WO2024020063A2 - Anti-hrf antibodies - Google Patents
Anti-hrf antibodies Download PDFInfo
- Publication number
- WO2024020063A2 WO2024020063A2 PCT/US2023/028081 US2023028081W WO2024020063A2 WO 2024020063 A2 WO2024020063 A2 WO 2024020063A2 US 2023028081 W US2023028081 W US 2023028081W WO 2024020063 A2 WO2024020063 A2 WO 2024020063A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- amino acid
- antigen binding
- binding fragment
- acid sequence
- Prior art date
Links
- 239000012634 fragment Substances 0.000 claims abstract description 250
- 241000282414 Homo sapiens Species 0.000 claims abstract description 100
- 230000003993 interaction Effects 0.000 claims abstract description 17
- 230000028709 inflammatory response Effects 0.000 claims abstract description 15
- 239000003112 inhibitor Substances 0.000 claims abstract description 10
- 239000000427 antigen Substances 0.000 claims description 248
- 108091007433 antigens Proteins 0.000 claims description 248
- 102000036639 antigens Human genes 0.000 claims description 248
- 230000027455 binding Effects 0.000 claims description 234
- 238000009739 binding Methods 0.000 claims description 232
- 210000004027 cell Anatomy 0.000 claims description 164
- 238000000034 method Methods 0.000 claims description 146
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 136
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 125
- 102000040430 polynucleotide Human genes 0.000 claims description 108
- 108091033319 polynucleotide Proteins 0.000 claims description 108
- 239000002157 polynucleotide Substances 0.000 claims description 108
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 95
- 229920001184 polypeptide Polymers 0.000 claims description 81
- 239000013598 vector Substances 0.000 claims description 80
- 230000014509 gene expression Effects 0.000 claims description 59
- 239000000203 mixture Substances 0.000 claims description 52
- 206010020751 Hypersensitivity Diseases 0.000 claims description 48
- 208000026935 allergic disease Diseases 0.000 claims description 33
- 108060003951 Immunoglobulin Proteins 0.000 claims description 28
- 102000018358 immunoglobulin Human genes 0.000 claims description 28
- 230000001105 regulatory effect Effects 0.000 claims description 26
- 208000004262 Food Hypersensitivity Diseases 0.000 claims description 25
- 235000020932 food allergy Nutrition 0.000 claims description 25
- 238000002965 ELISA Methods 0.000 claims description 24
- 206010016946 Food allergy Diseases 0.000 claims description 24
- 230000002163 immunogen Effects 0.000 claims description 23
- 238000000746 purification Methods 0.000 claims description 23
- 239000003795 chemical substances by application Substances 0.000 claims description 21
- 210000004408 hybridoma Anatomy 0.000 claims description 20
- 239000003550 marker Substances 0.000 claims description 20
- 230000000295 complement effect Effects 0.000 claims description 19
- 108020004999 messenger RNA Proteins 0.000 claims description 19
- 238000011282 treatment Methods 0.000 claims description 19
- 239000013612 plasmid Substances 0.000 claims description 18
- 206010061218 Inflammation Diseases 0.000 claims description 17
- 230000007815 allergy Effects 0.000 claims description 17
- 230000004054 inflammatory process Effects 0.000 claims description 17
- 239000013603 viral vector Substances 0.000 claims description 17
- 208000006673 asthma Diseases 0.000 claims description 16
- 239000003814 drug Substances 0.000 claims description 16
- 239000003623 enhancer Substances 0.000 claims description 15
- 208000030961 allergic reaction Diseases 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 14
- 229940079593 drug Drugs 0.000 claims description 14
- 230000009610 hypersensitivity Effects 0.000 claims description 14
- 238000013518 transcription Methods 0.000 claims description 14
- 230000035897 transcription Effects 0.000 claims description 14
- 241000124008 Mammalia Species 0.000 claims description 13
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 238000009169 immunotherapy Methods 0.000 claims description 12
- 230000010076 replication Effects 0.000 claims description 12
- 239000000523 sample Substances 0.000 claims description 12
- 238000002560 therapeutic procedure Methods 0.000 claims description 12
- 241000238631 Hexapoda Species 0.000 claims description 9
- 239000012472 biological sample Substances 0.000 claims description 9
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 9
- 239000000178 monomer Substances 0.000 claims description 9
- 206010002198 Anaphylactic reaction Diseases 0.000 claims description 8
- 230000036783 anaphylactic response Effects 0.000 claims description 8
- 208000003455 anaphylaxis Diseases 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 230000002401 inhibitory effect Effects 0.000 claims description 8
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 8
- 230000000977 initiatory effect Effects 0.000 claims description 7
- 230000001177 retroviral effect Effects 0.000 claims description 7
- 238000013519 translation Methods 0.000 claims description 7
- 239000000539 dimer Substances 0.000 claims description 6
- 108020004566 Transfer RNA Proteins 0.000 claims description 5
- 239000013566 allergen Substances 0.000 claims description 5
- 210000003630 histaminocyte Anatomy 0.000 claims description 5
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims description 4
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 4
- 102000052866 Amino Acyl-tRNA Synthetases Human genes 0.000 claims description 4
- 108700028939 Amino Acyl-tRNA Synthetases Proteins 0.000 claims description 4
- PCDQPRRSZKQHHS-CCXZUQQUSA-N Cytarabine Triphosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-CCXZUQQUSA-N 0.000 claims description 4
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 claims description 4
- 230000001419 dependent effect Effects 0.000 claims description 4
- 238000003018 immunoassay Methods 0.000 claims description 4
- 102000006495 integrins Human genes 0.000 claims description 4
- 108010044426 integrins Proteins 0.000 claims description 4
- 230000008488 polyadenylation Effects 0.000 claims description 4
- XKMLYUALXHKNFT-UHFFFAOYSA-N rGTP Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O XKMLYUALXHKNFT-UHFFFAOYSA-N 0.000 claims description 4
- 230000028327 secretion Effects 0.000 claims description 4
- 230000008685 targeting Effects 0.000 claims description 4
- 210000005253 yeast cell Anatomy 0.000 claims description 4
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 claims description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 210000003651 basophil Anatomy 0.000 claims description 3
- 239000013592 cell lysate Substances 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 3
- 229940088597 hormone Drugs 0.000 claims description 3
- 238000007920 subcutaneous administration Methods 0.000 claims description 3
- GUGOEEXESWIERI-UHFFFAOYSA-N Terfenadine Chemical compound C1=CC(C(C)(C)C)=CC=C1C(O)CCCN1CCC(C(O)(C=2C=CC=CC=2)C=2C=CC=CC=2)CC1 GUGOEEXESWIERI-UHFFFAOYSA-N 0.000 claims description 2
- PGAVKCOVUIYSFO-UHFFFAOYSA-N [[5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound OC1C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-UHFFFAOYSA-N 0.000 claims description 2
- 230000001088 anti-asthma Effects 0.000 claims description 2
- 230000001387 anti-histamine Effects 0.000 claims description 2
- 230000003110 anti-inflammatory effect Effects 0.000 claims description 2
- 230000002590 anti-leukotriene effect Effects 0.000 claims description 2
- 239000000043 antiallergic agent Substances 0.000 claims description 2
- 239000000924 antiasthmatic agent Substances 0.000 claims description 2
- 239000000739 antihistaminic agent Substances 0.000 claims description 2
- 229940124748 beta 2 agonist Drugs 0.000 claims description 2
- 230000037406 food intake Effects 0.000 claims description 2
- 150000003431 steroids Chemical class 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 44
- 102100029887 Translationally-controlled tumor protein Human genes 0.000 abstract description 111
- 108090000623 proteins and genes Proteins 0.000 abstract description 108
- 101710175870 Translationally-controlled tumor protein homolog Proteins 0.000 abstract description 106
- 102000004169 proteins and genes Human genes 0.000 abstract description 65
- 150000001413 amino acids Chemical group 0.000 description 192
- 238000005516 engineering process Methods 0.000 description 65
- 235000018102 proteins Nutrition 0.000 description 63
- 235000001014 amino acid Nutrition 0.000 description 60
- 229940024606 amino acid Drugs 0.000 description 59
- 102100033400 4F2 cell-surface antigen heavy chain Human genes 0.000 description 41
- 101000800023 Homo sapiens 4F2 cell-surface antigen heavy chain Proteins 0.000 description 41
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 39
- 150000007523 nucleic acids Chemical class 0.000 description 35
- 201000010099 disease Diseases 0.000 description 33
- 239000013604 expression vector Substances 0.000 description 33
- 125000003729 nucleotide group Chemical group 0.000 description 31
- 239000002773 nucleotide Substances 0.000 description 27
- 102000039446 nucleic acids Human genes 0.000 description 26
- 108020004707 nucleic acids Proteins 0.000 description 26
- 108020004414 DNA Proteins 0.000 description 25
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 24
- 108010058846 Ovalbumin Proteins 0.000 description 22
- 229940092253 ovalbumin Drugs 0.000 description 22
- 241001465754 Metazoa Species 0.000 description 21
- 108020001507 fusion proteins Proteins 0.000 description 21
- 102000037865 fusion proteins Human genes 0.000 description 21
- -1 DNA or RNA Chemical class 0.000 description 19
- 229940088598 enzyme Drugs 0.000 description 19
- 239000003446 ligand Substances 0.000 description 19
- 241000894007 species Species 0.000 description 19
- 102000004190 Enzymes Human genes 0.000 description 18
- 108090000790 Enzymes Proteins 0.000 description 18
- 241000699670 Mus sp. Species 0.000 description 17
- 230000004927 fusion Effects 0.000 description 17
- 241000283973 Oryctolagus cuniculus Species 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 16
- 230000006870 function Effects 0.000 description 16
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 14
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 14
- 238000000338 in vitro Methods 0.000 description 14
- 208000024891 symptom Diseases 0.000 description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 description 13
- 125000000539 amino acid group Chemical group 0.000 description 13
- 238000009396 hybridization Methods 0.000 description 13
- 238000001727 in vivo Methods 0.000 description 13
- 210000004962 mammalian cell Anatomy 0.000 description 13
- 230000004048 modification Effects 0.000 description 13
- 238000012986 modification Methods 0.000 description 13
- 241000702421 Dependoparvovirus Species 0.000 description 12
- 108020004511 Recombinant DNA Proteins 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 102100034343 Integrase Human genes 0.000 description 11
- 241000700605 Viruses Species 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 102000005720 Glutathione transferase Human genes 0.000 description 10
- 108010070675 Glutathione transferase Proteins 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 239000002245 particle Substances 0.000 description 10
- 230000003612 virological effect Effects 0.000 description 10
- 208000002267 Anti-neutrophil cytoplasmic antibody-associated vasculitis Diseases 0.000 description 9
- 241001529936 Murinae Species 0.000 description 9
- 150000001720 carbohydrates Chemical class 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 239000007850 fluorescent dye Substances 0.000 description 9
- 230000013595 glycosylation Effects 0.000 description 9
- 238000006206 glycosylation reaction Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 206010012735 Diarrhoea Diseases 0.000 description 8
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 235000014633 carbohydrates Nutrition 0.000 description 8
- 239000003937 drug carrier Substances 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 229940072221 immunoglobulins Drugs 0.000 description 8
- 238000002823 phage display Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 238000003259 recombinant expression Methods 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 7
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 150000002482 oligosaccharides Chemical class 0.000 description 7
- 230000009261 transgenic effect Effects 0.000 description 7
- 241001430294 unidentified retrovirus Species 0.000 description 7
- 241000283707 Capra Species 0.000 description 6
- 108010061833 Integrases Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 6
- 230000036039 immunity Effects 0.000 description 6
- 238000007912 intraperitoneal administration Methods 0.000 description 6
- 238000002372 labelling Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000008194 pharmaceutical composition Substances 0.000 description 6
- 239000000546 pharmaceutical excipient Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 235000002639 sodium chloride Nutrition 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 230000014616 translation Effects 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 241000700159 Rattus Species 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 108700019146 Transgenes Proteins 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 5
- 229960003669 carbenicillin Drugs 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- 229920001542 oligosaccharide Polymers 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 230000002265 prevention Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 108010087819 Fc receptors Proteins 0.000 description 4
- 102000009109 Fc receptors Human genes 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 102000008100 Human Serum Albumin Human genes 0.000 description 4
- 108091006905 Human Serum Albumin Proteins 0.000 description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000002457 bidirectional effect Effects 0.000 description 4
- 239000011230 binding agent Substances 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 239000013611 chromosomal DNA Substances 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 230000002631 hypothermal effect Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 201000000050 myeloid neoplasm Diseases 0.000 description 4
- 238000004091 panning Methods 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 230000037081 physical activity Effects 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 230000002285 radioactive effect Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 150000008163 sugars Chemical class 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- 241000701447 unidentified baculovirus Species 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 3
- 108091035707 Consensus sequence Proteins 0.000 description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 101000653679 Homo sapiens Translationally-controlled tumor protein Proteins 0.000 description 3
- 108090000144 Human Proteins Proteins 0.000 description 3
- 102000003839 Human Proteins Human genes 0.000 description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 3
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 3
- 239000004353 Polyethylene glycol 8000 Substances 0.000 description 3
- 241000288906 Primates Species 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 238000000692 Student's t-test Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 230000021615 conjugation Effects 0.000 description 3
- 229940104302 cytosine Drugs 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000006806 disease prevention Effects 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 229940085678 polyethylene glycol 8000 Drugs 0.000 description 3
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 2
- WZXXZHONLFRKGG-UHFFFAOYSA-N 2,3,4,5-tetrachlorothiophene Chemical group ClC=1SC(Cl)=C(Cl)C=1Cl WZXXZHONLFRKGG-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 2
- 241001164825 Adeno-associated virus - 8 Species 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 241000710929 Alphavirus Species 0.000 description 2
- 244000105975 Antidesma platyphyllum Species 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 101100230428 Caenorhabditis elegans hil-5 gene Proteins 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 101710154606 Hemagglutinin Proteins 0.000 description 2
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 2
- 101000935587 Homo sapiens Flavin reductase (NADPH) Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- 238000012449 Kunming mouse Methods 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 241000282553 Macaca Species 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 2
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 2
- 241000282579 Pan Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 241000282405 Pongo abelii Species 0.000 description 2
- 101710176177 Protein A56 Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 101710157927 Translationally-controlled tumor protein Proteins 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 2
- 238000001261 affinity purification Methods 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 230000009285 allergic inflammation Effects 0.000 description 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 229940037003 alum Drugs 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 210000001742 aqueous humor Anatomy 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 238000002820 assay format Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 210000000234 capsid Anatomy 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- GSOLWAFGMNOBSY-UHFFFAOYSA-N cobalt Chemical compound [Co][Co][Co][Co][Co][Co][Co][Co] GSOLWAFGMNOBSY-UHFFFAOYSA-N 0.000 description 2
- 229910017052 cobalt Inorganic materials 0.000 description 2
- 239000010941 cobalt Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 235000009424 haa Nutrition 0.000 description 2
- 201000008109 hereditary spherocytosis type 4 Diseases 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 229940042743 immune sera Drugs 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 208000026278 immune system disease Diseases 0.000 description 2
- 230000000984 immunochemical effect Effects 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000000185 intracerebroventricular administration Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 210000005075 mammary gland Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 238000002515 oligonucleotide synthesis Methods 0.000 description 2
- 210000000287 oocyte Anatomy 0.000 description 2
- 239000000816 peptidomimetic Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 108700004029 pol Genes Proteins 0.000 description 2
- 210000004909 pre-ejaculatory fluid Anatomy 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 230000012846 protein folding Effects 0.000 description 2
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 150000005846 sugar alcohols Chemical class 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000001179 synovial fluid Anatomy 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 239000000811 xylitol Substances 0.000 description 2
- 235000010447 xylitol Nutrition 0.000 description 2
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 2
- 229960002675 xylitol Drugs 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- KGLPWQKSKUVKMJ-UHFFFAOYSA-N 2,3-dihydrophthalazine-1,4-dione Chemical class C1=CC=C2C(=O)NNC(=O)C2=C1 KGLPWQKSKUVKMJ-UHFFFAOYSA-N 0.000 description 1
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- ACEMKCOYIINOHN-UHFFFAOYSA-N 5-methyl-1h-pyrimidine-2,4-dione;pyrimidine Chemical compound C1=CN=CN=C1.CC1=CNC(=O)NC1=O ACEMKCOYIINOHN-UHFFFAOYSA-N 0.000 description 1
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 1
- AXXVSCRPHPIDIW-UHFFFAOYSA-N 7h-purin-6-amine;7h-purine Chemical compound C1=NC=C2NC=NC2=N1.NC1=NC=NC2=C1NC=N2 AXXVSCRPHPIDIW-UHFFFAOYSA-N 0.000 description 1
- 241001655883 Adeno-associated virus - 1 Species 0.000 description 1
- 241000580270 Adeno-associated virus - 4 Species 0.000 description 1
- 241001634120 Adeno-associated virus - 5 Species 0.000 description 1
- 241000972680 Adeno-associated virus - 6 Species 0.000 description 1
- 241001164823 Adeno-associated virus - 7 Species 0.000 description 1
- 241000649045 Adeno-associated virus 10 Species 0.000 description 1
- 241000649046 Adeno-associated virus 11 Species 0.000 description 1
- 241000649047 Adeno-associated virus 12 Species 0.000 description 1
- 241000300529 Adeno-associated virus 13 Species 0.000 description 1
- 108010000239 Aequorin Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 1
- 244000303258 Annona diversifolia Species 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 102100024775 Beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase Human genes 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 208000031636 Body Temperature Changes Diseases 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 240000001432 Calendula officinalis Species 0.000 description 1
- 235000005881 Calendula officinalis Nutrition 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000000989 Complement System Proteins Human genes 0.000 description 1
- 108010069112 Complement System Proteins Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 108010013369 Enteropeptidase Proteins 0.000 description 1
- 102100029727 Enteropeptidase Human genes 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108091092566 Extrachromosomal DNA Proteins 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000724791 Filamentous phage Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101710177291 Gag polyprotein Proteins 0.000 description 1
- 241001663880 Gammaretrovirus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 241000282575 Gorilla Species 0.000 description 1
- 241000288105 Grus Species 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 241001272567 Hominoidea Species 0.000 description 1
- 101000690301 Homo sapiens Aldo-keto reductase family 1 member C4 Proteins 0.000 description 1
- 101001037140 Homo sapiens Immunoglobulin heavy variable 3-23 Proteins 0.000 description 1
- 101001138127 Homo sapiens Immunoglobulin kappa variable 1-13 Proteins 0.000 description 1
- 101001138089 Homo sapiens Immunoglobulin kappa variable 1-39 Proteins 0.000 description 1
- 101001116548 Homo sapiens Protein CBFA2T1 Proteins 0.000 description 1
- 241000701109 Human adenovirus 2 Species 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241000282620 Hylobates sp. Species 0.000 description 1
- 241000282596 Hylobatidae Species 0.000 description 1
- 102000009438 IgE Receptors Human genes 0.000 description 1
- 108010073816 IgE Receptors Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- 102100040220 Immunoglobulin heavy variable 3-23 Human genes 0.000 description 1
- 102100020772 Immunoglobulin kappa variable 1-13 Human genes 0.000 description 1
- 102100020910 Immunoglobulin kappa variable 1-39 Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 102000008192 Lactoglobulins Human genes 0.000 description 1
- 108010060630 Lactoglobulins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 101000800524 Loxosceles intermedia Translationally-controlled tumor protein homolog Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 101710150582 Monomethylamine corrinoid protein 1 Proteins 0.000 description 1
- 101000851936 Mus musculus Endothelial PAS domain-containing protein 1 Proteins 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 102000008763 Neurofilament Proteins Human genes 0.000 description 1
- 108010088373 Neurofilament Proteins Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 230000004989 O-glycosylation Effects 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 241000701945 Parvoviridae Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 208000005228 Pericardial Effusion Diseases 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 230000010799 Receptor Interactions Effects 0.000 description 1
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 1
- 208000004756 Respiratory Insufficiency Diseases 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000710961 Semliki Forest virus Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000710960 Sindbis virus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- PJANXHGTPQOBST-VAWYXSNFSA-N Stilbene Natural products C=1C=CC=CC=1/C=C/C1=CC=CC=C1 PJANXHGTPQOBST-VAWYXSNFSA-N 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 239000012163 TRI reagent Substances 0.000 description 1
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 101710195626 Transcriptional activator protein Proteins 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000007818 agglutination assay Methods 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- GZCGUPFRVQAUEE-KVTDHHQDSA-N aldehydo-D-mannose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-KVTDHHQDSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- CEGOLXSVJUTHNZ-UHFFFAOYSA-K aluminium tristearate Chemical compound [Al+3].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CEGOLXSVJUTHNZ-UHFFFAOYSA-K 0.000 description 1
- 229940063655 aluminum stearate Drugs 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 108010087667 beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 230000004993 binary fission Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004952 blastocoel Anatomy 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 210000002939 cerumen Anatomy 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 210000001268 chyle Anatomy 0.000 description 1
- 210000004913 chyme Anatomy 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000008119 colloidal silica Substances 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000003271 compound fluorescence assay Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000012926 crystallographic analysis Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000002726 cyst fluid Anatomy 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- NIJJYAXOARWZEE-UHFFFAOYSA-N di-n-propyl-acetic acid Natural products CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- GXGAKHNRMVGRPK-UHFFFAOYSA-N dimagnesium;dioxido-bis[[oxido(oxo)silyl]oxy]silane Chemical compound [Mg+2].[Mg+2].[O-][Si](=O)O[Si]([O-])([O-])O[Si]([O-])=O GXGAKHNRMVGRPK-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 108700004025 env Genes Proteins 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- IINNWAYUJNWZRM-UHFFFAOYSA-L erythrosin B Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 IINNWAYUJNWZRM-UHFFFAOYSA-L 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthrene Natural products C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 229960002449 glycine Drugs 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 125000005179 haloacetyl group Chemical group 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000012188 high-throughput screening assay Methods 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 102000054751 human RUNX1T1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002349 hydroxyamino group Chemical group [H]ON([H])[*] 0.000 description 1
- 239000012216 imaging agent Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000004201 immune sera Anatomy 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 230000009851 immunogenic response Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 108091008042 inhibitory receptors Proteins 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000000832 lactitol Substances 0.000 description 1
- 235000010448 lactitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-JVCRWLNRSA-N lactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-JVCRWLNRSA-N 0.000 description 1
- 229960003451 lactitol Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- DLBFLQKQABVKGT-UHFFFAOYSA-L lucifer yellow dye Chemical compound [Li+].[Li+].[O-]S(=O)(=O)C1=CC(C(N(C(=O)NN)C2=O)=O)=C3C2=CC(S([O-])(=O)=O)=CC3=C1N DLBFLQKQABVKGT-UHFFFAOYSA-L 0.000 description 1
- 238000007422 luminescence assay Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 239000000391 magnesium silicate Substances 0.000 description 1
- 229940099273 magnesium trisilicate Drugs 0.000 description 1
- 229910000386 magnesium trisilicate Inorganic materials 0.000 description 1
- 235000019793 magnesium trisilicate Nutrition 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229960001855 mannitol Drugs 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 description 1
- 210000005060 membrane bound organelle Anatomy 0.000 description 1
- 210000004914 menses Anatomy 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 230000004879 molecular function Effects 0.000 description 1
- 238000000302 molecular modelling Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 210000005044 neurofilament Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000007935 oral tablet Substances 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 210000001819 pancreatic juice Anatomy 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 108010087558 pectate lyase Proteins 0.000 description 1
- 210000004912 pericardial fluid Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 238000011338 personalized therapy Methods 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 210000004910 pleural fluid Anatomy 0.000 description 1
- 101150088264 pol gene Proteins 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 229920001993 poloxamer 188 Polymers 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000002600 positron emission tomography Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001566 pro-viral effect Effects 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000004908 prostatic fluid Anatomy 0.000 description 1
- 229950008679 protamine sulfate Drugs 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 210000004915 pus Anatomy 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 201000004193 respiratory failure Diseases 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 235000002020 sage Nutrition 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 210000002374 sebum Anatomy 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 208000013220 shortness of breath Diseases 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- PJANXHGTPQOBST-UHFFFAOYSA-N stilbene Chemical compound C=1C=CC=CC=1C=CC1=CC=CC=C1 PJANXHGTPQOBST-UHFFFAOYSA-N 0.000 description 1
- 235000021286 stilbenes Nutrition 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000003461 sulfonyl halides Chemical class 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 238000003325 tomography Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000012285 ultrasound imaging Methods 0.000 description 1
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 1
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229940096973 urethral suppository Drugs 0.000 description 1
- 239000006217 urethral suppository Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- MSRILKIQRXUYCT-UHFFFAOYSA-M valproate semisodium Chemical compound [Na+].CCCC(C(O)=O)CCC.CCCC(C([O-])=O)CCC MSRILKIQRXUYCT-UHFFFAOYSA-M 0.000 description 1
- 229960000604 valproic acid Drugs 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
- 210000004916 vomit Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- 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
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- HRF Histamine-releasing factor
- This disclosure provides antibodies and antigen binding fragments thereof that bind to HFR or a fragment thereof.
- This disclosure also provides compositions for use of the antibodies and fragments thereof, and compositions for manufacturing same.
- the provided antibodies or fragments thereof can be used to diagnose, treat, or monitor allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation. Other uses, diagnostic or therapeutic, also are described herein.
- exemplary antibodies that bind to HRF or a fragment thereof bind to HRF N19, HRF GST-N19, or HRF-2CA, are disclosed herein, or an equivalent of each thereof.
- the fragment of an HRF comprises, or consists essentially of, or yet further consists of GST-N19 or N19.
- the HRF or fragment thereof is human HRF or a fragment thereof.
- a designated antibody and its equivalent comprises the same CDRs.
- the antibody is a rabbit monoclonal antibody.
- humanized antibodies are also provided. [0008] Additionally, recombinant anti-HRF antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the disclosure.
- Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques such as, for example, the methods herein as well as described in U.S. Pat. No.7,112,421; Better et al. (1988) Science 240:1041-1043; or Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443.
- the antibodies and fragments thereof are described by specific CDR amino acid sequences and comprise an antibody or an antigen binding fragment thereof comprising one or more of as described herein.
- the antibodies and antigen binding domains thereof can also be described by variable domains as described herein and equivalents thereof.
- the equivalent is at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or more identical across the full length of the polypeptide.
- the Clustal Omega alignment program can be used to determine percent identity. Other methods are known in the art.
- an antibody or an antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more or all three of the following sequences: a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; or a heavy-chain CDR 3 (HCDR3) that comprise the amino acid sequence ARVSASYTSDGDAIIHSFAL.
- CDR heavy-chain complementarity determining region 1
- HCDR2 heavy-chain CDR 2
- HCDR3 heavy-chain CDR 3
- an antibody or an antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more or all three of the following sequences: a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS or a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG.
- LC light-chain complementarity determining region
- an antibody or antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more of: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; b) a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; c) a heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; d) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; e) a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; or f) a light-chain CDR) 3 (LCDR3) that comprises
- antibody or antigen binding fragment thereof comprising: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; a heavy- chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; and b) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; and a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGS
- an antibody or an antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more of the following amino acid sequences: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; b) QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; c) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS;
- an antibody or antigen binding fragment thereof comprising, consisting of, or consisting essentially of: a) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVK; b) a heavy chain variable region comprising the amino acid sequence: QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTA
- an antibody or an antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more of the following amino acid sequences: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVK; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; c) AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; c) AQV
- an antibody or antigen binding fragment comprising, consisting of, or consisting essentially of: a) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVK; b) a heavy chain variable region comprising the amino acid sequence: QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTST
- an antibody or antigen binding fragment comprising, consisting of, or consisting essentially of a heavy chain region comprising one of the following amino acid sequences: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLP EPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPAT NTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVD VSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKE FKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSV
- an antibody or antigen binding fragment comprising, consisting of, or consisting essentially of a light chain region comprising one of the following amino acid sequences: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVV QSFNRGDC; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAP
- a humnaized antibody or antigen binding fragment that comprises, or consists of, or consists essentially of one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises, consists of or consists essentially of the amino acid sequence: QSSQSVYDNNNLA; a light-chain (CDR) 2 (LCDR2) that comprises, consists of or consists essentially of the amino acid sequence: DASKLPS; or a light-chain CDR 3 (LCDR3) that comprises, consists of or consists essentially of the amino acid sequence: AGAVSGSNV.
- LC light-chain
- CDR1 light-chain complementarity determining region
- LCDR2 light-chain 2
- LCDR3 light-chain CDR 3
- a humanized antibody or antigen binding fragment thereof comprising, consisting of, or consisting essentially of: [0022] a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises, consists of or consists essentially of the amino acid sequence TVSGFSLSSGAVS; a heavy-chain CDR 2 (HCDR2) that comprises, consists of or consists essentially the amino acid sequence: IGVISSRDIAYFATWAKG; a heavy-chain CDR 3 (HCDR3) that comprises, consists of or consists essentially of the amino acid sequence ARVSASYTSDGDAIIHSFAL.
- HCDR1 heavy-chain complementarity determining region 1
- HCDR2 heavy-chain CDR 2
- HCDR3 heavy-chain CDR 3
- a humanized antibody or an antigen binding fragment thereof comprising, consists of, or consists essentially of a light-chain variable region comprising, consisting of, or consisting essentially of the following amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
- the humanized antibody or an antigen binding fragment thereof comprises, consists of or consists essentially of a heavy-chain variable region comprising, consisting of, or consisting essentially of the following amino acid sequence: [0025] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYT
- the antibody or antigen binding fragment thereof specifically recognizes and binds to one or more of: HRF, HRF-2CA or N19 or GST-N19, or an immunogenic fragment thereof.
- the HRF, HRF-2CA, N19 or GST-N19 are human proteins, fragments or variants thereof. Examples of such are provided herein.
- the HRF comprises or consists of the amino acid sequence of SEQ ID NO: 1.
- the antibody or antigen binding fragment is isolated, purified or recombinant.
- the antibody or antigen binding fragment is monospecific.
- the antibody or antigen binding fragment is multispecific, such as bispecific, e.g., binding to two or more epitopes.
- the two or more epitopes are all epitopes of an HRF or a fragment thereof.
- at least one of the two or more epitopes is an epitope of an HRF or a fragment thereof.
- at least one of the two or more epitopes are of a protein other than an HRF.
- provided is an antibody or antigen binding fragment thereof that competes with any one of an antibody or antigen binding fragment as disclosed herein for binding to HRF thereof, or a fragment of the HRF.
- the antibody or antigen binding fragment comprises a fragment crystallizable region (Fc region).
- the Fc region is a human Fc region.
- the Fc region comprises, or alternatively consists essentially of, or yet further consists of one or more of: an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgM Fc region, or an IgE Fc region.
- the Fc region comprises, or alternatively consists essentially of, or yet further consists of one or more of: an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG4 Fc region.
- the antibody or antigen binding fragment is post- translationally modified optionally glycosylated, hydroxylated, methylated, lapidated, acetylated, SUMOylated, phosphorylated, PEGylated, or any combination thereof.
- the antibody or antigen binding fragment further comprises a detectable or purification marker.
- the antibodies or fragments thereof as described herein can be used for various in vitro molecular biology applications such as, enzyme-linked immunosorbent assays (ELISA), Western blots, immunohistochemistry, immunocytochemistry, flow cytometry and fluorescence-activated cell sorting (FACS), immunoprecipitation, or enzyme-linked immunospot assays.
- ELISA enzyme-linked immunosorbent assays
- FACS fluorescence-activated cell sorting
- the antibodies or fragments thereof can be packaged in kits with or without additional reagents known to those of skill in the art for practicing any of the molecular biology techniques as disclosed herein.
- the polynucleotides are useful to replicate or detect encoding polynucleotides.
- Host cells are useful to replicate the polynucleotides or encode the antibodies or fragments thereof. These can be provided alone or in a composition, optionally comprising a pharmaceutically acceptable carrier.
- a polynucleotide encoding the antibody or antigen binding fragment thereof In another aspect, provided herein is a vector comprising, consisting of, or consisting essentially of the polynucleotide encoding the antibody or antigen binding fragment thereof.
- a cell comprising, consisting of, or consisting essentially of one or more of the antibody or antigen binding fragment thereof, the polynucleotide encoding the antibody or antigen binding fragment thereof, or the vector comprising the polynucleotide.
- a hybridoma expressing the antibody or antigen binding fragment thereof.
- a method of producing the antibody or antigen binding fragment thereof comprising culturing a cell comprising a polynucleotide encoding he antibody or antigen binding fragment thereof.
- the present disclosure provides a method of preventing or treating a disease, such as allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, in a subject in need thereof, comprising, or consisting essentially of, or yet further consisting of administering to the subject, optionally a therapeutically or prophylactically effective amount of, a pharmaceutical composition comprising, or consisting essentially of, or yet further consisting of one or more of the antibodies or antigen binding fragments as described herein.
- a disease such as allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation
- Such a method can comprise, or consists essentially of, or yet further consists of administration of any dose of the antibodies described herein effective for ameliorating or treating symptoms of allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation.
- Methods for determining if the disease has been treated or prevented are known in the art and include a reduction in symptoms, or severity of symptoms or the presence of neutralizing antibodies in the subject being treated.
- FIG.1A Anti-N19 Fabs inhibited interactions between C38-2 IgE (concentrations indicated) and immobilized mouse HRF by ELISA.
- D-A10 and H8-6 are Fabs that, respectively, bind or do not to HRF, serving as a positive or a negative control.
- FIG.1B Purified anti-N19 mAb SPF7-1 and isotype control IgG1 were analyzed by SDS-PAGE under reducing and nonreducing conditions and stained with Coomassie brilliant blue G-250. Mr, molecular weight marker in kilodaltons.
- FIG.1C The anti-N19 mAb SPF7-1 suppressed hypothermia induced by OVA gavage in OVA-sensitized mice.
- FIGS.2A – 2C PEGylated N19 peptide suppressed OVA-induced food allergy in mice.
- FIG.2A List of the tested N19 peptides and recombinant HRF inhibitors.
- B,C N19-PEG peptide suppressed diarrhea (FIG.2B) and hypothermia (FIG.2C) induced by OVA gavage in OVA-sensitized mice.
- FIGS.3A-3B Body temperature changes compared between SPF7, L2H1, and L2H2 humanized antibodies indicate L2H1 and L2H2 clones exhibiting inhibitory effects on ovalbumin (OVA)-induced food allergy (FIG.3A).
- OVA ovalbumin
- FIG.3B MMCP-1 levels in serum were quantified by an ELISA (enzyme-linked immunosorbent assay) kit from BioLegend (a San Diego company), according to the manufacturer's instructions (FIG.3B).
- FIG.4 Binding of humanized SPF7 clones was compared to that of the parent rabbit IgG SPF7.
- Microtiter wells were coated with a recombinant HRF protein at 1 ⁇ g/mL in PBS at 4 ⁇ C overnight. The wells were washed with PBS and blocked with 1% BSA/PBS. The IgGs were serially diluted and incubated with the coated antigen at RT for 1 h. The bound IgGs were detected with peroxidase conjugated goat anti-human IgG Fc ⁇ specific or anti-rabbit IgG Fc ⁇ specific antibodies. The humanized clones showed equivalent binding to the antigen as the parent rabbit IgG.
- FIG.4 shows EC50 maximal effective concentration values demonstrating that humanized antibodies to HRF are similar to that of the parent antibody SPF7.
- DETAILED DESCRIPTION Definitions [0042] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and/or separating the subject matter described. [0043] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of this invention will be limited only by the appended claims.
- compositions, and methods include the recited elements, but not exclude others.
- Consisting essentially of when used to define compounds, compositions, and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.
- comparative terms as used herein can refer to certain variation from the reference.
- such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 folds, or about 3 folds, or about 4 folds, or about 5 folds, or about 6 folds, or about 7 folds, or about 8 folds, or about 9 folds, or about 10 folds, or about 20 folds, or about 30 folds, or about 40 folds, or about 50 folds, or about 60 folds, or about 70 folds, or about 80 folds, or about 90 folds, or about 100 folds or more higher than the reference.
- such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference. [0051] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof.
- a range includes each individual member.
- “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
- “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
- “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
- the phrase “therapeutically effective” is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder or on the effecting of a clinical endpoint.
- an antibody or antigen binding fragment thereof is administered to a subject in a therapeutically effective amount.
- the term “therapeutically acceptable” refers to those compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
- an antibody or antigen binding fragment thereof is administered to a subject in a therapeutically acceptable amount.
- the terms “first” “second” “third” “fourth” or similar in a component name are used to distinguish and identify more than one component sharing certain identity in their names. For example, “first antibody” and “second antibody” are used to distinguishing two antibodies.
- antibody includes both full-length immunoglobulins and antibody fragments that bind to the same antigens. Non-limiting examples include a monoclonal, polyclonal, chimeric, humanized, or single chain antibody. In one embodiment, the disclosure provides an isolated antibody and antigen binding fragments thereof that bind to HRF, e.g., human HRF or a fragment thereof. In some embodiments, the fragment is an immunogenic fragment.
- isolated as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule.
- isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
- isolated is also used herein to refer to polypeptides, proteins and/or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
- isolated means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
- an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype.
- a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require “isolation” to distinguish it from its naturally occurring counterpart.
- the term “engineered” or “recombinant” refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain or the parental host strain of the referenced species. In some embodiments, the term “engineered” or “recombinant” refers to being synthetized by human intervention.
- the term “recombinant protein” refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous HRF.
- polynucleotide “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
- polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
- a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
- modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
- the sequence of nucleotides can be interrupted by non-nucleotide components.
- a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
- the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
- a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
- A adenine
- C cytosine
- G guanine
- T thymine
- U uracil
- polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
- complementary sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other.
- the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other.
- sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other.
- a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide.
- Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.
- a “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
- the term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins.
- expression refers to the process by which polynucleotides are transcribed into mRNA or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins.
- polynucleotide is derived from genomic DNA
- expression may include splicing of the mRNA in a eukaryotic cell.
- a “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
- the gene product may refer to an mRNA or other RNA, such as an interfering RNA, generated when a gene is transcribed.
- encode refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed to produce the mRNA for the polypeptide or a fragment thereof, and optionally translated to produce the polypeptide or a fragment thereof.
- the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
- an amino acid sequence coding sequence refers to a nucleotide sequence encoding the amino acid sequence.
- Under transcriptional control which is also used herein as “directing expression of” or any grammatical variation thereof, is a term well understood in the art and indicates that transcription and optionally translation of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription.
- “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.
- “directing the replication of” or any grammatical variation thereof is a term well understood in the art and indicates that replication of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to a regulatory sequence, such as an origin of replication or a primer.
- a regulatory sequence such as an origin of replication or a primer.
- a promoter is an example of an expression control element or a regulatory sequence.
- Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. Polymerase II and III are examples of promoters.
- a regulatory sequence is bidirectional, i.e., acting as a regulatory sequence for the coding sequences on both sides of the regulatory sequence. Such bidirectional regulatory sequences may comprises, or consists essentially of, or consists of a bidirectional promoter (see for example Trinklein ND, et al. An abundance of bidirectional promoters in the human genome. Genome Res.2004 Jan;14(1):62-6).
- promoter refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example.
- a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
- promoters include the EF1alpha promoter and the CMV promoter.
- the EF1alpha sequence is known in the art (see, e.g., addgene.org/11154/sequences/; ncbi.nlm.nih.gov/nuccore/J04617, each last accessed on March 13, 2019, and Zheng and Baum (2014) Int’l. J. Med. Sci. 11(5):404-408).
- An enhancer is a regulatory element that increases the expression of a target sequence.
- a “promoter/enhancer” is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions.
- the enhancer/promoter may be "endogenous” or “exogenous” or “heterologous.”
- An “endogenous" enhancer/promoter is one which is naturally linked with a given gene in the genome.
- an “exogenous” or “heterologous” enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
- the term “enhancer”, as used herein, denotes sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed.
- An enhancer may enhance transcription from a single promoter or simultaneously from more than one promoter.
- any truncated, mutated or otherwise modified variants of a wild-type enhancer sequence are also within the above definition.
- the term “vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and optionally integrate into the target cell’s genome.
- Non-limiting examples of vectors include a plasmid, a nanoparticle, a liposome, a virus, a cosmid, a phage, a BAC, a YAC, etc.
- plasmid vectors may be prepared from commercially available vectors.
- viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art.
- the viral vector is a lentiviral vector.
- the viral vector is a retroviral vector.
- a “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Many plasmids are commercially available for such uses.
- the gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
- MCS multiple cloning site
- Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produce HRFs to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass- producing a gene or the protein it then codes for.
- a “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
- the DNA viruses constitute classes I and II.
- the RNA viruses and retroviruses make up the remaining classes.
- Class III viruses have a double-stranded RNA genome.
- Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA
- Class V viruses have a negative single-stranded RNA genome used as a template for mRNA synthesis.
- Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
- examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
- Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy.
- Multiplicity of infection refers to the number of viral particles that are added per cell during infection.
- RNA usually a dimer RNA comprising a cap at the 5’ end and a polyA tail at the 3’ end flanked by LTRs
- proteins such as a protease.
- U.S. Patent No. 6,924,123 discloses that certain retroviral sequence facilitate integration into the target cell genome. This patent teaches that each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes.
- LTRs long terminal repeats
- the LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral genes.
- Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
- the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5.
- U3 is derived from the sequence unique to the 3' end of the RNA.
- R is derived from a sequence repeated at both ends of the RNA
- U5 is derived from the sequence unique to the 5'end of the RNA.
- gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
- the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
- RT reverse transcriptase
- I integrase
- the vector RNA genome is expressed from a DNA construct encoding it, in a host cell.
- the components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the "packaging system", which usually includes either or both of the gag/pol and env genes) expressed in the host cell.
- the set of sequences required for the production of the viral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways.
- AAV adeno-associated virus
- AAV adeno-associated virus
- AAV refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered, AAV serotypes are known in the art.
- Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant or synthetic serotypes, e.g., AAV-DJ and AAV PHP.B.
- the AAV particle comprises, alternatively consists essentially of, or yet further consists of three major viral proteins: VP1, VP2 and VP3.
- the AAV refers to of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. These vectors are commercially available or have been described in the patent or technical literature.
- “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
- the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
- the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
- a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. [0085] Hybridization reactions can be performed under conditions of different “stringency”.
- a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature.
- a moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC.
- Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art.
- a non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg 2+ normally found in a cell.
- Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
- Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
- high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1x SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1x SSC, 0.1x SSC, or deionized water.
- hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
- SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
- hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides
- the reaction is called “annealing” and those polynucleotides are described as “complementary.”
- a double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
- “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
- Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.
- the identity is calculated between two peptides or polynucleotides over their full-length, or over the shorter sequence of the two, or over the longer sequence of the two.
- a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in Ausubel et al. eds.
- BLAST BLAST
- the polynucleotide as disclosed herein is a RNA or an analog thereof. In some embodiments, the polynucleotide as disclosed herein is a DNA or an analog thereof. In some embodiments, the polynucleotide as disclosed herein is a hybrid of DNA and RNA or an analog thereof. [0091] In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide encodes the same sequence encoded by the reference.
- an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide hybridizes to the reference, a complement reference, a reverse reference, or a reverse-complement reference, optionally under conditions of high stringency.
- an equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 70% sequence identity, or at least 75% sequence identity, or at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence, or alternatively at least 99 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively an equivalent nucleic acid hybridizes under conditions of high stringency to a reference polynucleotide or its complementary.
- the equivalent must encode the same protein or a functional equivalent of the protein that optionally can be identified through one or more assays described herein.
- the equivalent of a polynucleotide would encode a protein or polypeptide of the same or similar function as the reference or parent polynucleotide.
- the term “transduce” or “transduction” refers to the process whereby a foreign nucleotide sequence is introduced into a cell. In some embodiments, this transduction is done via a vector, viral or non-viral.
- protein protein
- peptide and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
- the subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
- a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence.
- amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
- amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence.
- Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST.
- the term “edit distance” refers to the minimum number of insertions, deletions or substitutions required to transform a first sequence of a first nucleotide or peptide into a second sequence of a second nucleotide or polypeptide. Accordingly, in some embodiments, the edit distance between two sequences can be presented by the minimum number of insertions, deletions or substitutions.
- Various tools are available for calculating an edit distance, such as a sequence alignment program aligning two sequences and noting the differences therebetween, including insertions, deletions or substitutions.
- the Clustal Omega alignment program is used to determine sequence identity.
- antibody collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits, llama and mice, as well as non-mammalian species, such as shark immunoglobulins.
- the term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10 3 M -1 greater, at least 10 4 M -1 greater or at least 10 5 M -1 greater than a binding constant for other molecules in a biological sample).
- the term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, murine or humanized non-primate antibodies), heteroconjugate antibodies (such as, bispecific antibodies).
- antibody refers to a single-chain variable fragment (scFv or ScFV).
- an antibody refers to more than one single-chain variable fragments (scFv or ScFV) linked with each other, optionally via a peptide linker or another suitable component as disclosed herein.
- an antibody is a monoclonal antibody.
- an antibody is a monospecific antibody or a multispecific antibody, such as a bispecific antibody or a trispecific antibody.
- the species of the antibody can be a human or non-human, e.g., mammalian.
- an antigen binding fragment of an antibody contains at least one variable domain optionally covalently linked to at least one constant domain.
- Non-limiting, exemplary configurations of variable and constant domains that are found within an antigen-binding fragment of an antibody of the present invention include: (i) VH- CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH—CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH- CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
- variable and constant domains are either directly linked to one another or are linked by a full or partial hinge or linker region.
- a hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
- an antigen binding fragment of an antibody of the present disclosure can comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed herein in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
- an epitope refers to contiguous or non-contiguous amino acid residues in an antigen, such as those adjacent to each other in a three-dimensional structure of the antigen, wherein those residues are recognized and bound by an antibody or another component of the immune system.
- the term “multispecific” refers to capability of binding to more than one epitopes or antigens which are different from each other.
- the term “multispecific” refers to comprising, or consisting essentially of, or consisting of more than one antigen binding sequences or antigen ligands, optionally linked together by a peptide linker or another component as disclosed herein.
- the term “multispecific” refers to comprising, or consisting essentially of, or consisting of more than one antigen binding sequences (such as scFv), optionally linked together by a peptide linker or another component as disclosed herein.
- the more than one (such as two) epitopes are located in the same antigen. Alternatively, the more than one (such as two) epitopes are from at least two antigens.
- the ligand refers a ligand of the antigen.
- a multispecific antibody comprises, or consists essentially of, or consists of at least two antigen binding sequences. In some embodiments, a multispecific antibody comprises, or consists essentially of, or consists of at least one antigen binding sequence and at least one ligand (such as a polypeptide comprising or consisting of a binding domain of the antigen’s receptor).
- a bispecific antibody refers to an antibody capable of binding to two epitopes or antigens which are different from each other.
- a bispecific antibody comprises, or consists essentially of, or consists of two antigen binding sequences or antigen ligands, optionally linked together by a peptide linker or another component as disclosed herein.
- a bispecific antibody comprises, or consists essentially of, or consists of two antigen binding sequences (such as scFv), optionally linked together by a peptide linker or another component as disclosed herein.
- a bispecific antibody comprises, or consists essentially of, or consists of one antigen binding sequence recognizing and binding the first epitope and one ligand recognizing and binding the antigen comprising the second epitope.
- the two epitopes are located in the same antigen.
- the two epitopes are from two antigens which are different from each other.
- the ligand refers to a ligand of the antigen, such as a polypeptide comprising or consisting of a binding domain of the antigen’s receptor.
- a bispecific antibody comprises, or consists essentially of, or consists of at least two antigen binding sequences.
- a bispecific antibody comprises, or consists essentially of, or consists of at least one antigen binding sequence and at least one ligand.
- the term “monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
- an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
- Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains").
- the heavy and the light chain variable regions specifically bind the antigen.
- Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions" or "CDRs".
- framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference).
- the Kabat database is now maintained online.
- the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
- the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, largely adopts a ⁇ -sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure.
- framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
- the CDRs are primarily responsible for binding to an epitope of an antigen.
- the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located (heavy chain regions labeled CDRH or HCDR and light chain regions labeled CDRL or LCDR).
- CDRH or HCDR and light chain regions labeled CDRL or LCDR heavy chain regions labeled CDRH or HCDR and light chain regions labeled CDRL or LCDR.
- a HCDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found
- a LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
- a single-chain variable fragment also referred to herein as a fragment or an antigen binding fragment of an antibody
- scFv or ScFV is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, optionally connected with a short linker peptide of about 10 to about 25 amino acids.
- the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.
- the HRF retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
- a fragment crystallizable (Fc) region refers to the tail region of an antibody that stabilizes the antibody, such as a bispecific antibody, and optionally interacts with (such as binds) an Fc receptor on an immune cell or on a platelet or that binds a complement protein.
- the polypeptide or an equivalent thereof can be followed by an additional 50 amino acids, or alternatively about 40 amino acids, or alternatively about 30 amino acids, or alternatively about 20 amino acids, or alternatively about 10 amino acids, or alternatively about 5 amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the carboxy- terminus (C-terminus).
- polypeptide or an equivalent thereof can further comprises an additional 50 amino acids, or alternatively about 40 amino acids, or alternatively about 30 amino acids, or alternatively about 20 amino acids, or alternatively about 10 amino acids, or alternatively about 5 amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the amine-terminus (N-terminus).
- An equivalent of a reference polypeptide comprises, consists essentially of, or alternatively consists of an polypeptide having at least 80%, or at least 85 %, or at least 90%, or at least 95%, or at least about 96%, or at least 97%, or at least 98%, or at least 99% amino acid identity to the reference polypeptide (as determined, in one aspect using the Clustal Omega alignment program), such as the antibody or antigen binding fragment thereof as disclosed herein, or a polypeptide that is encoded by a polynucleotide that hybridizes under conditions of high stringency to the complementary sequence of a polynucleotide encoding the reference polypeptide, such as an antibody or antigen binding fragment thereof as disclosed herein, optionally wherein conditions of high stringency comprises incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1x SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1x SSC,
- Alternative embodiments include one or more of the CDRs (e.g., CDR1, CDR2, CDR3) from the LC variable region substituted with appropriate CDRs from other antibody CDRs, or an equivalent of each thereof.
- the CDR1 and CDR2 from the LC variable region can be combined with the CDR3 of another antibody’s LC variable region, and in some aspects, can include an additional 50 amino acids, or alternatively about 40 amino acids, or alternatively about 30 amino acids, or alternatively about 20 amino acids, or alternatively about 10 amino acids, or alternatively about 5 amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the carboxy-terminus.
- the term “equivalent” or “biological equivalent” of an antibody means the ability of the antibody to selectively bind its epitope protein or a fragment thereof as measured by ELISA or other suitable methods is substantively maintained, for example, at a level of at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or more.
- Biologically equivalent antibodies include, but are not limited to, those antibodies, peptides, antibody fragments, antibody variant, antibody derivative and antibody mimetics that bind to the same epitope as the reference antibody.
- a first sequence (nucleic acid sequence or amino acid) is compared to a second sequence, and the identity percentage or edit distance between the two sequences can be calculated.
- the first sequence can be referred to herein as an equivalent and the second sequence can be referred to herein as a reference sequence.
- the identity percentage is calculated based on the full- length sequence of the first sequence. In other embodiments, the identity percentage is calculated based on the full-length sequence of the second sequence.
- an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity, or at least about 85 % homology or identity, or alternatively at least about 90 % homology or identity, or alternatively at least about 95 % homology or identity, or alternatively 98 % or 99% homology or identity (in one aspect, as determined using the Clustal Omega alignment program) and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
- an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complementary sequence.
- an antibody as disclosed herein comprises, or consists essentially of, or yet further consists of an anybody variant.
- the term “antibody variant” intends to include antibodies produced in a species other than a mouse. It also includes antibodies containing post-translational modifications to the linear polypeptide sequence of the antibody or a fragment thereof. It further encompasses fully human antibodies.
- an antibody as disclosed herein comprises, or consists essentially of, or yet further consists of an antibody derivative.
- antibody derivative is intended to encompass molecules that bind an epitope as defined above and which are modifications or derivatives of a native monoclonal antibody of this disclosure.
- Derivatives include, but are not limited to, for example, bispecific, multispecific, heterospecific, trispecific, tetraspecific, multispecific antibodies, diabodies, chimeric, recombinant and humanized.
- the term “specific binding” or “binding” means the contact between an antibody and an antigen with a binding affinity of at least 10 ⁇ 6 M.
- antibodies bind with affinities of at least about 10 ⁇ 7 M, and preferably at least about 10 ⁇ 8 M, at least about 10 ⁇ 9 M, at least about 10 ⁇ 10 M, at least about 10 ⁇ 11 M, or at least about 10 ⁇ 12 M.
- the term “antigen” refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor.
- Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins.
- antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.
- the antigen as referred to herein is HRF or an immunogenic fragment.
- Exemplary mammalian HRF sequences include human and non-human HRF sequences.
- Ig immunoglobulin
- HRF binds are associated with immune disorders and diseases such as those associated with allergies (food or other antigens), asthma, hypersensitivity reactions and inflammation.
- the term “equivalent” in the context of describing a peptide, a polypeptide, or a protein encompass the wild-type sequence, sequences of variants, and sequences comprising one or more modifications.
- an equivalent of a HRF monomer can encompass a HRF variant as provided in WO 2020/102108, published May 22, 2020.
- HRF monomer of SEQ ID NO: 4 can also encompass at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4 published in WO 2020/102108, published May 22, 2020.
- HRF monomer of SEQ ID NO: 4 as provided in WO 2020/102108, published May 22, 2020 can further encompass at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4 published in WO 2020/102108, published May 22, 2020, while retaining one or more modifications, optionally amino acid substitution(s) at C28 and/or C172 according to SEQ ID NO: 4 published in WO 2020/102108, published May 22, 2020.
- antigen of a binding moiety such as an antibody, an antigen binding fragment thereof, can be provided herein in a format of “antigen” followed by the binding moiety (such as an anti-HRF antibody), or having “anti” or “anti-” before the antigen and the binding moiety after the antigen (such as an anti-HRF antibody), or the binding moiety followed by “to” or “directed to” and then the antigen (such as an antibody to HRF).
- a fragment of a protein can be an immunogenic fragment.
- immunogenic fragment refers to such a polypeptide fragment, which at least partially retains the immunogenicity of the protein from which it is derived.
- the immunogenic fragment is at least about 3 amino acid (aa) long, or at least about 4 aa long, or at least about 5 aa long, or at least about 6 aa long, or at least about 7 aa long, or at least about 8 aa long, or at least about 9 aa long, or at least about 10, aa long, or at least about 15, aa long, or at least about 20 aa long, or at least about 25 aa long, or at least about 30 aa long, or at least about 35 aa long, or at least about 40 aa long, or at least about 50 aa long, or at least about 60 aa long, or at least about 70 aa long, or at least about 80 aa long, or at least about 90 aa long, or at least about 100 aa long, or at least about 120 aa long, or at least about 150 aa long, or at least about 200, or longer.
- an immunogenic fragment of HRF protein comprises, or alternatively consists essentially of, or yet further consists of antibodies and subsequences thereof that bind to a HRF/TCTP sequence that includes or consists of a region of HRF that binds to an Ig, such as an IgE.
- a sequence of HRF to which antibodies or subsequences thereof bind include or consist of amino acids (MIIYRDLISHDEMFSDIYK (HRF N19)) or a GST fusion of such (GST- MIIYRDLISHDEMFSDIYK (HRF GST-N19)) or the amino acids (QETSFTKEAYKKYIKDYMKSIKGKLEEQRPERVKPFMTGAAEQIKHILANFKNYQFF IGEN) MNP of mammalian HRF, or an equivalent of each thereof.
- Such antibodies can also bind to any subsequence of the HRF/TCTP sequence that includes or consists of a region of HRF that binds to an Ig, such as an IgE.
- a subsequence is a portion of amino acids 1-19 (MIIYRDLISHDEMFSDIYK (HRF N19) or a GST fusion of such (GST- MIIYRDLISHDEMFSDIYK (HRF GST-N19)) or a portion of amino acids (QETSFTKEAYKKYIKDYMKSIKGKLEEQRPERVKPFMTGAAEQIKHILANFKNYQFF IGEN) MNP of mammalian HRF, or a portion of (MIIYRDLISHDEMFSDIYKIREIADGLCLEVEGKMVSRTEGNIDDSLIGGNASAEGPE GEGTESTVITGVDIVMNHHLQETSFTKEAYKKYIKDYMKSIKGKLEEQRPERVKPFM TGAA
- the terms “antigen binding fragment,” “fragment,” and “antibody fragment” are used interchangeably to refer to any fragment that comprises a portion of a full-length antibody, generally at least the antigen binding portion or the variable region thereof.
- antibody fragments include, but are not limited to, diabodies, single-chain antibody molecules, multi-specific antibodies, Fab, Fab’, F(ab') 2 , Fv or scFv.
- the term “antigen binding domain” refers to any protein or polypeptide domain that can specifically bind to an antigen target.
- Epitopes typically are short amino acid sequences, e.g. about five to 15 amino acids in length.
- Epitopes can be contiguous or non-contiguous.
- a non-contiguous amino acid sequence forms due to protein folding.
- an epitope can include a non-contiguous amino acid sequence, such as a 5 amino acid sequence and an 8 amino acid sequence, which are not contiguous with each other, but form an epitope due to protein folding.
- Techniques for identifying epitopes are known to the skilled artisan and include screening overlapping oligopeptides for binding to antibody (for example, U.S. Patent No.4,708,871), phage display peptide library kits, which are commercially available for epitope mapping (New England BioLabs).
- Epitopes may also be identified by inference when epitope length peptide sequences are used to immunize animals from which antibodies that bind to the peptide sequence are obtained and can be predicted using computer programs, such as BEPITOPE (Odorico et al., J. Mol. Recognit.16:20 (2003)) [00126] Methods of producing polyclonal and monoclonal antibodies are known in the art. For example, HRF, or a subsequence thereof, or an immunogenic fragment thereof, optionally conjugated to a carrier such as keyhole limpet hemocyanin (KLH) or ovalbumin (e.g., BSA), or mixed with an adjuvant such as Freund’s complete or incomplete adjuvant, and used to immunize an animal.
- KLH keyhole limpet hemocyanin
- BSA ovalbumin
- an adjuvant such as Freund’s complete or incomplete adjuvant
- splenocytes from immunized animals that respond to HRF can be isolated and fused with myeloma cells. Monoclonal antibodies produced by the hybridomas can be screened for reactivity with HRF or an immunogenic fragment thereof.
- Animals that may be immunized include mice, rats, rabbits, goats, sheep, cows or steer, guinea pigs or primates. Initial and any optional subsequent immunization may be through intravenous, intraperitoneal, intramuscular, or subcutaneous routes. Subsequent immunizations may be at the same or at different concentrations of HRF, or a subsequence thereof, preparation, and may be at regular or irregular intervals.
- Human antibodies can be produced by immunizing human transchromosomic KM mice TM (WO 02/43478) or HAC mice (WO 02/092812). KM mice TM and HAC mice express human immunoglobulin genes. Using conventional hybridoma technology, splenocytes from immunized mice that were high responders to the antigen can be isolated and fused with myeloma cells. A monoclonal antibody can be obtained that binds to the antigen. An overview of the technology for producing human antibodies is described in Lonberg and Huszar (Int. Rev. Immunol.13:65 (1995)).
- Transgenic animals with one or more human immunoglobulin genes that do not express endogenous immunoglobulins are described, for example in, U.S. Patent No.5,939,598. Additional methods for producing human polyclonal antibodies and human monoclonal antibodies are described (see, e.g., Kuroiwa et al., Nat. Biotechnol.20:889 (2002); WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S.
- Antibodies can also be generated using other techniques including hybridoma, recombinant, and phage display technologies, or a combination thereof (see U.S.
- Patent Nos.4,902,614, 4,543,439, and 4,411,993 see, also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.1988).
- Antibodies of the disclosure and disclosure methods employing antibodies include mammalian, primatized, humanized, fully human antibodies and chimeras.
- a mammalian antibody is an antibody produced by a mammal, transgenic or non-transgenic, or a non-mammalian organism engineered to produce a mammalian antibody, such as a non- mammalian cell (bacteria, yeast, insect cell), animal or plant.
- a mammalian cell bacteria, yeast, insect cell
- the term “human” when used in reference to an antibody, means that the amino acid sequence of the antibody is fully human, i.e., human heavy and human light chain variable and human constant regions. Thus, all of the amino acids are human or exist in a human antibody.
- An antibody that is non-human may be made fully human by substituting the non-human amino acid residues with amino acid residues that exist in a human antibody.
- Amino acid residues present in human antibodies, CDR region maps and human antibody consensus residues are known in the art (see, e.g., Kabat, Sequences of Proteins of Immunological Interest, 4 th Ed.US Department of Health and Human Services. Public Health Service (1987); Chothia and Lesk (1987).
- a consensus sequence of human VH subgroup III, based on a survey of 22 known human V H III sequences, and a consensus sequence of human VL kappa-chain subgroup I, based on a survey of 30 known human kappa I sequences is described in Padlan Mol. Immunol.31:169 (1994); and Padlan Mol. Immunol.28:489 (1991).
- Human antibodies therefore include antibodies in which one or more amino acid residues have been substituted with one or more amino acids present in any other human antibody.
- the term “humanized” when used in reference to an antibody means that the amino acid sequence of the antibody has non-human amino acid residues (e.g., mouse, rat, goat, rabbit, etc.) of one or more complementarity determining regions (CDRs) that specifically bind to the desired antigen in an acceptor human immunoglobulin molecule, and one or more human amino acid residues in the Fv framework region (FR), which are amino acid residues that flank the CDRs.
- CDRs complementarity determining regions
- Antibodies of the disclosure and disclosure methods employing antibodies include those to as “primatized” antibodies, which are “humanized” except that the acceptor human immunoglobulin molecule and framework region amino acid residues may be any primate amino acid residue (e.g., ape, gibbon, gorilla, chimpanzees orangutan, macaque), in addition to any human residue. Human FR residues of the immunoglobulin can be replaced with corresponding non-human residues.
- Residues in the CDR or human framework regions can therefore be substituted with a corresponding residue from the non-human CDR or framework region donor antibody to alter, generally to improve, antigen affinity or specificity, for example.
- a humanized antibody may include residues, which are found neither in the human antibody nor in the donor CDR or framework sequences. For example, a FR substitution at a particular position that is not found in a human antibody or the donor non-human antibody may be predicted to improve binding affinity or specificity human antibody at that position.
- Antibody framework and CDR substitutions based upon molecular modeling are well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Patent No.5,585,089; and Riechmann et al., Nature 332:323 (1988)).
- the term “chimeric” and grammatical variations thereof, when used in reference to an antibody, means that the amino acid sequence of the antibody contains one or more portions that are derived from, obtained or isolated from, or based upon two or more different species.
- a portion of the antibody may be human (e.g., a constant region) and another portion of the antibody may be non-human (e.g., a murine heavy or murine light chain variable region).
- an example of a chimeric antibody is an antibody in which different portions of the antibody are of different species origins. Unlike a humanized or primatized antibody, a chimeric antibody can have the different species sequences in any region of the antibody.
- Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; W091/09967; U.S. Patent Nos.
- Chimeric antibodies in which a variable domain from an antibody of one species is substituted for the variable domain of another species are described, for example, in Munro, Nature 312:597 (1984); Neuberger et al., Nature 312:604 (1984); Sharon et al., Nature 309:364 (1984); Morrison et al., Proc. Nat’l. Acad. Sci. USA 81:6851 (1984); Boulianne et al., Nature 312:643 (1984); Capon et al., Nature 337:525 (1989); and Traunecker et al., Nature 339:68 (1989).
- Suitable techniques that additionally may be employed in antibody methods include affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, size exclusion, purification on protein A column, or any combination of these techniques.
- the antibody isotype can be determined using an ELISA assay, for example, a human Ig can be identified using mouse Ig-absorbed anti-human Ig.
- the term “culturing” refers to the in vitro or ex vivo propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.
- Eukaryotic cells comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus.
- the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, canine, bovine, porcine, murine, rat, avian, reptilian and human.
- Prokaryotic cells that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells’ genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 ⁇ m in diameter and 10 ⁇ m long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coli bacterium, and Salmonella bacterium.
- a “hybridoma” refers to the product of a cell-fusion between a cultured neoplastic lymphocyte and a primed B- or T-lymphocyte which expresses the specific immune potential of the parent cell, such as an antibody.
- the term “disease” or “disorder” as used herein refers to allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation.
- the term “disease” or “disorder” as used herein refers to a status of being diagnosed with allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, a status of being suspect of having allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, or a status of at high risk of having allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation.
- ARDS acute respiratory distress syndrome
- ARDS is a type of respiratory failure characterized by rapid onset of widespread inflammation in the lungs. Symptoms include shortness of breath, rapid breathing, and bluish skin coloration.
- the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
- the term “mammal” includes both human and non-human mammals.
- the term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein.
- Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig).
- a mammal is a human.
- a mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero).
- a mammal can be male or female.
- a subject is a human.
- a subject has or is diagnosed of having or is suspected of having a disease.
- a subject as referred to herein has been treated with a standard care for the disease.
- a subject as referred to herein is concurrently treated with a standard care of the disease.
- a subject as referred to herein will be treated with a standard care of the disease.
- standard of care or “SOC” refers to the diagnostic and treatment process that a clinician should follow for a certain type of patient, illness, or clinical circumstance.
- SOC may include administration of drugs that are being used in clinical practice for the treatment of allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, other than those used as part of another clinical trial.
- “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
- treatment is an approach for obtaining beneficial or desired results, including clinical results.
- beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
- treatment excludes prophylaxis.
- the terms “treating,” “treatment,” and the like, as used herein, mean ameliorating a disease, so as to reduce, ameliorate, or eliminate its cause, its progression, its severity, or one or more of its symptoms, or otherwise beneficially alter the disease in a subject.
- Reference to “treating,” or “treatment” of a patient is intended to include prophylaxis.
- Treatment may also be preemptive in nature, i.e., it may include prevention of disease in a subject exposed to or at risk for the disease. Prevention of a disease may involve complete protection from disease, for example as in the case of prevention of infection with a pathogen, or may involve prevention of disease progression.
- prevention of a disease may not mean complete foreclosure of any effect related to the diseases at any level, but instead may mean prevention of the symptoms of a disease to a clinically significant or detectable level. Prevention of diseases may also mean prevention of progression of a disease to a later stage of the disease.
- the term “passive immunity” refers to the transfer of immunity from one subject to another through the transfer of antibodies. Passive immunity may occur naturally, as when maternal antibodies are transferred to a fetus. Passive immunity may also occur artificially as when antibody compositions are administered to non-immune subjects. Antibody donors and recipients may be human or non-human subjects.
- Antibodies may be polyclonal or monoclonal, may be generated in vitro or in vivo, and may be purified, partially purified, or unpurified depending on the embodiment.
- passive immunity is conferred on a subject in need thereof through the administration of antibodies or antigen-binding fragments that specifically recognize or bind to a particular antigen, such as an HRF, HRF N19, HRF GST-N19 or HRF-2CA.
- passive immunity is conferred through the administration of an isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen, such as an HRF.
- Immunogen broadly refers to the antigen-specific responses of lymphocytes to foreign substances.
- immunogen and “immunogenic” refer to molecules with the capacity to elicit an immune response. All immunogens are antigens, however, not all antigens are immunogenic.
- An immune response disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro.
- macromolecules including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to be immunogenic.
- Detectable label refers to nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen.
- immunogens are not limited to full- length molecules, but may include partial molecules.
- Detectable label refers to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, antibody or composition described herein.
- label or a detectable label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histidine tags (N-His), magnetically active isotopes, e.g., 115 Sn, 117 Sn and 119 Sn, a non-radioactive isotopes such as 13 C and 15 N, polynucleotide or protein such as an antibody so as to generate a “labeled” composition.
- N-terminal histidine tags N-His
- magnetically active isotopes e.g., 115 Sn, 117 Sn and 119 Sn
- a non-radioactive isotopes such as 13 C and 15 N
- polynucleotide or protein such as an antibody so as to generate a “labeled” composition.
- the term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequence
- the label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
- the labels can be suitable for small scale detection or more suitable for high-throughput screening.
- suitable labels include, but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
- the label may be simply detected, or it may be quantified.
- a response that is simply detected generally comprises a response whose existence merely is confirmed
- a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, or other property.
- the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
- luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence.
- Detectable luminescence response generally comprises a change in, or an occurrence of a luminescence signal.
- Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed).
- Examples of luminescent probes include, but are not limited to, aequorin and luciferases.
- the term “immunoconjugate” comprises an antibody or an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
- a second agent such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
- fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl- coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, and Texas Red.
- suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).
- the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
- Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
- the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
- a purification label or maker refers to a label that may be used in purifying the molecule or component that the label is conjugated to, such as an epitope tag (including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag), an affinity tag (including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)), or a fluorescent tag.
- an epitope tag including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag
- an affinity tag including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)
- fluorescent tag including but not limited to
- a detectable marker can be used to produce a detectable signal upon binding of two moieties, such as an antibody and its antigen.
- one of the two moieties is immobilized, the mobilized moiety is the provided for binding, and unbound mobilized moiety is removed by washing with a suitable solution.
- any detectable marker can be directly or indirectly conjugated to the mobilized moiety, and the detectable signal obtained after the washing step indicates binding between the two moieties.
- a detectable signal can be generated if two moieties are in the proximity with each other.
- a detectable marker such as a fluorescent protein
- FRET fluorescence resonance energy transfer
- ELISA enzyme-linked immunosorbant assay. Numerous methods and applications for carrying out an ELISA are well known in the art, and provided in many sources (See, e.g., Crowther, “Enzyme-Linked immunosorbant Assay (ELISA),” in Molecular Biomethods Handbook, Rapley et al.
- an ELISA is a “direct ELISA”, where a target-binding molecule, such as a cell, cell lysate, or isolated protein, is first bound and immobilized to a microtiter plate well.
- a target-binding molecule such as a cell, cell lysate, or isolated protein
- an ELISA is a “sandwich ELISA”, where a target-binding molecule is attached to the substrate by capturing it with an antibody that has been previously bound to the microtiter plate well.
- the ELISA method detects an immobilized ligand-receptor complex (binding) by use of fluorescent detection of fluorescently labeled ligands or an antibody-enzyme conjugate, where the antibody is specific for the antigen of interest, such as a phage virion, while the enzyme portion allows visualization and quantitation by the generation of a colored or fluorescent reaction product.
- the conjugated enzymes commonly used in the ELISA include horseradish peroxidase, urease, alkaline phosphatase, glucoamylase or O-galactosidase. The intensity of color development is proportional to the amount of antigen present in the reaction well.
- a lateral flow immunoassay refers to an assay format in which a sample is applied to a lateral flow matrix.
- the sample flows along the lateral flow matrix, and one or more analyte components to be detected in the sample react with at least one reagent which is provided in or added to the lateral flow matrix.
- At least one reagent is typically immobilized in the device for reaction with the analyte component to be detected or a reagent thereof, and labels are typically employed to measure the extent of reaction with an immobilized reagent. See, e.g., U.S. patents and patent application publications: U.S. Pat.
- Exemplary samples include, but are not limited to, cell sample, tissue sample, biopsy, liquid samples such as blood and other liquid samples of biological origin, including, but not limited to, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspir
- contacting means direct or indirect binding or interaction between two or more.
- a particular example of direct interaction is binding.
- a particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity.
- Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.
- administering or administration.
- administering or a grammatical variation thereof also refers to more than one doses with certain interval.
- the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer.
- one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more.
- Suitable dosage formulations and methods of administering the agents are known in the art.
- Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
- Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application.
- the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.
- administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
- parenteral e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection, or implant
- composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
- inert for example, a detectable agent or label
- active such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
- Carriers also include pharmaceutical excipients and additiveHRFs, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra- oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
- Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
- Representative amino acid components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
- Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
- monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like
- disaccharides such as lactose, sucrose
- a composition as disclosed herein can be a pharmaceutical composition.
- a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
- “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein.
- Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
- buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen
- Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
- the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of long- term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.
- compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage.
- unit dose or "dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen.
- the quantity to be administered both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
- solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
- the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.
- a combination as used herein intends that the individual active ingredients of the compositions are separately formulated for use in combination, and can be separately packaged with or without specific dosages. The active ingredients of the combination can be administered concurrently or sequentially.
- an antibody or antigen binding fragment thereof is administered in an effective amount.
- An “effective amount” is an amount sufficient to effect beneficial or desired results.
- An effective amount can be administered in one or more administrations, applications or dosages.
- Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific agent employed, bioavailability of the agent, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the agent that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo.
- “Therapeutically effective amount” of an agent refers to an amount of the agent that is an amount sufficient to obtain a pharmacological response; or alternatively, is an amount of the agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient.
- a therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.
- an antibody or an antigen binding fragment thereof comprising one or more of the following sequences: a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; or a heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL.
- CDR heavy-chain complementarity determining region
- HCDR1 that comprises the amino acid sequence GFSLSSGA
- HCDR2 heavy-chain CDR 2
- HCDR3 heavy-chain CDR 3
- an antibody or antigen binding fragment that comprises one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain (CDR) 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; or a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG.
- antibody or antigen binding fragment thereof comprising one or more of: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; a heavy- chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; b) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; or a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, A
- antibody or antigen binding fragment thereof comprising: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; a heavy- chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; and b) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; and a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGS
- the antibody or an antigen binding fragment thereof comprises a heavy-chain variable region comprising one of the following amino acid sequences: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS; b) QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS; c) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS; d)
- an antibody or an antigen binding fragment thereof comprising a light-chain variable region comprising one of the following amino acid sequences: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFGGGTE VVVK; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; c) AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; c) AQVLTQTASPVSA
- an antibody or antigen binding fragment thereof comprising: a) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFGGGTE VVVK; b) a heavy chain variable region comprising the amino acid sequence: QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASY
- providedherein is an antibody or antigen binding fragment thereof, comprising a heavy chain region comprising one of the following amino acid sequences: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG TLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSK PTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQV RTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQ PLEPKVYTMGPPREELSSRSVSLTCMINGFYPS
- the antibody or antigen biding fragment comprises a light- chain region comprising one of the following amino acid sequences: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFGGGTE VVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIEN SKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKY
- an antibody or antigen binding fragment that comprises one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSSQSVYDNNNLA; a light-chain (CDR) 2 (LCDR2) that comprises one of the following amino acid sequences: DASKLPS; or a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGAVSGSNV.
- antibody or antigen binding fragment thereof comprising: [00183] a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence TVSGFSLSSGAVS; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: IGVISSRDIAYFATWAKG; a heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL.
- CDR heavy-chain complementarity determining region
- HCDR1 that comprises the amino acid sequence TVSGFSLSSGAVS
- HCDR2 heavy-chain CDR 2
- HCDR3 heavy-chain CDR 3
- an antibody or an antigen binding fragment thereof comprising a light-chain variable region comprising the following amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
- the antibody or an antigen binding fragment thereof comprises a heavy-chain variable region comprising the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK.
- the equivalent of the antibody or antigen binding fragment is at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or more identical to the HC and/or LC reference sequence. Methods to determine such are known in the art and disclosed herein.
- the antibody or antigen binding fragment specifically recognizes and binds to HRF or an immunogenic fragment thereof.
- the immunogenic fragment comprises or consists of the amino acid sequence of N19 or GST-N19.
- the HRF comprises or consists of human HRF.
- the antibody or antigen binding fragment of this disclosure is isolated or recombinant, or alternatively is chimeric, humanized, a single chain, or a humanized single chain.
- Non-limiting examples of an antigen binding fragment are a Fab, F(ab’)2, Fab’, scFv, or Fv.
- the antibody or antigen binding fragment of this disclosure comprises, or consists essentially or, or yet further consists of a light chain constant domain.
- Non-limiting examples of such is a constant domain of a human ⁇ light chain, a constant domain of a human ⁇ light chain and a constant domain of a ⁇ 1 or ⁇ 2 or ⁇ 3 or ⁇ 4 light chain.
- the antibody or antigen binding fragment comprises a light chain constant domain.
- the constant domain is a human consists domain. Additionally or alternatively, the constant domain comprises, or alternatively consists essentially of, or yet further consists of a constant domain of a ⁇ light chain, optionally a human ⁇ light chain.
- the constant domain of the ⁇ light chain comprises, or alternatively consists essentially of, or yet further consists of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
- the constant domain comprises, or alternatively consists essentially of, or yet further consists of a constant domain of a ⁇ light chain, optionally a human ⁇ light chain.
- the constant domain comprises, or alternatively consists essentially of, or yet further consists of a constant domain of a ⁇ 1 or ⁇ 2 or ⁇ 3 or ⁇ 4 light chain, optionally a human ⁇ 1 or ⁇ 2 or ⁇ 3 or ⁇ 4 light chain.
- exemplary antibodies that bind to HRF or a fragment thereof, e.g., HRF N19, HRF GST-N19, or HRF-2CA, are disclosed herein, or an equivalent of each thereof.
- a designated antibody and its equivalent comprise the same CDRs.
- each of these is a rabbit monoclonal antibody.
- humanized antibodies are also provided.
- recombinant anti-HRF antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the disclosure.
- Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques such as, for example, the methods described in U.S. Pat. No. 7,112,421; Better et al. (1988) Science 240:1041-1043; or Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443.
- the fragment comprises a fragment crystallizable region (Fc region).
- Non-limiting examples of such include an region that comprises, or consists essentially of, or yet further consists of one or more of: an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgM Fc region, an IgE Fc region, an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG4 Fc region.
- the antibody or antigen binding fragment can be post-translationally modified optionally glycosylated, hydroxylated, methylated, lapidated, acetylated, SUMOylated, phosphorylated, PEGylated, or any combination thereof and/or further comprise a detectable or purification marker.
- polynucleotides encoding the one or more antibody or antigen binding fragment of this disclosure or a polynucleotide complementary thereto.
- the polynucleotide can be RNA, DNA or a hybrid polynucleotide.
- the polynucleotide can be comprised within a vector.
- the polynucleotide and/or vector further comprises a regulatory sequence that directs the expression or replication of the antibody or antigen binding fragment or polynucleotide, respectively.
- Non-limiting examples of such include without limitation a secretion signal, promoter, an enhancer, or a polyadenylation sequence.
- the regulatory sequence directs the replication or expression of the polynucleotide.
- Non-limiting examples of the vectors include a non-viral vector, optionally a plasmid, or a viral vector, optionally an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, or a plant viral vector.
- Also provided are a single or plurality of cells comprising one or more of: the antibody or antigen binding fragment, the polynucleotide, or the vector as described herein.
- the polynucleotide or cell is as described in the Experimental Section and incorporated herein by reference.
- the cell can be a prokaryotic cell, optionally an Escherichia coli cell.
- Non-limiting examples of a eukaryotic cell includes a mammal cell, an insect cell, a yeast cell or a human cell, e.g. a 293 cell or a cell as described herein.
- a hybridoma expressing the antibody or antigen binding fragment as described herein.
- the cells and vectors can be used to produce the antibody or antigen binding fragment as described herein by culturing a cell comprising a polynucleotide encoding the antibody or the antigen binding fragment under conditions suitable for expression of the antibody or antigen binding fragment.
- the polynucleotide is introduced into to the cell prior to the culturing step.
- the method can further comprise purifying they antigen or antigen binding fragment from the cell or culture medium.
- the hybridoma expressing the antibody can be cultured under conditions suitable for expression of the antibody or antigen binding fragment and optionally purifying or isolating the antibody produced thereby.
- This disclosure also provides a method of producing the antibody or antigen binding fragment of this disclosure by contacting the polynucleotide or the vector encoding same with an RNA polymerase, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine-5'-triphosphate (GTP), and uridine triphosphate (UTP) under conditions suitable for transcription to messenger RNA, and contacting the transcribed messenger RNA with a ribosome, tRNAs, an aminoacyl-tRNA synthetase, and initiation, elongation and termination factors under conditions suitable for translation to the antibody or antigen binding fragment.
- ATP adenosine triphosphate
- CTP cytidine triphosphate
- GTP guanosine-5'-triphosphate
- UDP uridine triphosphate
- the method comprises contacting the transcribed messenger RNA with a cell lysate comprising the ribosome, tRNAs, aminoacyl-tRNA synthetase, and initiation, elongation and termination factors under conditions.
- the antibody or antigen binding fragment can be purified or isolated from the reaction mixture.
- Compositions are provided that comprise a carrier and one or more of: the antibody or antigen binding fragment, the polynucleotide, the vector, the cell, or the hybridoma as described herein that can optionally be detectably labeled.
- the composition further comprises a carrier, such as a pharmaceutically acceptable carrier.
- compositions can comprise two or more of the antibodies or antigen binding fragments that can be the same or different from each other.
- the two or more of the antibodies or antigen binding fragments recognize and binds to at least two different epitopes.
- the composition further comprise one or more peptides or proteins identified as N19, GST-19 or HRF-2CA or polynucleotides encoding or a vector or host cell containing same, and optionally wherein the carrier is a pharmaceutical acceptable carrier.
- Methods of Use are methods for one or more of: a) targeting HRF N19-Ig interactions; b) inhibiting HRF N19 or HRF-Ig interactions; c) inhibiting Ig E-dependent activation of mast cells or basophils; d) the treatment of a condition from the group of: an allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation in a subject in need thereof, comprising, or consisting essentially of, or yet further consisting of administering an effective amount of the antibody or antigen binding fragment or compositions as described herein.
- the agent binds an HRF-reactive immunoglobulin (Ig).
- the agent modulates binding of an HRF monomer, an HRF dimer, or an HRF multimer with the HRF-reactive Ig.
- the method can be performed in vitro or in vivo. When performed in vitro, it provides a screen for alternative or equivalent compositions or for new combination therapies. When performed in vivo in an animal, it is a useful animal model for new therapies or personalized therapies. It also can be performed in a animal, mammal, murine, canine, feline, or human patient, for therapeutic purposes.
- the method further comprises administering an effective amount of a second drug.
- the second drug comprises an anti-inflammatory, anti-asthmatic or anti-allergy drug.
- Non-limiting examples include a hormone, a steroid, an anti-histamine, anti-leukotriene, anti-IgE, anti- ⁇ 4 integrin, anti- ⁇ 2 integrin, anti-CCR3 antagonist, ⁇ 2 agonist or anti-selectin.
- the second drug comprises an allergen immunotherapy.
- Non-limiting examples of the allergen immunotherapy is oral immunotherapy (OIT), subcutaneous immunotherapy (SIT), or sublingual immunotherapy (SLIT).
- the second therapy comprises an inhibitor of binding of HRF to an Ig molecule.
- the inhibitor is a peptide or polypeptide that inhibits the binding of an HRF monomer, an HRF dimer, or a HRF multimer with an HRF-reactive immunoglobulin (Ig).
- the second therapy is administered concurrently, prior to, or after the first therapy.
- the therapy and/or the second drug is administered via ingestion, via inhalation, topically, or a combination thereof.
- administration of a pharmaceutical composition comprising one or more of the antibodies, fragments or composition as described herein can be made to a subject in need thereof, such as allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation.
- the pharmaceutical compositions as described herein can be administered alone or in combination with other therapies deemed appropriate by a clinician or practitioner.
- the pharmaceutical compositions described herein may reduce the number of days of the subject having allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation symptoms by one or more days, such as reducing the days of having symptoms by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days.
- the subject is selected for the administration if the antibody or antigen binding fragment binds to a component, IgE or HRF detected in a biological sample isolated from the subject.
- other assays including commercially available tests, can be utilized to test the subject for inflammation or allergy.
- a method comprising, or consisting essentially of, or yet further consisting of contacting an antibody or antigen binding fragment of the detection system as disclosed herein with a biological sample isolated from a subject.
- the method further comprises contacting the detectable marker of the detection system as disclosed herein with the antibody or antigen binding fragment or polypeptide.
- binding of the antibody or antigen binding fragment or polypeptide with a component of the biological sample indicates the subject has or had a allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation.
- a method for detecting allergy optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation, HRF or an immunogenic fragment of the HRF.
- the method comprises, or consists essentially of, or yet further consists of contacting the antibody or antigen binding fragment of the detection system with a sample.
- the method further comprises contacting the detectable marker with the antibody or antigen binding fragment.
- binding of the antibody or antigen binding fragment with a component of the sample indicates presence of a HRF or an immunogenic fragment in the sample.
- a detection system comprising the antibody or antigen binding fragment as described herein and a detectable marker for producing a detectable signal upon binding of the antibody or antigen binding fragment thereof with HRF or an immunogenic fragment thereof.
- the system is an enzyme-linked immunosorbent assay (ELISA) or a lateral flow immunoassay.
- ELISA enzyme-linked immunosorbent assay
- a lateral flow immunoassay comprising contacting the antibody or antigen binding fragment of the detection system with a biological sample isolated from a subject, wherein binding of the antibody or antigen binding fragment thereof with a component of the biological sample indicates the subject expresses HRF.
- kits comprising, or consisting essentially of, or yet further consisting of an instruction for use in a method as disclosed herein, and one or more of: an antibody or antigen binding fragment or polypeptide as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell of as disclosed herein, a hybridoma as disclosed herein, a composition as disclosed herein, or a system as disclosed herein.
- HRF Histamine-releasing factor
- Ig immunoglobulin
- HRF can be present as a monomer and disulfide-linked oligomers. HRF directly binds to a subset of IgE and IgG molecules via low- affinity interactions between Ig-Fab portion and two Ig-binding sites within HRF, the amino- terminal 19 residues (N19) and the helical domain H3. It was also shown that a fusion protein GST-N19 and a recombinant monomeric HRF mutant with two cysteine residues replaced with alanine (HRF-2CA) work as strong inhibitors for in vitro HRF-IgE interactions and in vivo allergic inflammation.
- HRF-2CA a fusion protein GST-N19 and a recombinant monomeric HRF mutant with two cysteine residues replaced with alanine
- HRF dimers can activate murine mast cells through high-affinity IgE receptors (FceRI) 3 .
- FceRI high-affinity IgE receptors
- mAb antibodies
- OVA ovalbumin
- rabbits were immunized with GST-N19.
- Phage-display libraries expressing Fabs were generated from spleens of rabbits with high serum titers of anti-GST- N19. Phages were selected by 4 rounds of panning with immobilized GST-N19 and against immobilized GST.
- Fab sequences were randomly selected out of seventy-nine N19- specific Fab-displaying phages, and cloned into an expression vector pTT5, expressed in HEK293 cells and purified by Ni- NTA agarose column. These Fabs all bound to immobilized GST-N19 (data not shown), and they were confirmed for their ability to inhibit interactions of the HRF-reactive IgE, C38-2, with recombinant HRF by enzyme-linked immunosorbent assays (ELISA). Among them, three Fabs (A8-1, C4-4 and F7-1) with strong HRF-binding ability are shown in Fig.1A.
- the bindings of pre-immune sera and post-immune sera to the GST-N19, GST, and a recombinant human HRF-His 6 were compared by ELISA and the rabbit that showed the best binding to the immunogen and HRF-His 6 was selected for the library construction.
- the bone marrow and spleen cells were obtained and homogenized in TRI reagent (Molecular Research Center). Total RNA was isolated from the homogenized cells according to the manufacturer’s protocol. Then, the messenger RNA was purified using NucleoTrap mRNA (Macherey-Nagel) according to the manufacturer’s protocol.
- First strand cDNA was synthesized using PowerScribe MMLV RT (Monserate Biotechnology Group) and the antibody genes were amplified as described in US Patent No.9,890,414.
- the amplified antibody genes were digested with restriction enzymes and sequentially ligated with a Fab expression vector.
- the libraries were made by the electroporation of NEB 10-beta E. coli cells (New England Biolabs) with the ligated DNA. Selection of N19-specific clones [00227] For the amplification of the libraries in E. coli, 2 ⁇ g of each library DNA were electroporated into XL1-Blue E.
- coli cells (Monserate Biotechnology Group) and phage production was induced with VCS M13 helper phage in the presence of 1mM isopropyl ⁇ -D- 1- thiogalactopyranoside (IPTG) and antibiotics (carbenicillin, tetracycline, and kanamycin) at 30 ⁇ C overnight. Phage was precipitated from the bacterial supernatant with 4% PEG (Polyethylene Glycol-8000)/0.5M NaCl and re-suspended in 1% BSA/PBS. Excess 5-fold GST was added to the phage as a soluble competitor to remove binders to GST.
- IPTG isopropyl ⁇ -D- 1- thiogalactopyranoside
- antibiotics carbenicillin, tetracycline, and kanamycin
- the microtiter wells were coated overnight with GST-N19 at 5 ⁇ g/mL in PBS and blocked with 1% BSA/PBS.
- the wells were incubated with precipitated phage at 37 ⁇ C for 1.5 h and unbound phage was washed 3 times in the first round of panning and 5 times in the 2 nd , the 3 rd , and the 4 th round with PBS after 5 min incubation. Bound phage was eluted and freshly grown ER2738 cells were infected with eluted phage.
- antibiotics carbenicillin, tetracycline, and kanamycin
- microtiter wells were coated with 5 ⁇ g/mL GST- N19 protein in PBS at 4 ⁇ C overnight. After washing 3 times with PBS and blocking with 1% BSA/PBS, the culture plate was spun down and the wells were incubated with the supernatant containing Fab at 37 ⁇ C for 2 h. The wells were washed 3 times with PBS and the bound Fab was detected with HRP-conjugated goat anti-rabbit IgG F(ab’) 2 (Thermo Fisher Scientific Cat# 31461) at 37 ⁇ C for 1 h. The wells were washed 3 times with PBS and developed with TMB substrate (EMD Millipore Cat# ES022-50ml) and stopped with 2 N sulfuric acid.
- TMB substrate EMD Millipore Cat# ES022-50ml
- the signal was read with a plate reader at 450 nm.
- the light and heavy chains of Fab clones specific to N19 peptide were amplified by PCR, treated with ExoSAP-IT (Thermo Fisher Scientific Cat# 78201.1.ML), and submitted for sequence analysis.
- Cloning of N19-specific clones into a mammalian IgG expression vector [00228] The amplified light and heavy chains of the selected clones were digested with restriction enzymes and ligated with a fragment containing a CMV promoter into a mammalian IgG expression vector: a modified pTT5 vector (National Research Council Canada) for the bi-cistronic expression and purification.
- the ligated DNA was electroporated into NEB 10-beta cells and the cells were titrated onto LB agar plates containing 100 ⁇ g/mL of carbenicillin and 20 mM glucose. The single colonies were inoculated into 1.2 mL of Super Broth medium containing 50 ⁇ g/mL of carbenicillin and grown overnight at 37 ⁇ C. The cells were spun down and the DNA were purified from the cell pellets using spin columns (Qiagen Cat# 27104). The purified DNA were submitted for sequencing.
- Each 10 ⁇ g of IgG-encoding plasmid was added to 500 ⁇ L F17 freestyle media and vortexed gently 3 times for 1 sec.
- Thirty ⁇ L of the PEI transfection reagent Transporter5 Transfection Reagent, 1 mg/mL, Polysciences
- the two were combined and the cocktail was again vortexed 3 times and incubated at room temperature for 10 min.
- the 1 mL cocktail was added to the 9 mL HEK293 cell culture, swirled gently and incubated at 37 ⁇ C, 5% CO2, with 140 rpm agitation.
- mice were i.p. sensitized with OVA (50 ⁇ g/mouse) plus alum on days 0 and 14.
- OVA 50 ⁇ g/mouse
- mice were i.g. challenged with OVA (25 mg) or PBS (control).
- OVA 25 mg
- PBS control
- mice were starved for 3 h, then i.p. pretreated with anti-N19 mAb or isotype control (100 ⁇ g/mouse).
- peptides 100 ⁇ g
- mice were i.g. gavaged 30 min before each OVA challenge in starved mice.
- mice were intraperitoneally sensitized with OVA (25 or 50 ⁇ g/mouse) plus alum on days 0 and 14. From day 28, mice were intragastrically (i.g.) challenged with OVA (25 or 50 mg) or PBS (con- trol) 3 times a week.
- mice were starved for 3 hours, then i.g. pretreated with HRF inhibitor (or control protein (rabbit anti-HRF-N19 mAb SPF7-1 mAb or humanized mAb, or HRF-2CA) or control protein (isotype control or PBS).
- HRF inhibitor or control protein (rabbit anti-HRF-N19 mAb SPF7-1 mAb or humanized mAb, or HRF-2CA) or control protein (isotype control or PBS).
- HRF inhibitor or control protein (rabbit anti-HRF-N19 mAb SPF7-1 mAb or humanized mAb, or HRF-2CA) or control protein (isotype control or PBS).
- HRF inhibitor or control protein (rabbit anti-HRF-N19 mAb SPF7-1 mAb or humanized mAb, or HRF-2CA) or control protein (isotype control or PBS).
- mice were challenged i.g. with 25 or 50 mg of OVA. Body temperature and physical activity were
- the antibody of the present technology is an anti-HRF, anti-GST-N19 or anti-N19 monoclonal antibody produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
- Hybridoma techniques include those known in the art and taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981).
- the antibodies of the present technology can be produced through the application of recombinant DNA and phage display technology.
- the antibodies can be prepared using various phage display methods known in the art.
- phage display methods functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them.
- Phages with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g., human or murine) by selecting directly with an antigen, typically an antigen bound or captured to a solid surface or bead.
- Phages used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains that are recombinantly fused to either the phage gene III or gene VIII protein.
- methods can be adapted for the construction of Fab expression libraries (See, e.g., Huse, et al.,. Science 246: 1275-1281, 1989) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for an HRF, E.G., GST-N19 OR N19 polypeptide, e.g., a polypeptide or derivatives, fragments, analogs or homologs thereof.
- phage display methods that can be used to make the antibodies of the present technology include those disclosed in Huston et al., Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J.
- the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.
- techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.
- hybrid antibodies or hybrid antibody fragments that are cloned into a display vector can be selected against the appropriate antigen in order to identify variants that maintain good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle.
- a display vector can be selected against the appropriate antigen in order to identify variants that maintain good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle.
- Other vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and/or screening.
- the antibodies of the present technology can be produced through the application of recombinant DNA technology.
- Recombinant polynucleotide constructs encoding an antibody of the present technology typically include an expression control sequence operably-linked to the coding sequences of anti-HRF, e.g. anti-GST-N19 or anti-N19 antibody chains, including naturally-associated or heterologous promoter regions.
- another aspect of the technology includes vectors containing one or more nucleic acid sequences encoding an anti-HRF, e.g., GST-N19 or N19 antibody of the present technology.
- the nucleic acid containing all or a portion of the nucleotide sequence encoding the anti-HRF, e.g., GST-N19 or N19 antibody is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al., U.S. Pat. Nos.6,291,160 and 6,680,192.
- expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
- plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
- the present technology is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
- viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
- Such viral vectors permit infection of a subject and expression of a construct in that subject.
- the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells.
- the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding the anti-HRF, e.g., GST-N19 or N19 antibody, and the collection and purification of the anti-HRF, e.g., GST-N19 or N19 antibody, e.g., cross-reacting anti-HRF, e.g., GST-N19 or N19 antibodies.
- anti-HRF e.g., GST-N19 or N19 antibody
- cross-reacting anti-HRF e.g., GST-N19 or N19 antibodies.
- expression vectors contain selection markers, e.g., ampicillin-resistance or hygromycin- resistance, to permit detection of those cells transformed with the desired DNA sequences.
- Vectors can also encode signal peptide, e.g., pectate lyase, useful to direct the secretion of extracellular antibody fragments. See U.S. Pat. No.5,576,195.
- the recombinant expression vectors of the present technology comprise a nucleic acid encoding a protein with anti-HRF antibody protein or fragment thereof, e.g., in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operably-linked to the nucleic acid sequence to be expressed.
- operably-linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
- regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, e.g., in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
- Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
- Typical regulatory sequences useful as promoters of recombinant polypeptide expression include, e.g., but are not limited to, promoters of 3- phosphoglycerate kinase and other glycolytic enzymes.
- Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
- a polynucleotide encoding an anti-HRF, e.g., GST-N19 or N19 antibody of the present technology is operably- linked to an ara B promoter and expressible in a host cell. See U.S. Pat.5,028,530.
- the expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., anti-HRF, e.g., GST-N19 OR N19 antibody, etc.).
- nucleic acids as described herein (e.g., anti-HRF, e.g., GST-N19 OR N19 antibody, etc.).
- anti-HRF e.g., GST-N19 OR N19 antibody-expressing host cells, which contain a nucleic acid encoding one or more anti-HRF, e.g., GST-N19 or N19 antibodies or fragments thereof.
- the recombinant expression vectors of the present technology can be designed for expression of an anti-HRF, e.g., GST-N19 or N19 antibody or fragment thereof in prokaryotic or eukaryotic cells.
- an anti-HRF e.g., GST-N19 OR N19 antibody or fragment thereof can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
- the recombinant expression vector can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase.
- T7 promoter regulatory sequences and T7 polymerase Methods useful for the preparation and screening of polypeptides having a predetermined property, via expression of stochastically generated polynucleotide sequences has been previously described. See U.S. Pat. Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641. [00242] Expression of polypeptides in prokaryotes is most often carried out in E.
- Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide.
- Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
- a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide.
- enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
- Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
- E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
- the antiHRF, anti-GST-N19 or antiN19 antibody or fragment thereof expression vector is a yeast expression vector.
- yeast Saccharomyces cerevisiae examples include pYepSec1 (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).
- an anti-HRF e.g., GST-N19 OR N19 antibody can be expressed in insect cells using baculovirus expression vectors.
- Baculovirus vectors available for expression of polypeptides include the pAc series (Smith, et al., Mol. Cell. Biol.3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
- a nucleic acid encoding an anti-HRF antibody e.g., GST-N19 or N19 antibody or fragment thereof of the present technology is expressed in mammalian cells using a mammalian expression vector.
- mammalian expression vectors include, e.g., but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J.6: 187-195, 1987).
- the expression vector's control functions are often provided by viral regulatory elements.
- commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
- the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art.
- tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev.1: 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol.43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J.8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl.
- albumin promoter liver-specific; Pinkert, et al., Genes Dev.1: 268-277, 1987
- lymphoid-specific promoters Calame and Eaton, Adv. Immunol.43: 235-275, 1988
- pancreas-specific promoters Esdlund, et al., 1985. Science 230: 912- 916
- mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No.264,166
- Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the ⁇ -fetoprotein promoter (Campes and Tilghman, Genes Dev.3: 537-546, 1989).
- host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- a host cell can be any prokaryotic or eukaryotic cell.
- an anti- HRF, or anti-GST-N19 or anti- N19 antibody or fragment thereof can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells.
- Mammalian cells are a suitable host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987).
- a number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cells are non-human.
- Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Queen et al., Immunol. Rev.89: 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. Co et al., J Immunol.148: 1149, 1992. Other suitable host cells are known to those skilled in the art.
- Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
- transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics or viral-based transfection.
- Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (See generally, Sambrook et al., Molecular Cloning).
- Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL.2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
- the vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. [00250]
- For stable transfection of mammalian cells it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome.
- a gene that encodes a selectable marker is generally introduced into the host cells along with the gene of interest.
- selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
- Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the anti-HRF, e.g., GST-N19 OR N19 antibody or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
- a host cell that includes an anti HRF antibody, or anti- GST-N19 or anti-N19 antibody or fragment of each thereof of the present technology can be used to produce (i.e., express) recombinant antibodies, anti-HRF, or anti-GST-N19 or anti- N19 antibody.
- the method comprises culturing the host cell (into which a recombinant expression vector encoding the antibody or fragment thereof has been introduced) in a suitable medium such that the anti- antibody or fragment thereof is produced.
- the method further comprises the step of isolating the antibody or fragment thereof from the medium or the host cell.
- anti-HRF anti-GST-N19 or anti- N19 antibody or fragment thereof
- the anti-HRF, or anti-GST-N19 or anti- N19 antibody or fragment thereof can be purified according to standard procedures of the art, including HPLC purification, column chromatography, gel electrophoresis and the like.
- the anti-HRF e.g., GST-N19 OR N19 antibody or fragment thereof is produced in a host organism by the method of Boss et al., U.S. Pat. No.4,816,397.
- anti-HRF, e.g., GST-N19 or N19 antibody chains are expressed with signal sequences and are thus released to the culture media.
- the anti-HRF e.g., GST-N19 or N19 antibody chains
- the anti-HRF e.g., GST-N19 or N19 antibody chains
- mild detergent e.g., sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium EDTA, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sulfate, sodium sulfate, sodium bicarbonate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium
- Polynucleotides encoding antiHRF, e.g., GST-N19 or N19 antibodies or fragment thereof, e.g., the anti-HRF, e.g., GST-N19 or N19 antibody or fragment thereof coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. See, e.g., U.S. Pat. Nos.5,741,957, 5,304,489, and 5,849,992.
- transgenes include coding sequences for light and/or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or ⁇ -lactoglobulin.
- transgenes can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.
- Single-Chain Antibodies e.g., GST-N19 or N19 antibody of the present technology is a single-chain anti-HRF, e.g., GST-N19 or N19 antibody.
- techniques can be adapted for the production of single-chain antibodies specific to an HRF, e.g., GST-N19 or N19 protein (See, e.g., U.S. Pat. No.4,946,778).
- HRF e.g., GST-N19 or N19 protein
- Examples of techniques which can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Pat. Nos.4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.
- the anti-HRF, e.g., GST-N19 or N19 antibody of the present technology is a chimeric anti-HRF, e.g., GST-N19 or N19 antibody.
- the anti-HRF, e.g., GST-N19 or N19 antibody of the present technology is a humanized anti-HRF, e.g., GST-N19 or N19 antibody.
- the donor and acceptor antibodies are monoclonal antibodies from different species.
- the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody.
- Recombinant anti-HRF e.g.,GST-N19 or N19 antibodies
- chimeric and humanized monoclonal antibodies comprising both human and non-human portions
- Recombinant anti-HRF can be made using standard recombinant DNA techniques, and are within the scope of the present technology.
- the anti-HRF e.g., GST-N19 or N19 antibody of the present technology in humans
- chimeric or humanized anti-HRF E.G., GST-N19 or N19 antibodies.
- Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
- Such useful methods include, e.g., but are not limited to, methods described in International Application No. PCT/US86/02269; U.S. Pat. No.5,225,539; European Patent No.184187; European Patent No.171496; European Patent No.173494; PCT International Publication No. WO 86/01533; U.S. Pat. Nos. 4,816,567; 5,225,539; European Patent No.125023; Better, et al., 1988. Science 240: 1041- 1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J.
- antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0239 400; WO 91/09967; U.S. Pat.
- a cDNA encoding a murine anti-HRF, E.G., GST-N19 OR N19 monoclonal antibody is digested with a restriction enzyme selected specifically to remove the sequence encoding the Fc constant region, and the equivalent portion of a cDNA encoding a human Fc constant region is substituted
- the present technology provides the construction of humanized anti-HRF, E.G., GST-N19 or N19 antibodies that are unlikely to induce a human anti-mouse antibody (hereinafter referred to as “HAMA”) response, while still having an effective antibody effector function.
- HAMA human anti-mouse antibody
- the terms “human” and “humanized”, in relation to antibodies, relate to any antibody which is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject.
- the present technology provides for a humanized anti-HRF, E.G., GST-N19 OR N19 antibodies, heavy and light chain immunoglobulins.
- Humanization of SPF7 Clone SPF7 was humanized by grafting its CDRs into human germline frameworks IGKV1-13*02/IGKJ4*01 or IGKV1-39*01/IGKJ4*01 and JGHV3-53*04/IGHJ4*03 or IGHV3-23*04/IGHJ4*03 (SEQ ID No.1-4).
- L1H1 hSPF7L1/hSPF7H1
- L1H2 hSPF7L1/hSPF7H2
- L2H1 hSPF7L2/hSPF7H1
- L2H2 hSPF7L2/hSPF7H2
- hSPF7L1 1 AlaIleGlnLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThr GCGATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGGGTCACC 1 ---------!---------------------------------------------------------------------------------------------!
- the humanized antibody or antigen binding fragment that comprises one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises the amino acid sequence: QSSQSVYDNNNLA; a light-chain (CDR) 2 (LCDR2) that comprises the amino acid sequence: DASKLPS; or a light-chain CDR 3 (LCDR3) that comprises the amino acid sequence
- the humanized antibody or antigen binding fragment thereof comprising: [00264] a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence TVSGFSLSSGAVS; a heavy-chain CDR 2 (HCDR2) that comprises the amino acid sequence: IGVISSRDIAYFATWAKG; a heavy- chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL.
- HC heavy-chain complementarity determining region 1
- HCDR2 heavy-chain CDR 2
- HCDR3 heavy-chain CDR 3
- the humanized antibody or an antigen binding fragment thereof comprises a light-chain variable region comprising the following amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
- the humanized antibody or an antigen binding fragment thereof comprises a heavy-chain variable region comprising the following amino acid sequence: [00267] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSPGK.
- CDR Antibodies Generally the donor and acceptor antibodies used to generate the anti-HRF, or anti-GST-N19 or anti-N19 CDR antibody are monoclonal antibodies from different species; typically the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody.
- the graft may be of a single CDR (or even a portion of a single CDR) within a single VH or VL of the acceptor antibody, or can be of multiple CDRs (or portions thereof) within one or both of the VH and VL.
- DNA sequences encoding the hybrid variable domains described herein can be produced by oligonucleotide synthesis and/or PCR.
- the nucleic acid encoding CDR regions can also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes.
- the framework regions of the variable chains of the originating species antibody can be changed by site- directed mutagenesis.
- libraries of hybrids can be assembled having members with different combinations of individual framework regions.
- Such libraries can be electronic database collections of sequences or physical collections of hybrids.
- This process typically does not alter the acceptor antibody’s FRs flanking the grafted CDRs.
- one skilled in the art can sometimes improve antigen binding affinity of the resulting anti-HRF, or GST-N19 or N19 CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody.
- Suitable locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (See, e.g., US 5,585,089, especially columns 12-16).
- BsAbs Bispecific Antibodies
- a bispecific antibody is an antibody that can bind simultaneously to two targets that have a distinct structure, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen.
- BsAbs can be made, for example, by combining heavy chains and/or light chains that recognize different epitopes of the same or different antigen.
- a bispecific binding agent binds one antigen (or epitope) on one of its two binding arms (one VH/VL pair), and binds a different antigen (or epitope) on its second arm (a different VH/VL pair).
- bispecific binding agent has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds.
- Bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) of the present technology have at least one arm that specifically binds to, for example, HRF or a fragment thereof and at least one other arm that specifically binds to a second target antigen.
- the second target antigen is an antigen or epitope of a B-cell, a T-cell, a myeloid cell, a plasma cell, or a mast-cell.
- a variety of bispecific fusion proteins can be produced using molecular engineering.
- BsAbs have been constructed that either utilize the full immunoglobulin framework (e.g., IgG), single chain variable fragment (scFv), or combinations thereof.
- the bispecific fusion protein is divalent, comprising, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen.
- the bispecific fusion protein is tetravalent, comprising, for example, an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFv for a second antigen.
- BsAbs composed of two scFv units in tandem have been shown to be a clinically successful bispecific antibody format.
- BsAbs comprise two single chain variable fragments (scFvs) in tandem have been designed such that an scFv that binds a tumor antigen (e.g., HRF or GST-N19 or N19) is linked with an scFv that engages T cells (e.g., by binding CD3).
- a tumor antigen e.g., HRF or GST-N19 or N19
- T cells e.g., by binding CD3
- Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng.10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the needed dual specificities. See, e.g., Coloma et al., Nature Biotech.15:159-163 (1997).
- bispecific fusion proteins can be produced using molecular engineering.
- Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in similar manner. Recombinant methods can be used to produce a variety of fusion proteins.
- a BsAb according to the present technology comprises an immunoglobulin, which immunoglobulin comprises a heavy chain and a light chain, and an scFv.
- the scFv is linked to the C-terminal end of the heavy chain of any HRF, or GST-N19 or N19 immunoglobulin disclosed herein.
- scFvs are linked to the C-terminal end of the light chain of any HRF or fragment thereof, e.g. GST-N19 or N19 immunoglobulin disclosed herein.
- scFvs are linked to heavy or light chains via a linker sequence. Appropriate linker sequences necessary for the in-frame connection of the heavy chain Fd to the scFv are introduced into the VL and Vkappa domains through PCR reactions. The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain.
- scFv-CH1 construct is excised and ligated into a vector containing a DNA sequence encoding the V H region of an HRF or GST-N19 or N19 antibody.
- the resulting vector can be used to transfect an appropriate host cell, such as a mammalian cell for the expression of the bispecific fusion protein.
- a linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.
- a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., first and/or second antigen binding sites).
- a linker is employed in a BsAb described herein based on specific properties imparted to the BsAb such as, for example, an increase in stability.
- a BsAb of the present technology comprises a G 4 S linker.
- a BsAb of the present technology comprises a (G4S)n linker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.
- the anti-HRF or anti-GST-N19 or anti- N19 antibodies of the present technology comprise a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region (or the parental Fc region), such that said molecule has an altered affinity for an Fc receptor (e.g., an Fc ⁇ R), provided that said variant Fc region does not have a substitution at positions that make a direct contact with Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000).
- an Fc receptor e.g., an Fc ⁇ R
- an antiHRF or anti-GST-N19 or anti-N19 antibody of the present technology has an altered affinity for activating and/or inhibitory receptors, having a variant Fc region with one or more amino acid modifications.
- Glycosylation Modifications include amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F/G) loop.
- an antiHRF or anti-GST-N19 or anti-N19 antibody of the present technology has an altered affinity for activating and/or inhibitory receptors, having a variant Fc region with one or more amino acid modifications.
- anti-HRF or anti-GST-N19 or N19 antibodies of the present technology have an Fc region with variant glycosylation as compared to a parent Fc region.
- variant glycosylation includes the absence of fucose; in some embodiments, variant glycosylation results from expression in GnT1-deficient CHO cells.
- the antibodies of the present technology may have a modified glycosylation site relative to an appropriate reference antibody that binds to an antigen of interest (e.g., HRF, GST-N19 or N19), without altering the functionality of the antibody, e.g., binding activity to the antigen.
- glycosylation sites include any specific amino acid sequence in an antibody to which an oligosaccharide (i.e., carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach.
- Oligosaccharide side chains are typically linked to the backbone of an antibody via either N-or O-linkages.
- N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue.
- O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine.
- an Fc- glycoform that lacks certain oligosaccharides including fucose and terminal N- acetylglucosamine may be produced in special CHO cells and exhibit enhanced ADCC effector function.
- the carbohydrate content of an immunoglobulin-related composition disclosed herein is modified by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, e.g., U.S. Patent No.6,218,149; EP 0359096B1 ; U.S. Patent Publication No. US 2002/0028486; International Patent Application Publication WO 03/035835; U.S.
- the carbohydrate content of an antibody is modified by deleting one or more endogenous carbohydrate moieties of the antibody.
- the present technology includes deleting the glycosylation site of the Fc region of an antibody, by modifying position 297 from asparagine to alanine.
- Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function.
- Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N- acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed.
- Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al., 1999, Nat. Biotechnol.17: 176-180; Davies et al., 2001, Biotechnol.
- the anti-HRF, or anti-GST-N19 or anti- N19 antibody of the present technology is a fusion protein.
- the antibodies of the present technology when fused to a second protein, can be used as an antigenic tag.
- domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions.
- the fusion does not necessarily need to be direct, but can occur through linker sequences.
- fusion proteins of the present technology can also be engineered to improve characteristics of the antibodies. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the antibody to improve stability and persistence during purification from the host cell or subsequent handling and storage.
- peptide moieties can be added to an antibody to facilitate purification. Such regions can be removed prior to final preparation of the antibody. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
- the anti-HRF or anti-GST-N19 or anti-N19 antibody of the present technology can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide.
- the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif), among others, many of which are commercially available.
- hexa-histidine provides for convenient purification of the fusion protein.
- HA hemagglutinin protein
- HA hemagglutinin protein
- any of these above fusion proteins can be engineered using the polynucleotides or the polypeptides of the present technology.
- the fusion proteins described herein show an increased half-life in vivo.
- Fusion proteins having disulfide-linked dimeric structures due to the IgG can be more efficient in binding and neutralizing other molecules compared to the monomeric secreted protein or protein fragment alone. Fountoulakis et al., J.
- EP-A-O 464533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or a fragment thereof.
- the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, e.g., improved pharmacokinetic properties. See EP-A 0232262.
- deleting or modifying the Fc part after the fusion protein has been expressed, detected, and purified, may be desired.
- the Fc portion can hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
- human proteins such as hIL-5
- Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
- the antibody or fragment thereof of the present technology is coupled with a label moiety, i.e., detectable group.
- the particular label or detectable group conjugated to the antibody or fragment is not a critical aspect of the technology, so long as it does not significantly interfere with the specific binding of the antibody or fragment thereof of the present technology to the binding partner.
- the detectable group can be any material having a detectable physical or chemical property.
- Such detectable labels have been well- developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology.
- a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
- Labels useful in the practice of the present technology include magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 14 C, 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F, 11 C, 15 O, (for Positron emission tomography), 99m TC, 111 In (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and the like) beads.
- fluorescent dyes e.g., fluorescein isothiocyanate, Texas
- Non-radioactive labels are often attached by indirect means.
- a ligand molecule e.g., biotin
- the ligand then binds to an anti- ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- an anti- ligand e.g., streptavidin
- a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- a number of ligands and anti-ligands can be used.
- a ligand has a natural anti-ligand, e.g., biotin, thyroxine, and cortisol
- a natural anti-ligand e.g., biotin, thyroxine, and cortisol
- any haptenic or antigenic compound can be used in combination with an antibody, e.g., an anti-HRF, or anti- GST-N19 or anti-N19 antibody.
- the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
- Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
- Fluorescent compounds useful as labeling moieties include, but are not limited to, e.g., fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like.
- Chemiluminescent compounds useful as labeling moieties include, but are not limited to, e.g., luciferin, and 2,3- dihydrophthalazinediones, e.g., luminol.
- Means of detecting labels are well known to those of skill in the art.
- means for detection include a scintillation counter or photographic film as in autoradiography.
- the label is a fluorescent label
- it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence.
- the fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
- enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
- simple colorimetric labels can be detected simply by observing the color associated with the label.
- conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
- Some assay formats do not require the use of labeled components.
- agglutination assays can be used to detect the presence of the target antibodies, e.g., the anti- HRF, or anti- GST-N19 or anti- N19 antibodies.
- antigen-coated particles are agglutinated by samples comprising the target antibodies.
- none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
Abstract
Monoclonal antibodies reactive to human histamine releasing factor (HRF) protein and fragments thereof are provided for use diagnostically and therapeutically. These monoclonal antibodies are shown to be strong inhibitors of HRF-IGE interactions and the inflammatory response.
Description
ANTI-HRF ANTIBODIES CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No.63/390,603, filed July 19, 2022, the contents of which are hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT [0002] This invention was made with government support under Grant Nos. AI146042, AI124734, and HL124283 awarded by the US National Institutes of Health (NIH). The government has certain rights in the invention. BACKGROUND [0003] The prevalence of food allergy has been dramatically increasing for the last few decades. Six to eight percent of children under the age of three have food allergies and nearly four percent of adults have them. Symptoms of food allergy range from itching, hives, and diarrhea to life-threatening anaphylaxis. Currently, there is no cure for this disease, although allergen-specific immunotherapy can successfully treat some patients. [0004] Histamine-releasing factor (HRF), also known as translationally controlled tumor protein and fortilin, is a highly conserved protein required for fundamental intracellular functions such as proliferation and survival 1. Since it is secreted during allergic reactions, it is implicated in allergic diseases2. However, to the best of Applicant’s knowledge, effective monoclonal antibodies that specifically recognize HRF for diagnosis and treatment are unknown and a need exists in the art. This disclosure provides such antibodies. SUMMARY [0005] This disclosure provides antibodies and antigen binding fragments thereof that bind to HFR or a fragment thereof. This disclosure also provides compositions for use of the antibodies and fragments thereof, and compositions for manufacturing same. [0006] The provided antibodies or fragments thereof can be used to diagnose, treat, or monitor allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation. Other uses, diagnostic or therapeutic, also are described herein. [0007] In one aspect, exemplary antibodies that bind to HRF or a fragment thereof bind to HRF N19, HRF GST-N19, or HRF-2CA, are disclosed herein, or an equivalent of
each thereof. Additionally or alternatively, the fragment of an HRF comprises, or consists essentially of, or yet further consists of GST-N19 or N19. In one aspect, the HRF or fragment thereof is human HRF or a fragment thereof. In some embodiments, a designated antibody and its equivalent comprises the same CDRs. In some embodiments, the antibody is a rabbit monoclonal antibody. In other embodiments, humanized antibodies are also provided. [0008] Additionally, recombinant anti-HRF antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the disclosure. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques such as, for example, the methods herein as well as described in U.S. Pat. No.7,112,421; Better et al. (1988) Science 240:1041-1043; or Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443. [0009] In some embodiments the antibodies and fragments thereof are described by specific CDR amino acid sequences and comprise an antibody or an antigen binding fragment thereof comprising one or more of as described herein. [0010] The antibodies and antigen binding domains thereof can also be described by variable domains as described herein and equivalents thereof. In some aspects, the equivalent is at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or more identical across the full length of the polypeptide. When determining equivalency, the Clustal Omega alignment program can be used to determine percent identity. Other methods are known in the art. [0011] In one aspect, provided herein is an antibody or an antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more or all three of the following sequences: a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; or a heavy-chain CDR 3 (HCDR3) that comprise the amino acid sequence ARVSASYTSDGDAIIHSFAL. [0012] In one aspect, provided herein is an antibody or an antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more or all three of the
following sequences: a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS or a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG. [0013] In one aspect, provided herein is an antibody or antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more of: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; b) a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; c) a heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; d) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; e) a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; or f) a light-chain CDR) 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG. [0014] In a further aspect, provided herein is antibody or antigen binding fragment thereof comprising: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; a heavy- chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; and b) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; and a light-chain CDR 3 (LCDR3) that comprises one of the
following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG. [0015] In one aspect, provided herein is an antibody or an antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more of the following amino acid sequences: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; b) QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; c) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; d) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGALVTVSS; e) QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; f) QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; g) QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; h) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS; or i) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG
DAIIHSFALWGQGTLVTVSS. Also provided is an antibody or antigen binding fragment thereof, comprising, consisting of, or consisting essentially of: a) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVK; b) a heavy chain variable region comprising the amino acid sequence: QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; c) a heavy chain variable region comprising the amino acid sequence: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; d) a heavy chain variable region comprising the amino acid sequence: QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGALVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI
YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; e) a heavy chain variable region comprising the amino acid sequence: QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGG GTEVVVK; f) a heavy chain variable region comprising the amino acid sequence: QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFG GGTEVVVK; g) a heavy chain variable region comprising the amino acid sequence: QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK; h) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI
YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; or i) a heavy chain variable region comprising the amino acid sequence: QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK. [0016] In one aspect. provided herein is an antibody or an antigen binding fragment thereof comprising, consisting of, or consisting essentially of one or more of the following amino acid sequences: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVK; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; c) AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; d) ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; e) AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGG GTEVVVK; f) AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFG GGTEVVVK;
g) AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK; h) AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; or i) ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK. [0017] In one aspect, provided herein is an antibody or antigen binding fragment, comprising, consisting of, or consisting essentially of: a) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVK; b) a heavy chain variable region comprising the amino acid sequence: QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; c) a heavy chain variable region comprising the amino acid sequence: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence:
AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; d) a heavy chain variable region comprising the amino acid sequence: QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGALVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; e) a heavy chain variable region comprising the amino acid sequence: QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGG GTEVVVK; f) a heavy chain variable region comprising the amino acid sequence: QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFG GGTEVVVK; g) a heavy chain variable region comprising the amino acid sequence: QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence:
AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK; h) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; or i) a heavy chain variable region comprising the amino acid sequence: QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK. [0018] In one aspect, provided herein is an antibody or antigen binding fragment, comprising, consisting of, or consisting essentially of a heavy chain region comprising one of the following amino acid sequences: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLP EPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPAT NTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVD VSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKE FKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGF YPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFT CSVMHEALHNHYTQKSISRSPGK;
b) QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; c) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; d) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGALVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLP EPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPAT NTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVD VSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKE FKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGF YPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFT CSVMHEALHNHYTQKSISRSPGK; e) QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS
QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; f) QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; g) QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; h) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL PEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPA TNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVV DVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRG KEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMIN GFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDV FTCSVMHEALHNHYTQKSISRSPGK; or
i) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL PEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPA TNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVV DVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRG KEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMIN GFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDV FTCSVMHEALHNHYTQKSISRSPGK. [0019] In one aspect, provided herein is an antibody or antigen binding fragment, comprising, consisting of, or consisting essentially of a light chain region comprising one of the following amino acid sequences: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVV QSFNRGDC; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTT QTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSF NRGDC; c) AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTT QTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSF NRGDC; d) ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTT QTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTRGTTSVVQSF NRGDC;
e) AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGG GTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGT TQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQS FNRGDC; f) AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFG GGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSAS PIVQSFNRGDC; g) AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGT TQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQS FNRGDC; h) AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTT QTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTLGTTSVVQSF NRGDC; or i) ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGT TQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQS FNMGDC. [0020] In one aspect, provided herein is a humnaized antibody or antigen binding fragment that comprises, or consists of, or consists essentially of one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises, consists of or consists essentially of the amino acid sequence: QSSQSVYDNNNLA; a light-chain (CDR) 2 (LCDR2) that comprises, consists of or consists essentially of the amino acid sequence: DASKLPS; or a light-chain CDR 3
(LCDR3) that comprises, consists of or consists essentially of the amino acid sequence: AGAVSGSNV. [0021] In a further aspect, provided herein is a humanized antibody or antigen binding fragment thereof comprising, consisting of, or consisting essentially of: [0022] a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises, consists of or consists essentially of the amino acid sequence TVSGFSLSSGAVS; a heavy-chain CDR 2 (HCDR2) that comprises, consists of or consists essentially the amino acid sequence: IGVISSRDIAYFATWAKG; a heavy-chain CDR 3 (HCDR3) that comprises, consists of or consists essentially of the amino acid sequence ARVSASYTSDGDAIIHSFAL. [0023] Also provided is a humanized antibody or an antigen binding fragment thereof, comprising, consists of, or consists essentially of a light-chain variable region comprising, consisting of, or consisting essentially of the following amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. [0024] In a further aspect, the humanized antibody or an antigen binding fragment thereof, comprises, consists of or consists essentially of a heavy-chain variable region comprising, consisting of, or consisting essentially of the following amino acid sequence: [0025] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. [0026] In some embodiments, the antibody or antigen binding fragment thereof specifically recognizes and binds to one or more of: HRF, HRF-2CA or N19 or GST-N19, or an immunogenic fragment thereof. In one aspect, the HRF, HRF-2CA, N19 or GST-N19 are human proteins, fragments or variants thereof. Examples of such are provided herein. In some embodiments, the HRF comprises or consists of the amino acid sequence of SEQ ID NO: 1. [0027] In some embodiments, the antibody or antigen binding fragment is isolated, purified or recombinant.
[0028] In some embodiments, the antibody or antigen binding fragment is monospecific. In other embodiments, the antibody or antigen binding fragment is multispecific, such as bispecific, e.g., binding to two or more epitopes. In further embodiments, the two or more epitopes are all epitopes of an HRF or a fragment thereof. In other embodiments, at least one of the two or more epitopes is an epitope of an HRF or a fragment thereof. In further embodiments, at least one of the two or more epitopes are of a protein other than an HRF. [0029] In some embodiments, provided is an antibody or antigen binding fragment thereof that competes with any one of an antibody or antigen binding fragment as disclosed herein for binding to HRF thereof, or a fragment of the HRF. Methods to determine competition between 2 antibodies or antigen binding fragments thereof are known in the art. [0030] In some embodiments, the antibody or antigen binding fragment comprises a fragment crystallizable region (Fc region). In further embodiments, the Fc region is a human Fc region. Additionally or alternatively, the Fc region comprises, or alternatively consists essentially of, or yet further consists of one or more of: an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgM Fc region, or an IgE Fc region. In yet further embodiments, the Fc region comprises, or alternatively consists essentially of, or yet further consists of one or more of: an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG4 Fc region. [0031] In some embodiments, the antibody or antigen binding fragment is post- translationally modified optionally glycosylated, hydroxylated, methylated, lapidated, acetylated, SUMOylated, phosphorylated, PEGylated, or any combination thereof. [0032] In some embodiments, the antibody or antigen binding fragment further comprises a detectable or purification marker. [0033] Polynucleotides encoding the antibodies, fragments and complements thereof are further provided as well as vectors and host cells containing same. [0034] In some embodiments, the antibodies or fragments thereof as described herein can be used for various in vitro molecular biology applications such as, enzyme-linked immunosorbent assays (ELISA), Western blots, immunohistochemistry, immunocytochemistry, flow cytometry and fluorescence-activated cell sorting (FACS), immunoprecipitation, or enzyme-linked immunospot assays. In some embodiments, the antibodies or fragments thereof can be packaged in kits with or without additional reagents known to those of skill in the art for practicing any of the molecular biology techniques as disclosed herein. The polynucleotides are useful to replicate or detect encoding
polynucleotides. Host cells are useful to replicate the polynucleotides or encode the antibodies or fragments thereof. These can be provided alone or in a composition, optionally comprising a pharmaceutically acceptable carrier. [0035] In another aspect, provided herein is a polynucleotide encoding the antibody or antigen binding fragment thereof. In yet another aspect, provided herein is a vector comprising, consisting of, or consisting essentially of the polynucleotide encoding the antibody or antigen binding fragment thereof. In yet another aspect, provided herein is a cell comprising, consisting of, or consisting essentially of one or more of the antibody or antigen binding fragment thereof, the polynucleotide encoding the antibody or antigen binding fragment thereof, or the vector comprising the polynucleotide. In yet another aspect, provided herein is a hybridoma expressing the antibody or antigen binding fragment thereof. [0036] In yet another aspect, provided herein is a method of producing the antibody or antigen binding fragment thereof comprising culturing a cell comprising a polynucleotide encoding he antibody or antigen binding fragment thereof. [0037] In another aspect, the present disclosure provides a method of preventing or treating a disease, such as allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, in a subject in need thereof, comprising, or consisting essentially of, or yet further consisting of administering to the subject, optionally a therapeutically or prophylactically effective amount of, a pharmaceutical composition comprising, or consisting essentially of, or yet further consisting of one or more of the antibodies or antigen binding fragments as described herein. Such a method can comprise, or consists essentially of, or yet further consists of administration of any dose of the antibodies described herein effective for ameliorating or treating symptoms of allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation. Methods for determining if the disease has been treated or prevented are known in the art and include a reduction in symptoms, or severity of symptoms or the presence of neutralizing antibodies in the subject being treated. BRIEF DESCRIPTION OF THE DRAWINGS [0038] FIGS.1A – 1C: An anti-N19 mAb suppressed food allergy in mice. (FIG. 1A) Anti-N19 Fabs inhibited interactions between C38-2 IgE (concentrations indicated) and immobilized mouse HRF by ELISA. D-A10 and H8-6 are Fabs that, respectively, bind or do not to HRF, serving as a positive or a negative control. (FIG.1B) Purified anti-N19 mAb
SPF7-1 and isotype control IgG1 were analyzed by SDS-PAGE under reducing and nonreducing conditions and stained with Coomassie brilliant blue G-250. Mr, molecular weight marker in kilodaltons. (FIG.1C) The anti-N19 mAb SPF7-1 suppressed hypothermia induced by OVA gavage in OVA-sensitized mice. Temperature changes were compared between SPF7-1- and isotype control-treated mice by Student’s t-test. *, **, ***: p<0.05, p<0.001, p<0.0001. [0039] FIGS.2A – 2C: PEGylated N19 peptide suppressed OVA-induced food allergy in mice. (FIG.2A) List of the tested N19 peptides and recombinant HRF inhibitors. (B,C) N19-PEG peptide suppressed diarrhea (FIG.2B) and hypothermia (FIG.2C) induced by OVA gavage in OVA-sensitized mice. Asterisks, * and ** indicates p<0.05 and p<0.01, respectively, compared with PBS control in Student’s t-test. [0040] FIGS.3A-3B: Body temperature changes compared between SPF7, L2H1, and L2H2 humanized antibodies indicate L2H1 and L2H2 clones exhibiting inhibitory effects on ovalbumin (OVA)-induced food allergy (FIG.3A). MMCP-1 levels in serum were quantified by an ELISA (enzyme-linked immunosorbent assay) kit from BioLegend (a San Diego company), according to the manufacturer's instructions (FIG.3B). [0041] FIG.4: Binding of humanized SPF7 clones was compared to that of the parent rabbit IgG SPF7. Microtiter wells were coated with a recombinant HRF protein at 1 µg/mL in PBS at 4 ˚C overnight. The wells were washed with PBS and blocked with 1% BSA/PBS. The IgGs were serially diluted and incubated with the coated antigen at RT for 1 h. The bound IgGs were detected with peroxidase conjugated goat anti-human IgG Fcγ specific or anti-rabbit IgG Fcγ specific antibodies. The humanized clones showed equivalent binding to the antigen as the parent rabbit IgG. FIG.4 shows EC50 maximal effective concentration values demonstrating that humanized antibodies to HRF are similar to that of the parent antibody SPF7. DETAILED DESCRIPTION Definitions [0042] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and/or separating the subject matter described. [0043] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of this invention will be limited only by the appended claims. [0044] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety or in part as referenced herein. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure. [0045] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).
[0046] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. [0047] As used herein, the term “comprising” is intended to mean that the compounds, compositions, and methods include the recited elements, but not exclude others. “Consisting essentially of” when used to define compounds, compositions, and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology. [0048] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. [0049] The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1 % of the specified amount. [0050] As used herein, comparative terms as used herein, such as high, low, increase, decrease, reduce, or any grammatical variation thereof, can refer to certain variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 folds, or about 3 folds, or about 4 folds, or about 5 folds, or about 6 folds, or about 7 folds, or about 8 folds, or about 9 folds, or about 10 folds, or about 20 folds, or about 30 folds, or about 40 folds, or about 50 folds, or about 60 folds, or about 70 folds, or about 80 folds, or about 90 folds, or about 100 folds or more higher than the reference. In some embodiments, such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%,
or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference. [0051] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. [0052] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. [0053] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). [0054] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. [0055] The terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose. [0056] The phrase “therapeutically effective” is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder or on the effecting of a clinical endpoint. In some embodiments, an antibody or antigen binding fragment thereof is administered to a subject in a therapeutically effective amount. [0057] The term “therapeutically acceptable” refers to those compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use. In some embodiments, an antibody or antigen binding fragment thereof is administered to a subject in a therapeutically acceptable amount. [0058] In some embodiments, the terms “first” “second” “third” “fourth” or similar in a component name are used to distinguish and identify more than one component sharing certain identity in their names. For example, “first antibody” and “second antibody” are used to distinguishing two antibodies.
[0059] The term “antibody” as used herein, includes both full-length immunoglobulins and antibody fragments that bind to the same antigens. Non-limiting examples include a monoclonal, polyclonal, chimeric, humanized, or single chain antibody. In one embodiment, the disclosure provides an isolated antibody and antigen binding fragments thereof that bind to HRF, e.g., human HRF or a fragment thereof. In some embodiments, the fragment is an immunogenic fragment. [0060] The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides, proteins and/or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. [0061] In some embodiments, the term “engineered” or “recombinant” refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain or the parental host strain of the referenced species. In some embodiments, the term “engineered” or “recombinant” refers to being synthetized by human intervention. As used herein, the term “recombinant protein” refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous HRF. [0062] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for
example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. [0063] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. [0064] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some embodiments, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the
field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch. [0065] A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. [0066] The term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins. As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. [0067] A “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated. In some embodiments, the gene product may refer to an mRNA or other RNA, such as an interfering RNA, generated when a gene is transcribed. [0068] The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed to produce the mRNA for the polypeptide or a fragment thereof, and optionally translated to produce the polypeptide or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. Further, as used herein an amino acid sequence coding sequence refers to a nucleotide sequence encoding the amino acid sequence. [0069] “Under transcriptional control”, which is also used herein as “directing expression of” or any grammatical variation thereof, is a term well understood in the art and indicates that transcription and optionally translation of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. [0070] “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.
[0071] In some embodiments, “directing the replication of” or any grammatical variation thereof is a term well understood in the art and indicates that replication of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to a regulatory sequence, such as an origin of replication or a primer. [0072] The term “a regulatory sequence”, “an expression control element” or “promoter” as used herein, intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed or replicated, and facilitates the expression or replication of the target polynucleotide. [0073] A promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. Polymerase II and III are examples of promoters. In some embodiments, a regulatory sequence is bidirectional, i.e., acting as a regulatory sequence for the coding sequences on both sides of the regulatory sequence. Such bidirectional regulatory sequences may comprises, or consists essentially of, or consists of a bidirectional promoter (see for example Trinklein ND, et al. An abundance of bidirectional promoters in the human genome. Genome Res.2004 Jan;14(1):62-6). [0074] The term “promoter” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example. A “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. Non-limiting examples of promoters include the EF1alpha promoter and the CMV promoter. The EF1alpha sequence is known in the art (see, e.g., addgene.org/11154/sequences/; ncbi.nlm.nih.gov/nuccore/J04617, each last accessed on March 13, 2019, and Zheng and Baum (2014) Int’l. J. Med. Sci. 11(5):404-408). The CMV promoter sequence is known in the art (see, e.g., snapgene.com/resources/plasmid- files/?set=basic_cloning_vectors&plasmid=CMV_promoter, last accessed on March 13, 2019, and Zheng and Baum (2014), supra.). [0075] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter/enhancer" is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer/promoter may be
"endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer/promoter is one which is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter. [0076] As used herein, the term “enhancer”, as used herein, denotes sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed. An enhancer may enhance transcription from a single promoter or simultaneously from more than one promoter. As long as this functionality of improving transcription is retained or substantially retained (e.g., at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or higher of wild- type activity, that is, activity of a full-length sequence), any truncated, mutated or otherwise modified variants of a wild-type enhancer sequence are also within the above definition. [0077] In some embodiments, the term “vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and optionally integrate into the target cell’s genome. Non-limiting examples of vectors include a plasmid, a nanoparticle, a liposome, a virus, a cosmid, a phage, a BAC, a YAC, etc. In some embodiments, plasmid vectors may be prepared from commercially available vectors. In other embodiments, viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector. In one embodiment, the viral vector is a retroviral vector. [0078] A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this
location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produce HRFs to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass- producing a gene or the protein it then codes for. [0079] A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. As is known to those of skill in the art, there are 6 classes of viruses. The DNA viruses constitute classes I and II. The RNA viruses and retroviruses make up the remaining classes. Class III viruses have a double-stranded RNA genome. Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA Class V viruses have a negative single-stranded RNA genome used as a template for mRNA synthesis. Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol.5:434-439 and Ying, et al. (1999) Nat. Med.5(7):823-827. As used herein, Multiplicity of infection (MOI) refers to the number of viral particles that are added per cell during infection. [0080] A retrovirus such as a gammaretrovirus and/or a lentivirus comprises (a) envelope comprising lipids and glycoprotein, (b) a vector genome, which is a RNA (usually a dimer RNA comprising a cap at the 5’ end and a polyA tail at the 3’ end flanked by LTRs) derived to the target cell, (c) a capsid, and (d) proteins, such as a protease. U.S. Patent No. 6,924,123 discloses that certain retroviral sequence facilitate integration into the target cell genome. This patent teaches that each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral genes. Encapsidation of the retroviral
RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome. The LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from the sequence unique to the 5'end of the RNA. The sizes of the three elements can vary considerably among different retroviruses. For the viral genome and the site of poly (A) addition (termination) is at the boundary between R and U5 in the right hand side LTR. U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins. [0081] With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome. [0082] For the production of viral vector particles, the vector RNA genome is expressed from a DNA construct encoding it, in a host cell. The components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the "packaging system", which usually includes either or both of the gag/pol and env genes) expressed in the host cell. The set of sequences required for the production of the viral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways. The techniques involved are known to those skilled in the art. [0083] The term “adeno-associated virus” or “AAV” as used herein refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered, AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant or synthetic serotypes, e.g., AAV-DJ and AAV PHP.B. The AAV particle comprises, alternatively consists essentially of, or yet further consists of three major viral proteins: VP1, VP2 and VP3. In one embodiment, the AAV refers to of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,
AAV13, AAV PHP.B, or AAV rh74. These vectors are commercially available or have been described in the patent or technical literature. [0084] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. [0085] Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg2+ normally found in a cell. [0086] Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1x SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1x SSC, 0.1x SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
[0087] When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called “annealing” and those polynucleotides are described as “complementary.” A double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. “Complementarity” or “homology” (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules. [0088] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. In some embodiments, the identity is calculated between two peptides or polynucleotides over their full-length, or over the shorter sequence of the two, or over the longer sequence of the two. [0089] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: www.ncbi.nlm.nih.gov/cgi-bin/BLAST. [0090] In some embodiments, the polynucleotide as disclosed herein is a RNA or an analog thereof. In some embodiments, the polynucleotide as disclosed herein is a DNA or an
analog thereof. In some embodiments, the polynucleotide as disclosed herein is a hybrid of DNA and RNA or an analog thereof. [0091] In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide encodes the same sequence encoded by the reference. In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide hybridizes to the reference, a complement reference, a reverse reference, or a reverse-complement reference, optionally under conditions of high stringency. [0092] Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 70% sequence identity, or at least 75% sequence identity, or at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence, or alternatively at least 99 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively an equivalent nucleic acid hybridizes under conditions of high stringency to a reference polynucleotide or its complementary. In one aspect, the equivalent must encode the same protein or a functional equivalent of the protein that optionally can be identified through one or more assays described herein. In addition or alternatively, the equivalent of a polynucleotide would encode a protein or polypeptide of the same or similar function as the reference or parent polynucleotide. [0093] The term “transduce” or “transduction” refers to the process whereby a foreign nucleotide sequence is introduced into a cell. In some embodiments, this transduction is done via a vector, viral or non-viral. [0094] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
[0095] As used herein, an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence. Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST. [0096] As used herein, the term “edit distance” refers to the minimum number of insertions, deletions or substitutions required to transform a first sequence of a first nucleotide or peptide into a second sequence of a second nucleotide or polypeptide. Accordingly, in some embodiments, the edit distance between two sequences can be presented by the minimum number of insertions, deletions or substitutions. Various tools are available for calculating an edit distance, such as a sequence alignment program aligning two sequences and noting the differences therebetween, including insertions, deletions or substitutions. Non-limiting suitable alignment programs include Clustal Omega (accessible at www.ebi.ac.uk/Tools/msa/clustalo/), Needle (EMBOSS, accessible at /www.ebi.ac.uk/Tools/psa/emboss_needle/), Stretcher (EMBOSS, accessible at www.ebi.ac.uk/Tools/psa/emboss_stretcher/), Water (EMBOSS, accessible at www.ebi.ac.uk/Tools/psa/emboss_water), Matcher (EMBOSS, accessible at www.ebi.ac.uk/Tools/psa/emboss_matcher), LALIGN (accessible at www.ebi.ac.uk/Tools/psa/lalign) and BLAST (accessible at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_ PROG_DEF=blastn&BLAST_SPEC=GlobalAln&LINK_LOC=BlastHomeLink and www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?LINK_LOC=BlastHomeLink). In aspects of this disclosure, the Clustal Omega alignment program is used to determine sequence identity. [0097] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits, llama and mice, as well as non-mammalian species, such as shark immunoglobulins. [0098] Unless specifically noted otherwise, the term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103 M-1
greater, at least 104 M-1 greater or at least 105 M-1 greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, murine or humanized non-primate antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Owen et al., Kuby Immunology, 7th Ed., W.H. Freeman & Co., 2013; Murphy, Janeway’s Immunobiology, 8th Ed., Garland Science, 2014; Male et al., Immunology (Roitt), 8th Ed., Saunders, 2012; Parham, The Immune System, 4th Ed., Garland Science, 2014. In some embodiments, the term “antibody” refers to a single-chain variable fragment (scFv or ScFV). In some embodiments, the term “antibody” refers to more than one single-chain variable fragments (scFv or ScFV) linked with each other, optionally via a peptide linker or another suitable component as disclosed herein. In some embodiments, an antibody is a monoclonal antibody. In some embodiments, an antibody is a monospecific antibody or a multispecific antibody, such as a bispecific antibody or a trispecific antibody. The species of the antibody can be a human or non-human, e.g., mammalian. [0099] In certain embodiments, an antigen binding fragment of an antibody contains at least one variable domain optionally covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that are found within an antigen-binding fragment of an antibody of the present invention include: (i) VH- CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH—CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH- CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains are either directly linked to one another or are linked by a full or partial hinge or linker region. A hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen binding fragment of an antibody of the present disclosure can comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed herein in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). [00100] As used herein, an epitope refers to contiguous or non-contiguous amino acid residues in an antigen, such as those adjacent to each other in a three-dimensional structure of
the antigen, wherein those residues are recognized and bound by an antibody or another component of the immune system. [00101] As used herein, the term “multispecific” refers to capability of binding to more than one epitopes or antigens which are different from each other. In some embodiments, the term “multispecific” refers to comprising, or consisting essentially of, or consisting of more than one antigen binding sequences or antigen ligands, optionally linked together by a peptide linker or another component as disclosed herein. In further embodiments, the term “multispecific” refers to comprising, or consisting essentially of, or consisting of more than one antigen binding sequences (such as scFv), optionally linked together by a peptide linker or another component as disclosed herein. In some embodiments, the more than one (such as two) epitopes are located in the same antigen. Alternatively, the more than one (such as two) epitopes are from at least two antigens. In some embodiments, the ligand refers a ligand of the antigen. In some embodiments, a multispecific antibody comprises, or consists essentially of, or consists of at least two antigen binding sequences. In some embodiments, a multispecific antibody comprises, or consists essentially of, or consists of at least one antigen binding sequence and at least one ligand (such as a polypeptide comprising or consisting of a binding domain of the antigen’s receptor). [00102] Accordingly, a bispecific antibody (abbreviated as BsAb) refers to an antibody capable of binding to two epitopes or antigens which are different from each other. In some embodiments, a bispecific antibody comprises, or consists essentially of, or consists of two antigen binding sequences or antigen ligands, optionally linked together by a peptide linker or another component as disclosed herein. In further embodiments, a bispecific antibody comprises, or consists essentially of, or consists of two antigen binding sequences (such as scFv), optionally linked together by a peptide linker or another component as disclosed herein. In some embodiments, a bispecific antibody comprises, or consists essentially of, or consists of one antigen binding sequence recognizing and binding the first epitope and one ligand recognizing and binding the antigen comprising the second epitope. In some embodiments, the two epitopes are located in the same antigen. Alternatively, the two epitopes are from two antigens which are different from each other. In some embodiments, the ligand refers to a ligand of the antigen, such as a polypeptide comprising or consisting of a binding domain of the antigen’s receptor. In some embodiments, a bispecific antibody comprises, or consists essentially of, or consists of at least two antigen binding sequences. In
some embodiments, a bispecific antibody comprises, or consists essentially of, or consists of at least one antigen binding sequence and at least one ligand. [00103] As used herein, the term “monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies. [00104] In terms of antibody structure, an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa ( ^). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, largely adopts a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. [00105] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located (heavy chain regions labeled CDRH or HCDR and light chain regions labeled CDRL or LCDR). Thus, a HCDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found, whereas a LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
[00106] As used herein, a single-chain variable fragment (scFv or ScFV), also referred to herein as a fragment or an antigen binding fragment of an antibody, and is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, optionally connected with a short linker peptide of about 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. The HRF retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. [00107] As used herein, a fragment crystallizable (Fc) region refers to the tail region of an antibody that stabilizes the antibody, such as a bispecific antibody, and optionally interacts with (such as binds) an Fc receptor on an immune cell or on a platelet or that binds a complement protein. [00108] The polypeptide or an equivalent thereof, can be followed by an additional 50 amino acids, or alternatively about 40 amino acids, or alternatively about 30 amino acids, or alternatively about 20 amino acids, or alternatively about 10 amino acids, or alternatively about 5 amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the carboxy- terminus (C-terminus). Additionally or alternatively, the polypeptide or an equivalent thereof can further comprises an additional 50 amino acids, or alternatively about 40 amino acids, or alternatively about 30 amino acids, or alternatively about 20 amino acids, or alternatively about 10 amino acids, or alternatively about 5 amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the amine-terminus (N-terminus). [00109] An equivalent of a reference polypeptide comprises, consists essentially of, or alternatively consists of an polypeptide having at least 80%, or at least 85 %, or at least 90%, or at least 95%, or at least about 96%, or at least 97%, or at least 98%, or at least 99% amino acid identity to the reference polypeptide (as determined, in one aspect using the Clustal Omega alignment program), such as the antibody or antigen binding fragment thereof as disclosed herein, or a polypeptide that is encoded by a polynucleotide that hybridizes under conditions of high stringency to the complementary sequence of a polynucleotide encoding the reference polypeptide, such as an antibody or antigen binding fragment thereof as disclosed herein, optionally wherein conditions of high stringency comprises incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1x SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1x SSC, 0.1x SSC, or deionized water.
[00110] Alternative embodiments include one or more of the CDRs (e.g., CDR1, CDR2, CDR3) from the LC variable region substituted with appropriate CDRs from other antibody CDRs, or an equivalent of each thereof. Accordingly, and as an example, the CDR1 and CDR2 from the LC variable region can be combined with the CDR3 of another antibody’s LC variable region, and in some aspects, can include an additional 50 amino acids, or alternatively about 40 amino acids, or alternatively about 30 amino acids, or alternatively about 20 amino acids, or alternatively about 10 amino acids, or alternatively about 5 amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the carboxy-terminus. [00111] In some embodiments, the term “equivalent” or “biological equivalent” of an antibody means the ability of the antibody to selectively bind its epitope protein or a fragment thereof as measured by ELISA or other suitable methods is substantively maintained, for example, at a level of at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or more. Biologically equivalent antibodies include, but are not limited to, those antibodies, peptides, antibody fragments, antibody variant, antibody derivative and antibody mimetics that bind to the same epitope as the reference antibody. Additionally or alternatively, the equivalent and the reference antibody shares the same set of CDRs but other amino acids are modified. [00112] In some embodiments, a first sequence (nucleic acid sequence or amino acid) is compared to a second sequence, and the identity percentage or edit distance between the two sequences can be calculated. In further embodiments, the first sequence can be referred to herein as an equivalent and the second sequence can be referred to herein as a reference sequence. In yet further embodiments, the identity percentage is calculated based on the full- length sequence of the first sequence. In other embodiments, the identity percentage is calculated based on the full-length sequence of the second sequence. [00113] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents
thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity, or at least about 85 % homology or identity, or alternatively at least about 90 % homology or identity, or alternatively at least about 95 % homology or identity, or alternatively 98 % or 99% homology or identity (in one aspect, as determined using the Clustal Omega alignment program) and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complementary sequence. [00114] In some embodiments, an antibody as disclosed herein comprises, or consists essentially of, or yet further consists of an anybody variant. The term “antibody variant” intends to include antibodies produced in a species other than a mouse. It also includes antibodies containing post-translational modifications to the linear polypeptide sequence of the antibody or a fragment thereof. It further encompasses fully human antibodies. [00115] In some embodiments, an antibody as disclosed herein comprises, or consists essentially of, or yet further consists of an antibody derivative. The term “antibody derivative” is intended to encompass molecules that bind an epitope as defined above and which are modifications or derivatives of a native monoclonal antibody of this disclosure. Derivatives include, but are not limited to, for example, bispecific, multispecific, heterospecific, trispecific, tetraspecific, multispecific antibodies, diabodies, chimeric, recombinant and humanized. [00116] As used herein, the term “specific binding” or “binding” means the contact between an antibody and an antigen with a binding affinity of at least 10−6 M. In certain embodiments, antibodies bind with affinities of at least about 10−7 M, and preferably at least about 10−8 M, at least about 10−9 M, at least about 10−10 M, at least about 10−11 M, or at least about 10−12 M. [00117] As used herein, the term “antigen” refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune
disease, allergy and graft rejection, toxins, and other miscellaneous antigens. In some embodiments, the antigen as referred to herein is HRF or an immunogenic fragment. [00118] Exemplary mammalian HRF sequences include human and non-human HRF sequences. Exemplary Human (NM_003295), Mouse (NM_009429), Rat (NM_053867), Rabbit (NM_001082129), Guinea Pig (NM_001173082), Chimpanzee (NM_001098546), Monkey (NM_001095869), Dog (NM_851473), Pig (NM_214373) and Bovine (NM_001014388). Additional sequences are found in Table 1 of WO 2020/102108, published May 22, 2020. [00119] Exemplary HRF sequences that bind to an immunoglobulin (Ig) include HRF that binds to one or more of IgM, IgG, IgE, IgA, or IgD. Particular IgE to which HRF binds are associated with immune disorders and diseases such as those associated with allergies (food or other antigens), asthma, hypersensitivity reactions and inflammation. [00120] As used herein, the term “equivalent” in the context of describing a peptide, a polypeptide, or a protein encompass the wild-type sequence, sequences of variants, and sequences comprising one or more modifications. For example, an equivalent of a HRF monomer can encompass a HRF variant as provided in WO 2020/102108, published May 22, 2020. An equivalent of a HRF monomer of SEQ ID NO: 4 as provided in WO 2020/102108, published May 22, 2020, can also encompass at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4 published in WO 2020/102108, published May 22, 2020. An equivalent of a HRF monomer of SEQ ID NO: 4 as provided in WO 2020/102108, published May 22, 2020 can further encompass at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4 published in WO 2020/102108, published May 22, 2020, while retaining one or more modifications, optionally amino acid substitution(s) at C28 and/or C172 according to SEQ ID NO: 4 published in WO 2020/102108, published May 22, 2020. [00121] In some embodiments, antigen of a binding moiety, such as an antibody, an antigen binding fragment thereof, can be provided herein in a format of “antigen” followed by the binding moiety (such as an anti-HRF antibody), or having “anti” or “anti-” before the antigen and the binding moiety after the antigen (such as an anti-HRF antibody), or the binding moiety followed by “to” or “directed to” and then the antigen (such as an antibody to HRF).
[00122] In some embodiments, a fragment of a protein can be an immunogenic fragment. As used herein, the term “immunogenic fragment” refers to such a polypeptide fragment, which at least partially retains the immunogenicity of the protein from which it is derived. In some embodiments, the immunogenic fragment is at least about 3 amino acid (aa) long, or at least about 4 aa long, or at least about 5 aa long, or at least about 6 aa long, or at least about 7 aa long, or at least about 8 aa long, or at least about 9 aa long, or at least about 10, aa long, or at least about 15, aa long, or at least about 20 aa long, or at least about 25 aa long, or at least about 30 aa long, or at least about 35 aa long, or at least about 40 aa long, or at least about 50 aa long, or at least about 60 aa long, or at least about 70 aa long, or at least about 80 aa long, or at least about 90 aa long, or at least about 100 aa long, or at least about 120 aa long, or at least about 150 aa long, or at least about 200, or longer. In some embodiments, an immunogenic fragment of HRF protein comprises, or alternatively consists essentially of, or yet further consists of antibodies and subsequences thereof that bind to a HRF/TCTP sequence that includes or consists of a region of HRF that binds to an Ig, such as an IgE. [00123] In a particular embodiment, a sequence of HRF to which antibodies or subsequences thereof bind include or consist of amino acids (MIIYRDLISHDEMFSDIYK (HRF N19)) or a GST fusion of such (GST- MIIYRDLISHDEMFSDIYK (HRF GST-N19)) or the amino acids (QETSFTKEAYKKYIKDYMKSIKGKLEEQRPERVKPFMTGAAEQIKHILANFKNYQFF IGEN) MNP of mammalian HRF, or an equivalent of each thereof. Such antibodies can also bind to any subsequence of the HRF/TCTP sequence that includes or consists of a region of HRF that binds to an Ig, such as an IgE. In a particular embodiment, a subsequence is a portion of amino acids 1-19 (MIIYRDLISHDEMFSDIYK (HRF N19) or a GST fusion of such (GST- MIIYRDLISHDEMFSDIYK (HRF GST-N19)) or a portion of amino acids (QETSFTKEAYKKYIKDYMKSIKGKLEEQRPERVKPFMTGAAEQIKHILANFKNYQFF IGEN) MNP of mammalian HRF, or a portion of (MIIYRDLISHDEMFSDIYKIREIADGLCLEVEGKMVSRTEGNIDDSLIGGNASAEGPE GEGTESTVITGVDIVMNHHLQETSFTKEAYKKYIKDYMKSIKGKLEEQRPERVKPFM TGAAEQIKHILANFKNYQFFIGENMNPDGMVALLDYREDGVTPYMIFFKDGLEMEK C), wherein the subsequence is between 5-171 amino acid residues in length, e.g., 5-10, 10- 20, 20-50, 100-150, or 150-171 amino acid residues in length, or an equivalent of each thereof.
[00124] As used herein, the terms “antigen binding fragment,” “fragment,” and “antibody fragment” are used interchangeably to refer to any fragment that comprises a portion of a full-length antibody, generally at least the antigen binding portion or the variable region thereof. Examples of antibody fragments include, but are not limited to, diabodies, single-chain antibody molecules, multi-specific antibodies, Fab, Fab’, F(ab')2, Fv or scFv. In some embodiments, the term “antigen binding domain” refers to any protein or polypeptide domain that can specifically bind to an antigen target. [00125] Epitopes typically are short amino acid sequences, e.g. about five to 15 amino acids in length. Epitopes can be contiguous or non-contiguous. A non-contiguous amino acid sequence epitope forms due to protein folding. For example, an epitope can include a non-contiguous amino acid sequence, such as a 5 amino acid sequence and an 8 amino acid sequence, which are not contiguous with each other, but form an epitope due to protein folding. Techniques for identifying epitopes are known to the skilled artisan and include screening overlapping oligopeptides for binding to antibody (for example, U.S. Patent No.4,708,871), phage display peptide library kits, which are commercially available for epitope mapping (New England BioLabs). Epitopes may also be identified by inference when epitope length peptide sequences are used to immunize animals from which antibodies that bind to the peptide sequence are obtained and can be predicted using computer programs, such as BEPITOPE (Odorico et al., J. Mol. Recognit.16:20 (2003)) [00126] Methods of producing polyclonal and monoclonal antibodies are known in the art. For example, HRF, or a subsequence thereof, or an immunogenic fragment thereof, optionally conjugated to a carrier such as keyhole limpet hemocyanin (KLH) or ovalbumin (e.g., BSA), or mixed with an adjuvant such as Freund’s complete or incomplete adjuvant, and used to immunize an animal. Using conventional hybridoma technology, splenocytes from immunized animals that respond to HRF can be isolated and fused with myeloma cells. Monoclonal antibodies produced by the hybridomas can be screened for reactivity with HRF or an immunogenic fragment thereof. [00127] Animals that may be immunized include mice, rats, rabbits, goats, sheep, cows or steer, guinea pigs or primates. Initial and any optional subsequent immunization may be through intravenous, intraperitoneal, intramuscular, or subcutaneous routes. Subsequent immunizations may be at the same or at different concentrations of HRF, or a subsequence thereof, preparation, and may be at regular or irregular intervals.
[00128] Human antibodies can be produced by immunizing human transchromosomic KM miceTM (WO 02/43478) or HAC mice (WO 02/092812). KM miceTM and HAC mice express human immunoglobulin genes. Using conventional hybridoma technology, splenocytes from immunized mice that were high responders to the antigen can be isolated and fused with myeloma cells. A monoclonal antibody can be obtained that binds to the antigen. An overview of the technology for producing human antibodies is described in Lonberg and Huszar (Int. Rev. Immunol.13:65 (1995)). Transgenic animals with one or more human immunoglobulin genes (kappa or lambda) that do not express endogenous immunoglobulins are described, for example in, U.S. Patent No.5,939,598. Additional methods for producing human polyclonal antibodies and human monoclonal antibodies are described (see, e.g., Kuroiwa et al., Nat. Biotechnol.20:889 (2002); WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Patent Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598). Antibodies can also be generated using other techniques including hybridoma, recombinant, and phage display technologies, or a combination thereof (see U.S. Patent Nos.4,902,614, 4,543,439, and 4,411,993; see, also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.1988). [00129] Antibodies of the disclosure and disclosure methods employing antibodies include mammalian, primatized, humanized, fully human antibodies and chimeras. A mammalian antibody is an antibody produced by a mammal, transgenic or non-transgenic, or a non-mammalian organism engineered to produce a mammalian antibody, such as a non- mammalian cell (bacteria, yeast, insect cell), animal or plant. [00130] The term “human” when used in reference to an antibody, means that the amino acid sequence of the antibody is fully human, i.e., human heavy and human light chain variable and human constant regions. Thus, all of the amino acids are human or exist in a human antibody. An antibody that is non-human may be made fully human by substituting the non-human amino acid residues with amino acid residues that exist in a human antibody. Amino acid residues present in human antibodies, CDR region maps and human antibody consensus residues are known in the art (see, e.g., Kabat, Sequences of Proteins of Immunological Interest, 4th Ed.US Department of Health and Human Services. Public Health Service (1987); Chothia and Lesk (1987). A consensus sequence of human VH subgroup III,
based on a survey of 22 known human VH III sequences, and a consensus sequence of human VL kappa-chain subgroup I, based on a survey of 30 known human kappa I sequences is described in Padlan Mol. Immunol.31:169 (1994); and Padlan Mol. Immunol.28:489 (1991). Human antibodies therefore include antibodies in which one or more amino acid residues have been substituted with one or more amino acids present in any other human antibody. [00131] The term “humanized” when used in reference to an antibody, means that the amino acid sequence of the antibody has non-human amino acid residues (e.g., mouse, rat, goat, rabbit, etc.) of one or more complementarity determining regions (CDRs) that specifically bind to the desired antigen in an acceptor human immunoglobulin molecule, and one or more human amino acid residues in the Fv framework region (FR), which are amino acid residues that flank the CDRs. Such antibodies typically have reduced immunogenicity and therefore a longer half-life in humans as compared to the non-human parent antibody from which one or more CDRs were obtained or are based upon. [00132] Antibodies of the disclosure and disclosure methods employing antibodies include those to as “primatized” antibodies, which are “humanized” except that the acceptor human immunoglobulin molecule and framework region amino acid residues may be any primate amino acid residue (e.g., ape, gibbon, gorilla, chimpanzees orangutan, macaque), in addition to any human residue. Human FR residues of the immunoglobulin can be replaced with corresponding non-human residues. Residues in the CDR or human framework regions can therefore be substituted with a corresponding residue from the non-human CDR or framework region donor antibody to alter, generally to improve, antigen affinity or specificity, for example. A humanized antibody may include residues, which are found neither in the human antibody nor in the donor CDR or framework sequences. For example, a FR substitution at a particular position that is not found in a human antibody or the donor non-human antibody may be predicted to improve binding affinity or specificity human antibody at that position. Antibody framework and CDR substitutions based upon molecular modeling are well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Patent No.5,585,089; and Riechmann et al., Nature 332:323 (1988)). [00133] The term “chimeric” and grammatical variations thereof, when used in reference to an antibody, means that the amino acid sequence of the antibody contains one or more portions that are derived from, obtained or isolated from, or based upon two or more
different species. For example, a portion of the antibody may be human (e.g., a constant region) and another portion of the antibody may be non-human (e.g., a murine heavy or murine light chain variable region). Thus, an example of a chimeric antibody is an antibody in which different portions of the antibody are of different species origins. Unlike a humanized or primatized antibody, a chimeric antibody can have the different species sequences in any region of the antibody. [00134] Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; W091/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunol.28:489 (1991); Studnicka et al., Protein Engineering 7:805 (1994); Roguska. et al., Proc. Nat’l. Acad. Sci. USA 91:969 (1994)), and chain shuffling (U.S. Patent No.5,565,332). Human consensus sequences (Padlan, Mol. Immunol.31:169 (1994); and Padlan, Mol. Immunol.28:489 (1991)) have previously used to produce humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol.151:2623 (1993)). [00135] Methods for producing chimeric antibodies are known in the art (e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191 (1989); and U.S. Patent Nos.5,807,715; 4,816,567; and 4,816,397). Chimeric antibodies in which a variable domain from an antibody of one species is substituted for the variable domain of another species are described, for example, in Munro, Nature 312:597 (1984); Neuberger et al., Nature 312:604 (1984); Sharon et al., Nature 309:364 (1984); Morrison et al., Proc. Nat’l. Acad. Sci. USA 81:6851 (1984); Boulianne et al., Nature 312:643 (1984); Capon et al., Nature 337:525 (1989); and Traunecker et al., Nature 339:68 (1989). [00136] Suitable techniques that additionally may be employed in antibody methods include affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, size exclusion, purification on protein A column, or any combination of these techniques. The antibody isotype can be determined using an ELISA assay, for example, a human Ig can be identified using mouse Ig-absorbed anti-human Ig. [00137] The term “culturing” refers to the in vitro or ex vivo propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.
[00138] “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, canine, bovine, porcine, murine, rat, avian, reptilian and human. [00139] “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells’ genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2µm in diameter and 10 µm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coli bacterium, and Salmonella bacterium. [00140] As used herein, a “hybridoma” refers to the product of a cell-fusion between a cultured neoplastic lymphocyte and a primed B- or T-lymphocyte which expresses the specific immune potential of the parent cell, such as an antibody. [00141] In one embodiment, the term “disease” or “disorder” as used herein refers to allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation. In one embodiment, the term “disease” or “disorder” as used herein refers to a status of being diagnosed with allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, a status of being suspect of having allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, or a status of at high risk of having allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation. [00142] As used herein, “acute respiratory distress syndrome” or “ARDS” is a type of respiratory failure characterized by rapid onset of widespread inflammation in the lungs. Symptoms include shortness of breath, rapid breathing, and bluish skin coloration.
[00143] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals. [00144] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, a subject has or is diagnosed of having or is suspected of having a disease. [00145] In some embodiments, a subject as referred to herein has been treated with a standard care for the disease. In some embodiments, a subject as referred to herein is concurrently treated with a standard care of the disease. In some embodiments, a subject as referred to herein will be treated with a standard care of the disease. As used herein, “standard of care” or “SOC” refers to the diagnostic and treatment process that a clinician should follow for a certain type of patient, illness, or clinical circumstance. SOC may include administration of drugs that are being used in clinical practice for the treatment of allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, other than those used as part of another clinical trial. [00146] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the
condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. In one aspect, treatment excludes prophylaxis. [00147] In some embodiments, the terms “treating,” “treatment,” and the like, as used herein, mean ameliorating a disease, so as to reduce, ameliorate, or eliminate its cause, its progression, its severity, or one or more of its symptoms, or otherwise beneficially alter the disease in a subject. Reference to “treating,” or “treatment” of a patient is intended to include prophylaxis. Treatment may also be preemptive in nature, i.e., it may include prevention of disease in a subject exposed to or at risk for the disease. Prevention of a disease may involve complete protection from disease, for example as in the case of prevention of infection with a pathogen, or may involve prevention of disease progression. For example, prevention of a disease may not mean complete foreclosure of any effect related to the diseases at any level, but instead may mean prevention of the symptoms of a disease to a clinically significant or detectable level. Prevention of diseases may also mean prevention of progression of a disease to a later stage of the disease. [00148] The term “passive immunity” refers to the transfer of immunity from one subject to another through the transfer of antibodies. Passive immunity may occur naturally, as when maternal antibodies are transferred to a fetus. Passive immunity may also occur artificially as when antibody compositions are administered to non-immune subjects. Antibody donors and recipients may be human or non-human subjects. Antibodies may be polyclonal or monoclonal, may be generated in vitro or in vivo, and may be purified, partially purified, or unpurified depending on the embodiment. In some embodiments described herein, passive immunity is conferred on a subject in need thereof through the administration of antibodies or antigen-binding fragments that specifically recognize or bind to a particular antigen, such as an HRF, HRF N19, HRF GST-N19 or HRF-2CA. In some embodiments, passive immunity is conferred through the administration of an isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen, such as an HRF. [00149] “Immune response” broadly refers to the antigen-specific responses of lymphocytes to foreign substances. The terms “immunogen” and “immunogenic” refer to molecules with the capacity to elicit an immune response. All immunogens are antigens, however, not all antigens are immunogenic. An immune response disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro. The skilled artisan will understand that a variety of macromolecules,
including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to be immunogenic. The skilled artisan will further understand that nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen. The artisan will further understand that immunogens are not limited to full- length molecules, but may include partial molecules. [00150] “Detectable label”, “label”, “detectable marker” or “marker” are used interchangeably, including, but not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, antibody or composition described herein. [00151] As used herein, the term “label” or a detectable label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histidine tags (N-His), magnetically active isotopes, e.g., 115Sn, 117Sn and 119Sn, a non-radioactive isotopes such as 13C and 15N, polynucleotide or protein such as an antibody so as to generate a “labeled” composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected, or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and
described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed). Examples of luminescent probes include, but are not limited to, aequorin and luciferases. [00152] As used herein, the term “immunoconjugate” comprises an antibody or an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody. [00153] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl- coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). [00154] In some embodiments, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent. [00155] As used herein, a purification label or maker refers to a label that may be used in purifying the molecule or component that the label is conjugated to, such as an epitope tag (including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag), an affinity tag (including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)), or a fluorescent tag. [00156] In some embodiments, a detectable marker can be used to produce a detectable signal upon binding of two moieties, such as an antibody and its antigen. In some embodiments, one of the two moieties is immobilized, the mobilized moiety is the provided for binding, and unbound mobilized moiety is removed by washing with a suitable solution. Accordingly, any detectable marker can be directly or indirectly conjugated to the mobilized moiety, and the detectable signal obtained after the washing step indicates binding between the two moieties. In other embodiments, a detectable signal can be generated if two moieties
are in the proximity with each other. For example, one part of a detectable marker, such as a fluorescent protein, can be directly or indirectly conjugated to the first moiety while the other part of the detectable marker is directly or indirectly conjugated to the second moiety, and two parts of the detectable markers, when in the proximity with each other, generate a detectable signal. Alternatively, fluorescence resonance energy transfer (FRET) can also be used. [00157] As used herein, the term “ELISA” refers to enzyme-linked immunosorbant assay. Numerous methods and applications for carrying out an ELISA are well known in the art, and provided in many sources (See, e.g., Crowther, “Enzyme-Linked immunosorbant Assay (ELISA),” in Molecular Biomethods Handbook, Rapley et al. [eds.], pp.595-617, Humana Press, Inc., Totowa, N.J. [1998]). In some embodiments, an ELISA is a “direct ELISA”, where a target-binding molecule, such as a cell, cell lysate, or isolated protein, is first bound and immobilized to a microtiter plate well. In an alternative embodiment, an ELISA is a “sandwich ELISA”, where a target-binding molecule is attached to the substrate by capturing it with an antibody that has been previously bound to the microtiter plate well. The ELISA method detects an immobilized ligand-receptor complex (binding) by use of fluorescent detection of fluorescently labeled ligands or an antibody-enzyme conjugate, where the antibody is specific for the antigen of interest, such as a phage virion, while the enzyme portion allows visualization and quantitation by the generation of a colored or fluorescent reaction product. The conjugated enzymes commonly used in the ELISA include horseradish peroxidase, urease, alkaline phosphatase, glucoamylase or O-galactosidase. The intensity of color development is proportional to the amount of antigen present in the reaction well. [00158] A lateral flow immunoassay refers to an assay format in which a sample is applied to a lateral flow matrix. The sample flows along the lateral flow matrix, and one or more analyte components to be detected in the sample react with at least one reagent which is provided in or added to the lateral flow matrix. At least one reagent is typically immobilized in the device for reaction with the analyte component to be detected or a reagent thereof, and labels are typically employed to measure the extent of reaction with an immobilized reagent. See, e.g., U.S. patents and patent application publications: U.S. Pat. Nos.5,602,040; 5,622,871; 5,656,503; 6,187,598; 6,228,660; 6,818,455; 2001/0008774; 2005/0244986; 6,352,862; 2003/0207465; 2003/0143755; 2003/0219908; U.S. Pat. Nos.5,714,389; 5,989,921; 6,485,982; 11/035,047; 5,656,448; 5,559,041; 5,252,496; 5,728,587; 6,027,943;
6,506,612; 6,541,277; 6,737,277 B1; 5,073,484; 5,654,162; 6,020,147; 4,956,302; 5,120,643; 6,534,320; 4,942,522; 4,703,017; 4,743,560; 5,591,645; and RE 38,430. [00159] As used herein, a biological sample, or a sample, is obtained from a subject. Exemplary samples include, but are not limited to, cell sample, tissue sample, biopsy, liquid samples such as blood and other liquid samples of biological origin, including, but not limited to, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. [00160] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration. [00161] “Administration” or “delivery” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be
determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer. [00162] The term administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The disclosure is not limited by the route of administration, the formulation or dosing schedule. [00163] A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. [00164] Carriers also include pharmaceutical excipients and additiveHRFs, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra- oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include
but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. [00165] A composition as disclosed herein can be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. [00166] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. [00167] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of long- term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. [00168] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be
administered, both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein. [00169] A combination as used herein intends that the individual active ingredients of the compositions are separately formulated for use in combination, and can be separately packaged with or without specific dosages. The active ingredients of the combination can be administered concurrently or sequentially. [00170] In some embodiments, an antibody or antigen binding fragment thereof is administered in an effective amount. An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific agent employed, bioavailability of the agent, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the agent that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. [00171] “Therapeutically effective amount” of an agent refers to an amount of the agent that is an amount sufficient to obtain a pharmacological response; or alternatively, is an amount of the agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient.
A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. Modes for Carrying Out the Disclosure [00172] Provided herein is an antibody or an antigen binding fragment thereof comprising one or more of the following sequences: a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; or a heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL. [00173] In one aspect, provided herein is an antibody or antigen binding fragment that comprises one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain (CDR) 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; or a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG. [00174] In a further aspect, provided herein is antibody or antigen binding fragment thereof comprising one or more of: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; a heavy- chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; b) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; or a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG. [00175] In a further aspect, provided herein is antibody or antigen binding fragment thereof comprising:
a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; a heavy- chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; and b) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; and a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG. [00176] In a further aspect, the antibody or an antigen binding fragment thereof, comprises a heavy-chain variable region comprising one of the following amino acid sequences: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS; b) QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS; c) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS; d) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGALVTVSS; e) QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS; f) QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS;
g) QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS; h) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHS FALWGQGTLVTVSS; or i) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIH SFALWGQGTLVTVSS. [00177] Also provided is an antibody or an antigen binding fragment thereof, comprising a light-chain variable region comprising one of the following amino acid sequences: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFGGGTE VVVK; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; c) AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; d) ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; e) AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGGGTEVV VK; f) AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFGGGTE VVVK; g) AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK;
h) AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; or i) ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK. [00178] Also provided is an antibody or antigen binding fragment thereof, comprising: a) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFGGGTE VVVK; b) a heavy chain variable region comprising the amino acid sequence: QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; c) a heavy chain variable region comprising the amino acid sequence: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; d) a heavy chain variable region comprising the amino acid sequence: QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGALVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI
YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; e) a heavy chain variable region comprising the amino acid sequence: QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGGGTEVV VK; f) a heavy chain variable region comprising the amino acid sequence: QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFGGGTE VVVK; g) a heavy chain variable region comprising the amino acid sequence: QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; h) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHS FALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK; or i) a heavy chain variable region comprising the amino acid sequence: QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIH
SFALWGQGTLVTVSS, and a light-chain variable region comprising the amino acid sequence: ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VK. [00179] In a further aspect, providedherein is an antibody or antigen binding fragment thereof, comprising a heavy chain region comprising one of the following amino acid sequences: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG TLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSK PTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQV RTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQ PLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLD SDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; b) QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG TLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSK PTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQV RTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQ PLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLD SDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; c) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG TLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSK PTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQV RTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQ PLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLD SDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; d) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF
ALWGQGALVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNS GTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCS KPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQ VRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVL DSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; e) QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG TLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSK PTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQV RTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQ PLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLD SDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; f) QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG TLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSK PTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQV RTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQ PLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLD SDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; g) QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSF ALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG TLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSK PTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQV RTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQ PLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLD SDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; h) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHS FALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNS
GTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCS KPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQ VRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVL DSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK; or i) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHS FALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNS GTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCS KPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQ VRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVL DSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK. [00180] In a further aspect, the antibody or antigen biding fragment comprises a light- chain region comprising one of the following amino acid sequences: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFGGGTE VVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIEN SKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSK TPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC; c) AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSK TPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC; d) ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSK TPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTRGTTSVVQSFNRGDC; e) AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGGGTEVV
VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSK TPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC; f) AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFGGGTE VVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIEN SKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSASPIVQSFNRGDC; g) AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSK TPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC; h) AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSK TPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTLGTTSVVQSFNRGDC; or i) ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVV VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSK TPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNMGDC. [00181] In one aspect, provided herein is an antibody or antigen binding fragment that comprises one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSSQSVYDNNNLA; a light-chain (CDR) 2 (LCDR2) that comprises one of the following amino acid sequences: DASKLPS; or a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGAVSGSNV. [00182] In a further aspect, provided herein is antibody or antigen binding fragment thereof comprising: [00183] a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence TVSGFSLSSGAVS; a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: IGVISSRDIAYFATWAKG; a heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL. [00184] Also provided is an antibody or an antigen binding fragment thereof, comprising a light-chain variable region comprising the following amino acid sequence:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. [00185] In a further aspect, the antibody or an antigen binding fragment thereof, comprises a heavy-chain variable region comprising the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. [00186] In one aspect, the equivalent of the antibody or antigen binding fragment is at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or more identical to the HC and/or LC reference sequence. Methods to determine such are known in the art and disclosed herein. [00187] In one aspect of this disclosure, the antibody or antigen binding fragment specifically recognizes and binds to HRF or an immunogenic fragment thereof. In one aspect, the immunogenic fragment comprises or consists of the amino acid sequence of N19 or GST-N19. In another aspect, the HRF comprises or consists of human HRF. [00188] The antibody or antigen binding fragment of this disclosure is isolated or recombinant, or alternatively is chimeric, humanized, a single chain, or a humanized single chain. Non-limiting examples of an antigen binding fragment are a Fab, F(ab’)2, Fab’, scFv, or Fv. [00189] In one aspect, the antibody or antigen binding fragment of this disclosure comprises, or consists essentially or, or yet further consists of a light chain constant domain. Non-limiting examples of such is a constant domain of a human κ light chain, a constant domain of a human λ light chain and a constant domain of a λ1 or λ2 or λ3 or λ4 light chain. [00190] In some embodiments, the antibody or antigen binding fragment comprises a light chain constant domain. In further embodiments, the constant domain is a human consists domain. Additionally or alternatively, the constant domain comprises, or alternatively consists essentially of, or yet further consists of a constant domain of a κ light chain, optionally a human κ light chain. In yet further embodiments, the constant domain of the κ
light chain comprises, or alternatively consists essentially of, or yet further consists of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. In some embodiments, the constant domain comprises, or alternatively consists essentially of, or yet further consists of a constant domain of a λ light chain, optionally a human λ light chain. In further embodiments, the constant domain comprises, or alternatively consists essentially of, or yet further consists of a constant domain of a λ1 or λ2 or λ3 or λ4 light chain, optionally a human λ1 or λ2 or λ3 or λ4 light chain. [00191] In one aspect, exemplary antibodies that bind to HRF or a fragment thereof, e.g., HRF N19, HRF GST-N19, or HRF-2CA, are disclosed herein, or an equivalent of each thereof. In some embodiments, a designated antibody and its equivalent comprise the same CDRs. In some embodiments, each of these is a rabbit monoclonal antibody. In other embodiments, humanized antibodies are also provided. [00192] Additionally, recombinant anti-HRF antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the disclosure. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques such as, for example, the methods described in U.S. Pat. No. 7,112,421; Better et al. (1988) Science 240:1041-1043; or Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443. [00193] In one aspect, the fragment comprises a fragment crystallizable region (Fc region). Non-limiting examples of such include an region that comprises, or consists essentially of, or yet further consists of one or more of: an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgM Fc region, an IgE Fc region, an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG4 Fc region. [00194] The antibody or antigen binding fragment can be post-translationally modified optionally glycosylated, hydroxylated, methylated, lapidated, acetylated, SUMOylated, phosphorylated, PEGylated, or any combination thereof and/or further comprise a detectable or purification marker. [00195] The antibodies and fragments thereof as described herein are useful diagnostically, therapeutically or as reagents for the isolation and purification of their binding partners.
Polynucleotides, Vectors and Host Cells [00196] Also provided are polynucleotides encoding the one or more antibody or antigen binding fragment of this disclosure or a polynucleotide complementary thereto. The polynucleotide can be RNA, DNA or a hybrid polynucleotide. The polynucleotide can be comprised within a vector. In one aspect, the polynucleotide and/or vector further comprises a regulatory sequence that directs the expression or replication of the antibody or antigen binding fragment or polynucleotide, respectively. Non-limiting examples of such include without limitation a secretion signal, promoter, an enhancer, or a polyadenylation sequence. [00197] In a further aspect, the regulatory sequence directs the replication or expression of the polynucleotide. [00198] Non-limiting examples of the vectors, include a non-viral vector, optionally a plasmid, or a viral vector, optionally an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, or a plant viral vector. [00199] Also provided are a single or plurality of cells comprising one or more of: the antibody or antigen binding fragment, the polynucleotide, or the vector as described herein. [00200] In one aspect, the polynucleotide or cell is as described in the Experimental Section and incorporated herein by reference. The cell can be a prokaryotic cell, optionally an Escherichia coli cell. Non-limiting examples of a eukaryotic cell includes a mammal cell, an insect cell, a yeast cell or a human cell, e.g. a 293 cell or a cell as described herein. [00201] Also provided is a hybridoma expressing the antibody or antigen binding fragment as described herein. [00202] The cells and vectors can be used to produce the antibody or antigen binding fragment as described herein by culturing a cell comprising a polynucleotide encoding the antibody or the antigen binding fragment under conditions suitable for expression of the antibody or antigen binding fragment. In one aspect, the polynucleotide is introduced into to the cell prior to the culturing step. These methods are known in the art. The method can further comprise purifying they antigen or antigen binding fragment from the cell or culture medium. [00203] Alternatively, the hybridoma expressing the antibody can be cultured under conditions suitable for expression of the antibody or antigen binding fragment and optionally purifying or isolating the antibody produced thereby. [00204] This disclosure also provides a method of producing the antibody or antigen binding fragment of this disclosure by contacting the polynucleotide or the vector encoding
same with an RNA polymerase, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine-5'-triphosphate (GTP), and uridine triphosphate (UTP) under conditions suitable for transcription to messenger RNA, and contacting the transcribed messenger RNA with a ribosome, tRNAs, an aminoacyl-tRNA synthetase, and initiation, elongation and termination factors under conditions suitable for translation to the antibody or antigen binding fragment. In one aspect, the method comprises contacting the transcribed messenger RNA with a cell lysate comprising the ribosome, tRNAs, aminoacyl-tRNA synthetase, and initiation, elongation and termination factors under conditions. The antibody or antigen binding fragment can be purified or isolated from the reaction mixture. [00205] Compositions are provided that comprise a carrier and one or more of: the antibody or antigen binding fragment, the polynucleotide, the vector, the cell, or the hybridoma as described herein that can optionally be detectably labeled. In one aspect, the composition further comprises a carrier, such as a pharmaceutically acceptable carrier. The compositions can comprise two or more of the antibodies or antigen binding fragments that can be the same or different from each other. In one aspect, the two or more of the antibodies or antigen binding fragments recognize and binds to at least two different epitopes. [00206] In one aspect, the composition further comprise one or more peptides or proteins identified as N19, GST-19 or HRF-2CA or polynucleotides encoding or a vector or host cell containing same, and optionally wherein the carrier is a pharmaceutical acceptable carrier. Methods of Use [00207] Provided herein are methods for one or more of: a) targeting HRF N19-Ig interactions; b) inhibiting HRF N19 or HRF-Ig interactions; c) inhibiting Ig E-dependent activation of mast cells or basophils; d) the treatment of a condition from the group of: an allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation in a subject in need thereof, comprising, or consisting essentially of, or yet further consisting of administering an effective amount of the antibody or antigen binding fragment or compositions as described herein. On one aspect, the agent binds an HRF-reactive immunoglobulin (Ig). In another aspect, the agent modulates binding of an HRF monomer, an HRF dimer, or an HRF multimer with the HRF-reactive Ig.
[00208] In one aspect of the above for targeting or inhibiting, the method can be performed in vitro or in vivo. When performed in vitro, it provides a screen for alternative or equivalent compositions or for new combination therapies. When performed in vivo in an animal, it is a useful animal model for new therapies or personalized therapies. It also can be performed in a animal, mammal, murine, canine, feline, or human patient, for therapeutic purposes. [00209] In a further aspect, the method further comprises administering an effective amount of a second drug. In one aspect, the second drug comprises an anti-inflammatory, anti-asthmatic or anti-allergy drug. Non-limiting examples include a hormone, a steroid, an anti-histamine, anti-leukotriene, anti-IgE, anti- ^4 integrin, anti-β2 integrin, anti-CCR3 antagonist, β2 agonist or anti-selectin. Alternatively, the second drug comprises an allergen immunotherapy. Non-limiting examples of the allergen immunotherapy is oral immunotherapy (OIT), subcutaneous immunotherapy (SIT), or sublingual immunotherapy (SLIT). In another aspect, the second therapy comprises an inhibitor of binding of HRF to an Ig molecule. In a yet further aspect, the inhibitor is a peptide or polypeptide that inhibits the binding of an HRF monomer, an HRF dimer, or a HRF multimer with an HRF-reactive immunoglobulin (Ig). [00210] The second therapy is administered concurrently, prior to, or after the first therapy. [00211] In a further aspect, the therapy and/or the second drug is administered via ingestion, via inhalation, topically, or a combination thereof. [00212] In some embodiments, administration of a pharmaceutical composition comprising one or more of the antibodies, fragments or composition as described herein can be made to a subject in need thereof, such as allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation. In some embodiments, the pharmaceutical compositions as described herein can be administered alone or in combination with other therapies deemed appropriate by a clinician or practitioner. In some embodiments, the pharmaceutical compositions described herein may reduce the number of days of the subject having allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation symptoms by one or more days, such as reducing the days of having symptoms by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days.
[00213] In some embodiments, the subject is selected for the administration if the antibody or antigen binding fragment binds to a component, IgE or HRF detected in a biological sample isolated from the subject. In addition or alternatively, other assays including commercially available tests, can be utilized to test the subject for inflammation or allergy. [00214] In a further aspect, provided is a method comprising, or consisting essentially of, or yet further consisting of contacting an antibody or antigen binding fragment of the detection system as disclosed herein with a biological sample isolated from a subject. In some embodiments, the method further comprises contacting the detectable marker of the detection system as disclosed herein with the antibody or antigen binding fragment or polypeptide. In some embodiments, binding of the antibody or antigen binding fragment or polypeptide with a component of the biological sample indicates the subject has or had a allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation. Detection Systems [00215] In yet a further aspect, provided is a method for detecting allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation, HRF or an immunogenic fragment of the HRF. The method comprises, or consists essentially of, or yet further consists of contacting the antibody or antigen binding fragment of the detection system with a sample. In some embodiments, the method further comprises contacting the detectable marker with the antibody or antigen binding fragment. In further embodiments, binding of the antibody or antigen binding fragment with a component of the sample indicates presence of a HRF or an immunogenic fragment in the sample. [00216] Thus, provided herein is a detection system comprising the antibody or antigen binding fragment as described herein and a detectable marker for producing a detectable signal upon binding of the antibody or antigen binding fragment thereof with HRF or an immunogenic fragment thereof. In one embodiment, the system is an enzyme-linked immunosorbent assay (ELISA) or a lateral flow immunoassay. [00217] Further provided is a comprising contacting the antibody or antigen binding fragment of the detection system with a biological sample isolated from a subject, wherein binding of the antibody or antigen binding fragment thereof with a component of the biological sample indicates the subject expresses HRF.
Kits [00218] In one aspect, provided is a kit comprising, or consisting essentially of, or yet further consisting of an instruction for use in a method as disclosed herein, and one or more of: an antibody or antigen binding fragment or polypeptide as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell of as disclosed herein, a hybridoma as disclosed herein, a composition as disclosed herein, or a system as disclosed herein. EXAMPLES [00219] The following example is put forth so as to provide those of ordinary skill in the art with a complete description of how to make and use the present disclosure, and is not intended to limit the scope of what the inventors regard as their disclosure nor is it intended to represent that the experiment below is all or the only experiment that could be performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. [00220] The following merely illustrates the principles of the disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the disclosure and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the disclosure and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the disclosure as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present disclosure, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present disclosure is embodied by the appended claims.
Experimental [00221] Histamine-releasing factor (HRF), also known as translationally controlled tumor protein and fortilin, is a highly conserved protein required for fundamental intracellular functions such as proliferation and survival 1. Since it is secreted during allergic reactions, it is implicated in allergic diseases 2. Recent studies demonstrated that HRF amplifies allergic inflammation by promoting immunoglobulin (Ig) E-dependent activation of mast cells and basophils in animal models of anaphylaxis, asthma and food allergy 3, 4, 5. HRF can be present as a monomer and disulfide-linked oligomers. HRF directly binds to a subset of IgE and IgG molecules via low- affinity interactions between Ig-Fab portion and two Ig-binding sites within HRF, the amino- terminal 19 residues (N19) and the helical domain H3. It was also shown that a fusion protein GST-N19 and a recombinant monomeric HRF mutant with two cysteine residues replaced with alanine (HRF-2CA) work as strong inhibitors for in vitro HRF-IgE interactions and in vivo allergic inflammation. Thus, it is likely that HRF dimers, but not monomers, can activate murine mast cells through high-affinity IgE receptors (FceRI) 3. Provided herein are antibodies (mAb) and an optimal peptide inhibitor, which target the N19-Ig interactions, using an ovalbumin (OVA) sensitization/OVA challenge model of food allergy. [00222] Briefly, rabbits were immunized with GST-N19. Phage-display libraries expressing Fabs were generated from spleens of rabbits with high serum titers of anti-GST- N19. Phages were selected by 4 rounds of panning with immobilized GST-N19 and against immobilized GST. Twelve Fab sequences were randomly selected out of seventy-nine N19- specific Fab-displaying phages, and cloned into an expression vector pTT5, expressed in HEK293 cells and purified by Ni- NTA agarose column. These Fabs all bound to immobilized GST-N19 (data not shown), and they were confirmed for their ability to inhibit interactions of the HRF-reactive IgE, C38-2, with recombinant HRF by enzyme-linked immunosorbent assays (ELISA). Among them, three Fabs (A8-1, C4-4 and F7-1) with strong HRF-binding ability are shown in Fig.1A. Fab F7-1 with the strongest activity to inhibit HRF-IgE interactions was converted to a full-length IgG mAb SPF7- 1 (Fig.1B). SPF7-1 mAb i.p. administered suppressed OVA-induced hypothermia (Fig.1C) and reversed muted physical activity (data not shown). [00223] GST-N19 and HRF-2CA showed strong inhibitory activities of HRF function both in vitro and in vivo unlike weaker effects of synthetic N19 peptide 3, 4. In consideration of oral administration that requires resilience of the peptides to digestion, several chemical
modifications at the amino-terminal and/or carboxyl-terminal ends (Fig.2A) were tested to improve resistance to hydrolytic digestion and/or aqueous solubility in the stomach. These peptides were gavaged via an oral route 30 min before each OVA challenge in OVA- sensitized BALB/c mice. Unlike synthetic non-modified N19 peptide and the other modified peptides, N19 peptide PEGylated at the carboxyl terminus showed consistent suppression of OVA-challenge- induced diarrhea (Fig.2B) and hypothermia (Fig.2C) and reversed low physical activity (data not shown). [00224] In sum, Applicant demonstrates that HRF inhibitors, anti-N19 mAb and N19- PEG, which target HRF-Ig interactions, strongly suppress food allergy in a murine model. Materials and Methods Rabbit immunization and library construction [00225] Two New Zealand White Rabbits were immunized with GST-N19 fusion protein four times with 2-weeks intervals (first subcutaneous injection of 200 µg of protein with Complete Freund’s Adjuvant followed by 3 subcutaneous injections of 100 µg of protein with Incomplete Freund’s Adjuvant). The bindings of pre-immune sera and post-immune sera to the GST-N19, GST, and a recombinant human HRF-His 6 were compared by ELISA and the rabbit that showed the best binding to the immunogen and HRF-His6 was selected for the library construction. [00226] The bone marrow and spleen cells were obtained and homogenized in TRI reagent (Molecular Research Center). Total RNA was isolated from the homogenized cells according to the manufacturer’s protocol. Then, the messenger RNA was purified using NucleoTrap mRNA (Macherey-Nagel) according to the manufacturer’s protocol. First strand cDNA was synthesized using PowerScribe MMLV RT (Monserate Biotechnology Group) and the antibody genes were amplified as described in US Patent No.9,890,414. The amplified antibody genes were digested with restriction enzymes and sequentially ligated with a Fab expression vector. The libraries were made by the electroporation of NEB 10-beta E. coli cells (New England Biolabs) with the ligated DNA. Selection of N19-specific clones [00227] For the amplification of the libraries in E. coli, 2 µg of each library DNA were electroporated into XL1-Blue E. coli cells (Monserate Biotechnology Group) and phage production was induced with VCS M13 helper phage in the presence of 1mM isopropyl β-D- 1- thiogalactopyranoside (IPTG) and antibiotics (carbenicillin, tetracycline, and kanamycin)
at 30˚C overnight. Phage was precipitated from the bacterial supernatant with 4% PEG (Polyethylene Glycol-8000)/0.5M NaCl and re-suspended in 1% BSA/PBS. Excess 5-fold GST was added to the phage as a soluble competitor to remove binders to GST. For panning, the microtiter wells were coated overnight with GST-N19 at 5 µg/mL in PBS and blocked with 1% BSA/PBS. The wells were incubated with precipitated phage at 37˚C for 1.5 h and unbound phage was washed 3 times in the first round of panning and 5 times in the 2nd, the 3rd, and the 4th round with PBS after 5 min incubation. Bound phage was eluted and freshly grown ER2738 cells were infected with eluted phage. Phage was amplified overnight for the next round with M13K07 helper phage (New England Biolabs Cat# N0315S) in the presence of 1 mM IPTG and antibiotics (carbenicillin, tetracycline, and kanamycin) at 30˚C overnight. To screen the panning output, the bacterial cells infected with eluted phage were titrated on LB agar plates containing carbenicillin and glucose. For the screening of individual clones by ELISA, 95 bacterial colonies were grown in 1.2 mL culture and Fab expression was induced in the presence of 1 mM IPTG at 30˚C. The microtiter wells were coated with 5 µg/mL GST- N19 protein in PBS at 4˚C overnight. After washing 3 times with PBS and blocking with 1% BSA/PBS, the culture plate was spun down and the wells were incubated with the supernatant containing Fab at 37˚C for 2 h. The wells were washed 3 times with PBS and the bound Fab was detected with HRP-conjugated goat anti-rabbit IgG F(ab’) 2 (Thermo Fisher Scientific Cat# 31461) at 37˚C for 1 h. The wells were washed 3 times with PBS and developed with TMB substrate (EMD Millipore Cat# ES022-50ml) and stopped with 2 N sulfuric acid. The signal was read with a plate reader at 450 nm. The light and heavy chains of Fab clones specific to N19 peptide were amplified by PCR, treated with ExoSAP-IT (Thermo Fisher Scientific Cat# 78201.1.ML), and submitted for sequence analysis. Cloning of N19-specific clones into a mammalian IgG expression vector [00228] The amplified light and heavy chains of the selected clones were digested with restriction enzymes and ligated with a fragment containing a CMV promoter into a mammalian IgG expression vector: a modified pTT5 vector (National Research Council Canada) for the bi-cistronic expression and purification. The ligated DNA was electroporated into NEB 10-beta cells and the cells were titrated onto LB agar plates containing 100 µg/mL of carbenicillin and 20 mM glucose. The single colonies were inoculated into 1.2 mL of Super Broth medium containing 50 µg/mL of carbenicillin and grown overnight at 37˚C. The cells were spun down and the DNA were purified from the cell pellets using spin columns (Qiagen Cat# 27104). The purified DNA were submitted for sequencing.
Expression and purification of rabbit IgG [00229] Nine mL of F17 Freestyle media (Gibco) containing 4 mM glutamine (Corning) and 0.1% Kolliphor P-188 (Sigma) was inoculated at 1.0 x 106 HEK293 cells/mL. The HEK293 cells were incubated at 37˚C, 5% CO2 with 140 rpm agitation. The cells were transfected when the cell density was measured at 1.8 - 3.0 x 106 cells/mL (2.50 x 106 cells/mL at 96.9 % viability). Each 10 µg of IgG-encoding plasmid was added to 500 µL F17 freestyle media and vortexed gently 3 times for 1 sec. Thirty µL of the PEI transfection reagent (Transporter5 Transfection Reagent, 1 mg/mL, Polysciences) was added to a separate 470 µL of F17 freestyle media and vortexed gently 3 times for 1 sec. The two were combined and the cocktail was again vortexed 3 times and incubated at room temperature for 10 min. The 1 mL cocktail was added to the 9 mL HEK293 cell culture, swirled gently and incubated at 37˚C, 5% CO2, with 140 rpm agitation. [00230] Approximately 24 h posttransfection, 25 µL of 200 mM valproic acid (Sigma) and 125 µL of 40% Tryptone N1 (Organotechnie) were added to the flask. The cells were incubated at 37˚C for 6 days. For IgG purification, cell culture supernatant was harvested on day 6 post-transfection. IgG was purified using Protein A/G column chromatography. The purity was confirmed by protein gel electrophoresis. Peptides [00231] Peptides were synthesized andpurified to >90% purity by HPLC by A&A Labs, San Diego. Food allergy experiments [00232] Food allergy experiments were performed according to Applicant’s prior published study3. Briefly, BALB/c mice were i.p. sensitized with OVA (50 µg/mouse) plus alum on days 0 and 14. On days 28, 30, 32, and 34, mice were i.g. challenged with OVA (25 mg) or PBS (control). One day (day 27) before the 1st challenge and one day (day 33) after the 3rd challenge, mice were starved for 3 h, then i.p. pretreated with anti-N19 mAb or isotype control (100 µg/mouse). Alternatively, peptides (100 µg) were i.g. gavaged 30 min before each OVA challenge in starved mice. Body temperature and physical activity on each OVA challenge were monitored for 50 min after OVA challenge 6. Feces were monitored for 1.5 h after OVA challenges. Diarrhea was scored as follows: 0 = normal and dry pellet; 1 = normal and wetter pellet; 2 = wet and less-shaped pellet; 3 = complete diarrhea. Student’s t- test was used to compare temperature drops and diarrhea scores between groups.
[00233] Mice were intraperitoneally sensitized with OVA (25 or 50 μg/mouse) plus alum on days 0 and 14. From day 28, mice were intragastrically (i.g.) challenged with OVA (25 or 50 mg) or PBS (con- trol) 3 times a week. Before each challenge, mice were starved for 3 hours, then i.g. pretreated with HRF inhibitor (or control protein (rabbit anti-HRF-N19 mAb SPF7-1 mAb or humanized mAb, or HRF-2CA) or control protein (isotype control or PBS). 30 min later, mice were challenged i.g. with 25 or 50 mg of OVA. Body temperature and physical activity were monitored for 60 min after the OVA challenge. The development of diarrhea was monitored 90 minutes after the OVA challenge. Diarrhea severity (30) was based on states of stool: solid (score 0), funicular (score 1), slurry (score 2), and watery (score 3). [00234] Generation of Antibodies [00235] Hybridoma Technique. In some embodiments, the antibody of the present technology is an anti-HRF, anti-GST-N19 or anti-N19 monoclonal antibody produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma techniques include those known in the art and taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art. [00236] Phage Display Technique. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA and phage display technology. For example, the antibodies, can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them. Phages with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g., human or murine) by selecting directly with an antigen, typically an antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains that are recombinantly fused to either the phage gene III or gene VIII protein. In addition, methods can be adapted for the construction of Fab expression libraries (See, e.g., Huse, et al.,. Science 246: 1275-1281, 1989) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for an HRF, E.G.,
GST-N19 OR N19 polypeptide, e.g., a polypeptide or derivatives, fragments, analogs or homologs thereof. Other examples of phage display methods that can be used to make the antibodies of the present technology include those disclosed in Huston et al., Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol.24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191- 280, 1994; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; WO 96/06213; WO 92/01047 (Medical Research Council et al.); WO 97/08320 (Morphosys); WO 92/01047 (CAT/MRC); WO 91/17271 (Affymax); and U.S. Pat. Nos.5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. Methods useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides via disulfide bonds have been described by Lohning, U.S. Pat. No.6,753,136. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988. [00237] Generally, hybrid antibodies or hybrid antibody fragments that are cloned into a display vector can be selected against the appropriate antigen in order to identify variants that maintain good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). However, other vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and/or screening. [00238] Expression of Recombinant Anti-HRF, e.g. anti-GST-N19 or anti- N19 Antibodies. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA technology. Recombinant polynucleotide
constructs encoding an antibody of the present technology typically include an expression control sequence operably-linked to the coding sequences of anti-HRF, e.g. anti-GST-N19 or anti-N19 antibody chains, including naturally-associated or heterologous promoter regions. As such, another aspect of the technology includes vectors containing one or more nucleic acid sequences encoding an anti-HRF, e.g., GST-N19 or N19 antibody of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, the nucleic acid containing all or a portion of the nucleotide sequence encoding the anti-HRF, e.g., GST-N19 or N19 antibody is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al., U.S. Pat. Nos.6,291,160 and 6,680,192. [00239] In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present technology is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Such viral vectors permit infection of a subject and expression of a construct in that subject. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding the anti-HRF, e.g., GST-N19 or N19 antibody, and the collection and purification of the anti-HRF, e.g., GST-N19 or N19 antibody, e.g., cross-reacting anti-HRF, e.g., GST-N19 or N19 antibodies. See generally, U.S.2002/0199213. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin-resistance or hygromycin- resistance, to permit detection of those cells transformed with the desired DNA sequences. Vectors can also encode signal peptide, e.g., pectate lyase, useful to direct the secretion of extracellular antibody fragments. See U.S. Pat. No.5,576,195.
[00240] The recombinant expression vectors of the present technology comprise a nucleic acid encoding a protein with anti-HRF antibody protein or fragment thereof, e.g., in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operably-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, e.g., in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. Typical regulatory sequences useful as promoters of recombinant polypeptide expression (e.g., anti-HRF, e.g., GST-N19 or N19 antibody), include, e.g., but are not limited to, promoters of 3- phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. In one embodiment, a polynucleotide encoding an anti-HRF, e.g., GST-N19 or N19 antibody of the present technology is operably- linked to an ara B promoter and expressible in a host cell. See U.S. Pat.5,028,530. The expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., anti-HRF, e.g., GST-N19 OR N19 antibody, etc.). [00241] Another aspect of the present technology pertains to anti-HRF, e.g., GST-N19 OR N19 antibody-expressing host cells, which contain a nucleic acid encoding one or more anti-HRF, e.g., GST-N19 or N19 antibodies or fragments thereof. The recombinant expression vectors of the present technology can be designed for expression of an anti-HRF, e.g., GST-N19 or N19 antibody or fragment thereof in prokaryotic or eukaryotic cells. For
example, an anti-HRF, e.g., GST-N19 OR N19 antibody or fragment thereof can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase. Methods useful for the preparation and screening of polypeptides having a predetermined property, via expression of stochastically generated polynucleotide sequences has been previously described. See U.S. Pat. Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641. [00242] Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide. [00243] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of distinct active peptide or protein domains to yield multifunctional polypeptides via polypeptide fusion has been described by Pack et al., U.S. Pat. Nos.6,294,353; 6,692,935. One strategy to maximize recombinant polypeptide expression, e.g., an anti-HRF, e.g., GST-N19 OR N19 antibody, in E. coli is to express the polypeptide in host bacteria with an impaired capacity to
proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (See, e.g., Wada, et al., 1992. Nucl. Acids Res.20: 2111-2118). Such alteration of nucleic acid sequences of the present technology can be carried out by standard DNA synthesis techniques. [00244] In another embodiment, the antiHRF, anti-GST-N19 or antiN19 antibody or fragment thereof expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, an anti-HRF, e.g., GST-N19 OR N19 antibody can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides, e.g., anti-HRF, e.g., GST-N19 OR N19 antibody, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol.3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). [00245] In yet another embodiment, a nucleic acid encoding an anti-HRF antibody, e.g., GST-N19 or N19 antibody or fragment thereof of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, e.g., but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J.6: 187-195, 1987). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that are useful for expression of the anti-HRF, e.g., GST- N19 OR N19 antibody of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL.2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. [00246] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-
specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev.1: 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol.43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J.8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1989), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912- 916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No.264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the α-fetoprotein promoter (Campes and Tilghman, Genes Dev.3: 537-546, 1989). [00247] Another aspect of the present methods pertains to host cells into which a recombinant expression vector of the present technology has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. [00248] A host cell can be any prokaryotic or eukaryotic cell. For example, an anti- HRF, or anti-GST-N19 or anti- N19 antibody or fragment thereof can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are a suitable host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Queen et al., Immunol. Rev.89: 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40,
adenovirus, bovine papillomavirus, and the like. Co et al., J Immunol.148: 1149, 1992. Other suitable host cells are known to those skilled in the art. [00249] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (See generally, Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL.2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. [00250] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the anti-HRF, e.g., GST-N19 OR N19 antibody or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). [00251] A host cell that includes an anti HRF antibody, or anti- GST-N19 or anti-N19 antibody or fragment of each thereof of the present technology, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) recombinant antibodies, anti-HRF, or anti-GST-N19 or anti- N19 antibody. In one embodiment, the method comprises culturing the host cell (into which a recombinant expression vector encoding the antibody or fragment thereof has been introduced) in a suitable medium such that the anti- antibody or fragment thereof is produced. In another embodiment, the method further
comprises the step of isolating the antibody or fragment thereof from the medium or the host cell. Once expressed, collections of the antibody or fragment thereof are purified from culture media and host cells. The anti-HRF, or anti-GST-N19 or anti- N19 antibody or fragment thereof can be purified according to standard procedures of the art, including HPLC purification, column chromatography, gel electrophoresis and the like. In one embodiment, the anti-HRF, e.g., GST-N19 OR N19 antibody or fragment thereof is produced in a host organism by the method of Boss et al., U.S. Pat. No.4,816,397. Usually, anti-HRF, e.g., GST-N19 or N19 antibody chains are expressed with signal sequences and are thus released to the culture media. However, if the anti-HRF, e.g., GST-N19 or N19 antibody chains are not naturally secreted by host cells, the anti-HRF, e.g., GST-N19 or N19 antibody chains can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification technique, column chromatography, ion exchange purification technique, gel electrophoresis and the like (See generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982). [00252] Polynucleotides encoding antiHRF, e.g., GST-N19 or N19 antibodies or fragment thereof, e.g., the anti-HRF, e.g., GST-N19 or N19 antibody or fragment thereof coding sequences, can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. See, e.g., U.S. Pat. Nos.5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences for light and/or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or β-lactoglobulin. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes. [00253] Single-Chain Antibodies. In one embodiment, the anti-HRF, e.g., GST-N19 or N19 antibody of the present technology is a single-chain anti-HRF, e.g., GST-N19 or N19 antibody. According to the present technology, techniques can be adapted for the production of single-chain antibodies specific to an HRF, e.g., GST-N19 or N19 protein (See, e.g., U.S. Pat. No.4,946,778). Examples of techniques which can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Pat. Nos.4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.
[00254] Chimeric and Humanized Antibodies. In one embodiment, the anti-HRF, e.g., GST-N19 or N19 antibody of the present technology is a chimeric anti-HRF, e.g., GST-N19 or N19 antibody. In one embodiment, the anti-HRF, e.g., GST-N19 or N19 antibody of the present technology is a humanized anti-HRF, e.g., GST-N19 or N19 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody. [00255] Recombinant anti-HRF, e.g.,GST-N19 or N19 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques, and are within the scope of the present technology. For some uses, including in vivo use of the anti-HRF, e.g., GST-N19 or N19 antibody of the present technology in humans as well as use of these agents in in vitro detection assays, it is possible to use chimeric or humanized anti-HRF, E.G., GST-N19 or N19 antibodies. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, e.g., but are not limited to, methods described in International Application No. PCT/US86/02269; U.S. Pat. No.5,225,539; European Patent No.184187; European Patent No.171496; European Patent No.173494; PCT International Publication No. WO 86/01533; U.S. Pat. Nos. 4,816,567; 5,225,539; European Patent No.125023; Better, et al., 1988. Science 240: 1041- 1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol.139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res.47: 999-1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst.80: 1553-1559; Morrison (1985) Science 229: 1202- 1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988. Science 239: 1534; Morrison, Science 229: 1202, 1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Pat. No.5,807,715; and Beidler, et al., 1988. J. Immunol.141: 4053-4060. For example, antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0239 400; WO 91/09967; U.S. Pat. No.5,530,101; 5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0592106; EP 0519596; Padlan E. A., Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994), and chain shuffling (U.S. Pat. No.5,565,332). In one
embodiment, a cDNA encoding a murine anti-HRF, E.G., GST-N19 OR N19 monoclonal antibody is digested with a restriction enzyme selected specifically to remove the sequence encoding the Fc constant region, and the equivalent portion of a cDNA encoding a human Fc constant region is substituted (See Robinson et al., PCT/US86/02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86/01533; Cabilly et al. U.S. Patent No.4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80: 1553-1559; U.S. Pat. No.6,180,370; U.S. Pat. Nos.6,300,064; 6,696,248; 6,706,484; 6,828,422. [00256] In one embodiment, the present technology provides the construction of humanized anti-HRF, E.G., GST-N19 or N19 antibodies that are unlikely to induce a human anti-mouse antibody (hereinafter referred to as “HAMA”) response, while still having an effective antibody effector function. As used herein, the terms “human” and “humanized”, in relation to antibodies, relate to any antibody which is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject. In one embodiment, the present technology provides for a humanized anti-HRF, E.G., GST-N19 OR N19 antibodies, heavy and light chain immunoglobulins. [00257] Humanization of SPF7. Clone SPF7 was humanized by grafting its CDRs into human germline frameworks IGKV1-13*02/IGKJ4*01 or IGKV1-39*01/IGKJ4*01 and JGHV3-53*04/IGHJ4*03 or IGHV3-23*04/IGHJ4*03 (SEQ ID No.1-4). They were produced as L1H1 (hSPF7L1/hSPF7H1), L1H2 (hSPF7L1/hSPF7H2), L2H1 (hSPF7L2/hSPF7H1), and L2H2 (hSPF7L2/hSPF7H2) in HEK293 cells and purified using protein A column chromatography. The purified IgGs were tested by ELISA against a recombinant human HRF protein. They all showed equivalent binding to the antigen compared to the parent rabbit IgG (FIG 1). [00258] hSPF7L1 1: AlaIleGlnLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThr GCGATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGGGTCACC 1 ---------!---------!---------!---------!---------!---------! 60 CGCTAGGTCAACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCCCAGTGG 1: IleThrCysGlnSerSerGlnSerValTyrAspAsnAsnAsnLeuAlaTrpTyrGlnGln
ATCACGTGCCAGTCTAGTCAGTCCGTTTACGATAATAACAATTTAGCCTGGTACCAGCAG 61 ---------!---------!---------!---------!---------!---------! 120 TAGTGCACGGTCAGATCAGTCAGGCAAATGCTATTATTGTTAAATCGGACCATGGTCGTC 1: LysProGlyLysAlaProLysLeuLeuIleTyrAspAlaSerLysLeuProSerGlyVal AAACCAGGCAAGGCGCCGAAACTCCTGATCTATGATGCCTCCAAGTTGCCAAGTGGGGTC 121 ---------!---------!---------!---------!---------!---------! 180 TTTGGTCCGTTCCGCGGCTTTGAGGACTAGATACTACGGAGGTTCAACGGTTCACCCCAG 1: ProSerArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIleSerSerLeu CCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG 181 ---------!---------!---------!---------!---------!---------! 240 GGTAGTTCCAAGTCGCCGTCACCTAGACCCTGTCTAAAGTGAGAGTGGTAGTCGTCGGAC 1: GlnProGluAspPheAlaThrTyrTyrCysAlaGlyAlaValSerGlySerAsnValPhe CAGCCTGAGGACTTCGCGACGTACTACTGCGCTGGAGCAGTTTCTGGCAGTAATGTATTC 241 ---------!---------!---------!---------!---------!---------! 300 GTCGGACTCCTGAAGCGCTGCATGATGACGCGACCTCGTCAAAGACCGTCATTACATAAG 1: GlyGlyGlyThrLysValGluIleLysArgThrValAlaAlaProSerValPheIlePhe GGTGGTGGTACAAAGGTCGAGATCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTC 301 ---------!---------!---------!---------!---------!---------! 360 CCACCACCATGTTTCCAGCTCTAGTTTGCCTGACACCGACGTGGTAGACAGAAGTAGAAG 1: ProProSerAspGluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsn CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC 361 ---------!---------!---------!---------!---------!---------! 420 GGCGGTAGACTACTCGTCAACTTTAGACCTTGACGGAGACAACACACGGACGACTTATTG 1: PheTyrProArgGluAlaLysValGlnTrpLysValAspAsnAlaLeuGlnSerGlyAsn TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC 421 ---------!---------!---------!---------!---------!---------! 480 AAGATAGGGTCTCTCCGGTTTCATGTCACCTTCCACCTATTGCGGGAGGTTAGCCCATTG 1: SerGlnGluSerValThrGluGlnAspSerLysAspSerThrTyrSerLeuSerSerThr TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC 481 ---------!---------!---------!---------!---------!---------! 540 AGGGTCCTCTCACAGTGTCTCGTCCTGTCGTTCCTGTCGTGGATGTCGGAGTCGTCGTGG 1: LeuThrLeuSerLysAlaAspTyrGluLysHisLysValTyrAlaCysGluValThrHis CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT 541 ---------!---------!---------!---------!---------!---------! 600 GACTGCGACTCGTTTCGTCTGATGCTCTTTGTGTTTCAGATGCGGACGCTTCAGTGGGTA 1: GlnGlyLeuSerSerProValThrLysSerPheAsnArgGlyGluCys CAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 601 ---------!---------!---------!---------!-------- 648 GTCCCGGACTCGAGCGGGCAGTGTTTCTCGAAGTTGTCCCCTCTCACA [00259] hSPF7L2 1: AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThr GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGGGTCACC 1 ---------!---------!---------!---------!---------!---------! 60 CTGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCCCAGTGG
1: IleThrCysGlnSerSerGlnSerValTyrAspAsnAsnAsnLeuAlaTrpTyrGlnGln ATCACGTGCCAGTCTAGTCAGTCCGTTTACGATAATAACAATTTAGCCTGGTACCAGCAG 61 ---------!---------!---------!---------!---------!---------! 120 TAGTGCACGGTCAGATCAGTCAGGCAAATGCTATTATTGTTAAATCGGACCATGGTCGTC 1: LysProGlyLysAlaProLysLeuLeuIleTyrAspAlaSerLysLeuProSerGlyVal AAACCAGGCAAGGCGCCGAAACTCCTGATCTATGATGCCTCCAAGTTGCCAAGTGGGGTC 121 ---------!---------!---------!---------!---------!---------! 180 TTTGGTCCGTTCCGCGGCTTTGAGGACTAGATACTACGGAGGTTCAACGGTTCACCCCAG 1: ProSerArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIleSerSerLeu CCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG 181 ---------!---------!---------!---------!---------!---------! 240 GGTAGTTCCAAGTCGCCGTCACCTAGACCCTGTCTAAAGTGAGAGTGGTAGTCGTCGGAC 1: GlnProGluAspPheAlaThrTyrTyrCysAlaGlyAlaValSerGlySerAsnValPhe CAGCCTGAGGACTTCGCGACGTACTACTGCGCTGGAGCAGTTTCTGGCAGTAATGTATTC 241 ---------!---------!---------!---------!---------!---------! 300 GTCGGACTCCTGAAGCGCTGCATGATGACGCGACCTCGTCAAAGACCGTCATTACATAAG 1: GlyGlyGlyThrLysValGluIleLysArgThrValAlaAlaProSerValPheIlePhe GGTGGTGGTACAAAGGTCGAGATCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTC 301 ---------!---------!---------!---------!---------!---------! 360 CCACCACCATGTTTCCAGCTCTAGTTTGCCTGACACCGACGTGGTAGACAGAAGTAGAAG 1: ProProSerAspGluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsn CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC 361 ---------!---------!---------!---------!---------!---------! 420 GGCGGTAGACTACTCGTCAACTTTAGACCTTGACGGAGACAACACACGGACGACTTATTG 1: PheTyrProArgGluAlaLysValGlnTrpLysValAspAsnAlaLeuGlnSerGlyAsn TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC 421 ---------!---------!---------!---------!---------!---------! 480 AAGATAGGGTCTCTCCGGTTTCATGTCACCTTCCACCTATTGCGGGAGGTTAGCCCATTG 1: SerGlnGluSerValThrGluGlnAspSerLysAspSerThrTyrSerLeuSerSerThr TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC 481 ---------!---------!---------!---------!---------!---------! 540 AGGGTCCTCTCACAGTGTCTCGTCCTGTCGTTCCTGTCGTGGATGTCGGAGTCGTCGTGG 1: LeuThrLeuSerLysAlaAspTyrGluLysHisLysValTyrAlaCysGluValThrHis CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT 541 ---------!---------!---------!---------!---------!---------! 600 GACTGCGACTCGTTTCGTCTGATGCTCTTTGTGTTTCAGATGCGGACGCTTCAGTGGGTA 1: GlnGlyLeuSerSerProValThrLysSerPheAsnArgGlyGluCys CAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 601 ---------!---------!---------!---------!-------- 648 GTCCCGGACTCGAGCGGGCAGTGTTTCTCGAAGTTGTCCCCTCTCACA [00260] hSPF7H1 1: GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGlySerLeuArgLeu GAGGTCCAGCTCGTTGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGGCTC 1 ---------!---------!---------!---------!---------!---------! 60 CTCCAGGTCGAGCAACTCAGACCCCCTCCGAACCAGGTCGGACCTCCCAGGGACTCCGAG
1: SerCysThrValSerGlyPheSerLeuSerSerGlyAlaValSerTrpValArgGlnAla TCCTGTACTGTCTCTGGATTCAGCCTCAGTAGCGGCGCGGTGAGCTGGGTAAGACAGGCA 61 ---------!---------!---------!---------!---------!---------! 120 AGGACATGACAGAGACCTAAGTCGGAGTCATCGCCGCGCCACTCGACCCATTCTGTCCGT 1: ProGlyLysGlyLeuGluTrpIleGlyValIleSerSerArgAspIleAlaTyrPheAla CCAGGGAAGGGACTGGAGTGGATCGGCGTTATCTCTAGTCGTGATATTGCATACTTTGCC 121 ---------!---------!---------!---------!---------!---------! 180 GGTCCCTTCCCTGACCTCACCTAGCCGCAATAGAGATCAGCACTATAACGTATGAAACGG 1: ThrTrpAlaLysGlyArgPheThrIleSerArgHisAsnSerLysAsnThrLeuTyrLeu ACATGGGCTAAGGGCCGTTTCACCATTTCAAGACATAATTCAAAGAACACACTGTACCTG 181 ---------!---------!---------!---------!---------!---------! 240 TGTACCCGATTCCCGGCAAAGTGGTAAAGTTCTGTATTAAGTTTCTTGTGTGACATGGAC 1: GlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArgValSerAla CAGATGAACAGCCTGCGTGCGGAGGACACGGCCGTGTACTACTGTGCTAGAGTCTCCGCC 241 ---------!---------!---------!---------!---------!---------! 300 GTCTACTTGTCGGACGCACGCCTCCTGTGCCGGCACATGATGACACGATCTCAGAGGCGG 1: SerTyrThrSerAspGlyAspAlaIleIleHisSerPheAlaLeuTrpGlyGlnGlyThr TCTTACACTTCCGATGGTGACGCTATTATCCATTCATTTGCACTGTGGGGTCAAGGAACC 301 ---------!---------!---------!---------!---------!---------! 360 AGAATGTGAAGGCTACCACTGCGATAATAGGTAAGTAAACGTGACACCCCAGTTCCTTGG 1: LeuValThrValSerSerAlaSerThrLysGlyProSerValPheProLeuAlaProSer CTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC 361 ---------!---------!---------!---------!---------!---------! 420 GACCAGTGGCAGAGGAGTCGGAGGTGGTTCCCGGGTAGCCAGAAGGGGGACCGTGGGAGG 1: SerLysSerThrSerGlyGlyThrAlaAlaLeuGlyCysLeuValLysAspTyrPhePro TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC 421 ---------!---------!---------!---------!---------!---------! 480 AGGTTCTCGTGGAGACCCCCGTGTCGCCGGGACCCGACGGACCAGTTCCTGATGAAGGGG 1: GluProValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyValHisThrPhePro GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG 481 ---------!---------!---------!---------!---------!---------! 540 CTTGGCCACTGCCACAGCACCTTGAGTCCGCGGGACTGGTCGCCGCACGTGTGGAAGGGC 1: AlaValLeuGlnSerSerGlyLeuTyrSerLeuSerSerValValThrValProSerSer GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC 541 ---------!---------!---------!---------!---------!---------! 600 CGACAGGATGTCAGGAGTCCTGAGATGAGGGAGTCGTCGCACCACTGGCACGGGAGGTCG 1: SerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLysProSerAsnThrLysVal AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG 601 ---------!---------!---------!---------!---------!---------! 660 TCGAACCCGTGGGTCTGGATGTAGACGTTGCACTTAGTGTTCGGGTCGTTGTGGTTCCAC 1: AspLysLysValGluProLysSerCysAspLysThrHisThrCysProProCysProAla GACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCA 661 ---------!---------!---------!---------!---------!---------! 720 CTGTTCTTTCAACTCGGGTTTAGAACACTGTTTTGAGTGTGTACGGGTGGCACGGGTCGT
1: ProGluLeuLeuGlyGlyProSerValPheLeuPheProProLysProLysAspThrLeu CCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC 721 ---------!---------!---------!---------!---------!---------! 780 GGACTTGAGGACCCCCCTGGCAGTCAGAAGGAGAAGGGGGGTTTTGGGTTCCTGTGGGAG 1: MetIleSerArgThrProGluValThrCysValValValAspValSerHisGluAspPro ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT 781 ---------!---------!---------!---------!---------!---------! 840 TACTAGAGGGCCTGGGGACTCCAGTGTACGCACCACCACCTGCACTCGGTGCTTCTGGGA 1: GluValLysPheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLysPro GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG 841 ---------!---------!---------!---------!---------!---------! 900 CTCCAGTTCAAGTTGACCATGCACCTGCCGCACCTCCACGTATTACGGTTCTGTTTCGGC 1: ArgGluGluGlnTyrAsnSerThrTyrArgValValSerValLeuThrValLeuHisGln CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG 901 ---------!---------!---------!---------!---------!---------! 960 GCCCTCCTCGTCATGTTGTCGTGCATGGCACACCAGTCGCAGGAGTGGCAGGACGTGGTC 1: AspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaPro GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCC 961 ---------!---------!---------!---------!---------!---------! 1020 CTGACCGACTTACCGTTCCTCATGTTCACGTTCCAGAGGTTGTTTCGGGAGGGTCGGGGG 1: IleGluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeu ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG 1021 ---------!---------!---------!---------!---------!---------! 1080 TAGCTCTTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTGGTGTCCACATGTGGGAC 1: ProProSerArgAspGluLeuThrLysAsnGlnValSerLeuThrCysLeuValLysGly CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC 1081 ---------!---------!---------!---------!---------!---------! 1140 GGGGGTAGGGCCCTACTCGACTGGTTCTTGGTCCAGTCGGACTGGACGGACCAGTTTCCG 1: PheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyr TTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC 1141 ---------!---------!---------!---------!---------!---------! 1200 AAGATAGGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCTCTTGTTGATG 1: LysThrThrProProValLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThr AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC 1201 ---------!---------!---------!---------!---------!---------! 1260 TTCTGGTGCGGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGATGTCGTTCGAGTGG 1: ValAspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMetHisGluAla GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT 1261 ---------!---------!---------!---------!---------!---------! 1320 CACCTGTTCTCGTCCACCGTCGTCCCCTTGCAGAAGAGTACGAGGCACTACGTACTCCGA 1: LeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerProGlyLys CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 1321 ---------!---------!---------!---------!-------- 1368 GACGTGTTGGTGATGTGCGTCTTCTCGGAGAGGGACAGAGGCCCATTT
[00261] hSPF7H2 1: GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGlySerLeuArgLeu GAGGTCCAGCTCGTTGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGGCTC 1 ---------!---------!---------!---------!---------!---------! 60 CTCCAGGTCGAGCAACTCAGACCCCCTCCGAACCAGGTCGGACCTCCCAGGGACTCCGAG 1: SerCysThrValSerGlyPheSerLeuSerSerGlyAlaValSerTrpValArgGlnAla TCCTGTACTGTCTCTGGATTCAGCCTCAGTAGCGGCGCGGTGAGCTGGGTAAGACAGGCA 61 ---------!---------!---------!---------!---------!---------! 120 AGGACATGACAGAGACCTAAGTCGGAGTCATCGCCGCGCCACTCGACCCATTCTGTCCGT 1: ProGlyLysGlyLeuGluTrpIleGlyValIleSerSerArgAspIleAlaTyrPheAla CCAGGGAAGGGACTGGAGTGGATCGGCGTTATCTCTAGTCGTGATATTGCATACTTTGCC 121 ---------!---------!---------!---------!---------!---------! 180 GGTCCCTTCCCTGACCTCACCTAGCCGCAATAGAGATCAGCACTATAACGTATGAAACGG 1: ThrTrpAlaLysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeu ACATGGGCTAAGGGCCGTTTCACCATTTCAAGAGATAATTCAAAGAACACACTGTACCTG 181 ---------!---------!---------!---------!---------!---------! 240 TGTACCCGATTCCCGGCAAAGTGGTAAAGTTCTCTATTAAGTTTCTTGTGTGACATGGAC 1: GlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArgValSerAla CAGATGAACAGCCTGCGTGCGGAGGACACGGCCGTGTACTACTGTGCTAGAGTCTCCGCC 241 ---------!---------!---------!---------!---------!---------! 300 GTCTACTTGTCGGACGCACGCCTCCTGTGCCGGCACATGATGACACGATCTCAGAGGCGG 1: SerTyrThrSerAspGlyAspAlaIleIleHisSerPheAlaLeuTrpGlyGlnGlyThr TCTTACACTTCCGATGGTGACGCTATTATCCATTCATTTGCACTGTGGGGTCAAGGAACC 301 ---------!---------!---------!---------!---------!---------! 360 AGAATGTGAAGGCTACCACTGCGATAATAGGTAAGTAAACGTGACACCCCAGTTCCTTGG 1: LeuValThrValSerSerAlaSerThrLysGlyProSerValPheProLeuAlaProSer CTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC 361 ---------!---------!---------!---------!---------!---------! 420 GACCAGTGGCAGAGGAGTCGGAGGTGGTTCCCGGGTAGCCAGAAGGGGGACCGTGGGAGG 1: SerLysSerThrSerGlyGlyThrAlaAlaLeuGlyCysLeuValLysAspTyrPhePro TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC 421 ---------!---------!---------!---------!---------!---------! 480 AGGTTCTCGTGGAGACCCCCGTGTCGCCGGGACCCGACGGACCAGTTCCTGATGAAGGGG 1: GluProValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyValHisThrPhePro GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG 481 ---------!---------!---------!---------!---------!---------! 540 CTTGGCCACTGCCACAGCACCTTGAGTCCGCGGGACTGGTCGCCGCACGTGTGGAAGGGC 1: AlaValLeuGlnSerSerGlyLeuTyrSerLeuSerSerValValThrValProSerSer GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC 541 ---------!---------!---------!---------!---------!---------! 600 CGACAGGATGTCAGGAGTCCTGAGATGAGGGAGTCGTCGCACCACTGGCACGGGAGGTCG 1: SerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLysProSerAsnThrLysVal AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG 601 ---------!---------!---------!---------!---------!---------! 660 TCGAACCCGTGGGTCTGGATGTAGACGTTGCACTTAGTGTTCGGGTCGTTGTGGTTCCAC
1: AspLysLysValGluProLysSerCysAspLysThrHisThrCysProProCysProAla GACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCA 661 ---------!---------!---------!---------!---------!---------! 720 CTGTTCTTTCAACTCGGGTTTAGAACACTGTTTTGAGTGTGTACGGGTGGCACGGGTCGT 1: ProGluLeuLeuGlyGlyProSerValPheLeuPheProProLysProLysAspThrLeu CCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC 721 ---------!---------!---------!---------!---------!---------! 780 GGACTTGAGGACCCCCCTGGCAGTCAGAAGGAGAAGGGGGGTTTTGGGTTCCTGTGGGAG 1: MetIleSerArgThrProGluValThrCysValValValAspValSerHisGluAspPro ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT 781 ---------!---------!---------!---------!---------!---------! 840 TACTAGAGGGCCTGGGGACTCCAGTGTACGCACCACCACCTGCACTCGGTGCTTCTGGGA 1: GluValLysPheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLysPro GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG 841 ---------!---------!---------!---------!---------!---------! 900 CTCCAGTTCAAGTTGACCATGCACCTGCCGCACCTCCACGTATTACGGTTCTGTTTCGGC 1: ArgGluGluGlnTyrAsnSerThrTyrArgValValSerValLeuThrValLeuHisGln CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG 901 ---------!---------!---------!---------!---------!---------! 960 GCCCTCCTCGTCATGTTGTCGTGCATGGCACACCAGTCGCAGGAGTGGCAGGACGTGGTC 1: AspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaPro GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCC 961 ---------!---------!---------!---------!---------!---------! 1020 CTGACCGACTTACCGTTCCTCATGTTCACGTTCCAGAGGTTGTTTCGGGAGGGTCGGGGG 1: IleGluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeu ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG 1021 ---------!---------!---------!---------!---------!---------! 1080 TAGCTCTTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTGGTGTCCACATGTGGGAC 1: ProProSerArgAspGluLeuThrLysAsnGlnValSerLeuThrCysLeuValLysGly CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC 1081 ---------!---------!---------!---------!---------!---------! 1140 GGGGGTAGGGCCCTACTCGACTGGTTCTTGGTCCAGTCGGACTGGACGGACCAGTTTCCG 1: PheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyr TTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC 1141 ---------!---------!---------!---------!---------!---------! 1200 AAGATAGGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCTCTTGTTGATG 1: LysThrThrProProValLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThr AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC 1201 ---------!---------!---------!---------!---------!---------! 1260 TTCTGGTGCGGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGATGTCGTTCGAGTGG 1: ValAspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMetHisGluAla GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT 1261 ---------!---------!---------!---------!---------!---------! 1320 CACCTGTTCTCGTCCACCGTCGTCCCCTTGCAGAAGAGTACGAGGCACTACGTACTCCGA
1: LeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerProGlyLys CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 1321 ---------!---------!---------!---------!-------- 1368 GACGTGTTGGTGATGTGCGTCTTCTCGGAGAGGGACAGAGGCCCATTT [00262] In one aspect, the humanized antibody or antigen binding fragment that comprises one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises the amino acid sequence: QSSQSVYDNNNLA; a light-chain (CDR) 2 (LCDR2) that comprises the amino acid sequence: DASKLPS; or a light-chain CDR 3 (LCDR3) that comprises the amino acid sequence: AGAVSGSNV. [00263] In a further aspect, the humanized antibody or antigen binding fragment thereof comprising: [00264] a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence TVSGFSLSSGAVS; a heavy-chain CDR 2 (HCDR2) that comprises the amino acid sequence: IGVISSRDIAYFATWAKG; a heavy- chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL. [00265] The humanized antibody or an antigen binding fragment thereof comprises a light-chain variable region comprising the following amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. [00266] The humanized antibody or an antigen binding fragment thereof, comprises a heavy-chain variable region comprising the following amino acid sequence: [00267] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. [00268] CDR Antibodies. Generally the donor and acceptor antibodies used to generate the anti-HRF, or anti-GST-N19 or anti-N19 CDR antibody are monoclonal antibodies from different species; typically the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a
“humanized” antibody. The graft may be of a single CDR (or even a portion of a single CDR) within a single VH or VL of the acceptor antibody, or can be of multiple CDRs (or portions thereof) within one or both of the VH and VL. Frequently, all three CDRs in all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, though one need replace only as many as necessary to permit adequate binding of the resulting CDR-grafted antibody to HRF, or a fragment thereof, e.g., GST-N19 or N19 protein. Methods for generating CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Pat. No.5,585,089; U.S. Pat. No.5,693,761; U.S. Pat. No.5,693,762; and Winter U.S.5,225,539; and EP 0682040. Methods useful to prepare VH and VL polypeptides are taught by Winter et al., U.S. Pat. Nos.4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP0451216; and EP0120694. [00269] After selecting suitable framework region candidates from the same family and/or the same family member, either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions. Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on the target species and CDRs from the originating species) can be produced by oligonucleotide synthesis and/or PCR. The nucleic acid encoding CDR regions can also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes. Alternatively, the framework regions of the variable chains of the originating species antibody can be changed by site- directed mutagenesis. [00270] Since the hybrids are constructed from choices among multiple candidates corresponding to each framework region, there exist many combinations of sequences which are amenable to construction in accordance with the principles described herein. Accordingly, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids. [00271] This process typically does not alter the acceptor antibody’s FRs flanking the grafted CDRs. However, one skilled in the art can sometimes improve antigen binding affinity of the resulting anti-HRF, or GST-N19 or N19 CDR-grafted antibody by replacing
certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (See, e.g., US 5,585,089, especially columns 12-16). Or one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or a human consensus FR. Techniques for making these modifications are known in the art. Particularly if the resulting FR fits a human consensus FR for that position, or is at least 90% or more identical to such a consensus FR, doing so may not increase the antigenicity of the resulting modified anti-HRF, or GST-N19 or N19 CDR-grafted antibody significantly compared to the same antibody with a fully human FR. [00272] Bispecific Antibodies (BsAbs). A bispecific antibody is an antibody that can bind simultaneously to two targets that have a distinct structure, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen. BsAbs can be made, for example, by combining heavy chains and/or light chains that recognize different epitopes of the same or different antigen. In some embodiments, by molecular function, a bispecific binding agent binds one antigen (or epitope) on one of its two binding arms (one VH/VL pair), and binds a different antigen (or epitope) on its second arm (a different VH/VL pair). By this definition, a bispecific binding agent has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds. [00273] Bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) of the present technology have at least one arm that specifically binds to, for example, HRF or a fragment thereof and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of a B-cell, a T-cell, a myeloid cell, a plasma cell, or a mast-cell. [00274] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs have been constructed that either utilize the full immunoglobulin framework (e.g., IgG), single chain variable fragment (scFv), or combinations thereof. In some embodiments, the bispecific fusion protein is divalent, comprising, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, comprising, for example, an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFv for a second antigen. BsAbs composed
of two scFv units in tandem have been shown to be a clinically successful bispecific antibody format. In some embodiments, BsAbs comprise two single chain variable fragments (scFvs) in tandem have been designed such that an scFv that binds a tumor antigen (e.g., HRF or GST-N19 or N19) is linked with an scFv that engages T cells (e.g., by binding CD3). In this way, T cells are recruited to a tumor site such that they can mediate cytotoxic killing of the tumor cells. See e.g., Dreier et al., J. Immunol.170:4397-4402 (2003); Bargou et al., Science 321 :974- 977 (2008)). [00275] Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng.10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the needed dual specificities. See, e.g., Coloma et al., Nature Biotech.15:159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering. [00276] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In some certain embodiments, a BsAb according to the present technology comprises an immunoglobulin, which immunoglobulin comprises a heavy chain and a light chain, and an scFv. In some certain embodiments, the scFv is linked to the C-terminal end of the heavy chain of any HRF, or GST-N19 or N19 immunoglobulin disclosed herein. In some certain embodiments, scFvs are linked to the C-terminal end of the light chain of any HRF or fragment thereof, e.g. GST-N19 or N19 immunoglobulin disclosed herein. In various embodiments, scFvs are linked to heavy or light chains via a linker sequence. Appropriate linker sequences necessary for the in-frame connection of the heavy chain Fd to the scFv are introduced into the VL and Vkappa domains through PCR reactions. The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and ligated into a vector containing a DNA sequence encoding the VH region of an HRF or GST-N19 or N19 antibody. The resulting vector can be used to transfect an appropriate host cell, such as a mammalian cell for the expression of the bispecific fusion protein. [00277] In some embodiments, a linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is
characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., first and/or second antigen binding sites). In some embodiments, a linker is employed in a BsAb described herein based on specific properties imparted to the BsAb such as, for example, an increase in stability. In some embodiments, a BsAb of the present technology comprises a G4S linker. In some certain embodiments, a BsAb of the present technology comprises a (G4S)n linker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. [00278] Fc Modifications. In some embodiments, the anti-HRF or anti-GST-N19 or anti- N19 antibodies of the present technology comprise a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region (or the parental Fc region), such that said molecule has an altered affinity for an Fc receptor (e.g., an Fc ^R), provided that said variant Fc region does not have a substitution at positions that make a direct contact with Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that make a direct contact with an Fc receptor such as an Fc ^R, include amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F/G) loop. [00279] In some embodiments, an antiHRF or anti-GST-N19 or anti-N19 antibody of the present technology has an altered affinity for activating and/or inhibitory receptors, having a variant Fc region with one or more amino acid modifications. [00280] Glycosylation Modifications. In some embodiments, anti-HRF or anti-GST-N19 or N19 antibodies of the present technology have an Fc region with variant glycosylation as compared to a parent Fc region. In some embodiments, variant glycosylation includes the absence of fucose; in some embodiments, variant glycosylation results from expression in GnT1-deficient CHO cells. [00281] In some embodiments, the antibodies of the present technology, may have a modified glycosylation site relative to an appropriate reference antibody that binds to an antigen of interest (e.g., HRF, GST-N19 or N19), without altering the functionality of the antibody, e.g., binding activity to the antigen. As used herein, "glycosylation sites" include any specific amino acid sequence in an antibody to which an oligosaccharide (i.e., carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach.
[00282] Oligosaccharide side chains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine. For example, an Fc- glycoform that lacks certain oligosaccharides including fucose and terminal N- acetylglucosamine may be produced in special CHO cells and exhibit enhanced ADCC effector function. [00283] In some embodiments, the carbohydrate content of an immunoglobulin-related composition disclosed herein is modified by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, e.g., U.S. Patent No.6,218,149; EP 0359096B1 ; U.S. Patent Publication No. US 2002/0028486; International Patent Application Publication WO 03/035835; U.S. Patent Publication No.2003/0115614; U.S. Patent No.6,218,149; U.S. Patent No.6,472,511; all of which are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In some certain embodiments, the present technology includes deleting the glycosylation site of the Fc region of an antibody, by modifying position 297 from asparagine to alanine. [00284] Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N- acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al., 1999, Nat. Biotechnol.17: 176-180; Davies et al., 2001, Biotechnol. Bioeng.74:288-294; Shields et al., 2002, J. Biol. Chem.277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem.278:3466-3473; U.S. Patent No.6,602,684; U.S. Patent Application Serial No.10/277,370; U.S. Patent Application Serial No.10/113,929; International Patent Application Publications WO 00/61739A1 ; WO 01/292246A1; WO 02/311140A1; WO 02/30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton,
N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication WO 00/061739; U.S. Patent Application Publication No.2003/0115614; Okazaki et al., 2004, JMB, 336: 1239-49. [00285] Fusion Proteins. In one embodiment, the anti-HRF, or anti-GST-N19 or anti- N19 antibody of the present technology is a fusion protein. The antibodies of the present technology, when fused to a second protein, can be used as an antigenic tag. Examples of domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but can occur through linker sequences. Moreover, fusion proteins of the present technology can also be engineered to improve characteristics of the antibodies. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the antibody to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties can be added to an antibody to facilitate purification. Such regions can be removed prior to final preparation of the antibody. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art. The anti-HRF or anti-GST-N19 or anti-N19 antibody of the present technology can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In select embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984. [00286] Thus, any of these above fusion proteins can be engineered using the polynucleotides or the polypeptides of the present technology. Also, in some embodiments, the fusion proteins described herein show an increased half-life in vivo. [00287] Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can be more efficient in binding and neutralizing other molecules compared to the monomeric secreted protein or protein fragment alone. Fountoulakis et al., J. Biochem.270: 3958-3964, 1995.
[00288] Similarly, EP-A-O 464533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or a fragment thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, e.g., improved pharmacokinetic properties. See EP-A 0232262. Alternatively, deleting or modifying the Fc part after the fusion protein has been expressed, detected, and purified, may be desired. For example, the Fc portion can hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, e.g., human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995. [00289] Labeled Anti-HRF or anti-GSTN19 or anti- N19 antibodies and fragments thereof. In one embodiment, the antibody or fragment thereof of the present technology is coupled with a label moiety, i.e., detectable group. The particular label or detectable group conjugated to the antibody or fragment is not a critical aspect of the technology, so long as it does not significantly interfere with the specific binding of the antibody or fragment thereof of the present technology to the binding partner. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well- developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 14C, 35S, 125I, 121I, 131I, 112In, 99mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18F, 11C, 15O, (for Positron emission tomography), 99mTC, 111In (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and the like) beads. Patents that describe the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated herein by reference in their entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene OR.).
[00290] The label can be coupled directly or indirectly to the desired component of an assay according to methods well known in the art. As indicated above, a wide variety of labels can be used, with the choice of label depending on factors such as required sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions. [00291] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti- ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, e.g., biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally-occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., an anti-HRF, or anti- GST-N19 or anti-N19 antibody. [00292] The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties, include, but are not limited to, e.g., fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties, include, but are not limited to, e.g., luciferin, and 2,3- dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal-producing systems which can be used, see U.S. Pat. No.4,391,904. [00293] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels can be detected simply by observing the color associated with the
label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead. [00294] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies, e.g., the anti- HRF, or anti- GST-N19 or anti- N19 antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection. Equivalents [00295] Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosure embodied therein herein disclosed can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. [00296] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. [00297] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. [00298] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. Several references are identified by an Arabic number, and the full bibliographic citation or these references are provided below. In case of conflict, the present specification, including definitions, will control. Sequences of HuHRF Clone Amino Acids. Bolded text indicates CDRs: SPA8-1LC ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLIYGASILA
SGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFGGGTEVVVKGD PVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQN SADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SPA8-1HC QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDIAYF ATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWGQ GTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNG VRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARP PLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPK VYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSY FLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SPC4-4LC AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLIYDASKL PSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVVVKGDPV APTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSA DCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SPC4-4HC QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDIAY FATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWGQ GTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNG VRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARP PLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPK VYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSY FLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SPD9-2LC AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLIYDASKL PSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVVVKGDPV
APTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSA DCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SPD9-2HC QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDIAYF ATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWGQG TLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGV RTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPP ELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPP LREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKV YTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYF LYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SPF7-1LC ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLIYDASKL PSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVVVKGDPV APTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSA DCTYNLSSTLTLTSTQYNSHKEYTCKVTRGTTSVVQSFNRGDC SPF7-1HC QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDIAY FATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWGQ GALVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNG VRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARP PLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPK VYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSY FLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SPF8-1LC AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLLIYDASKL ASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGGGTEVVVKGDPV APTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSA
DCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SPF8-1HC QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDIAYF ATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWGQ GTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNG VRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARP PLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPK VYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSY FLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SPF8-4LC AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIFGASTLR YGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFGGGTEVVVKG DPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQ NSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSASPIVQSFNRGDC SPF8-4HC QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDIAYF ATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWGQ GTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNG VRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARP PLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPK VYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSY FLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SPF8-4 AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLIYDASKL PSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVVVKGDPV APTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSA DCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
SPF8-4 QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDIAY FATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWGQ GTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNG VRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARP PLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPK VYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSY FLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SPH4-3LC AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLIYDASKLP SGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVVVKGDPVA PTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSAD CTYNLSSTLTLTSTQYNSHKEYTCKVTLGTTSVVQSFNRGDC SPH4-3HC QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDNTY FATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWG QGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTN GVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCP PPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTAR PPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEP KVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGS YFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SPH8-6LC ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLIYDASKL PSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGGTEVVVKGDPV APTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSA DCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNMGDC
SPH8-6HC QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVISSRDNTY FATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDGDAIIHSFALWG QGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTN GVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCP PPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTAR PPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEP KVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGS YFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
REFERENCES 1. Bommer, U.A. Cellular function and regulation of the translationally controlled tumor protein TCTP. The Open Allergy Journal 5, 19-32 (2012). 2. MacDonald, S.M., Rafnar, T., Langdon, J. & Lichtenstein, L.M. Molecular identification of an IgE- dependent histamine-releasing factor. Science 269, 688-690 (1995). 3. Ando, T. et al. Histamine-releasing factor enhances food allergy. The Journal of clinical investigation 127, 4541-4553 (2017). 4. Kashiwakura, J. et al. Histamine-releasing factor has a proinflammatory role in mouse models of asthma and allergy. The Journal of clinical investigation 122, 218-228 (2012). 5. Kashiwakura, J.I. et al. The basophil-IL-4-mast cell axis is required for food allergy. Allergy (2019). 6. Kawakami, Y., Sielski, R. & Kawakami, T. Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation. J. Vis. Exp., e58391 (2018). 7. Kawakami, Y et al. Novel inhibitors of histamine-releasing factor suppress food allergy in a murine model. Allergology International., 71, 147-149 (2022).
Claims
WHAT IS CLAIMED IS 1. An antibody or an antigen binding fragment thereof comprising one or more of the following sequences: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; b) a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; or c) a heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL.
2. An antibody or antigen binding fragment comprising one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA; b) a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; or c) a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG.
3. An antibody or antigen binding fragment thereof comprising one or more of: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence GFSLSSGA; b) a heavy-chain CDR 2 (HCDR2) that comprises one of the following amino acid sequences: ISSRDIAY or ISSRDITY; c) a heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL; d) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises one of the following sequences: QSVYGNNN, QSVYDNNN, QSVYNNNN or ASVFDNNA;
e) a light-chain CDR 2 (LCDR2) that comprises one of the following amino acid sequences: GAS or DAS; or f) a light-chain CDR 3 (LCDR3) that comprises one of the following amino acid sequences: AGGYSSSSENG, AGAVSGSNV, AGAFSGSNF or AGGYTNNADNG.
4. An antibody or an antigen binding fragment thereof, comprising a heavy-chain variable region comprising one of the following amino acid sequences or an equivalent thereof: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; b) QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; c) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; d) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGALVTVSS; e) QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; f) QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS; g) QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS;
h) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS; or i) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS.
5. An antibody or an antigen binding fragment thereof, comprising a light-chain variable region comprising one of the following amino acid sequences or an equivalent thereof: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVK; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; c) AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; d) ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; e) AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGG GTEVVVK; f) AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFG GGTEVVVK; g) AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK;
h) AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK; or i) ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK.
6. An antibody or antigen binding fragment thereof, comprising: a) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVK or an equivalent thereof; b) a heavy chain variable region comprising the amino acid sequence: QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK or an equivalent thereof; c) a heavy chain variable region comprising the amino acid sequence: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK or an equivalent thereof;
d) a heavy chain variable region comprising the amino acid sequence: QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGALVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK or an equivalent thereof; e) a heavy chain variable region comprising the amino acid sequence: QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGG GTEVVVK or an equivalent thereof; f) a heavy chain variable region comprising the amino acid sequence: QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFG GGTEVVVK or an equivalent thereof; g) a heavy chain variable region comprising the amino acid sequence: QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK or an equivalent thereof;
h) a heavy chain variable region comprising the amino acid sequence: QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVK or an equivalent thereof; or i) a heavy chain variable region comprising the amino acid sequence: QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSS or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVK or an equivalent thereof.
7. An antibody or antigen biding fragment thereof, comprising a heavy chain region comprising one of the following amino acid sequences or an equivalent thereof: a) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLP EPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPAT NTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVD VSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKE FKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGF YPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFT CSVMHEALHNHYTQKSISRSPGK; b) QELVESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS
QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; c) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISRTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; d) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVI SSRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGD AIIHSFALWGQGALVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLP EPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPAT NTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVD VSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKE FKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGF YPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFT CSVMHEALHNHYTQKSISRSPGK; e) QSLEESRGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK;
f) QSLEESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; g) QTVKESEGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDIAYFATWAKGRFTISKTSTTVDLKITSPTPEDTATYFCARVSASYTSDGDA IIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVS QDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFK CKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK; h) QSLGESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL PEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPA TNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVV DVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRG KEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMIN GFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDV FTCSVMHEALHNHYTQKSISRSPGK; or i) QTVKESGGRLVTPGTPLTLTCTVSGFSLSSGAVSWVRQAPGKGLEWIGVIS SRDNTYFATWAKGRFTISKTSSTTVDLKITSPTPEDTATYFCARVSASYTSDG DAIIHSFALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL PEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPA TNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVV
DVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRG KEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMIN GFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDV FTCSVMHEALHNHYTQKSISRSPGK.
8. The antibody or antigen biding fragment of claim 7, comprising a light-chain region comprising one of the following amino acid sequences or an equivalent thereof: a) ALVMTQTPSPVSAAVGGTVTISCQASQSVYGNNNLSWYQQKPGQPPKLLI YGASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSSSENGFG GGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVV QSFNRGDC; b) AQGLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTT QTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSF NRGDC; c) AQVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTT QTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSF NRGDC; d) ALVLTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTT QTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTRGTTSVVQSF NRGDC; e) AQGMTQTPSPVSAAVGGKVTISCQSSQSVYNNNNLAWYQQKPGQPPKLL IYDASKLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAFSGSNFFGG GTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGT TQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQS FNRGDC;
f) AAVLTQTPSPVSAAVGGTVTISCQSSASVFDNNALSWYQQKPGQPPKLLIF GASTLRYGVPSRFSGSGSGTQFTLTISDVQCADAATYYCAGGYTNNADNGFG GGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSAS PIVQSFNRGDC; g) AQGMTQTPSPVSAAVGGKVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGT TQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQS FNRGDC; h) AIEMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLLI YDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGGG TEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTT QTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTLGTTSVVQSF NRGDC; or i) ALVMTQTASPVSAAVGGTVTINCQSSQSVYDNNNLAWYRQKPGQPPKLL IYDASKLPSGVPSRFKGSGSGTQFTLTISDLECADAATYFCAGAVSGSNVFGG GTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGT TQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQS FNMGDC.
9. A humanized antibody or an antigen binding fragment thereof comprising one or more of the following sequences: a) a light-chain (LC) complementarity determining region (CDR) 1 (LCDR1) that comprises the amino acid sequence QSSQSVYDNNNLA; a light-chain (CDR) 2 (LCDR2) that comprises the amino acid sequence DASKLPS; or a light-chain CDR 3 (LCDR3) that comprises the amino acid sequence AGAVSGSNV.
10. A humanized antibody or an antigen binding fragment thereof comprising one or more of the following sequences: a) a heavy-chain (HC) complementarity determining region (CDR) 1 (HCDR1) that comprises the amino acid sequence TVSGFSLSSGAVS; a heavy-chain CDR 2 (HCDR2) that comprises the amino acid sequence IGVISSRDIAYFATWAKG; a
heavy-chain CDR 3 (HCDR3) that comprises the amino acid sequence ARVSASYTSDGDAIIHSFAL.
11. A humanized antibody or an antigen binding fragment thereof, comprising a light- chain variable region comprising the following amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC or an equivalent thereof.
12. A humanized antibody or an antigen binding fragment thereof, comprising a light- chain variable region comprising the following amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK or an equivalent thereof.
13. A humanized antibody or antigen binding fragment thereof, comprising: a) a heavy chain variable region comprising the amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK or an equivalent thereof, and a light-chain variable region comprising the amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC or an equivalent thereof.
14. The antibody or antigen binding fragment of any one or more of claims 4-8 and 11- 13, wherein the equivalent is at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or more identical to the HC and/or LC reference sequence across the complete sequence.
15. The antibody or antigen binding fragment of any one or more of claims 1-13, specifically recognizing and binding to HRF or an immunogenic fragment thereof.
16. The antibody or antigen binding fragment of claim 15, wherein the immunogenic fragment comprises or consists of the amino acid sequence of N19 or GST-N19.
17. The antibody or antigen binding fragment of claim 15, wherein the HRF comprises or consists of the amino acid sequence of SEQ ID NO: 1.
18. The antibody or antigen binding fragment of any one of claims 1-17, wherein the antibody or antigen binding fragment is isolated or recombinant.
19. The antibody or antigen binding fragment of any one of claims 1-18, wherein the antibody or antigen binding fragment thereof is chimeric, humanized, a single chain, or a humanized single chain.
20. The antigen binding fragment of any one of claims 1-19, wherein the antigen binding fragment is a Fab, F(ab’)2, Fab’, scFv, or Fv.
21. The antibody or antigen binding fragment of any one of claims 1-20, comprising a light chain constant domain.
22. The antibody or antigen binding fragment of claim 21, wherein the light chain constant domain comprises a constant domain of a human κ light chain.
23. The antibody or antigen binding fragment of claim 21, wherein the light chain constant domain comprises a constant domain of a human λ light chain.
24. The antibody or antigen binding fragment of claim 23, wherein the light chain constant domain comprises a constant domain of a λ1 or λ2 or λ3 or λ4 light chain.
25. The antibody or antigen binding fragment of any one of claims 1-18, wherein the fragment comprises a fragment crystallizable region (Fc region).
26. The antibody or antigen binding fragment of claim 25, wherein the Fc region comprises one or more of: an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgM Fc region, or an IgE Fc region.
27. The antibody or antigen binding fragment of claim 25 or 26, wherein the Fc region comprises one or more of: an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG4 Fc region.
28. The antibody or antigen binding fragment of any one of claims 1-27, wherein the antibody or antigen binding fragment is post-translationally modified optionally glycosylated, hydroxylated, methylated, lapidated, acetylated, SUMOylated, phosphorylated, PEGylated, or any combination thereof.
29. The antibody or antigen binding fragment of any one of claims 1-28, further comprising a detectable or purification marker.
30. A polynucleotide encoding the antibody or antigen binding fragment of any one of claims 1-16, or a polynucleotide complementary thereto.
31. A vector comprising the polynucleotide of claim 30.
32. The vector of claim 31, further comprising a regulatory sequence that directs the expression and/or secretion of the antibody or antigen binding fragment.
33. The vector of claim 32, wherein the regulatory sequence comprises one or more of: a promoter, a secretion sequence, an enhancer, or a polyadenylation sequence.
34. The vector of any one of claims 31-33, further comprising a regulatory sequence that directs the replication or expression of the polynucleotide.
35. The vector of any one of claims 31-34, wherein the vector is a non-viral vector, optionally a plasmid.
36. The vector of any one of claims 31-35, wherein the vector is a viral vector, optionally an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, or a plant viral vector.
37. A cell comprising one or more of: the antibody or antigen binding fragment of any one of claims 1-26, the polynucleotide of claim 27, or the vector of any one of claims 28-33.
38. The cell of claim 37, wherein the cell is a prokaryotic cell, optionally an Escherichia coli cell.
39. The cell of claim 37, wherein the cell is a eukaryotic cell, optionally a mammal cell, an insect cell, or a yeast cell.
40. A hybridoma expressing the antibody or antigen binding fragment of any one of claims 1-29.
41. A method of producing the antibody or antigen binding fragment of any one of claims 1-29, comprising culturing a cell comprising a polynucleotide encoding the antibody or the antigen binding fragment under conditions suitable for expression of the antibody or antigen binding fragment.
42. The method of claim 41, further comprising introducing the polynucleotide to the cell prior to the culturing step.
43. A method of producing the antibody or antigen binding fragment of any one of claims 1-29, comprising culturing the hybridoma of claim 40 under conditions suitable for expression of the antibody or antigen binding fragment.
44. A method of producing the antibody or antigen binding fragment of any one of claims 1-29, comprising contacting the polynucleotide of claim 40 or the vector of any one of claims 31-36 with an RNA polymerase, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine-5'-triphosphate (GTP), and uridine triphosphate (UTP) under conditions suitable for transcription to messenger RNA, and contacting the transcribed messenger RNA with a ribosome, tRNAs, an aminoacyl-tRNA synthetase, and/or initiation, elongation and termination factors under conditions suitable for translation to the antibody or antigen binding fragment.
45. The method of claim 44, comprising contacting the transcribed messenger RNA with a cell lysate comprising the ribosome, tRNAs, aminoacyl-tRNA synthetase, and/or initiation, elongation and termination factors under conditions.
46. The method of any one of claims 41-45, further comprising isolating the expressed antibody or antigen binding fragment.
47. A composition comprising a carrier and one or more of: the antibody or antigen binding fragment of any one of claims 1-29, the polynucleotide of claim 30, the vector of any one of claims 31-36, the cell of any one of claims 37-39, or the hybridoma of claim 40.
48. The composition of claim 47, comprising two or more of the antibodies or antigen binding fragments of any one of claims 1-29.
49. The composition of claim 45, wherein the two or more of the antibodies or antigen binding fragments recognize and bind to at least two different epitopes.
50. The composition of any one of claims 47-49, further comprising N19, GST-N19 or HRF-2CA, and optionally wherein the carrier is a pharmaceutical acceptable carrier.
51. A method for one or more of: a) targeting HRF N19-Ig interactions; b) inhibiting HRF N19 or HRF-Ig interactions; c) inhibiting Ig E-dependent activation of mast cells or basophils; or d) the treatment of a condition from the group of: an allergy, optionally, a food allergy or an allergic reaction, hypersensitivity, asthma, anaphylaxis, inflammatory response or inflammation in a subject in need thereof, comprising contacting a tissue or administering an effective amount of the antibody or antigen binding fragment of any one of claims 1-28 to the subject or the composition of any of claims 47-49.
52. The method of claim 51, wherein the agent binds an HRF-reactive immunoglobulin (Ig).
53. The method of claim 51 or 52, wherein the agent modulates binding of an HRF monomer, an HRF dimer, or an HRF multimer with the HRF-reactive Ig.
54. The method of any one of the claims 51-53, wherein the method further comprises administering an effective amount of a second drug.
55. The method of claim 54, wherein the second drug comprises an anti-inflammatory, anti-asthmatic or anti-allergy drug.
56. The method of claim 55, wherein the second drug comprises a hormone, a steroid, an anti-histamine, anti-leukotriene, anti-IgE, anti- ^4 integrin, anti-β2 integrin, anti- CCR3 antagonist, β2 agonist or anti-selectin.
57. The method of claim 54, wherein the second drug comprises an allergen immunotherapy.
58. The method of claim 57, wherein the allergen immunotherapy is oral immunotherapy (OIT), subcutaneous immunotherapy (SIT), or sublingual immunotherapy (SLIT).
59. The method of any one of the claims 55-58, wherein the second drug is administered via ingestion, via inhalation, topically, or a combination thereof.
60. The method of claim 54, wherein the second therapy comprises an inhibitor of binding of HRF to an Ig molecule.
61. The method of claim 60, wherein the inhibitor is a peptide or polypeptide that inhibits the binding of an HRF monomer, an HRF dimer, or a HRF multimer with an HRF- reactive immunoglobulin (Ig).
62. The method of any one of the claims 55 to 61, wherein the second therapy is administered prior to, or after the sample is isolated from the subject.
63. A detection system comprising the antibody or antigen binding fragment of any one of claims 1-28 and a detectable marker producing a detectable signal upon binding of the antibody or antigen binding fragment thereof with HRF or an immunogenic fragment thereof.
64. The detection system of claim 63, wherein the system is an enzyme-linked immunosorbent assay (ELISA) or a lateral flow immunoassay.
65. A method comprising contacting the antibody or antigen binding fragment of the detection system of claim 63 or 64 with a biological sample isolated from a subject, wherein binding of the antibody or antigen binding fragment thereof with a component of the biological sample indicates the subject expresses HRF.
66. A kit comprising any one of the antibody or antigen binding fragment of any one of claims 1-29, the polynucleotide of claim 30, the vector of any one of claims 31-36, the cell of any one of claims 37-39, or the hybridoma of claim 40.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263390603P | 2022-07-19 | 2022-07-19 | |
US63/390,603 | 2022-07-19 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2024020063A2 true WO2024020063A2 (en) | 2024-01-25 |
WO2024020063A3 WO2024020063A3 (en) | 2024-03-07 |
Family
ID=89618455
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2023/028081 WO2024020063A2 (en) | 2022-07-19 | 2023-07-18 | Anti-hrf antibodies |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2024020063A2 (en) |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011123697A2 (en) * | 2010-03-31 | 2011-10-06 | La Jolla Institute For Allergy And Immunology | Histamine-releasing factor (hrf), hrf-receptor and methods of modulating inflammation |
JOP20180042A1 (en) * | 2017-04-24 | 2019-01-30 | Kite Pharma Inc | Humanized Antigen-Binding Domains and Methods of Use |
US20220056124A1 (en) * | 2018-09-05 | 2022-02-24 | The Board Of Regents Of The University Of Texas System | Monoclonal antibodies against endotrophin and the use thereof |
CN114364703A (en) * | 2019-04-19 | 2022-04-15 | 豪夫迈·罗氏有限公司 | Anti-merk antibodies and methods of use thereof |
-
2023
- 2023-07-18 WO PCT/US2023/028081 patent/WO2024020063A2/en unknown
Also Published As
Publication number | Publication date |
---|---|
WO2024020063A3 (en) | 2024-03-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11292836B2 (en) | Anti-CD47 antibodies and uses thereof | |
US20210347904A1 (en) | Antibodies against tl1a and uses thereof | |
TWI796328B (en) | B7-h3 antibody, antigen-binding fragment thereof and medical application thereof | |
CN112250763B (en) | Antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use | |
CA3101670C (en) | Anti-il-23p19 antibody and use thereof | |
WO2020151762A1 (en) | Novel bispecific antibody molecule and bispecific antibody simultaneously combining pd-l1 and lag-3 | |
CN112513090A (en) | Antibodies that bind human IL-4R, antigen-binding fragments thereof, and medical uses thereof | |
TW200844114A (en) | Binding members for IgE molecules | |
CN113966345A (en) | Natriuretic peptide receptor 1 antibodies and methods of use | |
US20020146753A1 (en) | Autoantibodies to glucose-6-phosphate isomerase and their participation in autoimmune disease | |
AU2020270888A1 (en) | CD19 antibodies and methods of using the same | |
US20230295274A1 (en) | Anti-sars coronavirus-2 spike protein antibodies | |
WO2024020063A2 (en) | Anti-hrf antibodies | |
US20240059762A1 (en) | Anti-sars coronavirus-2 nucleocapsid protein antibodies | |
BR112020005766A2 (en) | il-5 antibody, antigen-binding fragment, and medical application of the same | |
US20230365658A1 (en) | REPAIRED THERAPEUTIC AND PROPHYLACTIC ANTIBODIES AGAINST SARS-CoV-2 VARIANTS | |
WO2022166977A1 (en) | Anti-human il-36r antibody and application thereof | |
US20240141053A1 (en) | Antibodies against tl1a and uses thereof | |
US20230391854A1 (en) | Monoclonal antibody for coronavirus spike protein, and use thereof | |
CN117946265A (en) | Anti-CX 3CL1 antibody and application thereof | |
TW202221039A (en) | Antibodies to canine and feline oncostatin m receptor beta and uses thereof | |
KR20220012297A (en) | Anti-hepatitis B virus antibodies and uses thereof | |
CN114075284A (en) | CD47 binding molecules and uses thereof | |
CN115304672A (en) | Coronavirus antibody and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23843640 Country of ref document: EP Kind code of ref document: A2 |