NZ749020B2 - Cytosol-penetrating antibody and use thereof - Google Patents
Cytosol-penetrating antibody and use thereof Download PDFInfo
- Publication number
- NZ749020B2 NZ749020B2 NZ749020A NZ74902017A NZ749020B2 NZ 749020 B2 NZ749020 B2 NZ 749020B2 NZ 749020 A NZ749020 A NZ 749020A NZ 74902017 A NZ74902017 A NZ 74902017A NZ 749020 B2 NZ749020 B2 NZ 749020B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- cytosol
- antibody
- chain variable
- variable region
- cells
- Prior art date
Links
- 102000004965 antibodies Human genes 0.000 title claims abstract description 515
- 108090001123 antibodies Proteins 0.000 title claims abstract description 515
- 210000004027 cells Anatomy 0.000 claims abstract description 360
- 210000000172 Cytosol Anatomy 0.000 claims abstract description 78
- 210000001163 Endosomes Anatomy 0.000 claims abstract description 61
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 235000001014 amino acid Nutrition 0.000 claims description 213
- 150000001413 amino acids Chemical class 0.000 claims description 212
- 102100013834 SLC3A2 Human genes 0.000 claims description 157
- 101710007458 SLC3A2 Proteins 0.000 claims description 157
- 239000000427 antigen Substances 0.000 claims description 61
- 108091007172 antigens Proteins 0.000 claims description 61
- 102000038129 antigens Human genes 0.000 claims description 61
- 102000004169 proteins and genes Human genes 0.000 claims description 53
- 108090000623 proteins and genes Proteins 0.000 claims description 53
- 235000018102 proteins Nutrition 0.000 claims description 51
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 50
- 239000000126 substance Substances 0.000 claims description 45
- 239000000203 mixture Substances 0.000 claims description 42
- 229960005261 Aspartic Acid Drugs 0.000 claims description 41
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 41
- 235000003704 aspartic acid Nutrition 0.000 claims description 41
- 230000002378 acidificating Effects 0.000 claims description 39
- 229960002989 Glutamic Acid Drugs 0.000 claims description 31
- 235000013922 glutamic acid Nutrition 0.000 claims description 31
- 239000004220 glutamic acid Substances 0.000 claims description 31
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 30
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 29
- 229960000310 ISOLEUCINE Drugs 0.000 claims description 26
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 25
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 25
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 claims description 21
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims description 18
- 239000003814 drug Substances 0.000 claims description 15
- 108020004707 nucleic acids Proteins 0.000 claims description 14
- 150000007523 nucleic acids Chemical class 0.000 claims description 14
- 239000002502 liposome Substances 0.000 claims description 12
- 229910052721 tungsten Inorganic materials 0.000 claims description 9
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 229940079593 drugs Drugs 0.000 claims description 7
- 229910052739 hydrogen Inorganic materials 0.000 claims description 7
- 150000003384 small molecules Chemical class 0.000 claims description 7
- 229920000160 (ribonucleotides)n+m Polymers 0.000 claims description 6
- 102000018358 Immunoglobulins Human genes 0.000 claims description 6
- 108060003951 Immunoglobulins Proteins 0.000 claims description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 5
- 231100000765 Toxin Toxicity 0.000 claims description 5
- 239000002105 nanoparticle Substances 0.000 claims description 5
- 239000003053 toxin Substances 0.000 claims description 5
- 108020003112 toxins Proteins 0.000 claims description 5
- 108020004459 Small Interfering RNA Proteins 0.000 claims description 3
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims 1
- 210000000170 Cell Membrane Anatomy 0.000 abstract description 44
- 230000001939 inductive effect Effects 0.000 abstract description 21
- 230000001413 cellular Effects 0.000 abstract description 6
- 102000018697 Membrane Proteins Human genes 0.000 abstract description 4
- 108010052285 Membrane Proteins Proteins 0.000 abstract description 4
- 238000009114 investigational therapy Methods 0.000 abstract description 4
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 103
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 57
- 230000000149 penetrating Effects 0.000 description 54
- DEGAKNSWVGKMLS-UHFFFAOYSA-N Calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 46
- 229960002378 Oftasceine Drugs 0.000 description 46
- 239000012593 Hanks’ Balanced Salt Solution Substances 0.000 description 41
- 238000000034 method Methods 0.000 description 41
- 101700027814 CDR3 Proteins 0.000 description 40
- 238000004624 confocal microscopy Methods 0.000 description 38
- 230000001225 therapeutic Effects 0.000 description 37
- 238000005406 washing Methods 0.000 description 37
- 230000001086 cytosolic Effects 0.000 description 35
- 230000002209 hydrophobic Effects 0.000 description 33
- 230000003993 interaction Effects 0.000 description 33
- 101710033922 KRAS Proteins 0.000 description 32
- 230000001965 increased Effects 0.000 description 32
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 description 29
- 230000001419 dependent Effects 0.000 description 29
- 230000000694 effects Effects 0.000 description 27
- 239000002609 media Substances 0.000 description 27
- 235000005772 leucine Nutrition 0.000 description 25
- 239000011148 porous material Substances 0.000 description 25
- 101700073818 CDR1 Proteins 0.000 description 23
- 102100002977 CDR1 Human genes 0.000 description 23
- JKMHFZQWWAIEOD-UHFFFAOYSA-N HEPES Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 23
- 239000007995 HEPES buffer Substances 0.000 description 23
- 238000000746 purification Methods 0.000 description 23
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical group NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 22
- 230000004807 localization Effects 0.000 description 22
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 21
- 239000012528 membrane Substances 0.000 description 21
- 230000002401 inhibitory effect Effects 0.000 description 20
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 19
- 210000004940 Nucleus Anatomy 0.000 description 18
- 238000004458 analytical method Methods 0.000 description 18
- 150000002632 lipids Chemical class 0.000 description 18
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 18
- PRDFBSVERLRRMY-UHFFFAOYSA-N Bisbenzimide Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 17
- 239000003929 acidic solution Substances 0.000 description 17
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 16
- 229960005190 Phenylalanine Drugs 0.000 description 16
- 230000012202 endocytosis Effects 0.000 description 16
- 108010045030 monoclonal antibodies Proteins 0.000 description 15
- 102000005614 monoclonal antibodies Human genes 0.000 description 15
- 150000003904 phospholipids Chemical class 0.000 description 15
- 230000035693 Fab Effects 0.000 description 14
- 102100003684 HPSE Human genes 0.000 description 14
- 102200006539 KRAS G12D Human genes 0.000 description 14
- 229920001850 Nucleic acid sequence Polymers 0.000 description 14
- 235000004279 alanine Nutrition 0.000 description 14
- 201000011510 cancer Diseases 0.000 description 14
- 108010037536 heparanase Proteins 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 108010007562 Adalimumab Proteins 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 13
- 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 13
- 239000004475 Arginine Substances 0.000 description 12
- 108060001277 CDR2 Proteins 0.000 description 12
- 102100008744 CDR2 Human genes 0.000 description 12
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 12
- 108010088992 Heparan Sulfate Proteoglycans Proteins 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 12
- 125000003275 alpha amino acid group Chemical group 0.000 description 12
- 230000001388 anti-tubulin Effects 0.000 description 12
- 201000010099 disease Diseases 0.000 description 12
- 230000001264 neutralization Effects 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 12
- 102000019679 Cell-Penetrating Peptides Human genes 0.000 description 11
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 11
- 239000004471 Glycine Chemical group 0.000 description 11
- 239000008194 pharmaceutical composition Substances 0.000 description 11
- 230000002829 reduced Effects 0.000 description 11
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 10
- 238000010367 cloning Methods 0.000 description 10
- 230000000295 complement Effects 0.000 description 10
- 238000005755 formation reaction Methods 0.000 description 10
- 230000003834 intracellular Effects 0.000 description 10
- 230000032258 transport Effects 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 9
- 229920001985 Small interfering RNA Polymers 0.000 description 9
- 238000000684 flow cytometry Methods 0.000 description 9
- 108020003175 receptors Proteins 0.000 description 9
- 102000005962 receptors Human genes 0.000 description 9
- 239000004055 small Interfering RNA Substances 0.000 description 9
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- QGWNDRXFNXRZMB-UUOKFMHZSA-N Guanosine diphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O QGWNDRXFNXRZMB-UUOKFMHZSA-N 0.000 description 8
- 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 description 8
- 210000003712 Lysosomes Anatomy 0.000 description 8
- 108020005091 Replication Origin Proteins 0.000 description 8
- 229960002964 adalimumab Drugs 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- 230000001868 lysosomic Effects 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 8
- UQABYHGXWYXDTK-UUOKFMHZSA-N GppNP Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)NP(O)(O)=O)[C@@H](O)[C@H]1O UQABYHGXWYXDTK-UUOKFMHZSA-N 0.000 description 7
- 102000004243 Tubulin Human genes 0.000 description 7
- 108090000704 Tubulin Proteins 0.000 description 7
- 210000004102 animal cell Anatomy 0.000 description 7
- 125000000511 arginine group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 7
- 210000004602 germ cell Anatomy 0.000 description 7
- 229910052751 metal Inorganic materials 0.000 description 7
- 239000002184 metal Substances 0.000 description 7
- 230000037361 pathway Effects 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 102200006531 KRAS G12V Human genes 0.000 description 6
- 230000000903 blocking Effects 0.000 description 6
- 230000003436 cytoskeletal Effects 0.000 description 6
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 6
- 230000035440 response to pH Effects 0.000 description 6
- 230000001131 transforming Effects 0.000 description 6
- 125000002306 tributylsilyl group Chemical group C(CCC)[Si](CCCC)(CCCC)* 0.000 description 6
- NFGXHKASABOEEW-UHFFFAOYSA-N (+)-methoprene Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 5
- XDHNQDDQEHDUTM-JQWOJBOSSA-N Bafilomycin Chemical compound CO[C@H]1\C=C\C=C(C)\C[C@H](C)[C@H](O)[C@H](C)\C=C(/C)\C=C(OC)\C(=O)O[C@@H]1[C@@H](C)[C@@H](O)[C@H](C)[C@]1(O)O[C@H](C(C)C)[C@@H](C)[C@H](O)C1 XDHNQDDQEHDUTM-JQWOJBOSSA-N 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 101710033925 HRAS Proteins 0.000 description 5
- 108010070144 Single-Chain Antibodies Proteins 0.000 description 5
- 102000005632 Single-Chain Antibodies Human genes 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 229910052755 nonmetal Inorganic materials 0.000 description 5
- 235000021317 phosphate Nutrition 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- 229920000023 polynucleotide Polymers 0.000 description 5
- 239000002157 polynucleotide Substances 0.000 description 5
- 230000005588 protonation Effects 0.000 description 5
- 102220067383 rs199960045 Human genes 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 102000004121 Annexin A5 Human genes 0.000 description 4
- 108090000672 Annexin A5 Proteins 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 108020004999 Messenger RNA Proteins 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 201000011231 colorectal cancer Diseases 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 235000004554 glutamine Nutrition 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 229920002106 messenger RNA Polymers 0.000 description 4
- 239000002082 metal nanoparticle Substances 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000003287 optical Effects 0.000 description 4
- LVZSQWIWCANHPF-UHFFFAOYSA-N p-nitrophenyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 LVZSQWIWCANHPF-UHFFFAOYSA-N 0.000 description 4
- 210000001236 prokaryotic cell Anatomy 0.000 description 4
- 230000002441 reversible Effects 0.000 description 4
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 229960001230 Asparagine Drugs 0.000 description 3
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 229920002873 Polyethylenimine Polymers 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 101710040291 RAB11A Proteins 0.000 description 3
- 102100001402 RAB11A Human genes 0.000 description 3
- QDLHCMPXEPAAMD-QAIWCSMKSA-N Wortmannin Chemical compound C1([C@]2(C)C3=C(C4=O)OC=C3C(=O)O[C@@H]2COC)=C4[C@@H]2CCC(=O)[C@@]2(C)C[C@H]1OC(C)=O QDLHCMPXEPAAMD-QAIWCSMKSA-N 0.000 description 3
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 3
- 230000033115 angiogenesis Effects 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 3
- 230000021164 cell adhesion Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 108091006028 chimera Proteins 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000011258 core-shell material Substances 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 108091006031 fluorescent proteins Proteins 0.000 description 3
- 102000034387 fluorescent proteins Human genes 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 108010019130 nitrophorin Proteins 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000006174 pH buffer Substances 0.000 description 3
- 230000020477 pH reduction Effects 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000002194 synthesizing Effects 0.000 description 3
- HVYWMOMLDIMFJA-DPAQBDIFSA-N (3β)-Cholest-5-en-3-ol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- -1 Alexa Chemical compound 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M CHEMBL593252 Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 229940088598 Enzyme Drugs 0.000 description 2
- 102100014838 FCGRT Human genes 0.000 description 2
- 102200134361 IL1F10 A51D Human genes 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 101700048185 LAMP1 Proteins 0.000 description 2
- 102100005918 LAMP1 Human genes 0.000 description 2
- 239000000232 Lipid Bilayer Substances 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 108091007229 NSP3 Papain-like protease domain Proteins 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 229940055729 Papain Drugs 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 108091005771 Peptidases Proteins 0.000 description 2
- 102000035443 Peptidases Human genes 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 102200118332 RPA2 S33D Human genes 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 102200136100 TSG101 M95A Human genes 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H Tricalcium 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 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 108010067390 Viral Proteins Proteins 0.000 description 2
- 102000016350 Viral Proteins Human genes 0.000 description 2
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 2
- 230000003115 biocidal Effects 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000875 corresponding Effects 0.000 description 2
- 230000004059 degradation Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000002068 genetic Effects 0.000 description 2
- 125000003372 histidine group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 2
- 238000010191 image analysis Methods 0.000 description 2
- 238000010921 in-depth analysis Methods 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000002132 lysosomal Effects 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000006011 modification reaction Methods 0.000 description 2
- 229960000060 monoclonal antibodies Drugs 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 230000036961 partial Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 2
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 2
- 239000011435 rock Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 239000002691 unilamellar liposome Substances 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- GJFNRSDCSTVPCJ-UHFFFAOYSA-N 1,8-Bis(dimethylamino)naphthalene Chemical compound C1=CC(N(C)C)=C2C(N(C)C)=CC=CC2=C1 GJFNRSDCSTVPCJ-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- WHJKCPTVEYZNOG-UHFFFAOYSA-N 6-(hydroxymethyl)-5-methoxy-2-[4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane-3,4-diol Chemical group COCC1OC(OC)C(OC)C(OC)C1OC1C(O)C(O)C(OC)C(CO)O1 WHJKCPTVEYZNOG-UHFFFAOYSA-N 0.000 description 1
- 101710006746 7.5K Proteins 0.000 description 1
- 102100011550 ACTB Human genes 0.000 description 1
- 101700033661 ACTB Proteins 0.000 description 1
- 101710032514 ACTI Proteins 0.000 description 1
- AOJJSUZBOXZQNB-TZSSRYMLSA-N ADRIAMYCIN Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 1
- 102100009049 AGBL1 Human genes 0.000 description 1
- 101710003512 AGBL1 Proteins 0.000 description 1
- 102220457890 AHSG M95V Human genes 0.000 description 1
- 102000034451 ATPases Human genes 0.000 description 1
- 108091006096 ATPases Proteins 0.000 description 1
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 241000432074 Adeno-associated virus Species 0.000 description 1
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 206010061424 Anal cancer Diseases 0.000 description 1
- 241000796533 Arna Species 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920000195 Bacterial small RNA Polymers 0.000 description 1
- 241000723736 Black beetle virus Species 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 101700001721 CT11 Proteins 0.000 description 1
- 101700000366 CT12 Proteins 0.000 description 1
- JHLNERQLKQQLRZ-UHFFFAOYSA-N Calcium silicate Chemical compound [Ca+2].[Ca+2].[O-][Si]([O-])([O-])[O-] JHLNERQLKQQLRZ-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 229940107161 Cholesterol Drugs 0.000 description 1
- 206010008943 Chronic leukaemia Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 229920002676 Complementary DNA Polymers 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-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
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-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
- CKLJMWTZIZZHCS-UHFFFAOYSA-N DL-aspartic acid Chemical compound OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- DYYUXMKNXUZBMO-UHFFFAOYSA-N DND-99 dye Chemical compound C1=CC(CCC(=O)NCCN(C)C)=[N+]([B-](N23)(F)F)C1=CC2=CC=C3C1=CC=CN1 DYYUXMKNXUZBMO-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N Deoxyribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- 229960004679 Doxorubicin Drugs 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 210000002472 Endoplasmic Reticulum Anatomy 0.000 description 1
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 1
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 1
- 241001522878 Escherichia coli B Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
- 230000036081 Excretion rate Effects 0.000 description 1
- 101710003435 FCGRT Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 229920002024 GDNA Polymers 0.000 description 1
- 102200037379 GHRL Q90L Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 208000005017 Glioblastoma Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101710014266 Gzmf Proteins 0.000 description 1
- 102200107225 HLA-A T97I Human genes 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 206010073071 Hepatocellular carcinoma Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 229940088597 Hormone Drugs 0.000 description 1
- 108010064750 Humanized Monoclonal Antibodies Proteins 0.000 description 1
- 102000015434 Humanized Monoclonal Antibodies Human genes 0.000 description 1
- 229940072221 IMMUNOGLOBULINS Drugs 0.000 description 1
- 108010047852 Integrin alphaVbeta3 Proteins 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N Iron(III) oxide Chemical compound O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 102200006532 KRAS G13D Human genes 0.000 description 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- GUBGYTABKSRVRQ-UUNJERMWSA-N Lactose Natural products O([C@@H]1[C@H](O)[C@H](O)[C@H](O)O[C@@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 GUBGYTABKSRVRQ-UUNJERMWSA-N 0.000 description 1
- 229920000126 Latex Polymers 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 102100011960 MIR7-3HG Human genes 0.000 description 1
- 101710008473 MIR7-3HG Proteins 0.000 description 1
- 102200085547 MNDA Y58D Human genes 0.000 description 1
- 206010025650 Malignant melanoma Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108020004388 MicroRNAs Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 208000002154 Non-Small-Cell Lung Carcinoma Diseases 0.000 description 1
- 108009000071 Non-small cell lung cancer Proteins 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010025310 Other lymphomas Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 210000001672 Ovary Anatomy 0.000 description 1
- 102220397730 PBX3 M95I Human genes 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 208000008443 Pancreatic Carcinoma Diseases 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- UNJJBGNPUUVVFQ-ZJUUUORDSA-N Phosphatidylserine Chemical compound CCCC(=O)O[C@H](COC(=O)CC)COP(O)(=O)OC[C@H](N)C(O)=O UNJJBGNPUUVVFQ-ZJUUUORDSA-N 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N Propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 102000006270 Proton Pumps Human genes 0.000 description 1
- 108010083204 Proton Pumps Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 230000025458 RNA interference Effects 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010038038 Rectal cancer Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 229920001914 Ribonucleotide Polymers 0.000 description 1
- 206010061934 Salivary gland cancer Diseases 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 229940098362 Serratia marcescens Drugs 0.000 description 1
- 208000000587 Small Cell Lung Carcinoma Diseases 0.000 description 1
- 108020004688 Small Nuclear RNA Proteins 0.000 description 1
- 108020003224 Small Nucleolar RNA Proteins 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 206010041823 Squamous cell carcinoma Diseases 0.000 description 1
- 206010041826 Squamous cell carcinoma of lung Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 210000002784 Stomach Anatomy 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 1
- 229920001949 Transfer RNA Polymers 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 206010046431 Urethral cancer Diseases 0.000 description 1
- 206010046766 Uterine cancer Diseases 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- 230000001464 adherent Effects 0.000 description 1
- 201000005188 adrenal gland cancer Diseases 0.000 description 1
- 230000001058 adult Effects 0.000 description 1
- 150000001295 alanines Chemical class 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminum Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000031016 anaphase Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003302 anti-idiotype Effects 0.000 description 1
- 239000000611 antibody drug conjugate Substances 0.000 description 1
- 108091008116 antibody drug conjugates Proteins 0.000 description 1
- 230000000890 antigenic Effects 0.000 description 1
- 201000011165 anus cancer Diseases 0.000 description 1
- 230000000975 bioactive Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000003139 buffering Effects 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L cacl2 Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003247 decreasing Effects 0.000 description 1
- 230000000593 degrading Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 150000002019 disulfides Chemical class 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N edta Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000003511 endothelial Effects 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000001747 exhibiting Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000037320 fibronectin Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 230000002496 gastric Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000003899 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 230000003301 hydrolyzing Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000000968 intestinal Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229910000460 iron oxide Inorganic materials 0.000 description 1
- 125000000012 isoleucine group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N 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
- 239000004816 latex Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 201000005249 lung adenocarcinoma Diseases 0.000 description 1
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated Effects 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 229920001239 microRNA Polymers 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000007758 minimum essential media Substances 0.000 description 1
- 102000035365 modified proteins Human genes 0.000 description 1
- 108091005569 modified proteins Proteins 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 108010068617 neonatal Fc receptor Proteins 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 201000002575 ocular melanoma Diseases 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 150000002902 organometallic compounds Chemical class 0.000 description 1
- 230000003204 osmotic Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000000849 parathyroid Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological Effects 0.000 description 1
- 230000004963 pathophysiological condition Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000000546 pharmaceutic aid Substances 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000002285 radioactive Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 229920002973 ribosomal RNA Polymers 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 230000002987 rna-interference Effects 0.000 description 1
- 102220288199 rs201175894 Human genes 0.000 description 1
- 102220288200 rs201175894 Human genes 0.000 description 1
- 230000003248 secreting Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 108091006008 signal transducing proteins Proteins 0.000 description 1
- 102000034377 signal transducing proteins Human genes 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 201000003708 skin melanoma Diseases 0.000 description 1
- 201000002314 small intestine cancer Diseases 0.000 description 1
- 229920001255 small nuclear ribonucleic acid Polymers 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 108010018381 streptavidin-binding peptide Proteins 0.000 description 1
- 238000000547 structure data Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 210000001519 tissues Anatomy 0.000 description 1
- 230000000699 topical Effects 0.000 description 1
- 230000002588 toxic Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 102000003995 transcription factors Human genes 0.000 description 1
- 108090000464 transcription factors Proteins 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 102000035402 transmembrane proteins Human genes 0.000 description 1
- 108091005683 transmembrane proteins Proteins 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 201000005102 vulva cancer Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-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 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
- C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
-
- 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
- C07K2317/522—CH1 domain
-
- 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
- C07K2317/524—CH2 domain
-
- 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
- C07K2317/526—CH3 domain
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/80—Immunoglobulins specific features remaining in the (producing) cell, i.e. intracellular antibodies or intrabodies
- C07K2317/82—Immunoglobulins specific features remaining in the (producing) cell, i.e. intracellular antibodies or intrabodies functional in the cytoplasm, the inner aspect of the cell membrane, the nucleus or the mitochondria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Abstract
The present invention relates to a cytosol-penetrating antibody comprising an endosomal escape motif is in a CD3 and a use thereof, and specifically, to a light chain variable region and/or heavy chain variable region comprising a endosomal escape motif is in a CDR3 positioned in a cytosol, a cytosol-penetrating antibody containing same, a method for producing same, and a use thereof, wherein the light chain variable region and/or heavy chain variable region exhibit notably enhanced endosomal escape efficiency as a result of an investigation of a structural mechanism for inducing endosomal escape which is the escape from an endosome to a cytosol after cellular internalization through a membrane protein of a cell membrane of a living cell. ol-penetrating antibody containing same, a method for producing same, and a use thereof, wherein the light chain variable region and/or heavy chain variable region exhibit notably enhanced endosomal escape efficiency as a result of an investigation of a structural mechanism for inducing endosomal escape which is the escape from an endosome to a cytosol after cellular internalization through a membrane protein of a cell membrane of a living cell.
Description
CYTOSOL-PENETRATING ANTIBODY AND USE THEREOF
TECHNICAL FIELD
The present disclosure relates to a cytosol-penetrating
antibody and the use thereof, and more particularly to an
endosomal escape motif which can increase the endosomal
escape efficacy of a cytosol-penetrating antibody
(Cytotransmab) so as to have a significantly improved ability
to escape from endosomes into the cytosol after cellular
internalization into living cells through a cell membrane
protein, a light-chain variable region and/or heavy-chain
variable region comprising the same, a cytosol-penetrating
antibody comprising the same, a method for producing the same,
and the use thereof.
BACKGROUND ART
General antibodies and macromolecular bio-drugs have
limitations in that they cannot pass the hydrophobic cell
membrane, and thus cannot bind to and inhibit various
disease-related substances. In addition, conventional
antibodies cannot directly penetrate living cells due to
their large size and hydrophilic nature.
Thus, most conventional antibodies specifically target
extracellularly secreted proteins or cell membrane proteins.
Further, generally, commercial antibodies binding
specifically to intracellular substances which are used in
experiments for studies on mechanisms such as the growth,
specific inhibition, etc. of cells, cannot be used directly
to treat living cells, and in order for these antibodies to
bind to intracellular substances, a pretreatment process for
forming pores in the cell membrane by a cell membrane
permeabilization process using the amphipathic glycoside
saponin is necessarily required.
A number of therapeutic antibodies that target cell
membrane proteins or extracellularly secreted proteins due to
their property of binding to target proteins with high
specificity and high affinity have been developed. Antibodies
that target cell membrane proteins can bind to cell membrane
proteins, and then enter the cells via an endosomal pathway
through a receptor-mediated endocytosis process.
This process includes various pathways after the early
endosome stage. That is, 1) most antibodies can be
transported from early endosomes to late endosomes and
lysosomes, and can be completely degraded by proteases under
acidic conditions; and 2) some antibodies can bind to FcRn
(neonatal Fc receptor) in early endosomes under acidic
conditions and come out of the cells through the recycling
endosome pathway.
Thus, most antibodies bind strongly to the target
membrane proteins and are mostly degraded through the
lysosomal pathway. In the endosomal pathway, endosomes are
matured while the inside thereof is gradually acidified by
proton pumps. It is known that the pH of early endosomes is
about 5.5-6.5, the pH of late endosomes is about 4.5-5.5, and
the pH of lysosomes is about pH 3.5-4.5 (Quadir MA et al.,
2014; Li S et al., 2014). Many proteinases in endosomes are
activated, and endocytosed proteins are degraded in endosomes.
Consequently, when antibodies move through the endosomal
pathway after receptor-mediated endocytosis, they should be
separated from the target membrane proteins and form pores in
endosomes in order to escape from early or late endosomes
into the cytosol before trafficking to lysosomes.
Among naturally occurring intracellular substances,
viruses and toxins are known to actively penetrate living
cells through endocytosis. “Endosomal escape”, a process of
escaping from endosomes into the cytosol, is essential so
that a substance that penetrated into cells by endocytosis
exhibits activity in the cytosol.
Although the endosomal escape mechanism has not yet been
clearly found, three hypotheses for the endosomal escape are
known to date.
The first hypothesis is a mechanism by which a pore is
formed in the endosomal membrane. In this hypothesis,
substances such as cationic amphiphilic peptides in the
endosomal membrane bind to a negatively charged cellular
lipid bilayer to cause internal stress or inner membrane
contraction to thereby form a barrel-stave pore or a toroidal
channel (Jenssen et al., 2006), which is called a pore
formation mechanism.
The second hypothesis is a mechanism by which the
endosome bursts as a consequence of the proton-sponge effect.
In this hypothesis, due to the high buffering effect of a
substance having a protonated amino group, the osmotic
pressure of the endosome can be increased so that the
endosomal membrane can be degraded (Lin and Engbersen, 2008).
In the third hypothesis, a specific motif, which
maintains a hydrophilic coil shape in a neutral environment
but is changed into a hydrophobic helical structure in an
acidic environment such as endosome, is fused to the
endosomal membrane so that viruses and toxins including a
motiff escape from the endosome, which is called a lipid
membrane fusion mechanism. These three hypothese has been
proposed as endosome escape mechanisms after endocytosis of a
viral protein and a toxic protein derived from
plants/bacteria, but such endosome escape mechanism in
antibodies has not been specifically identified yet.
The common phenomenon observed in the above-described
endosomal escape mechanism is that endosomal escape occurs
under acidic pH conditions which are endosomal and lysosomal
environments. Proteins whose function changes depending on pH
have the property of changing their structure depending on pH.
Negatively charged amino acids (aspartic acid (D) and glutamic
acid (E)) and hydrophobic amino acids (methionine (M), leucine
(L), and isoleucine (I)) do not interact under neutral pH
conditions. However, as pH decreases, the carboxylic acids
(COO-) in the side chains of the negatively charged amino acids
become hydrophobic by protonation (Korte et al., 1992), and then
can hydrophobically interact with the surrounding hydrophobic
amino acids. As a result, the distance between the two amino
acids becomes closer, and the overall structure and function of
the protein change. The phenomenon that causes this change is
known as the Tanford transition (Qin et al., 1998).
As one example, nitrophorin 4, a nitrogen transporting
enzyme, has an open structure under neutral pH conditions.
However, as the pH decreases from neutral pH (pH 7.4) to weakly
acidic pH (pH 6.0), the structure of nitrophorin 4 changes to a
closed structure by the hydrophobic interaction of aspartic acid
and leucine, and thus nitrophorin 4 functions to transport
nitrogen (Di Russo et al., 2012).
However, this pH-dependent structural change has not yet
been found in antibodies. In particular, this change has not
yet been observed in antibodies that undergo endocytosis.
As one example, an antibody engineering improvement
technology for inducing pH-dependent antigen binding among
conventional antibody technologies is a method of screening
pH-dependent antigen-binding antibodies from libraries
introduced either with histidine (H) of CDRs (complementary
determining regions) or with random mutations including
histidine (Bonvin P et al., 2015). However, the two methods
all cause no structural change, and have a limitation in that
library-based screening should be performed in order to
induce pH-dependent antigen binding.
In order to increase the effect of a substance that
exhibits its activity in the cytosol, the amount of the
substance located in the cytosol should ultimately increase.
Hence, studies have been conducted to increase the endosomal
escape ability. Such studies have been conducted mainly on
cell-penetrating peptides (CPPs). Although it has been
reported that some cell-penetrating peptides localize in the
cytosol through an endosomal escape pathway, there is no
detailed study on an exact endosomal escape mechanism, and it
is known that only about 0.1 to 4% of endocytosed peptides
localize to the cytosol due to very low endosomal escape
efficiency.
Under this technical background, the present inventors
have identified an endosomal escape motif capable of
increasing the endosomal escape efficacy of a cytosol-
penetrating antibody (Cytotransmab) that penetrates cells and
localizes in the cytosol (Choi et al., 2014), and have found
that it is possible to develop a light-chain or heavy-chain
variable region including an endosomal escape motif having an
increased ability to escape from endosomes, and an antibody
or an antigen-binding fragment thereof comprising the same.
In addition, the present inventors have found that a
cytosol-penetrating antibody having endosomal escape ability
can be produced by grafting this endosomal escape motif into
other kinds of light-chain or heavy-chain variable regions,
thereby completing the present disclosure.
The information disclosed in the Background Art section
is only for the enhancement of understanding of the
background of the present disclosure, and therefore may not
contain information that forms a prior art that would already
be known to a person of ordinary skill in the art.
DISCLOSURE OF INVENTION
TECHNICAL PROBLEM
It is an object of the present disclosure to provide a
cytosol-penetrating antibody having endosome escape ability
or an antigen-binding fragment thereof.
Another object of the present disclosure is to provide a
nucleic acid encoding the antibody or antigen-binding
fragment thereof.
Still another object of the present disclosure is to
provide a vector comprising the above-described nucleic acid,
a cell transformed with the above-described vector, and a
method of producing the above-described antibody or antigen-
binding fragment thereof.
Yet another object of the present disclosure is to
provide an antibody-drug conjugate comprising the above-
described antibody or antigen-binding fragment thereof.
A further object of the present disclosure is to provide
a composition for delivering an active substance into cytosol,
comprising the above-described cytosol-penetrating antibody
or antigen-binding fragment thereof.
A still further object of the present disclosure is to
provide a method for producing the above-described cytosol-
penetrating antibody or antigen-binding fragment thereof.
TECHNICAL SOLUTION
To achieve the above object, the present disclosure
provides a cytosol-penetrating antibody or an antigen-binding
fragment thereof comprising a light-chain variable region
and/or heavy-chain variable region that comprises a sequence
represented by the following formula in its CDR3:
X1-X2-X3-Z1
wherein X1-X2-X3 is an endosomal escape motif, and each
of X1, X2 and X3 is selected from the group consisting of
tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine
(F);
Z1 is selected from the group consisting of methionine (M),
isoleucine (I), leucine (L), histidine (H), aspartic acid (D),
and glutamic acid (E);
the light-chain variable region and/or heavy-chain variable
region comprising Z1 induces a change in properties of the
antibody under endosomal acidic pH conditions; and
the antibody exhibits an ability to escape from endosomes
into the cytosol through the change in properties of the antibody.
In the cytosol-penetrating antibody or antigen-binding
fragment thereof according to the present disclosure, the first
amino acid of the light-chain variable region and/or heavy-chain
variable region may be aspartic acid (D) or glutamic acid (E).
The present disclosure also provides a nucleic acid
encoding the above-described cytosol-penetrating antibody or
antigen-binding fragment thereof.
The present disclosure also provides a vector comprising
the above-described nucleic acid.
The present disclosure also provides a cell transformed
with the above-described vector.
The present disclosure also provides a composition for
delivering an active substance into cytosol, comprising the
above-described cytosol-penetrating antibody or antigen-
binding fragment thereof.
The present disclosure also provides a method for
producing the above-described cytosol-penetrating antibody or
antigen-binding fragment thereof, the method comprising a step
of grafting the endosomal escape motif X1-X2-X3-Z1 (wherein
X1-X2-X3 is selected from the group consisting of tryptophan
(W), tyrosine (Y), histidine (H), and phenylalanine (F)) into
the CDR3 of a light-chain and/or heavy-chain variable region.
ADVANTAGEOUS EFFECTS
The cytosol-penetrating antibody or antigen-binding
fragment thereof comprising the light-chain variable region
and/or heavy-chain variable region comprising the endosomal
escape motif according to the present disclosure penetrates
living cells and localizes in the cytosol, and ultimately the
antibody or antigen-binding fragment thereof can be penetrate
living cells and localize in the cytosol without having to use
a special external protein delivery system.
The cytosol-penetrating antibody or antigen-binding
fragment thereof according to the present disclosure is a
cytosol-penetrating antibody or antigen-binding fragment
thereof comprising a light-chain variable region or a heavy-
chain variable region that easily interacts with and binds to
various human light-chain variable regions or heavy-chain
variable regions (VHs), and has the ability to escape from
endosomes into the cytosol. The antibody or antigen-binding
fragment thereof can penetrate cells and localize in the
cytosol, and does not show non-specific cytotoxicity for
target cells.
Based on the endosomal escape mechanism for the high
efficiency cytosol-penetrating antibody or an antigen-binding
fragment thereof according to the present disclosure, a design
of antibody libraries for improving the endosomal escape
ability and mutants can be performed.
The endosomal escape motif included in the cytosol-
penetrating antibody or antigen-binding fragment thereof
according to the present disclosure is introduced into other
antibodies so that it can be expected to impart the endosomal
escape ability.
In addition, the cytosol-penetrating antibody or
antigen-binding fragment thereof according to the present
disclosure can be utilized as a carrier that delivers an
active substance into the cytosol of a living cell, and can
also be utilized as a pharmaceutical composition for treatment
and prevention of diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
shows of a pulse-chase experiment and confocal
microscopy observation performed to observe the transport
process and stability of the cytosol-penetrating antibody
(cytotransmab) TMab4 or cell-penetrating peptide TAT
introduced into cells.
shows the results of confocal microscopy
observation of the cytosol-penetrating ability of the
cytosol-penetrating antibody TMab4 or the cell-penetrating
peptide TAT in the presence or absence of an inhibitor
thereof.
is a bar graph showing the results of
quantifying the FITC (green fluorescence) fluorescence of the
confocal micrographs shown in .
shows the results of observing the cytosolic
localization of the cytosol-penetrating antibody TMab4 or the
cell-penetrating peptide TAT by confocal microscopy using
calcein in the presence or absence of an inhibitor thereof.
is a bar graph showing the results of
quantifying the calcein fluorescence of the confocal
micrographs shown in .
shows the results of Western blot analysis
performed to confirm siRNA (short interfering RNA)-induced
inhibition of heparanase expression.
shows the results of confocal microscopy
observation of cytosol penetrating antibody/lysosome merging
caused by inhibition of heparanase expression.
shows the results of confocal microscopy
observation performed to confirm the cytosolic localization
of a cytosol-penetrating antibody, which is caused by
inhibition of heparanase expression.
is a schematic view showing an overall
trafficking process ranging from cellular internalization of
a cytosol-penetrating antibody to localization of the
antibody in the cytosol.
shows the results of observing a fluorescence-
labeled cytosol-penetrating antibody in Ramos cells by
confocal microscopy in order to examine whether the antibody
can be introduced through the cell membrane depending on pH
or whether the antibody can induce cell membrane permeation
of other substances.
shows the results of observing Ramos cells by an
optical microscope in order to examine whether a cytosol-
penetrating antibody can form pores and take up trypan blue
having no membrane-permeating ability, depending on pH.
is a graph quantitatively comparing the number
of cells that have taken up trypan blue.
FIG 7a shows the results of optical microscopic
observation performed to confirm whether cell membrane pores
produced by a cytosol-penetrating antibody at pH 5.5 is
temporary and reversible.
is a graph quantitatively comparing the number of
cells that have taken up trypan blue uptake.
shows the results of analyzing the cell membrane
binding of a cytosol-penetrating antibody and control antibody
adalimumab by flow cytometry (FACS) at varying pHs.
shows the results of analyzing the cell membrane
flip-flop inducing abilities of a cytosol-penetrating antibody
and control antibody adalimumab by flow cytometry (FACS) at
varying pHs.
is a schematic view showing a pore formation model
of a cytosol-penetrating antibody, expected based on the above-
described experiments.
shows the results of predicting the pH-dependent
structural change of a cytosol-penetrating antibody on the basis
of the WAM modeling structure of the light-chain variable region
of the cytosol-penetrating antibody, and shows amino acids,
which are involved in the structural change, and amino acids
which are exposed by the structural change.
is a graph quantitatively comparing the number of
cells that have taken up trypan blue depending on pH by
mutants constructed by substituting the 1 amino acid aspartic
acid and 95 amino acid methionine of a light-chain variable
region (VL), which induce a change in
properties of a cytosol-penetrating antibody at acidic pH,
with alanine, glutamic acid and leucine, respectively.
is a graph quantitatively comparing the number
of cells that have taken up trypan blue depending on pH by
mutants constructed by substituting particular amino acids of
the CDR3 of the light-chain variable region (VL) of a
cytosol-penetrating antibody, which can possibly be involved
in endosomal escape, with alanine.
a shows the results of confocal microscopy
performed to analyze the cytosol-penetrating ability of
mutants constructed by substituting the CDR1 and CDR2 of the
light-chain variable region (VL) of a cytosol-penetrating
antibody, which bind to HSPG receptor and are involved in
cytosol-penetrating ability, with human germline sequences.
b shows a graph quantitatively comparing the
number of cells that have taken up trypan blue depending on
pH by mutants constructed by substituting the CDR1 and CDR2
of the light-chain variable region (VL) of a cytosol-
penetrating antibody, which bind to HSPG receptor and are
involved in cytosol-penetrating ability, with human germline
sequences.
a shows the results of 12% SDS-PAGE analysis
under reducing or non-reducing conditions after purification
of cytosol-penetrating antibody mutants expected to have
improved endosomal escape ability.
b shows the results of confocal microscopy
performed to examine whether the cytosol-penetrating ability
of cytosol-penetrating antibody mutants expected to have
improved endosomal escape ability is maintained.
is a graph quantitatively comparing the number
of cells that have taken up trypan blue depending on pH by a
cytosol-penetrating antibody wild-type and cytosol-
penetrating antibody mutants expected to have improved
endosomal escape ability.
a shows the results of observing the cytosolic
localization of a cytosol-penetrating antibody wild-type and
cytosol-penetrating antibody mutants expected to have
improved endosomal escape ability, by confocal microscopy
using calcein.
b is a bar graph showing the results of
quantifying the calcein fluorescence of the confocal
micrographs shown in a.
is a schematic view showing a process in which
GFP fluorescence by enhanced split-GFP complementation is
observed when a cytosol-penetrating antibody wild-type and a
mutant having improved endosomal escape ability localizes in
the cytosol.
shows the results of 12% SDS-PAGE analysis under
reducing or non-reducing conditions after purification of a
GFP11-SBP2-fused cytosol-penetrating antibody wild-type and a
GFP11-SBP2-fused mutant having improved endosomal escape
ability.
a shows the results of confocal microscopy
performed to examine the GFP fluorescence of a GFP11-SBP2-
fused cytosol-penetrating antibody wild-type and a GFP11-
SBP2-fused mutant having improved endosomal escape ability by
enhanced split-GFP complementation.
b is a graph showing the results of quantifying
the GFP fluorescence of the confocal micrographs shown in 20a.
a is a graph showing the results of flow
cytometry (FACS) performed to analyze the cell membrane
binding of mutants obtained by substitution with arginine,
isoleucine and glycine, which are amino acids having
properties opposite to those of tryptophan.
b is a graph quantitatively comparing the number
of cells that have taken up trypan blue depending on pH by
mutants obtained by substitution with arginine, isoleucine
and glycine, which are amino acids having properties opposite
to those of tryptophan.
c is a bar graph showing the results of observing
the cytosolic localization of mutants obtained by
substitution with arginine, isoleucine and glycine, which are
amino acids having properties opposite to those of tryptophan
by confocal microscopy using calcein and quantifying the
calcein fluorescence of the confocal micrographs.
a is a schematic view showing a process of
constructing an intact IgG-format anti-tubulin cytosol-
penetrating antibody to be used to examine the activity of
cytosol-penetrating antibody mutants having improved
endosomal escape ability.
b shows the results of 12% SDS-PAGE analysis
under reducing or non-reducing conditions after purification
of an intact IgG-format anti-tubulin cytosol-penetrating
antibody.
c shows the results of confocal microscopy
performed to examine whether an intact IgG-format anti-
tubulin cytosol-penetrating antibody would merge with
cytoskeletal tubulin localized in the cytosol.
a is a schematic view showing a process of
constructing an intact IgG-format RAS-targeting cytosol-
penetrating antibody to be used to examine the activity of
mutants having improved endosomal escape ability.
b shows the results of 12% SDS-PAGE analysis
under reducing or non-reducing conditions after purification
of intact IgG-format RAS-targeting cytosol-penetrating
antibodies.
c shows the results of enzyme linked
immunosorbent assay performed to measure the affinities of
antibodies for GppNHp-bound K-RAS G12D and GDP-bound K-RAS G12D,
which are K-RAS mutants.
shows the results of confocal microscopy
observation performed to examine whether intact IgG-format RAS-
targeting cytosol-penetrating antibodies would merge with
intracellular H-RAS G12V mutants.
a is a graph showing the results of quantitatively
comparing the number of cells that have taken up trypan blue
depending on pH by mutants constructed by substituting the 1
amino acid aspartic acid of the light-chain variable region (VL)
of a cytosol-penetrating antibody, which induce a change in
properties of the cytosol-penetrating antibody at acidic pH 5.5,
with various amino acids.
b is a graph showing the results of quantitatively
comparing the number of cells that have taken up trypan blue
depending on pH by mutants constructed by substituting 95 amino
acid methionine of the light-chain variable region (VL) of a
cytosol-penetrating antibody, which induce a change in
properties of the cytosol-penetrating antibody at acidic pH 5.5,
with various amino acids.
a shows a graph showing quantitatively comparing
the number of cells that have taken up trypan blue depending on
pH by mutants designed for the purpose of inducing an additional
change in properties in response to pH.
b shows a bar graph showing the results of
observing the cytosolic localization of mutants designed for
the purpose of inducing an additional change in properties in
response to pH by confocal microscopy using calcein and
quantifying the calcein fluorescence of the confocal
micrographs.
is a graph quantitatively comparing the number
of cells that taken up trypan blue depending on pH by mutants
obtained by changing the amino acid number of the CDR3 of the
light-chain variable region of a cytosol-penetrating antibody.
a shows a process of constructing an intact IgG-
format RAS-targeting cytosol-penetrating antibody in which an
improved endosomal escape motif is introduced into the light-
chain variable region of a conventional therapeutic antibody.
b shows the results of fluorescence microscopic
observation performed to examine whether the HSPG binding
affinity and cytosol-penetrating ability of an intact IgG-
format RAS-targeting cytosol-penetrating antibody in which an
improved endosomal escape motif is introduced into the light-
chain variable region of a conventional therapeutic antibody
would be reduced or eliminated.
c shows a graph quantitatively comparing the
number of cells that taken up trypan blue at acidic pH by an
intact IgG-format RAS-targeting cytosol-penetrating antibody
in which an improved endosomal escape motif is introduced
into the light-chain variable region of a conventional
therapeutic antibody.
a shows the results of ELISA performed to measure
the affinities of an intact IgG-format RAS-targeting cytosol-
penetrating antibody, in which an improved endosomal escape
motif is introduced into the light-chain variable region of a
conventional therapeutic antibody, for GppNHp-bound K-RAS
G12D and GDP-bound K-RAS G12D, which are K-RAS mutants.
b shows a schematic view showing a process of
constructing an intact IgG-format RAS-targeting cytosol-
penetrating antibody in which an improved endosomal escape
motif is introduced into the RGD10 peptide-fused light-chain
variable region of a conventional therapeutic antibody.
c shows the results of confocal microscopy
performed to examine whether an intact IgG-format RAS-
targeting cytosol-penetrating antibody in which an improved
endosomal escape motif is introduced into the RGD10 peptide-
fused light-chain variable region of a conventional
therapeutic antibody would merge with intracellular activated
H-RAS G12V mutants.
a shows a process of constructing a cytosol-
penetrating antibody having a light-chain variable region
from which endosomal escape ability has been removed and a
heavy-chain variable region into which an improved endosomal
escape motif has been introduced.
b shows a graph quantitatively comparing the
number of cells that have taken up trypan blue depending on
pH by a cytosol-penetrating antibody having a light-chain
variable region from which endosomal escape ability is
removed and a heavy-chain variable region into which an
improved endosomal escape motif is introduced.
c shows the results of confocal microscopy
performed to observe the GFP fluorescence by enhanced split-
GFP complementation of a GFP11-SBP2-fused cytosol-penetrating
antibody having a light-chain variable region from which
endosomal escape ability has been removed, and a heavy-chain
variable region into which an improved endosomal escape motif
has been introduced.
d shows the results of confocal microscopy
performed using calcein in order to observe the cytosolic
localization of a cytosol-penetrating antibody having a
light-chain variable region from which endosomal escape
ability has been removed and a heavy-chain variable region
into which an improved endosomal escape motif has been
introduced.
a is a graph quantitatively comparing the number
of cells that taken up trypan blue depending on pH by mutants
constructed by substituting the 1 amino acid glutamic acid
of the heavy-chain variable region (VH) of a cytosol-
penetrating antibody, which induces a change in properties of
the antibody at acidic pH 5.5, with various amino acids.
b is a graph quantitatively comparing the number
of cells that taken up trypan blue depending on pH by mutants
constructed by substituting 102 amino acid leucine of the
heavy-chain variable region (VH) of a cytosol-penetrating
antibody, which induces a change in properties of the
antibody at acidic pH 5.5, with various amino acids.
a shows a graph quantitatively comparing the
number of cells that have taken up trypan blue depending on
pH by intact IgG-format cytosol-penetrating antibodies having
a light-chain variable region and/or a heavy-chain variable
region introduced with an endosomal escape motif having three
tryptophan residues.
b shows a bar graph showing the results of
observing the cytosolic localization of intact IgG-format
cytosol-penetrating antibodies having a light-chain variable
region and/or a heavy-chain variable region introduced with
an endosomal escape motif having three tryptophan residues by
confocal microscopy using calcein and quantifying the calcein
fluorescence of the confocal micrographs.
a shows a schematic view showing a process of
constructing an intact IgG-format cytosol-penetrating
antibody in which an improved endosomal escape motif has been
introduced into a heavy-chain variable region thereof and an
improved endosomal escape motif has been introduced into a
light-chain variable region of a conventional therapeutic
antibody fused with an EpCAM-targeting peptide.
b shows a bar graph showing the results of
observing the cytosolic localization of an intact IgG-format
cytosol-penetrating antibody, in which an improved endosomal
escape motif has been introduced into a heavy-chain variable
region thereof and an improved endosomal escape motif has
been introduced into a light-chain variable region of a
conventional therapeutic antibody fused with an EpCAM-
targeting peptide, by confocal microscopy using calcein and
quantifying the calcein fluorescence of the confocal
micrographs.
c shows a graph quantitatively comparing the
number of cells that have taken up trypan blue depending on
pH by an intact IgG-format cytosol-penetrating antibody in
which an improved endosomal escape motif has been introduced
into a heavy-chain variable region thereof and an improved
endosomal escape motif has been introduced into a light-chain
variable region of a conventional therapeutic antibody fused
with an EpCAM-targeting peptide.
a is a schematic view showing a process of
constructing an intact IgG-format cytosol-penetrating
antibody in which an improved endosomal escape motif has been
introduced into the heavy-chain variable region of a
conventional therapeutic antibody.
b is a graph quantitatively comparing the number of
cells that have taken up trypan blue depending on pH by an intact
IgG-format cytosol-penetrating antibody in which an improved
endosomal escape motif has been introduced into the heavy-chain
variable region of a conventional therapeutic antibody.
is a graph quantitatively comparing the number of
cells that have taken up trypan blue depending on pH by an intact
IgG-format cytosol-penetrating antibody comprising a light-
chain variable region and/or a heavy-chain variable region
introduced with aspartic acid.
a shows the results of observing a crystal of CT-
59 Fab, formed under Index G1 conditions, by RI1000 (Rock
Imager1000; automatic protein crystal image analysis system).
b shows the three-dimensional structure of CT-59
refined and validated using the pymol program. The 1 amino acid
aspartic acid (D) and 95 amino acid methionine (M) are shown
nd th
in yellow, and the 92 to 94 amino acids are shown in orange.
BEST MODE FOR CARRYING OUT THE INVENTION
Unless defined otherwise, all the technical and scientific
terms used herein have the same meaning as those generally
understood by one of ordinary skill in the art to which the
invention pertains. Generally, the nomenclature used herein and
the experiment methods, which will be described below, are those
well known and commonly employed in the art.
In one aspect, the present disclosure is directed to a
cytosol-penetrating antibody or an antigen-binding fragment
thereof comprising a light-chain variable region and/or heavy-
chain variable region that comprises a sequence represented by
the following formula in its CDR3:
X1-X2-X3-Z1
wherein X1-X2-X3 is an endosomal escape motif, and each of
X1, X2 and X3 is selected from the group consisting of tryptophan
(W), tyrosine (Y), histidine (H) and phenylalanine (F);
Z1 is selected from the group consisting of methionine (M),
isoleucine (I), leucine (L), histidine (H), aspartic acid (D),
and glutamic acid (E);
the light-chain variable region and/or heavy-chain variable
region comprising Z1 induces a change in properties of the
antibody under endosomal acidic pH conditions; and
the antibody exhibits an ability to escape from endosomes
into the cytosol through the change in properties of the antibody.
"Endosomal escape" in the present disclosure may mean
actively penetrating living cells by endocytosis, and then
escaping from endosomes into the cytosol under acidic conditions.
"Endosomal escape motif" in the present disclosure
includes a one-dimensional structure comprising a specific amino
acid sequence having the property of inducing endosomal escape
under acidic conditions, and a three-dimensional structure
formed thereby. "Endosomal escape motif" may be used
interchangeably with "motif having endosomal escape ability".
An antibody comprising a light-chain variable region (VL) or
heavy-chain variable region (VH) that comprises an "endosomal
escape motif" is capable of "penetrating the cytosol". "Cytosol-
penetrating antibody" means that an antibody that penetrated
cells by endocytosis escapes from endosomes into the cytosol
under acidic conditions. "Cytosol-penetrating antibody" may be
used interchangeably with "antibody having cytosol-penetrating
ability".
In the present disclosure, Z1 included in the endosomal
escape motif X1-X2-X3-Z1 may be located at the 95 amino acid
of the light-chain variable region or the 102 amino acid of
the heavy-chain variable region, as numbered by the Kabat
numbering system, and is the hydrophobic amino acid methionine
(M), isoleucine (I) or leucine (L), the negatively charged amino
acid aspartic acid (D) or glutamic acid (E), or the positively
charged amino acid histidine (H). The 1 amino acid of the
light-chain variable region or heavy-chain variable region of
the cytosol-penetrating antibody according to the present
disclosure can interact with negatively charged aspartic acid
(D) or glutamic acid (E) and Z1, which is the 95 amino acid of
the light-chain variable region or the 102 amino acid of the
heavy-chain variable region, under endosomal acidic pH
conditions, thereby inducing a change in the properties of the
antibody and allowing the antibody to have the ability to escape
from endosomes into the cytosol.
In the present disclosure, the 1 amino acid of the light-
chain variable region and/or heavy-chain variable region of the
cytosol-penetrating antibody of the cytosol-penetrating
antibody or antigen-binding fragment thereof may interact with
Z1 under endosomal acidic pH conditions to induce a change in
properties of the cytosol-penetrating antibody.
In addition, as pH 7.4 changes to endosomal acidic pH 5.5,
the interaction between Z1 of the endosomal escape motif and the
1 amino acid of the light-chain variable region and/or heavy-
chain variable region changes. Namely, when Z1 is composed of
the hydrophobic amino acid methionine (M), isoleucine (I) or
leucine (L) or the negatively charged amino acid aspartic acid
(D) or glutamic acid (E), the carboxylic acid in the side chain
of the negatively charged amino acid becomes hydrophobic by
partial protonation under the acidic conditions, and thus Z1
hydrophobically interacts with aspartic acid (D) or glutamic
acid (E), which is the 1 amino acid of the light-chain variable
region or heavy-chain variable region.
In addition, regarding induction of a pH-dependent change
in properties of the antibody by interaction between Z1 of the
endosomal escape motif and the 1 amino acid of the light-chain
variable region or heavy-chain variable region, when Z1 is
composed of the hydrophobic amino acid methionine (M),
isoleucine (I) or leucine (L), it does not interact with the
negatively charged amino acid aspartic acid (D) or glutamic acid
(E), which is the 1 amino acid of the light-chain variable
region or heavy-chain variable region, under neutral pH
conditions. However, as pH decreases, the negatively charged
amino acid becomes hydrophobic by protonation, and thus
hydrophobically interacts with Z1. As a result, the distance
between the two amino acids becomes closer, thereby inducing a
change in the structure and function of the protein. This
phenomenon is known as the Tanford transition.
In addition, when Z1 is composed of histidine (H), as pH
changes from 7.4 to 5.5, the net charge of the amino acid side
chains becomes positive, and Z1 electrostatically interacts with
aspartic acid (D) or glutamic acid (E), which is the 1 amino
acid of the light-chain variable region or heavy-chain variable
region.
In an example of the present disclosure, in order to
confirm whether a pH-dependent change in the properties of the
st th
antibody is induced by a pair of the 1 and 95 amino acids of
the light-chain variable region, endosomal escape ability was
analyzed using alanine substitution mutants. As a result, the
alanine substitution mutations showed no pH-dependent endosomal
escape ability. In addition, endosomal escape ability was
analyzed using mutations obtained by substituting the 95 amino
acid with 20 different amino acids, and as a result, mutants in
which the 95 amino acid of the light-chain variable region of
the cytosol-penetrating antibody according to the present
disclosure is composed of methionine (M), leucine (L),
isoleucine (I), aspartic acid (D), glutamic acid (E) and
histidine (H) showed pH-dependent endosomal escape ability.
In an example of the present disclosure, regarding a pair
st th
of the 1 and 102 amino acids of the heavy-chain variable
region, which induces a pH-dependent change in the properties
of the antibody, which has been found through the alanine
substitution mutation experiment in the same manner as that in
the above example, endosomal escape ability was analyzed using
mutations obtained by substituting the 102 amino acid with 13
different amino acids, and as a result, mutants in which the
102 amino acid of the heavy-chain variable region of the
cytosol-penetrating antibody according to the present disclosure
is composed of methionine (M), leucine (L), isoleucine (I),
aspartic acid (D), glutamic acid (E) and histidine (H) showed
pH-dependent endosomal escape ability.
In addition, in one embodiment of the present disclosure,
the cytosol-penetrating antibody may further comprise, between
X3 and Z1, an amino acid sequence represented by (a1-...-
an)(where n is an integer ranging from 1 to 10). In one
embodiment of the present disclosure, when the cytosol-
penetrating antibody further comprises, between X3 and Z1, an
amino acid sequence represented by (a1-...-an)(where n is an
integer ranging from 1 to 10), a change in the properties of the
endosomal escape motif can be promoted while the length of the
CDR3 increases.
In the present disclosure, the endosomal escape motif has
a structure of X1-X2-X3-Z1 included in the light-chain variable
region; the heavy-chain variable region; or the light-chain
variable region and heavy-chain variable region, and each of X1,
X2 and X3 is selected from the group consisting of tryptophan
(W), tyrosine (Y), histidine (H) and phenylalanine (F).
In the present disclosure, the endosomal escape motif
X1-X2-X3 can react at intracellular endosomal weakly acidic
INTENTIONALLY LEFT BLANK
pH, for example, a pH of 5.5 to 6.5, which is early endosomal
pH, and thus Z1 can interact with the 1 amino acid of the
light-chain variable region or heavy-chain variable region,
thereby changing the properties of the antibody and
significantly increasing the endosomal escape efficiency of
the antibody.
In the present disclosure, the endosomal escape motif X1,
X2 and X3 are selected from the group consisting of amino
acids that easily interact with the hydrophilic head portion
and hydrophobic tail portion of 1-palmitoyloleoyl-sn-
glycerophosphatidylcholine (POPC) which is the major
phospholipid component of the inner endosomal membrane.
Specifically, the average binding activity of 20
different amino acids for 1-palmitoyloleoyl-sn-glycero
phosphatidylcholine (POPC) is higher in the order of
tryptophan (W), phenylalanine (F), tyrosine (Y), leucine (L),
isoleucine (I), cysteine (C), and methionine (M).
Specifically, the binding activity of 20 different amino
acids for the hydrophilic head portion of 1-palmitoyl
oleoyl-sn-glycerophosphatidylcholine (POPC) is higher in
the order of arginine (R), tryptophan (W), tyrosine (Y),
histidine (H), asparagine (N), glutamine (Q), lysine (K),
and phenylalanine (F). In addition, the binding activity of
different amino acids for the hydrophobic head portion of
1-palmitoyloleoyl-sn-glycerophosphatidylcholine (POPC)
is higher in the order of tryptophan (W), phenylalanine (F),
leucine (L), methionine (M), isoleucine (I), valine (V), and
tyrosine (Y).
In the present disclosure, amino acids constituting X1,
X2 and X3 of the endosomal escape motif may include tyrosine
(Y) and histidine (H), which constitute a wild-type cytosol-
penetrating antibody. Thus, these amino acids may include
tryptophan (W) and phenylalanine (F), which have a higher
average binding affinity than tyrosine (Y) for 1-palmitoyl
oleoyl-sn-glycerophosphatidylcholine (POPC).
In an example of the present disclosure, amino acids
that easily interact with 1-palmitoyloleoyl-sn-glycero
phosphatidylcholine (POPC) was examined through literature
search. Furthermore, tryptophan (W) having high binding
affinity for the hydrophilic head portion and hydrophobic
tail portion was introduced into X1, X2 and X3 of the
endosomal escape motif, and endosomal escape ability was
analyzed. As a result, an improved cytosol-penetrating
antibody according to the present disclosure showed a higher
pH-dependent endosomal escape ability than the wild-type
cytosol-penetrating antibody including tyrosine (Y), tyrosine
(Y) and histidine (H) in X1, X2 and X3, respectively.
In another example, in order to examine whether
interaction with the head portion or tail portion of 1-
palmitoyloleoyl-sn-glycerophosphatidylcholine (POPC) is
important for endosomal escape, mutants were constructed by
introducing arginine (R) which easily binds only to the head
portion, isoleucine (I) which easily binds only to the tail
portion, and glycine (G) which shows significantly low
interaction with the lipid, into X1, X2 and X3 of the
endosomal escape motif, and endosomal escape ability was
analyzed. As a result, two mutants, excluding a cytosol-
penetrating antibody introduced with tryptophan (W) according
to the present disclosure, all showed significantly reduced
endosomal escape ability. This suggests that interactions
with the hydrophilic head and hydrophobic tail of the lipid
are all involved in endosomal escape.
In still another example, the endosomal escape motif of
the light-chain variable region may comprise one or more
tryptophans, or one or two tryptophans.
In order to increase the effect of a substance that
exhibits its activity in the cytosol, the amount of the
substance located in the cytosol should ultimately increase.
Hence, studies have been conducted to increase endosomal
escape ability. Such studies have been conducted mainly on
cell-penetrating peptides (CPPs). In particular, interaction
with the lipid membrane is essential for passage through the
cell lipid membrane, a strategy for enhancing this
interaction has been introduced. As one example, tryptophan
was added to the N-terminus of a cytosol-penetrating peptide
rich in arginine and to the middle portion of the peptide.
However, this approach has not been attempted on
antibodies. Tryptophan (W) is an amino acid showing high
interaction with the hydrophilic head portion and hydrophobic
tail portion of 1-palmitoyloleoyl-sn-glycero
phosphatidylcholine (POPC) which is the major phospholipid
component of the cell membrane. Thus, it can improve
interaction with the inner endosomal membrane and induce
endosomal escape.
Specifically, in an example of the present disclosure,
the endosomal escape motif X1-X2-X3 of the light-chain
variable region and/or heavy-chain variable region may
comprise a sequence selected from the group consisting of W-
W-W, W-W-H, W-Y-W, Y-W-W, W-Y-H, and Y-W-H (where W is
tryptophan, Y is tyrosine, H is histidine).
In an example of the present disclosure, it was found
that the endosomal escape motif X1-X2-X3 of the light-chain
variable region and/or heavy-chain variable region increases
the endosomal escape ability through a change in the
properties of the antibody by induction of the interaction
under endosomal acidic pH conditions.
As used herein, the term “endosomal acidic pH” refers to
a pH range of 6.0 to 4.5, which satisfies early endosomal and
late endosomal pH conditions and in which the side-chain
properties of aspartic acid (D) and glutamic acid (E) may change.
The CDR3 of the light-chain variable region comprising the
endosomal escape motif may comprise one or more sequences
selected from the following group consisting of:
QQYWWHMYT (SEQ ID NO: 8);
QQYWYWMYT (SEQ ID NO: 9);
QQYYWWMYT (SEQ ID NO: 10);
QQYWYHMYT (SEQ ID NO: 11);
QQYYWHMYT (SEQ ID NO: 12); and
QQYWWWMYT (SEQ ID NO: 51).
The light-chain variable region comprising the endosomal
escape motif may comprise a sequence having a homolog of at
least 80%, for example, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100%, to a light-chain variable region sequence selected from
the group consisting of, for example, SEQ ID NOS: 1 to 5, 13 to
23, 25 to 37, 50, and 60 to 64.
Improved endosomal escape efficiency can also be achieved
at the same level even when the endosomal escape motif is
included in the heavy-chain variable region. Specifically, the
heavy-chain variable region may include X1-X2-X3-Z1 (wherein
each of X1, X2 and X3 is selected from the group consisting of
tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine
(F)) in its CDR3, and Z1 can interact with the 1 amino acid
of the heavy-chain variable region
under endosomal acidic pH conditions, thus changing the
properties of the cytosol-penetrating antibody and enabling
the antibody to have the ability to escape from endosomes
into the cytosol.
The CDR3 of the heavy-chain variable region comprising
the endosomal escape motif may comprise one or more sequences
selected from the following group consisting of SEQ ID NOS: 46
to 49, and 53:
GWYWMDL (SEQ ID NO: 46);
GWYWFDL (SEQ ID NO: 47);
GWYWGFDL (SEQ ID NO: 48);
YWYWMDL (SEQ ID NO: 49); and
GWWWMDL (SEQ ID NO: 53).
the light-chain variable region comprise a sequence
having a homolog of at least 80% to a light-chain variable
region sequence selected from the group consisting of SEQ ID
NOS: 1 to 5, 13 to 23, 25 to 37, 50, and 60 to 64.
The heavy-chain variable region comprising the endosomal
escape motif may comprise a sequence having a homolog of at
least 80%, for example, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100%, to a heavy-chain variable region sequence selected from
the group consisting of, for example, SEQ ID NOS: 39 to 42,
52, and 54 to 59.
In addition, in one embodiment, the sequence may further
comprise Z2 linked to X1, and thus may be represented by the
following formula:
Z2-X1-X2-X3-Z1,
wherein Z2 is selected from the group consisting of
glutamine (Q), leucine (L) histidine (H).
As described above, the sequence is represented by Z2-X1-
X2-X3-Z1, the 1 amino acid of the light-chain variable region
and/or heavy-chain variable region interacts with Z1 and/or Z2
to induce pH-dependent endosomal escape under endosomal acidic
pH conditions.
The CDR3 of the light-chain variable region comprising the
endosomal escape motif may comprise a sequences of SEQ ID NO:
24 as set forth below:
QHYWYWMYT (SEQ ID NO: 24).
As used herein, “antibody” is meant to include an intact
antibody form that specifically binds to a target as well as
an antigen-binding fragment of the antibody.
The complete antibody is a structure having two full-
length light chains and two full-length heavy chains, and each
light chain is linked by a disulfide bond with a heavy chain.
A constant region of the heavy chain has gamma ( γ), mu ( μ),
alpha ( α), delta ( δ), and epsilon ( ε) types. Sub-classes have
gamma 1 ( γ1), gamma 2 ( γ2), gamma 3 ( γ3), gamma 4 ( γ4), alpha
1 (α1), and alpha 2 (α2) types. A constant region of the light
chain has kappa (κ) and lambda (λ) types.
An antigen binding fragment or an antibody fragment of
an antibody refers to a fragment having an antigen binding
function and includes Fab, F(ab′), F(ab′)2, Fv, and the like.
Fab of the antibody fragments has a structure including
variable regions of a light chain and a heavy chain, a
constant region of the light chain, and a first constant
region (CH1 domain) of the heavy chain with one antigen-
binding site. Fab′ differs from Fab in that it has a hinge
region containing one or more cysteine residues at the C-
terminal of the heavy chain CH1 domain. The F(ab′)2 antibody
is produced when the cysteine residue of the hinge region of
the Fab′ forms a disulfide bond. Recombinant techniques for
generating Fv fragments with minimal antibody fragments
having only a heavy-chain variable region and a light-chain
variable region are described in PCT International
Publication Nos. WO88/001649, WO88/006630, WO88/07085,
WO88/07086, and WO88/09344. A two-chain Fv has a non-covalent
bonding between a heavy-chain variable region and a light-
chain variable region. A single chain Fv (scFv) is connected
to a heavy-chain variable region and a light-chain variable
region via a peptide linker by a covalent bond or directly at
the C-terminal. Thus, the single chain Fv (scFv) has a
structure such as a dimer like the two-chain Fv. Such an
antibody fragment can be obtained using a protein hydrolyzing
enzyme (for example, when the whole antibody is cleaved with
papain, Fab can be obtained, and when whole antibody is cut
with pepsin, F(ab′)2 fragment can be obtained), and it can
also be produced through gene recombinant technology.
In one embodiment, the antibody according to the present
disclosure may be an Fv form (e.g., scFv) or a whole antibody
form. The cytosol-penetrating antibody according to the
present disclosure may be an IgG, IgM, IgA, IgD or IgE type.
For example, it may be an IgG1, IgG2, IgG3, IgG4, IgM, IgE,
IgA1, IgA5, or IgD type. Most preferably, it may be an
intact IgG-format monoclonal antibody.
Further, the heavy chain constant region can be selected
from any one isotype of gamma (γ), mu (μ), alpha (α), delta
(δ), and epsilon (ε). Sub-classes have gamma 1 (γ1), gamma 2
(γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2
(α2) types. A constant region of the light chain has kappa
(κ) and lambda (λ) types.
The term “heavy chain” as used herein refers to a full-
length heavy chain and fragments thereof including a variable
region domain VH including an amino acid sequence with
sufficient variable region sequence to confer specificity to
an antigen and three constant region domains CH1, CH2, and
CH3. The term “light chain” as used herein refers to a full-
length heavy chain and fragments thereof including a variable
region domain VL including an amino acid sequence with
sufficient variable region sequence to confer specificity to
an antigen and a constant region domain CL.
In the present disclosure, the antibody includes
monoclonal antibodies, multispecific antibodies, human
antibodies, humanized antibodies, chimeric antibodies,
single-chain Fvs (scFV), single chain antibodies, Fab
fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and
anti-idiotype (anti-Id) antibodies, and epitope-binding
fragments of these antibodies, but is not limited thereto.
An “Fv” fragment is an antibody fragment that contains
complete antigen recognition and binding sites. Such region
includes a heavy chain variable domain and a light chain
variable domain, for example, dimers substantially tightly
covalently associated with scFv.
"Fab" fragment contains the variable and constant domain
of the light-chain and the variable and first constant domain
(CH1) of the heavy chain. F(ab')2 antibody fragment generally
includes a pair of Fab fragments covalently linked by hinge
cysteine near their carboxy-terminus.
“Single chain Fv” or “scFv” antibody fragment comprises
VH and VL domains of the antibody. Such domains are within a
single polypeptide chain. The Fv polypeptide may further
include a polypeptide linker between the VH domain and the VL
domain such that the scFv can form the desired structure for
antigen binding.
The monoclonal antibody refers to an antibody obtained
from a substantially homogeneous population of antibodies,
i.e., the same except for possible naturally occurring
mutations that may be present in trace amounts of individual
antibodies that occupy the population. The monoclonal
antibody is highly specific and is derived against a single
antigenic site.
The non-human (e.g. murine) antibody of the “humanized”
form is a chimeric antibody containing minimal sequence
derived from non-human immunoglobulin. In most cases, the
humanized antibody is a human immunoglobulin (receptor
antibody) that has been replaced by a residue from the
hypervariable region of a non-human species (donor antibody),
such as a mouse, rat, rabbit, and non-human primate, having
specificity, affinity, and ability to retain a residue from
the hypervariable region of the receptor.
“Human antibody” is a molecule derived from human
immunoglobulin and means that all of the amino acid sequences
constituting the antibody including the complementarity
determining region and the structural region are composed of
human immunoglobulin.
A heavy chain and/or light chain is partly identical or
homologous to the corresponding sequence in an antibody
derived from a particular species or belonging to a
particular antibody class or subclass, while the remaining
chain(s) are identical or homologous to corresponding
sequences in an antibody derived from another species or
belonging to another antibody class or subclass “chimeric”
antibodies (immunoglobulins) as well as a fragment of such
antibody exhibiting the desired biological activity.
“Antibody variable domain” as used herein refers to the
light and heavy chain regions of an antibody molecule
including the amino acid sequences of a complementarity
determining region (CDR; i.e., CDR1, CDR2, and CDR3) and a
framework region (FR). VH refers to a variable domain of the
heavy chain. VL refers to a variable domain of the light
chain.
“Complementarity determining region” (CDR; i.e., CDR1,
CDR2, and CDR3) refers to the amino acid residue of the
antibody variable domain, which is necessary for antigen
binding. Each variable domain typically has three CDR regions
identified as CDR1, CDR2, and CDR3.
“Framework region” (FR) is a variable domain residue
other than a CDR residue. Each variable domain typically has
four FRs identified as FR1, FR2, FR3, and FR4.
In another aspect, the present disclosure is directed to
a composition for delivering an active substance into cytosol,
comprising the cytosol-penetrating antibody or antigen
binding fragment thereof.
The active substance may be a type fused or bonded to
the antibody, and the active substance may be one or more
selected from the group consisting of, for example, peptides,
proteins, toxins, antibodies, antibody fragments, RNAs,
siRNAs, DNAs, small molecule drugs, nanoparticles, and
liposomes, but is not limited thereto.
The proteins may be antibodies, antibody fragments,
immuoglubulin, peptides, enzymes, growth factors, cytokines,
transcription factors, toxins, antigen peptides, hormones,
carrier proteins, motor function proteins, receptors,
signaling proteins, storage proteins, membrane proteins,
transmembrane proteins, internal proteins, external proteins,
secretory proteins, viral proteins, glycoproteins, cleaved
proteins, protein complexes, chemically modified proteins, or
the like.
The RNA or ribonucleic acid is based on ribose, a kind
of pentose, is a kind of nucleic acid consisting of a chain
of nucleotides, has a single-stranded structure, and is
formed by transcription of a portion of DNA. In one
embodiment, the RNA may be selected from the group consisting
of rRNA, mRNA, tRNA, miRNA, snRNA, snoRNA, and aRNA, but is
not limited thereto.
The siRNA (Small interfering RNA) is a small RNA
interference molecule composed of dsRNA, and functions to
bind to and degrade an mRNA having a target sequence. It is
used as a disease treating agent or has an activity of
inhibiting expression of a protein translated from a target
mRNA by degrading the target mRNA. Due to this activity, it
is widely used herein.
The DNA or deoxyribonucleic acid is a kind of nucleic
acid, is composed of a backbone chain comprising
monosaccharide deoxyribose linked by phosphate, together with
two types of nucleobases (purines and pyrimidines), and
stores the genetic information of cells.
As used herein, the term “small-molecule drugs” refers
to organic compounds, inorganic compounds or organometallic
compounds that have a molecular weight of less than about
1000 Da and are active as therapeutic agents against diseases.
The term is used in a broad sense herein. The small-molecule
drugs herein encompass oligopeptides and other biomolecules
having a molecular weight of less than about 1000 Da.
In the present disclosure, a nanoparticle refers to a
particle including substances ranging between 1 and 1,000 nm
in diameter. The nanoparticle may be a metal nanoparticle, a
metal/metal core shell complex consisting of a metal
nanoparticle core and a metal shell enclosing the core, a
metal/non-metal core shell consisting of a metal nanoparticle
core and a non-metal shell enclosing the core, or a non-
metal/metal core shell complex consisting of a non-metal
nanoparticle core and a metal shell enclosing the core.
According to an embodiment, the metal may be selected from
gold, silver, copper, aluminum, nickel, palladium, platinum,
magnetic iron and oxides thereof, but is not limited thereto,
and the non-metal may be selected from silica, polystyrene,
latex and acrylate type substances, but is not limited
thereto.
In the present disclosure, liposomes include at least
one lipid bilayer enclosing the inner aqueous compartment,
which is capable of being associated by itself. Liposomes may
be characterized by membrane type and size thereof. Small
unilamellar vesicles (SUVs) may have a single membrane and
may range between 20 and 50 nm in diameter. Large unilamellar
vesicles (LUVs) may be at least 50 nm in diameter.
Oliglamellar large vesicles and multilamellar large vesicles
may have multiple, usually concentric, membrane layers and
may be at least 100 nm in diameter. Liposomes with several
nonconcentric membranes, i.e., several small vesicles
contained within a larger vesicle, are referred to as
multivesicular vesicles.
The term “fusion” or “binding” refers to unifying two
molecules having the same or different function or structure,
and the methods of fusing may include any physical, chemical
or biological method capable of binding the tumor tissue-
penetrating peptide to the protein, small-molecule drug,
nanoparticle or liposome. Preferably, the fusion may be made
by a linker peptide, and for example, the linker peptide may
mediate the fusion with the bioactive molecules at various
locations of an antibody light-chain variable region of the
present disclosure, an antibody, or fragments thereof.
In still another aspect, the present disclosure provides
a pharmaceutical composition for prevention or treatment of
cancer, comprising: the above-described cytosol-penetrating
antibody or antigen binding fragment thereof; and an active
substance to be delivered into cytosol by the cytosol-
penetrating antibody or antigen binding fragment thereof.
The use of the active substance can impart the property
of penetrating cells and localizing in the cytosol, without
affecting the high specificity and affinity of antibodies for
antigens, and thus can localize in the cytosol which is
currently classified as a target in disease treatment based
on small-molecule drugs, and at the same time, can exhibit
high effects on the treatment and diagnosis of tumor and
disease-related factors that show structurally complex
interactions through a wide and flat surface between protein
and protein.
The use of the pharmaceutical composition for prevention
or treatment of cancer can impart the property of enabling
the antibody to penetrate cells and remain in the cytosol,
without affecting the high specificity and affinity of the
antibody for antigens, and thus the antibody can localize in
the cytosol which is currently classified as a target in
disease treatment based on small-molecule drugs, and at the
same time, can be expected to exhibit high effects on the
treatment and diagnosis of tumor and disease-related factors
that show structurally complex interactions through a wide
and flat surface between protein and protein.
In one example of the present disclosure, the
pharmaceutical composition can selectively inhibit KRas
mutants, which are major drug resistance-associated factors
in the use of various conventional tumor therapeutic agents,
and at the same time, can be used in combination with
conventional therapeutic agents to thereby exhibit effective
anticancer activity.
The cancer may be selected from the group consisting of
squamous cell carcinoma, small cell lung cancer, non-small
cell lung cancer, adenocarcinoma of lung, squamous cell
carcinoma of lung, peritoneal cancer, skin cancer, skin or
ocular melanoma, rectal cancer, anal cancer, esophageal
cancer, small intestine cancer, endocrine cancer, parathyroid
cancer, adrenal cancer, soft tissue sarcoma, urethral cancer,
chronic or acute leukemia, lymphoma, hepatoma,
gastrointestinal cancer, pancreatic cancer, glioblastoma,
cervical cancer, ovarian cancer, liver cancer, bladder cancer,
liver tumor, breast cancer, colon cancer, colorectal cancer,
endometrial cancer or uterine cancer, salivary gland cancer,
kidney cancer, liver cancer, prostate cancer, vulva cancer,
thyroid cancer, liver cancer and head and neck cancer.
When the composition is prepared as a pharmaceutical
composition for preventing or treating cancer or
angiogenesis-related diseases, the composition may include a
pharmaceutically acceptable carrier. The pharmaceutically
acceptable carrier contained in the composition is typically
used in the formulation. Examples of the pharmaceutically
acceptable carrier included in the composition may include,
but are not limited to, lactose, dextrose, sucrose, sorbitol,
mannitol, starch, acacia rubber, calcium phosphate, alginate,
gelatin, calcium silicate, minute crystalline cellulose,
polyvinyl pyrrolidone, cellulose, water, syrup, methyl
cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate,
talc, magnesium stearate and mineral oil, etc., but are not
limited thereto. In addition to the above ingredients, the
pharmaceutical composition may further include a lubricant, a
wetting agent, a sweetener, a flavoring agent, an emulsifier,
a suspension, a preservative, etc.
The pharmaceutical composition for preventing or
treating cancer or angiogenesis-related diseases may be
administered orally or parenterally. Such a parenteral
administration includes intravenous injection, subcutaneous
injection, intramuscular injection, intraperitoneal injection,
endothelial administration, topical administration, nasal
administration, intrapulmonary administration, intrarectal
administration, etc. Because a protein or peptide is digested
when administered orally, it is preferred that a composition
for oral administration is formulated to coat an active
substance or to be protected against degradation in stomach.
Also, the pharmaceutical composition may be administered by
any device which can transport active substances to target
cells.
Proper dose of the pharmaceutical composition for
preventing or treating cancer or angiogenesis-related
diseases may vary according to various factors such as method
for formulating, administration method, age, weight, gender,
pathological state of patient, food, administration time,
administration route, excretion rate and reaction sensitivity,
etc. Preferably, a proper dose of the composition is within
the range of 0.001 and 100 mg/kg based on an adult. The term
"pharmaceutically effective dose" as used herein refers to an
amount sufficient to prevent or treat cancer or angiogenesis-
related diseases.
The composition may be formulated with pharmaceutically
acceptable carriers and/or excipients according to a method
that can be easily carried out by those skilled in the art,
and may be provided in a unit-dose form or enclosed in a
multiple-dose vial. Here, the formulation of the
pharmaceutical composition may be in the form of a solution,
a suspension, syrup or an emulsion in oily or aqueous medium,
or may be extracts, powders, granules, tablets or capsules,
and may further include a dispersion agent or a stabilizer.
Also, the composition may be administered individually or in
combination with other therapeutic agents, and may be
administered sequentially or simultaneously with conventional
therapeutic agents. Meanwhile, the composition includes an
antibody or an antigen-binding fragment, and thus may be
formulated into immuno liposome. Liposome including an
antibody may be prepared according to a method well known in
the pertinent art. The immuno liposome is a lipid composition
including phosphatidylcholine, cholesterol and
polyethyleneglycol-derived phosphatidylethanolamine, and may
be prepared by reverse phase evaporation method. For example,
a Fab' fragment of antibody may be conjugated to liposome
through disulphide exchange reaction. Liposome may further
include chemical therapeutic agents such as Doxorubicin.
In yet another aspect, the present disclosure is
directed to a pharmaceutical composition for diagnosis of
cancer, comprising: the above-described cytosol-penetrating
antibody or antigen binding fragment thereof; and an active
substance to be delivered into cytosol by the cytosol-
penetrating antibody or antigen binding fragment thereof.
The term "diagnosis" as used herein refers to
demonstrating the presence or characteristic of a
pathophysiological condition. Diagnosing in the present
disclosure refers to demonstrating the onset and progress of
cancer.
The intact immunoglobulin-format antibody and a fragment
thereof may bind to a fluorescent substance for molecular
imaging in order to diagnose cancer through images.
The fluorescent substance for molecular imaging refers
to all substances generating fluorescence. Preferably, red
or near-infrared fluorescence is emitted, and more preferably,
a fluorescence with high quantum yield is emitted. However,
the fluorescence is not limited thereto.
Preferably, the fluorescent substance for molecular
imaging is a fluorescent substance, a fluorescent protein or
other substances for imaging, which may bind to the tumor
tissue-penetrating peptide that specifically binds to the
intact immunoglobulin-format antibody and a fragment thereof,
but is not limited thereto.
Preferably, the fluorescent substance is fluorescein,
BODYPY, tetramethylrhodamine, Alexa, cyanine, allopicocyanine,
or a derivative thereof, but is not limited thereto.
Preferably, the fluorescent protein is Dronpa protein,
enhanced green fluorescence protein (EGFP), red fluorescent
protein (DsRFP), Cy5.5, which is a cyanine fluorescent
substance presenting near-infrared fluorescence, or other
fluorescent proteins, but is not limited thereto.
Preferably, other substances for imaging are ferric
oxide, radioactive isotope, etc., but are not limited thereto,
and they may be applied to imaging equipment such as MR, PET.
In a further another aspect, the present disclosure is
directed to a nucleic acid encoding the above-described
antibody or antigen-binding fragment thereof.
The nucleic acid is a polynucleotide, and the term
"polynucleotide" as used herein refers to a
deoxyribonucleotide or ribonucleotide polymer present in a
single-stranded or double-stranded form. It includes RNA
genome sequence, DNA (gDNA and cDNA), and RNA sequence
transcribed therefrom. Unless otherwise described, it also
includes an analog of the natural polynucleotide.
The polynucleotide includes not only a nucleotide
sequence encoding the above-described light-chain variable
region (VL) and heavy-chain variable region (VH) having
improved endosomal escape ability, but also a complementary
sequence thereto. The complementary sequence includes a
sequence fully complementary to the nucleotide sequence and a
sequence substantially complementary to the nucleotide
sequence. For example, this complementary sequence may
include a sequence that may be hybridized with a nucleotide
sequence encoding a light-chain variable region (VL) and
heavy-chain variable region (VH) having any one sequence
selected from the group consisting of SEQ ID NOS: 1 to 5, 13
to 23, 25 to 37, 50, and 60 to 64, and SEQ ID NOS: 39 to 42,
52, and 54 to 59 under stringent conditions known in the
pertinent art.
The polynucleotide includes not only a nucleotide
sequence encoding the above-described light-chain region
(kds), but also a complementary sequence thereto. The
complementary sequence includes a sequence fully
complementary to the nucleotide sequence and a sequence
substantially complementary to the nucleotide sequence. For
example, this means a sequence that may be hybridized with a
nucleotide sequence encoding an amino acid sequence of any
one of SEQ ID NO:1 to SEQ ID NO: 3 under stringent conditions
known in the pertinent art.
The nucleic acid may be modified. The modification
includes the addition, deletion, or non-conservative
substitution or conservative substitution of nucleotides. The
nucleic acid encoding the amino acid sequence is interpreted
to include a nucleotide sequence that has a substantial
identity to the nucleotide sequence. The substantial identity
may refer to a sequence having a homology of at least 80%, a
homology of at least 90%, or a homology of at least 95% when
aligning the nucleotide sequence to correspond to any other
sequence as much as possible and analyzing the aligned
sequence using an algorithm generally used in the pertinent
art.
The DNA encoding the antibody can be easily separated or
synthesized using conventional procedures (for example, using
an oligonucleotide probe capable of specifically binding to
DNA encoding the heavy chain and the light chain of the
antibody).
In a still further aspect, the present disclosure is
directed to a method for producing the above-described
cytosol-penetrating antibody or antigen binding fragment
thereof, comprising a step of grafting the endosomal escape
motif X1-X2-X3-Z1 (wherein X1-X2-X3 is selected from the
group consisting of tryptophan (W), tyrosine (Y), histidine
(H), and phenylalanine (F)) into the CDR3 of a light chain
and/or heavy-chain variable region.
The present disclosure can provide an antibody or
antigen-binding fragment thereof having a cytosol-penetrating
ability by substituting the light-chain variable region (VL)
of a conventional antibody with a light-chain variable region
(VL) having improved endosomal escape ability and
substituting the heavy-chain variable region (VH) of the
conventional antibody with a heavy-chain variable region (VH)
having improved endosomal escape ability.
In one embodiment, a method of producing an intact
immunoglobulin-format antibody, which penetrates cells and
localizes in the cytosol, by use of a cytosol-penetrating
light-chain variable region (VL) having improved endosomal
escape ability and a cytosol-penetrating heavy-chain variable
region having endosomal escape ability, comprises the steps
of: obtaining a nucleic acid, in which a light-chain variable
region (VL) in a light chain comprising the light-chain
variable region (VL) and a light chain constant region is
substituted with a light-chain variable region (VL) having
endosomal escape ability or a heavy-chain variable region
(VH) and a heavy chain constant region (CH) are substituted
with a heavy-chain variable region (VH) having endosomal
escape ability, cloning the nucleic acid into a vector, and
transforming the vector into a host cell to express the
antibody or an antigen binding fragment thereof; and
recovering the expressed antibody or an antigen binding
fragment thereof.
The above-described method makes it possible to produce
an intact immunoglobulin-format antibody having increased
endosomal escape ability and cytosol-penetrating ability.
Furthermore, transformation with a vector expressing a heavy
chain comprising a heavy-chain variable region capable of
recognizing a specific protein in cells makes it possible to
express an antibody which is able to penetrate cells and
localize in the cytosol to bind to the specific protein. The
vector may be either a vector system that co-expresses the
heavy chain and the light chain in a single vector or a
vector system that expresses the heavy chain and the light
chain in separate vectors. In the latter case, the two
vectors may be introduced into a host cell by co-
transformation and targeted transformation.
In the present disclosure, the vector may be either a
vector system that co-expresses the heavy chain and the light
chain in a single vector or a vector system that expresses
the heavy chain and the light chain in separate vectors. In
the latter case, the two vectors may be introduced into a
host cell by co-transformation and targeted transformation.
The term "vector" as used herein refers to a means for
expressing a target gene in a host cell. For example, the
vector may include plasmid vector, cosmid vector,
bacteriophage vector, and virus vectors such as adenovirus
vector, retrovirus vector, and adeno-associated virus vector.
The vector that may be used as the recombinant vector may be
produced by operating plasmid (for example, pSC101, pGV1106,
pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9,
pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and
pUC19, etc.), phages (for example, λgt4λB, λ-Charon, λΔz1 and
M13, etc.), or virus (for example, CMV, SV40, etc.) commonly
used in the pertinent art.
The light-chain variable region, the light-chain
constant region (CL), the heavy-chain variable region (VH),
and the heavy-chain constant region (CH1-hinge-CH2-CH3) of
the present disclosure in the recombinant vector may be
operatively linked to a promoter. The term "operatively
linked" as used herein means a functional linkage between a
nucleotide expression control sequence (such as a promoter
sequence) and a second nucleotide sequence. Accordingly, the
control sequence may control the transcription and/or
translation of the second nucleotide sequence.
The recombinant vector may be generally constructed as a
vector for cloning or a vector for expression. As the vector
for expression, vectors generally used for expressing foreign
protein from plants, animals or microorganisms in the
pertinent art may be used. The recombinant vector may be
constructed by various methods known in the pertinent art.
The recombinant vector may be constructed to be a vector
that employs a prokaryotic cell or an eukaryotic cell as a
host. For example, when the vector used is an expression
vector and employs a prokaryotic cell as a host, the vector
generally includes a strong promoter which may promote
transcription (for example, pLλ promoter, trp promoter, lac
promoter, tac promoter, T7 promoter, etc.), a ribosome
binding site for initiation of translation, and termination
sequences for transcription/translation. When the vector
employs an eukaryotic cell as a host, a replication origin
operating in the eukaryotic cell included in the vector may
include an f1 replication origin, an SV40 replication origin,
a pMB1 replication origin, an adeno replication origin, an
AAV replication origin, a CMV replication origin and a BBV
replication origin, etc., but is not limited thereto. In
addition, a promoter derived from a genome of a mammal cell
(for example, a metalthionine promoter) or a promoter derived
from a virus of a mammal cell (for example, an adenovirus
anaphase promoter, a vaccinia virus 7.5K promoter, a SV40
promoter, a cytomegalo virus (CMV) promoter, or a tk promoter
of HSV) may be used, and the promoter generally has a
polyadenylated sequence as a transcription termination
sequence.
Another aspect of the present disclosure provides a host
cell transformed with the recombinant vector.
Any kind of host cell known in the pertinent art may be
used as a host cell. Examples of a prokaryotic cell include
strains belonging to the genus Bascillus such as E. coli
JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E.
coli X 1776, E. coli W3110, Bascillus subtilus and Bascillus
thuringiensis, Salmonella typhimurium, intestinal flora and
strains such as Serratia marcescens and various Pseudomonas
Spp., etc. In addition, when the vector is transformed in an
eukaryotic cell, a host cell such as yeast (Saccharomyce
cerevisiae), an insect cell, a plant cell, and an animal cell,
for example, SP2/0, CHO (Chinese hamster ovary) K1, CHO DG44,
PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RN, and MDCK
cell line, etc., may be used.
\Another aspect of the present disclosure may provide a
method for producing an intact immunoglobulin-format antibody
that penetrates cells and localizes in the cytosol, the
method comprising a step of culturing the above-described
host cell.
A recombinant vector may be inserted into a host cell
using an insertion method well known in the pertinent art.
For example, when a host cell is a prokaryotic cell, the
transfer may be carried out according to CaCl method or an
electroporation method, etc., and when a host cell is an
eukaryotic cell, the vector may be transferred into a host
cell according to a microscope injection method, calcium
phosphate precipitation method, an electroporation method, a
liposome-mediated transformation method, and a gene
bombardment method, etc., but the transferring method is not
limited thereto. When using microorganisms such as E. coli,
etc. the productivity is higher than using animal cells.
However, although it is not suitable for production of intact
Ig form of antibodies due to glycosylation, it may be used
for production of antigen binding fragments such as Fab and
The method for selecting the transformed host cell may
be readily carried out according to a method well known in
the pertinent art using a phenotype expressed by a selected
label. For example, when the selected label is a specific
antibiotic resistance gene, the transformant may be readily
selected by culturing the transformant in a medium containing
the antibiotic.
EXAMPLES
Hereinafter, the present disclosure will be described in
further detail with reference to examples. It will be obvious
to a person having ordinary skill in the art that these
examples are illustrative purposes only and are not to be
construed to limit or change the scope of the present
disclosure.
Example 1: Expression and Purification of Cytosol-
Penetrating Antibody (Cytotransmab)
In order to elucidate the endosomal escape mechanism of
a cytosol-penetrating antibody and to improve the endosomal
escape mechanism, the cytosol-penetrating antibody was
purified.
Specifically, in order to construct a heavy-chain
expression vector for producing an intact IgG-format
monoclonal antibody, a DNA encoding a heavy chain comprising
an antibody heavy-chain variable region (humanized hT0 VH;
SEQ ID NO: 38) and a heavy-chain constant region (CH1-hinge-
CH2-CH3), which has a secretion signal peptide-encoding DNA
fused to the 5′ end, was cloned into a pcDNA3.4 vector
(Invitrogen) by NotI/HindIII.
Furthermore, in order to construct a vector that
expresses a light chain, a DNA encoding a light chain
comprising a cytosol-penetrating light-chain variable region
(hT4 VL; SEQ ID NO: 65) and light-chain constant region (CL),
which has a secretion signal peptide-encoding DNA fused to
the 5′ end, was cloned into a pcDNA3.4 vector (Invitrogen) by
use of NotI/HindIII.
The light-chain and heavy-chain expression vectors were
transiently transfected, and the proteins were expressed and
purified. In a shaking flask, HEK293-F cells suspension-
growing in serum-free FreeStyle 293 expression medium
(Invitrogen) were transfected with a mixture of plasmid and
polyethylenimine (PEI) (Polyscience). After 200 mL
transfection in a shaking flask (Corning), HEK293-F cells
were seeded into 100 ml of medium at a density of 2.0 x
cells/ml, and cultured at 150 rpm and in 8% CO . To
produce each monoclonal antibody, a suitable heavy-chain and
light-chain plasmid were diluted in 10 ml of FreeStyle 293
expression medium (Invitrogen) (125 μg heavy chain, 125 μg
light chain, a total of 250 μg (2.5 μg/ml)), and the dilution
was mixed with 10 ml of medium containing 750 μg (7.5 μg/ml)
of PEI, and the mixture was incubated at room temperature for
minutes. The incubated medium mixture was added to 100 ml
of the seeded cell culture which was then cultured at 150 rpm
in 8% CO for 4 hours, after which 100 ml of FreeStyle 293
expression was added to the cell culture, followed by culture
for 6 days.
In accordance with the standard protocol, the protein
was purified from the collected cell culture supernatant. The
antibody was applied to a Protein A Sepharose column (GE
Healthcare), and washed with PBS (pH 7.4). The antibody was
eluted using 0.1 M glycine buffer (pH 3.0), and then
immediately neutralized with 1M Tris buffer. The eluted
antibody fraction was concentrated while the buffer was
replaced with PBS (pH 7.4) by dialysis. The purified protein
was quantified by measuring the absorbance at 280 nm and the
absorption coefficient.
Example 2: Observation of Trafficking after Endocytosis
of Cytosol-Penetrating Antibody
Trafficking from endocytosis of the developed cytosol-
penetrating antibody to localization into the cytosol was
observed. This may be an important clue to the mechanism of
endosomal escape into the cytosol.
shows of a pulse-chase experiment and confocal
microscopy observation performed to observe the transport
process and stability of the cytosol-penetrating antibody
(cytotransmab) TMab4 or cell-penetrating peptide TAT
introduced into cells.
Specifically, a cover slip was added to 24-well plates,
and 2.5 x 10 HeLa cells per well were added to 0.5 ml of 10%
FBS-containing medium and cultured for 12 hours under the
conditions of 5% CO and 37°C. When the cells were stabilized,
the cells were transiently transfected with pcDNA3.4-flag-
rab11. To maximize the efficiency of transient transfection,
Opti-MEM media (Gibco) was used. 500 ng of pcDNA3.4-flag-
rab11 to be transiently transfected was incubated with μl of
Opti-MEM media and 2 μl of Lipofectamine 2000 (Invitrogen,
USA) in a tube at room temperature for 20 minutes, and then
added to each well. Additionally, 450 μl of antibiotic-free
DMEM medium was added to each well which was then incubated
at 37°C in 5% CO for 6 hours, after which the medium was
replaced with 500 μl of 10% FBS-containing DMEM medium,
followed by incubation for 24 hours. Next, each well was
treated with 3 μM of TMab4 in 0.5 ml of fresh medium for 30
minutes, and then washed rapidly three times with PBS and
incubated in medium at 37°C for 0, 2 and 6 hours. Thereafter,
the medium was removed, and each well was washed with PBS,
and then proteins attached to the surface were removed with
weakly acidic solution (200 mM glycine, 150 mM NaCl pH 2.5).
After washing with PBS, the cells were fixed in 4%
paraformaldehyde at 25°C for 10 minutes.
After washing with PBS, each well was incubated with PBS
buffer containing 0.1% saponin, 0.1% sodium azide and 1% BSA
at 25°C for 10 minutes to form pores in the cell membranes.
After washing with PBS, each well was incubated with PBS
buffer containing 2% BSA at 25°C for 1 hour to eliminate
nonspecific binding. Then, the cells were stained with an
FITC (green fluorescence) or TRITC (red fluorescence)-labeled
antibody (Sigma) that specifically recognizes human Fc. Rab5
was incubated with anti-rab5 against the early endosome
marker rab5. Each well was incubated with anti-flag
antibodies against a flag-tag of rab11, a recycling endosome
marker, at 25°C for 1 hour, and was then incubated with TRITC
(red fluorescence) or FITC (green fluorescence)-labeled
secondary antibody at 25°C for 1 hour. To observe late
endosomes and lysosomes, the cells being incubated were
treated with 1 mM LysoTracker Red DND-99 at 30 minutes before
cell fixation. The nucleus was blue-stained with Hoechst33342
and observed with a confocal microscope. As a result, it was
shown that, unlike TAT, TMab4 was located in early endosomes
up to 2 hours, and then was not transported to lysosomes or
recycling endosomes.
Example 3: Evaluation of the Effect of Acidification in
Early Endosomes on Endosomal Escape
To obtain more clear evidence that the cytosol-
penetrating antibody of the present disclosure escapes from
early endosomes, an experiment was performed using inhibitors.
Specifically, the inhibitors used were wortmannin that
inhibits maturation from early endosomes to late endosomes,
bafilomycin that prevents endosomal oxidation by inhibiting
ATPase hydrogen pump, and brefeldin A that inhibits transport
from endosomes to endoplasmic reticulum and Golgi.
shows the results of confocal microscopy
observation of the cytosol-penetrating ability of the
cytosol-penetrating antibody TMab4 or the cell-penetrating
peptide TAT according to the present disclosure in the
presence or absence of an inhibitor thereof.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2. When the cells were
stabilized, the cells were incubated with each of 100 nM
wortmannin, 200 nM bafilomycin and 7 μM brefeldin A for 30
minutes. Next, the cells were incubated with each of PBS, 2
μM TMab4 and 2 μM TAT at 37°C for 6 hours. The cells were
washed with PBS and weakly acidic solution in the same manner
as described in Example 2, and then subjected to cell
fixation, cell perforation and blocking processes. The TMab4-
treated cells were stained with an FITC (green fluorescence)-
labeled antibody that specifically recognizes human Fc. The
nucleus was blue-stained with Hoechst 33342 and observed with
a confocal microscope. In the case of TMab4, green
fluorescence localized in the cytosol was not observed only
in the bafilomycin-treated cells, and spot-shaped
fluorescence appeared.
is a bar graph showing the results of
quantifying the FITC (green fluorescence) fluorescence of the
confocal micrographs shown in .
Specifically, using Image J software (National
Institutes of Health, USA), 20 cells were selected in each
condition, and then the obtained mean values of fluorescence
are graphically shown.
shows the results of observing the cytosolic
localization of the cytosol-penetrating antibody TMab4 or the
cell-penetrating peptide TAT according to the present
disclosure by confocal microscopy using calcein in the
presence or absence of an inhibitor thereof.
Specifically, HeLa cells were prepared in the same as
described in Example 2, and were incubated in serum-free
medium with each of 200 nM wortmannin, 200 nM bafilomycin and
7 μM brefeldin A for 30 minutes. Next, the cells were
incubated with each of PBS, 2 μM TMab4 and 20 μM TAT at 37°C
for 6 hours. After 4 hours, each well containing PBS or the
antibody was treated with 150 μM calcein and incubated at
37°C for 2 hours. In the same manner as described in Example
2, the cells were washed with PBS and weakly acidic solution,
and then fixed.
The nucleus was blue-stained with Hoechst 33342 and
observed with a confocal microscope. As a result, green
calcein fluorescence appeared, indicating that calcein did
escape from endosomes into the cytosol by the cytosol-
penetrating antibody TMab4 of the present disclosure and TAT.
However, in the case of TMab4, green calcein fluorescence
localized in the cytosol could not be observed only in the
bafilomycin-treated cells, unlike the cells treated with
other inhibitors.
is a bar graph showing the results of
quantifying the calcein fluorescence of the confocal
micrographs shown in .
Specifically, as shown in , using Image J
software (National Institutes of Health, USA), 20 cells were
selected in each condition, and then the obtained mean values
of fluorescence are graphically shown.
Example 4: Evaluation of the Effect of HSPG Degradation
in Early Endosomes on Endosomal Escape
The cytosol-penetrating antibody is endocytosed by
binding to HSPG on the cell surface. At this time, it is
endocytosed with pro-heparanase. Pro-heparanase is activated
with endosomal acidification (Gingis-Velitski et al., 2004).
Activated heparanase degrades HSPG, and thus the cytosol-
penetrating antibody can be freely localized in the cytosol.
shows the results of Western blot analysis
performed to confirm siRNA (short interfering RNA)-induced
inhibition of heparanase expression.
Specifically, 1 x 10 HeLa cells were added to each well
of 6-well plates and cultured in 1 ml of 10% FBS-containing
medium at 37°C in 5% CO for 12 hours. After 24 hours of
culture, each well was transiently transfected with siRNA.
For transient transfection, 500 ng of each of a control siRNA
having no targeting ability and an siRNA targeting inhibition
of heparanase expression was incubated with 500 μl of Opti-
MEM media (Gibco) and 3.5 μl of Lipofectamine 2000
(Invitrogen, USA) in a tube at room temperature for 20
minutes, and then added to each well. 500 μl of antibiotic-
free DMEM medium was added to each well which was then
incubated at 37°C in 5% CO for 6 hours. Next, the medium was
preplaced with 1 ml of 10% FBS-containing DMEM medium,
followed by incubation for 72 hours.
After incubation, lysis buffer (10 mM Tris-HCl pH 7.4,
100mM NaCl, 1% SDS, 1mM EDTA, Inhibitor cocktail(sigma)) was
added to each well to a cell lysate. The cell lysate was
quantified using a BCA protein assay kit (Pierce). The gel
subjected to SDS-PAGE was transferred to a PVDF membrane,
incubated with the antibody (SantaCruz)(which recognize
heparanase and β-actin, respectively) at 25°C for 2 hours,
and then incubated with HRP-conjugated secondary antibody
(SantaCruz) at 25°C for 1 hour, followed by detection.
Analysis was performed using ImageQuant LAS4000 mini (GE
Healthcare).
shows the results of confocal microscopy
observation of cytosol penetrating antibody/lysosome merging
caused by inhibition of heparanase expression.
Specifically, HeLa cells with inhibited inhibition of
heparanase expression and control HeLa cells were prepared in
the same manner as described in Example 2. The cells were
treated with each of 3 μM TMab4 and 20 μM FITC-TAT at 37°C
for 30 minutes, washed rapidly three times with PBS, and then
incubated in medium at 37°C for 2 hours. In the same manner
as described in Example 2, the cells were washed with PBS and
weakly acidic solution, and then subjected to cell fixation,
cell perforation and blocking processes.
The TMab4-treated cells were stained with an FITC (green
fluoescence)-labeled antibody that specifically recognizes
human Fc. The cells were incubated with anti-LAMP-1 (santa
cruz) against the lysosome marker LAMP-1 at 25°C for 1 hour,
and incubated with TRITC (red fluorescence)-labeled secondary
antibody at 25°C for 1 hour. The nucleus was blue-stained
with Hoechst 33342 and observed with a confocal microscope.
In the case of TMab4, merging with LAMP-1 was observed when
heparanase expression was inhibited.
shows the results of confocal microscopy
observation performed to confirm the cytosolic localization
of a cytosol-penetrating antibody, which is caused by
inhibition of heparanase expression.
Specifically, HeLa cells with inhibited inhibition of
heparanase expression and control HeLa cells were prepared in
the same manner as described in Example 2. The cells were
treated with each of 2 μM TMab4 and 20 μM FITC-TAT at 37°C
for 6 hours. After 4 hours, each well containing PBS or the
antibody was treated with 150 μM calcein and incubated at
37°C for 2 hours. In the same manner as described in Example
2, the cells were washed with PBS and weakly acidic solution,
and then fixed. The nucleus was blue-stained with Hoechst
33342 and observed with a confocal microscope. In the cells
with inhibited expression of heparanase, calcein fluorescence
that localized to the cytosol by TMab4 could not be observed.
is a schematic view showing an overall
trafficking process ranging from cellular internalization of
a cytosol-penetrating antibody according to the present
disclosure to localization of the antibody in the cytosol.
Example 5: Observation of Introduction of Cytosol-
Penetrating Intact IgG-Format Monoclonal Antibody through
Cell Membrane at Varying pHs
In order for the cytosol-penetrating antibody of the
present disclosure to localize in the cytosol after
endocytosis, an endosomal escape process is essential. Until
now, there has been no report on endosomal escape of
antibodies. To elucidate the endosomal escape mechanism, an
experiment was performed at simulated endosomal pH.
The components of the inner phospholipid layer of early
endosomes are similar to those of the outer phospholipid
layer of the cell membrane (Bissig and Gruenberg, 2013), and
the major component of the phospholipid layer is 1-palmitoyl-
2-oleoyl-sn-glycerophosphatidylcholine (POPC). Thus,
assuming that the outer phospholipid layer of the membrane of
Ramos cells expressing no HSPG is the same as the inner
phospholipid layer of early endosomes, an experiment was
performed.
shows the results of observing a fluorescence-
labeled cytosol-penetrating antibody in Ramos cells by
confocal microscopy in order to examine whether the antibody
can be introduced through the cell membrane depending on pH
or whether the antibody can induce cell membrane permeation
of other substances.
Specifically, a cover slip was added to 24-well plates,
and 200 μl of 0.01% poly-L-lysine solution was added to
attach suspending Ramos cells to the plate, followed by
incubation at 25°C for 20 minutes. After washing with PBS, 5
x 10 Ramos cells were added to each well and incubated in 0.5
ml of 10% FBS-containing medium at 37°C for 30 minutes. After
confirming cell adhesion, the cells were incubated in 200 μl
of pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) or pH
.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) with each of 10
μM PBS and TMab4 labeled directly with the fluorescent
reagent DyLight-488, 10 μM non-labeled TMab4 and 2 μM control
antibody adalimumab labeled directly with DyLight-488, at
37°C for 2 hours. Adalimumab used as the control antibody is
a therapeutic antibody that targets extracellular cytokines.
In the same manner as described in Example 2, the cells
were washed with PBS, and then fiaxed. The nucleus was blue-
stained with Hoechst 33342 and observed with a confocal
microscope. At pH 5.5, the fluorescence of TMab4 labeled
directly with DyLight-488 was observed. At pH 5.5, green
FITC fluorescence was observed in the cells treated with
TMab4 and adalimumab labeled directly with DyLight-488. It
was confirmed that the cytosol-penetrating antibody was
introduced through the cell membrane at acidic pH and could
introduce other substance as well as itself.
In addition, it was confirmed that the morphology of the
cell membrane was maintained, even though the substance was
introduced externally.
Example 6: Examination of Whether Cytosol-Penetrating
Antibody Forms Pores by Trypan Blue Uptake Depending on pH
Among known endosomal escape mechanisms, endosomal
perforation was expected to be the most promising endosomal
escape mechanism by which an intact IgG-format substance can
escape from endosomes while maintaining the morphology of
endosomes as shown in the experimental results.
Similar to Example 5, an experiment was performed in
order to observe the morphology of the cell membrane when the
cytosol-penetrating antibody passed through the cell membrane.
shows the results of observing Ramos cells by an
optical microscope in order to examine whether a cytosol-
penetrating antibody can form pores and take up trypan blue
having no membrane-permeating ability, depending on pH.
pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) or pH
5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5)
Specifically, 5 x 10 Ramos cells were attached to each
well of 24-well plates in the same manner as described in
Example 5. After confirming cell adhesion, the cells were
incubated with each of TMab4 and 1 μM and 10 μM of adalimumab
in 200 μl of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH
7.4(cytosol pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES
pH 5.5)(early endosomal pH)) at 37°C for 2 hours. After
careful washing with PBS, 200 μl of a mixture of 190 μl of
PBS and 10 μl of trypan blue was added to each well, and the
cells were observed with a microscope.
is a graph quantitatively comparing the number
of cells that have taken up trypan blue.
Specifically, the number of cells showing trypan blue
uptake was counted and expressed as percentage relative to
the total number of cells. A total of 400 or more cells were
counted, and the mean values are graphically shown.
As shown in , only at pH 5.5, the cells treated
with the cytosol-penetrating antibody TMab4 of the present
disclosure showed trypan blue uptake in a concentration-
dependent manner. In addition, it was shown that the
morphology of the cell membrane during the passage of the
cytosol-penetrating antibody was maintained.
Example 7: Observation of Temporary ad Reversible Pore
Formation by Cytosol-Penetrating Antibody
In the case of conventional peptides known to show a
pore formation mechanism by the endosomal escape mechanism,
it is known that the alpha-helical structure of the peptides
forms pores through the cell membrane.
However, since antibodies have no alpha-helical
structure, they were generally considered almost impossible
to form pores through the cell membrane. Thus, it was assumed
that the antibody would escape from endosomes after temporary
pore formation, and then the cell membrane would be
reversibly restored. To demonstrate this assumption, an
experiment was performed.
FIG 7a shows the results of optical microscopic
observation performed to confirm whether cell membrane pores
produced by a cytosol-penetrating antibody at pH 5.5 is
temporary and reversible.
Specifically, 5 x 10 Ramos cells were attached to each
well of 24-well plates in the same manner as described in
Example 5. After conforming cell adhesion, the cells were
incubated with 10 μM of TMab4 in 200 μl of pH 5.5 buffer
(HBSS (Welgene), 50 mM MES pH 5.5) at 37°C for 2 hours in
order to maintain an early endosomal pH of 5.5. The buffer
was replaced with fresh buffer, and the cells were incubated
for 2 hours so that the cells could be recovered. After
careful washing with PBS, 200 μl of a mixture of 190 μl of
PBS and 10 μl of trypan blue was added to each well, and the
cells were observed with a microscope.
is a graph quantitatively comparing the number
of cells that have taken up trypan blue uptake. Specifically,
a total of 400 or more cells were counted, and the mean
values are graphically shown. As shown in , at pH 5.5,
the cells treated with TMab4 having endosomal escape ability
according to the present disclosure did take up trypan blue
immediately after addition of TMab4, but the cells subjected
to recovery in the medium did not take up blue uptake. Namely,
it was confirmed that pore formation by the cytosol-
penetrating antibody was a temporary and reversible
phenomenon.
Example 8: Observation of Membrane Binding and Lipid
Membrane Flip-Flop of Cytosol-Penetrating Intact IgG-Format
Monoclonal Antibody at Varying pHs
The pore formation mechanism is a mechanism by which
pores are formed while maintaining the overall morphology of
the cell membrane and a substance escapes from endosomes into
the cytosol through the pores. For pore formation, it is
known that a substance interacts with the inner phospholipid
layer of endosomes, and then membrane pores are formed by a
flip-flop mechanism (H. D. Herce et al., 2009).
Thus, in order for endosomal escape occurs by pore
formation in early endosomes, an antibody should first bind
to the cell membrane by endosomal acidification. To confirm
this, an experiment was performed.
shows the results of analyzing the cell membrane
binding of a cytosol-penetrating antibody and control
antibody adalimumab by flow cytometry (FACS) at varying pHs.
Specifically, 1x10 Ramos were prepared for each sample.
The cells were washed with PBS, and then incubated with each
of 5 μM TMab4 and 5 μM adalimumab in each of pH 7.4 buffer
(TBS, 2% BSA, 50 mM HEPES pH 7.4)(for maintaining a cytosolic
pH of 7.4) and pH 5.5 buffer (TBS, 2% BSA, 50 mM MES pH 5.5)
(for maintaining an early endosomal pH) at 4°C for 1 hour.
The cells were washed with each pH buffer, and then the cells
treated with each of TMab4 or adalimumab were incubated with
FITC (green fluorescence)-labeled antibody (which
specifically recognizes human Fc) at 4°C for 30 minutes. The
cells were washed with PBS, and then analyzed by flow
cytometry. As a result, it was shown that, at pH 5.5, only
TMab4 did bind to the cell membrane.
shows the results of analyzing the cell membrane
flip-flop inducing abilities of a cytosol-penetrating antibody
and control antibody adalimumab by flow cytometry (FACS) at
varying pHs.
Specifically, 1x10 Ramos cells were prepared for each
sample. The cells were washed with PBS, and then incubated
with each of 5 μM TMab4 and 5 μM adalimumab in each of pH 7.4
buffer (TBS, 2% BSA, 50 mM HEPES pH 7.4) (for maintaining a
cytosolic pH of 7.4) and pH 5.5 buffer (TBS, 2% BSA, 50 mM
MES pH 5.5) (for maintaining an early endosomal pH of 5.5) at
4°C for 1 hour.
The cells were washed with each pH buffer, and then
incubated with FITC (green fluorescence)-labeled Annexin-V at
°C for 15 minutes. Annexin-V is a substance that targets
phosphatidylserine, a lipid present only in the cell membrane,
and only when cell membrane lipid flip-flop occurs, the lipid
can be exposed to the outside and Annexin-V can bind thereto.
After washing with PBS, the cells were analyzed by flow cytometry.
As a result, it was confirmed that, at pH 5.5, Annexin-V did
bind only to TMab4.
is a schematic view showing a pore formation model
of a cytosol-penetrating antibody, expected based on the above-
described experiments.
Example 9: Logic of Prediction of pH-Dependent Change in
Properties
The reason why the cytosol-penetrating antibody according
to the present disclosure showed different cytosol penetration
properties depending on pH was assumed to be because a pH-
dependent change in interaction between antibody residues led
to a change in the properties.
To demonstrate this assumption, literature search was
performed. As a result, it was confirmed that as pH decreases
from 7.4 (neutral pH) to 5.0, aspartic acid (D) and glutamic
acid (E) among amino acids lose negative charge by protonation
and becomes hydrophobic (Korte et al., 1992).
Specifically, aspartic acid (D) and glutamic acid (E),
which have become hydrophobic, hydrophobically interact with
methionine (M), leucine (L) and isoleucine (I), which are
originally hydrophobic amino acids. The phenomenon that the
surrounding amino acids induce structural modification through
this newly formed interaction is defined as the Tanford
transition (Qin et al., 1998). To confirm this pH-dependent
change in the properties, an experiment was performed (Di Russo
et al., 2012).
In a hT4 VL structure which is a cytosol-penetrating light-
chain variable region, hydrophobic amino acids, methionine (M),
isoleucine (I) and leucine (L), which surround histidine (H),
aspartic acid (D) and glutamic acid (E), which can show a
difference between pH 7.4 and pH 5.0, were examined.
Among these amino acids, candidate amino acids where the
distance between the side chains of two amino acids was less
st th
than 6-7Å were identified, and a pair of the 1 and 95 amino
acids from the N-terminus were selected as candidate amino
acids capable of showing the Tanford transition effect.
st th th
Among the pair of the 1 and 95 amino acids, the 95
amino acid is an amino acid present in the sequence VL-CDR3 of
the cytosol-penetrating light-chain variable region hT4 VL.
It was confirmed that the 95 amino acid could induce a
change in the VL-CDR3 loop structure through a phenomenon,
such as the Tanford transition, by interaction with the 1
amino acid.
It was confirmed that, in the cytosol-penetrating light-
chain variable region hT4 VL, the amino acids of the VL-CDR3
st th
loop which was structurally changed by the 1 and 95 amino
acids include a very high proportion of tyrosine (Y) which
easily interacts with 1-palmitoyloleoyl-sn-glycero
phosphatidylcholine (POPC) which is the major component of
the inner phospholipid layer of early endosomes (Morita et
al., 2011).
shows the results of predicting the pH-dependent
structural change of a cytosol-penetrating antibody on the
basis of the WAM modeling structure of the light-chain
variable region of the cytosol-penetrating antibody, and
shows amino acids, which are involved in the structural
change, and amino acids which are exposed by the structural
change.
In order to confirm the pH-dependent change in
st th
properties induced by the 1 and 95 amino acids and the
endosomal escape resulting from the change, mutants were
st th
constructed by substituting the 1 and 95 amino acids with
alanine (A).
In addition, in order to confirm the pH-dependent change
st th
in properties induced by the 1 and 95 amino acids and the
endosomal escape resulting from the change, mutants were
st th
constructed by substituting the 1 and 95 amino acids with
glutamic acid (E) and leucine having properties similar
thereto.
Table 1 shows the names and sequences of mutants
constructed using an overlap PCR technique.
[Table 1]
In the same manner as described in Example 1, cloning,
expression in HEK293F cell lines, and purification were
performed.
Example 10: Observation of pH-Dependent Change in
Properties of Cytosol-Penetrating Antibody
is a graph quantitatively comparing the number of
cells that have taken up trypan blue at varying pHs by mutants
(TMab4-D1A), (TMab4-M95A), (TMab4-D1E), and (TMab4-M95L)
constructed by substituting the 1 amino acid aspartic acid (D),
th st
the 95 amino acid methionine (M), the 1 amino acid aspartic
acid (D), and the 95 amino acid methionine (M) of a light-chain
variable region (VL), which are involved in induction of a
structural change of a cytosol-penetrating antibody at acidic
pH, with alanine (A), alanine (A), glutamic acid (E), and leucine
(L), respectively.
Specifically, Ramos cells were attached to plates in the
same manner as described in Example 5. Then, the cells were
incubated with 10 μM of each of TMab4, Adalimumab, TMab4-D1A,
TMab4-M95A, TMab4-D1E and TMab4-M95L in 200 μl of each of pH 7.4
buffer (HBSS(Welgene), 50 mM HEPES pH 7.4)(for maintaining a
cytosolic pH of 7.4) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES
pH 5.5)(for maintaining an early endosomal pH of 5.5) at 37°C
for 2 hours.
After careful washing with PBS, 200 μl of a mixture of
190 μl of PBS and 10 μl of trypan blue was added to each well,
and the cells were observed with a microscope. The number of
cells showing trypan blue uptake was counted and expressed as
percentage relative to the total number of cells. A total of
400 or more cells were counted, and the mean values are
graphically shown.
It was confirmed that the mutants, TMab4-D1A and TMab4-
M95A, showed little or no trypan blue uptake, unlike TMab4.
TMab4-D1E and TMab4-M95L showed trypan blue uptake similar to
that of TMab4. This suggests that the 1 amino acid and the
95 amino acid play an important role in endosomal escape.
Example 11: Investigation of Amino Acids and Motifs
Contributing to Endosomal Escape Ability of Cytosol-
Penetrating Antibody
Through the experimental examples obtained in the above
Examples, it was found that the pH-dependent change in the
properties of the antibody occurred by interaction with the
st th
1 and 95 antibodies of the cytosol-penetrating antibody and
that endosomal escape was induced by the change in the
properties.
In order to confirm endosomal escape induced by the pH-
dependent change in the properties, mutants were constructed
by substituting amino acids of VL-CDR3, which were expected
to interact with phospholipid, with alanine (A).
Specifically, based on the results of structural
modeling analysis, mutants were constructed by simultaneously
nd rd th
substituting the 92 , 93 and 94 amino acids, which were
most likely to be exposed to the surface, with alanine (A).
Table 2 below shows the names and sequences of mutants
constructed using an overlap PCR technique.
[Table 2]
In the same manner as described in Example 1, cloning,
expression in HEK293F cell lines, and purification were
performed.
is a graph quantitatively comparing the number
of cells that have taken up trypan blue depending on pH by
nd rd th
mutants constructed by substituting the 92 , 93 , and 94
amino acids of the CDR3 of the light-chain variable region
(VL) of a cytosol-penetrating antibody, which can possibly be
involved in endosomal escape, with alanine.
Specifically, Ramos cells were attached to plates in the
same manner as described in Example 5. Then, the cells were
incubated with each of buffer and 10 μM of TMab4, TMab4-Y91A,
TMab4-Y92A, TMab4-Y93A, TMab4-H94A, TMab4-AAA and TMab4-Y96A
in 200 ㎕ of each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES
pH 7.4)(for maintaining a cytosolic pH of 7.4) and pH 5.5
buffer (HBSS(Welgene), 50 mM MES pH 5.5)(for maintaining an
early endosomal pH of 5.5) at 37°C for 2 hours.
After careful washing with PBS, 200 μl of a mixture of
190 μl of PBS and 10 μl of trypan blue was added to each well,
and the cells were observed with a microscope. The number of
cells showing trypan blue uptake was counted and expressed as
percentage relative to the total number of cells. A total of
400 or more cells were counted, and the mean values are
graphically shown. It was shown that TMab4-Y92A, TMab4-Y93A
and TMab4-H94A showed significantly reduced trypan blue
uptake compared to TMab4. In particular, TMab4-AAA showed
little or no trypan blue uptake. However, TMab4-Y91A and
TMab4-Y96A showed trypan blue uptake similar to that of TMab4.
nd rd th
This suggests that the 92 , 93 and 94 amino acids greatly
contribute to endosomal escape.
Example 12: Confirmation of Contribution of CDR1 and
CDR2 of Cytosol-Penetrating Antibody Light-Chain Variable
Region (VL) to Endosomal Escape
The above-described experimental results demonstrated
that the CDR3 of the light-chain variable region (VL) is
involved in endosomal escape. Then, in order to elucidate the
effect of the CDR1 and CDR2 of the light-chain variable
region (VL), which are involved in endocytosis, on endosomal
escape, an experiment was performed.
The CDR1 and CDR2 of the light-chain variable region
(VL) were substituted with CDR sequences which have the same
amino acid number or do not include the cationic patch
sequence of CDR1 involved in endocytosis, among human
germline sequences. At this time, amino acids known to be
important for the stability of the existing light-chain
variable region were conserved.
Table 3 below shows the names and sequences of mutants
constructed using genetic synthesis.
[Table 3]
In the same manner as described in Example 1, cloning,
expression in HEK293F cell lines, and purification were
performed.
a shows the results of confocal microscopy
performed to analyze the cytosol-penetrating ability of
mutants constructed by substituting the CDR1 and CDR2 of the
light-chain variable region (VL) of a cytosol-penetrating
antibody, which bind to HSPG receptor and are involved in
cytosol-penetrating ability, with human germline sequences.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2. When the cells were
stabilized, the cells were incubated with each of PBS and 2
μM TMab4, TMab4-01, TMab4-02 and TMab4-03 at 37°C for 6 hours.
The cells were washed with PBS and weakly acidic solution in
the same manner as described in Example 2, and then subjected
to cell fixation, cell perforation and blocking processes.
TMab4 was stained with an Alexa-488 (green
fluorescence)-labeled antibody that specifically recognizes
human Fc. The nucleus was blue-stained with Hoechst33342 and
observed with a confocal microscope. All the three mutants
showed reduced intracellular fluorescence compared to wild-
type TMab4. In particular, in the case of TMab4-03, little or
no intracellular fluorescence was observed.
b shows a graph quantitatively comparing the
number of cells that have taken up trypan blue depending on pH
by mutants constructed by substituting the CDR1 and CDR2 of
the light-chain variable region (VL) of a cytosol-penetrating
antibody, which bind to HSPG receptor and are involved in
cytosol-penetrating ability, with human germline sequences.
Specifically, Ramos cells were attached to plates in the
same manner as described in Example 5. Then, the cells were
incubated with each of buffer and 10 μM of TMab4, TMab4-01,
TMab4-02 and TMab4-03 in 200 ㎕ of each of pH 7.4 buffer
(HBSS(Welgene), 50 mM HEPES pH 7.4)(for maintaining a
cytosolic pH of 7.4) and pH 5.5 buffer (HBSS(Welgene), 50 mM
MES pH 5.5)(for maintaining an early endosomal pH of 5.5) at
37°C for 2 hours. After careful washing with PBS, 200 μl of a
mixture of 190 μl of PBS and 10 μl of trypan blue was added
to each well, and the cells were observed with a microscope.
The number of cells showing trypan blue uptake was counted
and expressed as percentage relative to the total number of
cells. A total of 400 or more cells were counted, and the
mean values are graphically shown. As a result, the mutants,
TMab4-01 and TMab4-03, showed trypan blue uptake similar to
that of TMab4. Namely, it was demonstrated that, in the
light-chain variable region, the region involved in
endocytosis (VL-CDR1 and VL-CDR2) is distinguished from the
region involved in endosomal escape (VL-CDR3).
Example 13: Logic of Improvement in Endosomal Escape
Ability of Cytosol-Penetrating Antibody
nd rd th
The 92 , 93 and 94 amino acids are expected to
increase solvent accessibility for binding to the inner
phospholipid membrane of early endosomes, which is the early
mechanism of endosomal escape, through the change in
st th
properties of VL-CDR3 by interaction with the 1 and 95
amino acids of the cytosol-penetrating light-chain variable
region. These amino acids are tyrosine (Y), tyrosine (Y) and
histidine (H), respectively.
These amino acids easily interact with 1-palmitoyl
oleoyl-sn-glycerophosphatidylcholine (POPC) which is the
major component of the inner phospholipid layer of early
endosomes.
In order to confirm that the three amino acids expected
to be exposed due to a change in pH conditions interact with
the inner phospholipid layer of early endosomes and are
involved in endosomal escape and to increase the proportion
of cytosol-penetrating antibody that escapes from endosomes,
nd rd th
mutants for the 92 , 93 and 94 amino acids were
constructed.
For mutant construction, literature search was performed,
and as a result, amino acids that easily interact with 1-
palmitoyloleoyl-sn-glycerophosphatidylcholine (POPC)
were selected (Morita et al., 2011). The mutant design was
made such that the selected amino acids are introduced into
nd rd th
the 92 , 93 and 94 amino acids.
Specifically, the average binding activity of 20
different amino acids for 1-palmitoyloleoyl-sn-glycero
phosphatidylcholine (POPC) is higher in the order of
tryptophan (W), phenylalanine (F), tyrosine (Y), leucine (L),
isoleucine (I), cysteine (C), and methionine (M).
Specifically, the binding activity of 20 different amino
acids for the hydrophilic head portion of 1-palmitoyl
oleoyl-sn-glycerophosphatidylcholine (POPC) is higher in
the order of arginine (R), tryptophan (W), tyrosine (Y),
histidine (H), asparagine (N), glutamine (Q), lysine (K),
and phenylalanine (F). In addition, the binding activity of
20 different amino acids for the hydrophobic head portion of
1-palmitoyloleoyl-sn-glycerophosphatidylcholine (POPC)
is higher in the order of tryptophan (W), phenylalanine (F),
leucine (L), methionine (M), isoleucine (I), valine V), and
tyrosine (Y).
Based on such results, it was confirmed that tryptophan
(W) is an amino acid that most easily interacts with POPC
which is the major component of the inner phospholipid layer
of early endosomes (Morita et al., 2011). Thus, in the
present disclosure, a strategy of substituting one or two
amino acids with tryptophan (W) was adopted.
Tables 4, 5 and 6 below show the sequences of the
designed mutant light-chain variable regions expected to
improve the endosomal escape ability of the human antibody
having cytosol-penetrating ability. Table 4 below shows the
full-length sequences of the light-chain variable regions of
the human antibody according to the Kabat numbering system,
and Tables 5 and 6 below show the CDR1 and CDR2 sequences or
CDR3 sequences of the antibody sequences shown in Table 4.
[Table 4]
[Table 5]
[Table 6]
Example 14: Expression and Purification of Cytosol-
Penetrating Antibody Mutants Expected to Have Increased
Endosomal Escape Ability and Confirmation of Maintenance of
Cytosol-Penetrating Ability
For animal cell expression of cytosol-penetrating
antibody mutants expected to have increased endosomal escape
ability, vectors expressing the light chain were constructed
as described in Example 1 above. To this end, DNA encoding a
light chain comprising the cytosol-penetrating light-chain
variable region (hT4 VL) or the mutant antibody’s light-chain
variable region (hT4-WWH VL, hT4-WYW VL, hT4-YWW VL, hT4-WYH
VL, hT4-YWH VL) and light chain constant region (CL), which
has a secretion signal peptide-encoding DNA fused to the 5′
end, was cloned into a pcDNA3.4 vector (Invitrogen) by use of
NotI/HindIII.
Next, a humanized hT0 VH-encoding animal expression
vector and the constructed animal expression vector encoding
the light chain comprising the light-chain variable region
expected to have increased endosomal escape ability were
transiently transfected into HEK293F protein-expressing cells.
Next, purification of the cytosol-penetrating antibody mutant
expected to have increased endosomal escape ability was
performed in the same manner as described in Example 1.
a shows the results of 12% SDS-PAGE analysis
under reducing or non-reducing conditions after purification
of cytosol-penetrating antibody mutants expected to have
improved endosomal escape ability.
Specifically, under non-reducing conditions, a molecular
weight of about 150 kDa was observed, and under reducing
conditions, the heavy chain showed a molecular weight of 50
kDa, and the light chain showed a molecular weight of 25 kDa.
This suggests that the expression and purified cytosol-
penetrating antibody mutants expected to have increased
endosomal escape ability are present as monomers in a
solution state and do not form dimers or oligomers through
non-natural disulfide bonds.
b shows the results of confocal microscopy
performed to examine whether the cytosol-penetrating ability
of cytosol-penetrating antibody mutants expected to have
improved endosomal escape ability is maintained.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2 above. When the cells were
stabilized, the cells were incubated with each of PBS and 2
μM TMab4, TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and
TMab4-YWH at 37°C for 6 hours.
The cells were washed with PBS and weakly acidic
solution in the same manner as described in Example 2, and
then subjected to cell fixation, cell perforation and
blocking processes. Each antibody was stained with an FITC
(green fluorescence)-labeled antibody that specifically
recognizes human Fc. It was found that in all the five
mutants, the cytosol-penetrating ability was maintained.
Example 15: Confirmation of pH Dependence of Cytosol-
Penetrating Antibody Mutants Expected to Have Increased
Endosomal Escape Ability
is a graph quantitatively comparing the number
of cells that have taken up trypan blue depending on pH by a
cytosol-penetrating antibody wild-type and cytosol-
penetrating antibody mutants expected to have improved
endosomal escape ability.
Specifically, Ramos cells were attached to plates in the
same manner as described in Example 5. Then, the cells were
incubated with each of 1 μM TMab4, Adalimumab, TMab4-WWH,
TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH in 200 ㎕ of
each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH
7.4(cytosol pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES
pH 5.5(early endosomal pH)) (early endosomal pH) at 37°C for
2 hours. After careful washing with PBS, 200 μl of a mixture
of 190 μl of PBS and 10 μl of trypan blue was added to each
well, and the cells were observed with a microscope. The
number of cells showing trypan blue uptake was counted and
expressed as percentage relative to the total number of cells.
A total of 400 or more cells were counted, and the mean
values are graphically shown. Among the five mutants, TMab4-
WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH showed increased
trypan blue uptake, and among them, TMab4-WYW showed pH-
dependent trypan blue uptake.
TMab4-WYW, which showed increased pH-dependent trypan
blue uptake while retaining the cytosol-penetrating ability
of the wild-type antibody, was selected as a final clone.
Example 16: Confirmation of Improvement in Cytosol
Localization of Cytosol-Penetrating Antibody Mutant Having
Increased Endosomal Escape Ability
a shows the results of observing the cytosolic
localization of a cytosol-penetrating antibody wild-type and
cytosol-penetrating antibody mutants expected to have
improved endosomal escape ability, by confocal microscopy
using calcein.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2. The cells were incubated
with PBS or 0.1 μM, 0.5 μM and 1 μM of each of TMab4 and
TMab4-WYW in serum-free medium at 37°C for 6 hours. After 4
hours, each well containing PBS or the antibody was treated
with 150 μM calcein and incubated at 37°C for 2 hours. The
cells were washed with PBS and weakly acidic solution in the
same manner as described in Example 2, and then fixed. The
nucleus was blue-stained with Hoechst33342 and observed with
a confocal microscope. It was confirmed that TMab4-WYW showed
green calcein fluorescence with higher intensity even at
lower concentration than TMab4.
b is a bar graph showing the results of
quantifying the calcein fluorescence of the confocal
micrographs shown in a.
Specifically, using Image J software (National
Institutes of Health, USA), 20 cells were selected in each
condition, and then the obtained mean values of fluorescence
are graphically shown.
Example 17: Confirmation of Cytosol Localization of
Cytosol-Penetrating Monoclonal Antibody by Enhanced Split-GFP
Complementation Assay
is a schematic view showing a process in which
GFP fluorescence by enhanced split-GFP complementation is
observed when a cytosol-penetrating antibody wild-type and a
mutant having improved endosomal escape ability localizes in
the cytosol.
Specifically, an enhanced split-GFP complementation
system was used to confirm that the cytosol-penetrating
antibody would localize to the cytosol. When the green
fluorescence protein GFP is split into a fragment 1-10 and a
fragment 11, the fluorescent property is removed, and when
the two fragments become closer to each other and are
combined with each other, the fluorescent property can be
restored (Cabantous et al., 2005).
Based on this property, the GFP fragment 1-10 was
expressed in the cytosol, and the GFP fragment 11 was fused
to the C-terminus of the cytosol-penetrating antibody. In
addition, for complementation between the GFP fragments,
streptavidin and streptavidin-binding peptide 2 (SBP2) having
high affinity were fused to the GFP fragments. Thus, the fact
that GFP fluorescence indicates that the cytosol-penetrating
antibody localizes in the cytosol.
Example 18: Expression and Purification of Cytosol-
Penetrating Antibody Fused with GFP11-SBP2
For expression of a GFP11-SBP2-fused cytosol-penetrating
antibody in animal cells, GFP11-SBP2 was genetically fused to
the C-terminus of the heavy chain by three GGGGS linkers.
Next, the animal expression vector encoding the cytosol-
penetrating light chain or the cytosol-penetrating light
chain having increased endosomal escape ability, and the
animal expression vector encoding the GFP11-SBP2-fused heavy
chain, were transiently co-transfected. Next, purification of
the GFP11-SBP2-fused cytosol-penetrating antibody was
performed in the same manner as described in Example 1.
shows the results of 12% SDS-PAGE analysis under
reducing or non-reducing conditions after purification of a
GFP11-SBP2-fused cytosol-penetrating antibody wild-type and a
GFP11-SBP2-fused mutant having improved endosomal escape
ability.
Specifically, under non-reducing conditions, a molecular
weight of about 150 kDa was observed, and under reducing
conditions, the heavy chain showed a molecular weight of 50
kDa, and the light chain showed a molecular weight of 25 kDa.
This suggests that the expressed purified GFP11-SBP2-fused
cytosol-penetrating antibody is present as a monomer in a
solution state and does not form a dimer or an oligomer by a
non-natural disulfide bond.
Example 19: Examination of GFP Fluorescence with Cytosol
Localization of GFP11-SBP2-Fused Cytosol-Penetrating Antibody
a shows the results of confocal microscopy
performed to examine the GFP fluorescence of a GFP11-SBP2-
fused cytosol-penetrating antibody wild-type and a GFP11-
SBP2-fused mutant having improved endosomal escape ability by
enhanced split-GFP complementation.
Specifically, transformed HeLa cells stably expressing
SA-GFP1-10 were prepared in the same manner as described in
Example 2. When the cells were stabilized, the cells were
incubated with PBS or 0.2, 0.4, 0.6, 0.8, 1.6 or 3.2 μM of
each of TMab4-GFP11-SBP2 and TMab4-WYW-GFP11-SBP2 at 37°C for
6 hours.
In the same manner as described in Example 2, the cells
were washed with PBS and weakly acidic solution, and then
fixed. The nucleus was blue-stained with Hoechst 33342 and
observed with a confocal microscope. It was observed that
TMab4-WYW showed green GFP fluorescence with higher intensity
at lower concentration than TMab4.
b is a graph showing the results of quantifying
the GFP fluorescence of the confocal micrographs shown in a.
Specifically, using Image J software (National
Institutes of Health, USA), 20 cells were selected in each
condition, and then the obtained mean values of fluorescence
are graphically shown.
In order to quantitatively express and compare the
intracytosolic concentrations and endosomal escape
efficiencies of the GFP11-SBP2-fused intact IgG-format
antibody and the cytosol-penetrating antibody having
increased endosomal escape ability, an experiment was
performed.
Table 7 below shows the intracytosolic concentrations
and endosomal escape efficiencies of the GFP11-SBP2-fused
intact IgG-format antibody and the cytosol-penetrating
antibody having increased endosomal escape ability.
[Table 7]
Example 20: In-Depth Analysis of Interaction between
Cytosol-Penetrating Antibody Having Increased Endosomal
Escape Ability and Lipid
nd th
It was found that when the 92 and 94 amino acids of
the light-chain variable region CDR3 of the wild-type
cytosol-penetrating antibody were substituted with tryptophan,
the endosomal escape ability was increased.
In order to determine whether this increase in the
endosomal escape ability is due to improved interaction with
any part of the lipid, an experiment was performed.
Tryptophan (W) is an amino acid that easily interacts with
both the hydrophilic head and hydrophobic tail of the lipid.
When tryptophan is substituted with arginine (R) (which
easily interacts with the hydrophilic head), isoleucine (I)
(which easily interacts with the hydrophilic tail) or glycine
(G) (which very weakly interacts with the lipid) and the
activities are compared, it can be seen that the interaction
with any part of the lipid plays an important role.
In order to analyze in depth the interaction between the
cytosol-penetrating antibody having increased endosomal
escape ability and the lipid, mutants were constructed by
substituting tryptophan with each of arginine (R), isoleucine
(I) and glycine (G).
Table 8 below shows the names and sequences of the
mutants constructed using an overlap PCR technique.
[Table 8]
In the same manner as described in Example 1, cloning,
expression in HEK293F cell lines, and purification were
performed.
Example 21: In-Depth Analysis of Interaction between
Cytosol-Penetrating Antibody and Lipid
a is a graph showing the results of flow
cytometry (FACS) performed to analyze the cell membrane
binding of mutants obtained by substitution with arginine,
isoleucine and glycine, which are amino acids having
properties opposite to those of tryptophan.
Specifically, 1 x 10 Ramos cells were prepared for each
well. The cells were washed with PBS, and then incubated with
each of 3 μM TMab4, TMab4-WYW, TMab4-RYR, TMab4-IYI and
TMab4-GYG in each of pH 7.4 buffer (TBS, 2% BSA, 50 mM HEPES
pH 7.4(cytosolic pH)), and pH 5.5 buffer (TBS, 2% BSA, 50 mM
MES pH 5.5(early endosomal pH)) at 4°C for 1 hour. After
washing with each pH buffer, TMab4, TMab4-WYW, TMab4-RYR,
TMab4-IYI and TMab4-GYG were incubated with an FITC (green
fluorescence)-labeled antibody (which specifically recognizes
human Fc) at 4°C for 30 minutes. After washing with PBS, the
cells were analyzed by flow cytometry, and as a result, it
was found that, at pH 5.5, only TMab4 did bind to the cell
membrane.
b is a graph quantitatively comparing the number
of cells that have taken up trypan blue depending on pH by
mutants obtained by substitution with arginine, isoleucine
and glycine, which are amino acids having properties opposite
to those of tryptophan.
Specifically, Ramos cells were attached to plates in the
same manner as described in Example 5. Then, the cells were
incubated with each of 1 μM TMab4, TMab4-WYW, TMab4-RYR,
TMab4-IYI and TMab4-GYG in 200 μl of each of pH 7.4 buffer
(HBSS(Welgene), 50 mM HEPES pH 7.4 (cytosolic pH)) and pH 5.5
buffer (HBSS(Welgene), 50 mM MES pH 5.5(early endosomal pH))
at 4°C for 2 hours. After careful washing with PBS, 200 μl of
a mixture of 190 μl of PBS and 10 μl of trypan blue was added
to each well, and the cells were observed with a microscope.
The number of cells showing trypan blue uptake was counted
and expressed as percentage relative to the total number of
cells. A total of 400 or more cells were counted, and the
mean values are graphically shown. As a result, TMab4-RYR,
TMab4-IYI and TMab4-GYG showed reduced trypan blue uptake
compared to TMab4-WYW.
c is a bar graph showing the results of observing
the cytosolic localization of mutants obtained by
substitution with arginine, isoleucine and glycine, which are
amino acids having properties opposite to those of tryptophan
by confocal microscopy using calcein and quantifying the
calcein fluorescence of the confocal micrographs.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2. The cells were incubated
with each of 0.5 μM TMab4, TMab4-WYW, TMab4-RYR, TMab4-IYI
and TMab4-GYG 0.5 μM at 37°C for 6 hours. After 4 hours, each
well containing PBS or the antibody was treated with 150 μM
calcein 150 μM and incubated at 37°C for 2 hours. In the same
manner as described in Example 2, the cells were washed with
PBS and weakly acidic solution, and then fixed. The nucleus
was blue-stained with Hoechst33342 and observed with a
confocal microscope. In the cells treated with TMab4-RYR,
TMab4-IYI or TMab4-GYG, the green calcein fluorescence
localized in the cytosol was weaker than that in the cells
treated with TMab4-WYW.
Therefore, it was confirmed that interactions with all
the hydrophilic head and hydrophobic tail of the lipid were
involved in endosomal escape, and for this reason,
substitution with tryptophan increased the endosomal escape
ability.
Example 22: Expression and Purification of Intact IgG-
Format Anti-Tubulin Cytosol-Penetrating Antibody
The mutant having increased endosomal escape ability can
more effectively target a protein located in the cytosol,
because the amount of antibody located in the cytosol will
increase.
a is a schematic view showing a process of
constructing an intact IgG-format anti-tubulin cytosol-
penetrating antibody to be used to examine the activity of
cytosol-penetrating antibody mutants having improved
endosomal escape ability.
For the purpose of expression of an intact IgG-format
anti-tubulin cytosol-penetrating antibody in animal cells,
DNA encoding a heavy chain comprising the heavy-chain
variable region and heavy chain constant region (CH1-hinge-
CH2-CH3) binding specifically to cytoskeletal tubulin, which
has a secretion signal peptide-encoding DNA fused to the 5’
end, was cloned into a pcDNA3.4 vector (Invitrogen) by use of
NotI/HindIII (Laurence et al., 2011).
Next, the animal expression vector encoding the cytosol-
penetrating light chain or the cytosol-penetrating light
chain having increased endosomal escape ability, and the
constructed animal expression vector encoding the heavy chain
comprising the heavy-chain variable region that specifically
to tubulin, were transiently co-transfected into HEK293F
protein-expressing cells. Next, purification of the intact
IgG-format anti-tubulin cytosol-penetrating antibody was
performed in the same manner as described in Example 1.
b shows the results of 12% SDS-PAGE analysis
under reducing or non-reducing conditions after purification
of an intact IgG-format anti-tubulin cytosol-penetrating
antibody.
Specifically, under non-reducing conditions, a molecular
weight of about 150 kDa was observed, and under reducing
conditions, the heavy chain showed a molecular weight of 50
kDa, and the light chain showed a molecular weight of 25 kDa.
This suggests that the expressed purified intact IgG-format
anti-tubulin cytosol-penetrating antibody is present as a
monomer in a solution state and does not form a dimer or an
oligomer by a non-natural disulfide bond.
Example 23: Confirmation of Cytoskeletal Tubulin-
Specific Binding of Intact IgG-Format Anti-Tubulin Cytosol-
Penetrating Antibody
c shows the results of confocal microscopy
performed to examine whether an intact IgG-format anti-
tubulin cytosol-penetrating antibody would merge with
cytoskeletal tubulin localized in the cytosol.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2. The cells were incubated
with PBS or each of 3 μM TuT4 and TuT4-WYW in 500 μl of 10%
FBS-containing medium at 37°C for 6 hours. The cells were
washed with PBS and weakly acidic solution in the same manner
as described in Example 2, and then subjected to cell
fixation, cell perforation and blocking processes.
Cytoskeletal tubulin was incubated with anti-tubulin
antibody (Santa Cruz) at 25°C for 1 hour, and incubated with
TRITC (red fluorescence)-labeled secondary antibody at 25°C
for 1 hour. Each antibody was stained with an FITC (green
fluorescence)-labeled antibody that specifically recognizes
human Fc. The nucleus was blue-stained with Hoechst33342 and
observed with a confocal microscope.
As shown in c, with the cytosol portion in which
red fluorescent tubulin was localized, green fluorescent
TuT4-WYW was merged in a fibrillar shape, but TuT4 was not
merged.
Example 24: Expression and Purification of Intact IgG-
Format RAS-Targeting Cell-Penetrating Antibody and Analysis
of Affinities of K-RAS Mutants
In order to confirm whether the cytosol-penetrating
antibody can effectively target other intracytosolic proteins
in addition to cytoskeletal tubulin, an experiment was
performed.
a is a schematic view showing a process of
constructing an intact IgG-format RAS-targeting cytosol-
penetrating antibody to be used to examine the activity of
mutants having improved endosomal escape ability.
For the purpose of expression of an intact IgG-format
Ras-targeting cytosol-penetrating antibody in animal cells,
DNA encoding a heavy chain the heavy-chain variable region
(RT11 VH) and heavy chain constant region (CH1-hinge-CH2-CH3)
binding specifically to GTP-bound K-RAS, which has a
secretion signal-encoding DNA fused to the 5’ end, was cloned
into a pcDNA3.4 vector (Invitrogen) by use of NotI/HindIII as
described in Example 5.
Next, the animal expression vector encoding the cytosol-
penetrating light chain or the cytosol-penetrating light
chain having increased endosomal escape ability, and the
constructed animal expression vector encoding the heavy chain
comprising the heavy-chain variable region that binds
specifically to GTP-bound K-RAS, were transiently co-
transfected into HEK293F protein-expressing cells. Next,
purification of the intact IgG-format Ras-targeting cytosol-
penetrating antibody was performed in the same manner as
described above.
b shows the results of 12% SDS-PAGE analysis
under reducing or non-reducing conditions after purification
of intact IgG-format RAS-targeting cytosol-penetrating
antibodies.
Specifically, under non-reducing conditions, a molecular
weight of about 150 kDa was observed, and under reducing
conditions, the heavy chain showed a molecular weight of 50
kDa, and the light chain showed a molecular weight of 25 kDa.
This suggests that the expressed purified intact IgG-format
Ras-targeting cytosol-penetrating antibody is present as a
monomer in a solution state and does not form a dimer or an
oligomer by a non-natural disulfide bond.
c shows the results of enzyme linked
immunosorbent assay performed to measure the affinities of
antibodies for GppNHp-bound K-RAS G12D and GDP-bound K-RAS
G12D, which are K-RAS mutants.
Specifically, GTP is very easily hydrolyzed, and hence
it is difficult to maintain the morphology of GTP-bound K-RAS
G12D. Thus, in order to enable K-RAS to have an activated
structure, like GTP, a GTP-bound K-RAS G12D antigen was
constructed using GppNHp which is a non-hydrolyzable GTP
analogue. Each of a GppNHp-bound K-RAS G12D and a GDP-bound
K-RAS G12D, which are target molecules, was incubated in 96-
well EIA/RIA plates (COSTAR Corning) at 37°C for 1 hour,
followed by washing three times with 0.1% PBST (0.1 %
Tween20, pH 7.4, 137 mM NaCl, 12mM phosphate, 2.7 mM KCl)
(SIGMA) for 10 minutes each time. Each well was incubated
with 5% PBSS (5% Skim milk, pH7.4, 137 mM NaCl, 12mM
phosphate, 2.7 mM KCl) (SIGMA) for 1 hour, and then washed
three times with 0.1% PBST for 10 minutes. Next, each well
was incubated with each of the IgG-format cytosol-penetrating
antibodies (TMab4, RT11, and RT11-WYW), and then washed three
times with 0.1% PBST for 10 minutes. As a marker antibody,
goat alkaline phosphatase-conjugated anti-human mAb (SIGMA)
was used. Each well was treated with pNPP (p-nitrophenyl
palmitate) (Sigma), and the absorbance at 405 nm was measured.
Affinities for the K-RAS mutants were analyzed, and as a
result, it was shown that there was little or no difference
in affinity between wild-type RT11 and mutant RT11-WYW. TMab4
used as a negative control did not bind, and all the clones
did not bind to the GDP-bound K-RASs.
Example 25: Confirmation of Specific Binding between
Intact IgG-Format Anti-RAS Cytotransmab and GTP-Bound K-RAS
in Cells
shows the results of confocal microscopy
observation performed to examine whether intact IgG-format RAS-
targeting cytosol-penetrating antibodies would merge with
intracellular H-RAS G12V mutants.
Specifically, fibronectin (Sigma) was coated on a 24-well
plate, and then 0.5 ml of a dilution of 2 x 10 NIH3T3 cells
expressing mCherry (red fluorescence) H-RAS G12V was added to
each well and incubated at 37°C in 5% CO for 12 hours. Next,
the cells were treated with each of 2 μM TMab4, RT11 and RT11-
WYW and incubated at 37°C for 12 hours. Next, the cells were
stained in the same manner as described in Example and were
observed with a confocal microscope.
As shown in , with the inner cell membranes in which
red fluorescent activated RAS was located, green fluorescent
RT11 or RT11-WYW was merged, but TMab4 was not merged.
From the above results, it was found that the intact IgG-
format Ras-targeting cytosol-penetrating antibody did bind
specifically to activated RAS in cells. The degree of merging
was higher in the order of RT11-WYW and RT11.
Example 26: Analysis of Properties of D1-M95 Inducing
Structural Change Depending on pH
For more detailed analysis of the properties of the 1
amino acid aspartic acid (D) and 95 amino acid methionine
(M) of the light-chain variable region (VL), which induce a
change in the properties of the cytosol-penetrating antibody
depending on pH, mutants were constructed by substituting the
1 amino acid in the antibody backbone with each of glutamic
acid (E), alanine (A) and asparagine (N) present in the
germline sequences, and substituting the 95 amino acid in
the CDR3 with each of all the 20 amino acids.
When the mutants were constructed, the 87 amino acid
tyrosine was substituted with phenylalanine in order to
increase the protein expression yield that decreased by the
improved endosomal escape motif. Phenylalanine is an amino
acid that can easily interact with the aromatic ring amino
acids and hydrophobic amino acids located in the backbone of
the heavy-chain variable region, thus enhancing the interface
between the light-chain variable region and the heavy-chain
variable region. The light-chain variable region, in which
the 87 amino acid is substituted with phenylalanine and
which has the improved endosomal escape motif WYW at the 92 ,
rd th
93 and 94 amino acid, was named ‘hT4-3’. Thus, the cytosol-
penetrating intact IgG-format antibody comprising the light-
chain variable region was named ‘TMab4-3’.
In the same manner as described in Example 1, each of
the mutants was cloned, expressed in HEK293F cell lines, and
purified.
b is a graph showing the results of
quantitatively comparing the number of cells that have taken
up trypan blue depending on pH by mutants constructed by
substituting 95 amino acid methionine of the light-chain
variable region (VL) of a cytosol-penetrating antibody, which
induce a structural change of the cytosol-penetrating
antibody at acidic pH 5.5, with various amino acids.
Specifically, 1 x 10 adherent cells (pgsD-677)
expressing no HSPG receptor were incubated. On the next day,
in the same manner as described in Example 5, the cells were
incubated with each of 1 μM TMab4-3, TMab4-3 D1A, TMab4-3 D1E
and TMab4-3 D1N in 200 μl of each of pH 7.4 buffer
(HBSS(Welgene), 50 mM HEPES pH 7.4)(for maintaining cytosolic
pH) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES pH 5.5)(for
maintaining early endosomal pH) at 37°C for 2 hours. After
careful washing with PBS, 200 μl of a mixture of 190 μl of
PBS and 10 μl of trypan blue was added to each well, and the
cells were observed with a microscope. Next, after careful
washing with PBS, the cells were lysed by adding 50 μl of 1%
SDS (sodium dodecyl sulfate) to each well. The cells were
transferred to a 96-well plate, and the absorbance at 590 nm
was measured.
As a result, TMab4-3 D1E showed trypan blue uptake
similar to that of the wild-type, and the TMab4-3 D1A and
TMab4-3 D1N mutants showed reduced trypan blue uptake.
b is a graph showing the results of
quantitatively comparing the number of cells that have taken
up trypan blue depending on pH by mutants constructed by
substituting 95 amino acid methionine of the light-chain
variable region (VL) of a cytosol-penetrating antibody, which
induce a structural change of the cytosol-penetrating
antibody at acidic pH 5.5, with various amino acids.
Specifically, pgsD-677 cells were prepared in the same
manner as described in Example 26. Then, in the same manner
as in described Example 5, the cells were incubated with 1 μM
of each of TMab4-3 and nineteen TMab4-3 mutants in 200 μl of
each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH 7.4
(cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES
pH 5.5 (early endosomal pH)) at 37°C for 2 hours. After
careful washing with PBS, 200 μl of a mixture of 190 μl of
PBS and 10 μl of trypan blue was added to each well, and the
cells were observed with a microscope. Next, after careful
washing with PBS, the cells were lysed by adding 50 μl of 1%
SDS (sodium dodecyl sulfate) to each well.
The cells were transferred to a 96-well plate, and the
absorbance at 590 nm was measured. As a result, TMab4-3 M95L,
M95I and M95H showed trypan blue uptake similar to that of
TMab4-3, and TMab4-3 M95A, M95S, M95V, M95G and M95P mutants
showed reduced trypan blue uptake. In addition, TMab4-3 M59D
and M59E showed increased pH-dependent trypan blue uptake,
but TMab4-3 M95K and M95R mutants showed increased trypan blue
uptake at neutral pH.
Therefore, it was found that interaction between
hydrophobic amino acids having long side chains, negatively
charged amino acids, and histidine (H), is most effective for
inducing structural changes at acidic pH.
When the 95 amino acid of the light-chain variable region
is composed of the hydrophobic amino acid methionine (M),
isoleucine (I) or leucine (L), or the negatively charged amino
acid aspartic acid (D) or glutamic acid (E), it is expected that
the carboxylic acid in the side chain of the negatively charged
amino acid will become hydrophobic by partial protonation, and
thus the 95 amino acid will hydrophobically interacts with
aspartic acid (D) or glutamic acid (E), which is the 1 amino
acid of the light-chain variable region or heavy-chain variable
region (Du Z et al., 2011; Di Russo et al., 2012).
In addition, when the 95 amino acid of the light-chain
variable region is composed of histidine (H), it is expected
that as pH change from 7.4 to 5.5, the net charge of the amino
acid side chains will become positive, and the 95 amino acid
will induce endosomal escape by electrostatic interaction with
aspartic acid (D) or glutamic acid (E), which is the 1 amino
acid of the light-chain variable region or heavy-chain variable
region.
Example 27: Design of Mutants Introduced with Amino Acids
That ‘Induce Change in Properties in Response to pH’
In addition to D1-M95, the present inventors have attempted
to introduce amino acids capable of inducing endosomal escape
by changing their interaction depending on pH.
Based on the results of structural modeling analysis, the
th st st
90 and 91 amino acids capable of interacting with the 1
amino acid aspartic acid (D) were selected as possible
candidates. To enable interaction under acidic pH conditions,
the 90 amino acid was replaced with histidine (TMab4-3 Q90H),
and the 91 amino acid was substituted with histidine (TMab4-3
Y91H).
In addition, the 91 amino acid capable of additionally
interacting with the 2 hydrophobic amino acid was substituted
with aspartic acid (TMab4-3 Y91D).
In addition, the 2 amino acid was also substituted with
negatively charged glutamic acid (E) so that it could interact
st th
with the 1 negatively charged amino acid, and the 90 amino
acid was substituted with leucine (L) (TMab4-3 L2E Q90L) so
that it could interact with the 95 hydrophobic amino acid.
Furthermore, the 2 amino acid was also substituted with
glutamic acid (E) so that it could interact with the 1
negatively charged amino acid, and the 97 amino
acid was substituted with isoleucine (I) (TMab4-3 L2E T97I)
so that it could interact with the 95 hydrophobic amino acid.
Table 9 below shows the names and sequences of the
mutants constructed using an overlap PCR technique.
[Table 9]
In the same manner as described in Example 1, each of
the mutants was cloned, expressed in HEK293F cell lines, and
purified.
Example 28: Confirmation of Improvement in Endosomal
Escape Ability of Mutants Introduced with Amino Acids That
‘Induce Change in Properties in Response to pH’
a shows a graph showing quantitatively comparing
the number of cells that have taken up trypan blue depending
on pH by mutants designed for the purpose of ‘inducing an
additional change in properties in response to pH’.
Specifically, cells were prepared in the same manner as
described in Example 26. Then, in the same manner as
described in Example 5, the cells were incubated with 0.5 or
1 μM of each of seven mutants (including TMab4-3, TMab4-3
D1E-M95L, etc.) in 200 μl of each of pH 7.4 buffer
(HBSS(Welgene), 50 mM HEPES pH 7.4(cytosolic pH)) and pH 5.5
buffer (HBSS(Welgene), 50 mM MES pH 5.5(early endosomal pH))
at 37°C for 2 hours. After careful washing with PBS, 200 μl
of a mixture of 190 μl of PBS and 10 μl of trypan blue was
added to each well, and the cells were observed with a
microscope. Next, after careful washing with PBS, the cells
were lysed by adding 50 μl of 1% SDS (sodium dodecyl sulfate)
to each well. The cells were transferred to a 96-well plate,
and the absorbance at 590 nm was measured.
As a result, the TMab4-3 Q90H mutant showed higher
trypan blue uptake than TMab4-3 at 0.5 μM. Additionally,
using the TMab4-3 Q90H mutant showing a significant
difference from the wild-type, an experiment was performed.
b shows a bar graph showing the results of
observing the cytosolic localization of mutants designed for
the purpose of inducing an additional change in properties in
response to pH by confocal microscopy using calcein and
quantifying the calcein fluorescence of the confocal
micrographs.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2, and the cells were
incubated with 0.5 μM and 1 μM of each of TMab4-3 and TMab4-3
Q90H at 37°C for 6 hours. After 4 hours, each well containing
PBS or the antibody was treated with 150 μM calcein and
incubated at 37°C for 2 hours. The cells were washed with PBS
and weakly acidic solution in the same manner as described in
Example 2, and then fixed. The nucleus was blue-stained with
Hoechst33342 and observed with a confocal microscope.
As a result, in the cells treated with TMab4-3 Q90H,
green calcein fluorescence localized in the cytosol increased
compared to that in the cells treated with TMab4-3. Therefore,
it was confirmed that, in addition to the 95 amino acid of
the light-chain variable region of the cytosol-penetrating
th st
antibody, the 90 amino acid interacted with the 1 amino
acid and induced endosomal escape by a pH-dependent change in
the interaction.
Table 10 below the CDR3 sequence of the light-chain
variable region of the mutant having an increased ability to
escape from endosomes by inducing an additional change in the
properties depending on pH.
[Table 10]
Example 29: Investigation of Endosomal Escape Ability at
Varying Lengths of CDR3 of Light-Chain Variable Region
85% or more of the CDR3 of the light-chain variable
region consists of 9 amino acids. Depending on the number and
composition of amino acids, the CDR3 loop structure varies.
In the present disclosure, to analyze how the endosomal
escape ability changes depending on the number and
composition of amino acids, mutants comprising a CDR3
consisting of 8, 10 or 11 amino acids were constructed.
Table 11 below shows the names and sequences of the
mutants constructed using an overlap PCR technique.
[Table 11]
In the same manner as described in Example 1, each of
the mutants was cloned, expressed in HEK293F cell lines, and
purified.
is a graph quantitatively comparing the number
of cells that taken up trypan blue depending on pH by mutants
obtained by changing the amino acid number of the CDR3 of the
light-chain variable region of a cytosol-penetrating antibody.
Specifically, cells were prepared in the same manner as
described in Example 26. Then, in the same manner as
described in Example 5, the cells were incubated with 1 μM of
each of seven mutants (including TMab4-3, TMab4-3 L8-1, etc.)
in 200 μl of each of pH 7.4 buffer (HBSS(Welgene), 50 mM
HEPES pH 7.4(cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene),
50 mM MES pH 5.5(early endosomal pH)) at 37°C for 2 hours.
After careful washing with PBS, 200 μl of a mixture of 190 μl
of PBS and 10 μl of trypan blue was added to each well, and
the cells were observed with a microscope. Next, after
careful washing with PBS, the cells were lysed by adding 50
μl of 1% SDS (sodium dodecyl sulfate) to each well. The cells
were transferred to a 96-well plate, and the absorbance at
590 nm was measured.
As a result, in comparison with TMab4-3, the mutants
showed increased trypan blue uptake at neutral pH as the
number of amino acids increased. The reason is believed to
be as follows. As the number of amino acids increases, the
overall CDR3 loop structure is stretched, and the endosomal
escape motif WYW which binds to the cell membrane in order to
escape from endosomes is exposed to the outside, and thus
trypan blue uptake increases even at neutral pH.
In addition, in the experimental results, it was
confirmed that even when the distance between the 95 amino
acid, which induces endosomal escape by a pH-dependent change
nd rd th
in interaction, and the 92 , 93 or 94 amino acid, which
influences endosomal escape by binding to the phospholipid,
increases, the properties of the endosomal escape motif are
maintained.
Example 30: Logic of Possibility of Imparting Improved
Endosomal Escape Motif to Light-Chain Variable Region of
Conventional Therapeutic Antibody
Currently commercially available therapeutic antibodies
include many kinds of monoclonal antibodies that target cell
surface receptors, particularly cell surface receptors that
undergo endocytosis. However, these conventional antibodies
have disadvantages in that their binding to antigen is not
broken after endocytosis, and these antibodies do not
localize in the cytosol and are released out of the cells
because they have no endosomal escape ability. Thus, if
endosomal escape ability can be imparted to these receptor-
targeting antibodies that undergo endocytosis, there is an
advantage in that these antibodies can be used in a wider
range of applications.
In addition, the use of the stable backbone of
commercially available therapeutic antibodies can increase
the overall expression yield, and when the affinity of these
antibodies for HSPG, a non-tumor-specific receptor, is
eliminated, tumor tissue specificity can be imparted to these
antibodies.
To impart an improved endosomal escape motif, the
sequences of the light-chain variable regions of receptor-
targeting antibodies that undergo endocytosis were compared
with the sequence of the light-chain variable region of the
cytosol-penetrating antibody. As a result, candidate light-
chain variable regions were selected, which have a negatively
charged amino acid as the 1 amino acid and in which
backbone amino acids that can influence the CDR3 loop
structure are the same as those of the cytosol-penetrating
antibody.
Mutants were constructed by the CDR3 sequences of the
candidate light-chain variable regions with the CDR3 sequence
of the cytosol-penetrating antibody.
Table 12 shows the names and sequences of the mutants
constructed using a genesis synthesis technique.
[Table 12]
a shows a process of constructing an intact IgG-
format RAS-targeting cytosol-penetrating antibody in which an
improved endosomal escape motif is introduced into the light-
chain variable region of a conventional therapeutic antibody.
As shown in a, in the same manner as described in
Example 1, cloning of the light-chain variable region was
performed, and the resulting animal expression vector and he
animal expression vector encoding the heavy chain comprising
the heavy-chain variable region that binds specifically to
GTP-bound K-RAS were transiently co-transfected into HEK293F
protein-expressing cells. Next, purification of the resulting
intact IgG-format anti-RAS cytotransmab was performed in the
same manner as described in Example 1.
Example 31: Confirmation of Possibility of Imparting
Improved Endosomal Escape Motif to Light-Chain Variable
Region of Conventional Therapeutic Antibody
b shows the results of fluorescence microscopic
observation performed to examine whether the HSPG binding
affinity and cytosol-penetrating ability of an intact IgG-
format RAS-targeting cytosol-penetrating antibody in which an
improved endosomal escape motif is introduced into the light-
chain variable region of a conventional therapeutic antibody
would be reduced or eliminated.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2. When the cells were
stabilized, the cells were incubated with PBS or each of 1 μM
RT11-3, RT11-Neci-WYW, RT11-Nimo-WYW, RT11-Pani-WYW, RT11-
Pert-WYW, RT11-Lumr-WYW and RT11-Emib-WYW at 37°C for 6 hours.
The cells were washed with PBS and weakly acidic
solution in the same manner as described in Example 2, and
then subjected to cell fixation, cell perforation and
blocking processes. Each of the antibodies was stained with
an FITC (green fluorescence)-labeled antibody that
specifically recognizes human Fc. The nucleus was blue-
stained with Hoechst33342 and observed with a confocal
microscope. As a result, in all the six intact IgG-format
RAS-targeting cytosol-penetrating antibodies comprising the
monoclonal antibody backbone imparted with the improved
endosomal escape motif, no fluorescence was observed.
c shows a graph quantitatively comparing the
number of cells that taken up trypan blue at acidic pH by an
intact IgG-format RAS-targeting cytosol-penetrating antibody
in which an improved endosomal escape motif is introduced
into the light-chain variable region of a conventional
therapeutic antibody.
Specifically, Ramos cells were attached to plates in the
same manner as described in Example 5. Then, the cells were
incubated with each of 1 μM RT11-3, RT11-Neci-WYW, RT11-Nimo-
WYW, RT11-Pani-WYW, RT11-Pert-WYW, RT11-Lumr-WYW, and RT11-
Emib-WYW in 200 μl of pH 7.4 buffer (HBSS(Welgene), 50 mM
HEPES pH 7.4(cytosolic pH)) and pH 5.5 buffer(HBSS(Welgene),
50 mM MES pH 5.5(early endosomal pH)) at 37°C for 2 hours.
After careful washing with PBS, 200 μl of a mixture of 190 μl
of PBS and 10 μl of trypan blue was added to each well, and
the cells were observed with a microscope.
The number of cells showing trypan blue uptake was
counted and expressed as percentage relative to the total
number of cells. A total of 400 or more cells were counted,
and the mean values are graphically shown. As a result,
except for RT11-Pert, the five intact IgG-format RAS-
targeting cytosol-penetrating antibodies comprising the
therapeutic antibody backbone imparted with the improved
endosomal escape motif showed trypan blue uptake similar to
that of RT11-3.
Example 32: Confirmation of Maintenance of Specific
Binding between Intact IgG-Format RAS-Targeting Cytosol-
Penetrating Antibody Comprising Therapeutic Antibody Backbone
Imparted with Improved Endosomal Escape Motif and GTP-Bound
K-RAS
a shows the results of ELISA performed to measure
the affinities of an intact IgG-format RAS-targeting cytosol-
penetrating antibody, in which an improved endosomal escape
motif is introduced into the light-chain variable region of a
conventional therapeutic antibody, for GppNHp-bound K-RAS
G12D and GDP-bound K-RAS G12D, which are K-RAS mutants.
Specifically, each of a GppNHp-bound K-RAS G12D and a
GDP-bound K-RAS G12D, which are target molecules, was
incubated in 96-well EIA/RIA plates (COSTAR Corning) at 37°C
for 1 hour, followed by washing three times with 0.1% PBST
(0.1 % Tween20, pH 7.4, 137 mM NaCl, 12mM phosphate, 2.7 mM
KCl) (SIGMA) for 10 minutes. Each well was incubated with 5%
PBSS (5% Skim milk, pH7.4, 137 mM NaCl, 12mM phosphate, 2.7
mM KCl) (SIGMA) for 1 hour, and then washed three times with
0.1% PBST for 10 minutes. Next, each well was incubated with
each of the IgG-format RAS-targeting cytosol-penetrating
antibodies (RT11-3, RT11-Neci-WYW, RT11-Nimo-WYW, RT11-Pani-
WYW, RT11-Pert-WYW, RT11-Lumr-WYW, RT11-Emib-WYW), and then
washed three times with 0.1% PBST for 10 minutes. As a marker
antibody, goat alkaline phosphatase-conjugated anti-human mAb
(SIGMA) was used. Each well was treated with pNPP (p-
nitrophenyl palmitate) (Sigma), and the absorbance at 405 nm
was measured.
Affinities for the K-RAS mutants were analyzed. As a
result, except for RT11-Nimo, the five intact IgG-format RAS-
targeting cytosol-penetrating antibodies comprising the
therapeutic antibody backbone imparted with the endosomal
escape motif showed no difference in affinity from RT11-3,
and all the clones did not bind to the GDP-bound K-RASs used
as negative controls.
b shows a schematic view showing a process of
constructing an intact IgG-format RAS-targeting cytosol-
penetrating antibody in which an improved endosomal escape
motif is introduced into the RGD10 peptide-fused light-chain
variable region of a conventional therapeutic antibody.
Because the intact IgG-format RAS-targeting cytosol-
penetrating antibody imparted with the improved endosomal
escape motif showed no cell-penetrating ability, an RGD10
peptide specific for integrin αvβ3 which is overexpressed in
neovascular cells and various tumors was genetically fused to
the N-terminus of the light chain by two GGGGS linkers. The
RGD10 peptide has an affinity similar to that of a RGD4C
peptide, but has characteristics in that it has a single
disulfide bond formed by two cysteine residues and can be
genetically fused.
In addition, based on the results of analysis of
expression yield, endosomal escape ability, and affinity for
Ras, the RGD10 peptide was fused to the N-terminus of the
light-chain variable region of each of RT11-Pani-WYW and
RT11-Neci-WYW which are excellent candidate antibodies.
c shows the results of confocal microscopy
performed to examine whether an intact IgG-format RAS-
targeting cytosol-penetrating antibody in which an improved
endosomal escape motif is introduced into the RGD10 peptide-
fused light-chain variable region of a conventional
therapeutic antibody would merge with intracellular activated
H-RAS G12V mutants.
Specifically, 0.5 ml of a dilution of 2 x 10 human
colorectal cancer SW480 cells having a K-RAS G12V mutation
were added to each of a 24-well plate and incubated with each
of 1 μM RT11-i3, RT11-i-Neci-WYW and RT11-i-Pani-WYW at 37°C
in 5% CO for 12 hours. Next, in the same manner as described
in Example 2, antibody labeling and nucleus staining were
performed, and the cells were treated with a Ras-labeled
antibody at 37°C for 1 hour. Then, the cells were secondary
antibody and observed with a confocal microscope.
With the inner cell membrane in which the red
fluorescent activated RAS was located, green fluorescent
RT11-i3, RT11-i-Neci-WYW and RT11-i-Pani-WYW were merged.
The above experimental results indicate that the intact
IgG-format RAS-targeting cytosol-penetrating antibody
introduced with the improved endosomal escape motif binds
specifically to activated RAS in cells.
Example 33: Logic of Possibility of Imparting Improved
Endosomal Escape Motif to CDR of Heavy-Chain Variable Region
The heavy-chain variable region and the light-chain
variable region are structurally common in that they have a
beta-sheet structure as a backbone and are composed of three
CDR having a loop structure. Thus, it was considered that the
endosomal escape motif of the light-variable variable region,
which induces endosomal escape by a pH-dependent change in
interaction, can also be applied to the heavy-chain variable
region.
Whether this phenomenon is reproducible in the heavy-
chain variable region was analyzed through the sequence and
three-dimensional structure of the heavy-chain variable
region. As a result, the endosomal escape motif could be
grafted into the CDR3 at a distance that can interact with
the 1 amino acid glutamic acid (E) of the heavy-chain
variable region.
The number of amino acids in the CDR3 of the wild-type
heavy-chain variable region is 11, and the center of the loop
structure of the CDR3 is significantly exposed to the surface.
For this reason, it is considered that the pH-dependent
phenomenon occurring in the light-chain variable region
hardly occurs. For this reason, the amino acid number of the
CDR3 was reduced to 7 or 8 while maintaining a portion of the
sequence.
In addition, it was considered that an amino acid
capable of interacting with the 1 amino acid of the heavy-
chain variable region at early endosomal pH is the 102 amino
acid of the heavy-chain variable region and this amino acid
is located at a suitable distance. Thus, this amino acid was
substituted with leucine (L).
Mutants were constructed by introducing the improved
endosomal escape motif into the CDR3 of the heavy-chain
variable region.
Tables 13, 14 and 15 show the heavy-chain variable
region sequences obtained by grafting the designed endosomal
escape motif into the heavy-chain variable region. Table 13
below shows the full-length sequences of the human antibody
light-chain variable regions according to the Kabat numbering
system, and Tables 14 and 15 below show the CDR1 and CDR2
sequences or CDR3 sequences of the antibody sequences shown
in Table 13.
[Table 13]
[Table 14]
[Table 15]
a shows a process of constructing a cytosol-
penetrating antibody having a light-chain variable region
from which endosomal escape ability is removed and a heavy-
chain variable region into which an improved endosomal escape
motif is introduced.
In order to evaluate the endosomal escape ability of the
heavy-chain variable region introduced with the improved
nd th
endosomal escape motif, the 92 to 94 amino acids (WYW) of
the light-chain variable region, which are involved in
endosomal escape, AAA (three consecutive alanines), thereby
removing the function thereof. In the same manner as
described in Example 1, cloning of the heavy-chain variable
region was performed, and the resulting heavy chain together
with the light chain comprising the light-chain variable
region from which the endosomal escape motif has been removed
was expressed in HEK293F cell lines and purified.
In order to more clearly name the cytosol-penetrating
antibody comprising the heavy-chain variable region
introduced with the improved endosomal escape motif, TMab4 is
abbreviated as CT. In other words, TMab4-AAA is CT-AAA.
Example 34: Confirmation of Possibility of Imparting
Improved Endosomal Escape Motif to CDR of Heavy-Chain
Variable Region
b shows a graph quantitatively comparing the
number of cells that have taken up trypan blue depending on
pH by a cytosol-penetrating antibody having a light-chain
variable region from which endosomal escape ability is
removed and a heavy-chain variable region into which an
improved endosomal escape motif is introduced.
Specifically, as shown in b, Ramos cells were
attached to plates in the same manner as described in Example
. Then, the cells were incubated with each of 1 μM TMab4-WYW,
TMab4-AAA, CT01-AAA, CT02-AAA, CT03-AAA and CT04-AAA in 200
μl of each of pH 7.4 buffer(HBSS(Welgene), 50 mM HEPES pH
7.4(cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM
MES pH 5.5(early endosomal pH)) at 37°C for 2 hours. After
careful washing with PBS, 200 μl of a mixture of 190 μl of
PBS and 10 μl of trypan blue was added to each well, and the
cells were observed with a microscope. The number of cells
showing trypan blue uptake was counted and expressed as
percentage relative to the total number of cells. A total of
400 or more cells were counted, and the mean values are
graphically shown. It was observed that the CT01-AAA, CT02-
AAA, CT03-AAA and CT04-AAA all showed trypan blue uptake
equal to about half of TMab4-3. However, it was shown that
the CT04-AAA mutant showed trypan blue uptake even at neutral
c shows the results of confocal microscopy
performed to observe the GFP fluorescence by enhanced split-
GFP complementation of a GFP11-SBP2-fused cytosol-penetrating
antibody having a light-chain variable region from which
endosomal escape ability has been removed, and a heavy-chain
variable region into which an improved endosomal escape motif
has been introduced.
Specifically, transformed HeLa cells stably expressing
SA-GFP1-10 were prepared in the same manner as described in
Example 2. When the cells were stabilized, the cells were
incubated with PBS or each of 1.6 μM CT01-AAA-GFP11-SBP2,
CT02-AAA-GFP11-SBP2, CT03-AAA-GFP11-SBP2 and CT04-AAA-GFP11-
SBP2 at 37°C for 6 hours. The cells were washed with PBS and
weakly acidic solution in the same manner as described in
Example 2, and then fixed. The nucleus was blue-stained with
Hoechst33342 and observed with a confocal microscope. As a
result, green fluorescence was observed in the cells with
CT01-AAA-GFP11-SBP2, CT02-AAA-GFP11-SBP2, CT03-AAA-GFP11-SBP2
or CT04-AAA-GFP11-SBP2.
d shows the results of confocal microscopy
performed using calcein in order to observe the cytosolic
localization of a cytosol-penetrating antibody having a
light-chain variable region from which endosomal escape
ability has been removed and a heavy-chain variable region
into which an improved endosomal escape motif has been
introduced.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2. The cells were incubated
with each of 0.2 μM and 1 μM CT01-AAA, CT02-AAA and CT03-AAA
at 37°C for 6 hours. After 4 hours, each well containing PBS or
the antibody was treated with 150 μM calcein and incubated at
37°C for 2 hours. In the same manner as descried in Example 2,
the cells were washed with PBS and weakly acidic solution, and
then fixed. The nucleus was blue-stained with Hoechst33342 and
observed with a confocal microscope. In the cells treated with
CT01-AAA or CT02-AAA, green calcein fluorescence localized in
the cytosol was similar to that in the cells treated with TMab4.
However, in the cells treated with CT03-AAA, green calcein
fluorescence localized in the cytosol was weaker than that in
the cells treated with CT.
Therefore, it was confirmed that even when the improved
endosomal escape motif is imparted to the heavy-chain variable
region, the antibody can escape from endosomes and finally
localize in the cytosol.
Example 35: Analysis of Properties of E1-L102 of Heavy-
Chain Variable Region That Induces Structural Change Depending
on pH
For more detailed analysis of the 1 amino acid glutamic
acid and 102 amino acid leucine of the heavy-chain variable
region, which induce a structural change depending on pH,
mutants were constructed by substituting the 1 amino acid in
the antibody backbone with each of aspartic acid, alanine and
glutamine present in germline sequences, and
substituting the 102 amino acid in the CDR3 with each of 12
amino acids of the light-chain variable region, which showed
trypan blue uptake at neutral or acidic pH. In the same
manner as described in Example, each of the mutants was
cloned, expressed in HEK293F cell lines, and purified.
a is a graph quantitatively comparing the number
of cells that taken up trypan blue depending on pH by mutants
constructed by substituting the 1 amino acid glutamic acid
of the heavy-chain variable region (VH) of a cytosol-
penetrating antibody, which is involved in induction of a
structural change in properties of the antibody at acidic pH
.5, with various amino acids.
Specifically, 1x10 pgsD-677 cells were incubated in 24-
well plates in the same manner as described in Example 26. On
the next day, in the same manner as described in Example 5,
the cells were incubated with each of 1 μM CT01-AAA, CT01-AAA
E1A, CT01-AAA E1D and CT01-AAA E1Q in 200 μl of each of pH
7.4 buffer (HBSS(Welgene), 50 mM HEPES pH 7.4(cytosolic pH))
and pH 5.5 buffer(HBSS(Welgene), 50 mM MES pH 5.5(early
endosomal pH)) at 37°C for 2 hours. After careful washing
with PBS, 200 μl of a mixture of 190 μl of PBS and 10 μl of
trypan blue was added to each well, and the cells were
observed with a microscope. Next, after careful washing with
PBS, the cells were lysed by adding 50 μl of 1% SDS (sodium
dodecyl sulfate) to each well. The cells were transferred to
a 96-well plate, and the absorbance at 590 nm was measured.
As a result, CT01-AAA E1D showed trypan blue uptake similar
to that of the wild type, and the CT01-AAA E1A and CT01-AAA
E1Q mutants showed reduced trypan blue uptake compared to
that of the wild type.
a is a graph quantitatively comparing the number
of cells that taken up trypan blue depending on pH by mutants
constructed by substituting 102 amino acid leucine of the
heavy-chain variable region (VH) of a cytosol-penetrating
antibody, which is involved in induction of a structural
change of the antibody at acidic pH 5.5, with various amino
acids.
Specifically, pgsD-677 cells were prepared in the same
manner as described in Example 26. Then, in the same manner
as in described Example 5, the cells were incubated with 1 μM
of each of CT01-AAA and nineteen CT01-AAA L102X mutants in
200 μl of each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES
pH 7.4 (cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene), 50
mM MES pH 5.5 (early endosomal pH)) at 37°C for 2 hours.
After careful washing with PBS, 200 μl of a mixture of 190 μl
of PBS and 10 μl of trypan blue was added to each well, and
the cells were observed with a microscope. Next, after
careful washing with PBS, the cells were lysed by adding 50
μl of 1% SDS (sodium dodecyl sulfate) to each well. The cells
were transferred to a 96-well plate, and the absorbance at
590 nm was measured. As a result, compared to CT01-AAA, CT01-
AAA L102I, L102M, and L102H showed trypan blue uptake similar
to that of the wild type, and the CT01-AAA L102K and L102R
mutants showed increased trypan blue uptake at neutral pH.
This suggests that interaction between hydrophobic amino
acids having long side chains, negatively charged amino acids,
and histidine (H), is most effective so that the 102 amino
acid of the heavy-chain variable region induces endosomal can
escape through a change in interaction at early endosomal pH
5.5, like the 95 amino acid of the light-chain variable
region.
Example 36: Construction of Endosomal Escape Motif
Mutants Having Three Tryptophan Residues
In order to improve the endosomal escape ability of the
endosomal escape motif having two tryptophan residues, an
endosomal escape motif having a total of three tryptophan
nd th
residues was constructed by substituting the 92 to 94 amino
acids with tryptophan.
Tables 16 and 17 show light-chain variable region mutant
sequences obtained by introducing the endosomal escape motif
having three tryptophan residues. Specifically, Table 16
below shows the full-length sequence of the human antibody
light-chain variable region according to the Kabat numbering
system, and Table 17 below the CDR3 sequence of the antibody
sequence shown in Table 16.
[Table 16]
[Table 17]
Tables 18 and 19 show heavy-chain variable region mutant
sequences obtained by introducing the endosomal escape motif
having three tryptophan residues. Specifically, Table 18
below shows the full-length sequence of the human antibody
light-chain variable region according to the Kabat numbering
system, and Table 19 below the CDR3 sequence of the antibody
sequence shown in Table 18.
[Table 18]
[Table 19]
In order to evaluate the endosomal escape ability of the
heavy-chain variable region or heavy-chain variable region
comprising the endosomal escape motif having three tryptophan
residues, this heavy-chain variable region or heavy-chain
variable region and the heavy-chain variable region or light
variable region that does not comprise the endosomal escape
motif were expressed together in HEK293F cell lines in the
same manner as described in Example 1, and purified.
Example 37: Confirmation of Improvement in Endosomal
Escape Ability of Intact IgG-Format Cytosol-Penetrating
Antibody Comprising the Heavy-Chain Variable Region or Light-
Chain Variable Region Introduced with Endosomal Escape Motif
Having Two or Three Tryptophan Residues
As one strategy for improving the endosomal escape
ability, the endosomal escape motif was imparted to both the
heavy-chain variable region and the light-chain variable
region. Thus, a single intact IgG-format cytosol-penetrating
antibody includes a total of four endosomal escape motifs.
The heavy-chain variable region and the light-chain
variable region, which comprise the endosomal escape motif
having two or three tryptophan motifs, were expressed
together in HEK293F cell lines in the same manner as
described 1 above, and purified.
a shows a graph quantitatively comparing the
number of cells that have taken up trypan blue depending on
pH by intact IgG-format cytosol-penetrating antibodies having
a light-chain variable region and/or a heavy-chain variable
region introduced with an endosomal escape motif having three
tryptophan residues.
Specifically, pgsD-677 cells were prepared in the same
manner as described in Example 26. Then, in the same manner
as in described Example 5, the cells were incubated with 0.5
or 1 μM of each of CT-3, CT-3_WWW, CT01-AAA, CT01_WWW-AAA,
and CT01-3, and CT01_WWW-3_WWW in 200 μl of each of pH 7.4
buffer (HBSS(Welgene), 50 mM HEPES pH 7.4 (cytosolic pH)) and
pH 5.5 buffer (HBSS(Welgene), 50 mM MES pH 5.5 (early
endosomal pH)) at 37°C for 2 hours. After careful washing
with PBS, 200 μl of a mixture of 190 μl of PBS and 10 μl of
trypan blue was added to each well, and the cells were
observed with a microscope. Next, after careful washing with
PBS, the cells were lysed by adding 50 μl of 1% SDS (sodium
dodecyl sulfate) to each well. The cells were transferred to
a 96-well plate, and the absorbance at 590 nm was measured.
As a result, CT-3_WWW, CT01_WWW-AAA and CT01_WWW-3_WWW
showed significantly increased trypan blue uptake, compared
to the CT-3, CT01-AAA and CT01-3 comprising the existing
endosomal escape motif. In addition, compared to CT-3 and
CT01-AAA, the CT01-3 and CT01_WWW-3_WWW comprising the
endosomal escape motif in both the heavy-chain and light-
chain variable regions showed higher trypan blue uptake.
b shows a bar graph showing the results of
observing the cytosolic localization of intact IgG-format
cytosol-penetrating antibodies having a light-chain variable
region and/or a heavy-chain variable region introduced with
an endosomal escape motif having three tryptophan residues by
confocal microscopy using calcein and quantifying the calcein
fluorescence of the confocal micrographs.
Specifically, HeLa cells were prepared in the same
manner as described in Example 2. The cells were incubated
with 0.25, 0.5 and 1 μM of each of CT-3, CT-3_WWW, CT01-AAA,
CT01_WWW-AAA, CT01-3, and CT01_WWW-3_WWW at 37°C for 6 hours.
After 4 hours, each well containing PBS or the antibody was
treated with 150 μM calcein and incubated at 37°C for 2 hours.
In the same manner as described in Example 2The cells were
washed with PBS and weakly acidic solution and fixed. The
nucleus was blue-stained with Hoechst33342 and observed with
a confocal microscope.
As a result, compared to the cells treated with the CT-3,
CT01-AAA or CT01-3 comprising the existing endosomal escape
motif, the cells treated with CT-3_WWW, CT01_WWW-AAA or
CT01_WWW-3_WWW showed stronger green calcein fluorescence
that localized in the cytosol. In addition, compared to the
cells treated with CT-3 or CT01-AAA, the cells treated with
the CT01-3 or CT01_WWW-3_WWW comprising the endosomal escape
motif in both the heavy-chain and light-chain variable
regions showed stronger green calcein fluorescence that
localized in the cytosol.
It was confirmed that the endosomal escape motif having
three tryptophan motifs has improved endosomal escape motif
compared to the existing endosomal escape motif, and even
when the endosomal escape motif was imparted to the heavy-
chain variable region and the light-chain variable region,
the endosomal escape ability was improved.
Example 38: Confirmation of Improvement in Endosomal
Escape Ability of Intact IgG-Format Cytosol-Penetrating
Antibody Comprising Heavy-Chain Variable Region Introduced
with Improved Endosomal Escape Motif and Light-Chain Variable
Region Having Therapeutic Antibody Backbone Imparted with
Improved Endosomal Escape Ability
In order to confirm that the endosomal escape ability is
improved when the endosomal escape motif is imparted to both
the heavy-chain variable region and the light-chain variable
region, the heavy-chain variable region introduced with the
improved endosomal escape motif and the light-chain variable
region having the therapeutic antibody backbone imparted with
endosomal escape ability were expressed together in HEK293F
cell lines and purified.
Specifically, the intact IgG-format cytosol-penetrating
antibody comprising the heavy-chain variable region
introduced with the improved endosomal escape motif and the
light-chain variable region imparted with improved endosomal
ability showed no cell penetrating ability. For this reason,
an EpCAM-targeting cyclic peptide specific for EpCAM which is
overexpressed on the cell membrane surface in various tumors
including colorectal cancer was genetically fused to the N-
terminus of the antibody by two GGGGS linkers so that the
antibody could penetrate cells (US 2015/0246945 A1).
a shows a schematic view showing a process of
constructing an intact IgG-format cytosol-penetrating
antibody in which an improved endosomal escape motif has been
introduced into a heavy-chain variable region thereof and an
improved endosomal escape motif has been introduced into a
light-chain variable region of a conventional therapeutic
antibody fused with an EpCAM-targeting peptide.
Specifically, animal expression vectors encoding a heavy
chain comprising the heavy-chain variable region introduced
with the improved endosomal escape motif and a light chain
comprising the monoclonal antibody light-chain variable
region imparted with improved endosomal ability were
transiently co-transfected into HEK293F protein-expressing
cells in the same manner as described in Example 1. Next,
purification of the intact IgG-format cytosol-penetrating
antibody was performed in the same manner as described in
Example 1.
b shows a bar graph showing the results of
observing the cytosolic localization of an intact IgG-format
cytosol-penetrating antibody, in which an improved endosomal
escape motif has been introduced into a heavy-chain variable
region thereof and an improved endosomal escape motif has
been introduced into a light-chain variable region of a
conventional therapeutic antibody fused with an EpCAM-
targeting peptide, by confocal microscopy using calcein and
quantifying the calcein fluorescence of the confocal
micrographs.
Specifically, human colorectal cancer HCT116 cells
having a K-RAS G13D mutation were prepared in the same manner
as described in Example 2. The cells were incubated with 0.1,
0.25 and 0.5 μM of each of CT-ep41 and CT01-ep41 at 37°C for
6 hours. After 4 hours, each well containing PBS or the
antibody was treated with 150 μM calcein and incubated at
37°C for 2 hours. In the same manner as described in Example
2, the cells were washed with PBS and weakly acidic solution,
and then fixed. The nucleus was blue-stained with
Hoechst33342 and observed with a confocal microscope. In the
cells treated with varying concentrations of CT01-ep41, the
intensity of green calcein fluorescence localized in the
cytosol was stronger than that in the cells treated with CT-
ep41.
c shows a graph quantitatively comparing the
number of cells that have taken up trypan blue depending on
pH by an intact IgG-format cytosol-penetrating antibody in
which an improved endosomal escape motif has been introduced
into a heavy-chain variable region thereof and an improved
endosomal escape motif has been introduced into a light-chain
variable region of a conventional therapeutic antibody fused
with an EpCAM-targeting peptide.
Specifically, Ramos cells were attached to plates in the
same manner as described in Example 5. Then, the cells were
incubated with each of 1 μM CT-ep41 and CT01-ep41 0.5 in 200
μl of each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH
7.4) (for maintaining a cytosolic pH of 7.4) and pH 5.5
buffer (HBSS(Welgene), 50 mM MES pH 5.5)(for maintaining an
early endosomal pH of 5.5) at 37°C for 2 hours. After careful
washing with PBS, 200 μl of a mixture of 190 μl of PBS and 10
μl of trypan blue was added to each well, and the cells were
observed with a microscope. The number of cells showing
trypan blue uptake was counted and expressed as percentage
relative to the total number of cells. A total of 400 or more
cells were counted, and the mean values are graphically shown.
As a result, CT01-ep41 showed a concentration-dependent
increase in trypan blue uptake compared to CT-ep41.
Thus, it was confirmed that when the endosomal escape
motif was introduced into each of the heavy-chain variable
region and the light-chain variable region, the endosomal
escape ability was improved compared to when the endosomal
escape motif was present only in the light-chain variable
region.
Example 39: Logic of Possibility of Imparting Improved
Endosomal Escape Motif to Heavy-Chain Variable Region of
Conventional Therapeutic Antibody
Similar to the logic that the endosomal escape motif was
imparted to the light-chain variable region of conventional
therapeutic antibodies, the use of the stable backbones of
commercially available therapeutic antibody can be expected
to increase the overall expression yield. In order to examine
whether the endosomal escape motif can operate as a single
motif, the endosomal escape motif was also imparted to the
heavy-chain variable region of conventional therapeutic
antibodies.
Mutants were constructed by substituting the CDR3 of
candidate heavy-chain variable regions with the CDR3 of the
cytosol-penetrating antibody.
Table 20 below shows the names and sequences of the
mutants constructed using a gene synthesis technique.
[Table 20]
a is a schematic view showing a process of
constructing an intact IgG-format cytosol-penetrating
antibody in which an improved endosomal escape motif has been
introduced into the heavy-chain variable region of a
conventional therapeutic antibody.
In the same manner as described in Example 1, cloning of
the heavy-chain variable region was performed, and the
resulting heavy chain and the light chain comprising the
monoclonal antibody light-chain variable region introduced
with the improved endosomal escape motif were expressed
together in HEK293F cell lines and purified.
Example 40: Confirmation of Possibility of Imparting
Improved Endosomal Escape Motif to Heavy-Chain Variable
Region of Monoclonal Antibody
b is a graph quantitatively comparing the number
of cells that have taken up trypan blue depending on pH by an
intact IgG-format cytosol-penetrating antibody in which an
improved endosomal escape motif has been introduced into the
heavy-chain variable region of a conventional therapeutic
antibody.
Specifically, pgsD-677 cells were prepared in the same
manner as described in Example 26. Then, in the same manner
as in described Example 5, the cells were incubated with 0.5
or 1 μM of each of CT-3, CT-3_WWW, CT01-AAA, CT01_WWW-AAA,
and CT01-3, and CT01_WWW-3_WWW in 200 μl of each of pH 7.4
buffer (HBSS(Welgene), 50 mM HEPES pH 7.4 (cytosolic pH)) and
pH 5.5 buffer (HBSS(Welgene), 50 mM MES pH 5.5 (early
endosomal pH)) at 37°C for 2 hours. After careful washing
with PBS, 200 μl of a mixture of 190 μl of PBS and 10 μl of
trypan blue was added to each well, and the cells were
observed with a microscope. Next, after careful washing with
PBS, the cells were lysed by adding 50 μl of 1% SDS (sodium
dodecyl sulfate) to each well. The cells were transferred to
a 96-well plate, and the absorbance at 590 nm was measured.
All the mutants showed trypan blue uptake similar to that of
CT01-ep41.
Example 41: Construction of the Heavy-Chain Variable
Region and Light-Chain Variable Region Introduced with
Aspartic Acid for Improving Properties of Cytosol-Penetrating
Antibody
In order to improve the endosomal escape ability of the
cytosol-penetrating antibody, the endosomal escape motif was
introduced into the CRD3 of each of the heavy-chain variable
region and the light-chain variable region. Due to the
hydrophobic amino acids (tryptophan (W) and tyrosine (Y) of the
endosomal escape motif, the cytosol-penetrating antibody becomes
hydrophobic. To offset this hydrophobicity, mutants were
constructed by substituting an amino acid adjacent to the
endosomal escape motif with negatively charged aspartic acid.
The mutants were constructed with reference to studies where
aspartic acid was introduced into the backbone and CDR regions
of antibody variable regions to increase the overall stability
of the antibody and reduce protein aggregation caused by the
high hydrophobicity of the antibody (Perchiacca et al., 2011;
Dudgeon et al., 2012).
nd rd th
Since the 32 , 33 and 58 amino acids of the heavy-chain
variable region are adjacent to the endosomal escape motif of
each of the heavy-chain variable region and the heavy-chain
variable region, these amino acids were substituted with
aspartic acid. The resulting amino acids were named CT11 VH
(F32D, S33D) or CT12 VH (F32D, S33D, Y58D).
th th th
Since the 27b , 50 and 51 amino acids of the light-
chain variable region are adjacent to the endosomal escape motif
of each of the heavy-chain variable region and the heavy-chain
variable region, these amino acids were substituted with
aspartic acid. The resulting amino acids were named hT4-60 VL
(L27bD), hT4-61 VL (W50D), hT4-62 VL (W50D, A51D) or hT4-63 VL
(L27Bd, W50D, A51D).
Here, the heavy-chain variable region and light-chain
variable regions used as templates for the mutants are antibody
variable regions introduced with the endosomal escape motif
while showing high yields in animal cell expression systems, and
these regions were named CT01 VH and hT4-59 VL, respectively.
Tables 21 and 22 below the heavy-chain variable region and
light-chain variable mutant sequences obtained by introducing
aspartic acid into the backbone and CDR regions of the antibody
variable region.
[Table 21]
[Table 22]
In the same manner as described in Example 1, cloning of
each heavy-chain variable region was performed, and the
resulting heavy chain and the light chain comprising the
monoclonal antibody light-chain variable region introduced with
the improved endosomal escape motif were expressed together in
HEK293F cell lines and purified.
Example 42: Confirmation of Improvement in Endosomal
Escape Ability of Intact IgG-Format Cytosol-Penetrating Antibody
Comprising the Heavy-Chain Variable Region and/or Light-Chain
Variable Region Introduced with Aspartic Acid
is a graph quantitatively comparing the number of
cells that have taken up trypan blue depending on pH by an intact
IgG-format cytosol-penetrating antibody comprising a light-
chain variable region and/or a heavy-chain variable region
introduced with aspartic acid.
Specifically, pgsD-677 cells were prepared in the same
manner as described in Example 26. Then, in the same manner as
in described Example 5, the cells were incubated with 1 μM of
each of CT10-ep59, CT11-ep59, CT12-ep59, CT10-ep60, CT10-ep61,
CT10-ep62, CT10-ep63, and CT12-ep63 in 200 μl of each of pH 7.4
buffer (HBSS(Welgene), 50 mM HEPES pH 7.4 (cytosolic pH)) and
pH 5.5 buffer (HBSS(Welgene), 50 mM MES pH 5.5 (early endosomal
pH)) at 37°C for 2 hours. After careful washing with PBS, 200
μl of a mixture of 190 μl of PBS and 10 μl of trypan blue was
added to each well, and the cells were observed with a microscope.
Next, after careful washing with PBS, the cells were lysed by
adding 50 μl of 1% SDS (sodium dodecyl sulfate) to each well.
The cells were transferred to a 96-well plate, and the absorbance
at 590 nm was measured. All the mutants showed trypan blue uptake
similar to that of CT01-ep41 tested in the above Example. This
suggests that even when aspartic acid is introduced, the
endosomal escape ability is not reduced. Antibody stability
experiments for these antibodies will be carried out later.
Example 43: Analysis of Structure of Cytosol-Penetrating
Antibody
In order to identify the structures of the IgG-format
cytosol-penetrating antibodies, the CT-59 antibody showing a
very high production yield was used among the cytosol-
penetrating antibodies having endosomal escape ability at
endosomal acidic pH conditions. This antibody is an IgG-
format cytosol-penetrating antibody comprising hT0 VH and
hT4-59 VL as the heavy-chain variable region and the light-
chain variable region, respectively.
To identify the three-dimensional structure, IgG-format
CT-59 produced using HEK293 cells was treated with papain,
and then high-purity Fab was purified by protein A column and
size exclusion chromatography. Next, a crystal for structural
identification was formed using an Mosquito-LCP system under
screening buffer index G1 conditions (0.2 M NaCl, 0.1 M Tris,
pH 8.5, 25 % (w/v) PEG3350). When the cytosol-penetrating
antibody was mixed with the screening buffer, the final pH
was 8.1.
a shows the results of observing a crystal of CT-
59 Fab, formed under Index G1 conditions, by RI1000 (Rock
Imager1000; automatic protein crystal image analysis system).
X-ray diffraction data were collected at the 5C beamline
(Pohang Accelerator Laboratory(PAL)), and indexing and
scaling were performed using the HKL2000 package (HKL Research
Inc., USA), and then the Initial electron density map of CT-59
Fab was obtained by a molecular replacement (MR) method. The
three-dimensional structure data of a protein having a similar
to that of CT-59 is required to use the MR method, and a
structure model obtained through the FFAS site
(http://ffas.sanfordburnham.org/ffas-cgi/cgi/ffas.pl) was used
as a model. Initial phase information of CT-59 Fab was obtained
using CCP4. Based on the obtained initial phase information, a
model building operation was performed using COOT
(Crystallographic Object-Oriented Toolkit,
http://www.biop.ox.ac.uk/coot/)), and refinement and validation
operations were performed using Refmac5
(http://www.ccp4.ac.uk/html/refmac5.html) and PHENIX (Python-
based Hierarchical ENvironment for Integrated Xtallography,
http:// www.phenix-online.org/) software (see b).
As a result, at a final pH of pH 8.1, a three-dimensional
structure with a high resolution of 1.8Å was observed. It was
found that the distance between the 1 aspartic acid (D) of the
light-chain variable region of CT-59 and the side chain of the
95 methionine (M) of the light-chain variable region was 6.87
Although the present disclosure has been described in
detail with reference to the specific features, it will be
apparent to those skilled in the art that this description is
only for a preferred embodiment and does not limit the scope
of the present disclosure. Thus, the substantial scope of
the present disclosure will be defined by the appended claims
and equivalents thereof.
Claims (1)
- 【Claims 】 【Claim 1 】 A cytosol-penetrating antibody or antigen-binding fragment thereof comprising a light-chain variable region and/or heavy- chain variable region that comprises an endosomal escape motif in its CDR3, wherein the endosomal escape motif comprises a sequence represented by the following formula: X1-X2-X3-Z1 wherein X1-X2-X3 is an endosomal escape motif, and comprises a sequence selected from the group consisting of W-W- W, W-W-H, W-Y-W, Y-W-W, W-Y-H, and Y-W-H (where W is tryptophan, Y is tyrosine, H is histidine); Z1 is selected from the group consisting of methionine (M), isoleucine (I), leucine (L), histidine (H), aspartic acid (D), and glutamic acid (E); the 1 amino acid of the light-chain variable region and/or heavy-chain variable region is aspartic acid (D) or glutamic acid (E); the light-chain variable region and/or heavy-chain variable region comprising Z1 induces a change in properties of the antibody under endosomal acidic pH conditions; and the antibody exhibits an ability to escape from endosomes into the cytosol through the change in properties of the antibody. 【Claim 2 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 1, wherein the 1 amino acid of the light-chain variable region and/or heavy-chain variable region interacts with the Z1 under endosomal acidic pH conditions to induce a change in properties of the cytosol-penetrating antibody. 【Claim 3 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 1, wherein the light-chain variable region and/or heavy-chain variable region further comprises an amino acid sequence represented by (a1-...-an, where n is an integer ranging from 1 to 10) between X3 and Z1. 【Claim 4 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 1, wherein the sequence further comprises Z2 linked to X1, and thus is represented by the following formula: Z2-X1-X2-X3-Z1, wherein Z2 is selected from the group consisting of glutamine (Q), leucine (L) histidine (H). 【Claim 5 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 4, wherein the 1 amino acid of the light-chain variable region and/or heavy-chain variable region interacts with Z1 and/or Z2 under endosomal acidic pH conditions to induce a change in properties of the cytosol-penetrating antibody. 【Claim 6 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 4, wherein the light-chain variable region and/or heavy-chain variable region further comprises an amino acid sequence represented by (b1-...-bn, where n is an integer ranging from 1 to 10) between X1 and Z2. 【Claim 7 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 1, wherein the CDR3 of the light-chain variable region comprises the sequence selected from the group consisting of SEQ ID NOS: 8 to 12, 24 or 51. 【Claim 8 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 1, wherein the CDR3 of the heavy-chain variable region comprises the sequence selected from the group consisting of SEQ ID NOS: 46 to 49, and 53. 【Claim 9 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 1, wherein the light-chain variable region comprise a sequence selected from the group consisting of SEQ ID NOS: 1 to 5, 13 to 23, 25 to 37, 50, and 60 to 64. 【Claim 10 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 1, wherein the heavy-chain variable region comprise a sequence selected from the group consisting of SEQ ID NOS: 39 to 42, 52, and 54 to 59. 【Claim 11 】 The cytosol-penetrating antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is an intact immunoglobulin G-format antibody. 【Claim 12 】 A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 11. 【Claim 13 】 A vector comprising the nucleic acid of claim 12. 【Claim 14 】 An in vitro cell transformed with the vector of claim 13. 【Claim 15 】 Use of a composition comprising the cytosol-penetrating antibody or antigen-bind fragment thereof of any one of claims 1 to 13 in the manufacture of a medicament for delivering an active substance into cytosol. 【Claim 16 】 The use of claim 15, wherein the active substance comprises one or more selected from the group consisting of peptides, proteins, toxins, antibodies, antibody fragments, RNAs, siRNAs, DNAs, small molecule drugs, nanoparticles, and liposomes. 【Claim 17 】 A method for producing the cytosol-penetrating antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the method comprising a step of grafting the endosomal escape motif X1-X2-X3-Z1, wherein X1-X2-X3 is selected from the group consisting of W-W-W, W-W-H, W-Y-W, Y-W-W, W-Y-H, and Y-W-H (where W is tryptophan, Y is tyrosine, H is histidine) into the CDR3 of a light-chain and/or heavy-chain variable region.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2016-0065365 | 2016-05-27 | ||
KR1020160065365A KR102000000B1 (en) | 2016-05-27 | 2016-05-27 | Endosomal escape motif enabling antibody to escape from endosomes and uses thereof |
KR1020160065379A KR20170133933A (en) | 2016-05-27 | 2016-05-27 | Method for inducing conformational changes in the complementarity determining regions of antibody |
KR10-2016-0065379 | 2016-05-27 | ||
PCT/KR2017/005559 WO2017204606A1 (en) | 2016-05-27 | 2017-05-26 | Cytosol-penetrating antibody and use thereof |
KR10-2017-0065670 | 2017-05-26 | ||
KR1020170065670A KR20180129514A (en) | 2017-05-26 | 2017-05-26 | Cytosol-Penetrating Antibodies and Uses Thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ749020A NZ749020A (en) | 2020-11-27 |
NZ749020B2 true NZ749020B2 (en) | 2021-03-02 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3173428B1 (en) | Method for positioning, in cytoplasm, antibody having complete immunoglobulin form by penetrating antibody through cell membrane, and use for same | |
US11155641B2 (en) | Cytosol-penetrating antibody and use thereof | |
EP3173099B1 (en) | Method for suppressing ras activated in cell by using antibody having cytoplasm penetration capacity and complete immunoglobulin form, and use for same | |
US20200362010A1 (en) | Neuropilin-1 specific binding peptide, fusion protein fused with same, and use thereof | |
US20110223107A1 (en) | Antibodies that specifically block the biological activity of a tumor antigen | |
JP2021509020A (en) | Antibodies that permeate the cytoplasm of cells and suppress intracellularly activated RAS and their uses | |
US20190023781A1 (en) | Therapeutic drug for malignant tumors | |
KR102091195B1 (en) | Cytosol-Penetrating Antibodies and Uses Thereof | |
KR20180129514A (en) | Cytosol-Penetrating Antibodies and Uses Thereof | |
KR101906558B1 (en) | Novel Antibody Specific For TSPAN8 and Uses Thereof | |
KR20200088780A (en) | Enhanced cytosol-penetrating antibody | |
EP3466970A1 (en) | Cytosol-penetrating antibody and use thereof | |
NZ749020B2 (en) | Cytosol-penetrating antibody and use thereof | |
KR20180116204A (en) | Endosomal escape motif enabling antibody to escape from endosomes and uses thereof |