SEQ ID NO: 13).
11 [0027] Figure 8 schematically illustrates the cloning of the Hu1A4A1IgG1-furin and 12 Hu1A4A1IgG1-2A genes into an adenoviral vector respectively.
13 [0028] Figure 9 schematically illustrates expression and purification of the Hu1A4A1IgG1-furin 14 and Hu1A4A1IgG1-2A rAbs.
[0029] Figures 10 and 11 illustrate the results from the SDS-PAGE
separation of the produced 16 Hu1A4A1IgG1-furin rAb.
17 [0030] Figure 12 illustrates the results from the sodium dodecyl sulfate-polyacrylamide gel 18 electrophoresis (SDS-PAGE) separation of the produced Hu1A4A1IgG1-2A
rAb.
19 [0031] Figure 13 illustrates the results of the enzyme-linked immunosorbent assays (ELISA) for the reactivity of the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A rAbs.
21 [0032] Figure 14 schematically illustrates Hu1A4A1IgG1-2A was cleaved between the heavy 22 and light chains as expected, whereas Hu1A4A1IgG1-furin was not cleaved.
23 [0033] Figure 15 schematically illustrates the neutralization assay used in assessing the 24 neutralizing activity of the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A rAbs against VEEV.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 2 [0034] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein 3 refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody 4 composition displays a single binding specificity and affinity for a particular epitope.
[0035] The term "recombinant antibody", as used herein, refers to antibodies that are prepared, 6 expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an 7 animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes or a 8 hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the 9 antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means 11 that involve splicing of immunoglobulin gene sequences to other DNA
sequences.
12 [0036] As used herein the terms "expression vector" or "cloning vector" include vectors which 13 are designed to provide transcription of the nucleic acid sequence. The transcribed nucleic acid may 14 be translated into a polypeptide or protein product. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One 16 type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which 17 additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional 18 DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous 19 replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors are integrated into the genome of a host cell upon introduction 21 into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors 22 are capable of directing the expression of genes to which they are operatively-linked. Such vectors 23 are referred to herein as "expression vectors" or "cloning vectors". In general, expression vectors of 24 utility in recombinant DNA techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form 26 of vector. However, the invention is intended to include such other forms of expression vectors, 27 such as viral vectors or plant transformation vectors, binary or otherwise, which serve equivalent 28 functions.
29 [0037] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the 31 recombinant expression vectors include one or more regulatory sequences, selected on the basis of 32 the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 expressed. Within a recombinant expression vector, "operatively-linked"
or "operably-linked" is 2 intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a 3 manner that allows for expression of the nucleotide sequence (e.g., in an in vitro 4 transcription/translation system or in a host cell when the vector is introduced into the host cell).
[0038] The term "regulatory sequence" is intended to include promoters, enhancers and other 6 expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well 7 known in the art such as, for example, in Goeddel, Gene Expression Technology: Methods in 8 Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those 9 that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific 11 regulatory sequences) or inducible promoters (e.g., induced in response to abiotic factors such as 12 environmental conditions, heat, drought, nutrient status or physiological status of the cell or biotic 13 such as pathogen responsive). Examples of suitable promoters include for example constitutive 14 promoters, ABA inducible promoters, tissue specific promoters and abiotic or biotic inducible promoters. It will be appreciated by those skilled in the art that the design of the expression vector 16 can depend on such factors as the choice of the host cell to be transformed, the level of expression 17 of protein desired as well as timing and location of expression, etc.
The expression vectors of the 18 invention can be introduced into host cells to thereby produce proteins or peptides, including fusion 19 proteins or peptides, encoded by nucleic acids as described herein.
[0039] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is 21 understood that such terms refer not only to the particular subject cell but also to the progeny or 22 potential progeny of such a cell. Because certain modifications may occur in succeeding 23 generations due to either mutation or environmental influences, such progeny may not, in fact, be 24 identical to the parent cell, but are still included within the scope of the term as used herein.
[0040] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional 26 transformation or transfection techniques. As used herein, the terms "transformation" and 27 "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign 28 nucleic acid (e.g., DNA) into a host cell.
29 [0041] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention encoded in an open reading frame 31 of a polynucleotide of the invention. Accordingly, the invention further provides methods for 32 producing a polypeptide using the host cells of the invention. In one embodiment, the method 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 comprises culturing the host cell of invention (into which a recombinant expression vector encoding 2 a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is 3 produced. In another embodiment, the method further comprises isolating the polypeptide from the 4 medium or the host cell.
[0042] Figure 1 illustrates the external structure of the VEEV. As shown, the virus 10 includes a 6 nucleocapsid 12 enveloping the viral RNA genome. The envelope comprises glycoproteins El and 7 E2, arranged in the form of heterodimers 14. Protein E2, which is responsible for viral attachment to 8 the host cell, contains neutralizing epitopes.
9 [0043] As has been described in the prior art, the murine mAb 1A4A1 has been found to be specific to the VEEV E2 envelope protein and, further, has been found to have a strong neutralizing 11 function against VEEV. The murine mAb, however, causes a sometimes fatal allergenic reaction in 12 humans, resulting in the formation of human anti-mouse Abs (HAMA). It is for this reason that the 13 present inventors have sought to humanize the 1A4A1 mAb so as to provide an effective agent to 14 counter VEEV infection in humans. In the course of this research, humanized recombinant anti-VEEV monoclonal antibodies have recently been designed and developed [18].
Such recombinant 16 antibodies are described further herein and are the subject of Canadian patent application number 17 2,607,771 and U.S. patent application number 11/933,948, both filed on November 1, 2007. The 18 present invention provides methods and uses involving such antibodies for the prevention 19 (prophylaxis) and treatment against VEEV infection in mammals.
[0044] In vivo efficacy studies in mice have demonstrated that treatment with murine mAb 21 1A4A1 leads to protection of animals from a lethal peripheral challenge with virulent VEEV. Thus, 22 the present invention builds upon these findings by providing a humanized mAb 1A4A1 to reduce the 23 foreignness of murine mAb in humans. For doing this, the majority of the non-human protein 24 sequence (in one embodiment, more than 90%) of mAb 1A4A1 is replaced with a human Ab sequence and the resultant whole humanized mAb gene is then synthesized and cloned to an 26 expression vector such as an adenoviral vector. The recombinant adenoviral vector can be 27 delivered as a therapeutic agent for prophylaxis or treatment of VEEV
infection in humans. One 28 advantage of this method is that the vector can express the humanized Ab in the human body for a 29 long period of time. The humanized Ab can also be produced in cell culture and delivered directly as a therapeutic.
31 [0045] The humanization of the present anti-VEEV mAb 1A4A1 has not been done previously 32 and particularly not for the prophylaxis or treatment of VEEV infection.
The present invention 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 provides in one embodiment a humanized Ab, referred to herein as Hu1A4A1IgG1, that retains the 2 VEEV-binding specificity and neutralizing activity of murine 1A4A1 while not eliciting a HAMA
3 response. As described further below, the humanized Ab comprises an Ig framework of human 4 IgG1 and CDRs obtained from murine mAb 1A4A1. The rAb of the present invention is specific to an epitope of the E2 envelope glycoprotein of VEEV and, more specifically, to the E2c epitope 6 thereon.
7 [0046] The construction of the humanized Ab of the invention is schematically illustrated in 8 Figures 2a to 2d. Figure 2a illustrates schematically the structure of a murine Ab 16 containing 9 murine CDRs 18 on the respective variable regions. Figure 2b shows a human Ab 20 containing human CDRs 22. As shown in Figure 2c, a chimeric Ab 26 would comprise the murine variable 11 regions 24, containing the murine CDRs 18, joined to the constant regions of the human Ab. On the 12 other hand, Figure 2d illustrates a humanized Ab 28 according to an embodiment of the invention, 13 wherein only the murine CDRs 18 are grafted to the variable regions of the human Ab 20.
14 [0047] The substitution of the murine CDRs into the human Ig framework is illustrated also in Figures 3a to 3c. As shown, the humanized Ab variable region comprises the grafted CDRs, 18, 16 from the murine Ab.
17 [0048] The protein sequences of the rAbs of the invention include linker sequences. The 18 expressed rAbs of the invention have amino acid sequences as shown in SEQ ID NO:12 and SEQ
19 ID NO:14. The nucleic acid constructs used in transforming cells to express the above rAbs are shown in SEQ ID NO:11 and SEQ ID NO:13.
21 [0049] As illustrated further below, the humanized recombinant antibodies of the present 22 invention have been found to be effective as both a prophylaxis and a treatment against VEEV
23 infection.
24 [0050] Examples [0051] The following examples are provided to illustrate embodiments of the present invention.
26 The examples are not intended to limit the scope of the invention in any way.
27 [0052] Example 1: Construction of HulA4A11pG1 and in vitro studies 28 [0053] In the study described below, murine mAb 1A4A1 CDRs of VH, VL were grafted onto the 29 frameworks of germline variable and joining (V, J) gene segments of human Ig heavy and light chains, respectively, which were chosen based on the CDR similarities between human Igs and 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 murine mAb 1A4A1. Furthermore, the humanized VH and VL were, respectively, grafted onto 2 human gamma 1 heavy chain constant regions (CHs) and kappa 1 light chain constant region (CL) 3 to assemble the whole humanized Ab gene. The resultant whole humanized mAb gene was 4 synthesized and cloned to an adenoviral vector. After the humanized Ab was expressed in HEK 293 cells and purified with protein L column, the Ab was demonstrated to retain antigen-binding 6 specificity and neutralizing activity.
7 [0054] Materials and Methods 8 [0055] Humanization of murine mAb 1A4A1 9 [0056] Murine mAb 1A4A1 was provided by Dr. J.T. Roehrig (Division of Vector-borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Colins, CO, USA).
The VH and VL of 11 mAb 1A4A1 were cloned in a single chain variable fragment (ScFv) format, mA116 previously [7], 12 which showed to retain the same binding specificity as mAb 1A4A1 [11].
The humanization of VH
13 and VL of murine mAb 1A4A1 was done by Absalus Inc. (Mountain View, CA, USA). Briefly, in order 14 to select human VH and VL frameworks 1-3, the VH and VL amino acid sequences of murine 1A4A1 were separately subjected to IgBlast and IMGT searches against the entire human Ig germline V
16 gene segments and then human heavy and light chain germline V gene segments were selected 17 based on their highest CDR 1 and 2 similarities with those of murine 1A4A1 VH and VL without 18 consideration of framework similarity. Both human VH and VL framework 4 were selected, 19 respectively, from human heavy and light chain J gene segments based on the highest similarities between human J gene segments and murine 1A4A1 VH and VL CDR3. Finally, CDRs of murine 21 1A4A1 VH and VL were, respectively, grafted onto the frameworks of selected germline V and J
22 gene segments of human Ab heavy and light chains, resulting in humanized 1A4A1 (Hu1A4A1).
23 Furthermore, the Hu1A4A1 VH and VL were, respectively, grafted onto human gamma 1 heavy 24 chain CHs and kappa 1 light chain CL to assemble the whole humanized Ab gene, resulting in humanized 1A4A1IgG1 (Hu1A4A1IgG1). This process is illustrated in Figures 3 to 6.
26 [0057] Construction, expression and purification of Hu1A4A1IpG1 (Hu1A4A1loG1-furin and 27 Hu1A4A1IoG1-2A) 28 [0058] The Hu1A4A1IgG1 DNA sequence (-2 kb) is schematically illustrated in Figure 7. The 29 nucleic acid sequence of the Hu1A4A1IgG1-furin rAb is provided in SEQ ID
NO:11 and the nucleic acid sequence of the Hu1A4A1IgG1-2A rAb is provided in SEQ ID NO:13.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0059] The Hu1A4A1IgG1 DNA sequences were synthesized as follows.
As shown in Figure 7, 2 a light chain leader sequence was provided upstream from the light chain, followed by a furin or 2A
3 linker (discussed further below) before the heavy chain. The whole DNA
sequence flanked by Kpn I
4 and Hind III was synthesized by GenScript Corporation (Scotch Plaines, NJ, USA) and cloned into pUC57 vector, resulting in pUC57-Hu1A4A1IgG1-furin or pUC57-Hu1A4A1IgG1-2A.
6 [0060] Recombinant adenovirus vectors expressing either Hu1A4A1IgG1-furin or 7 Hu1A4A1IgG1-2A were constructed using AdEasy TM system (Qbiogene, Carlsbad, CA, USA) 8 according to the manufacturer's protocol. Briefly, the Kpn I¨Hind III
fragment of Hu1A4A1IgG1-furin 9 or Hu1A4A1IgG1-2A was ligated to a Kpn I¨Hind III-digested pShuttle-CMV
vector. The resulting pShuttle construct was co-transformed with the pAdEasy-1 vector into Escherichia coli BJ5183 cells 11 to produce recombinant adenoviral genomic constructs for Hu1A4A1IgG1-furin or Hu1A4AlIgG1-2A
12 proteins. The recombinant adenoviral constructs, pAd-Hu1A4A1IgG1-furin and pAd-Hu1A4A1IgG1-13 2A were linearized with Pac I and transfected into HEK 293 cells (American Type Culture Collection, 14 Manassas, VA, USA) cultured in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum (FBS) for amplification and then the amplified adenovirus was purified by a 16 chromatographic method. This procedure is illustrated in Figure 8.
17 [0061] As illustrated in Figure 9, the expression of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was 18 achieved by first infecting HEK 293 cells with the recombinant adenovirus pAd-Hu1A4A1IgG1-furin 19 or pAd-Hu1A4A1IgG1-2A at a multiplicity of infection (M01) of 1. The infected cells were cultured for one week and the culture supernatant was harvested. The expressed Hu1A4A1IgG1-furin or 21 Hu1A4A1IgG1-2A was purified using protein L agarose gel from Pierce (Brockville, Ont., Canada).
22 Briefly, culture supernatant was dialyzed against phosphate buffer saline (PBS) (Sigma¨Aldrich, 23 Oakville, Ont., Canada) for 12h and then concentrated using PEG
(Sigma¨Aldrich) to less than 50 24 ml. The concentrated sample was incubated with 2m1 protein L agarose gel at 4 C for 1 h. The gel and supernatant mixture was then loaded to an empty column, which was subsequently washed with 26 binding buffer. Bound Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was eluted with elution buffer. The 27 eluted Ab was further desalted using an excellulose column (Pierce) and then concentrated by a 28 Centracon TM YM-30 (Millipore Corp., Bedford, MA, USA).
29 [0062] The amino acid sequence of the expressed Hu1A4A1IgG1-furin is shown in SEQ ID
NO:12 and the amino acid sequence of the expressed Hu1A4A1IgG1-2A is shown in SEQ ID NO:14.
31 [0063] Cells that were transformed to express the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A
32 humanized antibodies have been deposited at the International Depositary Authority of Canada 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 (IDAC) (National Microbiology Laboratory, Winnipeg, Manitoba, Canada) under accession numbers 2 141107-01 and 141107-02, respectively.
3 [0064] SDS-PAGE
4 [0065] Abs were separated by 10% SDS-PAGE gels using a Mini-PROTEANTm II apparatus (Bio-Rad Laboratories, Mississauga, Ont., Canada). The bands were visualized by SimplyBlue TM
6 safestain staining (Invitrogen, Burlington, Ont., Canada). The molecular weights of the samples 7 were estimated by comparison to the relative mobility values of standards of known molecular 8 weights. The SDS-PAGE analyses of the purified Hu1A4A1IgG1-furin are illustrated in Figures 10 9 and 11. Figure 12 illustrates the SDS-PAGE analysis of the purified Hu1A4A1IgG1-2A. As shown, lanes 1 and 3 correspond to purified Hu1A4A1IgG1 and control human IgG1 in a non-reducing 11 condition and lanes 2 and 4 correspond to purified Hu1A4A1IgG1 and control human IgG1 in a 12 reducing condition.
13 [0066] ELISA
14 [0067] The reactivity of purified Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to VEEV E2 antigen was determined by ELISA. Nunc Maxisorp7m flat bottomed 96-well plates (Canadian Life 16 Technologies, Burlington, Ont., Canada) were coated overnight at 4 C
with recombinant VEEV E2 17 antigen at a concentration of 10pg/m1 in carbonate bicarbonate buffer, pH 9.6. The plates were 18 washed five times with PBS containing 0.1% TweenTm-20 (PBST) and then blocked in 2% bovine 19 serum albumin for 2h at room temperature. After five washes with PBST, the plates were incubated for 2h at room temperature with various concentrations of Hu1A4A1IgG1-furin, Hu1A4A1IgG1-2A or 21 1A4A1 Abs diluted in PBST. Following five washes with PBST, the plates were incubated for 2h at 22 room temperature with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG fragment 23 crystallizable portion or HRP-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch 24 Laboratories Inc., West Grove, PA, USA) diluted 1:5000 in PBST. Finally, the plates were washed five times with PBST and developed for 10 min at room temperature with a 3,3',5,5'-26 tetramethylbenzidine substrate (Kirkegaard and Perry Laboratories). The reactions were read at an 27 absorbance of 650 nm by a microplate autoreader (Molecular Devices, Sunnyvale, CA, USA). The 28 results of the ELISA Hu1A4A1IgG1-antigen binding assay are illustrated in Figure 13.
29 [0068] Neutralization assay in vitro [0069] Neutralizing activity of each of Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A against VEEV
31 (strain TC-83) was analyzed by a plaque reduction assay. Briefly, each Ab was serially two-fold 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 diluted (1:32, 1:64, 1:128, etc.) and mixed with an equal volume containing 50 plaque-forming units 2 of virus per 100 pl. Afterwards, the mixtures were incubated for 1 h at room temperature, 200 pl of 3 the mixture was inoculated in duplicate into wells of six-well plates containing confluent Vero cell 4 monolayers and incubated at 37 C for 1 h. At the end of the incubation, the virus/Ab mixtures were removed from the wells before the wells were overlaid by tragacanth gum and then incubated for 2 6 days. The wells were stained with 0.3% crystal violet and plaques were counted. Neutralization titre 7 was expressed as the highest Ab dilution that inhibited 50% of virus plaques. This procedure is 8 illustrated in Figure 15.
9 [0070] Results and Discussion [0071] Different approaches have been developed to humanize murine Abs in order to reduce 11 the antigenicity of murine Abs in humans [9,10]. One widely used approach is CDR-grafting, which 12 involves the grafting of all murine CDRs onto a human Ab frameworks. The human Ab frameworks 13 are chosen based on their similarities to the frameworks of the murine Ab to be humanized. The 14 CDR-grafting approach has been proven successful in some cases. However, in many more instances, this humanization process could result in CDR conformation changes, which affect the 16 antigen-binding affinity. To restore the affinity, additional work for back-mutation of several murine 17 framework amino acids, which are deemed to be critical for CDR loop conformation, have to be 18 done.
19 [0072] Recently, Hwang et al. [12] employed an approach which consisted of grafting CDRs onto human germline Ab frameworks based on the CDR sequence similarities between the murine 21 and human Abs while basically ignoring the frameworks. Because the selection of the human 22 frameworks is driven by the sequence of the CDRs, this strategy minimizes the differences between 23 the murine and human CDRs. This approach has the potential to generate humanized Abs that 24 retain their binding affinity to their cognate antigen. Further, since all residues in frameworks are from human Ab germline sequences, the potential immunogenicity of non-human Abs is highly 26 reduced.
27 [0073] Using the above approach, and as disclosed herein, the present inventors humanized an 28 anti-VEEV murine mAb 1A4A1. The amino acid sequences of VH and VL from murine 1A4A1 were 29 first aligned with human Ig germline V and J genes. As shown in Figure 5, the human heavy chain V
gene segment H5-51 and J gene segment JH4 were selected to provide the frameworks for the 31 murine 1A4A1 VH. Similarly, as shown in Figure 6, for the murine 1A4A1 VL, the human light chain 32 V gene segment L15 and J gene segment Jk3 were selected.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0074] The identities of the CDR1 and CDR2 amino acid sequences between murine 1A4A1 VH
2 and the human H5-51 gene segment were 20% and 47%, respectively, while the identity of the 3 CDR3 between murine 1A4A1 VH and the JH4 gene segment was 33%. For the light chain, the 4 identities of the CDR1 and CDR2 between murine 1A4A1 VL and the human L15 gene segment were 27% and 14%, respectively, while the identity of the CDR3 between murine 1A4A1 VL and 6 human Jk3 gene segment was 22%. The CDRs of murine 1A4A1 VH were then grafted onto the 7 frameworks of selected human Ig germline H5-51 and JH4 gene segments, while the CDRs of 8 murine 1A4A1 VL were grafted onto human L15 and Jk3 gene segments. The hu1A4A1 VH was 9 further grafted onto the human gamma 1 heavy chain CHs to form a complete heavy chain, while the VL was grafted onto the human kappa 1 light chain CL to form a whole humanized light chain. This 11 procedure is schematically illustrated in Figures 5 and 6 with the end structure being illustrated in 12 Figure 7.
13 [0075] As shown in Figure 5, the murine 1A4A1 VH CDRs grafted onto the human framework 14 comprised the following amino acid sequences:
VH CDR1: DYHVH (SEQ ID NO: 1) 16 VH CDR2: MTYPGFDNTNYSETFKG (SEQ ID NO: 2) 17 VH CDR3: GVGLDY (SEQ ID NO: 3) 18 [0076] As shown in Figure 6, the murine 1A4A1 VL CDRs grafted onto the human framework 19 comprised the following amino acid sequences:
VL CDR1: KASQDVDTAVG (SEQ ID NO: 4) 21 VL CDR2: WSSTRHT (SEQ ID NO: 5) 22 VL CDR3: HQYSSYPFT (SEQ ID NO: 6) 23 [0077] As shown in Figure 5, the VH of the humanized Ab according to the present invention 24 comprises the following amino acid sequence:
Hu-VH:
27 KGQVTISADKSISTAYLQWSSLKASDTAMYYCARGVGLDYWGQGTLVTVSS (SEQ ID NO: 7).
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0078] Thus, as shown in Figure 6, the VL of the humanized Ab according to the present 2 invention comprises the following amino acid sequence:
3 Hu-VL:
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPFTFGPGTKVDIKR (SEQ ID NO: 8).
6 [0079] In order to express heavy and light chains in a monocistronic construct, a six-residue 7 peptide, RGRKRR (SEQ ID NO: 9) containing the recognition site for the protease furin, designated 8 as "furin linker", or a twenty-four-residue peptide of the foot-and-mouth-disease virus (FMDV)-9 derived 2A self-processing sequence, APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:
10), designated as "2A linker", was incorporated between the two chains. The location of the furin or 2A
11 linker within the nucleic acid constructs of the Abs is illustrated in Figure 7. Furin is a ubiquitous 12 subtilisin-like proprotein convertase, which is the major processing enzyme of the secretory pathway 13 [13]. The furin minimal cleavage site is R¨X¨X¨R; however, the enzyme prefers the site R¨X-14 (K/R)¨R. An additional R at the P6 position appears to enhance cleavage.
The FMDV-derived 2A
linker is able to cleave at its own C terminus between the last two residues through an enzyme-16 independent but undefined mechanism, probably by ribosomal skip, during protein translation. To 17 get the expressed Ab to be secreted to culture media, a leader sequence was added upstream to 18 the Ab gene. Figure 7 illustrates the synthesized DNA sequence, of approximately 2 kb, including 19 the human Ab kappa light chain L15 leader sequence, the humanized light chain (VL + CL), the furin or 2A linker, and the humanized heavy chain (VH + CHI + CH2 + CH3). This sequence was then 21 cloned into an adenoviral vector. The unique restriction sites, as also shown in Figure 7, flanking the 22 V regions, which allow for efficient V region replacement and at the heavy chain V¨C region junction 23 for generation of fragment antigen-binding portion of Ab (Fab), were also designed.
24 [0080] Protein G and A columns are widely used for a quick purification for Abs because of protein G and A binding to the Fc portion of lg. However, protein G and A
cannot only bind to 26 human lg, but also bind to bovine lg, therefore they cannot be used for purification of Hu1A4A1IgG1-27 furin or Hu1A4A1IgG1-2A in our study since pAd-Hu1A4A1IgG1-furin or pAd-Hu1A4A1IgG1-2A-28 infected HEK 293 cells were cultured in the medium with 5% FBS
containing a high percentage of 29 bovine lg. Unlike protein G and A, protein L binds lg through interactions with the light chains.
Protein L only binds to lg containing light chains of type kappa 1, 3 and 4 in human and kappa 1 in 31 mouse. Most importantly, protein L does not bind to bovine lg. Since our humanized Ab has human 32 kappa 1 chain, we chose a protein L column to purify Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 eliminate co-purification of bovine lg. In this way, the purity of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2 2A was relatively high in SDS-PAGE as shown in Figures 10, 11 and 12.
3 [0081] When the purified product was subjected to 10% SDS-PAGE, Hu1A4A1IgG1-furin and 4 Hu1A4A1IgG1-2A showed up in a different way. As illustrated in Figure 12, Hu1A4A1IgG1-2A
showed the same patterns as a control human IgG1, one band of ¨150kDa in non-reducing condition 6 (intact disulfide bridges) and two bands, 50kDa for heavy chains and 25kDa for light chains (broken 7 disulfide bridges) in reducing condition, indicating that the 2A linker underwent self-processing 8 perfectly. On the other hand, Hu1A4A1IgG1-furin showed only one clear band of ¨75kDa in 9 reducing condition observed as illustrated in Figures 10 and 11, indicating that the furin linker was not cleaved. However, in another study (data not shown), the same furin linker sequence was 11 cleaved in another Fab construct expressed in a mammalian system. This indicated the 12 conformation of expressed Hu1A4A1IgG1-furin probably rendered the furin linker inaccessible to 13 furin or that the sequence surrounding the furin linker influenced furin cleavage.
14 [0082] The specific binding reactivities of purified Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A to VEEV E2 antigen were examined by ELISA. As illustrated in Figure 13, both versions of the 16 Hu1A4A1IgG1 were found to bind to VEEV E2 in a dose-dependent manner, similar to the binding to 17 VEEV E2 of its parental murine 1A4A1, indicating this non-cleaved Ab was still reactive to VEEV E2 18 antigen in ELISA. Furthermore, both versions were evaluated for their ability to block VEEV infection 19 in Vero cells using a standard plaque-reduction assay. The Hu1A4A1IgG1-fruin showed a neutralizing activity with 50% plaque reduction neutralization titre at 0.78 pg/ml, whereas 21 Hu1A4A1IgG1-2A showed a much higher neutralization titre at 0.1 pg/ml.
22 [0083] From the above results, it is concluded that the murine 1A4A1 Ab was successfully 23 humanized. As illustrated in Figure 14, the expressed and purified Ab of Hu1A4A1IgG1-2A was 24 cleaved between the heavy and light chains as expected; however, Hu1A4A1IgG1-furin was not cleaved. Nevertheless, the present inventors have exhibited that both versions of the Hu1A4A1IgG1 26 retained the antigen binding specificity and virus neutralizing activity. Thus, the Hu1A4A1IgG1-furin 27 or Hu1A4A1IgG1-2A discussed and characterized herein would serve as an effective prophylactic 28 and therapeutic agent against VEEV infection.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0084] Example 2: In vivo study - Protection or pre-exposure prophylaxis of mice from 2 VEEV challenge by passive immunization with Hu1A4A110G1-furin or Hu1A4A1IgG1-2A
3 [0085] Materials and methods 4 [0086] Passive immunization (Pre-exposure prophylaxis) [0087] Balb/c mice aged 6-8 weeks were injected intraperitoneally (i.p) with 50 pg of 6 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in 100 pl PBS, human IgG in 100 pl PBS (positive control) 7 or 100 pl PBS alone (negative control) 24 h prior to VEEV challenge.
8 [0088] VEEV challenge 9 [0089] Each mouse was challenged subcutaneously (s.c.) with 30-50 plaque forming units (pfu) of virulent VEEV (Trinidad donkey, TRD) in 50 pl of Leibovitz L15 maintenance medium (L15MM) 24 11 h after passive immunization. The challenge dose approximated to 100 x 50% lethal dose (LD50).
12 Mice were examined frequently for signs of illness for 14 days, and humane endpoints were used.
13 [0090] Results 14 [0091] Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A clearance in mice [0092] To determine the half-life of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in mouse serum, 16 groups of 4 mice, were injected i.p. with 50 pg, each mouse, of either Hu1A4A1IgG1-furin or 17 Hu1A4A1IgG1-2A, or human anti-VEEV IgG and bled from the vein at increasing time intervals after 18 injection. The quantity of Ab present in serum samples was estimated by immunoassay.
19 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A had a similar half-life as human anti-VEEV IgG, around 10 days.
21 [0093] Protection of mice from VEEV challenge bv passive immunization with Hu1A4A1IgG1-22 furin or HulA4AllaG1 -2A
23 [0094] Groups of 8 mice were injected i.p. with the Hu1A4A1IgG1-furin, Hu1A4A1IgG1-2A, 24 human IgG (positive control) or PBS alone (negative control) and 24h later challenged s.c. with 100 x LD50 of VEEV. None of the mice treated only with human IgG (positive control) or PBS alone 26 (negative control) survived. All the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A
treated mice survived 27 the VEEV challenge without any clinical signs at 14 days post-challenge.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0095] Discussion 2 [0096] Passive immunization of the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in mice (50 3 pg/mouse) 24 h before virulent VEEV challenge provided 100% protection against 100xLD50 4 challenge of VEEV when mice were treated with 50 pg/each mouse of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A. The mice were also found to be asymptomatic throughout the 14 day 6 observation period. These results indicate that the humanized anti-VEEV
rAbs of the present 7 invention have pre-exposure prophylactic capacity against VEEV
infections. The half-lives of the 8 humanized anti-VEEV rAbs in mice was around 10 days suggesting that the humanized anti-VEEV
9 rAbs of the invention would be an effective prophylactic against VEEV for at least several weeks.
Thus, the rAbs of the invention have been demonstrated to have functionality as an immunization 11 agent against VEEV infection.
12 [0097] Example 3: In vivo study - Treatment or post-exposure therapy of mice after VEEV
13 challenae by passive immunization with Hu1A4A1IaG1-furin or Hu1A4A1laG1-14 [0098] Materials and methods [0099] Post-exposure Therapy 16 [00100] Balb/c mice aged 6-8 weeks were challenged s.c. with 100 x LD50 of virulent VEEV in 17 50 pl of L15MM per mouse. At 24 h post-challenge, mice were injected i.p with 50 pg of 18 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in 100 pl PBS, or 100 pl PBS alone.
Mice were examined 19 frequently for signs of illness for 20 days, and humane endpoints were used.
[00101] Results 21 [00102] The half-lives of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was determined above in 22 Example 2.
23 [00103] Treatment of mice after VEEV challenge using Hu1A4A1loG1-furin or Hu1A4A1loG1-2A
24 [00104] Groups of 8 mice were challenged s.c. with 100 x LD50 of VEEV.
Twenty-four hours later, the infected mice were administered i.p. with Hu1A4A1IgG1.-furin, Hu1A4A1IgG1-2A or PBS
26 alone (50 ug/mouse). All the Hu1A4A1IgG1-2A-treated mice survived throughout the observation 27 period (20 days post-challenge) with minor clinical signs. All Hu1A4A1IgG1-furin or PBS-treated 28 mice died.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [00105] Discussion 2 [00106] Passive immunization of the Hu1A4A1IgG1-2A in mice (50 pg/mouse) 24 h after virulent 3 VEEV challenge provided 100% protection against 100xLD50 challenge of VEEV with only minor 4 clinical signs, indicating the Hu1A4A1IgG1-2A has post-exposure therapeutic capacity against VEEV
infections. Unfortunately, Hu1A4A1IgG1-furin did not show any post-exposure therapy capacity.
6 One possible reason for this finding may be that the antigen binding capacity of uncut 7 Hu1A4A1IgG1-furin is inferior to the cleaved Hu1A4A1IgG1-2A.
9 [00107] Bibliography [00108] One or more of the following documents have been referred to in the present disclosure.
11 [00109] [1] Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC.
Venezuelan equine 12 encephalitis. Annu Rev Entomol, 2004;49:141-74.
13 [00110] [2] Rivas F, Diaz LA, Cardenas VM, Daza E, Bruzon L, Alcala A, et al. Epidemic 14 Venezuelan equine encephalitis in La Guajira, Colombia, 1995. J Infect Dis, 1997;175:828-32.
[00111] [3] Pittman PR, Makuch RS, Mangiafico JA, Cannon TL, Gibbs PH, Peters CJ. Long-term 16 duration of detectable neutralizing antibodies after administration of live-attenuated VEE vaccine and 17 following booster vaccination with inactivated VEE vaccine. Vaccine, 1996; 14:337-43.
18 [00112] [4] Jahrling PB, Stephenson EH. Protective efficacies of live attenuated and 19 formaldehyde-inactivated Venezuelan equine encephalitis virus vaccines against aerosol challenge in hamsters. J Clin Microbiol, 1984; 19:429-31.
21 [00113] [5] France JK, Wyrick BC, Trent DW. Biochemical and antigenic comparison of the 22 envelope glycoproteins of Venezuelan equine encephalomyelitis virus strains. J Gen Virol, 1979;
23 44:725-40.
24 [00114] [6] Roehrig JT, Day JW, Kinney RM. Antigenic analysis of the surface glycoproteins of a Venezuelan equine encephalomyelitis virus (TC-83) using monoclonal antibodies.
Virology, 26 1982;118:269-78.
21989275.2 CA 2,704,119 Blakes Ref: 67853100022 1 [00115] [7] Roehrig JT, Mathews JH. The neutralization site on the E2 glycoprotein of 2 Venezuelan equine encephalomyelitis (TC-83) virus is composed of multiple conformationally stable 3 epitopes. Virology, 1985; 142:347-56.
4 [00116] [8] Schroff RW, Foon KA, Beatty SM, Oldham RK, Morgan Jr AC.
Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy.
Cancer Res, 1985;
6 45:879-85.
7 [00117] [9] Verhoeyen M, Milstein C, Winter G. Reshaping human antibodies: grafting an 8 antilysozyme activity. Science, 1988; 239:1534-6.
9 [00118] [10] Dall'Acqua WF, Damschroder MM, Zhang J, Woods RM, Widjaja L, Yu J, et al.
Antibody humanization by framework shuffling. Methods, 2005; 36:43-60.
1'1 [00119] [11] Hu WG, Alvi AZ, Fulton RE, Suresh MR, Nagata LE.
Genetic engineering of 12 streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine 13 encephalitis virus. Hybrid Hybridomics, 2002; 21:415-20.
14 [00120] [12] Hwang WY, Almagro JC, Buss TN, Tan P, Foote J. Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods, 2005; 36:35-42.
16 [00121] [13] van den Ouweland AM, van Duijnhoven HL, Keizer GD, Dorssers LC, Van de Ven 17 WJ. Structural homology between the human fur gene product and the subtilisin-like protease 18 encoded by yeast KEX2. Nucleic Acids Res, 1990; 18:664.
19 [00122] [14] Fulton RE, Nagata, L, Alvi, A; US Patent No.
6,818,748, Nov. 16, 2004.
[00123] [15] Johnson KM, Martin DH. Venezuelan equine encephalitis. Adv.
Vet Sci Comp Med.
21 1974; 18(0):79-116.
22 [00124] [16] Groot H, The health and economic importance of Venezuelan equine encephalitis 23 (VEE) in Venezuelan encephalitis, Scientific publication no. 243, 1972, pp. 7-16, Pan American 24 Health Organization, Washington DC.
[00125] [17] Phillpotts RJ, Jones LD, Howard SC, Monoclonal antibody protects mice against 26 infection and disease when given either before or up to 24h after airborne challenge with virulent 27 Venezuelan equine encephalitis virus. Vaccine, 2002 Feb 22; 20 (11-12):
1497-504.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [00126] [18] Hu WG, Chau D, Wu J, Jager S, Nagata L, Humanization and mammalian 2 expression of murine monoclonal antibody against Venezuelan equine encephalitis virus. Vaccine, 3 2007; 25:3210-3214.
[00127] Although the invention has been described with reference to certain specific 6 embodiments, various modifications thereof will be apparent to those skilled in the art without 7 departing from the scope of the invention as outlined in the claims appended hereto. Any examples 8 provided herein are included solely for the purpose of illustrating the invention and are not intended 9 to limit the invention in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the invention and are not intended to be drawn to scale or to limit the 11 invention in any way.
21989275.2
The FMDV-derived 2A
linker is able to cleave at its own C terminus between the last two residues through an enzyme-16 independent but undefined mechanism, probably by ribosomal skip, during protein translation. To 17 get the expressed Ab to be secreted to culture media, a leader sequence was added upstream to 18 the Ab gene. Figure 7 illustrates the synthesized DNA sequence, of approximately 2 kb, including 19 the human Ab kappa light chain L15 leader sequence, the humanized light chain (VL + CL), the furin or 2A linker, and the humanized heavy chain (VH + CHI + CH2 + CH3). This sequence was then 21 cloned into an adenoviral vector. The unique restriction sites, as also shown in Figure 7, flanking the 22 V regions, which allow for efficient V region replacement and at the heavy chain V¨C region junction 23 for generation of fragment antigen-binding portion of Ab (Fab), were also designed.
24 [0080] Protein G and A columns are widely used for a quick purification for Abs because of protein G and A binding to the Fc portion of lg. However, protein G and A
cannot only bind to 26 human lg, but also bind to bovine lg, therefore they cannot be used for purification of Hu1A4A1IgG1-27 furin or Hu1A4A1IgG1-2A in our study since pAd-Hu1A4A1IgG1-furin or pAd-Hu1A4A1IgG1-2A-28 infected HEK 293 cells were cultured in the medium with 5% FBS
containing a high percentage of 29 bovine lg. Unlike protein G and A, protein L binds lg through interactions with the light chains.
Protein L only binds to lg containing light chains of type kappa 1, 3 and 4 in human and kappa 1 in 31 mouse. Most importantly, protein L does not bind to bovine lg. Since our humanized Ab has human 32 kappa 1 chain, we chose a protein L column to purify Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 eliminate co-purification of bovine lg. In this way, the purity of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2 2A was relatively high in SDS-PAGE as shown in Figures 10, 11 and 12.
3 [0081] When the purified product was subjected to 10% SDS-PAGE, Hu1A4A1IgG1-furin and 4 Hu1A4A1IgG1-2A showed up in a different way. As illustrated in Figure 12, Hu1A4A1IgG1-2A
showed the same patterns as a control human IgG1, one band of ¨150kDa in non-reducing condition 6 (intact disulfide bridges) and two bands, 50kDa for heavy chains and 25kDa for light chains (broken 7 disulfide bridges) in reducing condition, indicating that the 2A linker underwent self-processing 8 perfectly. On the other hand, Hu1A4A1IgG1-furin showed only one clear band of ¨75kDa in 9 reducing condition observed as illustrated in Figures 10 and 11, indicating that the furin linker was not cleaved. However, in another study (data not shown), the same furin linker sequence was 11 cleaved in another Fab construct expressed in a mammalian system. This indicated the 12 conformation of expressed Hu1A4A1IgG1-furin probably rendered the furin linker inaccessible to 13 furin or that the sequence surrounding the furin linker influenced furin cleavage.
14 [0082] The specific binding reactivities of purified Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A to VEEV E2 antigen were examined by ELISA. As illustrated in Figure 13, both versions of the 16 Hu1A4A1IgG1 were found to bind to VEEV E2 in a dose-dependent manner, similar to the binding to 17 VEEV E2 of its parental murine 1A4A1, indicating this non-cleaved Ab was still reactive to VEEV E2 18 antigen in ELISA. Furthermore, both versions were evaluated for their ability to block VEEV infection 19 in Vero cells using a standard plaque-reduction assay. The Hu1A4A1IgG1-fruin showed a neutralizing activity with 50% plaque reduction neutralization titre at 0.78 pg/ml, whereas 21 Hu1A4A1IgG1-2A showed a much higher neutralization titre at 0.1 pg/ml.
22 [0083] From the above results, it is concluded that the murine 1A4A1 Ab was successfully 23 humanized. As illustrated in Figure 14, the expressed and purified Ab of Hu1A4A1IgG1-2A was 24 cleaved between the heavy and light chains as expected; however, Hu1A4A1IgG1-furin was not cleaved. Nevertheless, the present inventors have exhibited that both versions of the Hu1A4A1IgG1 26 retained the antigen binding specificity and virus neutralizing activity. Thus, the Hu1A4A1IgG1-furin 27 or Hu1A4A1IgG1-2A discussed and characterized herein would serve as an effective prophylactic 28 and therapeutic agent against VEEV infection.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0084] Example 2: In vivo study - Protection or pre-exposure prophylaxis of mice from 2 VEEV challenge by passive immunization with Hu1A4A110G1-furin or Hu1A4A1IgG1-2A
3 [0085] Materials and methods 4 [0086] Passive immunization (Pre-exposure prophylaxis) [0087] Balb/c mice aged 6-8 weeks were injected intraperitoneally (i.p) with 50 pg of 6 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in 100 pl PBS, human IgG in 100 pl PBS (positive control) 7 or 100 pl PBS alone (negative control) 24 h prior to VEEV challenge.
8 [0088] VEEV challenge 9 [0089] Each mouse was challenged subcutaneously (s.c.) with 30-50 plaque forming units (pfu) of virulent VEEV (Trinidad donkey, TRD) in 50 pl of Leibovitz L15 maintenance medium (L15MM) 24 11 h after passive immunization. The challenge dose approximated to 100 x 50% lethal dose (LD50).
12 Mice were examined frequently for signs of illness for 14 days, and humane endpoints were used.
13 [0090] Results 14 [0091] Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A clearance in mice [0092] To determine the half-life of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in mouse serum, 16 groups of 4 mice, were injected i.p. with 50 pg, each mouse, of either Hu1A4A1IgG1-furin or 17 Hu1A4A1IgG1-2A, or human anti-VEEV IgG and bled from the vein at increasing time intervals after 18 injection. The quantity of Ab present in serum samples was estimated by immunoassay.
19 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A had a similar half-life as human anti-VEEV IgG, around 10 days.
21 [0093] Protection of mice from VEEV challenge bv passive immunization with Hu1A4A1IgG1-22 furin or HulA4AllaG1 -2A
23 [0094] Groups of 8 mice were injected i.p. with the Hu1A4A1IgG1-furin, Hu1A4A1IgG1-2A, 24 human IgG (positive control) or PBS alone (negative control) and 24h later challenged s.c. with 100 x LD50 of VEEV. None of the mice treated only with human IgG (positive control) or PBS alone 26 (negative control) survived. All the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A
treated mice survived 27 the VEEV challenge without any clinical signs at 14 days post-challenge.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0095] Discussion 2 [0096] Passive immunization of the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in mice (50 3 pg/mouse) 24 h before virulent VEEV challenge provided 100% protection against 100xLD50 4 challenge of VEEV when mice were treated with 50 pg/each mouse of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A. The mice were also found to be asymptomatic throughout the 14 day 6 observation period. These results indicate that the humanized anti-VEEV
rAbs of the present 7 invention have pre-exposure prophylactic capacity against VEEV
infections. The half-lives of the 8 humanized anti-VEEV rAbs in mice was around 10 days suggesting that the humanized anti-VEEV
9 rAbs of the invention would be an effective prophylactic against VEEV for at least several weeks.
Thus, the rAbs of the invention have been demonstrated to have functionality as an immunization 11 agent against VEEV infection.
12 [0097] Example 3: In vivo study - Treatment or post-exposure therapy of mice after VEEV
13 challenae by passive immunization with Hu1A4A1IaG1-furin or Hu1A4A1laG1-14 [0098] Materials and methods [0099] Post-exposure Therapy 16 [00100] Balb/c mice aged 6-8 weeks were challenged s.c. with 100 x LD50 of virulent VEEV in 17 50 pl of L15MM per mouse. At 24 h post-challenge, mice were injected i.p with 50 pg of 18 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in 100 pl PBS, or 100 pl PBS alone.
Mice were examined 19 frequently for signs of illness for 20 days, and humane endpoints were used.
[00101] Results 21 [00102] The half-lives of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was determined above in 22 Example 2.
23 [00103] Treatment of mice after VEEV challenge using Hu1A4A1loG1-furin or Hu1A4A1loG1-2A
24 [00104] Groups of 8 mice were challenged s.c. with 100 x LD50 of VEEV.
Twenty-four hours later, the infected mice were administered i.p. with Hu1A4A1IgG1.-furin, Hu1A4A1IgG1-2A or PBS
26 alone (50 ug/mouse). All the Hu1A4A1IgG1-2A-treated mice survived throughout the observation 27 period (20 days post-challenge) with minor clinical signs. All Hu1A4A1IgG1-furin or PBS-treated 28 mice died.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [00105] Discussion 2 [00106] Passive immunization of the Hu1A4A1IgG1-2A in mice (50 pg/mouse) 24 h after virulent 3 VEEV challenge provided 100% protection against 100xLD50 challenge of VEEV with only minor 4 clinical signs, indicating the Hu1A4A1IgG1-2A has post-exposure therapeutic capacity against VEEV
infections. Unfortunately, Hu1A4A1IgG1-furin did not show any post-exposure therapy capacity.
6 One possible reason for this finding may be that the antigen binding capacity of uncut 7 Hu1A4A1IgG1-furin is inferior to the cleaved Hu1A4A1IgG1-2A.
9 [00107] Bibliography [00108] One or more of the following documents have been referred to in the present disclosure.
11 [00109] [1] Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC.
Venezuelan equine 12 encephalitis. Annu Rev Entomol, 2004;49:141-74.
13 [00110] [2] Rivas F, Diaz LA, Cardenas VM, Daza E, Bruzon L, Alcala A, et al. Epidemic 14 Venezuelan equine encephalitis in La Guajira, Colombia, 1995. J Infect Dis, 1997;175:828-32.
[00111] [3] Pittman PR, Makuch RS, Mangiafico JA, Cannon TL, Gibbs PH, Peters CJ. Long-term 16 duration of detectable neutralizing antibodies after administration of live-attenuated VEE vaccine and 17 following booster vaccination with inactivated VEE vaccine. Vaccine, 1996; 14:337-43.
18 [00112] [4] Jahrling PB, Stephenson EH. Protective efficacies of live attenuated and 19 formaldehyde-inactivated Venezuelan equine encephalitis virus vaccines against aerosol challenge in hamsters. J Clin Microbiol, 1984; 19:429-31.
21 [00113] [5] France JK, Wyrick BC, Trent DW. Biochemical and antigenic comparison of the 22 envelope glycoproteins of Venezuelan equine encephalomyelitis virus strains. J Gen Virol, 1979;
23 44:725-40.
24 [00114] [6] Roehrig JT, Day JW, Kinney RM. Antigenic analysis of the surface glycoproteins of a Venezuelan equine encephalomyelitis virus (TC-83) using monoclonal antibodies.
Virology, 26 1982;118:269-78.
21989275.2 CA 2,704,119 Blakes Ref: 67853100022 1 [00115] [7] Roehrig JT, Mathews JH. The neutralization site on the E2 glycoprotein of 2 Venezuelan equine encephalomyelitis (TC-83) virus is composed of multiple conformationally stable 3 epitopes. Virology, 1985; 142:347-56.
4 [00116] [8] Schroff RW, Foon KA, Beatty SM, Oldham RK, Morgan Jr AC.
Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy.
Cancer Res, 1985;
6 45:879-85.
7 [00117] [9] Verhoeyen M, Milstein C, Winter G. Reshaping human antibodies: grafting an 8 antilysozyme activity. Science, 1988; 239:1534-6.
9 [00118] [10] Dall'Acqua WF, Damschroder MM, Zhang J, Woods RM, Widjaja L, Yu J, et al.
Antibody humanization by framework shuffling. Methods, 2005; 36:43-60.
1'1 [00119] [11] Hu WG, Alvi AZ, Fulton RE, Suresh MR, Nagata LE.
Genetic engineering of 12 streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine 13 encephalitis virus. Hybrid Hybridomics, 2002; 21:415-20.
14 [00120] [12] Hwang WY, Almagro JC, Buss TN, Tan P, Foote J. Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods, 2005; 36:35-42.
16 [00121] [13] van den Ouweland AM, van Duijnhoven HL, Keizer GD, Dorssers LC, Van de Ven 17 WJ. Structural homology between the human fur gene product and the subtilisin-like protease 18 encoded by yeast KEX2. Nucleic Acids Res, 1990; 18:664.
19 [00122] [14] Fulton RE, Nagata, L, Alvi, A; US Patent No.
6,818,748, Nov. 16, 2004.
[00123] [15] Johnson KM, Martin DH. Venezuelan equine encephalitis. Adv.
Vet Sci Comp Med.
21 1974; 18(0):79-116.
22 [00124] [16] Groot H, The health and economic importance of Venezuelan equine encephalitis 23 (VEE) in Venezuelan encephalitis, Scientific publication no. 243, 1972, pp. 7-16, Pan American 24 Health Organization, Washington DC.
[00125] [17] Phillpotts RJ, Jones LD, Howard SC, Monoclonal antibody protects mice against 26 infection and disease when given either before or up to 24h after airborne challenge with virulent 27 Venezuelan equine encephalitis virus. Vaccine, 2002 Feb 22; 20 (11-12):
1497-504.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [00126] [18] Hu WG, Chau D, Wu J, Jager S, Nagata L, Humanization and mammalian 2 expression of murine monoclonal antibody against Venezuelan equine encephalitis virus. Vaccine, 3 2007; 25:3210-3214.
[00127] Although the invention has been described with reference to certain specific 6 embodiments, various modifications thereof will be apparent to those skilled in the art without 7 departing from the scope of the invention as outlined in the claims appended hereto. Any examples 8 provided herein are included solely for the purpose of illustrating the invention and are not intended 9 to limit the invention in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the invention and are not intended to be drawn to scale or to limit the 11 invention in any way.
21989275.2
Claims (12)
1. A humanized recombinant antibody for use in the prophylaxis or treatment against infection from Venezuelan equine encephalitis virus, VEEV, in a mammal, - wherein said antibody comprises a human Ig framework having grafted thereon complementarity determining regions, CDRs, from the murine monoclonal antibody 1A4A1;
- and wherein said antibody is produced by expressing a protein encoded by a nucleic acid construct having a nucleic acid sequence according to SEQ ID NO: 13.
- and wherein said antibody is produced by expressing a protein encoded by a nucleic acid construct having a nucleic acid sequence according to SEQ ID NO: 13.
2. The humanized recombinant antibody for use according to claim 1, wherein said antibody has an amino acid sequence according to SEQ ID NO: 14.
3. The humanized recombinant antibody for use according to claim 1 or claim 2, wherein said antibody is encoded by an expression vector.
4. The humanized recombinant antibody for use according to claim 1 or claim 2, wherein said antibody is expressed by an isolated transformed host cell.
5. A use of a humanized recombinant antibody in inhibiting Venezuelan equine encephalitis virus (VEEV) infection in a human, the humanized recombinant antibody comprising a human Ig framework and having grafted thereon complementarity determining regions, CDRs, from the murine monoclonal antibody 1A4A1, wherein:
- the antibody is capable of inhibiting VEEV infection when administered prior to or after VEEV exposure; and, - the antibody is encoded by the nucleic acid sequence according to SEQ ID NO:
13.
- the antibody is capable of inhibiting VEEV infection when administered prior to or after VEEV exposure; and, - the antibody is encoded by the nucleic acid sequence according to SEQ ID NO:
13.
6. The use according to claim 5, wherein said antibody has a VH region having the amino acid sequence according to SEQ ID NO: 7.
7. The use according to claim 5, wherein said antibody has a VL region having the amino acid sequence according to SEQ ID NO: 8.
8. The use according to claim 5, wherein said antibody has the amino acid sequence according to SEQ ID NO: 14.
9. The use according to any one of claims 5 to 8, wherein said antibody is encoded by an expression vector.
10. The use according to any one of claims 5 to 9, wherein said antibody is expressed by an isolated transformed host cell.
11. A use of a humanized recombinant antibody in the passive transfer of immunity, to a human, against Venezuelan equine encephalitis virus (VEEV) infection, after exposure to VEEV, the humanized recombinant antibody comprising a human Ig framework and having grafted thereon complementarity determining regions, CDRs, from the murine monoclonal antibody 1A4A1, wherein the antibody is encoded by the nucleic acid sequence according to SEQ ID NO:
13.
13.
12. The use according to claim 11 wherein the antibody has the amino acid sequence according to SEQ ID NO: 14.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2704119A CA2704119C (en) | 2007-11-01 | 2008-11-03 | Humanized anti-venezuelan equine encephalitis virus recombinant antibodies |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002607771A CA2607771A1 (en) | 2007-11-01 | 2007-11-01 | Humanized anti-venezuelan equine encephalitis virus recombinant antibody |
CA2,607,771 | 2007-11-01 | ||
PCT/CA2008/001940 WO2009055936A1 (en) | 2007-11-01 | 2008-11-03 | Humanized anti-venezuelan equine encephalitis virus recombinant antibodies |
CA2704119A CA2704119C (en) | 2007-11-01 | 2008-11-03 | Humanized anti-venezuelan equine encephalitis virus recombinant antibodies |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2704119A1 CA2704119A1 (en) | 2009-05-07 |
CA2704119C true CA2704119C (en) | 2014-12-30 |
Family
ID=40589892
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002607771A Abandoned CA2607771A1 (en) | 2007-11-01 | 2007-11-01 | Humanized anti-venezuelan equine encephalitis virus recombinant antibody |
CA2704119A Expired - Fee Related CA2704119C (en) | 2007-11-01 | 2008-11-03 | Humanized anti-venezuelan equine encephalitis virus recombinant antibodies |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002607771A Abandoned CA2607771A1 (en) | 2007-11-01 | 2007-11-01 | Humanized anti-venezuelan equine encephalitis virus recombinant antibody |
Country Status (3)
Country | Link |
---|---|
CA (2) | CA2607771A1 (en) |
GB (1) | GB2467491C (en) |
WO (1) | WO2009055936A1 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8501157B2 (en) | 2007-11-01 | 2013-08-06 | Her Majesty the Queen in right of Canada as represented by The Ministry of National Defence | Humanized anti-Venezuelan equine encephalitis virus recombinant antibodies |
GB0916630D0 (en) * | 2009-09-22 | 2009-11-04 | Secr Defence | Antibody |
RS55161B1 (en) | 2010-11-04 | 2017-01-31 | Boehringer Ingelheim Int | Anti-il-23 antibodies |
EP2844284A1 (en) | 2012-05-03 | 2015-03-11 | Boehringer Ingelheim International GmbH | Anti-il-23p19 antibodies |
EP3708679A1 (en) | 2014-07-24 | 2020-09-16 | Boehringer Ingelheim International GmbH | Biomarkers useful in the treatment of il-23a related diseases |
BR112017004169A2 (en) | 2014-09-03 | 2017-12-05 | Boehringer Ingelheim Int | compound directed to il-23a and tnf-alpha and uses thereof |
WO2016208695A1 (en) * | 2015-06-24 | 2016-12-29 | Jcrファーマ株式会社 | Anti-human transferrin receptor antibody permeating blood-brain barrier |
KR20180020279A (en) | 2015-06-24 | 2018-02-27 | 제이씨알 파마 가부시키가이샤 | A fusion protein comprising BDNF |
CN107586335B (en) * | 2016-07-06 | 2020-12-29 | 中国科学院微生物研究所 | Humanized monoclonal antibody and application |
BR112019009316A2 (en) | 2016-12-26 | 2019-08-06 | Japan Chem Res | human transferrin anti-receptor antibody, fusion protein, DNA fragment, expression vector, mammalian cell, pharmacologically active compound and human transferrin anti-receptor antibody complex, uses of human transferrin anti-receptor antibody and fusion protein, and, methods for treating a central nervous system disorder and a central nervous system disorder accompanying pompe disease, hurler syndrome or hurler-scheie syndrome. |
-
2007
- 2007-11-01 CA CA002607771A patent/CA2607771A1/en not_active Abandoned
-
2008
- 2008-11-03 WO PCT/CA2008/001940 patent/WO2009055936A1/en active Application Filing
- 2008-11-03 CA CA2704119A patent/CA2704119C/en not_active Expired - Fee Related
- 2008-11-03 GB GB201009145A patent/GB2467491C/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
GB2467491C (en) | 2013-03-27 |
CA2607771A1 (en) | 2009-05-01 |
GB2467491A (en) | 2010-08-04 |
CA2704119A1 (en) | 2009-05-07 |
GB2467491B (en) | 2013-03-20 |
WO2009055936A1 (en) | 2009-05-07 |
GB2467491A8 (en) | 2010-12-22 |
GB201009145D0 (en) | 2010-07-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2704119C (en) | Humanized anti-venezuelan equine encephalitis virus recombinant antibodies | |
US20090117105A1 (en) | Humanized anti-venezuelan equine encephalitis virus recombinant antibody | |
Goodchild et al. | A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus | |
CN110317267B (en) | Bispecific antibodies against rabies virus and uses thereof | |
EP3572125A2 (en) | Monoclonal antibodies for ebola and marburg viruses | |
US20170158753A1 (en) | Monoclonal antibodies directed against envelope glycoproteins from multiple filovirus species | |
Hu et al. | A recombinant humanized monoclonal antibody completely protects mice against lethal challenge with Venezuelan equine encephalitis virus | |
US20180237502A1 (en) | Pan-ebola and pan-filovirus protective epitopes, antibodies, and antibody cocktails | |
Bennett et al. | DNA vaccination protects against botulinum neurotoxin type F | |
Hu et al. | Humanization and mammalian expression of a murine monoclonal antibody against Venezuelan equine encephalitis virus | |
Unkauf et al. | Generation of recombinant antibodies against toxins and viruses by phage display for diagnostics and therapy | |
Goncalvez et al. | Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses | |
WO2022216223A1 (en) | Vaccine and/or antibody for viral infection | |
WO2020186687A1 (en) | Human antibody specifically binding four serotypes of dengue viruses | |
US10081672B2 (en) | Anti-ricin antibodies and uses thereof | |
EP4353744A1 (en) | Antibody against respiratory syncytial virus and use thereof | |
CN114957479B (en) | Anti-H1N 1 influenza virus bispecific neutralizing antibody Bis-Hu11-1 and application thereof | |
US8501157B2 (en) | Humanized anti-Venezuelan equine encephalitis virus recombinant antibodies | |
US11440951B2 (en) | Therapeutic antibodies to Marburg virus | |
Lamarre et al. | A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice | |
EP3525813B1 (en) | Broadly neutralizing antibody targeting the ebolavirus glycoprotein internal fusion loop | |
KR20240001181A (en) | Antibodies for treatment and prevention of COVID-19 and new variants | |
CN114751987A (en) | Multispecific antibody for neutralizing coronavirus |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20131003 |
|
MKLA | Lapsed |
Effective date: 20171103 |