OA18689A - Agents, Uses and Methods for the Treatment of Synucleinopathy - Google Patents
Agents, Uses and Methods for the Treatment of Synucleinopathy Download PDFInfo
- Publication number
- OA18689A OA18689A OA1201800011 OA18689A OA 18689 A OA18689 A OA 18689A OA 1201800011 OA1201800011 OA 1201800011 OA 18689 A OA18689 A OA 18689A
- Authority
- OA
- OAPI
- Prior art keywords
- seq
- synuclein
- antibody
- amino acid
- alpha
- Prior art date
Links
- 201000003356 synucleinopathy Diseases 0.000 title claims abstract description 20
- 102000004965 antibodies Human genes 0.000 claims abstract description 508
- 108090001123 antibodies Proteins 0.000 claims abstract description 508
- 206010061536 Parkinson's disease Diseases 0.000 claims abstract description 64
- 201000002832 Lewy body dementia Diseases 0.000 claims abstract description 26
- 208000009829 Lewy Body Disease Diseases 0.000 claims abstract description 24
- 206010067889 Dementia with Lewy body Diseases 0.000 claims abstract description 22
- 208000001089 Multiple System Atrophy Diseases 0.000 claims abstract description 13
- 208000008404 Lewy Body Variant of Alzheimer Disease Diseases 0.000 claims abstract description 11
- 206010001897 Alzheimer's disease Diseases 0.000 claims abstract description 9
- 102000003802 alpha-Synuclein Human genes 0.000 claims description 361
- 108090000185 alpha-Synuclein Proteins 0.000 claims description 361
- 230000027455 binding Effects 0.000 claims description 284
- 241000282414 Homo sapiens Species 0.000 claims description 181
- 239000000427 antigen Substances 0.000 claims description 179
- 102000038129 antigens Human genes 0.000 claims description 175
- 108091007172 antigens Proteins 0.000 claims description 175
- 150000001413 amino acids Chemical class 0.000 claims description 134
- 102000005614 monoclonal antibodies Human genes 0.000 claims description 100
- 108010045030 monoclonal antibodies Proteins 0.000 claims description 100
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 79
- 201000010099 disease Diseases 0.000 claims description 43
- 150000007523 nucleic acids Chemical class 0.000 claims description 26
- 108020004707 nucleic acids Proteins 0.000 claims description 25
- 239000008194 pharmaceutical composition Substances 0.000 claims description 21
- 239000000969 carrier Substances 0.000 claims description 10
- 206010018048 Gaucher's disease Diseases 0.000 claims description 9
- 238000002560 therapeutic procedure Methods 0.000 claims description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 235000001014 amino acid Nutrition 0.000 description 139
- 210000004027 cells Anatomy 0.000 description 112
- 241000699666 Mus <mouse, genus> Species 0.000 description 71
- 108010037850 glycylvaline Proteins 0.000 description 52
- LSPKYLAFTPBWIL-BYPYZUCNSA-N Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(O)=O LSPKYLAFTPBWIL-BYPYZUCNSA-N 0.000 description 44
- 230000014509 gene expression Effects 0.000 description 42
- 102000004169 proteins and genes Human genes 0.000 description 42
- 108090000623 proteins and genes Proteins 0.000 description 42
- 235000018102 proteins Nutrition 0.000 description 41
- 230000005062 synaptic transmission Effects 0.000 description 38
- STKYPAFSDFAEPH-LURJTMIESA-N gly-val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CN STKYPAFSDFAEPH-LURJTMIESA-N 0.000 description 37
- 241000880493 Leptailurus serval Species 0.000 description 36
- 239000000203 mixture Substances 0.000 description 36
- ZOKVLMBYDSIDKG-CSMHCCOUSA-N Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN ZOKVLMBYDSIDKG-CSMHCCOUSA-N 0.000 description 33
- XZKQVQKUZMAADP-IMJSIDKUSA-N Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(O)=O XZKQVQKUZMAADP-IMJSIDKUSA-N 0.000 description 33
- 210000004556 Brain Anatomy 0.000 description 31
- 230000000694 effects Effects 0.000 description 31
- 239000002953 phosphate buffered saline Substances 0.000 description 31
- 241000700159 Rattus Species 0.000 description 30
- 238000006467 substitution reaction Methods 0.000 description 29
- 241000699660 Mus musculus Species 0.000 description 27
- 239000000243 solution Substances 0.000 description 27
- 108090001095 Immunoglobulin G Proteins 0.000 description 26
- 102000004851 Immunoglobulin G Human genes 0.000 description 26
- VYZAGTDAHUIRQA-WHFBIAKZSA-N L-alanyl-L-glutamic acid Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O VYZAGTDAHUIRQA-WHFBIAKZSA-N 0.000 description 26
- GVRKWABULJAONN-UHFFFAOYSA-N Valyl-Threonine Chemical compound CC(C)C(N)C(=O)NC(C(C)O)C(O)=O GVRKWABULJAONN-UHFFFAOYSA-N 0.000 description 26
- 230000001225 therapeutic Effects 0.000 description 26
- 210000001320 Hippocampus Anatomy 0.000 description 25
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 25
- 102000019355 Synucleins Human genes 0.000 description 25
- 108050006783 Synucleins Proteins 0.000 description 25
- 108010049041 glutamylalanine Proteins 0.000 description 25
- 239000002609 media Substances 0.000 description 25
- 210000002569 neurons Anatomy 0.000 description 25
- 108010061238 threonyl-glycine Proteins 0.000 description 25
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 24
- 150000001875 compounds Chemical class 0.000 description 24
- 241000894007 species Species 0.000 description 24
- 125000000539 amino acid group Chemical group 0.000 description 23
- 239000007924 injection Substances 0.000 description 23
- 108010064235 lysylglycine Proteins 0.000 description 22
- JZDHUJAFXGNDSB-WHFBIAKZSA-N Glu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O JZDHUJAFXGNDSB-WHFBIAKZSA-N 0.000 description 21
- CKHWEVXPLJBEOZ-UHFFFAOYSA-N Threoninyl-Valine Chemical compound CC(C)C(C(O)=O)NC(=O)C(N)C(C)O CKHWEVXPLJBEOZ-UHFFFAOYSA-N 0.000 description 21
- 108010031491 threonyl-lysyl-glutamic acid Proteins 0.000 description 21
- QXRNAOYBCYVZCD-BQBZGAKWSA-N (2S)-6-amino-2-[[(2S)-2-aminopropanoyl]amino]hexanoic acid Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN QXRNAOYBCYVZCD-BQBZGAKWSA-N 0.000 description 20
- BUQICHWNXBIBOG-LMVFSUKVSA-N Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)N BUQICHWNXBIBOG-LMVFSUKVSA-N 0.000 description 20
- WOUIMBGNEUWXQG-VKHMYHEASA-N Ser-Gly Chemical compound OC[C@H](N)C(=O)NCC(O)=O WOUIMBGNEUWXQG-VKHMYHEASA-N 0.000 description 20
- 239000000523 sample Substances 0.000 description 20
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 19
- 108010079364 N-glycylalanine Proteins 0.000 description 19
- 108010089804 glycyl-threonine Proteins 0.000 description 19
- 230000002401 inhibitory effect Effects 0.000 description 19
- JBCLFWXMTIKCCB-VIFPVBQESA-N Gly-Phe Chemical compound NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-VIFPVBQESA-N 0.000 description 18
- 102000018358 Immunoglobulins Human genes 0.000 description 18
- 108060003951 Immunoglobulins Proteins 0.000 description 18
- XGDCYUQSFDQISZ-BQBZGAKWSA-N Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(O)=O XGDCYUQSFDQISZ-BQBZGAKWSA-N 0.000 description 18
- JKHXYJKMNSSFFL-IUCAKERBSA-N Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN JKHXYJKMNSSFFL-IUCAKERBSA-N 0.000 description 18
- KRNYOVHEKOBTEF-YUMQZZPRSA-N Val-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(O)=O KRNYOVHEKOBTEF-YUMQZZPRSA-N 0.000 description 18
- 230000001965 increased Effects 0.000 description 18
- 238000002347 injection Methods 0.000 description 18
- 238000004519 manufacturing process Methods 0.000 description 18
- 239000004365 Protease Substances 0.000 description 17
- 108010070643 prolylglutamic acid Proteins 0.000 description 17
- 108010073969 valyllysine Proteins 0.000 description 17
- 108091005771 Peptidases Proteins 0.000 description 16
- 238000009825 accumulation Methods 0.000 description 16
- 230000037396 body weight Effects 0.000 description 16
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 16
- 238000003384 imaging method Methods 0.000 description 16
- 238000010899 nucleation Methods 0.000 description 16
- 210000004408 Hybridomas Anatomy 0.000 description 15
- 108010087924 alanylproline Proteins 0.000 description 15
- 238000003776 cleavage reaction Methods 0.000 description 15
- 102000033147 ERVK-25 Human genes 0.000 description 14
- 230000035693 Fab Effects 0.000 description 14
- YBTCBQBIJKGSJP-BQBZGAKWSA-N Glu-Pro Chemical compound OC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O YBTCBQBIJKGSJP-BQBZGAKWSA-N 0.000 description 14
- OLIFSFOFKGKIRH-WUJLRWPWSA-N Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CN OLIFSFOFKGKIRH-WUJLRWPWSA-N 0.000 description 14
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 14
- HGNRJCINZYHNOU-LURJTMIESA-N Lys-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(O)=O HGNRJCINZYHNOU-LURJTMIESA-N 0.000 description 14
- ILVGMCVCQBJPSH-WDSKDSINSA-N Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CO ILVGMCVCQBJPSH-WDSKDSINSA-N 0.000 description 14
- GJNDXQBALKCYSZ-RYUDHWBXSA-N Val-Phe Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 GJNDXQBALKCYSZ-RYUDHWBXSA-N 0.000 description 14
- 230000002776 aggregation Effects 0.000 description 14
- 238000004220 aggregation Methods 0.000 description 14
- 238000000338 in vitro Methods 0.000 description 14
- 108010051242 phenylalanylserine Proteins 0.000 description 14
- WPWUFUBLGADILS-WDSKDSINSA-N Ala-Pro Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O WPWUFUBLGADILS-WDSKDSINSA-N 0.000 description 13
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 13
- MTCFGRXMJLQNBG-REOHCLBHSA-N L-serine Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 13
- NFNVDJGXRFEYTK-YUMQZZPRSA-N Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O NFNVDJGXRFEYTK-YUMQZZPRSA-N 0.000 description 13
- STTYIMSDIYISRG-WDSKDSINSA-N Val-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(O)=O STTYIMSDIYISRG-WDSKDSINSA-N 0.000 description 13
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 13
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 13
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 13
- 108010015792 glycyllysine Proteins 0.000 description 13
- 108010009298 lysylglutamic acid Proteins 0.000 description 13
- 108010017391 lysylvaline Proteins 0.000 description 13
- 238000001690 micro-dialysis Methods 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 13
- -1 159Gd) Chemical compound 0.000 description 12
- DKEXFJVMVGETOO-LURJTMIESA-N Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CN DKEXFJVMVGETOO-LURJTMIESA-N 0.000 description 12
- 210000003523 Substantia Nigra Anatomy 0.000 description 12
- HSRXSKHRSXRCFC-WDSKDSINSA-N Val-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(O)=O HSRXSKHRSXRCFC-WDSKDSINSA-N 0.000 description 12
- 108010068265 aspartyltyrosine Proteins 0.000 description 12
- 239000003795 chemical substances by application Substances 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 108010081551 glycylphenylalanine Proteins 0.000 description 12
- 230000003053 immunization Effects 0.000 description 12
- 238000006366 phosphorylation reaction Methods 0.000 description 12
- 230000000865 phosphorylative Effects 0.000 description 12
- 108010031719 prolyl-serine Proteins 0.000 description 12
- IPWKGIFRRBGCJO-IMJSIDKUSA-N Ala-Ser Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](CO)C([O-])=O IPWKGIFRRBGCJO-IMJSIDKUSA-N 0.000 description 11
- 241000283707 Capra Species 0.000 description 11
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 11
- RNKSNIBMTUYWSH-YFKPBYRVSA-N L-prolylglycine Chemical compound [O-]C(=O)CNC(=O)[C@@H]1CCC[NH2+]1 RNKSNIBMTUYWSH-YFKPBYRVSA-N 0.000 description 11
- AFWBWPCXSWUCLB-WDSKDSINSA-N Pro-Ser Chemical compound OC[C@@H](C([O-])=O)NC(=O)[C@@H]1CCC[NH2+]1 AFWBWPCXSWUCLB-WDSKDSINSA-N 0.000 description 11
- 101700063973 lgg-1 Proteins 0.000 description 11
- 239000003446 ligand Substances 0.000 description 11
- 229960000060 monoclonal antibodies Drugs 0.000 description 11
- 229920001184 polypeptide Polymers 0.000 description 11
- 108010053725 prolylvaline Proteins 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- BUDNAJYVCUHLSV-ZLUOBGJFSA-N (3S)-3-[[(2S)-2-aminopropanoyl]amino]-4-[[(1S)-1-carboxy-2-hydroxyethyl]amino]-4-oxobutanoic acid Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O BUDNAJYVCUHLSV-ZLUOBGJFSA-N 0.000 description 10
- XNSKSTRGQIPTSE-UHFFFAOYSA-N Arginyl-Threonine Chemical compound CC(O)C(C(O)=O)NC(=O)C(N)CCCNC(N)=N XNSKSTRGQIPTSE-UHFFFAOYSA-N 0.000 description 10
- VGRHZPNRCLAHQA-UHFFFAOYSA-N Aspartyl-Asparagine Chemical compound OC(=O)CC(N)C(=O)NC(CC(N)=O)C(O)=O VGRHZPNRCLAHQA-UHFFFAOYSA-N 0.000 description 10
- BBBXWRGITSUJPB-YUMQZZPRSA-N Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(O)=O BBBXWRGITSUJPB-YUMQZZPRSA-N 0.000 description 10
- IKAIKUBBJHFNBZ-LURJTMIESA-N Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CN IKAIKUBBJHFNBZ-LURJTMIESA-N 0.000 description 10
- RZEQTVHJZCIUBT-UHFFFAOYSA-N Serinyl-Arginine Chemical compound OCC(N)C(=O)NC(C(O)=O)CCCNC(N)=N RZEQTVHJZCIUBT-UHFFFAOYSA-N 0.000 description 10
- YKRQRPFODDJQTC-UHFFFAOYSA-N Threoninyl-Lysine Chemical compound CC(O)C(N)C(=O)NC(C(O)=O)CCCCN YKRQRPFODDJQTC-UHFFFAOYSA-N 0.000 description 10
- VEYJKJORLPYVLO-RYUDHWBXSA-N Val-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 VEYJKJORLPYVLO-RYUDHWBXSA-N 0.000 description 10
- 230000001684 chronic Effects 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 230000002829 reduced Effects 0.000 description 10
- 230000002588 toxic Effects 0.000 description 10
- 108010009962 valyltyrosine Proteins 0.000 description 10
- NALWOULWGHTVDA-UWVGGRQHSA-N Asp-Tyr Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NALWOULWGHTVDA-UWVGGRQHSA-N 0.000 description 9
- 229960001230 Asparagine Drugs 0.000 description 9
- UQHGAYSULGRWRG-WHFBIAKZSA-N Glu-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CO)C(O)=O UQHGAYSULGRWRG-WHFBIAKZSA-N 0.000 description 9
- CIOWSLJGLSUOME-BQBZGAKWSA-N Lys-Asp Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O CIOWSLJGLSUOME-BQBZGAKWSA-N 0.000 description 9
- NYQBYASWHVRESG-MIMYLULJSA-N Phe-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 NYQBYASWHVRESG-MIMYLULJSA-N 0.000 description 9
- 210000002381 Plasma Anatomy 0.000 description 9
- SBMNPABNWKXNBJ-UHFFFAOYSA-N Serinyl-Lysine Chemical compound NCCCCC(C(O)=O)NC(=O)C(N)CO SBMNPABNWKXNBJ-UHFFFAOYSA-N 0.000 description 9
- 235000009582 asparagine Nutrition 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 230000003291 dopaminomimetic Effects 0.000 description 9
- 238000005755 formation reaction Methods 0.000 description 9
- 108010079547 glutamylmethionine Proteins 0.000 description 9
- 230000002163 immunogen Effects 0.000 description 9
- QLROSWPKSBORFJ-BQBZGAKWSA-N pro glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 QLROSWPKSBORFJ-BQBZGAKWSA-N 0.000 description 9
- 108010077112 prolyl-proline Proteins 0.000 description 9
- 108010029020 prolylglycine Proteins 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 230000000638 stimulation Effects 0.000 description 9
- 231100000331 toxic Toxicity 0.000 description 9
- LIWMQSWFLXEGMA-WDSKDSINSA-N Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)N LIWMQSWFLXEGMA-WDSKDSINSA-N 0.000 description 8
- VBKIFHUVGLOJKT-UHFFFAOYSA-N Asparaginyl-Threonine Chemical compound CC(O)C(C(O)=O)NC(=O)C(N)CC(N)=O VBKIFHUVGLOJKT-UHFFFAOYSA-N 0.000 description 8
- UKGGPJNBONZZCM-WDSKDSINSA-N Aspartyl-L-proline Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O UKGGPJNBONZZCM-WDSKDSINSA-N 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- LRKCBIUDWAXNEG-CSMHCCOUSA-N Leu-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LRKCBIUDWAXNEG-CSMHCCOUSA-N 0.000 description 8
- MDSUKZSLOATHMH-IUCAKERBSA-N Leu-Val Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C([O-])=O MDSUKZSLOATHMH-IUCAKERBSA-N 0.000 description 8
- YQAIUOWPSUOINN-IUCAKERBSA-N Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN YQAIUOWPSUOINN-IUCAKERBSA-N 0.000 description 8
- JPNRPAJITHRXRH-UHFFFAOYSA-N Lysyl-Asparagine Chemical compound NCCCCC(N)C(=O)NC(C(O)=O)CC(N)=O JPNRPAJITHRXRH-UHFFFAOYSA-N 0.000 description 8
- KAKJTZWHIUWTTD-VQVTYTSYSA-N Met-Thr Chemical compound CSCC[C@H]([NH3+])C(=O)N[C@@H]([C@@H](C)O)C([O-])=O KAKJTZWHIUWTTD-VQVTYTSYSA-N 0.000 description 8
- BIYXEUAFGLTAEM-WUJLRWPWSA-N Thr-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(O)=O BIYXEUAFGLTAEM-WUJLRWPWSA-N 0.000 description 8
- IQHUITKNHOKGFC-MIMYLULJSA-N Thr-Phe Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IQHUITKNHOKGFC-MIMYLULJSA-N 0.000 description 8
- PDSLRCZINIDLMU-QWRGUYRKSA-N Tyr-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PDSLRCZINIDLMU-QWRGUYRKSA-N 0.000 description 8
- OYOQKMOWUDVWCR-RYUDHWBXSA-N Tyr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OYOQKMOWUDVWCR-RYUDHWBXSA-N 0.000 description 8
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 8
- 108010093581 aspartyl-proline Proteins 0.000 description 8
- 239000011324 bead Substances 0.000 description 8
- 238000004166 bioassay Methods 0.000 description 8
- 239000003085 diluting agent Substances 0.000 description 8
- 239000006185 dispersion Substances 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 230000000763 evoked Effects 0.000 description 8
- 230000003619 fibrillary Effects 0.000 description 8
- 102000004963 gamma-Synuclein Human genes 0.000 description 8
- 108090001121 gamma-Synuclein Proteins 0.000 description 8
- VPZXBVLAVMBEQI-VKHMYHEASA-N gly ala Chemical compound OC(=O)[C@H](C)NC(=O)CN VPZXBVLAVMBEQI-VKHMYHEASA-N 0.000 description 8
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 8
- 238000002649 immunization Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- RZVAJINKPMORJF-UHFFFAOYSA-N p-acetaminophenol Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 8
- 230000005298 paramagnetic Effects 0.000 description 8
- 108010026333 seryl-proline Proteins 0.000 description 8
- 108010033670 threonyl-aspartyl-tyrosine Proteins 0.000 description 8
- 210000001519 tissues Anatomy 0.000 description 8
- IOUPEELXVYPCPG-UHFFFAOYSA-N val-gly Chemical compound CC(C)C(N)C(=O)NCC(O)=O IOUPEELXVYPCPG-UHFFFAOYSA-N 0.000 description 8
- 108010021889 valylvaline Proteins 0.000 description 8
- 230000003612 virological Effects 0.000 description 8
- SITLTJHOQZFJGG-XPUUQOCRSA-N α-Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(O)=O SITLTJHOQZFJGG-XPUUQOCRSA-N 0.000 description 8
- IIFDPDVJAHQFSR-WHFBIAKZSA-N Asn-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O IIFDPDVJAHQFSR-WHFBIAKZSA-N 0.000 description 7
- LLEUXCDZPQOJMY-AAEUAGOBSA-N Glu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)N)C(O)=O)=CNC2=C1 LLEUXCDZPQOJMY-AAEUAGOBSA-N 0.000 description 7
- FUESBOMYALLFNI-VKHMYHEASA-N Gly-Asn Chemical compound NCC(=O)N[C@H](C(O)=O)CC(N)=O FUESBOMYALLFNI-VKHMYHEASA-N 0.000 description 7
- 108010064750 Humanized Monoclonal Antibodies Proteins 0.000 description 7
- 102000015434 Humanized Monoclonal Antibodies Human genes 0.000 description 7
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 7
- 210000004558 Lewy Bodies Anatomy 0.000 description 7
- QTZXSYBVOSXBEJ-WDSKDSINSA-N Met-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O QTZXSYBVOSXBEJ-WDSKDSINSA-N 0.000 description 7
- IMTUWVJPCQPJEE-IUCAKERBSA-N Met-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN IMTUWVJPCQPJEE-IUCAKERBSA-N 0.000 description 7
- ROHDXJUFQVRDAV-UWVGGRQHSA-N Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 ROHDXJUFQVRDAV-UWVGGRQHSA-N 0.000 description 7
- 239000002202 Polyethylene glycol Substances 0.000 description 7
- RWCOTTLHDJWHRS-YUMQZZPRSA-N Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RWCOTTLHDJWHRS-YUMQZZPRSA-N 0.000 description 7
- LDEBVRIURYMKQS-UHFFFAOYSA-N Serinyl-Threonine Chemical compound CC(O)C(C(O)=O)NC(=O)C(N)CO LDEBVRIURYMKQS-UHFFFAOYSA-N 0.000 description 7
- 108010070144 Single-Chain Antibodies Proteins 0.000 description 7
- 102000005632 Single-Chain Antibodies Human genes 0.000 description 7
- NHUHCSRWZMLRLA-UHFFFAOYSA-N Sulfizole Chemical compound CC1=NOC(NS(=O)(=O)C=2C=CC(N)=CC=2)=C1C NHUHCSRWZMLRLA-UHFFFAOYSA-N 0.000 description 7
- 210000000225 Synapses Anatomy 0.000 description 7
- BQBCIBCLXBKYHW-CSMHCCOUSA-N Thr-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@@H]([NH3+])[C@@H](C)O BQBCIBCLXBKYHW-CSMHCCOUSA-N 0.000 description 7
- XCTHZFGSVQBHBW-IUCAKERBSA-N Val-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@@H]([NH3+])C(C)C XCTHZFGSVQBHBW-IUCAKERBSA-N 0.000 description 7
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 7
- 108010092854 aspartyllysine Proteins 0.000 description 7
- 102000003799 beta-Synuclein Human genes 0.000 description 7
- 108090000182 beta-Synuclein Proteins 0.000 description 7
- 210000003618 cortical neuron Anatomy 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 108010087823 glycyltyrosine Proteins 0.000 description 7
- 230000035800 maturation Effects 0.000 description 7
- 238000010172 mouse model Methods 0.000 description 7
- 230000001575 pathological Effects 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 230000004481 post-translational protein modification Effects 0.000 description 7
- 210000002243 primary neuron Anatomy 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- 108010036320 valylleucine Proteins 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- UCSJYZPVAKXKNQ-HZYVHMACSA-N 1-[(1S,2R,3R,4S,5R,6R)-3-carbamimidamido-6-{[(2R,3R,4R,5S)-3-{[(2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methylamino)oxan-2-yl]oxy}-4-formyl-4-hydroxy-5-methyloxolan-2-yl]oxy}-2,4,5-trihydroxycyclohexyl]guanidine Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- IJYZHIOOBGIINM-WDSKDSINSA-N Arg-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N IJYZHIOOBGIINM-WDSKDSINSA-N 0.000 description 6
- OAMLVOVXNKILLQ-BQBZGAKWSA-N Asp-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC(O)=O OAMLVOVXNKILLQ-BQBZGAKWSA-N 0.000 description 6
- 108090000206 Autoantibodies Proteins 0.000 description 6
- 102000003852 Autoantibodies Human genes 0.000 description 6
- 210000001175 Cerebrospinal Fluid Anatomy 0.000 description 6
- TWVKGYNQQAUNRN-ACZMJKKPSA-N Ile-Ser Chemical compound CC[C@H](C)[C@H]([NH3+])C(=O)N[C@@H](CO)C([O-])=O TWVKGYNQQAUNRN-ACZMJKKPSA-N 0.000 description 6
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 6
- YSZNURNVYFUEHC-BQBZGAKWSA-N Lys-Ser Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(O)=O YSZNURNVYFUEHC-BQBZGAKWSA-N 0.000 description 6
- DZMGFGQBRYWJOR-YUMQZZPRSA-N Met-Pro Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O DZMGFGQBRYWJOR-YUMQZZPRSA-N 0.000 description 6
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 6
- HWMGTNOVUDIKRE-UWVGGRQHSA-N Phe-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 HWMGTNOVUDIKRE-UWVGGRQHSA-N 0.000 description 6
- SSJMZMUVNKEENT-IMJSIDKUSA-N Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CO SSJMZMUVNKEENT-IMJSIDKUSA-N 0.000 description 6
- NFDYGNFETJVMSE-BQBZGAKWSA-N Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CO NFDYGNFETJVMSE-BQBZGAKWSA-N 0.000 description 6
- OBTCMSPFOITUIJ-FSPLSTOPSA-N Val-Asp Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O OBTCMSPFOITUIJ-FSPLSTOPSA-N 0.000 description 6
- BNQVUHQWZGTIBX-IUCAKERBSA-N Val-His Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CC1=CN=CN1 BNQVUHQWZGTIBX-IUCAKERBSA-N 0.000 description 6
- 230000001154 acute Effects 0.000 description 6
- 108010047857 aspartylglycine Proteins 0.000 description 6
- 230000000875 corresponding Effects 0.000 description 6
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 239000003937 drug carrier Substances 0.000 description 6
- 230000002708 enhancing Effects 0.000 description 6
- 210000004744 fore-foot Anatomy 0.000 description 6
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 6
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 6
- 230000001771 impaired Effects 0.000 description 6
- 108010054155 lysyllysine Proteins 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 108010038745 tryptophylglycine Proteins 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- TUTIHHSZKFBMHM-UHFFFAOYSA-N 4-amino-5-[(3-amino-1-carboxy-3-oxopropyl)amino]-5-oxopentanoic acid Chemical compound OC(=O)CCC(N)C(=O)NC(CC(N)=O)C(O)=O TUTIHHSZKFBMHM-UHFFFAOYSA-N 0.000 description 5
- RDIKFPRVLJLMER-BQBZGAKWSA-N Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)N RDIKFPRVLJLMER-BQBZGAKWSA-N 0.000 description 5
- KWBQPGIYEZKDEG-FSPLSTOPSA-N Asn-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CC(N)=O KWBQPGIYEZKDEG-FSPLSTOPSA-N 0.000 description 5
- CPMKYMGGYUFOHS-FSPLSTOPSA-N Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CC(O)=O CPMKYMGGYUFOHS-FSPLSTOPSA-N 0.000 description 5
- DYDKXJWQCIVTMR-UHFFFAOYSA-N Aspartyl-Methionine Chemical compound CSCCC(C(O)=O)NC(=O)C(N)CC(O)=O DYDKXJWQCIVTMR-UHFFFAOYSA-N 0.000 description 5
- 102000003895 Calpain-1 Human genes 0.000 description 5
- 108090000236 Calpain-1 Proteins 0.000 description 5
- 210000003722 Extracellular Fluid Anatomy 0.000 description 5
- SXGAGTVDWKQYCX-BQBZGAKWSA-N Glu-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(O)=O SXGAGTVDWKQYCX-BQBZGAKWSA-N 0.000 description 5
- JLXVRFDTDUGQEE-YFKPBYRVSA-N Gly-Arg Chemical compound NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N JLXVRFDTDUGQEE-YFKPBYRVSA-N 0.000 description 5
- IEFJWDNGDZAYNZ-BYPYZUCNSA-N Gly-Glu Chemical compound NCC(=O)N[C@H](C(O)=O)CCC(O)=O IEFJWDNGDZAYNZ-BYPYZUCNSA-N 0.000 description 5
- XBGGUPMXALFZOT-VIFPVBQESA-N Gly-Tyr Chemical compound NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-VIFPVBQESA-N 0.000 description 5
- LESXFEZIFXFIQR-LURJTMIESA-N Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(O)=O LESXFEZIFXFIQR-LURJTMIESA-N 0.000 description 5
- 241000282567 Macaca fascicularis Species 0.000 description 5
- JMEWFDUAFKVAAT-UHFFFAOYSA-N Methionyl-Asparagine Chemical compound CSCCC(N)C(=O)NC(C(O)=O)CC(N)=O JMEWFDUAFKVAAT-UHFFFAOYSA-N 0.000 description 5
- JXWLMUIXUXLIJR-QWRGUYRKSA-N Phe-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 JXWLMUIXUXLIJR-QWRGUYRKSA-N 0.000 description 5
- LTFSLKWFMWZEBD-IMJSIDKUSA-N Ser-Asn Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CC(N)=O LTFSLKWFMWZEBD-IMJSIDKUSA-N 0.000 description 5
- FFOKMZOAVHEWET-UHFFFAOYSA-N Serinyl-Cysteine Chemical compound OCC(N)C(=O)NC(CS)C(O)=O FFOKMZOAVHEWET-UHFFFAOYSA-N 0.000 description 5
- BECPPKYKPSRKCP-ZDLURKLDSA-N Thr-Glu Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O BECPPKYKPSRKCP-ZDLURKLDSA-N 0.000 description 5
- CUTPSEKWUPZFLV-UHFFFAOYSA-N Threoninyl-Cysteine Chemical compound CC(O)C(N)C(=O)NC(CS)C(O)=O CUTPSEKWUPZFLV-UHFFFAOYSA-N 0.000 description 5
- LWFWZRANSFAJDR-JSGCOSHPSA-N Trp-Val Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(O)=O)=CNC2=C1 LWFWZRANSFAJDR-JSGCOSHPSA-N 0.000 description 5
- ZSXJENBJGRHKIG-UHFFFAOYSA-N Tyrosyl-Serine Chemical compound OCC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 ZSXJENBJGRHKIG-UHFFFAOYSA-N 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 230000000240 adjuvant Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 5
- 229960002685 biotin Drugs 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 108091006028 chimera Proteins 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 238000006481 deamination reaction Methods 0.000 description 5
- 230000003247 decreasing Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 239000005090 green fluorescent protein Substances 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 108010036413 histidylglycine Proteins 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 230000003834 intracellular Effects 0.000 description 5
- 108010012058 leucyltyrosine Proteins 0.000 description 5
- 101700079891 lgg-2 Proteins 0.000 description 5
- 125000005647 linker group Chemical group 0.000 description 5
- 108010044655 lysylproline Proteins 0.000 description 5
- 230000001404 mediated Effects 0.000 description 5
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 5
- 108010012581 phenylalanylglutamate Proteins 0.000 description 5
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 229910052717 sulfur Inorganic materials 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 108010051110 tyrosyl-lysine Proteins 0.000 description 5
- 230000003442 weekly Effects 0.000 description 5
- XPJBQTCXPJNIFE-ZETCQYMHSA-N (2S)-2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]-4-methylpentanoate Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)CN XPJBQTCXPJNIFE-ZETCQYMHSA-N 0.000 description 4
- CCQOOWAONKGYKQ-BYPYZUCNSA-N (2S)-2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]propanoate Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 4
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 4
- VWHGTYCRDRBSFI-ZETCQYMHSA-N 2-[[2-[[(2S)-2-amino-4-methylpentanoyl]amino]acetyl]amino]acetic acid Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 4
- 108060000679 ATG12 Proteins 0.000 description 4
- RJUHZPRQRQLCFL-IMJSIDKUSA-N Asn-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(O)=O RJUHZPRQRQLCFL-IMJSIDKUSA-N 0.000 description 4
- KLKHFFMNGWULBN-VKHMYHEASA-N Asn-Gly Chemical compound NC(=O)C[C@H](N)C(=O)NCC(O)=O KLKHFFMNGWULBN-VKHMYHEASA-N 0.000 description 4
- JHFNSBBHKSZXKB-VKHMYHEASA-N Asp-Gly Chemical compound OC(=O)C[C@H](N)C(=O)NCC(O)=O JHFNSBBHKSZXKB-VKHMYHEASA-N 0.000 description 4
- YZQCXOFQZKCETR-UWVGGRQHSA-N Asp-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YZQCXOFQZKCETR-UWVGGRQHSA-N 0.000 description 4
- RGGVDKVXLBOLNS-UHFFFAOYSA-N Asparaginyl-Tryptophan Chemical compound C1=CC=C2C(CC(NC(=O)C(CC(N)=O)N)C(O)=O)=CNC2=C1 RGGVDKVXLBOLNS-UHFFFAOYSA-N 0.000 description 4
- NXTYATMDWQYLGJ-UHFFFAOYSA-N Cysteinyl-Leucine Chemical compound CC(C)CC(C(O)=O)NC(=O)C(N)CS NXTYATMDWQYLGJ-UHFFFAOYSA-N 0.000 description 4
- 210000004002 Dopaminergic cell Anatomy 0.000 description 4
- 229940088598 Enzyme Drugs 0.000 description 4
- FYYSIASRLDJUNP-WHFBIAKZSA-N Glu-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(O)=O FYYSIASRLDJUNP-WHFBIAKZSA-N 0.000 description 4
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 4
- YSWHPLCDIMUKFE-QWRGUYRKSA-N Glu-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 YSWHPLCDIMUKFE-QWRGUYRKSA-N 0.000 description 4
- 230000036499 Half live Effects 0.000 description 4
- VHOLZZKNEBBHTH-YUMQZZPRSA-N His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CNC=N1 VHOLZZKNEBBHTH-YUMQZZPRSA-N 0.000 description 4
- WSDOHRLQDGAOGU-UHFFFAOYSA-N Histidinyl-Asparagine Chemical compound NC(=O)CC(C(O)=O)NC(=O)C(N)CC1=CN=CN1 WSDOHRLQDGAOGU-UHFFFAOYSA-N 0.000 description 4
- 229940100601 Interleukin-6 Drugs 0.000 description 4
- 102000004889 Interleukin-6 Human genes 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 4
- 101710019425 LARS2 Proteins 0.000 description 4
- MLTRLIITQPXHBJ-BQBZGAKWSA-N Leu-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC(N)=O MLTRLIITQPXHBJ-BQBZGAKWSA-N 0.000 description 4
- LHSGPCFBGJHPCY-STQMWFEESA-N Leu-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-STQMWFEESA-N 0.000 description 4
- NVGBPTNZLWRQSY-UWVGGRQHSA-N Lys-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN NVGBPTNZLWRQSY-UWVGGRQHSA-N 0.000 description 4
- AIXUQKMMBQJZCU-IUCAKERBSA-N Lys-Pro Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O AIXUQKMMBQJZCU-IUCAKERBSA-N 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 210000002241 Neurites Anatomy 0.000 description 4
- BXNGIHFNNNSEOS-UWVGGRQHSA-N Phe-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 BXNGIHFNNNSEOS-UWVGGRQHSA-N 0.000 description 4
- HMNSRTLZAJHSIK-YUMQZZPRSA-N Pro-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 HMNSRTLZAJHSIK-YUMQZZPRSA-N 0.000 description 4
- UEKYKRQIAQHOOZ-KBPBESRZSA-N Pro-Trp Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)[O-])C(=O)[C@@H]1CCC[NH2+]1 UEKYKRQIAQHOOZ-KBPBESRZSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- LAFKUZYWNCHOHT-WHFBIAKZSA-N Ser-Glu Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O LAFKUZYWNCHOHT-WHFBIAKZSA-N 0.000 description 4
- LZLREEUGSYITMX-UHFFFAOYSA-N Serinyl-Tryptophan Chemical compound C1=CC=C2C(CC(NC(=O)C(CO)N)C(O)=O)=CNC2=C1 LZLREEUGSYITMX-UHFFFAOYSA-N 0.000 description 4
- VPZKQTYZIVOJDV-LMVFSUKVSA-N Thr-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(O)=O VPZKQTYZIVOJDV-LMVFSUKVSA-N 0.000 description 4
- HPYDSVWYXXKHRD-VIFPVBQESA-N Tyr-Gly Chemical compound [O-]C(=O)CNC(=O)[C@@H]([NH3+])CC1=CC=C(O)C=C1 HPYDSVWYXXKHRD-VIFPVBQESA-N 0.000 description 4
- UPJONISHZRADBH-XPUUQOCRSA-N Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O UPJONISHZRADBH-XPUUQOCRSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 4
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000111 anti-oxidant Effects 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 108010013835 arginine glutamate Proteins 0.000 description 4
- 108010036533 arginylvaline Proteins 0.000 description 4
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000002591 computed tomography Methods 0.000 description 4
- 230000001808 coupling Effects 0.000 description 4
- 108010060199 cysteinylproline Proteins 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 239000002612 dispersion media Substances 0.000 description 4
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 4
- 235000004554 glutamine Nutrition 0.000 description 4
- 108010050848 glycylleucine Proteins 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 230000001976 improved Effects 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 238000001990 intravenous administration Methods 0.000 description 4
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 4
- 108010057821 leucylproline Proteins 0.000 description 4
- 210000004962 mammalian cells Anatomy 0.000 description 4
- 230000001264 neutralization Effects 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 108010084572 phenylalanyl-valine Proteins 0.000 description 4
- 229910052700 potassium Inorganic materials 0.000 description 4
- 108010004914 prolylarginine Proteins 0.000 description 4
- DNIAPMSPPWPWGF-UHFFFAOYSA-N propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 4
- 108010071207 serylmethionine Proteins 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000007910 systemic administration Methods 0.000 description 4
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N (2S)-1-[(2S)-1-[(2S)-2-amino-3-phenylpropanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carboxylic acid Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 3
- BUZMZDDKFCSKOT-CIUDSAMLSA-N (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-carboxybutanoyl]amino]-4-carboxybutanoyl]amino]pentanedioic acid Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 3
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N (2S)-2-[[(2S)-2-[[(2S)-2-aminopropanoyl]amino]propanoyl]amino]-3-hydroxypropanoic acid Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 3
- NBTGEURICRTMGL-WHFBIAKZSA-N (2S)-2-[[2-[[(2S)-2-aminopropanoyl]amino]acetyl]amino]-3-hydroxypropanoic acid Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 3
- PVMPDMIKUVNOBD-CIUDSAMLSA-N (3S)-3-[[(2S)-2-amino-4-methylpentanoyl]amino]-4-[[(1S)-1-carboxy-2-hydroxyethyl]amino]-4-oxobutanoic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O PVMPDMIKUVNOBD-CIUDSAMLSA-N 0.000 description 3
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 3
- KTGFOCFYOZQVRJ-UHFFFAOYSA-N 2-[(2-amino-3-methylpentanoyl)amino]pentanedioic acid Chemical compound CCC(C)C(N)C(=O)NC(C(O)=O)CCC(O)=O KTGFOCFYOZQVRJ-UHFFFAOYSA-N 0.000 description 3
- RLMISHABBKUNFO-WHFBIAKZSA-N 2-[[(2S)-2-[[(2S)-2-azaniumylpropanoyl]amino]propanoyl]amino]acetate Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- CXISPYVYMQWFLE-VKHMYHEASA-N Ala-Gly Chemical compound C[C@H]([NH3+])C(=O)NCC([O-])=O CXISPYVYMQWFLE-VKHMYHEASA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- DAQIJMOLTMGJLO-YUMQZZPRSA-N Arg-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCNC(N)=N DAQIJMOLTMGJLO-YUMQZZPRSA-N 0.000 description 3
- HZYFHQOWCFUSOV-IMJSIDKUSA-N Asn-Asp Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(O)=O HZYFHQOWCFUSOV-IMJSIDKUSA-N 0.000 description 3
- SONUFGRSSMFHFN-IMJSIDKUSA-N Asn-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(O)=O SONUFGRSSMFHFN-IMJSIDKUSA-N 0.000 description 3
- FYRVDDJMNISIKJ-UWVGGRQHSA-N Asn-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FYRVDDJMNISIKJ-UWVGGRQHSA-N 0.000 description 3
- NTQDELBZOMWXRS-UHFFFAOYSA-N Aspartyl-Threonine Chemical compound CC(O)C(C(O)=O)NC(=O)C(N)CC(O)=O NTQDELBZOMWXRS-UHFFFAOYSA-N 0.000 description 3
- 210000001218 Blood-Brain Barrier Anatomy 0.000 description 3
- 108010032088 Calpain Proteins 0.000 description 3
- 102000007590 Calpain Human genes 0.000 description 3
- 210000000349 Chromosomes Anatomy 0.000 description 3
- HAYVTMHUNMMXCV-UHFFFAOYSA-N Cysteinyl-Alanine Chemical compound OC(=O)C(C)NC(=O)C(N)CS HAYVTMHUNMMXCV-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 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 3
- 108010074860 Factor Xa Proteins 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- 229960002743 Glutamine Drugs 0.000 description 3
- SCCPDJAQCXWPTF-VKHMYHEASA-N Gly-Asp Chemical compound NCC(=O)N[C@H](C(O)=O)CC(O)=O SCCPDJAQCXWPTF-VKHMYHEASA-N 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- LYCVKHSJGDMDLM-LURJTMIESA-N His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 LYCVKHSJGDMDLM-LURJTMIESA-N 0.000 description 3
- WRPDZHJNLYNFFT-UHFFFAOYSA-N Histidinyl-Threonine Chemical compound CC(O)C(C(O)=O)NC(=O)C(N)CC1=CN=CN1 WRPDZHJNLYNFFT-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- RCFDOSNHHZGBOY-ACZMJKKPSA-N Ile-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(O)=O RCFDOSNHHZGBOY-ACZMJKKPSA-N 0.000 description 3
- HYXQKVOADYPQEA-CIUDSAMLSA-N Ile-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HYXQKVOADYPQEA-CIUDSAMLSA-N 0.000 description 3
- 210000000987 Immune System Anatomy 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- QOOWRKBDDXQRHC-BQBZGAKWSA-N L-lysyl-L-alanine Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN QOOWRKBDDXQRHC-BQBZGAKWSA-N 0.000 description 3
- 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 3
- 229940067606 Lecithin Drugs 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 108060001084 Luciferase family Proteins 0.000 description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N Luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 3
- UGTZHPSKYRIGRJ-YUMQZZPRSA-N Lys-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O UGTZHPSKYRIGRJ-YUMQZZPRSA-N 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 229940049954 Penicillin Drugs 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- WEQJQNWXCSUVMA-RYUDHWBXSA-N Phe-Pro Chemical compound C([C@H]([NH3+])C(=O)N1[C@@H](CCC1)C([O-])=O)C1=CC=CC=C1 WEQJQNWXCSUVMA-RYUDHWBXSA-N 0.000 description 3
- FSXRLASFHBWESK-HOTGVXAUSA-N Phe-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 FSXRLASFHBWESK-HOTGVXAUSA-N 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 108010033725 Recombinant Proteins Proteins 0.000 description 3
- 102000007312 Recombinant Proteins Human genes 0.000 description 3
- PBUXMVYWOSKHMF-WDSKDSINSA-N Ser-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CO PBUXMVYWOSKHMF-WDSKDSINSA-N 0.000 description 3
- 210000003625 Skull Anatomy 0.000 description 3
- 229940083542 Sodium Drugs 0.000 description 3
- 229960005322 Streptomycin Drugs 0.000 description 3
- GXDLGHLJTHMDII-WISUUJSJSA-N Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(O)=O GXDLGHLJTHMDII-WISUUJSJSA-N 0.000 description 3
- DSGIVWSDDRDJIO-ZXXMMSQZSA-N Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DSGIVWSDDRDJIO-ZXXMMSQZSA-N 0.000 description 3
- UYKREHOKELZSPB-JTQLQIEISA-N Trp-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(O)=O)=CNC2=C1 UYKREHOKELZSPB-JTQLQIEISA-N 0.000 description 3
- TYYLDKGBCJGJGW-WMZOPIPTSA-N Trp-Tyr Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)C(O)=O)C1=CC=C(O)C=C1 TYYLDKGBCJGJGW-WMZOPIPTSA-N 0.000 description 3
- WITCOKQIPFWQQD-FSPLSTOPSA-N Val-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CC(N)=O WITCOKQIPFWQQD-FSPLSTOPSA-N 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 230000032683 aging Effects 0.000 description 3
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 3
- 108010047495 alanylglycine Proteins 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 3
- 229960000626 benzylpenicillin Drugs 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000011651 chromium Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 230000001149 cognitive Effects 0.000 description 3
- 230000021615 conjugation Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000002354 daily Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 229940079593 drugs Drugs 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 230000036749 excitatory postsynaptic potential Effects 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 229910052731 fluorine Inorganic materials 0.000 description 3
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- 230000003899 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 108010050475 glycyl-leucyl-tyrosine Proteins 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 239000012133 immunoprecipitate Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- 239000000787 lecithin Substances 0.000 description 3
- 235000010445 lecithin Nutrition 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 230000036961 partial Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 230000002572 peristaltic Effects 0.000 description 3
- 108010024654 phenylalanyl-prolyl-alanine Proteins 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000002600 positron emission tomography Methods 0.000 description 3
- 230000002335 preservative Effects 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 230000000750 progressive Effects 0.000 description 3
- 230000017854 proteolysis Effects 0.000 description 3
- 230000002797 proteolythic Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000010948 rhodium Substances 0.000 description 3
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 3
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 238000003892 spreading Methods 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 230000003956 synaptic plasticity Effects 0.000 description 3
- 108010071097 threonyl-lysyl-proline Proteins 0.000 description 3
- 239000010936 titanium Substances 0.000 description 3
- 108010080629 tryptophan-leucine Proteins 0.000 description 3
- 108010044292 tryptophyltyrosine Proteins 0.000 description 3
- 229910052721 tungsten Inorganic materials 0.000 description 3
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 229910052727 yttrium Inorganic materials 0.000 description 3
- 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 2
- ILDSIMPXNFWKLH-KATARQTJSA-N (2S)-2-[[(2S,3R)-2-[[(2S)-2-amino-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ILDSIMPXNFWKLH-KATARQTJSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N 2-mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 229940022698 Acetylcholinesterase Drugs 0.000 description 2
- 102000012440 Acetylcholinesterase Human genes 0.000 description 2
- 108010022752 Acetylcholinesterase Proteins 0.000 description 2
- 206010069754 Acquired gene mutation Diseases 0.000 description 2
- 108010000239 Aequorin Proteins 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- XAEWTDMGFGHWFK-IMJSIDKUSA-M Ala-Asp(1-) Chemical compound C[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CC([O-])=O XAEWTDMGFGHWFK-IMJSIDKUSA-M 0.000 description 2
- 210000004727 Amygdala Anatomy 0.000 description 2
- PSZNHSNIGMJYOZ-WDSKDSINSA-N Asp-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PSZNHSNIGMJYOZ-WDSKDSINSA-N 0.000 description 2
- DWBZEJHQQIURML-IMJSIDKUSA-N Asp-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(O)=O DWBZEJHQQIURML-IMJSIDKUSA-N 0.000 description 2
- ZARXTZFGQZBYFO-JQWIXIFHSA-N Asp-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(O)=O)N)C(O)=O)=CNC2=C1 ZARXTZFGQZBYFO-JQWIXIFHSA-N 0.000 description 2
- MQLZLIYPFDIDMZ-UHFFFAOYSA-N Asparaginyl-Isoleucine Chemical compound CCC(C)C(C(O)=O)NC(=O)C(N)CC(N)=O MQLZLIYPFDIDMZ-UHFFFAOYSA-N 0.000 description 2
- 229940009098 Aspartate Drugs 0.000 description 2
- BSWHERGFUNMWGS-UHFFFAOYSA-N Aspartyl-Isoleucine Chemical compound CCC(C)C(C(O)=O)NC(=O)C(N)CC(O)=O BSWHERGFUNMWGS-UHFFFAOYSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- 102100015648 BACE2 Human genes 0.000 description 2
- 101700051947 BACE2 Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108010071919 Bispecific Antibodies Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 229940095259 Butylated Hydroxytoluene Drugs 0.000 description 2
- 239000004322 Butylated hydroxytoluene Substances 0.000 description 2
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 2
- VDQQXEISLMTGAB-UHFFFAOYSA-N Chloramine-T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 2
- YXQDRIRSAHTJKM-IMJSIDKUSA-N Cys-Ser Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(O)=O YXQDRIRSAHTJKM-IMJSIDKUSA-N 0.000 description 2
- 229960002433 Cysteine Drugs 0.000 description 2
- RGTVXXNMOGHRAY-UHFFFAOYSA-N Cysteinyl-Arginine Chemical compound SCC(N)C(=O)NC(C(O)=O)CCCNC(N)=N RGTVXXNMOGHRAY-UHFFFAOYSA-N 0.000 description 2
- AYKQJQVWUYEZNU-UHFFFAOYSA-N Cysteinyl-Asparagine Chemical compound SCC(N)C(=O)NC(C(O)=O)CC(N)=O AYKQJQVWUYEZNU-UHFFFAOYSA-N 0.000 description 2
- WXOFKRKAHJQKLT-UHFFFAOYSA-N Cysteinyl-Lysine Chemical compound NCCCCC(C(O)=O)NC(=O)C(N)CS WXOFKRKAHJQKLT-UHFFFAOYSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytosar Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 2
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 2
- 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 2
- CKLJMWTZIZZHCS-UHFFFAOYSA-N DL-aspartic acid Chemical compound OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 2
- 229960000958 Deferoxamine Drugs 0.000 description 2
- 229950006137 Dexfosfoserine Drugs 0.000 description 2
- 210000001029 Dorsal striatum Anatomy 0.000 description 2
- 241001269524 Dura Species 0.000 description 2
- 229910052692 Dysprosium Inorganic materials 0.000 description 2
- 229910052691 Erbium Inorganic materials 0.000 description 2
- LVGKNOAMLMIIKO-QXMHVHEDSA-N Ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 2
- 229910052693 Europium Inorganic materials 0.000 description 2
- 210000001723 Extracellular Space Anatomy 0.000 description 2
- 102100011343 GLB1 Human genes 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- YBAFDPFAUTYYRW-YUMQZZPRSA-N Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(O)=O YBAFDPFAUTYYRW-YUMQZZPRSA-N 0.000 description 2
- MFBYPDKTAJXHNI-VKHMYHEASA-N Gly-Cys Chemical compound [NH3+]CC(=O)N[C@@H](CS)C([O-])=O MFBYPDKTAJXHNI-VKHMYHEASA-N 0.000 description 2
- 210000004326 Gyrus Cinguli Anatomy 0.000 description 2
- 101710017531 H4C15 Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- CZVQSYNVUHAILZ-UWVGGRQHSA-N His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CN=CN1 CZVQSYNVUHAILZ-UWVGGRQHSA-N 0.000 description 2
- HTOOKGDPMXSJSY-STQMWFEESA-N His-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 HTOOKGDPMXSJSY-STQMWFEESA-N 0.000 description 2
- 229910052689 Holmium Inorganic materials 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 2
- 102220384595 IDH3B N54Q Human genes 0.000 description 2
- 229940072221 IMMUNOGLOBULINS Drugs 0.000 description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N Imidazole Chemical compound C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- HFKJBCPRWWGPEY-BQBZGAKWSA-N L-arginyl-L-glutamic acid Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HFKJBCPRWWGPEY-BQBZGAKWSA-N 0.000 description 2
- JAQGKXUEKGKTKX-HOTGVXAUSA-N L-tyrosyl-L-tyrosine Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 JAQGKXUEKGKTKX-HOTGVXAUSA-N 0.000 description 2
- OTXBNHIUIHNGAO-UWVGGRQHSA-N Leu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN OTXBNHIUIHNGAO-UWVGGRQHSA-N 0.000 description 2
- VTJUNIYRYIAIHF-IUCAKERBSA-N Leu-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O VTJUNIYRYIAIHF-IUCAKERBSA-N 0.000 description 2
- 210000001165 Lymph Nodes Anatomy 0.000 description 2
- NPBGTPKLVJEOBE-IUCAKERBSA-N Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N NPBGTPKLVJEOBE-IUCAKERBSA-N 0.000 description 2
- 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 2
- BJFJQOMZCSHBMY-YUMQZZPRSA-N Met-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(O)=O BJFJQOMZCSHBMY-YUMQZZPRSA-N 0.000 description 2
- 241000087624 Monoclona Species 0.000 description 2
- UBQYURCVBFRUQT-UHFFFAOYSA-N N-benzoyl-Ferrioxamine B Chemical compound CC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCN UBQYURCVBFRUQT-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 210000001577 Neostriatum Anatomy 0.000 description 2
- 229920001850 Nucleic acid sequence Polymers 0.000 description 2
- 208000003715 Parkinsonian Disorders Diseases 0.000 description 2
- 206010034010 Parkinsonism Diseases 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 102000035443 Peptidases Human genes 0.000 description 2
- 229940072417 Peroxidase Drugs 0.000 description 2
- 108090000437 Peroxidases Proteins 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- JWBLQDDHSDGEGR-DRZSPHRISA-N Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 JWBLQDDHSDGEGR-DRZSPHRISA-N 0.000 description 2
- BZQFBWGGLXLEPQ-REOHCLBHSA-N Phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 229920000954 Polyglycolide Polymers 0.000 description 2
- FELJDCNGZFDUNR-WDSKDSINSA-N Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 FELJDCNGZFDUNR-WDSKDSINSA-N 0.000 description 2
- RVQDZELMXZRSSI-IUCAKERBSA-N Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 RVQDZELMXZRSSI-IUCAKERBSA-N 0.000 description 2
- HXNYBZQLBWIADP-UHFFFAOYSA-N Prolyl-Cysteine Chemical compound OC(=O)C(CS)NC(=O)C1CCCN1 HXNYBZQLBWIADP-UHFFFAOYSA-N 0.000 description 2
- 229910052772 Samarium Inorganic materials 0.000 description 2
- VBKBDLMWICBSCY-IMJSIDKUSA-N Ser-Asp Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O VBKBDLMWICBSCY-IMJSIDKUSA-N 0.000 description 2
- PPQRSMGDOHLTBE-UWVGGRQHSA-N Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PPQRSMGDOHLTBE-UWVGGRQHSA-N 0.000 description 2
- WBAXJMCUFIXCNI-WDSKDSINSA-N Ser-Pro Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O WBAXJMCUFIXCNI-WDSKDSINSA-N 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M Sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L Sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- IOWJRKAVLALBQB-IWGUZYHVSA-N Thr-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O IOWJRKAVLALBQB-IWGUZYHVSA-N 0.000 description 2
- WXVIGTAUZBUDPZ-DTLFHODZSA-N Thr-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 WXVIGTAUZBUDPZ-DTLFHODZSA-N 0.000 description 2
- QOLYAJSZHIJCTO-VQVTYTSYSA-N Thr-Pro Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(O)=O QOLYAJSZHIJCTO-VQVTYTSYSA-N 0.000 description 2
- WCRFXRIWBFRZBR-GGVZMXCHSA-N Thr-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WCRFXRIWBFRZBR-GGVZMXCHSA-N 0.000 description 2
- FPKOPBFLPLFWAD-UHFFFAOYSA-N Trinitrotoluene Chemical compound CC1=CC=C([N+]([O-])=O)C([N+]([O-])=O)=C1[N+]([O-])=O FPKOPBFLPLFWAD-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- GRQCSEWEPIHLBI-UHFFFAOYSA-N Tryptophyl-Asparagine Chemical compound C1=CC=C2C(CC(N)C(=O)NC(CC(N)=O)C(O)=O)=CNC2=C1 GRQCSEWEPIHLBI-UHFFFAOYSA-N 0.000 description 2
- KBUBZAMBIVEFEI-UHFFFAOYSA-N Tryptophyl-Histidine Chemical compound C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CN=CN1 KBUBZAMBIVEFEI-UHFFFAOYSA-N 0.000 description 2
- ONWMQORSVZYVNH-UHFFFAOYSA-N Tyrosyl-Asparagine Chemical compound NC(=O)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 ONWMQORSVZYVNH-UHFFFAOYSA-N 0.000 description 2
- 230000036462 Unbound Effects 0.000 description 2
- 101700053999 VAR1 Proteins 0.000 description 2
- IBIDRSSEHFLGSD-YUMQZZPRSA-N Val-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-YUMQZZPRSA-N 0.000 description 2
- GIAZPLMMQOERPN-YUMQZZPRSA-N Val-Pro Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(O)=O GIAZPLMMQOERPN-YUMQZZPRSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000002730 additional Effects 0.000 description 2
- 108010041407 alanylaspartic acid Proteins 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000890 antigenic Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 2
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 2
- 101700065210 asp1 Proteins 0.000 description 2
- 108010038633 aspartylglutamate Proteins 0.000 description 2
- 230000001580 bacterial Effects 0.000 description 2
- 230000003542 behavioural Effects 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000000903 blocking Effects 0.000 description 2
- 235000010354 butylated hydroxytoluene Nutrition 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 210000000692 cap cell Anatomy 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000001413 cellular Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000005591 charge neutralization Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000002759 chromosomal Effects 0.000 description 2
- 231100000762 chronic effect Toxicity 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 230000001054 cortical Effects 0.000 description 2
- 238000010192 crystallographic characterization Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 229940042399 direct acting antivirals Protease inhibitors Drugs 0.000 description 2
- 229960003638 dopamine Drugs 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- KBQHZAAAGSGFKK-UHFFFAOYSA-N dysprosium Chemical compound [Dy] KBQHZAAAGSGFKK-UHFFFAOYSA-N 0.000 description 2
- 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 2
- UYAHIZSMUZPPFV-UHFFFAOYSA-N erbium Chemical compound [Er] UYAHIZSMUZPPFV-UHFFFAOYSA-N 0.000 description 2
- 229940093471 ethyl oleate Drugs 0.000 description 2
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 2
- 201000008808 fibrosarcoma Diseases 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 108010013768 glutamyl-aspartyl-proline Proteins 0.000 description 2
- KZNQNBZMBZJQJO-YFKPBYRVSA-N gly pro Chemical compound NCC(=O)N1CCC[C@H]1C(O)=O KZNQNBZMBZJQJO-YFKPBYRVSA-N 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine zwitterion Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- KJZYNXUDTRRSPN-UHFFFAOYSA-N holmium Chemical compound [Ho] KJZYNXUDTRRSPN-UHFFFAOYSA-N 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000002998 immunogenetic Effects 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 238000000126 in silico method Methods 0.000 description 2
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 2
- 229910052738 indium Inorganic materials 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 description 2
- 229910052741 iridium Inorganic materials 0.000 description 2
- 108010027338 isoleucylcysteine Proteins 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- 150000002632 lipids Chemical group 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 238000011068 load Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000002934 lysing Effects 0.000 description 2
- 230000005291 magnetic Effects 0.000 description 2
- PWHULOQIROXLJO-UHFFFAOYSA-N manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000001471 micro-filtration Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 230000003278 mimic Effects 0.000 description 2
- ZOKXTWBITQBERF-UHFFFAOYSA-N molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 2
- 229910052750 molybdenum Inorganic materials 0.000 description 2
- 239000011733 molybdenum Substances 0.000 description 2
- 239000001788 mono and diglycerides of fatty acids Substances 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 230000001537 neural Effects 0.000 description 2
- 210000004255 neuroglia Anatomy 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- 230000003287 optical Effects 0.000 description 2
- 150000002895 organic esters Chemical class 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 229910052763 palladium Inorganic materials 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 229920000747 poly(lactic acid) polymer Polymers 0.000 description 2
- 229920000023 polynucleotide Polymers 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 229920005862 polyol Polymers 0.000 description 2
- 150000003077 polyols Chemical class 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000002708 random mutagenesis Methods 0.000 description 2
- 229910052761 rare earth metal Inorganic materials 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 2
- 229910052703 rhodium Inorganic materials 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- KZUNJOHGWZRPMI-UHFFFAOYSA-N samarium Chemical compound [Sm] KZUNJOHGWZRPMI-UHFFFAOYSA-N 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000002603 single-photon emission computed tomography Methods 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 2
- 229910052712 strontium Inorganic materials 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 description 2
- 229910052713 technetium Inorganic materials 0.000 description 2
- 230000004797 therapeutic response Effects 0.000 description 2
- JADVWWSKYZXRGX-UHFFFAOYSA-M thioflavin T Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C1=[N+](C)C2=CC=C(C)C=C2S1 JADVWWSKYZXRGX-UHFFFAOYSA-M 0.000 description 2
- 229910052718 tin Inorganic materials 0.000 description 2
- ATJFFYVFTNAWJD-UHFFFAOYSA-N tin hydride Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 2
- 230000000699 topical Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001052 transient Effects 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 238000007492 two-way ANOVA Methods 0.000 description 2
- 235000002374 tyrosine Nutrition 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 238000009777 vacuum freeze-drying Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- QCWXUUIWCKQGHC-UHFFFAOYSA-N zirconium Chemical compound [Zr] QCWXUUIWCKQGHC-UHFFFAOYSA-N 0.000 description 2
- 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 2
- FAWLNYODPPEZHK-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-trimethylstannylbenzoate Chemical compound C[Sn](C)(C)C1=CC=CC(C(=O)ON2C(CCC2=O)=O)=C1 FAWLNYODPPEZHK-UHFFFAOYSA-N 0.000 description 1
- JSZMKEYEVLDPDO-ACZMJKKPSA-N (2R)-2-[[(2S,3S)-2-amino-3-methylpentanoyl]amino]-3-sulfanylpropanoic acid Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CS)C(O)=O JSZMKEYEVLDPDO-ACZMJKKPSA-N 0.000 description 1
- CXRCVCURMBFFOL-FXQIFTODSA-N (2S)-1-[(2S)-2-[[(2S)-2-azaniumylpropanoyl]amino]propanoyl]pyrrolidine-2-carboxylate Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 description 1
- LQJAALCCPOTJGB-YUMQZZPRSA-N (2S)-1-[(2S)-2-amino-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carboxylic acid Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O LQJAALCCPOTJGB-YUMQZZPRSA-N 0.000 description 1
- DWYAUVCQDTZIJI-VZFHVOOUSA-N (2S)-2-[[(2S)-2-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]propanoyl]amino]-3-hydroxypropanoic acid Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DWYAUVCQDTZIJI-VZFHVOOUSA-N 0.000 description 1
- BEJKOYIMCGMNRB-GRHHLOCNSA-N (2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid;(2S)-2-amino-3-phenylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1.OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 BEJKOYIMCGMNRB-GRHHLOCNSA-N 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N (4S)-4-amino-5-[[(2S)-4-carboxy-1-(carboxymethylamino)-1-oxobutan-2-yl]amino]-5-oxopentanoic acid Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 1
- VHJLVAABSRFDPM-UHFFFAOYSA-N 1,4-dimercaptobutane-2,3-diol Chemical compound SCC(O)C(O)CS VHJLVAABSRFDPM-UHFFFAOYSA-N 0.000 description 1
- UGBLISDIHDMHJX-UHFFFAOYSA-N 1-(4-fluorophenyl)-4-[4-(2-methoxyphenyl)piperazin-1-yl]butan-1-one;hydrochloride Chemical compound [Cl-].COC1=CC=CC=C1N1CC[NH+](CCCC(=O)C=2C=CC(F)=CC=2)CC1 UGBLISDIHDMHJX-UHFFFAOYSA-N 0.000 description 1
- 101700083798 12KD Proteins 0.000 description 1
- GJSURZIOUXUGAL-UHFFFAOYSA-N 2-((2,6-Dichlorophenyl)imino)imidazolidine Chemical compound ClC1=CC=CC(Cl)=C1NC1=NCCN1 GJSURZIOUXUGAL-UHFFFAOYSA-N 0.000 description 1
- BHHGXPLMPWCGHP-UHFFFAOYSA-N 2-Phenethylamine Chemical class NCCC1=CC=CC=C1 BHHGXPLMPWCGHP-UHFFFAOYSA-N 0.000 description 1
- BUXAPSQPMALTOY-UHFFFAOYSA-N 2-[(2-amino-3-sulfanylpropanoyl)amino]pentanedioic acid Chemical compound SCC(N)C(=O)NC(C(O)=O)CCC(O)=O BUXAPSQPMALTOY-UHFFFAOYSA-N 0.000 description 1
- RFCVXVPWSPOMFJ-UHFFFAOYSA-N 2-[(2-azaniumyl-3-phenylpropanoyl)amino]-4-methylpentanoate Chemical compound CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 RFCVXVPWSPOMFJ-UHFFFAOYSA-N 0.000 description 1
- JHALWMSZGCVVEM-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CC1 JHALWMSZGCVVEM-UHFFFAOYSA-N 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N 2-[[(2S)-2-[[(2S)-2-azaniumyl-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetate Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- XUUXCWCKKCZEAW-YFKPBYRVSA-N 2-[[(2S)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N XUUXCWCKKCZEAW-YFKPBYRVSA-N 0.000 description 1
- XPDXVDYUQZHFPV-UHFFFAOYSA-N 5-Dimethylaminonaphthyl-5-sulfonyl chloride Chemical compound C1=CC=C2C(N(C)C)=CC=CC2=C1S(Cl)(=O)=O XPDXVDYUQZHFPV-UHFFFAOYSA-N 0.000 description 1
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 description 1
- GZAJOEGTZDUSKS-UHFFFAOYSA-N 6-amino-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(N)=CC=C21 GZAJOEGTZDUSKS-UHFFFAOYSA-N 0.000 description 1
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- ZTOJFFHGPLIVKC-CLFAGFIQSA-N ABTS Chemical compound S/1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C\1=N\N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-CLFAGFIQSA-N 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
- 101710027066 ALB Proteins 0.000 description 1
- 206010000117 Abnormal behaviour Diseases 0.000 description 1
- 241000432074 Adeno-associated virus Species 0.000 description 1
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 1
- CCUAQNUWXLYFRA-IMJSIDKUSA-N Ala-Asn Chemical compound C[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CC(N)=O CCUAQNUWXLYFRA-IMJSIDKUSA-N 0.000 description 1
- 108010076441 Ala-His-His Proteins 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- 206010002653 Anosmia Diseases 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 210000000702 Aorta, Abdominal Anatomy 0.000 description 1
- WYBVBIHNJWOLCJ-IUCAKERBSA-N Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCNC(N)=N WYBVBIHNJWOLCJ-IUCAKERBSA-N 0.000 description 1
- ROWCTNFEMKOIFQ-YUMQZZPRSA-N Arg-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCNC(N)=N ROWCTNFEMKOIFQ-YUMQZZPRSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- SJUXYGVRSGTPMC-UHFFFAOYSA-N Asparaginyl-Alanine Chemical compound OC(=O)C(C)NC(=O)C(N)CC(N)=O SJUXYGVRSGTPMC-UHFFFAOYSA-N 0.000 description 1
- 210000001130 Astrocytes Anatomy 0.000 description 1
- 210000003719 B-Lymphocytes Anatomy 0.000 description 1
- 210000004369 Blood Anatomy 0.000 description 1
- 210000001124 Body Fluids Anatomy 0.000 description 1
- 108091003117 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006100 Bradykinesia Diseases 0.000 description 1
- 210000004900 C-terminal fragment Anatomy 0.000 description 1
- 101710038039 CAMK2A Proteins 0.000 description 1
- 102100013916 CAMK2A Human genes 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M CHEMBL593252 Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 102100006400 CSF2 Human genes 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241001631457 Cannula Species 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 210000000170 Cell Membrane Anatomy 0.000 description 1
- 210000003169 Central Nervous System Anatomy 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 210000003467 Cheek Anatomy 0.000 description 1
- 241000256135 Chironomus thummi Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 230000037250 Clearance Effects 0.000 description 1
- 229920001405 Coding region Polymers 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010057668 Cognitive disease Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 241001573498 Compacta Species 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- ZSRSLWKGWFFVCM-WDSKDSINSA-N Cys-Pro Chemical compound SC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O ZSRSLWKGWFFVCM-WDSKDSINSA-N 0.000 description 1
- 229960001305 Cysteine Hydrochloride Drugs 0.000 description 1
- 229960000684 Cytarabine Drugs 0.000 description 1
- 210000000805 Cytoplasm Anatomy 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 230000035687 Dissociation Rate Constant Effects 0.000 description 1
- 210000001951 Dura Mater Anatomy 0.000 description 1
- 108010015776 EC 1.1.3.4 Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 230000036809 Fabs Effects 0.000 description 1
- 210000002683 Foot Anatomy 0.000 description 1
- 210000002454 Frontal Bone Anatomy 0.000 description 1
- 101710030624 GALNT5 Proteins 0.000 description 1
- 229940116332 GLUCOSE OXIDASE Drugs 0.000 description 1
- 102200099393 GOLGA6L2 N54H Human genes 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N Gadolinium Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- GYHNNYVSQQEPJS-UHFFFAOYSA-N Gallium Chemical compound [Ga] GYHNNYVSQQEPJS-UHFFFAOYSA-N 0.000 description 1
- 229940014259 Gelatin Drugs 0.000 description 1
- QOIGKGMMAGJZNZ-UHFFFAOYSA-N Gepirone Chemical compound O=C1CC(C)(C)CC(=O)N1CCCCN1CCN(C=2N=CC=CN=2)CC1 QOIGKGMMAGJZNZ-UHFFFAOYSA-N 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- PFMUCCYYAAFKTH-YFKPBYRVSA-N Gly-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)CN PFMUCCYYAAFKTH-YFKPBYRVSA-N 0.000 description 1
- 229940074045 Glyceryl Distearate Drugs 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- MMFKFJORZBJVNF-UWVGGRQHSA-N His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CN=CN1 MMFKFJORZBJVNF-UWVGGRQHSA-N 0.000 description 1
- 108091006038 His-tagged proteins Proteins 0.000 description 1
- 210000003284 Horns Anatomy 0.000 description 1
- 108091006822 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 206010053317 Hydrophobia Diseases 0.000 description 1
- 241000406221 Hypostomus robinii Species 0.000 description 1
- 102200066210 IL37 N54S Human genes 0.000 description 1
- 231100000608 Immunotoxin Toxicity 0.000 description 1
- 108010004484 Immunotoxins Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- WKXVAXOSIPTXEC-UHFFFAOYSA-N Isoleucyl-Aspartate Chemical compound CCC(C)C(N)C(=O)NC(C(O)=O)CC(O)=O WKXVAXOSIPTXEC-UHFFFAOYSA-N 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N Kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-Methionine Natural products CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 239000011786 L-ascorbyl-6-palmitate Substances 0.000 description 1
- QAQJMLQRFWZOBN-LAUBAEHRSA-N L-ascorbyl-6-palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](O)[C@H]1OC(=O)C(O)=C1O QAQJMLQRFWZOBN-LAUBAEHRSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- JXNRXNCCROJZFB-RYUDHWBXSA-N L-tyrosyl-L-arginine Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JXNRXNCCROJZFB-RYUDHWBXSA-N 0.000 description 1
- 229920000126 Latex Polymers 0.000 description 1
- XWOBNBRUDDUEEY-UWVGGRQHSA-N Leu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 XWOBNBRUDDUEEY-UWVGGRQHSA-N 0.000 description 1
- FMIIKPHLJKUXGE-GUBZILKMSA-N Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN FMIIKPHLJKUXGE-GUBZILKMSA-N 0.000 description 1
- QCZYYEFXOBKCNQ-STQMWFEESA-N Lys-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QCZYYEFXOBKCNQ-STQMWFEESA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229920002521 Macromolecule Polymers 0.000 description 1
- 210000001259 Mesencephalon Anatomy 0.000 description 1
- ADHNYKZHPOEULM-BQBZGAKWSA-N Met-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O ADHNYKZHPOEULM-BQBZGAKWSA-N 0.000 description 1
- OGGRSJFVXREKOR-CIUDSAMLSA-N Met-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCSC OGGRSJFVXREKOR-CIUDSAMLSA-N 0.000 description 1
- PESQCPHRXOFIPX-RYUDHWBXSA-N Met-Tyr Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-RYUDHWBXSA-N 0.000 description 1
- 230000035633 Metabolized Effects 0.000 description 1
- 210000000274 Microglia Anatomy 0.000 description 1
- 210000004080 Milk Anatomy 0.000 description 1
- 210000002161 Motor Neurons Anatomy 0.000 description 1
- 206010061296 Motor dysfunction Diseases 0.000 description 1
- 241000713333 Mouse mammary tumor virus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000002740 Muscle Rigidity Diseases 0.000 description 1
- ZTLGJPIZUOVDMT-UHFFFAOYSA-N N,N-dichlorotriazin-4-amine Chemical compound ClN(Cl)C1=CC=NN=N1 ZTLGJPIZUOVDMT-UHFFFAOYSA-N 0.000 description 1
- 229920002957 Naked DNA Polymers 0.000 description 1
- QEFYFXOXNSNQGX-UHFFFAOYSA-N Neodymium Chemical compound [Nd] QEFYFXOXNSNQGX-UHFFFAOYSA-N 0.000 description 1
- 229910052779 Neodymium Inorganic materials 0.000 description 1
- 206010053643 Neurodegenerative disease Diseases 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- KJONHKAYOJNZEC-UHFFFAOYSA-N Nitrazepam Chemical compound C12=CC([N+](=O)[O-])=CC=C2NC(=O)CN=C1C1=CC=CC=C1 KJONHKAYOJNZEC-UHFFFAOYSA-N 0.000 description 1
- 210000004940 Nucleus Anatomy 0.000 description 1
- 208000001292 Olivopontocerebellar Atrophy Diseases 0.000 description 1
- 241000283898 Ovis Species 0.000 description 1
- 229940055729 Papain Drugs 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- PYOHODCEOHCZBM-RYUDHWBXSA-N Phe-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 PYOHODCEOHCZBM-RYUDHWBXSA-N 0.000 description 1
- GKZIWHRNKRBEOH-HOTGVXAUSA-N Phe-Phe Chemical compound C([C@H]([NH3+])C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)C1=CC=CC=C1 GKZIWHRNKRBEOH-HOTGVXAUSA-N 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N Phenylalanyl-Lysine Chemical compound NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- 229940012957 Plasmin Drugs 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 241001272996 Polyphylla fullo Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229910052777 Praseodymium Inorganic materials 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- ZKQOUHVVXABNDG-IUCAKERBSA-N Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 ZKQOUHVVXABNDG-IUCAKERBSA-N 0.000 description 1
- IWIANZLCJVYEFX-RYUDHWBXSA-N Pro-Phe Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 IWIANZLCJVYEFX-RYUDHWBXSA-N 0.000 description 1
- 229940075579 Propyl Gallate Drugs 0.000 description 1
- 210000004129 Prosencephalon Anatomy 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 229920000320 RNA (poly(A)) Polymers 0.000 description 1
- 108020004412 RNA 3' Polyadenylation Signals Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 206010071390 Resting tremor Diseases 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N Rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 241000724205 Rice stripe tenuivirus Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 102200036626 SNCA A53T Human genes 0.000 description 1
- 229940100996 SODIUM BISULFATE Drugs 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- BXLYSRPHVMCOPS-UHFFFAOYSA-N Serinyl-Isoleucine Chemical compound CCC(C)C(C(O)=O)NC(=O)C(N)CO BXLYSRPHVMCOPS-UHFFFAOYSA-N 0.000 description 1
- 208000009106 Shy-Drager Syndrome Diseases 0.000 description 1
- 102000001555 Sialic Acid Binding Ig-like Lectin 3 Human genes 0.000 description 1
- 108010029180 Sialic Acid Binding Ig-like Lectin 3 Proteins 0.000 description 1
- 241000580858 Simian-Human immunodeficiency virus Species 0.000 description 1
- 229940054269 Sodium Pyruvate Drugs 0.000 description 1
- WBHQBSYUUJJSRZ-UHFFFAOYSA-M Sodium bisulfate Chemical compound [Na+].OS([O-])(=O)=O WBHQBSYUUJJSRZ-UHFFFAOYSA-M 0.000 description 1
- 229940091252 Sodium supplements Drugs 0.000 description 1
- 210000000952 Spleen Anatomy 0.000 description 1
- 208000003755 Striatonigral Degeneration Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L Sulphite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 210000002504 Synaptic Vesicles Anatomy 0.000 description 1
- JVHROZDXPAUZFK-UHFFFAOYSA-N TETA Chemical compound OC(=O)CN1CCCN(CC(O)=O)CCN(CC(O)=O)CCCN(CC(O)=O)CC1 JVHROZDXPAUZFK-UHFFFAOYSA-N 0.000 description 1
- 101700026084 THY1 Proteins 0.000 description 1
- 108060008443 TPPP Proteins 0.000 description 1
- 229910052771 Terbium Inorganic materials 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N Texas Red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- HYLXOQURIOCKIH-VQVTYTSYSA-N Thr-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N HYLXOQURIOCKIH-VQVTYTSYSA-N 0.000 description 1
- LUMXICQAOKVQOB-UHFFFAOYSA-N Threoninyl-Isoleucine Chemical compound CCC(C)C(C(O)=O)NC(=O)C(N)C(C)O LUMXICQAOKVQOB-UHFFFAOYSA-N 0.000 description 1
- 229910052775 Thulium Inorganic materials 0.000 description 1
- AOBORMOPSGHCAX-DGHZZKTQSA-N Tocofersolan Chemical compound OCCOC(=O)CCC(=O)OC1=C(C)C(C)=C2O[C@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C AOBORMOPSGHCAX-DGHZZKTQSA-N 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- LYMVXFSTACVOLP-ZFWWWQNUSA-N Trp-Leu Chemical compound C1=CC=C2C(C[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C([O-])=O)=CNC2=C1 LYMVXFSTACVOLP-ZFWWWQNUSA-N 0.000 description 1
- DZHDVYLBNKMLMB-ZFWWWQNUSA-N Trp-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 DZHDVYLBNKMLMB-ZFWWWQNUSA-N 0.000 description 1
- NLKUJNGEGZDXGO-XVKPBYJWSA-N Tyr-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NLKUJNGEGZDXGO-XVKPBYJWSA-N 0.000 description 1
- QJKMCQRFHJRIPU-XDTLVQLUSA-N Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 QJKMCQRFHJRIPU-XDTLVQLUSA-N 0.000 description 1
- AOLHUMAVONBBEZ-STQMWFEESA-N Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AOLHUMAVONBBEZ-STQMWFEESA-N 0.000 description 1
- CGWAPUBOXJWXMS-HOTGVXAUSA-N Tyr-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 CGWAPUBOXJWXMS-HOTGVXAUSA-N 0.000 description 1
- BMPPMAOOKQJYIP-WMZOPIPTSA-N Tyr-Trp Chemical compound C([C@H]([NH3+])C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C([O-])=O)C1=CC=C(O)C=C1 BMPPMAOOKQJYIP-WMZOPIPTSA-N 0.000 description 1
- 102000031061 Tyrosine 3-monooxygenases Human genes 0.000 description 1
- 108091000118 Tyrosine 3-monooxygenases Proteins 0.000 description 1
- MFEVVAXTBZELLL-UHFFFAOYSA-N Tyrosyl-Threonine Chemical compound CC(O)C(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 MFEVVAXTBZELLL-UHFFFAOYSA-N 0.000 description 1
- 210000003462 Veins Anatomy 0.000 description 1
- 208000001756 Virus Disease Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Vitamin C Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 229920002533 WHP Posttrascriptional Response Element Polymers 0.000 description 1
- 229910052769 Ytterbium Inorganic materials 0.000 description 1
- QIJRTFXNRTXDIP-JIZZDEOASA-N [(1R)-1-carboxy-2-sulfanylethyl]azanium;chloride;hydrate Chemical compound O.Cl.SC[C@H](N)C(O)=O QIJRTFXNRTXDIP-JIZZDEOASA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 108010081404 acein-2 Proteins 0.000 description 1
- 230000002378 acidificating Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001058 adult Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010025188 alcohol oxidase Proteins 0.000 description 1
- 229940087168 alpha Tocopherol Drugs 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N alpha-Tocopherol Natural products OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminum Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 230000001668 ameliorated Effects 0.000 description 1
- 230000001130 anti-lysozyme Effects 0.000 description 1
- 230000000840 anti-viral Effects 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000010385 ascorbyl palmitate Nutrition 0.000 description 1
- 239000000305 astragalus gummifer gum Substances 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium(0) Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal Effects 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 description 1
- 229910052797 bismuth Inorganic materials 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 210000003008 brain-resident macrophage Anatomy 0.000 description 1
- 230000001488 breeding Effects 0.000 description 1
- 235000019282 butylated hydroxyanisole Nutrition 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 150000007942 carboxylates Chemical group 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 101700018328 ccdB Proteins 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 201000008779 central nervous system disease Diseases 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229940099111 chloramine-T Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 230000035512 clearance Effects 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 229910052803 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229960005188 collagen Drugs 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000001447 compensatory Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000003636 conditioned culture media Substances 0.000 description 1
- 229920000407 conserved sequence Polymers 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- VOLSCWDWGMWXGO-UHFFFAOYSA-N cyclobuten-1-yl acetate Chemical compound CC(=O)OC1=CCC1 VOLSCWDWGMWXGO-UHFFFAOYSA-N 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000001627 detrimental Effects 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000002001 electrophysiology Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000000534 elicitor Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000002255 enzymatic Effects 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 230000002964 excitative Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- YCKRFDGAMUMZLT-UHFFFAOYSA-N fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000012520 frozen sample Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 229910052733 gallium Inorganic materials 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 230000002068 genetic Effects 0.000 description 1
- GNPVGFCGXDBREM-UHFFFAOYSA-N germanium Chemical compound [Ge] GNPVGFCGXDBREM-UHFFFAOYSA-N 0.000 description 1
- 229910052732 germanium Inorganic materials 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 150000002309 glutamines Chemical class 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 229940075507 glyceryl monostearate Drugs 0.000 description 1
- 150000002337 glycosamines Chemical group 0.000 description 1
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 1
- 108010074027 glycyl-seryl-phenylalanine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 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
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth media Substances 0.000 description 1
- 108010028295 histidylhistidine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002055 immunohistochemical Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 108010031614 immunorphin Proteins 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000003447 ipsilateral Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229910052743 krypton Inorganic materials 0.000 description 1
- DNNSSWSSYDEUBZ-UHFFFAOYSA-N krypton(0) Chemical compound [Kr] DNNSSWSSYDEUBZ-UHFFFAOYSA-N 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 101700072735 lys-1 Proteins 0.000 description 1
- 229960003646 lysine Drugs 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 101710023801 mab-20 Proteins 0.000 description 1
- 201000011614 malignant glioma Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 235000006109 methionine Nutrition 0.000 description 1
- 108010056582 methionylglutamic acid Proteins 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 238000000302 molecular modelling Methods 0.000 description 1
- 108010053480 monoclonal antibody 9E4 Proteins 0.000 description 1
- 230000000877 morphologic Effects 0.000 description 1
- 201000009457 movement disease Diseases 0.000 description 1
- 238000000569 multi-angle light scattering Methods 0.000 description 1
- 230000000869 mutational Effects 0.000 description 1
- 230000023837 negative regulation of proteolysis Effects 0.000 description 1
- 238000004848 nephelometry Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 230000000926 neurological Effects 0.000 description 1
- 230000000324 neuroprotective Effects 0.000 description 1
- 230000002887 neurotoxic Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 230000003472 neutralizing Effects 0.000 description 1
- 238000006396 nitration reaction Methods 0.000 description 1
- 230000003000 nontoxic Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 201000003497 olivopontocerebellar atrophy Diseases 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 210000000056 organs Anatomy 0.000 description 1
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 1
- 229910052762 osmium Inorganic materials 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 230000001717 pathogenic Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000002093 peripheral Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 230000000275 pharmacokinetic Effects 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 230000000896 plasminic Effects 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 108091008117 polyclonal antibodies Proteins 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 230000001242 postsynaptic Effects 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000003334 potential Effects 0.000 description 1
- PUDIUYLPXJFUGB-UHFFFAOYSA-N praseodymium Chemical compound [Pr] PUDIUYLPXJFUGB-UHFFFAOYSA-N 0.000 description 1
- 230000002035 prolonged Effects 0.000 description 1
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- ZTHYODDOHIVTJV-UHFFFAOYSA-N propyl 3,4,5-trihydroxybenzoate Chemical compound CCCOC(=O)C1=CC(O)=C(O)C(O)=C1 ZTHYODDOHIVTJV-UHFFFAOYSA-N 0.000 description 1
- 239000000473 propyl gallate Substances 0.000 description 1
- 235000010388 propyl gallate Nutrition 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- VBICKXHEKHSIBG-UHFFFAOYSA-N rac-1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 1
- 230000002285 radioactive Effects 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- 239000003638 reducing agent Substances 0.000 description 1
- 230000001105 regulatory Effects 0.000 description 1
- 230000000241 respiratory Effects 0.000 description 1
- IGLNJRXAVVLDKE-UHFFFAOYSA-N rubidium Chemical compound [Rb] IGLNJRXAVVLDKE-UHFFFAOYSA-N 0.000 description 1
- 229910052701 rubidium Inorganic materials 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- SIXSYDAISGFNSX-UHFFFAOYSA-N scandium Chemical compound [Sc] SIXSYDAISGFNSX-UHFFFAOYSA-N 0.000 description 1
- 229910052706 scandium Inorganic materials 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 125000003607 serino group Chemical group [H]OC(=O)C([H])(N([H])*)C([H])([H])O[H] 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 231100000486 side effect Toxicity 0.000 description 1
- 230000007958 sleep Effects 0.000 description 1
- 229910000342 sodium bisulfate Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- IGHGOYDCVRUTSU-UHFFFAOYSA-M sodium;2-hydroxypropane-1,2,3-tricarboxylic acid;hydroxide Chemical compound [OH-].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O IGHGOYDCVRUTSU-UHFFFAOYSA-M 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000003393 splenic Effects 0.000 description 1
- 108010005652 splenotritin Proteins 0.000 description 1
- 230000002269 spontaneous Effects 0.000 description 1
- 230000004936 stimulating Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000000576 supplementary Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained Effects 0.000 description 1
- 230000000946 synaptic Effects 0.000 description 1
- 230000003976 synaptic dysfunction Effects 0.000 description 1
- 230000003977 synaptic function Effects 0.000 description 1
- 101700042113 tap Proteins 0.000 description 1
- GZCRRIHWUXGPOV-UHFFFAOYSA-N terbium Chemical compound [Tb] GZCRRIHWUXGPOV-UHFFFAOYSA-N 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
- 229910052716 thallium Inorganic materials 0.000 description 1
- BKVIYDNLLOSFOA-UHFFFAOYSA-N thallium Chemical compound [Tl] BKVIYDNLLOSFOA-UHFFFAOYSA-N 0.000 description 1
- FRNOGLGSGLTDKL-UHFFFAOYSA-N thulium Chemical compound [Tm] FRNOGLGSGLTDKL-UHFFFAOYSA-N 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- RTAQQCXQSZGOHL-UHFFFAOYSA-N titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- 229910052719 titanium Inorganic materials 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- 238000003325 tomography Methods 0.000 description 1
- 229960001479 tosylchloramide sodium Drugs 0.000 description 1
- 230000031998 transcytosis Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- VOBHRQFELWTZFS-AWLRYRRCSA-K trisodium;(4Z)-3-oxo-4-[(4-sulfonatonaphthalen-1-yl)hydrazinylidene]naphthalene-2,7-disulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N/N=C3/C4=CC=C(C=C4C=C(C3=O)S(=O)(=O)[O-])S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 VOBHRQFELWTZFS-AWLRYRRCSA-K 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 108010021199 valyl-valyl-valine Proteins 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- NAWDYIZEMPQZHO-UHFFFAOYSA-N ytterbium Chemical compound [Yb] NAWDYIZEMPQZHO-UHFFFAOYSA-N 0.000 description 1
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 229910052726 zirconium Inorganic materials 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
Abstract
The invention relates to novel monoclonal antialpha-synuclein antibodies. The antibodies can be used for treating a synucleino pathy such as Parkinson's disease (including idiopathic and inherited forms of Parkinson's disease). Diffuse Lewy Body Disease (DLBD). Lewy body variant of Alzheimer's disease (LBV). Combined Alzheimer's and Parkinson disease. pure autonomie failure and multiple system atrophy.
Description
Agents, Uses and Methods for the Treatment of Synucleinopathy
Field of the Invention:
The présent invention relates to a novel class of monoclonaî antibody that specifically binds to alpha-synuclein, as well as to methods Of using these molécules and their alpha-synuclein binding fragments in the treatment and diagnosis of synucleinopathies.
Référencé To Sequence Listing:
This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in computer-readable media (file name: 0992_ST25.txt, created on 22 June 2016, and having a size of 44 kB), which file is herein incorporated by référencé in its entirety.
Background of the Invention:
Synucleinopathies, also known as Lewy body diseases (LBDs), are characterized by déposition of intracellular protein aggregates that are microscopically visible as Lewy bodies (LBs) and/or Lewy neurites, where the protein alpha-synuclein is the major component (Jellinger, Mov Disord. 2012 Jan;27(1):8-30; McKeith et al., Neurology (1996) 47:1113-24). Synucleinopathies include Parkinson's disease (PD) (including idiopathic and inherited forms of Parkinson's disease) and Diffuse Lewy Body (DLB) disease (also known as Dementia with Lewy Bodies (DLB), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease (CAPD), pure autonomie failure (PAF) and multiple System atrophy (MSA; e.g., Olivopontocerebellar Atrophy, Striatonigral Degeneration and Shy-Drager Syndrome)). Synucleinopathies frequently hâve degeneration of the dopaminergic nigrostriatal System, responsible forthe core motor déficits in Parkinsonism (rigidity, bradykinesia, resting tremor), but there is also widespread occurrence of Lewy bodies and dystrophie Lewy neurites in the central, peripheral and autonomie nervous System and brain régions and other organs associated with non-motor dysfunctions, such as dementia and autonomie nervous System déficits. Several of the non-motor signs and symptoms are thought to précédé motor symptoms in Parkinson’s disease and other synucleinopathies. Such early signs include, for example, REM sleep behaviour disorder (RBD) and loss of smell and constipation (Mahowald et al., Neurology (2010) 75:488-489). Synucleinopathies continue to be a common cause for movement disorders and cognitive détérioration in the aging population (Galasko et al., Arch. Neurol. (1994) 51:888-95).
A'pha-synuclein is a member of a family of proteins including beta- and gammasynuclein and synoretin. Alpha-synuclein is expressed in the normal state associated with synapses and is believed to play a rôle in regulating synaptic vesicle release and thereby affecting neural communication, plasticity, learning and memory.
Several studies hâve implicated alpha-synuclein with a central rôle in PD pathogenesis. The protein can aggregate to form intracellular insoluble fibrils in pathological conditions. For example, synuclein accumulâtes in LBs (Spillantini et al., Nature (1997) 388:839-40; Takeda et al., J. Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci. Lett. (1997) 239:45-8). Mutations in the alpha-synuclein gene as well as duplications and triplications of the gene co-segregate with rare familial forms of parkinsonism (Kruger et al., Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science (1997) 276:2045-7). An important finding has been that alpha-synuclein can be secreted into the extracellular fluid and be présent in plasma and cerebrospinal fluid (CSF). Several studies, for exampie by Pacheco et al. (2015) and others (Pacheco et al J Neurochem. 2015 Mar; 132(6):731-4; Conway et al., Proc Natl Acad Sci USA (2000) 97:571-576;
Voiles et al., J. Biochem. 42:7871-7878, 2003) hâve suggested that extracellularsynuclein plays a pathogenic rôle in the brain. They demonstrated that extraceliular alpha-synuclein oligomers possesses neurotoxicity toward brain neuronal plasma membranes. Another intriguing hypothesis based on the data of synuclein sécrétion is that a prion-like spread of alpha-synuclein underlies the progression of Parkinson's disease and other synucleinopathies (Lee et al. 2014, Nat Rev Neurol. 2014 Feb;10(2):92-8; Hansen and Li 2012, Trends Mol Med. 2012 May; 18(5):248-55). These findings hâve given rise to a hope that extracellular-synuclein could be targeted by immunotherapy (Vekrellis et al. 2011, Lancet Neurol. 2011 Nov; 10(11):1015-25).
Naturally occurring alpha-synuclein auto-antibodies hâve been shown to be présent in both PD patients and healthy Controls (Smith et al. 2012, PLoS One.
2012;7(12):e52285; Maetzler et al. 2014, PLoS One. 2014 Feb 21;9(2):e88604,
Papachroni et al. 2007 J Neurochem. 2007 May; 101 (3):749-56 and Woulfe et al. 2002, Neurology. 2002 May 14;58(9):1435-6), sometimes increased levels of auto-antibodies to alpha-synuclein in PD (Gruden et al. 2011, J Neuroimmunol. 2011 Apr;233(1-2):2217, Gruden et al. 2012,Neuroimmunomodulation. 2012; 19(6):334-42 and Yanamandra 2011, PLoS One. 2011 Apr 25;6(4):e18513) or decreased auto-antibodies to alphasynuclein in PD patients compared to heaîthy Controls hâve been reported (BesongAgbo et a: 2013 Neurology. 2013 Jan 8;80(2): 169-75). . The possibility that circulating anti-alpha-synuclein autoantibodies may serve a protective rôle with respect to alphasynuclein aggregation was suggested very early on after finding of the auto-antibodies (Woulfe et al. 2002, Neurology. 2002 May 14;58(9): 1435-6).
Over expression of alpha-synuclein in transgenic mice mimics some pathologica! aspects of Lewy body disease. Several different transgenic lines of mice overexpressing alpha-synuclein hâve been generated in the last ten years (described in reviews: Koehler et al 2014, PLoS One. 2013 May 31;8(5):e64649; Fleming and Chesselet, 2006,Behav Pharmacol. 2006 Sep; 17(5-6):383-91 ; Springer and Kahle 2006,Cuit Neurol Neurosci Rep. 2006 Sep;6(5):432-6). Mouse lines with Thy-1 and PDGF-beia promoters develop motor déficits and cognitive déficits and hâve been used to demonstrate a neuroprotective effect of antibodies directed against alphasynuclein in vivo. However, none of the transgenic lines hâve robust degeneration of dopaminergic neurons, and often the motor phenotypes are driven by expression in motor neurons, which do not normally degenerate in Parkinson’s disease. Therefore, it is not clear if positive outcome of a potential disease modifying treatment is mediated through effects on dopaminergic neurons or other central nervous System neurons.
One robust finding in the transgenic mouse models has been that chronic overexpression of human alpha-synuclein impairs synaptic function. Using studies in both in vitro and in vivo Systems it was shown that overexpression of wild-type (wt) human alpha-synuclein impaired synaptic transmission in hippocampus (Nemani et al. 2010, Neuron. 2010 Jan 14;65(1):66-79; Paumieret al. 2013, PLoS One. 2013 Aug 1;8(8):e70274). This was shown in the CA1 région of the hippocampus where both studies found reduced basal synaptic transmission. The mechanism behind this was assumed to be intracellular accumulation of alpha-synuclein leading to dysfunctional synaptic release. However, the recent findings about sécrétion of alpha-synuclein into extracellular space in synapses and the toxic effects of alpha-synuclein oligomers on synapse function opens for the possibility of a rôle of extracellular aipha-synuclein in synaptic dysfunction, and as such for the ability of therapeutic antibodies to rescue the déficit.
The use of viral vectors to over-express alpha-synuclein represents an important way 5 to model PD in rodents because this approach produces a relative fast progressive degeneration of nigrostriatal neurons, a feature not yet reproduced by genetic mutations in mice or rats (Kirik and Bjorklund, 2003, Trends Neurosci. 2003 Jul;26(7):386-92). Furthermore. viral gene delivery revealed the ability of wt alphasynuclein to induce nigrostriatal pathology (Kirik et al. 2002, J Neurosci. 2002 Apr 10 1 ;22(7):2780-91 ), a finding in agreement with evidence in familial forms of PD with alpha-synuclein dublications and triplications(Lee and Trojanowski, 2006, Neuron. 2006 Oct 5;52(1 ):33-8). In one study, it has been shown that a a pool of goat antibodies against the alpha-synuclein N-terminal protected against dopaminergic cell death and ameliorated behavioural déficits in a AAV-alpha-synuclein based rat model 15 of Parkinson's disease (Shahaduzzaman et al 2015, PLoS One. 2015 Feb 6;10(2):e0116841).
Prion like spreading of alpha-synuclein pathology has recently been shown to develop alpha-synuclein pathology and also develop dopaminergic cell death (Luk et al. 2012, Science. 2012 Nov 16;338(6109):949-53). This model has been used to show that 20 alpha-synuclein antibodies are able to ameliorate the pathology (Tran et al. 2014, Cell
Rep. 2014 Jun 26;7(6):2054-65). In this model antibody treatment was able to reduce accumulation of phosphorylated alpha-synuclein in several brain régions - including dopaminergic neurons in substantia nigra, and reduce development of motor déficit.
In addition to mutations, alternative splicing ofthe alpha-synuclein gene and 25 posttranslational modifications of the protein, such as phosphorylation, ubiquitination, nitration, and truncation can create alpha-synuclein protein forms that hâve enhanced capacity. to form aggregated and/or toxic forms of alpha-synuclein (Beyer and Ariza, Mol Neurobiol. 2013 Apr;47(2):509-24). However, the précisé pathological species of alpha-synuclein remains unknown. Varions misfolded/aggregated/secreted species 30 ranging from oligomers to fibrils, and different post-translational modifications hâve been associated with toxicity but there is no consensus on which is most important, if indeed there even is a single toxic species.
Overall the accumulation of alpha-synuclein with similar morphological and neurological alterations in animal models as diverse as humans, mice, and flies suggests that this molécule is central in the pathogenesis of Lewy body diseases.
Several different antibodies to alpha-synuclein hâve been shown to hâve therapeutic 5 effect in preclinical animal models. Both an antibody targeting an epitope involving alpha-synuclein residues 91-99 and antibodies targeting an epitope that involves alphasynuclein residues 118-126 hâve been shown to hâve an effect on motor and cognitive déficits in transgenic mice (Games et al. 2014, J Neurosci. 2014 Jul 9;34(28):9441-54). The most advanced of these antibodies is a humanized antibody based on the mouse 10 monoclonal antibody 9E4, which targets an epitope that involves alpha-synuclein residues 118-126, and which is now in clinical trials in phase I. A C-terminal antibody 274 which targets an epitope that involves alpha-synuclein residues 120-140 (Bae et al. 2012, J Neurosci. 2012 Sep 26;32(39): 13454-69) was also shown to hâve an effect in a preclinical model on spreading of the pathology from cell to cell. In addition to 15 these, antibodies targeting conformational species such as oligomers and fibrils of alpha-synuclein hâve been shown to be able to at least reduce the levels of these presumably toxic alpha-synuclein species (Lindstrom et ai. 2014, Neurobiol Dis. 2014 Sep;69:134-43 and Spencer et ai. 2014, Mol Ther. 2014 Oct;22(10):1753-67). These conformational antibodies that lower alpha-synuclein oligomer levels in vivo, such as 20 mab47 were also shown to target epitopes in the C-terminus of alpha-synuclein, from amino acid 121-125 (US20120308572). Other conformational, fîbril and oligomer spécifie antibodies also target C-terminal sequences (Vaikath et al. Neurobiol Dis. 2015;79:81-99). .
As the toxic form of alpha-synuclein is unknown, a therapeutic antibody should be 25 idéally able to bind to most of the alpha-synuclein species that are formed by alternative splicing or posttranslational modifications, such as truncations, as well as oîigomeric and fibrillary forms. One problem with current antibodies that hâve been tested as therapeutics in preclinical models, as discussed above, is that many of them target C-terminal epitopes, which are not found in some of the major truncated forms of 30 alpha-synuclein. For example, the amino acids that are important for binding of 9E4 are asparagine 122 and tyrosine 125 (according to an alanine scan presented in patent US20140127131), and this means that this antibody cannot bind alpha-synuclein which is truncated at amino acids 119, and 122, which are some ofthe majortruncated species in Parkinson brain tissue (Kellie et al. Soi Rep. 2014;4:5797). The same would be the case for the antibody 274 and antibody mab47 (US8,632,776). Also, amino terminal antibodies would possibly not be able to bind to some of the major truncated species that lack the first amino acids of alpha-synuclein, such as alpha-synuclein truncated to amino acids 5-140. For the 9E4 antibody, one suggested mechanism of action is the prévention of truncation at amino acids 119-122 in extracellular space, as the antibody will bind to the same région where the protease that will cleave alphasynuclein (Games et al. 2014, J Neurosci. 2014 Jul 9;34(28):9441-54). A similar mechanism of action could also be found with antibodies in dose proximity of the site, and therefore many antibodies around this région would be expected to hâve this activity.
There is some support for a toxic rôle ofthe truncated alpha-synuclein species in anima! models. Expression of truncated alpha-synuclein under the tyrosinehydroxylase promoter has been shown to lead to nigrostriatal pathology, which is normaliy not seen in transgenic alpha-synuclein models (Tofaris et al. 2006, J Neurosci. 2006 Apr 12;26(15):3942-50; Wakamatsu et al. 2006, Neurobiol Aging. 2008 Apr;29(4):574-85). For example, expression of amino acids 1-130 of a human alphasynuclein protein having the A53T mutation caused embryonic loss of dopaminergic neurons in the substantia nigra pars compacta whereas expression of the full length protein did not (Wakamatsu et al. 2006, Neurobiol Aging. 2008 Apr;29(4):574-85). Expression of a 120 amino acid alpha-synuclein molécule under the calcium/calmodulin-dependent protein kinase II alpha (CamKII-alpha) promoter was associated with alpha-synuclein aggregation and a progressive déficit in corticalhippocampal memory tests including the Barnes maze and novel object récognition (Hall et al. 2015, Exp Neurol. 2015 Feb;264:8-13). Also in the rat AAV model coexpression of C-terminal truncated alpha-synuclein enhanced full-length alphasynuclein-induced pathology (Ulusoy et al. 2010, Eur J Neurosci. 2010 Aug;32(3):40922).
In this invention, antibodies (such as “GM37” and “GM285”, described in the Examples) hâve been generated that can bind to the toxic alpha-synuclein fragment 1-119/122 and neutralize this truncated form of alpha-synuclein. The antibodies ofthe invention, such as GM37 and GM285, are capable of binding to other oligomeric forms of alphasynuclein and altering their uptake by other CNS résident cells in a manner that reduce the spreading of disease. Furthermore, the antibodies of the invention, such as GM37 and 285, were surprisingly found to be superior to prior art antibodies such as 9E4 in binding to different alpha-synuclein species in human brain, and has a surprising superior effect on clearing extracellular aîpha-synuclein and normalising impaired synaptic transmission induced by the presence of abnormal alpha-synuclein in vivo. Further illustrating their therapeutic capabilities, the antibodies of the invention, such as GM37 and 285, a-e able to prevent the appearance of a disease related motor phenotype in a rat model for Parkinson’s disease. Finally, antibodies GM37 and GM285 are able to inhibit seeding of aggregation and phosphorylation of endogenous alpha-synuclein induced by extracellular added recombinant pathological alphasynuclein seeds in primary mouse neurons. Antibodies such as GM37 and 285 can also inhibit seeding of alpha-synuclein pathology into dopaminergic neurons in vivo using a mouse model for Parkinson’s disease, further supporting the therapeutic capability of these antibodies in preventing the cell to cell propagation of pathology. Together these data strongly support the use of these novel antibodies, GM37 and GM285, as new therapeutic agents capable of modifying disease through inhibition of the mechanism by which the disease pathology spreads between the neurons Parkinson’s patients.
In a further aspect of the invention is provided 3 amino acid variants of the GM37 antibody. Ail the variants hâve similar functional readouts as the parent antibody, GM37, but with improved properties for manufacturability. The variants reduce the risk of post-translational modification occurring within the binding domain of the GM37 antibody and provide some improvement in the production of the antibody. This is advantageous because large scale clinical or commercial manufacturing of antibodies is complicated and expensive, and providing a homogenous product in pharmaceutical médicaments is crucial in particular for immunoglobulins and proteins.
Summary of the Invention:
The invention relates to novel monoclonal antibodies, and antigen-binding fragments thereof, capable of specifically binding an epitope within amino acids 112-117 in alphasynuclein (SEQ ID NO:9 (ILEDMP)). The epitope bound by the antibodies or antibodybinding fragments thereof of the invention, such as exemplary antibody “GM37, or “GM285”, is referred to herein as “the 112-117 epitope”. The antibodies of the present invention specifically bind to an epitope within the 112-117 epitope and may, according to one embodiment, compete with antibody GM37 or GM285 for binding to an epitope within amino acids 112-117. For example, antibodies or antigen- binding fragments thereof according to the invention may compete for binding to an epitope within amino acids 112-117 of human alpha-synuclein with a heavy chain consisting of a variable domain of SEQ ID NO:7 and a light chain consisting of a variable domain of SEQ ID NO:8. Such compétitive bnding inhibition can be determined using assays and methods well known in the art, for example using an unlabelled binding assay such as surface plasmon résonance (SPR) For example, immobilising human alpha-synuclein on a surface and incubating with or without the reference antibody ‘GM37’ prior to incubation with an antibody or binding fragment to be tested. Alternatively, a pair-wise mapping approach can be used, in which the reference antibody ‘GM37’ is immobîlised to the surface, human alpha-synuclein antigen is bound to the immobîlised antibody, and then a second antibody is tested for simultaneous binding ability to human alphasynuclein (see ‘BIAcore® Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05/2012; the disciosures of which are incorporated herein by reference).
More specifically the GM285 antibody binds an epitope within residues 112-117 of alpha-synuclein comprising residues 112-115 of alpha-synuclein (ILED; SEQ1D NO:19).
In one embodiment, the invention relates to monoclonal antibody GM37, its variants (e.g., GM37 Variant 1, GM37 Variant 2 and GM37 Variant 3), or GM285.
In particular, the invention provides a monoclonal antibody GM37, its variants (e.g., GM37 Variant 1, GM37 Variant 2 and GM37 Variant 3), or GM285, and encompasses such antibodies as well as dérivatives thereof that possess a sufficient number (e.g., 1, 2, or 3) light chain CDRs and a sufficient number (e.g., 1,2, or 3) heavy chain CDRs to form a binding site capable of specifically binding to human synuclein. Preferably, such antibodies will possess the three light chain CDRs and three heavy chain CDRs, as defined below. The numbering of amino acid residues in this région is according to IMGT®,the international ImMunoGeneTics information system® or, Kabat, E. A., Wu, T. T., Perry, H. M., Gottesmann, K. S. & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of
Health and Human Services; Chothia, C. & Lesk, A. M. (1987). Canonical structures For The Hypervariable domains Of Immunoglobulins. J. Mol. Biol. 196, 901-917.
In one embodiment, the monoclonal antibody or antigen-binding fragments thereof possesses a synuclein antigen-binding fragment comprising or consisting of:
(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:1 ; and/or (b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:2;
and/or (c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:3; and/or (d) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:4;
and/or (e) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and/or . (f) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:6; that is capable of specifically binding to human alpha-synuclein.
In another embodiment, the monoclonal antibody or antigen-binding fragments thereof possesses a synuclein antigen-binding fragment comprising or consisting of:
(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:1 ;
(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:33, 34 or 35;
(c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:3;
(d) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:4;
(e) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:6;
that is capable of specifically binding to human alpha- synuclein.
In yet another embodiment, the monoclonal antibody or antigen-binding fragments thereof possesses a synuclein antigen-binding fragment comprising or consisting of:
(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;
and/or (b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21;
and/or (c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22; and/or (d) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:23; and/or (e) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:24; and/or (f) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:25. that is capable of specifically binding to human alpha-synuclein.
In one embodiment, the monoclonal antibody or antigen-binding fragments thereof possesses a synuclein antigen-binding fragment comprising an amino acid sequence (in its CDRs, its variable domains, its framework residues or in its constant domains) that differs from that of naturally occurring anti-alpha-synuclein antibodies, and that exhibits (relative to such naturally occurring anti-alpha-synuclein antibodies):
(i) a différence in binding affinity (KD) for alpha-synuclein;
(ii) a différence in the capability of inhibiting protease truncation of alphasynuclein fibrils;
(iii) a différence in the capability of reversing impairment in basal synaptic transmission in F28-snca transgenic mice;
(iv) a différence in the capability of reducing levels of alpha-synuclein in the mouse hippocampus as measured by in vivo microdialysis; and/or (v) a différence in the capability, when administered chronically, to restore motor function in a rat model of Parkinson’s disease (vi) a différence in the ability to prevent seeding of alpha-synuclein (such as accumulation of insoluble phosphorylated alpha-synuclein in vitro and/or in â mouse model of Parkinson's disease); and/or (vii) a diffirence in the capability to bind truncated alpha-synuclein in a human brain.
The antibodies and antigen-binding fragments thereof of the invention may be used in a method to treat, diagnose or image synucleinopathies, such as Parkinson's disease ((PD), including idiopathic and inherited forms of Parkinson's disease), Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheîmer's disease (LBV), Gauchers Disease (GD), Combined Alzheîmer's and Parkinson disease (CAPD), pure autonomie failure and multiple System atrophy.
Brief Description of the Drawings
Fig. 1 shows immunization protocols for génération of hybridomas. The table outlines the différences ofthe immunogens and mouse strains used for the identification of GM37 and GM285. Different HCo17-Balb/c and HCo12/Balb/c mice were immunized independently (description of these mice are provided below). The hybridoma expressing GM37 was identified from mice immunized with full length alpha-synuclein containing amsno actds 1-140 fibrils and boosted with truncated alpha-synuclein fragments 1-60 and 1-119 of full length (FL) alpha-synuclein (SEQ ID NO 10). The hybridoma expressing antibody GM285 came from an immunization protocol in which HCo12-Balb/c mice were immunized with full length monomeric alpha-synuclein, amino acids 1-140 followed by a boostwith full length fibrillary alpha-synuclein (Example 1).
Fig. 2 (PANEL A-C) shows screening of GM37 for binding to alpha synuclein, alphasynuclein homologs and orthologs.
A) Binding of antibody GM37 to alpha-synuclein using a no wash solution based ELiSA (FMAT).
B) Using SPR (Fortebio) binding of antibody GM37 is spécifie for alpha-synuclein (Aipha Panel) and does not bind the other related synuclein family proteins, betasynuclein (Beta Panel) and gamma-synuclein (Gamma Panel). Measurements were performed using SPR (Fortebio Octetred) GM37 shows similar binding to alphasynuclein from cynomolgus monkey (Cyno Panel) and mouse (Mouse Panel). (Example 1).
C) Using SPR (Fortebio Octetred) binding of antibody GM285 is spécifie for alphasynuclein and does not bind the other related synuclein family proteins, beta-synuclein and gamma-synuclein. Measurements were performed using SPR (Fortebio Octetred) shows similar binding of GM285 to alpha-synuclein from cynomolgus monkey (Cyno) and mouse (Mouse)(Example 1).
Fig. 3 (Panels A-C) shows real time binding Affinity of GM37
A) Binding of antibody GM37 to alpha-synuclein measured in RU (Relative Units) (yaxis) over time (X-axis) as determined by SPR (BIAcore® 3000). Goat anti-human IgG was immobilized on the CM5 chip. GM37 was captured on the Goat antihuman IgG immobilized chip and sériés of concentrations of human alpha- synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested on binding to the surface. The sensor surface was regenerated between each cycle.
B) Signal from binding at different concentrations converted into a binding curve.
C) Calculated binding constants of antibody GM37 (denoted hlgG1-6004-037-C106S) (Example 2).
Fig. 4 (Panels A-C) shows real time binding Affmity of GM285
A) Binding of antibody GM285 to alpha-synuclein measured in RU (y-axis) over time (X-axis) as determined by SPR (BIAcore® 3000). Goat anti-human IgG was immobilized on the CM5 chip. GM285 was captured on the Goat anti-human IgG immobilized chip and sériés of concentrations of human alpha-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested on binding to the surface. The sensor surface was regenerated between each cycle.
B) Signal from binding at different concentrations converted into a binding curve.
C) Calculated binding constants of antibody GM285 (denoted hlgG 1-6004-285) (Example 2).
Fig. 5 (Panels A-C) shows real time binding of comparator antibody 9E4
A) Shows binding of 9E4 to alpha-synuclein measured in RU (y-axis) over time (Xaxis) as determined by SPR (BIAcore® 3000). Goat anti-human IgG was immobilized on the CM5 chip. 9E4 was captured on the chip by its binding to Goat anti-human IgG that had been immobilized to the chip. A sériés of concentrations of human alpha-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested for binding to the surface. The sensor surface was regenerated between each cycle.
B) Signal from binding at different concentrations converted into a binding curve.
C) Calculated binding constants for antibody 9E4. (Example 2).
Fig. 6 shows the amino acid sequence of alpha-synuclein. Major truncation sites (indicated by arrows) identified by mass spectrometry in human brain tissue (Kellie JF, Higgs RE, Ryder JW, Major A, Beach TG, Adler CH, Merchant K, Knierman MD. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by mass spectrometry. Sci Rep. 2014 Jul 23;4:5797. doi: 10.1038/srep05797)
Fig. 7 (Panels A-B) shows epitope mapping of antibody GM37 and GM285. ELISA data showing relative levels of binding of the antibodies to sequential peptides (20mers) derived from alpha-synuclein amino acid sequence 95-132 (the other nonbinding peptides are not shown).
A) GM37 epitope requires peptide sequence ILEDMP (SEQ ID NO:9) forfull binding.
B) GM285 requires peptide ILED (SEQ ID NO:19) forfull binding. (Example 3).
Fig. 8 (Panels A-B) shows a schematic représentation of truncated forms of alphasynuclein.
A) binding epitopes of GM37/285 (ILEDMP; SEQ ID NO:9) and 9E4 (NEAYE; SEQ ID NO:36) are shown in bold on the alpha-synuclein amino acid sequence (SEQ ID NO:10). Arrows indicates the c-terminal truncations sites from Fig 6.
B) Major truncated forms of alpha-synuclein that hâve been identifïed from human brain material. Size based on amino acid numbers is indicated on the right side. Full length alpha-synuclein is 140 amino acids. As can be deducted from the epitopes, GM37, it’s variants 1-3, and GM285 should bind full length and the 1119/122, 1-135 fragments. Antibody 9E4 will bind only to full length and 1-135 fragment. The spécifie nature of the smaller c-terminal fragments left after the truncations are not shown.
Fig. 9 shows that antibodies GM37 and GM285 immunoprecipitate full length alphasynuclein as well as truncated alpha-synuclein from human brain. Crude homogenates of human DLB brain were incubated with the test antibodies (Beads (No ab), B12human lgG1 control antibody not binding to alpha-synuclein, GM-37, GM37 variant 2, GM-285 and murine (m)9E4) and the immunodepleted supernatant and immunoprecipitated material was separated on SDS-PAGE. The western blot. shows the bands representing the full length and the different truncated forms of alphasynuclein being depleted from the supernatant and being immunoprecipitated with the antibodies (IP). As can be seen, the GM37, GM37v2 and GM285 antibody depleted the major alpha-synuclein species from the supernatant, and the IP shows these species, the truncated species 1-135, 1-119/122 and full length alpha synuclein. The 9E4 does not affect the 1-119/122 species but only IPs full length and 1-135 (Example 4).
Fig. 10 shows schematics of the proteolysis of alpha-synuclein fibrils cleaved by calpain at amino acid 119/122. Alpha-synuclein fibrils (PFF) are added to the culture with (PFF+) or without (PFF) test antibody. The presence of GM-37/285 inhibit the formation of the truncated alpha-synuclein in cells and secreted into the cell media.
Fig. 11A shows that GM37 inhibits the formation of the truncated band (12KD) in both the media and in cell lysâtes of primary mouse cortical cultures treated with PFFs. Proteins were separated by SDS-PAGE and western blotted to detect different species of alpha-synuclein. In cells treated only with PFF or the contre’ antibody (B12) two monomeric alpha-synuclein bands are detected at 12 and 14 kDa, representing truncated and full length alpha-synuclein, respectively. In the presence of GM-37 there is only a faint band at 12Kd indicating that the majority of the cleavage is blocked. This effect is also reflected in the in the media of the cells. The relative levels of accumulation may also be inhibited by the presence of GM-37 as reflected in the réduction in the relative intensity of the 14Kd band. Alternatively there may be reduced amount of the 14Kd band available for uptake by the cells. (Example 5).
Fig. 11B shows dose dépendent inhibition of proteolysis of alpha-synuclein fibrils by antibodies GM37, GM37 variant 2 and GM285. In cell lysâtes from primary mouse cortical cultures at low antibody concentration (0,1 ug/ml) there are both a band representing full length (FL) alpha-synulceîn and a band representing C-terminally truncated (CT) alpha-synuclein (indicated by arrows). Increasing antibody concentration to 1, 5 and 10 ug/ml leads to reduced proteolysis of alpha-synuclien fibrils in cells. This is observed with both antibody GM37, GM37v2 and GM285. Control samples are treated with a human lgG1 antibody B12 not recognising alpha-synuclein. There is also a control with no antibody added (No ab), and cells with no alphasynuclein fibrils added (No Asyn). The total amount of alpha-synuclein is also reduced in samples treated with 37, 37v2 and 285 compared to B12 or “no antibody” control, indicative that ail three antibodies reduce accumulation of alpha-synuclein in cells in concentration dépendent manner. The actin band on the top of the gel shows equal loading of the samples (Example 5).
Fig. 12 shows the impact of GM37 and GM285 on seeding of alpha-synuclein . aggregation and alpha-synuclein phosphorylation in mouse primary cortical neurons. 12A) Example of images of primary neurons stained for phosphorylated alphasynuclein, which appears as spots or punctate staining in cells when the cells are seeded with either 1 ng of pure seeds or crude seeds of alpha-synuclein^
12B) Western blot of proteins from primary cortical neurons separated in soluble and insoluble fractions. The blots were stained with human alpha-synuclein spécifie antibody (4B12/H a-syn), phospho-Ser-129alpha-synûclein spécifie antibody (ab51253/pS-a-Syn) and mouse alphasynuclein spécifie antibody (D37A2/M a-syn) and show that addition of the crude seeds in primary neurons leads to accumulation of endogenous mouse alpha-synuclein and phosphorylated alpha-synuclein and higher molecular weight multimers of alpha-synuclein in the insoluble fraction.
120) GM37, GM37 variant 2 and GM285 inhibit appearance of phosphorylated alphasynuclein quantitated as the number of alpha-synuclein phosphoserine 129 positive spots in cells by a Cellomics ARRAYSCAN™automated microscope. GM37, GM37v2 and GM285 reduce the amount of phosphorylated alphasynuclein spots in cells in dose dépendent manner.
12D) Western blot of the homogenates from primary cortical neurons treated at the highest dose of antibody (133 nM), and stained for actin, human alphasynuclein, phosphorylated alpha-synuclein and mouse alpha-synuclein shows that antibodies 37, 37v2 and 285 inhibit truncation of the alpha-synuclein crude seeds taken up by the cells in the insoluble fraction. Ail antibodies also inhibit the accumulation of phosphorylated, endogenous mouse and higher molecular weigth multimers of phosphorylated mouse alpha-synuclein in the insoluble fraction. The actin band on the top of the gel shows equal loading of the samples (Example 6).
Fig 13 shows basal synaptic transmission atthe Schaffer collateral-CA1 synapse in the hippocampus of F28-snca transgenic and age-matched control mice. Field excitatory post-synaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function ofthe stimulation intensity. Short-term synaptic plasticity was evaluated by induction of paired-pulse facilitation. The different intensities of stimulation were 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μΑ, and were applied successively in increasing order, with 2 to 3 repeats for each intensity. Basal synaptic transmission was found to be significantly impaired in F28-snca transgenic mice overexpressing wild-type alpha-synuclein compared to age-matched control mice (Example 7).
Fig 14 shows the effect of the systemic administration of a single dose of human 9E4 (15 mg/kg, i.p.) on the impairment in basal synaptic transmission atthe Schaffer collateral- CA1 synapse in the hippocampus of F28-snca transgenic mice. Field excitatory post-synaptic potentiels (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function of the stimulation intensity. Acute treatment with h9E4 induced a significant reversai of the impairment in basal synaptic transmission in F28snca transgenic mice (Tg-snca + h9E4 vs. Tg-snca + PBS, p=0.002). However, the reversai by h9E4 was only partial, as indicated by a significantly lower basal synaptic transmission compared to littermates treated with PBS (p=0.007) (Example 7).
Fig 15 shows the effect of the systemic administration of a single dose of human GM37 (15 mg/kg, i.p) or an isotype control antibody (B12) on the impairment in basal synaptic transmission at the Schaffer colIateral-CA1 synapse in the hippocampus of F28-snca transgenic mice. Field excitatory post-synaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function of the stimulation intensity. Acute treatment with GM37 induced full reversai of the impairment in basal synaptic transmission in F28-snca transgenic mice (Tg-snca + GM37 vs. Tg-snca + B12, p=0.004) (Example 7).
Fig 16 shows the effect of the systemic administration of a single dose of human GM285 (15 mg/kg, i.p) on the impairments in basal synaptic transmission atthe Schaffer collateral-CA1 synapse in the hippocampus of F28-snca transgenic mice. Field excitatory post-synaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function of the stimulation intensity. Acute treatment with GM285 induced full reversai of the impairment in basal synaptic transmission in F28-snca transgenic mice (Tg-snca + GM285 vs. Tg-snca + PBS, p=0.001) (Example 7).
Fig 17 (Panels A-B) shows the effect of the systemic administration (15 mg/kg, i.p.) of human 9E4, GM37 or isotype control antibody (anti-HEL) on the levels of human alphasynuclein in the interstitial fluid (isf) in the hippocampus of freely movîng F28-snca transgenic mice. The average of the two-three basal values (4h-6h) prior to antibody 5 treatment was taken as baseline and set to 100% for each animal. Différences were analyzed using a two-way analysis of variance (ANOVA) with repeated measures. The basai levels of human alpha-synuclein in hippocampus were 8.1 + 1.1 ng/ml (mean ± SEM, n = 25, not corrected for the in vitro dialysis probe recovery). The administration of GM37 induced a larger réduction in human alpha-synuclein in the hippocampus of 10 F28 mice compared to both the comparator antibody, human 9E4, and the control isotype, anti-HEL Timepoints that show significant différences in the levels of alphasynuclein between animais treated with GM37 or the control antibody are indicated by an asterisk. (Example 8).
Fig 18 shows a schematic représentation of the timeline for antibody treatment (down 15 arrcws), viral injections and behavioural assessment in the rat AAV human alpha. synuclein model shown in Fig. 19 (Example 9).
Fig 19 shows that antibody GM37 can reduce Parkinsonian motor déficits after chronic treatment in the rat AAV model. The effect of chronic treatment with GM37 or PBS in AAV-human- alpha-synuclein rats on motor asymmetry is assessed in the cylinder test. 20 Each rat was tested for the use of the forepaws by monitoring for 5 minutes. The percentage of use of the right forepaw (ipsilateral to the injection) and use of left (contralateral+ right forepaws) was calculated for each animal (as shown on the y-axis) *, ** p<0.05 and 0.01 compared to GFP-PBS rats. The rats treated with PBS still hâve a significant asymmetry in paw use, while animais treated with antibody GM37 hâve no 25 longer a significant déficit. (Example 9).
Fig 20A-20C shows that chronic treatment with antibody GM37 can reduce pathological alpha-synuclein phosphorylation induced by injection of pathological alpha-synuclein fibrillary seeds into the mouse striatum. Fig 20A shows a schematic indicating relative treatment fîmes with respect to seed injection and cell counting. The 30 antibody GM37 was administered one day before injection of recombinant alphasynuclein fibrillary seeds into dorsal striatum of mice, and then weekly for six weeks. Dosing regimen was either 15 mg/kg iv or 30 mg/kg îp. Fig 20B shows the exposure level of GM37 in plasma based on site of injection and dose. Weekly samples were taken before the injection of new antibody dose. Fig 20C shows the exposure level of GM37 in csf based on dose and injection site at the end of the study. Fig 20D compares the number of cells with phosphorylated alpha-synuclein positive inclusions 5 counted from every sixth section in substantia nigra after treatment with GM37 or PBS control. The mice treated with GM37 both 15 mg/kg iv and 30 mg/kg ip had a significant réduction in cells with phosphorylated alpha-synuclein inclusions compared to the PBS treated mice (Example 10).
Fig 21 shows alignment of human a (SEQ ID NO:10), β (SEQ ID NO:37) and γ (SEQ 10 ID NO:38) synuclein proteins. Amino acid residues different from a- synuclein are highlighted. Gaps are indicated by a dot. SwissProt numbers are in parenthesis.
Fig .22 shows alignment of alpha-synuclein orthologs (Cynomolgus monkey, SEQ ID NO:39; Rat, SEQ ID NO:40; Mouse, SEQ ID NO:41). Amino acid residues different from human alpha-synuclein (SEQ ID NO:10) are highlighted. SwissProt numbers are 15 shown in parenthesis.
Fig 23 shows transient expression of GM37 (named GM37 wild type (wt) and 3 GM37 variants, named GM37 var 1, 2 and 3. Asterisk indicates that the data are determined post protein A purification and neutralisation, t indicates that data are calculated from yield achieved post protein A and neutralization in relation to scale of expression culture (0.4L).
Fig 24 shows a compétition ELiSA measuring binding of four antibodies GM37 wt, GM37 var 1, GM37 var 2 and GM37 var 3 to human alpha-synuclein. Plates coated with aipha-synuclein are used to detect the amount of antibody remaining after preincubation in solution of each antibody (0.3 pg/ml) with increasing concentration of 25 alpha-synuclein (0-1000nM). Ail four antibodies show similar binding to alphasynuclein. .
Fig 25 shows a table comparing the binding rate kinetic parameters of GM37wt and variants 1-3 to immobilized recombinant human alpha-synuclein. The binding was measured using SPR and the rates were determined using a 1:1 binding algorithm 30 (BIAcore® T200).
Fig. 26 compares the effect of alpha-synuclein antibodies on phosphorylated alphasynuclein levels in murine primary neurons treated with pathological alpha-synuclein fibrillary seeds. Primary neurons were treated with seeds (10ng) in the presence or absence of four GM37, GM37 var 1, GM37 var 2 and GM37 var 3 (2pg). Neurons were fixed & stained after 3 weeks and analysed by Cellomics ARRAYSCAN™ for alphasynuclein phospho serine 129 positive spots. Cells treated with seeds alone or with seeds plus the isotype control antibody (B12) show significantly increased levers phosphorylation. Cells treated with GM37wt and the 3 variants are able to inhibit phosphorylation of alpha-synuclein, they ail show the same level of phosphorylation as cells that did not receive seeds. Data is shown as mean ±SD as determined from seven images per well in five wells. N=2.
Fig 27 compares température dépendent aggregation of wt GM37, var1, var2 and var3. A sample of each of the antibodies was subjected to a steady increase in température over time and the level of aggregation was simultaneously measured by multi-angle light scattering (Prometheus NT.48, NanoTemper Technologies). The température for onset of aggregation is similar for GM37 and GM37-variants, howeverthe lowest level of aggregation observed for GM37-Var2.
DETAILED DESCRIPTION OF THE INVENTION
Définitions
As used herein, the term “alpha-synuclein” is synonymous with “the alpha-synuclein protein” and refers to any of the alpha-synuclein protein isoforms (identified in, for example, UniProt as P37840, 1-3). The amino acid numbering of alpha-synuclein is given with respect to SEQ iD NO;10 as shown below, with méthionine (M) being amino acid résiduel :
SEQ ID NO:10:
MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA
The present invention relates to antibodies and to fragments of antibodies that are capable of specifically binding to alpha-synuclein, and in particular to human alphasynuclein. In particular, the antibodies and fragments thereof exhibit the ability to specifically bind to an epitope within 112-117 of human alpha-synuclein.
The term antibody (Ab) in the context of the présent invention refera to an immunoglobulin molécule or according to some embodiments of the invention, a fragment of an immunoglobulin molécule which has the abiiity to specifically bind to an epitope of a molecuie (“antigen”). Naturally occurring antibodies typically comprise a tetramer which is usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain, usually comprised of three domains (CH1, CH2 and CH3). Heavy chains can be of any isotype, including IgG (lgG1, igG2, igG3 and îgG4 subtypes), IgA (lgA1 and lgA2 subtypes), IgM and IgE. Each light chain is comprised of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (CL). Light chains include kappa chains and lambda chains. The heavy and light chain variable domain is typically responsîble for antigen récognition, while the heavy and light chain constant domain may médiate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effectorcells) and the first component (C1q) ofthe classical complément System. The VH and VL régions can be further subdivided into régions of hypervariability, termed “complementarity determining régions,” that are interspersed with régions of more conserved sequence, termed “framework régions (FR). Each VH and VL is composed of three CDR Domains and four FR Domains arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2FR3-CDR3-FR4. The variable domains of the heavy and light chains contain a binding domain that interacts with an antigen. Of particular relevance are antibodies and their antigen-binding fragments that hâve been “isolated” so as to exist in a physical milieu distinct from that in which it may occur in nature or that hâve been modified so as to differ from a naturally occurring antibody in amino acid sequence.
The term epitope means an antigenic déterminant capable of spécifie binding to an antibody. Epitopes usually consist of surface groupings of molécules such as amino acids or sugar side chains and usually hâve spécifie three dimensional structural characteristics, as well as spécifie charge characteristics. Conformational and linear epitopes are distinguished in that the binding to the former, but not the latter, is always lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not dîrectly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen-binding peptide). The term “112-117 epitope” refers to a région of human alpha-synuclein that contains at least 4 of the 6 amino acid residues of 112-117 human alpha-synuclein, which epitope does not include any residue from 1-111 (including any residue from 106-111) of human alpha-synuclein, nor any residue from 118-140 (including residue 118-120) of human alpha-synuclein. As used herein, an antibody :s saia to be capable of specifically binding to an epitope within the “112-117 epitope if it is capable of specifically binding to human alpha-synuclein by binding to at least 4 of the 6 amino acid residues of the 112-117 epitope.
As used herein, the term “antigen-binding fragment of an antibody” means a fragment, portion, région or domain of an antibody (regardless of how it is produced (e.g., via cleavage, recombinantly, synthetically, etc.)) that is capable of specifically binding to an epitope, and thus the term “antigen-binding” is intended to mean the same as “epitopebinding” so that, for example, an “antigen-binding fragment of an antibody” is intended to be the same as an “epitope-binding fragment of an antibody”. An antigen-binding fragment may contain 1, 2, 3, 4, 5 or ail 6 of the CDR Domains of such antibody and, although capable of specifically binding to such epitope, may exhibit a specificity, affinity or selectivity toward such epitope that differs from that of such antibody. Preferably, however, an antigen-binding fragment will contain ail 6 of the CDR Domains of such antibody. An antigen-binding fragment of an antibody may be part of, or comprise, a single polypeptide chain (e.g., an scFv), or may be part of, or comprise, two or more polypeptide chains, each having an amino-terminus and a carboxyl terminus (e.g., a diabody, a Fab fragment, a Fab2 fragment, etc.). Fragments of antibodies that exhibit antigen-binding ability can be obtained, for example, by protease cleavage of intact antibodies. More preferably, although the two domains of the Fv fragment, VL and VH, are naturally encoded by separate genes, or polynucleotides that encode such gene sequences (e.g., their encoding cDNA) can be joined, using recombinant methods, by a flexible linker that enables them to be made as a single protein chain in which the VL and VH régions associate to form monovalent antigenbinding molécules (known as single-chain Fv (scFv); see e.g., Bird et al. , (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85:58795883). Alternatively, by employing a flexible linker that is too short (e.g., less than about residues) to enable the VL and VH régions of a single polypeptide chain to associate together, one can form a bispecific antibody, diabody, or similar molécule (in which two such polypeptide chains associate together to form a bivalent antigen-binding molécule) (see for instance PNAS USA 90(14), 6444-8 (1993) for a description of diabodies). Examples of antigen-binding fragments encompassed within the present invention inciude (i) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in W02007059782; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge domain; (iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of a VL and VH domains, (v) a dAb fragment (Ward et al., Nature 341, 544546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends BiotechnoL 2003 Nov;2i(ll) :484-90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan;5_(l): III-24) and (vii) an isolated complementarity determining région (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linkerthat enabies them to be made as a single protein chain in which the VL and VH régions pair to form monovalent molécules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird étal., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). These and other useful antibody fragments in the context of the present invention are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype. As used herein, isotype refers to the immunoglobulin class (for instance lgG1, lgG2, lgG3 or lgG4) that is encoded by heavy chain constant domain genes. Such antibody fragments are obtained using conventional techniques known to those of skill in the art; suitable fragments capable of binding to a desired epitope may be readily screened for utility in the same manner as an intact antibody.
The term “bispecific antibody” refers to an antibody containing two independent antigen-binding fragments that each target independent targets. These targets can be epitopes présent on different proteins or different epitopes présent on the same target. Bispecific antibody molécules can be made using compensatory amino acid changes in the constant domains ofthe HCs of the parent monospecific bivalent antibody molécules. The resulting heterodimeric antibody contains one Fabs contributed from two different parent monospecific antibodies. Amino acid changes in the Fc domain leads to increased stability of the heterodimeric antibody with bispecificity that is stable over time. (Ridgway et al., Protein Engineering 9, 617-621 (1996), Gunasekaran et al., JBC 285, 19637-1(2010), Moore et al., MAbs 3:6 546-557 (2011), Strop étal., JMB 420, 204-219 (2012), Metz et al., Protein Engineering 25:10 571-580 (2012), Labrijn et al., PNAS 110:113, 5145 -5150 (2013), SpreterVon Kreudenstein et al., MAbs 5:5 646654 (2013)). Bispecific antibodies can also include molécules that are generated using ScFv fusions. Two monospecific scfv are then independently joined to Fc domains able to form stable heterodimers to generate a single bispecific molécule (Mabry et al., PEDS 23:3 115-127 (2010). Bispecific molécules hâve dual binding capabilities. For example, targeting both a therapeutic target and a transcytosing surface receptor for the purpose of delivering a therapeutic antibody across the blood brain barrier to treat a CNS disease.
The terms GM37, GM-37, GM37 wild type (wt), mab37 and 6004-37 are used interchangeably herein and ail refer to the same antibody.
The term antibody GM37 is intended to include an antibody or antigen-binding fragment thereof comprising or consisting of the Heavy Chain as given in CDR1-3 SEQ ID Nos:1-3 and the Light Chain CDR1-3 as given in SEQ ID Nos:4-6. In one embodiment, the antibody GM37 or antigen-binding fragment thereof may comprise or consist ofthe heavy chain variable domain of SEQ ID NO:7 and/or the light chain variable domain of SEQ ID NO:8. For example, the antibody GM37 may be an IgG antibody comprising a heavy chain consisting of a variable domain of SEQ ID NO:7 and a constant domain of SEQ ID NO:18 together with a light chain consisting of a variable domain of SEQ ID NO:8 and a kappa constant domain of SEQ ID NQ:17.
Deamination of proteins, and in these instance antibodies, can occur spontaneously during manufacturing and storage, but also in vivo, and makes the quality of the final pharmaceutical médicament difficult to control. The deamination may aiso in some instances affect the activity ofthe molécule. Deamination occurs at asparagine residues, but the location of the relevant asparagine may be difficult to predict with certainty, but may be influenced in some instances by an asparagine-glycine motif. Several possible deamination motifs are found on the GM37 antibody, however, one likely site of deamination was found to be at residue 54 ofthe heavy chain. The subséquent substitution of asparagine by another amino acid is not straight forward, but 3 variants of GM37 (GM37 variant (var) 1, 2 and 3) were found to retain the activity ofthe original GM37 (GM37 wild type (wt))
The term GM37 variants refers to the deaminated variants 1,2 or 3, wherein variant 1 has a N54S substitution, variant 2 has a N54Q substitution and variant 3 has a N54H compared to the GM37 antibody described herein above.
The antibody GM37 variant (var) 1, 2 and 3 are thus intended to include an antibody or antigen-binding fragment thereof comprising or consisting of the Heavy Chain as given in CDR1 and 3 SEQ ID Nos:1 and 3 from GM 37 and the Light Chain CDR1-3 from GM37 as given in SEQ ID Nos:4-6, but differing in their heavy chain CDR2 so that variant 1 has CDR 2 of SEQ !D NO:33, variant 2 has CDR 2 of SEQ ID NO:34 and variant 3 has CDR 2 of SEQ ID NO:35.
In one embodiment, the antibody GM37 variants or their antigen-binding fragments may comprise or consist of the heavy chain variable domain of SEQ ID NO:30, 31 and 32 for variant 1, 2 and 3, respectively, and the light chain variable domain of SEQ ID NO:8. The antibody GM37 may be an IgG antibody comprising a heavy chain consisting of a variable domain of SEQ ID NO:30, 31 or 32 and a constant domain of SEQ ID NO:18 together with a light chain consisting of a variable domain of SEQ ID NO:8 and a kappa constant domain of SEQ ID NO: 17.
The terms GM285, GM-285, mab285 and 6004-285 are used interchangeably herein and ali refer to the same antibody.
The term antibody GM285 is intended to include an antibody or antigen-binding fragment thereof comprising or consisting of the Heavy Chain as given in CDR1-3 SEQ ID NOs:20-22 and the Light Chain CDR1-3 as given in SEQ ID NOs:23-25. In one embodiment, the antibody GM37 or antigen-binding fragment thereof may comprise or consist of the heavy chain variable domain of SEQ ID NO:26 and/or the light chain variable domain of SEQ ID NO:27 For example, the antibody GM37 may be an IgG antibody comprising a heavy chain consisting of a variable domain of SEQ ID NO:26 and a constant domain of SEQ ID NO:28 together with a light chain consisting of a variable domain of SEQ ID NO:27 and a kappa constant domain of SEQ ID NO:29.
The GM285 antibody specifically binds an epitope within the sequence 112-115 (ILED; SEQ ID NO:19) of human alpha-synuclein (SEQ ID NO:10).
Unless otherwise specified herein, the numbering of amino acid residues in this région is according to IMGT®, the international ImMunoGeneTics information System® or, Kabat, E. A., Wu. T. T., Perry, H. M., Gottesmann, K. S. & Foeller, C. (1991).
Sequences of Proteins of Immunological Interest, 5th edit, NIH Publication no. 913242 U.S. Department of Health and HumanServices. Chothia, C. & Lesk, A. M.
(1987). Canonical structures for the hypervariable domains of immunoglobulins. J. Mol. Biol. 196, 901-917).
An anti-alpha-synuciein antibody or “aipha-synuclein antibody” (used interchangeably herein, depending on the context wherein its written) is an antibody or an antigenbinding fragment thereof which binds specifically to alpha-synuclein or an alphasynuclein fragment as defined herein above, in particular the sequence of alphasynuclein corresponding to SEQ ID NOs 9 and/or 19.
The term human antibody (which may be abbreviated to “humAb” or “HuMab), as used herein, is intended to include antibodies having variable and constant domains derived from human germline immunoglobulin sequences. The human antibodies ofthe invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo).
The terms monoclonal antibody or monoclonai antibody composition as used herein refer to a préparation of antibody molécules of single molecular composition. A conventions! monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. In certain embodiments a monoclonal antibody can be composed of more than one Fab domain thereby increasing the specificity to more than one target. The terms monoclonal antibody or monoclonal antibody composition are not intended to be limited by any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).
The term “humanized” refer to a molécule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a nonhuman species and a remaining immunoglobulin structure based upon the structure and /or sequence of a human immunoglobulin. The antigen-binding site may comprise either complété non-human antibody variable domains fused to human constant domains, or only the complementarity determining régions (CDRs) of such variable domains grafted to appropriate human framework régions of human variable domains. The framework resîdues of such humanized molécules may be wild type (e.g., fully human) or they may be modified to contain one or more amino acid substitutions not found in the human antibody whose sequence has served as the basis for humanization. Humanization lessens or éliminâtes the likelihood that a constant domain ofthe molécule will act as an immunogen in human individuals, but the possibility of an immune response to the foreign variable domain remains (LoBuglio, A. F. et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response, Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Another approach focuses not only on providing human-derived constant domains, but modifying the variable domains as well so as to reshape them as closely as possible to human form. It is known that the variable domains of both heavy and light chains contain three complementarity- determining régions (CDRs) which vary in response to the antigens in question and détermine binding capability, flanked by four framework régions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular antigen, the variable domains can be Teshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs présent in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) “Reshaping Human Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,” Science 239:15341536; Kettleborough, C. A. et al. (1991) “Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation,” Protein Engineering 4:773-3783; Maeda, H. et al. (1991) “Construction
Of Reshaped Human Antibodies With HIV-Neutralizing ActivityHuman Antibodies Hybridoma 2:124-134; Gorman, S< D. et al. (1991) “Reshapîng A Therapeutic CD4 Antibody, Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, P.R. et al. (1991) “Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial
Virus Infection in vivo, Bio/Technology 9:266-271; Co, M. S. et al. (1991) “Humanized Antibodies For Antiviral Therapy, Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. et al. (1992) “Humanizatton Of An Anti-p185her2 Antibody For Human Cancer Therapy,” Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M.S. et al. (1992) “Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen, J.
Immunol. 148:1149-1154. In some embodiments, humanized antibodies preserve al! CDR sequences (for example, a humanized mouse antibody which contains ail six CDRs from the mouse antibodies). In other embodiments, humanized antibodies hâve one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more
CDRs from the original antibody. The ability to humanize an antigen is well known (see, e.g., US Patents No. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).
As used herein, an antibody or an antigen-binding fragment thereof is said to “specifically” bind a région of another molécule (i.e., an epitope) if it reacts or .
associâtes more frequently, more rapidly, with greater duration and/or with greater affinity or avidity with that epitope relative to alternative epitopes. In one embodiment, the antibody, or antigen-binding fragment thereof, of the invention binds at least 10-fold more strongly to its target (human alpha synuclein) than to another molécule; preferably at least 50-fold more strongly and more preferably at least 100-fold more strongly. Preferably, the antibody, or antigen-binding fragment thereof, binds under physiological conditions, for example, in vivo. Thus, an antibody that is capable of “specifically binding” to an epitope within residues 112-117 (ILEDMP (SEQ ID NO:9)) of human alpha-synuclein encompasses an antibody or antigen-binding fragments thereof, that is capable of binding to an epitope within residues 112-117 of human alpha-synuclein with such specificity and/or under such conditions. Methods suitable for determining such binding will be known to those skilled in the art, and exemplary methods are described in the accompanying Examples. As used herein, the term binding in the context of the binding of an antibody to a predetermined antigen typically refers to binding with an affinity corresponding to a KD of about 10‘7 M or less, such as about 10'8 M or less, such as about 10'9 M or less when determined by for instance surface plasmon résonance (SPR) technology in either a BIAcore® 3000 or T200instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dépendent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly spécifie), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.
The term kd (sec -1 or 1/s), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.
The term ka (M-1 x sec-1 or 1/Msec), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.
The term KD (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the kd by the ka.
The term KA (M-1 or 1/M), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the ka by the kd.
in one embodiment, the invention relates to an antibody or antigen-binding fragments thereof, which exhibits one or more of the following properties:
i. a binding affinity (KD) for alpha-synuclein of between 0.5-10 nM, such as 1-5 nM or 1-2 nM;
ii. capability of inhibiting protease truncation of alpha-synuclein fibrils;
iii. capability of reversing impairment in basal synaptic transmission in F28snca transgenic mice;
. iv. capability of reducing levels of alpha-synuclein in the mouse hippocampus as measured by in vivo microdialysis;
v. capability, when administered chronically, to restore motor function in a rat model of Parkinson’s disease vi. Capability to prevent seeding of alpha-synuclein (such as accumulation of insoluble phosphorylated alpha-synuclein in vitro and/or in a mouse model of Parkinson’s disease); and/or vii. Capability to bind truncated alpha-synuclein in a human brain.
The binding affinity (KD) for alpha-synuclein may be determined using methods well known in the art, e.g. as described in Example 2.
The term “capability of inhibiting protease truncation of alpha-synuclein fibrils” includes 10 the capability of inhibiting calpain-1 induced formation of fragment 1-119-122 of human alpha synuclein in primary cortical neurons (see Example 5).
The term “capability of reversing impairment in basal synaptic transmission in F28-snca transgenic mice” includes the capability of reverse the impairment of synaptic transmission and plasticity in the CA1 area of the hippocampus in F28-snca transgenic mice, for example as indicated by evoked fEPSP slope as measured electrophysiologically (See Example 6).
The term “capability of reducing levels of alpha-synuclein in the mouse hippocampus as measured by in vivo microdialysis” includes the capability of reducing levels of human alpha synuclein in the hippocampus awake, freely-moving F28-snca transgenic mice, as measured using in vivo microdialysis (see Example 7).
The term “capability, when administered chronically, to restore motor function in a rat model of Parkinson’s disease” include the capability to reduce or eliminate motor asymmetry in a rat recombinant adeno-associated viral vector (rAAV) model of Parkinson’s Disease (see Example 8).
In some antibodies, only part of a CDR, namely the subset of CDR residues required for binding, termed the SDRs, are needed to retain binding in a humanized antibody. CDR residues not contacting the relevant epitope and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from régions of Kabat CDRs lying outside Chothia hypervariable loops (see,
Kabat et al. (1992) Sequences of Proteins of Immunological Interest, National
Institutes of Health Publication No. 91-3242; Chothia, C. et al. (1987) “Canonical Structures For The Hypervariable domains Of Imrnunoglobulins J. Mol. Biol. 196:901 917), by molecular modeling and/or empirically, or as described in Gonzales, N.R. et al. (2004) “SDF? Grafting OfA Murine Antibody Using Multiple Human Germline Templates To Minimize Its Immunogenicity, Mol. Immunoî. 41:863-872. In such humanized antibodies at positions in which one or more donor CDR residues is absent or in which an entire donor CDR is omitted, the amino acid occupying the position can be an amino acid occupying the corresponding position (by Kabat numberïng) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids in the CDRs to include refîects a balance of competing considérations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity. However, substitutions can also cause changes of affinity, and significant réductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.
The fact that a single amino acid alteration of a CDR residue can resuit in loss of functional binding (Rudikoff, S. etc. (1982) “Single Amino Acid Substitution Altering Antigen-Binding Specificity,” Proc. Natl. Acad. Sci. (USA) 79(6):1979-1983) provides a means for systematically identifying alternative functional CDR sequences. In one preferred method for obtaining such variant CDRs, a polynucleotide encoding the CDR is mutagenized (for example via random mutagenesis or by a site-directed method (e.g., polymerase chain-mediated amplification with primers that encode the mutated locus)) to produce a CDR having a substituted amino acid residue. By comparing the identity of the relevant residue in the original (functional) CDR sequence to the identity of the substituted (non-functional) variant CDR sequence, the BLOSUM62.iij substitution score for that substitution can be identified. The BLOSUM System provides a matrix of amino acid substitutions created by analyzîng a database of sequences for trusted alignments (Eddy, S.R. (2004) “Where Did The BLOSUM62 Alignaient Score Matrix Corne From?, Nature Biotech. 22(8): 1035-1036; Henikoff, J.G. (1992) “Amino acid substitution matrices from protein blocks,” Proc. Natl. Acad. Sci. (USA) 89:1091510919; Karlin, S. et al. (1990) “Methods ForAssessing The Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes, Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul, S.F. (1991) “Amino Acid Substitution Matrices From
An Information Theoretic Perspective,” J. Mol. Biol. 219, 555-565. Currëntly, the most advanced BLOSUM database isthe BLOSUM62 database (BLOSUM62.iij). Table 1 présents the BLOSUM62.iij substitution scores (the higher the score the more conservative the substitution and thus the more likely the substitution will not affect function). If an antigen-binding fragment comprising the résultant CDR fails to bind to alpha-synuclein, for example, then the BLOSUM62.iij substitution score is deemed to be insufficiently conservative, and a new candidate substitution is selected and produced having a higher substitution score. Thus, for example, if the original residue was glutamate (E), and the non-functional substftute residue was histidine (H), then the
BLOSUM62.iij substitution score will be 0, and more conservative changes (such as to aspartate, asparagine, glutamine, or lysine) are preferred.
Table 1 | ||||||||||||||||||||
A | R | N | D | C | Q | E | G | H | I | L | K | M | F | P | S | T | W | Y | V | |
A | +4 | -T | -2 | -2 | 0 | -1 | -1 | 0 | -2 | -1 | -1 | -1 | -1 | -2 | -1 | +1 | 0 | -3 | -2 | 0 |
R | -1 | +5 | 0 | -2 | -3 | +1 | 0 | -2 | 0 | -3 | -2 | +2 | -1 | -3 | -2 | -1 | -1 | , -3 | -2 | -3 |
N | -2 | 0 | +6 | +1 | -3 | 0 | 0 | 0 | +1 | -3 | -3 | 0 | -2 | -3 | -2 | +1 | 0 | -4 | -2 | -3 |
D | -2 | -2 | +1 | +6 | -3 | 0 | +2 | -1 | -1 | -3 | -4 | -1 | -3 | -3 | -1 | 0 | -1 | -4 | -3 | -3 |
C | 0 | -3 | -3 | -3 | +9 | -3 | -4 | -3 | -1 | -1 | -3 | -1 | -2 | -3 | -1 | -1 | -2 | -2 | -1 | |
Q | -1 | +1 | 0 | 0 | -3 | +5 | +2 | -2 | 0 | -3 | -2 | +1 | 0 | -3 | -1 | 0 | -1 | -2 | -1 | -2 |
E | -1 | 0 | 0 | +2 | -4 | +2 | +5 | -2 | 0 | -3 | -3 | +1 | -2 | -3 | -1 | 0 | -1 | -3 | -2 | -2 |
G | 0 | -2 | 0 | -1 | -3 | -2 | -2 | +6 | -2 | -4 | -4 | -2 | -3 | -3 | -2 | 0 | -2 | -2 | -3 | -3 |
H | -2 | 0 | +1 | -1 | -3 | 0 | 0 | -2 | +8 | -3 | -3 | -1 | -2 | -1 | -2 | -1 | -2 | -2 | +2 | -3 |
I | -1 | -3 | -3 | -3 | -1 | -3 | -3 | -4 | -3 | +4 | +2 | -3 | +1 | 0 | -3 | -2 | -1 | -3 | -1 | +3 |
L | -1 | -2 | -3 | -4 | -1 | -2 | -3 | -4 | -3 | +2 | +4 | -2 | +2 | 0 | -3 | -2 | -1 | -2 | -1 | +1 |
K | -1 | +2 | 0 | -1 | -3 | +1 | +1 | -2 | -1 | -3 | -2 | +5 | -1 | -3 | -1 | 0 | -1 | -3 | -2 | -2 |
M | -1 | -1 | -2 | -3 | -1 | 0 | -2 | -3 | -2 | +1 | +2 | -1 | +5 | 0 | -2 | -1 | -1 | -1 | -1 | +1 |
F | -2 | -3 | -3 | -3 | -2 | -3 | -3 | -3 | -1 | 0 | 0 | -3 | 0 | +6 | -4 | -2 | -2 | +1 | +3 | -1 |
P | -1 | -2 | -2 | -1 | -3 | -1 | -1 | -2 | -2 | -3 | -3 | -1 | -2 | -4 | +7 | -1 | -1 | -4 | -3 | -2 |
S | +1 | -1 | +1 | 0 | -1 | 0 | 0 | 0 | -1 | -2 | -2 | 0 | -1 | -2 | -1 | +4 | +1 | -3 | -2 | -2 |
T | 0 | -1 | 0 | -1 | -1 | -1 | -1 | -2 | -2 | -1 | -1 | -1 | -1 | -2 | -1 | +1 | +5 | -2 | -2 | 0 |
w | -3 | -3 | -4 | -4 | -2 | -2 | -3 | -2 | -2 | -3 | -2 | -3 | -1 | +1 | -4 | -3 | -2 | +11 | +2 | -3 |
Y | -2 | -2 | -2 | -3 | -2 | -1 | -2 | -3 | +2 | -1 | -1 | -2 | -1 | +3 | -3 | -2 | -2 | +2 | +7 | -1 |
V | 0 | -3 | -3 | -3 | -1 | -2 | -2 | -3 | -3 | +3 | +1 | -2 | +1 | -1 | -2 | -2 | 0 | -3 | -1 | +4 |
The invention thus contemplâtes the use of random mutagenesis to identify improved CDRs. In the context of the présent invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables:
Amino Acid Residue Classes For Conservative Substitutions:
Table 2 | |
Acidic Residues | Asp (D) and Glu (E) |
Basic Residues | Lys (K), Arg (R), and His (H) |
Hydrophilic Uncharged Residues | Ser (S), Thr (T), Asn (N), and Gin (Q) |
Aliphatic Uncharged Residues | Cly (G), Ala (A), Val (V), Leu (L), and Ile (D |
Non-polar Uncharged Residues | Cys (C), Met (M), and Pro (P) |
Aromatic Residues | Phe (F), Tyr (Y), and Trp (W) |
Alternative Conservative Amino Acid Residue Substitution Classes:
Table 3 | |||
1 | A | S | T |
2 | D | E | |
3 | N | Q | |
4 | R | K | |
5 | I | L | M |
6 | F | Y | W |
Alternative Physical and Functional Classifications of Amino Acid Residues:
Table 4 | |
Alcohol Group-Containing Residues | S and T |
Aliphatic Residues | I, L, V and M |
Cycloalkenyl-Associated Residues | F, H, W and Y |
Hydrophobie Residues | A, C, F, G, H, I, L, M, R, T, V, W and Y |
Negatively Charged Residues | D and E |
Polar Residues | C, D, E, H, K, N, Q, R, S and T |
Positively Charged Residues | H,K and R |
Small Residues | A, C, D, G, N, P, S, T and V |
Very Small Residues | A, G and S |
Residues Involved In Turn Formation | A, C, D, E, G, H, K, N, Q, R, S, P and T |
Flexible Residues | Q, T, K, S, G, P, D, Eand R |
More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
Additional groups of amino acids may also be formulated using the principles described 5 in, e.g., Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed. 1993),
W. H. Freeman and Company.
Phage display technology can alternatively be used to increase (or decrease) CDR affinity. This technology, referred to as affinity maturation, employs mutagenesis or “CDR walking” and re-selection uses the target antigen or an antigenic antigen-binding 10 fragment thereof to identify antibodies having CDRs that bind with higher (or lower) affinity to the antigen when compared with the initial or parental antibody (See, e.g.
Glaser et al. (1992) J. Immunology 149:3903). Mutagenizing entire codons ratherthan single nucléotides results in a semi-randomized répertoire of amino acid mutations.
Libraries can be constructed consisting of a pool of variant clones each of which differs 15 by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue. Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased or decreased 20 affinity to the antigen (e.g., ELISA) (See Wu et al. 1998, Proc. Natl. Acad. Sci. (U.S.A.)
95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDR walking which randomizes the Light Chain may be used possible (see, Schieret al., 1996, J. Mol. Bio. 263:551).
Methods for accomplishing such affinity maturation are described for example in: 25 Krause, J.C. et al. (2011) An Insertion Mutation That Distorts Antibody Binding Site
Architecture Enhances Function OfA Human Antibody, MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10; Kuan, C.T. et al. (2010) Affinity-Matured Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas, Int.
J. Cancer 10.1002/ijc.25645; Hackel, B.J. et al. (2010) Stability And CDR Composition
8/ases Enrich Binder Functionality Landscapes,” J. Mol. Biol. 4.01 (1 ):84-96;
Montgomery, D.L. et al. (2009) “Affinity Maturation And Characterization OfA Human Monoclonal Antibody Against HIV-1 gp41, MAbs 1(5)1462-474; Gustchina, E. et al. (2009) “Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop OfA Monoclonal Fab Derived From A Synthetic Naïve Human Antibody Library And Directed Against The Internai Trimeric Coiled-Coil OfGp41 Yields A Set OfFabs With Improved HIV-1 Neutralizatron Potency And Breadth Virology 393(1 );112-119; Finlay, W.J. étal. (2009) “Affinity Maturation OfA Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementanty-Determining Régions, J. Mol. Biol. 388(3):541-558; Bostrom, J. ét al. (2009) “Improving Antibody Binding Affinity And Specificity For Therapeutic Development, Methods Mol. Biol. 525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification, Mol. Immunol. 46(1):135-144; and Barderas, R. et al. (2008) “Affinity Maturation Of Antibodies Assisted By In Silico Modeling, Proc. Natl. Acad. Sci. (USA) 105(26):9029-9034.
Thus,.the sequence of CDR variants of encompassed antibodies or their antigenbinding fragments may differ from the sequence of the CDR of the parent antibody, GM37, GM37 var 1-3, or 285, through substitutions; for instance substituted 4 amino acid residue, 3 amino acid residue, 2 amino acid residue or 1 of the amino acid residues. According to an embodiment of the invention it is furthermore envisaged that the amino acids in the CDR régions may be substituted with conservative substitutions, as defined in the 3 tables above.
The term “treatment” or “treating” as used herein means ameliorating, slowing, attenuating or reversing the progress or severity of a disease or disorder, or ameliorating, slowing, attenuating or reversing one or more symptoms or side effects of such disease or disorder. For purposes of this invention, “treatment or “treating” further means an approach for obtaining bénéficiai or desired clinical results, where “bénéficiai or desired clinical results” include, without limitation, aliénation of a symptom, diminishment of the extent of a disorder or disease, stabilized (i.e., not worsening) disease or disorder state, delay or slowing of the progression a disease or disorder state, amelioration or palliation of a disease or disorder state, and remission of a disease or disorder, whether partial or total, détectable or undetectable.
An effective amount, when applied to an antibody or antigen-binding fragments thereof, of the invention, refers to an amount sufficient, at dosages and for periods of time necessary, to achîeve an întended biological effect or a desired therapeutic resuit including, without limitation, clinical results. The phrase “therapeutically effective amount” when applied to an antibody or antigen-binding fragments thereof, of the invention is întended to dénoté an amount of the antibody, or antigen-binding fragment thereof, that is sufficient to ameliorate, paiiiate. stabilize, reverse, slow, attenuate or delay the progression of a disorder or disease State, or of a symptom ofthe disorder or disease. In an embodiment, the method ofthe present invention provides for administration of the antibody, or antigen-binding fragment thereof, in combinations with other compounds. In such instances, the effective amount” is the amount ofthe combination sufficient to cause the întended biological effect.
A therapeutically effective amount of an anti-alpha-synuclein antibody or antigenbinding fragment thereof ofthe invention may vary according to factors such as the disease state, âge, sex, and weight of the individual, and the ability of the anti-alphasynuclein antibody to eiicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically bénéficiai effects.
As indicated above, the present invention particularly relates to a monoclonal antibody capable of specifically binding to an epitope within amino acids 112-117 of human alpha-synuclein (SEQ ID NO:9 (ILEDMP)). In one embodiment the antibody is capable of competing with the antibody GM37 for binding to an epitope within the 112-117 amino acids of alpha-synuclein.
The antibodies ofthe present invention, exemplified by GM37 its variants GM37 var 1-3 and GM285, and their alpha-synuclein binding fragments are capable of binding the toxic alpha-synuclein fragment consisting of residues 1-119/122 of alpha-synuclein and neutralizing its toxicity (for example, by extracellular binding to the alpha-synuclein fragment and thereby preventing it from being taken up by cells. Surprisingly the antibodies of the present invention, which are capable of binding to an epitope within amino acids 112-117 of alpha-synuclein are superior to prior art antibodies such as antibody 9E4 in binding to toxic alpha-synuclein species in human brain, and hâve superior effects on clearing extracellular alpha-synuclein and normalising an impaired synaptic transmission induced by alpha-synuclein in vivo. The antibodies ofthe invention are also able to ameliorate the appearance of a relevant motor phenotype in a rat model for Parkinson's disease.
The antibodies of the présent invention are preferably human or humanized antibodies.
The présent invention also provides a method of reducing alpha-synuclein aggregate formation in a patient, comprising administering to the patient in need of such treatment. a therapeutically effective amount of an antibody of the invention.
Further the antibodies may be in a composition together with a pharmaceutically acceptable carrier, diluent and/or stabilizer. The antibodies of the invention may .be 10 used in therapy. In particular, the antibodies ofthe invention may be used in treating synucleinopathies such as Parkinson's disease (including idiopathic inherited forms of Parkinson's disease), Gaucher’s Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease, pure autonomie failure and multiple system atrophy.
The treatment envisioned by the présent invention may be chronic and the patient may be treated at least 2 weeks, such as at least for 1 month, 6, months, 1 year or more. .
The antibodies of antigen-binding fragments thereof ofthe présent invention may be produced in different cell lines, such as a human cell line, a mammal non-human cell line, and insect cell line, for example a CHO cell line, HEK cell line, BHK-21 cell line, 20 murine cell line (such as a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line,
HKB-11 cell line, CAP cell line and HuH-7 human cell line (Dumont et al, 2015, Crit Rev Biotechnol. Sep 18:1-13., the contents which is included herein by reference).
The antibodies of the présent invention may for example be monoclonal antibodies produced by the hybridoma method first described by Kohler et al., Nature 256, 495 25 (1975), or may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. BioL 222, 581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas 30 prepared from murine splenic B lymphocyte cells obtained from mice immunized with an antigen of interest, for instance, in the form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest. Monocîonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, rabbits, dogs, sheep, goats, primates, etc.
In one embodiment, the antibody of the invention is a human antibody Human monocîonal antibodies directed against alpha-synuclein may be generated using transgenic or transchromosomal mice carrying parts of the human immune System rather than the mouse System. Such transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively.
The HuMAb mouse contains a human immunoglobulin gene minilocus that encodes unrearranged human heavy variable and constant (μ and Y) and light variable and constant (k) chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or IgK and in response to immunîzation, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N., Ann. N. Y. Acad. Sci 764 536-546 (1995)). The préparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.
The HCo7, HCo12, HCo17 and HCo20 mice hâve a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12,811820 (1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), and a KCo5 human kappa light chain transgene (as described in Fishwild et ai., Nature Biotechnology 14, 845-851 (1996)). Additionally, the HCo7 mice hâve a HCo7 human heavy chain transgene (as described in US 5,770,429), the HCo12 mice hâve a HCo12 human heavy chain transgene (as described in Example 2 of WO 01/14424), the HCo17 mice hâve a HCo17 human heavy chain transgene (as described in Example 2 of WO 01/09187) and the HCo20 mice hâve a HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain loci.
In the KM mouse strain, the endogenous mouse kappa light chain gene has been 10 homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in . . WO 02/43478. HCo12-Balb/c, HCo17-Balb/c and HCo20-Balb/c mice can be generated by Crossing HCo12, HCo17 and HÇo20 to KCo5[J/K](Balb) as described in WO 09/097006.
In the KM mouse strain, the endogenous mouse kappa light chain gene has been 20 homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain trans25 chromosome composed of chromosome 14 antigen-binding fragment hCF (SC20) as described in WO 02/43478.
Splénocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques. Human monoclonal or polyclonal antibodies of the présent invention, or antibodies of the 30 present invention originating from other species may also be generated transgenically through the génération of another non-human mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with the transgenic production in mammals, antibodies may be produced in, and recovered from, the milk of goats, cows, or other mammals. See for instance US 5,827,690, US 5,756,687, US 5,750,172 and US 5,741,957.
The antibody of the invention may be of any isotype. The choice of isotype typically will be guided by the desired effector fonctions such as ADCC induction. Exemplary isotypes are lgG1, lgG2, lgG3, and lgG4. Either of the human light chain constant domains, kappa or lambda, may be used. If desired. the class of an anti-alphasynuclein antibody of the présent invention may be switched by known methods. For example, an antibody of the présent invention that was orîginally IgM may be class switched to an IgG antibody of the présent invention. Further, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGI to lgG2. Thus, the effector function ofthe antibodies ofthe présent invention may be changed by isotype switching to, e.g., an igG 1, lgG2, lgG3 or lgG4 antibody for various therapeutic uses. In one embodiment an antibody ofthe présent invention is an lgG1 antibody, for instance an lgG1, rç. An antibody is said to be of a particular isotype if its amino acid sequence is most homologous to that isotype, relative to other isotypes.
In one embodiment, the antibody ofthe invention is a full-length antibody, preferably an IgG antibody, in particular an lgG1, rç antibody. In another embodiment, the antibody of the invention is an antibody fragment or a single-chain antibody.
Antibodies and antigen-binding fragments thereof may e.g. be obtained by antigenbinding fragmentation using conventional techniques, and antigen-binding fragments screened for utility in the same manner as described herein forwhole antibodies. For example, F(ab')2 antigen-binding fragments may be generated by treating antibody with pepsin. The resulting F(ab')2 antigen-binding fragment may be treated to reduce disuîfide bridges to produce Fab' antigen-binding fragments. Fab antigen-binding fragments may be obtained by treating an IgG antibody with papain; Fab' antigenbinding fragments may be obtained with pepsin digestion of IgG antibody. An F(ab') antigen-binding fragment may also be produced by binding Fab'-described below via a thioether bond or a disuîfide bond. A Fab' antigen-binding fragment is an antibody antigen-binding fragment obtained by cutting a disuîfide bond ofthe hinge domain of the F(ab')2. A Fab'- antigen-binding fragment may be obtained by treating an F(ab')2 antigen-binding fragment with a reducing agent, such as dithiothreitol. Antibody antigen-binding fragment may also be generated by expression of nucleic acids encoding such antigen-binding fragments in recombinant cells (see for instance Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimeric gene encoding a portion of an F(ab')2 antigen-binding fragment could include DNA sequences encoding the CH1 région and hinge domain of the H chain, followed by a transiational stop codon to yield such a truncated antibody antigen-binding fragment molécule.
In one embodiment, the anti-alpha-synuclein antibody is a monovalent antibody, preferably a monovalent antibody as described in W02007059782 (which is incorporated herein by reference in its entirety) having a délétion ofthe hinge domain. Accordingly, in one embodiment, the antibody is a monovalent antibody, wherein said anti-alpha-synuclein antibody is constructed by a method comprising: i) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucléotide sequence encoding the VL région of a selected antigen spécifie anti-alpha-synuclein antibody and a nucléotide sequence encoding the . constant CL région of an Ig, wherein said nucléotide sequence encoding the VL région of a selected antigen spécifie antibody and said nucléotide sequence encoding the CL région of an Ig are operably linked together, and wherein, in case of an lgG1 subtype, the nucléotide sequence encoding the CL région has been modified such that the CL région does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the CL région in the presence of polyclonal human IgG or when administered to an animal or human being; ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucléotide sequence encoding the VH région of a selected antigen spécifie antibody and a nucléotide sequence encoding a constant CH région of a human Ig, wherein the nucléotide sequence encoding the CH région has been modified such that the région corresponding to the hinge domain and, as required by the Ig subtype, other régions ofthe CH région, such as the CH3 région, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence ofthe CH région ofthe human Ig in the presence of polyclonal human IgG or when administered to an animal human being, wherein said nucléotide sequence encoding the VH région of a selected antigen spécifie antibody and said nucléotide sequence encoding the CH région of said Ig are operably linked together; iii) providing a cell expression system for producing said monovalent antibody; iv) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).
Similarly, in one embodiment, the anti- alpha-synuclein antibody is a monovalent antibody, which composes:
(i) a variable domain of an antibody ofthe invention as described herein or an antigenbinding part of the said région, and (ii) a CH domain of an immunoglobulin or a domain thereof comprising the CH2 and CH3 domains, wherein the CH domain or domain thereof has been modified such that the domain corresponding to the hinge domain and, if the immunoglobulin is not an lgG4 subtype, other domains of the CH domain, such as the CH3 domain, do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical CH domain or other covalent or stable non-covalent interheavy chain bonds with an identical CH domain in the presence of polyclonal human IgG.
In a further embodiment, the heavy chain ofthe monovalent anti-alpha-synuclein antibody has been modified such that the entire hinge domain has been deleted.
In anotherfurther embodiment, the sequence of said monovalent antibody has been modified so that it does not comprise any acceptor sites for N-linked glycosylation.
The invention also includes “Bispecific Antibodies,” wherein an anti-Alpha-synuclein binding région (e.g., a Alpha-synuclein-binding région of an anti-alpha-synuclein monoclonai antibody) is part of a bivalent or polyvalent bispecific scaffold that targets more than one epitope, (for example a second epitope could comprise an epitope of an active transport receptor, such that the Bispecific Antibody would exhibit improved transcytosis across a biological barrier, such as the Blood Brain Barrier). Thus, in another further embodiment, the monovalent Fab of an anti-synuclein antibody may be joined to an additional Fab or sefv that targets a different protein to generate a bispecific antibody. A bispecific antibody can hâve a dual function, for example a therapeutic function imparted by an anti-synuclein binding domain and a transport · function that can bind to a receptor molécule to enhance transfer cross a biological barrier, such as the blood brain barrier.
Anti-alpha-synuclein antibodies, and antigen-binding fragments thereof, of the invention also include single chain antibodies. Single chain antibodies are peptides in which the heavy and light chain Fv régions are connected. In one embodiment, the present invention provides a single-chain Fv (scFv) wherein the heavy and light chains in the Fv of an anti-alpha-synuclein antibody ofthe present invention are joined with a flexible peptide linker (typically of about 10, 12, 15 or more amino acid residues) in a single peptide chain. Methods of producing such antibodies are described in for instance US 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Veriag, New York, pp. 269-315 (1994), Bird et al., Science 242, 423-426 (1988), Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature 348, 552-554 (1990). The Single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used.
The anti-alpha-synuclein antibodies and antigen-binding fragments thereof described herein may be modified by inclusion of any suitable number of modified amino acids and/or associations with such conjugated substituents. Suitability in this context is generally determined by the ability to at least substantially retain the alpha-synuclein selectivity and/or the anti-alpha-synuclein specificity associated with the nonderivatized parent anti-alpha-synuclein antibody. The inclusion of one or more modified amino acids may be advantageous in, for example, increasing polypeptide sérum halflife, reducing polypeptide antigenicity, or increasing polypeptide storage stability. Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means. Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated (e. g., farnesylated, geranylgeranyiated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. Référencés adéquate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols On CDRom, Humana Press, Totowa, NJ. The modified amino acid may, for instance, be selected from a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent.
Anti-alpha-synuclein antibodies may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are iliustrated in for instance US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Additional illustrative polymers include polyoxyethylated polyoîs and polyethylene glycol (PEG) (e.g., a PEG with a moiecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000, e.g., about 3,000-12,000 g/mol).
The antibodies of the present invention may further be used in a diagnostic method or as a diagnostic imaging ligand.
In one embodiment, anti-alpha-synuclein antibodies comprising one or more radiolabeied amino acids are provided. A radiolabeied anti-alpha-synuclein antibody may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeied molécules is another possible feature). Non-limiting examples of such labels include, but are not limited to bismuth (213Bi), carbon (11C, 13C, 14C), chromium (51Cr), cobalt (57Co, 60Co), copper (64Cu), dysprosium (165Dy), erbium (169Er), fluorine (1SF), gadolinium (153Gd, 159Gd), gallium (68Ga, 67Ga), germanium (68Ge), gold (198Au), holmium (166Ho), hydrogen (3H), indium (111ln, 112ln, 113ln, 115ln), iodine (121l, 123l, 125l, 131l), iridium (192lr), iron (59Fe), krypton (81mKr), lanthanium (140La), lutelium (177Lu), manganèse (54Mn), molybdenum (99Mo), nitrogen (13N, 15N), oxygen (15O), palladium (103Pd), phosphorus (32P), potassium (42K), praseodymium (142Pr), prométhium (149Pm), rhénium (18eRe, 188Re), rhodium (105Rh), rubidium (81Rb, 8ZRb), ruthénium (82Ru, 97Ru), samarium (153Sm), scandium (47Sc), sélénium (75Se), sodium (24Na), strontium (85Sr, 89Sr, 92Sr), sulfur (35S), technetium (99Tc), thallium (2Û1TI), tin (1l3Sn, 117Sn), xénon (133Xe), ytterbium (169Yb, 175Yb, 177Yb), yttrium (90Y) and zinc (6SZn). Methods for preparing radiolabeied amino acids and related peptide dérivatives are known in the art (see for instance Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2nd édition, Chaîner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE35,500), US 5,648,471 and US
5,697,902. For example, a radioisotope may be conjugated by a chloramine T method (Lindegren, S. et al. (1998) “Chloramine-T In High-Specific-Activity Radioiodination Of Antibodies Using N-Succinimidyl-3-(Trimethylstannyl)Benzoate As An Intermediate,’’ Nucl. Med. Biol. 25(7):659-665; Kurth, M. et al. (1993) “Site-Specific Conjugation OfA
Radioiodinated Phenethylamine Dérivative To A Monoclonal Antibody Results In Increased Radioactivity Localization In Tumor, J. Med. Chem. 36(9):1255-1261 ; Rea, D.W. et al. (1990) “Site-specifically radioiodinated antibody for targeting tumors,” Cancer Res. 50(3 Suppl):857s-861s).
The invention also provides anti-alpha-synuclein antibodies and antigen-binding 10 fragments thereof that are detectably iabeled using a fluorescent label (such as a rare earth chelate (e.g., a europium chelate)), a fluorescein-type label (e.g., fluorescein, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxy fluorescein, dichlorotriazinylamine fluorescein), a rhodamine-type label (e.g., ALEXA FLUOR® 568 (Invitrogen), TAMRA® or dansyl chloride), VIVOTAG 680 XL FLUOROCHROME™ (Perkin Elmer), phycoerythrin; umbelîiferone, Lissamine; a cyanine; a phycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen) or an analogue thereof, ail of which are suitable for optical détection. Chemiluminescent labels may be employed (e.g., luminol, luciferase, luciferin, and aequorin). Such diagnosis and détection can also be accomplished by coupling the diagnostic molécule of the present invention to détectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, betagalactosidase, or acetylcholinesterase, or to prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin.
Chemiluminescent labels may be employed (e.g., luminol, luciferase, luciferin, and 25 aequorin). Such diagnosis and détection can also be accomplished by coupling the diagnostic molécule of the present invention to détectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, or to prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin. Paramagnetic labels can also be employed, and are preferably detected using Positron Emission Tomography (PET) or Single-Photon Emission Computed Tomography (SPECT). Such paramagnetic labels include, but are not limited to compounds containing paramagnetic ions of Aluminum (Al), Barium (Ba), Calcium (Ca), Cérium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu), Gandolinium (Gd), Holmium (Ho), Iridium (Ir), Lithium (Li), Magnésium (Mg), Manganèse (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os), Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthénium (Ru), Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm), Tin (Sn), Titanium (Ti), Tungsten (W), and Zirconium (Zi), and particularly, Co+2, CR+2, Cr+3, Cu+2, Fe+2, Fe+3, Ga+3, Mn+3, Ni+2, Ti+3, \Λ3, and V*4, positron emÆting metals using various positron émission tomographies, and non-radioactive paramagnetic métal ions.
Thus in one embodiment the anti-alpha-synuclein antibody ofthe invention may be labelled with a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotopic label or an enzyme label. The labelled antibody may be used in detecting or measuring the presence or amount of said alpha-synuclein in the brain of a subject. This method may comprise the détection or measurement of in vivo imaging of antialpha-synuclein antibody bound to said alpha-synuclein and may comprises ex vivo imaging of said anti-alpha-synuciein antibody bound to said alpha-synuclein.
In a further aspect, the invention relates to an expression vector encoding one or more polypeptide chains of an antibody of the invention or an antigen-binding fragment thereof. Such expression vectors may be used for recombinant production of the antibodies and antigen-binding fragments of the invention.
An expression vector in the context of the présent invention may be any suitable DNA or RNA vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control éléments). Examples of such vectors include dérivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, an anti-aipha-synuclein antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in, for instance, Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), a compacted nucleic acid vector (as described in for instance US 6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a midge minimally-sized nucleic acid vector (as described in, for instance, Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaPO4-precipitated construct (as described in, for instance, WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 2, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance US
5,589,466 and US 5,973,972).
In one embodiment, the vector is suitable for expression of anti-alpha-synuclein antibodies or antigen-binding fragments thereof in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vectors (Novagen,
Madison, Wl) and the like).
An expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et 15 a/., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley
InterScience New York (1987), Grant et al., Methods in Enzymol 153, 516-544 (1987), Mattanovich, D. et al. Methods Mol. Biol. 824, 329-358 (2012), Celik, E. et al.
Biotechnol. Adv. 30(5), 1108-1118 (2012), Li, P. et al. Appl. Biochem. Biotechnol.
142(2), 105-124 (2007), Bôer, E. et al. Appl. Microbiol. Biotechnol. 77(3), 513-523 20 (2007), van derVaart, J.M. Methods Mol. Biol. 178, 359-366 (2002), and Holliger, P.
Methods Mol. Biol. 178, 349-357 (2002)).
In an expression vector of the invention, anti-alpha-synuclein antibody-encoding nucleic acids may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating éléments. Examples of such éléments include strong 25 expression promoters (e.g., human CMV IE promoter/enhancer as well as RSV, SV40,
SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of réplication for plasmid product in E. coli, an antibiotic résistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such 30 as CMV IE (the skilled artisan will recognize that such terms are actually descriptors of a degree of gene expression under certain conditions).
The antibodies of antigen-binding fragments thereof ofthe present invention may be produced in different cell Unes, süch as a human cell line, a mammal non-human cell line, and insect cell line, for example a CHO cell line, HEK cell line, BHK-21 cell line, murine cell line (such as a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line, HKB-11 ceil line, CAP cell line and HuH-7 human cell line (Dumont et al, 2015, Crit Rev Biotechnol. Sep 18:1-13., the contents which is included herein by reference).
In an even further aspect, the invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody or an antigen-binding domain thereof of the invention as defined herein or a bispecific molécule of the invention as defined herein. Examples of host cells include yeast, bacteria, and mammalian cells, such as CHO or HEK cells. For example, in one embodiment, the present invention provides a celi comprising a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of an anti-alpha-synuclein antibody of the present invention or an antigen-binding fragment thereof. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of an anti-alpha-synuclein antibody of the invention.
In a further aspect, the invention relates to a method for producing an anti-alphasynuclein antibody of the invention, said method comprising the steps of a) culturing a hybridoma or a host cell of the invention as described herein above, and b) purifying the antibody ofthe invention from the culture media.
In one embodiment, the invention relates to a préparation that, as such term is used herein, comprises an anti-alpha-synuclein antibody as defined herein, and that is substantially free of naturally-arising antibodies that are either not capable of binding to alpha-synuclein or that do not materially alter the anti-alpha-synuclein functionality of the préparation. Thus, such a préparation does not encompass naturally-arising sérum, or a purified dérivative of such sérum, that comprises a mixture of an antialpha-synuclein antibody and another antibody that does not alter the functionality of the anti-alpha-synuclein antibody ofthe préparation; wherein such functionality is selected from the group consisting of:
(i) a binding affinity (KD) of the anti-aipha-synuclein antibody for alphasynuclein;
(ii) a capability of the anti-alpha-synuclein antibody of inhibiting protease truncation of alpha-synuclein fibrils;
(iii) a capability of the anti-alpha-synuclein antibody of reversing impairment in basal synaptic transmission in F28-snca transgenic mice;
;'iv) a capability of the anti-alpha-synuclein antibody of reducing levels of alpha-synuclein in the mouse hippocampus as measured by in vivo microdialysis; and (v) a capability, when administered chronically, of the anti-alpha-synuclein antibody to restore motor function in a rat model of Parkinson’s disease.
(vi) a capability to prevent seeding of alpha-synuclein (such as accumulation of insoluble phosphorylated alpha-synuclein in vitro and/or in a mouse model of Parkinson’s disease); and/or (vii) a capability to bind truncated alpha-synuclein in a human brain.
The invention particularly relates to préparations of such an anti-alpha-synuclein antibody having a structural change in its amino acid sequence (in any of its CDRs, variable domains, framework residues and/or constant domains) relative to the structure of a naturally-occurring anti-alpha-synuclein antibody, wherein said structural change causes the anti-alpha-synuclein monoclonal antibody to exhibit a markedly altered functionality (i.e., more than a 20% différence, more than a 40% différence, more than a 60% différence, more than an 80% différence, more than a 100% différence, more than a 150% différence, more than a 2-fold différence, more than a 4fold différence, more than a 5-fold différence, or more than a 10-fold différence in functionality) relative to the functionality exhibited by said naturally-occurring antialpha-synuclein antibody; wherein such functionality is: .
(i) a binding affinity (KD) of the anti-alpha-synuclein monoclonal antibody for alpha-synuclein;
(ii) a capability of the anti-alpha-synuclein monoclonal antibody of inhibiting protease truncation of alpha-synuclein fibrils;
(iii) a capability of the anti-alpha-synuclein monoclonal antibody of reversing impairment in basal synaptic transmission in F28-snca transgenic mice;
(iv) a capabiîity of the anti-alpha-synuclein monoclonal antibody of reducing levels of alpha-synuclein in the mouse hippocampus as measured by in vivo microdialysis; and/or (v) a capabiîity, when administered chronically, of the anti-alpha-synuclein monoclonal antibody to restore motor function in a rat model of Parkinson’s disease;
(vi) a capabii.'ty to prevent seeding of alpha-synuclein (such as accumulation of insoluble phosphorylated alpha-synuclein in vitro and/or in a mouse model of Parkinson’s disease); and/or (vii) a capabiîity to bind truncated alpha-synuclein in a human brain.
especially wherein such altered functionality is a resuit of the structural change and thus is inséparable from it.
The term “substantially free” of naturally-arising antibodies refers to the complété absence of such naturally-arising antibodies in such préparations, or of the inclusion of a concentration of such naturally-arising antibodies in such préparations that does not materially affect the alpha-synuclein-binding properties of the préparations. An antibody is said to be “isolated” if it has no naturally-arising counterpart or has been separated or purified from components which naturally accompany it.
The term “naturally-arising antibodies,” as it relates to such préparations, refers to antibodies (including naturally-arising autoantibodies) elicited within living humans or other animais, as a natural conséquence to the functioning of their immune Systems.
Thus, the préparations of the présent invention do not exclude, and indeed explicitly encompass, such préparations that contain an anti-alpha-synuclein antibody and a deliberately added additional antibody capable of binding to an epitope that is not possessed by alpha-synuclein. Such préparations particularly include embodiments thereof wherein the préparation exhibits enhanced efficacy in treating synucleinopathies such as Parkinson's disease (including idiopathic and inherited form of Parkinson's disease), Gaucher’s Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease, pure autonomie failure and multiple system atrophy.
In an even further aspect, the invention relates to a pharmaceutical composition comprising:
an anti-alpha-synuclein antibody or antigen-binding fragment thereof, both as defined herein or a préparation, as such term is defined herein, that comprises such an anti-alpha' synuclein antibody or antigen-binding fragment thereof, and a pharmaceuticaüy-acceptable carrier.
The pharmaceuticai compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2013.
Pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable forthe chosen compound ofthe present invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant négative impact on the desired biological properties of the chosen compound or pharmaceutical composition ofthe present invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.)) on epitope binding.
A pharmaceutical composition of the present invention may also include diluents, fillers, salts, buffers, détergents (e.g., a non-ionic detergent, such as Tween-20 orTween80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition. The diluent is selected to not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphatebuffered saline, Ringer’s solutions, dextrose solution, and Hank’s solution. In addition/ the pharmaceutical composition or formulation may also include other carriers, or nontoxic, nontherapeutic, non-immunogenic stabilizers and the like. The. compositions may also include large, slowly metabolized macromolecules, such as proteins, polysaccharides like chitosan, polylactic acids, polyglycolic acids and copolymers (e.g., latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).
The actual dosage levels of the active ingrédients in the pharmaceutîcal compositions of the present invention may be varied so as to obtain an amount of the active ingrédient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level will dépend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excrétion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the âge, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The pharmaceutîcal composition may be administered by any suitable route and mode, including: parentéral, topical, oral or intranasal means for prophylactic and/or therapeutic treatment. In one embodiment, a pharmaceutîcal composition of the present invention is administered parenterally. The phrases parentéral administration and administered parenterally as used herein means modes of administration other than enterai and topical administration, usually by injection, and include epidermal, . intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, épidural and intrasternal injection and infusion. Additional suitable routes of administering a compound of the present invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art. In one embodiment that pharmaceutîcal composition is administered by intravenous or subcutaneous injection or infusion.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a compound of the present invention.
Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutîcal compositions of the present invention include water, saline, phosphate buffered saline, éthanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oiis, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloïdal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.
Pharmaceutica!'y acceptable carriers include stérile aqueous solutions or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the présent invention is contemplated.
Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisülfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) métal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartane acid, phosphoric acid, and the like.
Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds ofthe present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery Systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodégradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the préparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems. J. R. Robinson, ed., Marcel Dekker, Inc., New York 1978.
In one embodiment, the compounds of the présent invention may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parentéral administration include stérile aqueous solutions or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions ofthe présent invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.
Pharmaceutical compositions for injection must typically be stérile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing for instance water, éthanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluîdity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonie agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays antibody absorption, for example, monostearate salts and gelatin. Stérile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingrédients e.g. as enumerated above, as required, followed by sterîlization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle that contains a basic dispersion medium and the required other ingrédients e.g. from those enumerated above. In the case of stérile powders for the préparation of stérile injectable solutions, examples of methods of préparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingrédient plus any additional desired ingrédient from a previously sterile-filtered solution thereof.
Stérile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingrédients enumerated above, as required, followed by sterîlization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle that contains a basic dispersion medium and the required other ingrédients from those enumerated above. In the case of stérile powders for the préparation of stérile injectable solutions, examples of methods of préparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingrédient plus any additional desired ingrédient from a previously sterile-filtered solution thereof.
Dosage regimens in the above methods of treatment and uses described herein are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parentéral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrète units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The spécification for the dosage unit forms of the present invention are dictated by and directly dépendent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inhérent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
The effective dosages and the dosage regimens for the anti aipha-synuclein antibodies dépend on the disease or condition to be treated and may be determined by the persons skilled in the art. On any given day that a dosage is given, the dosage may range from about 0.0001 to about 100 mg/kg, and more usually from about 0.01 to about 5 mg/kg, of the host body weight. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg body weight.
Exemplary dosages thus include: from about 0.1 to about 10 mg/kg/body weight, from about 0.1 to about 5 mg/kg/body weight, from about 0.1 to about 2 mg/kg/body weight, from about 0.1 to about 1 mg/kg/body weight, for instance about 0.15 mg/kg/body weight, about 0,2 mg/kg/body weight, about 0.5 mg/kg/body weight, about 1 mg/kg/body weight, about 1.5 mg/kg/body weight, about 2 mg/kg/body weight, about 5 10 mg/kg/body weight, or about 10 mg/kg/body weight.
A physician having ordinary skill in the art may readily détermine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the anti-alpha-synuclein antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the 15 desired therapeutic effect and gradually increase the dosage until the desired effect is , achieved. In general, a suitable daily dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally dépend upon the factors described above. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous. If desired, the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition as described above.
Labelled antibodies of the invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases or disorders. The invention provides for the détection or diagnosis of a neurodegenerative or cognitive disease or disorder, including but not limited to Parkinson’s disease, idiopathic Parkinson's disease, familiar Parkinson’s
Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson’s disease, pure autonomie failure or multiple system atrophy, comprising: (a) assaying the existence of alpha-synuclein species and fragments in cells or tissue samples of a subject using one or more antibodies that specifically bind to alpha-synuclein; and (b) comparing the level of the antigen with a control level, e.g. levels in normal tissue samples, whereby an increase in thé assayed level of antigen compared to the control level of antigen is indicative of the disease or disorder, or indicative of the severity of the disease or disorder.
Antibodies of the invention can be used to assay alpha-synuclein monomer, oligomers, fibrillary forms or fragments of alpha-synuclein in a biological sample using immunohistochemical methods well-known in the art. Other antibody-based methods useful for detecting protein include immunoassays such as the enzyme linked immunoassay (ELISA), radioimmunoassay (RIA) and mesoscale discovery platform based assays (MSD). Suitable antibody labels may be used in such kits and methods, and labels known in the art include enzyme labels, such as alkaline phosphatase and glucose oxidase; radioisotope labels, such as iodine (125l, 131l), carbon (14C), sulfur (35S), tritium (3H), indium (121ln), and technetium (99mTc); and luminescent labels, such as luminol and luciferase; and fluorescent labels, such as fluorescein and rhodamine.
The presence of labeled anti-alpha-synuclein antibodies or their alpha-synucleinbinding fragments may be detected in vivo for diagnosis purposes. In one embodiment, diagnosis comprises: a) administering to a subject an effective amount of such labeled molécule; b) waiting for a time interval following administration to allow the labeled molécule to concentrate at sites (if any) of Αβ déposition and to allow for unbound labeled molécule to be cleared to background level; c) determining a background level; and d) detecting the labeled molécule in the subject, such that détection of labeled molécule above the background level is indicative that the subject has the disease or disorder, or is indicative of the severity of the disease or disorder. In accordance with . such embodiment, the molécule is labeled with an imaging moiety suitable for détection using a particular imaging System known to those skîlled in the art. Background levels may be determined by various methods known in the art, including comparing the amount of labeled antibody detected to a standard value previously determined for a particular imaging system. Methods and Systems that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as positron émission tomography (PET), magnetic résonance imaging (MRI), and sonography.
In a further aspect, the invention relates to an antibody or antigen-binding fragments thereof, of the invention, for use in medicine.
In a further aspect, the invention relates to an antibody or antigen-binding fragments thereof, of the invention, for use in treating, diagnosing or imaging a synucleinopathy.
In one embodiment, the monocîonal antibody, or antigen-binding fragment thereof, is for use in treating Parkinson’s disease, idiopathic Parkinson’s disease, familiar forms of Parkinson’s Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimeris disease (LBV), Combined Alzheimeris and Parkinson’s disease, pure autonomie failure or multiple system atrophy.
In a further aspect, the invention relates to the use of the antibody, or antigen-binding fragment thereof, of the invention, in the manufacture of a médicament for treating, diagnosing or imaging a synucleinopathy.
In a further aspect, the invention relates to a treating, diagnosing or imaging Parkinson’s disease or other synucleinopathies, comprising administéring an effective dosage of an antibody of the invention, or an antigen-binding fragment thereof.
Preferably, in the uses and methods of those aspects of the invention, the treatment is chronic, and is preferably for at least 2 weeks, such as at least for 1 month, 6, months, 1 year or more.
In a further aspect, the invention provides a kit comprising the antibody, or antigen20 binding fragment thereof, of the invention.
SEQ ID N0:1 | GM37 CDR 1 Heavy Chain |
SEQ ID NO:2 | GM37 CDR 2 Heavy Chain |
SEQ ID N0:3 | GM37 CDR 3 Heavy Chain |
SEQ ID N0:4 | GM37 CDR 1 Light Chain |
SEQ ID NO:5 | GM37 CDR 2 Light Chain |
SEQ ID NO:6 | GM37 CDR 3 Light Chain |
SEQ ID NO:7 | GM37 Heavy Chain Variable Domain |
SEQ ID NO:8 | GM37 Light Chain Variable Domain |
SEQ ID NO:9 | Epitope 112-117 of Human Alpha-Synuciein |
SEQ IDNO:10 | Human Alpha-Synuciein |
SEQ ID NO:11 | A-Syn-AAKK-BAP |
SEQ ID NO:12 | A-Syn-BAAK-BAP |
SEQ ID N0:13 | A-Syn-BBAA-BAP |
SEQ ID N0:14 | A-Syn-BBKK-BAP |
SEQ ID NO:15 | A-Syn-120-140„Del-BAP |
SEQ ID N0:16 | Residues 1-119 of Human Alpha-Synuciein |
SEQ ID NO: 17 | Kappa Light Chain Constant domain |
SEQ ID NO:18 | lgG1 Heavy Chain Constant domain |
SEQ ID NO:19 | GM285 Epitope 112-115 |
SEQ ID NO:20 | GM285 CDR 1 Heavy Chain |
SEQ ID NO:21 | GM285 CDR 2 Heavy Chain |
SEQ ID NO:22 | GM285 CDR 3 Heavy Chain |
SEQ ÎD NO:23 | GM285 CDR 1 Light Chain . |
SEQ ID NO:24 | GM285 CDR 2 Light Chain |
SEQ ID NO:25 | GM285 CDR 3 Light Chain |
SEQ ID NO:26 | GM285 Heavy Chain Variable Domain |
SEQ ID NO:27 | GM285 Light Chain Variable Domain |
SEQ ID NO:28 | GM285 lgG1 Heavy Chain Constant domain |
SEQ ID NO:29 | GM285 Kappa Light Chain Constant domain |
SEQ ID NO:30 | GM37 Variant 1 Heavy Chain Variable Domain |
SEQ ID NO:31 | GM37 Variant 2 Heavy Chain Variable Domain |
SEQ ID NO:32 | GM37 Variant 3 Heavy Chain Variable Domain |
SEQ ID NO:33 | GM37 Variant 1 Heavy Chain CDR 2 |
SEQ ID NO:34 | GM37 Variant 2 Heavy Chain CDR 2 |
SEQ ID NO:35 | GM37 Variant 3 Heavy Chain CDR 2 |
SEQ ID NO:36 | 9E4 Binding Epitope |
SEQ ID NO:37 | Human Beta-Synuclein |
SEQ ID NO:38 | Human Gamma-Synuclein |
SEQ ID NO:39 | Alpha-Synuciein Ortholog for Cynomolgus Monkey |
SEQ ID NO:40 | Alpha-Synuciein Ortholog for Rat |
SEQ ID NO:41 | Alpha-Synuciein Ortholog for Mouse |
SEQ ID NO:42 | 9E4 HC |
SEQ ID NO:43 | 9E4 LC |
Embodiments ofthe invention
As would be apparent from the text and the Exampies the invention further relates to the below embodiments
1. A monoclonal antibody, or antigen-binding fragment thereof capable of specifically binding to an epitope within amino acids 112-117 on alphasynuclein (SEQ ID NO:9 (ILEDMP)).
2. The monoclonal antibody, or antigen-binding fragment thereof, according to Embodiment 1 which competes with the antibody GM37 for binding to said epitope.
3. The monoclonal antibody, or antigen-binding fragment thereof, according to Embodiment 1, which is GM37, GM37 variant 1, GM37 variant 2 or GM37 variant 3.
4. A monoclonal antibody, or antigen-binding fragment thereof capable of specifically binding to an epitope within amino acids 112-115 on alphasynuclein (SEQ ID NO:19 (ILED)).
5. The monoclonal antibody, or antigen-binding fragment thereof, according to Embodiment 1 or 4, which is GM285.
6. The monoclonal antibody, or antigen-binding fragment thereof, according to the previous Embodiments, wherein the antibody comprises or consists of an intact antibody.
7. The monoclonal antibody, or antigen-binding fragment thereof, according to any one of the preceding Embodiments comprising or consisting of an antigenbinding fragment selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)2 fragments) and domain antibodies (e.g. single VH variable domains or VL variable domains).
8. The monoclonal antibody, or antigen-binding fragment thereof, according to any one of the preceding Embodiments wherein the monoclonal antibody is selected from the group consisting of antibodies of subtype lgG1, lgG2, lgG3 and lgG4.
9. The monoclonal antibody, or antigen-binding fragment thereof, according to any one of the preceding Embodiments wherein the antibody or antigen-binding fragment exhibits one or more of the following properties:
(i) a bindng affinity (KD) for alpha-synuclein between 0.5-10 nM such as 1-5 nM or 1-2 nM;
(ii) capability of inhibiting protease truncation of alpha-synuclein fibrils;
(iii) capability of reversing impairment in basal synaptic transmission in F28snca transgenic mice;
(iv) capability of reducing levels of alpha-synuclein in the mouse hippocampus as measured by in vivo microdialysis;
(v) capability, when administered chronically, to restore motor function in a rat model of Parkinson’s disease;
(vi) Capability to prevent seeding of alpha-synuclein (such as accumulation of insoluble phosphorylated aîphasynuclein in vitra and/or in a mouse model of Parkinson’s disease); and/or (vii) Capability to bind truncated alpha-synuclein in a human brain.
10. The monoclonal antibody, or antigen-binding fragment thereof, according to any one of the preceding Embodiments that is human or humanized.
11. A monoclonal antibody or the monoclona! antibody according to Embodiments 1-3 and 6-10, or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising the following CDRs:
a) GFTFSSYAMT (SEQ ID NO:1) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; .
b) AIRS(N/S/Q/H) GDRTD YADSVKG (SEQ ID Nos:2, 33, 34, 35) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; or
c) AKNWAPFDS (SEQ ID NO:3) or an amino acid sequence having with no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.
12.
13.
14.
The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 11 comprising a heavy chain variable domain comprising the
CDRs of SEQ ID NOs:1 and 3 and one of SEQ ID NOs:2 and 33, 34 or 35.
The monoclonal antibody or antigen-binding fragment thereof according to
Embodiment 11 comprising or consisting of a heavy chain variable domain selected from the group consisting of:
a) EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQA PGKGLEWVSA · IRSNGDRTDY ADSVKGRFTI SRDNSQNTLY LQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS (SEQ ID NO:7),
b) EVQLLESGGG LVQTGGSLRL SCAASGETFS SYAMTWVRQA
PGKGLEWVSA IRSSGDRTDY ADSVKGRFTI SRDNSQNTLY LQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS (SEQ ID NO:30),
C) EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQA ' PGKGLEWVSA IRSQGDRTDY ADSVKGRFTI SRDNSQNTLY
LQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS (SEQ ID NO:31), or
d) EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQA PGKGLEWVSA IRSHGDRTDY ADSVKGRFTI SRDNSQNTLY LQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS (SEQ ID NO:32).
A monoclonal antibody or the monoclonal antibody according to Embodiments 1-3 and 6-13, or antigen-binding fragment thereof, comprising a light chain variable domain comprising the following CDRs:
a) ASQSVSSSYLA (SEQ ID NO:4) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;
b) GASSRAT (SEQ ID NO:5) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; or
c) QQYGSSPWT (SEQ ID NO:6) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.
15. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 14 comprising a light chain variable domain comprising the CDRs of SEQ ID NOs:4, 5 and 6.
16. The antibody or antigen-binding fragment thereof according to Embodiment 14 comprising a light chain variable domain comprising or consisting of the amino acid sequence:
EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK (SEQ ID NO:8).
17. The monocîonal antibody or antigen-binding fragment thereof according to Embodiment 14 comprising a light chain comprising or consisting of the amino acid sequence:
. EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK (SEQ ID NO:8).
18. The monoclonal antibody or antigen-binding fragment thereof according Embodiments 1-3 and 6-17 comprising a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:8 and heavy a chain variable domain comprising or consisting of the amino acids given in either SEQ ID No:7, 33, 34 or 35.
19. The monoclonal antibody or antigen-binding fragment thereof according to Embodiments 1-3 and 6-18 comprising a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:8 and heavy a chain variable domain comprising or consisting of the amino acids given in SEQ ID NO:34 having an increased thermal stability, such as an increased stability to prevent aggregate and unfold as shown in Fig. 27, being between 2%-10% more stable at températures above 65°C compared to GM37 wt, 2%-8% more stable at températures above 65°C compared to GM37 wt or 2%-5% more stable at températures above 65°C compared to GM37 wt.
20. A monoclonal antibody or the monoclonal antibody according to Embodiments 1-10, or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising the following CDRs:
a) AASGFTFSRFTMT (SEQ ID NO:20) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;
b) AISGSGGGTS YADSVKG (SEQ ID NO:21) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; or
c) AKNWAPFDY (SEQ ID NO:22) or an amino acid sequence having with no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.
21. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 20 comprising a heavy chain variable domain comprising the CDRs of SEQ ID NOs:20, 21 and 22.
22. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 20 comprising a heavy chain variable domain comprising or consisting of the amino acid sequence
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RFTMTWVRQA
PGKGLEWVSA ISGSGGGTSY ADSVKGRLTV SRDNSKNTLY
LQMNSLRAED TAVYYCAKNW APFDYWGQGT LVTVSS (SEQ ID NO 26).
23. A monoclonal antibody or the monoclonal antibody according to any one of Embodiments 1-10 and 20-22, or antigen-binding fragment thereof, comprising a light chain variable domain comprising the following CDRs:
d) RASQSVSRSYLA (SEQ ID NO:23) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;
e) GASSRAT (SEQ ID NO:24) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; or
f) QQYGSSPWT (SEQ ID NO:25) or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.
24. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 23 comprising a light chain variable domain comprising the CDRs of SEQ ID NOs:23, 24 and 25.
25. The antibody or antigen-binding fragment thereof according to Embodiment 24 comprising a light chain variable domain comprising or consisting of the amino acid sequence of:
EIVLTQSPGT LSLSPGERAT LSCRASQSVS RSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTVSRLE PEDFAVYYCQ QYGSSPWTFG qgtkveik (SEQ ID NO:27).
26. The monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding Embodiments comprising a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:27 and heavy a chain variable domain comprising or consisting of the amino acids given in either SEQ ID NO:26.
27. The monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding Embodiment comprising an Fc région.
28. The monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding Embodiment further comprising a moiety for increasing the in vivo half-life of the agent.
29. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 28 wherein the moiety for increasing the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids and dextran.
30. The monoclona! antibody or antigen-binding fragment thereof according to any one of the preceding Embodiments wherein the antibody polypeptide further comprises a détectable moiety.
31. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 30 wherein the détectable moiety is a fluorescent label, a chemiîuminescent label, a paramagnetic label, a radioisotopic label or an enzyme label.
32. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 30 or 31 wherein the détectable moiety comprises or consists of a radioisotope.
33. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 32 wherein the radioisotope is selected from the group consisting of 99mTc, 111 in, 67Ga, 68Ga, 72As,a9Zr, 123l and 201TI.
34. The monoclonal antibody or antigen-binding fragment thereof according to
Embodiment 30 wherein the détectable moiety comprises or consists of a paramagnetic isotope. .
35. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 34 wherein the paramagnetic isotope is selected from the group consisting of 157Gd, 55Mn, 162Dy, 52Cr and 5SFe.
36. The monoclonal antibody or antigen-binding fragment thereof according to any of Embodiments 30 to 35 wherein the détectable moiety is détectable by an imaging technique such as SPECT, PET, MRI, optical or ultrasound imaging.
37. The monoclonal antibody or antigen-binding fragment thereof according to any of Embodiments 30 to 36 wherein the détectable moiety is joined to the antibody or antigen-binding fragment thereof indirectly, via a linking moiety.
38. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 37 wherein the linking moiety is selected from the group consisting of dérivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), deferoxamine (DFO), dérivatives of diethylenetriaminepentaacetic avid (DTPA), dérivatives of S-2-(4-lsothiocyanatobenzyl)-1,4,7-triazacyclononane1,4,7-triacetic acid (NOTA) and dérivatives of 1,4,8,11-tetraazacyclodocedan1,4,8,11 -tetraacetic acid (TETA).
39. An isolated nucleic acid molécule encoding an antibody or antigen-binding fragment thereof according to any one of the preceding Embodiments or a component polypeptide chain thereof.
40. A nucleic acid molécule according to Embodiment 39 wherein the molécule is a cDNA molécule.
41. A nucleic acid molécule according to Embodiment 30 or 31 encoding an antibody heavy chain or variable domain thereof.
42. A nucleic acid molécule according to any one of Embodiments 39 to 41 encoding an antibody light chain or variable domain thereof.
43. A vector comprising a nucleic acid molécule according to any one of Embodiments 39 to 42.
44. A recombinant host cell comprising a nucleic acid molécule according to any one of Embodiments 39 to 42 or a vector according to Embodiment 43.
45. A method for producing an antibody or antigen-binding fragment according to any one of the Embodiments 1 to 27, the method comprising culturing a host cell as defined in Embodiment 44 under conditions which permit expression of the encoded antibody or antigen-binding fragment thereof.
46. A pharmaceutical composition comprising the monoclonal antibody or antigenbinding fragment according to any one of Embodiments 1 to 35 and a pharmaceutical acceptable carrier.
47. The monoclonal antibody or antigen-binding fragment thereof of Embodiments 1-35 for use in medicine.
48. The monoclonal antibody or antigen-binding fragment thereof of Embodiments 1-35 for use in treating, diagnosing or imaging a synucleinopathy.
49. The monoclonal antibody or antigen-binding fragment thereof according to Embodiment 48 for use in treating Parkinsoris disease (including idiopathic and inherited forms of Parkinson’s disease), Gaucher’s Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease, pure autonomie failure and multiple System atrophy.
50. Use of a monoclonal antibody or antigen-binding fragment thereof of Embodiments 1-35 in the manufacturing of a médicament for treating, diagnosing or imaging a synucleinopathy.
51. The use of a monoclonal antibody or antigen-binding fragment thereof according to Embodiment 50 in the manufacturing of a médicament for treating Parkinson’s disease (including idiopathic and inherited forms of Parkinson’s disease Parkinson’s disease), Gaucher’s Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease, pure autonomie failure and multiple System atrophy.
52. A method of treating, diagnosing or imaging a synucleinopathy in a subject, said method comprising administering the pharmaceutical composition of Embodiment 46 to said subject in an effective amount.
53. The antibody, or antigen-binding fragment thereof, for use according to Embodiment 48, or the use according to Embodiment 50, or the method according to Embodiment 52 for treating Parkinson’s disease (including idiopathic and inherited forms of Parkinson’s disease Parkinsoris disease), Gaucher’s Disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease, pure autonomie failure and multiple System atrophy.
54. The antibody, or antigen-binding fragment thereof, for use; or the use; or the method according to Embodiment 52 or 53, wherein the treatment is chronic
55. The antibody, or antigen-binding fragment thereof, for use; or the use; or the method according to Embodiment 52, wherein the chronic treatment is for at least 2 weeks, such as at ieast for 1 month, 6, months, 1 year or more.
56. The antibody, or antigen-binding fragment thereof, for use; or the use; or the method according to any one of Embodiments 47 to 55, wherein the subject is human.
57. A kit comprising the antibody or antigen-binding fragment thereof according to Embodiments 1-35
58. The kit according to Embodiment 57 for use in medicine
59. The monoclonal antibody or antigen-binding fragment thereof of Embodiments 30-35 for use in detecting or measuring the presence or amount of said alphasynuclein in the brain or a body fluid of a subject.
60. The monoclonal antibody or antigen-binding fragment thereof of Embodiments 59, wherein said détection or measurement comprises in vivo imaging of said anti-synuciein antibody bound to said alpha-synuclein.
61. The monoclonal antibody or antigen-binding fragment thereof of Embodiments 30-35 wherein said détection or measurement comprises ex vivo imaging of said anti-synuclein antibody bound to said alpha-synuclein.
EXAMPLES
Example 1: Antibody Screening
1. Immunogen and ligand production
The following proteins were acquired or produced for use as immunogens shown in Fig 5 1. The mice were immunized with three immunogens: full length recombinant human alpha-synuclein fibrils; human aipha-synuclein recombinant protein containing amino acids 1-60 (Rpeptide, Bogart, Georgia) and human alpha-synuclein recombinant protein containing amino acids 1-119. To make the fibrils from the full length the alphasynuclein a lyophilized product from Rpeptide, Bogart, Georgia (Catalog number S10 1001-2) was used. This was dissolved in 20 mM tris and 300 mM NaCl buffer at concentration of 1 mg/ml protein. To make the fibrils the protein solution was incubated 170 μΙ aliquots in 96 well plate with a 70 pm diameter ceramic bead in each well at 200 rpm in Vortemp 56 shaker incubator (Labnet International, Edison, NJ, USA), at 37°C for 7 days, and the formation of fibrils was followed by adding thioflavin T and measuring fluorescence in one of the wells. The recombinant alpha-synuclein containing amino acids 1-60 was dissolved in water to give a concentration of 1 mg/ml.
The recombinant alpha-synuclein containing amino acids 1-119 was made using the following construct: A synthetic gene coding for a 6 amino acid Histidine tag, followed by factor Xa cleavage site and sequence coding for human alpha-synuclein amino 20 acids 1-119:
. MAHHHHHHIE GRMDVFMKGL SKAKEGVVAA AEKTKQGVAE AAGKTKEGVL
YVGSKTKEGV VHGVATVAEK TKEQVTNVGG AWTGVTAVA QKTVEGAGSI AAATGFVKKD QLGKNEEGAP QEGILEDMPV D (SEQ ID NO:16) was synthezised by Genscript and cloned into Ndel-Xhol site in pET24a(+) expression 25 vector (Novagen).
The expression vector was transformed into E. coli BL21 and a single colony picked for expression using the ovemight express autoinduction system from Novagen (User protocol TB383 rev. H1005). The scale was 500 ml of final culture volume.
. Cells were harvested by centrifugation 10 min at 6000g and subsequently lyzed 30 using BugBuster protein extraction Reagent (User protocol TB245 Rev. 0304). After lysis the sample was cleared by centrifugation and the supernatant used for further purification.
The His-tagged protein was purified on a 5ml HisTrap column (GE healthcare) equilibrated in 20 mM Sodium phosphate pH7.5, 1 M NaCl (A-buffer). After sample application and wash using A-buffer the protein was eluted in a gradient to 0.25 M imidazole in A-buffer over 20 column volumes. Fractions of 5 ml were collected and analyzed by SDS-PAGE. Fractions with the protein of interest was pooled, concentrated and applied to an S200 (26/60) size exclusion column (GE healthcare) in 10 mM tris pH 7.4, 300 mM NaCl Again fractions were pooled according to presence in SDS-PAGE of a band with expected size.
To remove the N-terminal tag, the purified his-tagged alpha-synuclein 1-119 was incubated with factor Xa in a 1:50 ration using the Novagen kit (69037-3FRX). After overnight incubation, the factor Xa was removed batchwise using Xarrest agarose. The cleaved alpha-synuclein 1-119 was finally purified by permissive HisTrap chromatography as described above. From the flow through the purified alphasynuclein 1-119 was obtained and concentrated to -400 pg/ml using centricon concentration devises.
Alpha-synuclein (Rpeptide) was rehydrated in PBS at 2 mg/ml and peroxynitirite (100pL/mg protein) was added dropwise while mixing. The nitrosylated alphasynuclein was then dialyzed in 5L PBS and stored at -20°C.
Dopamine was used to oxidize alpha-synuclein. Equal volumes of a 200uM solution of Dopamine-HCL (Sigma P5244) prepared in 10mM PBS, pH7.4 and a 28μΜ solution of alpha-synuclein (Rpeptide) in 10mM PBS, pH7.4were combined. The resulting 14 uM alpha-synucIein/100 uM Dopamine were incubated at 37°C O/N (over night). The oxidized alpha-synuclein was then dialyzed in PBS and stored at 20°C.
Different native and chimeric versions of synuclein proteins were produced in order to screen a diverse library of anti-alpha-synuclein antibodies. Screening çonstructs included the following: human, mouse, rat and cynomolgus monkey alphasynuclein, human Beta-synuclein, Human Gamma-synuclein (fig 21 and 22) and lastly an alpha-synuclein dérivative that lacked residues 120-140 of alphasynuclein. In addition, a séries of 4 shuffle constructs: A-Syn-AAKK-BAP, A-SynBAAK-BAP, A-Syn-BBAA-BAP, A-Syn-BBKK-BAP (SEQ ID Nos: 11-14) were produced. These constructs contained linear stretches of human alpha-synuclein (A), human Beta-synuclein (B) and chicken alpha-synuclein (K). Gene were cloned containing a Biotin Acceptor Peptide (BAP) tag fused to the C-terminus ofthe ligands in orderto facilitate site spécifie biotinylation of each ofthe ligands. The bioytinylation allowed for attachment of the ligands to beads used in the soluble ELISA format. Mammalian expression vectors were constructed carrying the different alpha-synuclein BAP tag fusion constructs (ASynBAP). The ligands were expressed in HEK 293 cells using transierit transfection (Genmab A/S).
2. Immunization
Antibodies HuMab-Synuclein were derived from the immunizations of HuMAb mouse strain HCo17-BALB/c and HCo12-BALB/c mice, double knock out for the mouse immunoglobulin (Ig) heavy and mouse kappa light chain, which prevents the expression of antibodies that are completely murine (human monoclonal antibody; Medarex Inc., San José, CA, USA). The various mouse strains were made transgenic by the insertion of human Ig heavy and human Ig kappa light chain loci and differ in the number of human VH (variable domain of heavy chain) and VL (variable domain of light chain) genes.
Mice were immunized alternating intraperitoneally (IP) with 20 pg antigens and subcutaneously (SC, at the tailbase) with the same immunogen, with an interval of 14 days. A maximum of eight immunizations were performed, 4 IP and 4 SC.
The first immunization was performed with alpha-synuclein immunogens in complété Freund’s adjuvant (CFA; Difco Laboratories, Detroit, Ml, USA), the following immunizations in incomplète Freund’s adjuvant (IFA). When sérum titers were found to be sufficient (dilution of sérum of 1/50 or lower found positive in antigen spécifie screening assay as described in herein above on at least two sequential, biweekly, screening events), mice were additionally boosted twice intravenously (IV) with 10 pg alpha-synuclein immunogen protein in 100 pL PBS, four and three days before fusion.
The immunization protocols are shown in Fig 1.
Antibody 37 came from an immunization protocol where human full length aSynuclein-fibrils was used, alternating with alpha-synuclein C- terminally truncated forms with amino acids 1-60 and 1-119.
Antibody 285 came from an immunization protocol where Human a-Synucleinmonomer 1-140 was used for the first 4 immunizations. If there was no titer, the immunization was continued with fibrils (ip/sc), otherwise it was continued with monomer.
3. HuMab hybridoma génération
HuMAb mice with sufficient antigen-specific titer development as defined above were sacrificed and the spleen and lymph nodes flanking the abdominal aorta and caval vein were collected. Fusion of splénocytes and lymph node cells with a mouse myeloma cell line was done by electrofusion using a CEEF 50 Electrofusion System (Cyto Puise Sciences, Gien Burnie, MD, USA), essentially according to the manufacturées instructions. Fused cells were seeded in fusion medium containing 10% Fêta! Clone I Bovine sérum (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5 U/mL penicillin, 0.5 U/mL streptomycin (Cambrex), 50 μΜ 2-mercaptoethanol (Invitrogen), 600 ng/mL interleukin 6 (IL-6) (Strathmann), 1 x HAT (Sigma) and 0.5 mg/mL kanamycin (Invitrogen) in HyQ mADCF-Mab (Perbio). After ten days, supernatant was harvested and cells were refreshed with harvest medium, containing 10 % Fêtai Clone I Bovine sérum, 0.5 U/mL penicillin, 0.5 U/mL streptomycin, 600 ng/mL IL-6 and 1 x proHT (Cambrex) in HyQ mADCF-Mab. Supematants of the hybridoma cultures were screened by primary screening assays. Supematants were characterized for binding to eight different ligands. These included 4 orthologs: human, mouse, rat and cynomologus monkey, human alpha-synuclein Beta-synuclein and human Gamma-synuclein (SEQ ID NOs 37-41) and lastly they were tested for their ability to bind to a human alpha-synuclein dérivative that lacked residues 120-140 of alpha-synuclein.
The screening of anti-alpha-synuclein antibodies was performed using a high throughput suspension ELISA format using automated liquid handling Systems (Genmab A/S). The reading of the plates was performed by two Systems, the FMAT 8200 from Applied Biosystems was used to read 384 well plates and the
ImageXpress Vélos Cyto meter from Molecular Devices was used to reàd the 1536 well plates.
In the primary screen clones were characterized by their ability to bind 8 different ligands. These included a sériés of 4 shuffle constructs: A-Syn-AAKK-BAP, A-SynBAAK-BAP, A-Syn-BBAA-BAP, A-Syn-BBKK-BAP (SEQ ID NOs:11-14), alphasynuclein 120-140 deletion-BAP, nifrated human alpha-synuclein-ΒΑΡ and oxidized human alpha-synuclein-BAP.
In short, the sera or supernatant potentially containing alpha-synuclein spécifie antibodies were added to the beads to allow binding to alpha-Synuclein and/or alpha-synuciein derived constructs. The binding of the anti-alpha-synuclein antibodies is detected using a fluorescent conjugate, DyLight649 conjugated goat antihuman IgG, Fc spécifie. Two known mouse anti-alpha-synuclein antibodies, LB509 and Syn211, were included in screenings as positive Controls. To ensure spécifie détection of alpha-synuclein antibodies, an anti-alpha-synuclein sera pool is used as a négative control in the 384 well format titer screening while human ChromPure IgG is used in the 1536 well format 8-bead based assay.
Hybridoma cells from the best primary wells were seeded in semisolid medium made from 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2x complété medium (Hyclone, Wattham, USA). For each primary well, a well of a Genetix black 6-well plate was seeded. From each well, 25 sub clones were picked, using the CionePix system (Genetix). The sub clones were picked in harvest medium. After seven days, the supernatants of the sub clones were screened again for Synuclein-specific human IgG binding and the human IgG concentration was measured using Octet (Fortebio, Menlo Park, USA). From each primary well, the best sub clone was selected and expanded in expansion medium containing only 600 ng/mL IL-6, 0.5 U/mL penicillin, 0.5 U/mL streptomycin and 1 x proHT. The sub clones were expanded from one 96-well plate well to one 24-well plate well to four 24-well plate wells to six 6-well plate wells. Clones derived by this process were. designated as primary clones (PC).
Additional antibody binding studies were performed using Octet 384RED (Fortebio, Menlo Park, USA). HuMab antibody solutions of 2 pg/ml were made by dilution in sample diluent (ForteBio, art. No. 18-5028). Amine reactive sensors (ForteBio, art.no. 18-0008) were used for immobilization of HuMabs. Prior to coupling to amine reactive sensors, HuMabs were diluted in MES pH 6.0 buffer (18-5027). Coupling was performed at 30°C and 1000 rpm as follows: Amine reactive sensors were pre-wet in PBS and subsequently activated with EDC/NHS(ForteBio. Art.no. 18-1033/18-1034) activation solution (according to manufacturées instruction) for 300 seconds. Activated sensors were immobilized with HuMabs during 600 seconds.
The binding of 37 and 285 in Octet to recombinant human, cynomolgus and mouse alpha-synuclein, and lack of binding to human beta or gamma-synuclein is shown in Figure 2
4. Sequence analysis ofthe Synuclein-specific HuMab variable domains and cloning in expression vectors
Total RNA was prepared from 0.2 to 5x106 hybridoma cells and 5’-RAŒComplementary DNA (cDNA) was prepared from 100 ng total RNAS using the SMART RACE cDNA Amplification kit (Clontech), according to the manufacturais instructions. VH and VL coding ragions were amplified by PCR and cloned directiy, in frame, in the p33G1f and p33Kappa expression vectors (containing the human lgG1/kappa constant domain encoding sequences), by ligation independent cloning (Aslanidis, C. and P.J. de Jong, Nucleic Acids Res 1990; 18(20): 6069-74). For each antibody, 16 VL clones and 16 VH clones were sequenced. Clones with a correct Open Reading Frame (ORF) were selected for further study and expression. Vectors of ail combinations of heavy chains and light chains were transiently coexpressed in FreestyleTM 293-F cells using 293fectin.
In the case of GM37 sequencing ofthe VH région identified an extra cysteine in the CDR3 domain at position 106. In orderto eliminate the possibility of misfolding and potential loss of antibody activity due to disuîfide bond formation the cysteine was mutated to serine at position 106.
Comparator antibody 9E4 was generated based on the VH and VL sequence derived from hybridoma PTA-8221 (US patent 20080175838) (SEQ ID NO 42 and 43)
5. Expression/Purification of Antibodies
Antibodies were produced by transfection in HEK293 6E cells using the pTT5 vectors and PEIpro as a transient transfection agent (National Research Council of Canada). In short, The heavy and light chains were transfected into HEK293 cells using PEIpro (VWR), and cells were supplemented with TN1 (Sigma) 24 hours after transfection. Cells were grown until the viability approached 50%. and yield of antibody measured by easy IgG titre (Thermo). Culture supematant was filtered over 0.2 pm dead-end filters, loaded on 5 ml_ Protein A columns (rProtein A FF, Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3. The eluate was immediately neutralized with 2M Tris-HCI, pH 9 and dialyzed to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun), O/N. After dialysis, samples were sterile-filtered over 0.2 pm dead-end filters. Purity was determined by SDS-PAGE and concentration was measured by nephelometry and absorbance at 280 nm. Purified antibodies were aliquoted and stored at -80°C.
Example 2: Antibody Characterization Using Surface Plasmon
Résonance
Real time binding of the antibodies to alpha-synuclein was measured using a BIAcore® 3000. A capture surface was prepared by amine-coupling a polyclonal rabbit Anti-Mouse antibody (part of Mouse Antibody Capture Kit, GE Healthcare, Cat. no: BR-1008-38) in first flow cell (Fc1) and second flow cell (Fc2) of a CM5 chip (BIAcore®). The mouse antibody was captured in Fc2 at the concentration required to achieve a ligand level of around 500RU. The baseline was allowed to stabilize for 10min before injecting analyte (ASynBAP) in Fc1-2 at 30pl/min. ASynBAP was run at 100-3200nM and 25-3200RU, respectively. The highest concentration in each titration sériés was run in duplicate. The surface was regenerated with 10mM Glycine-HCI, pH 1.7 (30sec inject) to remove captured mouse antibody and analyte in the end of each cycle. HBS-EP (GE Healthcare, Cat. No: BR-1001-88) was used as running buffer and sample diluent in ail experiments and the assay was run at 25°C. AH samples were kept at 4°C before acquisition. .
The response recorded in Fc1, where capture antibody had been immobilized but no Alpha-Synuclein antibody captured, was subtracted from the response in Fc2. A
1:1 or 2:1 binding algorithm was fit to the dataset using BIAevaluation software version 4.1.1. Results can be seen in Figs. 3, 4 and 5 showing binding of antibody 37, 285 and 9E4 to human alpha-synuclein.
Example 3: Epitope Mapping
Epitope mapping ofthe antibodies to alpha-synuclein was done with arrays of overlapping linear peptides at Pepscan (Pepscan Zuidersluisweg 2 8243 RC Lelystad, The Netherlands). The binding of antibody to each of the synthesized 20 mer peptides was tested in a Pepscan based ELISA. The linear peptide array covering the entire coding sequence of alpha-synuclein, as well as ail peptides with 10 oxidized methionines or nitrosylated tyrosines, were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of an antibody peroxidase conjugale (SBA, cat. nr.
2010-05) for one hour at 25°C. After washing, the peroxidase substrate 2,2’-azinodi-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μΙ/ml of 3 percent H2O2 were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD) - caméra and an image processing System. For data processing the values were obtained from the CCD caméra range from 0 to 3000 mAU, similar to a standard 96-well plate ELISAreader. The results were quantified and stored into the Peplab database.
Occasionally a well contains an air-bubble resulting in a false-positive value, the cards are manually inspected and any values caused by an air-bubble are scored as 0. The binding data of antibody 37 and 285 to peptides containing the sequence ILEDMP or ILED respectively can be seen in Figure 7.
Example 4: Immunoprécipitation of alpha-synuclein from human brain homogenates of cingulate cortex from patients with dementia with Lewy bodies
The ability ofthe antibodies to bind to and pull down alpha-synuclein from crude homogenates of cingulate cortex from human DLB or healthy control (marked with *) was analyzed by immunoprécipitation. Frozen sample from human cingulate 30 cortex (obtained throughTissue Solutions Ltd, Scotland) was dissected in cryostat, and 100mg sample was added to 1600μ! cellytic M cell lysis reagent (Sigma C2978) containing protease inhibitors and phosphatase inhibitors (Roche). Brain tissue was homogenized until the sample is dissolved completely using Precellys bead homogenizer (Bertin technologies, France) 4x30 sec at 5000 rpm. The solution was centrifuged at 3000 x g and the supernatant was used as the crude homogenate for immunoprécipitation.
For immunoprécipitation 10 pg of antibody was mixed with magnetic Dynabeads protein G beads using manufacturer s instructions (Life Technologies, Paisley, UK). The crude brain homogenate was diluted 30 fold.in lysis buffer (Sigma). Antibody coupled dynabeads were mixed with 500 ul of diluted homogenate and incubated 90 minutes at room température under continuous mixing in a rotator. After incubation the beads were washed in washing buffer and the bound antigens were eluted using the non-denaturing elution buffer according to manufacturées instructions (Dynabeads G protocol, Life Technologies, Paisley UK). The yield of the immunoprécipitation was visualized by Western blotting with détection mouse monoclonal anti-human alpha-synuclein antibody, (4B12, Thermo Scientific). The patterns of bands representing different molecuiar weight forms of alpha-synuclein being pulled down differ between the 37, 37 variant 2 and 285 antibodies and the comparator antibody 9E4 in that the 37. 37v2 and 285 antibody can immunoprecipitate the major alpha-synuclein species, the full length alphasynuclein (FL asyn 1-140) and the C-terminal terminal truncated species (1-135 and 1-119/122), while antibody 9E4 cannot immunoprecipitate the truncated species 1-119/122. Figure 9.
Example 5: Inhibition of protease truncation of alpha-synuclein fibrils by antibodies in cell culture .
Recombinant alpha-synuclein monomers and fibrils can be taken up by primary neurons in culture. As shown schematically in Figure 10, after uptake of the alphasynuclein in neurons, it can be procèssed by intracellular proteases, such as Calpain I, with the major protease sensitive site at amino acid 119/122. To investigate truncation of alpha-synuclein by proteases mouse primary cortical neurons were prepared as described in Elvang et al. 2009 (Elvang et al.. J Neurochem. 2009; 110(5):1377-87) and treated with cytarabine on DIV3 (3 days in vitro) to inhibit astrocyte growth. On DIV4 (4 days in vitro), the neurons were treated with sonicated (5 min at 50 % power in cup horn sonicator) pre-formed alphasynuclein fibrils (PFFs) at an end concentration of 0.7 μΜ alone or together with antibodies in the indicated concentrations. After 24 hours of incubation, the media was harvested and the cells were lysed. Western Blots was run on both media and cell lysate using the 4B12 antibody (Fig 11 A) (Pierce MA1-90346) and a secondary anti-mouse antibody. After probing with 4B12 + anti-mouse, the blots were stripped and reprobed with an anti-humàn-IgG antibody. On blot with 4B12, it can be seen that in the media from cells treated with PFFs only, there were strong bands at 14 and 12 kDa, where 14 kDa represents the fuîi4ength alpha-synuclein (FL-asyn) and 12 kDa represents the C-terminally truncated fragment 1-119/122 (CT-asyn). In addition to that, there were higher molecular weight bands, most likely representing SDS-resistant oligomeric species. Co-treatment with the isotype control antibody B12 did not change this pattern of proteolysis or uptake.
In the media from cells treated with fibrils together with 37 there was mainly full length alpha-synuclein (14 kD) and only small amounts of the terminally truncated band (12 kD). In cell lysate inform cells treated with fibrils together with 37 there was fut! length alpha-synuclein only, indicating that 37 prevent cleavage of FLalpha-synuclein. Furthermore the total amount of FL-alpha-synuclein is reduced in relation to cells treated with PFFs only or B12 control antibody. It has been shown by several groups (Games et al, Am J of Pathol, Vol. 182, No. 3, March, 2013; Ritchie et al, Health, Vol. 4, Spécial Issue, 1167-1177, 2012; Mishizen-Eberz, Biochemistry, 2005, 44, 7818-7829; Dufty et al, Am J of Pathol, Vol. 170, No. 5, May 2007) that alpha-synuclein can be cleaved by Calpain-1. The cleavage site of Calpain-1 for fibrillized alpha-synuclein has been found to be in the région 114-122 (Mishizen-Eberz, J of Neurochem, 86, 836-847, 2003). In vivo in transgenic animais and human brain 1-119/122 seems to be the main cleavage product - in alpha-synuclein, the cleavage is likely after asparte 119 or asparagine 122, which is deamidated to aspartate and is cleaved by Calpain or another protease with similar cleavage specificity. These results indicate that antibody 37 is able to inhibit Ctermina! truncation of alpha-synuclein. The epitope of antibody 37 overlaps with the enzyme Calpain-1 binding site, so binding of 37 to alpha-synuclein could directly inhibit binding and cleavage mediated by Calpain-1 (Fig 10 and 11).
The epitope of 285 overlaps with the epitope of 37 and it would also be expected to inhibit the protease cleavage. The amino acid sequence of 37v2 only differs from 37 at one amino acid in CDR and has similar binding as 37, so it would also be expected to inhibit protease cleavage in similar mannerto 37. To investigate if the effect of the antibodies were dose-dependent, a 24-hour experiment with coaddition of PFFs and antibodies to primary cortical neurons was set up. The concentration of PFFs were stable (10 pg/ml), whereas the concentrations of control antibody B12, and antibodies 37, 37v2 and 285 that was tested was 10, 5, 1 and 0,1 ug/ml. Alpha-synuclein on Western Blots were detected with 1904/4B12 antibody (Aocam), which has an epitope in the région 103-108 and therefore binds to both the FL and the C-terminally truncated alpha-synuclein (Fig. 11 B). As can be seen from Figure 11 B, both GM37, 37v2 and GM285 hâve a dose dépendent inhibition of protease cleavage, with almost complété inhibition of the cleavage at high concentration of the antibody.
Example 6: Antîbody-Mediated Inhibition Of Seeding Of Alpha-Synuclein
Aggregation In Cell Culture
Several studies hâve shown that exogenous addition of recombinant alpha synuclein fibniîar aggregates can enter cells and recruit endogenous alpha• synuclein and induce alpha-synuclein aggregation and phosphorylation in vitro and in vivo, which resernble LB. (Volpiœlli-Daley et al. 2011, Luk et al. 2012a, Luk et al. · 2012b, Recasens et al. 2013, Peelaerts et al. 2015).To study seeding of endogenous mouse alpha-synuclein by recombinant alpha-synuclein seeds, mouse primary cortical neurons prepared as above are plated in 96 well plates (15,000 cells per well). On day 5 in vitro culture (DIV), 50% of media is changed and supplemented with cytosine arabinoside (final conc. of 1uM). On DIV 6, half of the media is changed with glia conditioned media along with alpha synuclein fibrillary material, either crude fibril seeds or pure seeds. The crude fibril seeds are made from recombinant monomeric human alpha-synuclein, which was isolated from bacteria and the monomers were filtered through an Amîcon Ultra 100.000 eut off filter (Millipore cat. No UFC510096) and adjusted to concentration of 1mg/ml in PBS, pH 7.4. To make fibril crude seeds, the monomer solution was incubated in thermomixer at 37C with continuous mixing (800rpm) until plateu is reached (evaluated by daily measures with Thioflavin S). To minimize évaporation a drop of minéral oil was added to cover the solution. The total time for incubation was 5-7 days, The pure seeds are made from crude fibril seeds that are centrifuged to purify them and the aggregated pellet is resuspended in fresh PBS and sonicated. The antibodies are added once on DIV 6 along with alpha-synuclein crude seeds. Half of the media in the primary neurôns is replaced with glia condîtioned media every week to maintain them up to DIV21. The neurons are fixed and stained for Phospho-synuclein using a rabbit antibody spécifie for phosphorylation of alphasynuclein at amino acid S129 (abcam 51253), followed by a fluorescently labelled anti-rabbit antibody, fluorescence is quantified using automated fluorescent microscopy, Cellomics Arrayscan. Nuclei were detected in one channel and defined the number of valid cells. Phosphorylated alpha-synuclein spots werè detected in another channel in a pre-defined ring-formed area surrounding the nucléus, thus representing the cytoplasm of the cells. The average number of spots per cell was calculated. Example of cell staining is shown in Fig 12A. Phosphorylated alpha synuclein spots are not seen in untreated neurons. Neurons incubated with crude or pure seeds (1 -1 Ong per well) induce phosphorylation of alpha synuclein (Fig. 12A). In neurites, phosphorylated synuclein appears as spots or punctate and some ofthe phospho-synuclein in the neurites appear elongated.
For fractionation studies cells were harvested in phosphate buffered saline solution (PBS) and centrifuged. Pellet was resuspended in 1% triton buffer with protease inhibitors. Samples were kept on ice for 15 min. followed by sonication. The samples were centrifuged at 100,000x g for 30 min. at 4C. The supernatant is collected and labelled as soluble fraction. The pellet was washed once in triton buffer and re-suspended in 1% SDS buffer followed by sonication. Samples were centrifuged again at 100,000xg for 30 min. The supernatant is collected as insoluble fraction. The protein concentrations were measured and samples were run on 4-12% SDS_PAGE gel, blotted on membranes and alpha synuclein and phosphorylated alpha synuclein (S129P) are detected by 4B12/1904 antibody (Thermo scientific: MA1-90346-human synuclein), S129P-asyn antibody (abcam 51253) and mouse synuclein antibody (cell signalling- D37A6), respectively.
Figure 12 B shows the Western blot of the soluble and insoluble fraction from the primary neurons with and without crude seeds. As can be seen from Figure 12B the addition ofthe seeds lead to accumulation of endogenous mouse alpha-synuclein and p-S129-alpha-synuclein and multimers of phosphorylated mouse alphasynuclein in the insoluble fraction of the cells.
To test if antibodies can inhibit seeding, alpha synuclein synuclein seeds were used at conc. of 6.6nM (10ng/ well). Différent concentration of antibody and alphasynuclein seeds were added together on DIV 6, to make a dose response (starting from highest antibody conc. at 133nM down to 133 pM). The neurons were again fixed and stained for Phospho-synuclein (abcam 51253) and fluorescence from cells was quantified using automated fluorescent microscopy, Cellomics arrayscan. The spots/puncta per cell were counted in Cellomics arrayscan. As can be seen from Figure 12C, both antibody 37, 37v2 and antibody 285 reduced alpha synuclein phosphorylation in neurons in a dose dépendent manner with similar maximal inhibition for 37, 37v2 and 285 (around 70-75%) and EC50 around 5 nM.
Fractionation of the cellular proteins to soluble and insoluble fraction after treatment with antibody at the highest concentration (133 nM) shows that both antibodies 37, 37v2 and 285 rnhibited the truncation of the recombinant crude seeds and accumulation of C terminally truncated fragment (CT a-syn), and reduced the accumulation of phosphorylated endogenous mouse alpha-synuclein and agsresated forms of mouse alpha-synuclein in the insoluble fraction, as seen in Figure 12D.
Exemple 7. Acute Electrophysiological Effects Of Alpha-Synuclein Antibodies in vivo
High expression levels of human alpha-synuclein are present in the hippocampus of F28-snca transgenic mice, a model overexpressing wildtype alpha-synuclein under the control of the mouse alpha-synuclein promotor (Westerlund M, et al. Mol Cell Neurosci. 2008 Dec; 39 (4):586-91). Assessment of synaptic transmission and plasticity in the CA1 area of the hippocampus in 4 to 6 months old male F28-snca transgenic and age-matched control mice was performed by in vivo electrophysiology. The data shows that basal synaptic transmission is signifïcantly impaired in F28-snca transgenic compared to age-matched control mice (Fig. 13).
F28-snca transgenic and age-matched control male mice (CRO breeding, Taconic Europe A/S) aged 4 to 6 months were single-housed in controlled température (22 ± 1.5°C) and humidity conditions (55-65%) and kept in a 12:12 hour light/dark cycle (lights on at 06:00h). Food and water were available ad libitum.
Animais were anesthetized with an intraperitoneal (i.p.) injection of urethane (1.2 g/kg). Mice were then mounted in a stereotaxîc frame, their température adjusted to 37.5°C via a heating pad, and the skull was exposed. A platinum wire was placed in the frontal bone to act as a reference, and an additional hole was drilled for insertion ofthe recording and stimulating électrodes in the hippocampus, at the following coordinates according to the atlas of Paxinos and Franklin (Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordnates 4th Edition, 2001): recording, 1.5-1.7 mm posteriorto Bregma, 1.0-1.2 mm latéral to the midline, 1.41 7 mm below the surface ofthe brain; stimulation, 1.8-2 0 mm posteriorto Bregma, 1.5-1.7 mm latéral to the midline, 1.5-1.7 mm below the surface ofthe brain.
Animais were left in the stereotaxic frame through the whole duration of the recordings and their level of anesthésia was regularly checked.
Field potentials (fEPSP) were evoked in the CA1 by electrical stimulation of the Schaffer collateral every 30 s, and the depth of the recording electrode was adjusted until a négative fEPSP was recorded in response to a unipolar square puise. The slope ofthe evoked fEPSP was measured between 30 and 70% ofthe maximum amplitude ofthe fEPSP.
Once an optimal fEPSP was induced, basal synaptic transmission was assessed by the relationship between stimulation intensity and slope of the evoked fEPSP (input-output relationship). The different intensifies of stimulation were 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μΑ, and were applied successively in increasing order, with 2 to 3 repeats for each intensity. Basal synaptic transmission was found to be significantly impaired in F28-snca transgenic compared to age-matched control mice.
The identified impairments in basal synaptic transmission in F28-snca transgenic mice were used to test the GM37, GM285 and comparator h9E4 for their ability to block the alpha synuclein mediated effect.
Recordings were performed in ail experiments 3 to 6 h following administration of a single dose of antibody at a dose of 15 mg/kg (i.p ). Basal synaptic transmission were recorded in both hippocampi in each animal when possible, and recorded as individual experiments.
Acute treatment with h9E4 induced a significant reversai of the impairment in basal synaptic transmission in F28-snca transgenic mice (Tg-snca + h9E4 vs. Tg-snca + PBS, p=0.002, Fig. 14). However, the reversai by h9E4 was only partial, as indicated by a significantly différentiation to basal synaptic transmission in littermates treated with PBS (p=0.007).
Acute treatment with GM37 induced a significant reversai of the impairment in basal synaptic transmission in F28-snca transgenic mice (Tg-snca + GM37 vs. Tg-snca + PBS, p=0.004, Fig. 15). Basal synaptic transmission in GM37-treated transgenic mice was not significantly different from basal synaptic transmission in PBS-treated littermates, indicating a full reversai of the impairment (Fig 15).
GM285 also induced a significant reversai of the impairment in basal synaptic transmission in F28-snca transgenic mice (Fig 16). Basal synaptic transmission in GM2854reated transgenic mice was not significantly different from basal synaptic transmission in PBS-treated littermates, indicating a full reversai of the impairment.
Example 8. Microdialysis to assess human alpha-synuclein in the brain of awake freely moving animais
The push-puil microdialysis method was used to assess the levels of human alphasynuclein in brain interstitial fluid (ISF). Mice were single-housed in controlled température (22 ± 1.5°C) and humidity conditions (55-65%) and kept on a 12:12 hour light/dark cycle (lights on at 06:00h). Food and water were available ad libitum. The current study was performed in the hippocampus of F28-snca transgenic mice (50-54 weeks old). To enable microdialysis in the hippocampus, mice were anaesthetized with isoflurane and an intracerebral guide cannula (CMA) was stereotaxically implanted into the brain, positioning the microdialysis probe in the hippocampus (co-ordinates of probe tip: 3.1 mm posterior and 2.8 mm latéral from bregma, and 1.3 mm relative dura mater) according to the atlas of Paxinos and Franklin 2001. Anchor screws and acrylic cernent were used for the fixation of the guide cannulas. After implantation of the cannula mice were allowed to recover from the surgery for 2-3 days before dialysis.
On the day of the experiment, a 2-mm, 1000 kDa cut-off CMA probe was inserted through the guide cannula. A probe was connected to a microdialysis peristaltic pump with two channels (MAB20; Microbiotech) and operated in push-pull mode.
The iniet tubing of the microdialysis probe was connected to a peristaltic pump perfusing the probe with artificial cerebrospinai fluid (aCSF; in mM: 147 NaCI, 2.7 KCI, 1.2 CaCI2, 0.85 MgCI2). The peristaltic pump was also connected to the outlet tubing in order to prevent perfusion fluid loss from the probe, by pulling the fluid through the tubing. As a perfusion buffer, 25% bovine albumin fraction V (Sigma) was diluted to 0.2 % with artificial CSF on the day of use and filtered through a 0.1pm membrane. The actua! f-ow rate of the pump was determined without having the probe connected. The sample tubes were weighed before and after sampling for a given time period and the flow rate was calculated. The pump was then set to a constant flow of 1 pL/min. A 120 min sampling regimen was used throughout the experiment period. To avoid interférence of tissue damage, the experimental window was set from 14 to 48 hr after probe implantation. 14-16 h after the start of the experiments, GM37, human 9E4 or isotype control(anti-HEL) were injected i.p. at 15 mg/kg, and an additional 6 samples (12h of collection) were collected. The dialysates were stored at -80 °C. Concentration of human alpha synuclein was determined by ELISA (Covance ELISA kit).
The average of the two-three basal values (4h-6h) prior to antibody treatment was taken as baseline and set to 100% for each animal. Data was evaluated using twoway analysis Of variance (ANOVA) with repeated measures to evaluate statistical relevance. The basal levels of human alpha-synuclein in hippocampus were 8.1 ± 1.1 ng/ml (mean ± SEM, n = 25, not corrected for the in vitro dialysis probe recovery). The administration of GM37 induced a larger réduction in human alphasynuclein in the hippocampus of F28 mice compared to both the comparator antibody, human 9E4, and the isotype control (anti-HEL). (Fig. 17).
Example 9: Chronic effects of alpha-synuclein antibodies in vivo.
Antibody GM37 ameliorate motor phenotype in rat Parkinson model.
Targeted overexpression of human alpha-synuclein to dopaminergic neurons in the rat midbrain can be achieved using a recombinant adeno-associated viral vector (rAAV) and is associated with a progressive loss of dopaminergic cells in the substantia nigra as well as motor impairments.
Adult female Sprague-Dawley rats (225-250g) were used to express human alphasynuclein in substantia nigra (SN) by injection with Adeno associated virus of
AAV2/5 serotype containing chicken beta-actin promoterwith enhancer éléments from the cytomégalovirus promoter, followed by human alpha-synuclein cDNA and WPRE element as previously described (Xu L, Daly T, Gao C, Flotte TR, Song S, Byrne BJ, Sands MS, Fonder KP (2001). In this model it has been shown that human alpha-synuclein expression leads to neurodegeneration of dopaminergic neurons. Maingay M, étal. CNS Spectr. 2005 Mar; 10(3):235-44). Totestthe effect of an alpha synuclein therapeutic antibody in this model antibody treatment was initiated 2 to 4 days prior to viral injections, and continued until the end of the study (Fig. 18). PBS administration at the same volume (5 ml/kg: IP) was used as a control. GM37 was dosed twice per week at a dose of 15 mg/kg (IP). The viral particles (rAAV2/5) containing the gene for human wt alpha-synuclein or green fluorescent protein (GFP) were injected unilaterally in the SN. Animais were anaesthetized with a combination of Hypnorm® and Dormicum® at 2.0 ml/kg s.c. and placed in a stereotaxic frame. Their température was adjusted to 37.5°C via a heating pad, and their skull was exposed. A hole was drilled above the right SN at the following coordinates, according to the atlas of Paxinos and Watson (Paxinos & Watson, 1998): 5.5 mm posterior and 2.0 mm latéral from Bregma. A single injection of 3 pL of rAAV2/5-alpha-syn or rAAV2/5-GFP was performed at a depth of 7.2 mm below the dura matter, and a flow rate of 0.2 pL/min using a Hamilton syringe connected to a stereotaxic injector. The needle was left in place an additional 5 min to allow diffusion of the vector in the SN. Following surgery, the animais were returned to their home cage, and placed in a heated environment where they were allowed to recover from anesthésia. Testing of motor asymmetry in the cylinder test was evaluated prior to AAV injections, as well as 3, 7 and 10 weeks following AAV injections. The data presented correspond to the ratio between use of the right forepaw compared to the total use of both the left and right forepaws. Each animal’s performance in the cylinder was filmed for a total 5 min, and manual scoring of the number of touches using the left and right forepaws fpr 5 minutes has been performed for the final testing day 10 weeks after virus injection. A significant impairment is présent in AAV-syn compared to AAV-GFP injected rats (p=0.012) at week 10. Atrend for a reversai for GM37 treated animais was shown, as their performance is different from GFP rats (p=0.163 and p=0.407 for gm37, respectively). This finding indicates that antibody GM37 is able to ameliorate the Parkinsonian motor phenotype in this rat model (Figs 18 and 19).
Exampie 10: Chronic effects of alpha synuclein antibodies in vivo.
Antibody GM37 inhibits seeding of endogenous mouse alpha synuclein aggregation and phosphorylation
Injection of alpha synuclein preformed fibrils made from recombinant protein into dorsal striatum of wild type mice recruit endogenous mouse alpha synuclein and induce formation of Ser-129 phosphorylated aggregates inside neurons in cortex, amygdala and substantia nigra (Luk et al. 2012, Science 2012 Nov 16;338(6109):949-53).To see if alpha-synuclein spécifie monoclonal antibody GM37 could reduce the appearance of alpha-synuclein fibril-induced phosphorylated alpha synuclein inclusion formation in vivo a total of 45 mice were used. Mice were dosed with GM 37 at 30 mg/kg i.p, GM 3715 mg/kg i.v. , or vehicle ip (PBS).One day later the mice were anesthetized and stereotactically injected in one hemisphere with 2 ul of recombinant human alpha-Syn crude seeds, made as described previously (Example 6) (total of 2 pg crude seeds per animal). To inject the crude seeds, the skull was opened by boring a hole and a single glass pipette was inserted (co-ordinates: +0.5 mm anterior to Bregma, +2.0 mm latéral to midline) into the right forebrain to target the inoculum to the dorsal neostriatum (+2.6 mm beneath the dura). Following recovery, the mice received weekly i.p. or i.v. injections of antibodies until sacrifice at 45 days. Groups of 15 mice/group were dosed either iv w. GM37 15mg/kg, ip with GM37 30mg/kg, or PBS (10 ml/kg) ip once weekly.
To measure the antibody concentration in plasma, cheek blood was drawn once weekly just prior to next injection, ie 7 days after last injection. Plasma was obtained by a 2000g spin, 15min incubation at RT, supernatant was subsequently frozen at -20°C. A CSF sample was taken at the end of the study and frozen at -20 °C. Plasma and CSF samples were analysed to détermine the concentration of Human IgG by MSD. In short, mouse anti-human IgG (clone MH16-1 (M1268) was used for capture, plasma or CSF was incubated in the well, followed by a sulfo-TAG goat anti-human as the détection antibody (MSD cat no: R32AJ-1). Plates were analysed from electrochemiluminesence by MSD.
The antibody levels in plasma are shown in Figure 20B and show a dose dépendent increase in antibody plasma concentration and accumulation of antibody in plasma during the six weeks. The antibody levels in csf are shown in Figure 20C, and show that around 0.1% of antibody level in plasma can be measured in csf.
Atday45, from the time of the injection ofthe alpha synuclein fibrillary seed, the mice were anesthetized, transcardially perfused with PBS, followed by perfusion with neutral buffered paraformaldéhyde (4%). The brains were removed and incubated ovemight for post-fixation in neutral buffered paraformaldéhyde. Immunohistochemistry was performed on 45 pm thick serial sections by Neuroscience associâtes. Briefly, Using MultiBrain® technology, up to 25 mouse brains were embedded together per block, into 3 blocks, freeze-sectioned at 45pm thickness in the coronal plane, and collected into cups containing antigen preserve solution. Every sixth section was stained with antibody to Ser-129 phosphorylated alpha-synuclein (Anti-alpha Synuclein (phospho S129) antibody [Psyn/81A] ab184674) to revea! Ser129 phosphorylated alpha synuclein reactive structures.
Quantitation of pSyn pathology was performed by manual counting immunoreactive positive cells from images at 10 x magnification from 5-7 sections covering the entire substantia nigra from every sixth section. The counting was performed blinded. Cell counts in amygdala and nigra were analysed by a one-way ANOVA followed by Bonferoni t-test, where the effect of GM37 antibody was compared to PBS treatment.
As can be seen from Fig 20C, treatment with antibody GM37 reduced the number of intra-cellular inclusions in Substantia Nigra significantly when compared to PBS control, with either ip or iv treatment. The data shows that antibody GM37 could hâve therapeutic effect in PD by blocking entry of extracellular pathological alpha-synuclein into neurons, by blocking its propagation between neurons and/or facîlitating clearance from the ISF by uptake into microglia. As this appearance of inclusions has been linked to loss of dopaminergic neurons and development of Parkinsonian motor déficits in animal models, treatment with antibody GM37 could hâve a therapeutic effect on loss of dopaminergic cells and development of motor déficits in PD.
Example 11: Manufactability of GM37 and GM37 variants
The anti-alpha-synuclein antibodies are produced in mammalian cell culture under conditions that mimic the production conditions that will be used for producing clinical grade material for use in patients. It is well known that proteins produced in this manner undergo post-translational modifications that can impact both therapeutic potency of the antibody as well as biophysical attributes that affect the stability of the antibody over time. Empirical knowledge ascertained from décades of studies identified a set of post-translational modifications known to provide risk for the developability of a spécifie molécule. These post-translational modifications hâve been shown to correlate with amino acid strings présent in the primary sequence of the heavy and light chain proteins. Algorithme hâve been generated that can identify these sequences and détermine the potential risk they will hâve on the manufacturability and developability of a therapeutic antibody.
In silico analysis of the primary sequence of the antibody can be used to de-risk a molécule for its potential to be developed as a therapeutic. In particular, detailed analysis of the VH and VL régions can identified unique amino acids that are deemed important for the molécules activity but also may be a potential risk for its stability over time. Sequence spécifie deamidation has been identified as a potential risk for protein structures. Protein deamidation can occur on the amide side chains of glutamines or asparagine residues and transform them into a carboxylate group (Lorenzo et al.
PLOSone, 001:10.1371, Dec. (2015)). Nonenzymatic deamidation at neutral pH occurs faster for asparagine and is therefore considered a higher risk than glutamine. The activity is further influenced by the subséquent amino acid in the sequence and can occur at a rate of days or years. The actual fate of the protein that undergoes deamidation needs to be evaluated experimentally to determine the impact ofthe change both on its stability and activity.
We identified a site for deamidation within the VH domain of GM37. Amino acid residues 54 is an asparagine(N) followed by a glycine(G) at position 55. The N54 is at high risk for spontaneous deamidation. To mitigate this risk we generated a set of 3 variants that replace the asparagine(N) with serine(S), glutamine(Q) or histidine(H). AH 3 variants were produced in mammalian cell culture using transient transfection methods (example 1.5). AH 3 variants showed similar expression and purification properties as GM37wt. (Fig. 23).
For each of the eight products 400 ml transient transfections were performed using CHOK1SV GS-KO cells which had been in culture for minimum 2 weeks. Cells were sub-cultured 24 hours prior to transfection. Ail transfections were carried out via electroporation using Gene Puise XCell (Bio-Rad). For each transfection, viable cells were resuspended in pre-warmed CD-CHO media supplemented with 6mM Lglutamine to 2.86x107 cells/ml. 40 pg of each established SGV DNA containing the appropriate heavy and light chains were aliquoted into each cuvette (Bio-Rad,
GenePulser cuvette, 0.4 cm gap, 165-2088) and 700 pl cell suspension added. Cells were electroporated at 300V, 900pF. Transfected cells were transferred to rep-warmed media in Erienmeyer flasks and the contents of the cuvettes rinsed twice with prewarmed media were also transferred to the flasks. Transfectant cultures were incubated in a shaking incubator at 36.5;C, 5% CO2, 85% humidity, 140 rpm for 6 days. Cell viability was measured at the time of harvest using a Cedex HiRes automated cell counter (Rosche).
In order to evaluate the importance of residue 54 in binding to human alpha-synuclein we analyzed the ability ofthe variants to bind in two different experiments. Using a compétition ELISA format we evaluated the impact the change at residue 54 would 15 hâve on the ability of GM37 to bind alpha-synuclein in solution. By evaluating the concentration of synuclein able to inhibit binding ofthe antibody to synuclein coated ELISA plates we showed ail three variants maintained the same binding as GM37wt and bind to alpha-synuclein with high affinity resulting in IC50s of 1-2nM (Fig. 24). A compétition assay was performed using preincubation of a fixed concentration (0.3 20 pg/ml) of each of the following antibodies, GM37 (named GM 37wt), GM37 variantl,
GM37 variant2 and GM37 variants with a range of 0-1000 nM human alpha-synuclein for 60 minutes at room température. The remaining unbound antibody was captured and measured on ELISA plates coated with 100 ng/ml of recombinant human alphasynuclein using an anti-human détection antibody by electrochemiluminesence (MSD, 25 Gathersburg, MD). The IC50s ofthe interaction are 1.9nM, 1.6nM, 2.1nM and 1.4nM for GM37 wt, GM37variant1, GM37variant2 and GM37variant3, respectively (as determined using Prism Graphpad®).
Using surface plasmin résonance (SPR), we evaluated the real time kinetics of binding of GM37 wt (2 batches) and the three variants (Example 2). The human alpha30 synuclein was captured to the slide(ligand) and the antibodies were each tested at multiple concentrations as analytes. Analysis of the binding curves in the presence of antibody at multiple concentrations showed that the on rates were the same for ail four antibodies, similarly when the antibody was removed from the buffer the off-rates measured showed no statistical différence between the antibodies. Using a 1:1 binding algorithm al! 4 antibodies hâve near identical binding constants (Fig. 25).
In order to evaluate the impact of the changes at N54 on the functional activity of GM37 we analyzed the abiiity of the antibodies to block synuclein seeding activity in a culture of primary neurons (Example 6). The level of seeding is measured using an antibody SDecific for phosphc-synuclein . Ail 3 antibodies were able to block seeding as measured by the phospho-synuclein signal (Fig. 26). Furthermore, the level of inhibition was the same for ail 4 antibodies. This call based data further confirms the binding data that amino acid 54 in the VH domain is not required for binding affinity to human alpha-synuclein or for inhibition of seeding in a primary cell based assay. Furthermore, we found that ail three of these antibodies were capable of production using standard expression and purification methods. Interestingly one ofthe variants N54Q showed improvement in production over the other variants, which is of great importance when the antibody is to be produced commercially on large scale. These data support the possibility of reducing the potential risk of deamidation by replacing asparagine (N) with another amino acid without concern over the loss of potency.
A samples of each of the antibodies wt GM37, var1, var2 and var3 was subjected to a steady increase in température over time and the level of aggregation was simultaneously measured by mufti-angle light scattering (Prometheus NT.48, NanoTemper Technologies). The température for onset of aggregation was found to be similar for GM37 and the GM37-variants, however the lowest level of aggregation observed for GM37-Var2 (Fig. 27).
SEQUENCE LISTING
<110> | H. Lundbeck A/S |
<120> | New Monoclonal Anti-Alpha-Synuclein Antibodies |
<130> | 0992 |
<160> | 43 |
<170> | Patentln version 3.5 |
<210> | 1 |
<211> | 10 |
<212> | PRT |
<213> | Artificial |
<220> <223> | GM37 Cdr 1 Heavy Chain |
<400> | 1 |
Gly Phe Thr Phe Ser Ser Tyr Ala Met Thr
10 <210> 2 <211> 17 <212> PRT <213> Artificial <220>
<223> GM37 CDR2 Heavy Chain <400> 2
Ala île Arg Ser Asn Gly Asp Arg Thr Asp Tyr Ala Asp Ser val Lys
10 15
Gly
<210> <211> <212> <213> | 3 9 PRT Arti fi ci al |
<220> <223> | GM37 CDR3 Heavy Chain |
<400> | 3 |
Ala Lys Asn Trp Ala Pro Phe Asp Ser 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial <220>
<223> GM37 CDR1 Light Chain <400> 4
Ala Ser Gin Ser val Ser Ser Ser
5 <210> 5 <211> 7 <212> PRT <213> Artificial <22O>
<223> GM37 CDR 2 Light Chain <400> 5
Gly Ala ser ser Arg Ala Thr
5 <210> 6 <211> 9 <212> PRT <213> Artificial <220>
<223> GM37 CDR 3 Light Chain <400> 6
Gin Gin Tyr Gly Ser Ser Pro Trp
5 <210> 7 <211> 116 <212> PRT <213> Artificial <220>
<223> GM37 CDR Heavy Chain <400> 7
Glu val Gin Leu Leu Glu Ser Gly
5
Tyr Leu Ala
Thr
Gly Gly Leu Val Gin Thr Gly Gly
15
Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe ser ser Tyr
30
Ala Met Thr Trp Val Arg Gin Ala
40
Pro Gly Lys Gly Leu Glu Trp Val
Ser Ala Ile Arg Ser Asn Gly Asp
55
Arg Thr Asp Tyr Ala Asp Ser Val
Lys 65 | Gly Arg Phe | Thr | Ile Ser Arg Asp Asn Ser | Gin | Asn | Thr | Leu | Tyr 80 | |
70 | 75 | ||||||||
Leu | Gin Met Asn | Ser | Leu | Arg Ala Glu Asp Thr | Ala | val | Tyr | Tyr | Cys |
85 | 90 | 95 | |||||||
Ala | Lys Asn Trp | Ala | Pro | Phe Asp ser Trp Gly | Gin | Gly | Thr | Leu | val |
100 | 105 | 110 | |||||||
Thr | val Ser Ser |
115 <210> 8 <211> 108 <212> PRT <213> Artificial <220>
<223> GM 37 Light Chain
<400> 8 Glu île val 1 | Leu | Thr 5 | G1 n | Ser | Pro | Gly | Thr 10 | Leu | Ser | Leu | Ser | pro 15 | Gly | ||
Glu | Arg | Ala | Thr 20 | Leu | ser | cys | Arg | Ala 25 | Ser | Gin | Ser | Val | Ser 30 | ser | Ser |
Tyr | Leu | Ala 35 | Trp | Tyr | Gin | Gin | Lys 40 | Pro | Gly | Gin | Al a | Pro 45 | Arg | Leu | Leu |
Ile | Tyr 50 | Gly | Ala | ser | Ser | Arg 55 | Al a | Thr | Gly | île | Pro 60 | ASp | Arg | Phe | Ser |
Gly 65 | ser | Gly | Ser | Gly | Thr 70 | Asp | Phe | Thr | Leu | Thr 75 | Ile | Ser | Arg | Leu | Glu 80 |
Pro | G1 u | Asp | Phe | Ala 85 | val | Tyr | Tyr | cys | Gin 90 | Gin | Tyr | Gly | ser | ser 95 | Pro |
Trp | Thr | Phe | Gly 100 | Gin | Gly | Thr | Lys | val 105 | Glu | île | Lys |
<210> | 9 |
<211> | 6 |
<212> | PRT |
<213> | Arti fi ci al |
<220> <223> | Epitope 112-117 |
<400> | 9 |
Ile Leu Glu Asp Met Pro 1 5 <210> 10 <211> 140 <212> PRT <213> Artificial <220>
<223> Alpha-synuclein <400> 10
Met Asp Val Phe Met Lys Gly Leu
5
Ser Lys Ala Lys Glu Gly val val
15
Ala Ala Ala Glu Lys Thr Lys Gin
Gly val Ala Glu Ala Ala Gly Lys
30
Thr Lys Glu Gly val Leu Tyr val
40
Gly Ser Lys Thr Lys Glu Gly Val val His Gly val Ala Thr val Ala
55
Glu Lys Thr Lys Glu Gin val Thr 60
Asn val Gly Gly Ala val val Thr
70
Gly val Thr Ala val Ala Gin Lys
80
Thr Val Glu Gly Ala Gly Ser île
Ala Ala Ala Thr Gly Phe val Lys
95
Lys Asp Gin Leu Gly Lys Asn Glu
100
Glu Gly Ala Pro Gin Glu Gly île
105 110
Leu Glu Asp Met Pro val Asp Pro
115 120
Asp Asn Glu Ala Tyr Glu Met Pro
125
Ser Glu Glu Gly Tyr Gin Asp Tyr
130 135 <210> 11 <211> 165 <212> PRT <213> Artificial <220>
<223> A-Syn-AAKK-BAP <400> 11
Met Asp Val Phe Met Lys Gly Leu
5
Glu Pro Glu Ala
140
Ser Lys Ala Lys Glu Gly val Val
15
Ala Ala Ala Glu Lys Thr Lys Gin Gly val Ala Glu Ala Ala Gly Lys
2530
Thr Lys Glu Gly val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly val
4045 val His Gly val Ala Thr val Ala Glu Lys Thr Lys Glu Gin val Thr
5560
Asn val Gly Gly Ala val val Thr Gly val Thr Ala val Ala Gin Lys 65 70 7580
Thr val Glu Gly Ala Gly Asn île Ala Ala Ala Thr Gly Leu Val Lys
9095
Lys Asp Gin Leu Ala Lys Gin Asn Glu Glu Gly Phe Leu Gin Glu Gly
100 105110
Met Val Asn Asn Thr Asp île Pro val Asp Pro Glu Asn Glu Ala Tyr
115 120125
Glu Met Pro Pro Glu Glu Glu Tyr Gin Asp Tyr Glu Pro Glu Ala Gly
130 135140
Ser Ala Gly Gly Ser Gly Gly Leu Asn Asp île Phe Glu Ala Gin Lys
145 150 155160
Ile Glu Trp His Glu
165 <210> 12 <211> 165 <212> PRT <213> Artificial <220>
<223> A-Syn-BAAK-BAP <400> 12
Met Asp val Phe Met Lys Gly Leu ser Met Ala Lys Glu Gly val val
1015
Ala Ala Ala Glu Lys Thr Lys Gin Gly val Thr Glu Ala Ala Glu Lys
2530
Thr Lys Glu Gly Val Leu Tyr val Gly ser Lys Thr Lys Glu Gly Val
4045 val His Gly Val Ala Thr val Ala Glu Lys Thr Lys Glu Gin Val Thr
5560
Asn 65 | val | Gly | Gly | Al a | val 70 | Val | Thr | Gly | Val | Thr 75 | Ala | val | Ala | Gin | Lys 80 |
Thr | val | Glu | Gly | Ala 85 | Gly | Ser | île | Ala | Ala 90 | Ala | Thr | Gly | Phe | val 95 | Lys |
Lys | Asp | Gin | Leu 100 | Ala | Lys | Gin | Asn | Glu 105 | Glu | Gly | Phe | Leu | Gin 110 | Glu | Gly |
Met | val | Asn 115 | Asn | Thr | Asp | Ile | Pro 120 | val | Asp | ΡΓΟ | Glu | Asn 125 | Glu | Ala | Tyr |
Glu | Met 130 | Pro | Pro | Glu | Glu | Glu 135 | Tyr | Gin | Asp | Tyr | Glu 140 | Pro | Glu | Ala | Gly |
ser 145 | Ala | Gly | Gly | Ser | Gly 150 | Gly | Leu | Asn | Asp | Ile 155 | Phe | Glu | Ala | Gin | Lys 160 |
Ile | Glu | Trp | His | Glu 165 | |||||||||||
<210> <211> <212> <213> . | 13 162 PRT Arti fi ci al | ||||||||||||||
<220> <223> , | A-Syr | l-BBAA-BAP | |||||||||||||
<400> | 13 | ||||||||||||||
Met 1 | Asp | Val | Phe | Met 5 | Lys | Gly | Leu | Ser | Met 10 | Ala | Lys | Glu | Gly | val 15 | val |
Ala | Ala | Ala | Glu 20 | Lys | Thr | Lys | Gin | Gly 25 | Val | Thr | Glu | Ala | Ala 30 | Glu | Lys |
Thr | Lys | Glu 35 | Gly | val | Leu | Tyr | val 40 | Gly | Ser | Lys | Thr | Arg 45 | Glu | Gly | val |
val | Gin 50 | Gly | Val | Ala | Ser | Val 55 | Ala | Glu | Lys | Thr | Lys 60 | Glu | Gin | Ala | ser |
Hi s 65 | Leu | Gly Gly | Ala | Val 70 | Val | Thr | Gly | val | Thr 75 | Ala | Val | Ala | Gin | Lys 80 | |
Thr | val | Glu | Gly | Ala 85 | Gly | Ser | île | Al a | Ala 90 | Ala | Thr | Gly | Phe | val 95 | Lys |
Lys Asp Gin Leu Gly Lys Asn Glu Glu Gly Ala Pro Gin Glu Gly Ile
100 105110
Leu Glu Asp Met Pro val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro
115 120125
Ser Glu Glu Gly Tyr Gin Asp Tyr Glu Pro Glu Ala Gly Ser Ala Gly
130 135140
Gly Ser Gly Gly Leu Asn Asp île Phe Glu Ala Gin Lys Ile Glu Trp
145 150 155160
His Glu
20.
<210> 14 <211> 165 <212> PRT <213> Artificial <220>
<223> A-Syn-BBKK-BAP <400> 14
Met Asp Val Phe Met Lys Gly Leu ser Met Ala Lys Glu Gly Val Val
1015
Ala Ala Ala Glu Lys Thr Lys Gin Gly val Thr Glu Ala Ala Glu Lys
2530
Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Arg Glu Gly Val
35 4045
Val Gin Gly val Ala ser Val Ala Glu Lys Thr Lys Glu Gin Ala ser
5560
His Leu Gly Gly Ala val Val Thr Gly Val Thr Ala Val Ala Gin Lys
70 7580
Thr val Glu Gly Ala Gly Asn Ile Ala Ala Ala Thr Gly Leu Val Lys
9095
Lys Asp Gin Leu Ala Lys Gin Asn Glu Glu Gly Phe Leu Gin Glu Gly
100 105110
Met val Asn Asn Thr Asp île Pro Val Asp Pro Glu Asn Glu Ala Tyr
115 120125
Glu Met Pro Pro Glu Glu Glu Tyr Gin Asp Tyr Glu Pro Glu Ala Gly
130 135140
Ser Ala Gly Gly ser Gly Gly Leu Asn Asp île Phe Glu Ala Gin Lys
145 150 155 160 île Glu Trp His Glu
165 <210> 15 <21I> 141 <212> PRT <213> Artificial <220>
<223> A-Syn-120-140_Del-BAP
<400> 15 | |||||||||||||||
Met 1 | Asp | val | Phe | Met 5 | Lys | Gly | Leu | Ser | Lys 10 | Ala | Lys | Glu | Gly | val 15 | val |
Ala | Ala | Ala | Glu 20 | Lys | Thr | Lys | Gin | Gly 25 | Val | Ala | Glu | Ala | Ala 30 | Gly | Lys |
Thr | Lys | Glu 35 | Gly | Val | Leu | Tyr | Val 40 | Gly | Ser | Lys | Thr | Lys 45 | Glu | Gly | Val |
val | Hl s 50 | Gly | val | Ala | Thr | val 55 | Ala | Glu | Lys | Thr | Lys 60 | Glu | Gin | val | Thr |
Asn 65 | val | Gly | Gly | Ala | val 70 | Val | Thr | Gly | val | Thr 75 | Ala | val | Ala | Gin | Lys 80 |
Thr | val | Glu | Gly | Al a 85 | Gly | Ser | île | Ala | Ala 90 | Ala | Thr | Gly | Phe | Val 95 | Lys |
Lys | Asp | Gin | Leu 100 | Gly | Lys | Asn | Glu | Glu 105 | Gly | Ala | Pro | Gin | Glu 110 | Gly | île |
Leu | Glu | Asp 115 | Met | Pro | val | Asp | Gly 120 | Ser | Ala | Gly | Gly | Ser 125 | Gly | Gly | Leu |
Asn | Asp 130 | île | Phe | Glu | Ala | Gin 13 5 | Lys | île | Glu | Trp | Hl s 140 | G1 u |
<210> 16 <211> 131 <212> PRT <213> Artificial <220>
<223> alpha-synuclein amino acids 1-119 <400> 16
Met Ala His His His His His His île Glu Gly Arg Met Asp Val Phe
1015
Met Lys Gly Leu Ser Lys Ala Lys Glu Gly val val Ala Ala Ala Glu
2530
Lys Thr Lys Gin Gly val Ala Glu Ala Ala Gly Lys Thr Lys Glu Gly
4045
Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val val His Gly Val
5560
Ala Thr Val Ala Glu Lys Thr Lys Glu Gin Val Thr Asn Val Gly Gly 65 70 7580
Ala Val Val Thr Gly Val Thr Ala Val Ala Gin Lys Thr Val Glu Gly
9095
Ala Gly Ser île Ala Ala Ala Thr Gly Phe val Lys Lys Asp Gin Leu
100 105110
Gly Lys Asn Glu Glu Gly Ala Pro Gin Glu Gly Ile Leu Glu Asp Met
115 120125
Pro Val Asp
130 <210> 17 <211> 106 <212> PRT <213> artificial <220>
<223> kappa (LC constant région) <400> 17
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro ser Asp Glu Gin
1015
Leu Lys Ser Gly Thr Ala ser val val cys Leu Leu Asn Asn Phe Tyr
2530
Pro Arg Glu Ala Lys val Gin Trp Lys val Asp Asn Ala Leu Gin Ser
4045
Gly Asn ser Gin Glu Ser val Thr Glu Gin Asp Ser Lys Asp Ser Thr
5560
Tyr 65 | Ser | Leu | Ser | Ser | Thr 70 | Leu | Thr | Leu | Ser | Lys 75 | Ala | Asp | Tyr | Glu | Lys 80 | |
5 | His | Lys | val | Tyr | Ala 85 | cys | Glu | Val | Thr | His 90 | Gin | Gly | Leu | Ser | Ser 95 | Pro |
10 | val | Thr | Lys | Ser 100 | Phe | Asn | Arg | Gly | Glu 105 | Cys |
<210> 18 <211> 329 <212> PRT <213> Artificial <220>
20 | <223> IgGl <400> 18 | (HC | Constant région) | |||||||||||||
25 | Ala 1 | Ser | Thr | Lys | Gly 5 | Pro | ser | val | phe | Pro 10 | Leu | Ala | Pro | ser | Ser 15 | Lys |
Ser | Thr | Ser | Gly 20 | Gly | Thr | Al a | Ala | Leu 25 | Gly | cys | Leu | Val | Lys 30 | ASp | Tyr | |
30 | Phe | Pro | Glu 35 | Pro | Val | Thr | Val | Ser 40 | Trp | Asn | ser | Gly | Ala 45 | Leu | Thr | ser |
35 | Gly | val 50 | His | Thr | phe | pro | Ala 55 | Val | Leu | Gin | Ser | Ser 60 | Gly | Leu | Tyr | Ser |
40 | Leu 65 | Ser | ser | val | val | Thr 70 | val | pro | ser | Ser | Ser 75 | Leu | Gly | Thr | Gin | Thr 80 |
45 | Tyr | Ile | cys | Asn | Val 85 | Asn | Hi s | Lys | Pro | Ser 90 | Asn | Thr | Lys | Val | ASp 95 | Lys |
Arg | val | Glu | Pro 100 | Lys | Ser | cys | ASp | Lys 105 | Thr | His | Thr | cys | Pro 110 | Pro | cys | |
50 | Pro | Al a | Pro 115 | Glu | Leu | Leu | Gly | Gly 120 | Pro | Ser | Val | Phe | Leu 125 | Phe | ΡΓΟ | Pro |
55 | Lys | Pro 130 | Lys | Asp | Thr | Leu | Met 135 | Ile | Ser | Arg | Thr | Pro 140 | Glu | val | Thr | Cys |
60 | val 145 | val | val | ASp | val | Ser 150 | Hi s | Glu | Asp | Pro | Glu 155 | Val | Lys | Phe | Asn | Trp 160 |
Tyr | val | Asp | Gly | val 165 | Glu | val | His | Asn | Al a 170 | Lys | Thr | Lys | Pro | Arg 175 | Glu |
100
Glu Gin Tyr Asn Ser Thr Tyr Arg Val val ser val Leu Thr Val Leu
180 185190
His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val ser Asn
195 200205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr île Ser Lys Ala Lys Gly
210 215220
Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235240
Met Thr Lys Asn Gin Val Ser Leu Thr cys Leu val Lys Gly Phe Tyr
245 250255
Pro Ser Asp Ile Ala val Glu Trp Glu ser Asn Gly Gin Pro Glu Asn
260 265270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280285
Leu Tyr Ser Lys Leu Thr val Asp Lys Ser Arg Trp Gin Gin Gly Asn
290 295300
Val Phe Ser cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315320
Gin Lys Ser Leu Ser Leu Ser Pro Gly
325 <210> 19 <211> 4 <212> PRT <213> Artificial <220>
<223> GM285 epitope 112-115 <400> 19
Ile Leu Glu Asp <210> 20 <211> 13 <212> PRT <213> Artificial <220>
<223> GM285 CDR1 Heavy Chain
101 <400> 20
5 | Ala Ala Ser Gly Phe Thr Phe ser Arq Phe Thr Met Thr | ||
1 | 5 | 10 | |
10 | <210> <211> <212> <213> | 21 17 PRT Artificial | |
<22O> <223> | GM285 CDR2 Heavy | Chai n | |
15 | <400> | 21 |
Ala Ile Ser Gly Ser Gly Gly Gly Thr Ser Tyr Ala Asp ser Val Lys 15 10 15
Gly
25 | <210> <211> <212> <213> | 22 9 PRT Arti fi ci al |
30 | <220> | |
<223> | GM285 CDR3 Heavy Chain | |
<400> | 22 | |
35 | Ala Lys | ; Asn Trp Ala Pro Phe Asp Tyr |
1 | 5 |
40 | <210> <211> <212> <213> | 23 12 PRT Artificial |
45 | <220> <223> | GM285 CDR1 Light Chain |
<400> 23 Arg Ala Ser Gin Ser val Ser Arg ser Tyr Leu Ala | ||
50 55 60 | 15 10 <210> 24 <211> 7 <212> PRT <213> Artificial <220> <223> GM285 CDR2 Light Chain <400> 24 Gly Ala Ser Ser Arg Ala Thr 1 5 |
102
<210> | 25 |
<211> | 9 |
<212> | PRT |
<213> | Artificial |
<220> <223> | GM285 CDR3 Light Chain |
<400> | 25 |
Gin Gin Tyr Gly Ser Ser Pro Trp Thr
1 | 5 |
<210> | 26 |
<211> | 116 |
<212> | PRT |
<213> | Artificial |
<220> <223> | GM285 VH |
<400> | 26 |
Glu 1 | val | Gin | Leu | Leu 5 | Glu | ser | Gly | Gly | Gly Leu 10 | val | Gin | Pro | Gly 15 | Gly |
ser | Leu | Arg | Leu 20 | Ser | cys | Ala | Ala | Ser 25 | Gly Phe | Thr | Phe | ser 30 | Arg | Phe |
Thr | Met | Thr 35 | Trp | val | Arg | Gin | Ala 40 | Pro | Gly Lys | Gly | Leu 45 | Glu | Trp | Val |
ser | Ala 50 | île | Ser | Gly | Ser | Gly 55 | Gly | Gly | Thr Ser | Tyr 60 | Ala | Asp | Ser | Val |
Lys 65 | Gly | Arg | Leu | Thr | val 70 | Ser | Arg | Asp | Asn Ser 75 | Lys | Asn | Thr | Leu | Tyr 80 |
Leu | G1 n | Met | Asn | Ser 85 | Leu | Arg | Ala | Glu | Asp Thr 90 | Al a | val | Tyr | Tyr 95 | cys |
Ala | Lys | Asn | Trp 100 | Al a | Pro | Phe | Asp | Tyr 105 | Trp Gly | Gin | Gly | Thr 110 | Leu | val |
Thr Val Ser Ser
115
<210> <211> <212> <213> | 27 108 PRT Arti fi ci al |
<220>
103 <223> GM285 VL <400> 27
Glu 1 | île val | Leu | Thr Gin Ser Pro Gly 5 | Thr 10 | Leu | Ser Leu | Ser | Pro 15 | Gly | ||||||
Glu | Arg | Ala | Thr 20 | Leu | Ser | Cys | Arg | Ala 25 | ser | Gin | Ser | val | Ser 30 | Arg | Ser |
Tyr | Leu | Ala 35 | Trp | Tyr | Gin | Gin | Lys 40 | Pro | Gly | Gin | Ala | Pro 45 | Arg | Leu | Leu |
île | Tyr 50 | Gly | Ala | ser | ser | Arg 55 | Ala | Thr | Gly | île | Pro 60 | ASp | Arg | Phe | Ser |
Gly 65 | Ser | Gly | Ser | Gly | Thr 70 | Asp | Phe | Thr | Leu | Thr 75 | Val | Ser | Arg | Leu | Glu 80 |
Pro | Glu | Asp | Phe | Ala 85 | val | Tyr | Tyr | cys | Gin 90 | Gin | Tyr | Gly | Ser | Ser 95 | Pro |
Trp | Thr | phe | Gly | Gin | Gly | Thr | Lys | val | Glu | île | Lys |
100 105 <210> 28 <211> 329 <212> PRT <213> Artificial <22O>
<223> GM285 IgGl constant | région |
<400> 28 | |
Ala Ser Thr Lys Gly Pro ser 1 5 | val Phe Pro Leu Ala Pro Ser Ser Lys 10 15 |
ser Thr ser Gly Gly Thr Ala 20 | Ala Leu Gly Cys Leu Val Lys Asp Tyr 25 30 |
Phe Pro Glu Pro Val Thr Val 35 | Ser Trp Asn Ser Gly Ala Leu Thr Ser 40 45 |
Gly val His Thr Phe Pro Ala 50 55 | val Leu Gin Ser Ser Gly Leu Tyr Ser 60 |
Leu ser ser val val Thr val 65 70 | Pro Ser Ser Ser Leu Gly Thr Gin Thr 75 80 |
Tyr île cys Asn val Asn His 85 | Lys Pro Ser Asn Thr Lys val Asp Lys 90 95 |
104
5 | Arg | val | Glu | Pro 100 | Lys | Ser | cys | Asp | Lys 105 | Thr | His | Thr | cys | Pro 110 | Pro | Cys |
Pro | Ala | Pro 115 | Glu | Leu | Leu | Gly | Gly 120 | Pro | Ser | Val | Phe | Leu 125 | Phe | Pro | Pro | |
10 | Lys | Pro 130 | Lys | Asp | Thr | Leu | Met 135 | Ile | Ser | Arg | Thr | Pro 140 | Glu | val | Thr | cys |
15. | val 145 | val | val | ASp | val | Ser 150 | His | Glu | Asp | Pro | G1 u 155 | Val | Lys | Phe | Asn | Trp 160 |
20 | Tyr | val | Asp | Gly | val 165 | Glu | val | His | Asn | Ala 170 | Lys | Thr | Lys | Pro | Arg 175 | Glu |
25 | Glu | G1 n | Tyr | Asn 180 | ser | Th η· | Tyr | Arg | val 185 | val | Ser | val | Leu | Thr 190 | val | Leu |
Hi s | G1 n | Asp 195 | Trp | Leu | Asn | Gly | Lys 200 | Glu | Tyr | Lys | Cys | Lys 205 | val | Ser | Asn | |
30 | Lys | Ala 210 | Leu | Pro | Ala | Pro | Ile 215 | Glu | Lys | Thr | île | Ser 220 | Lys | Ala | Lys | Gly |
35 | Gin 225 | Pro | Arg | Glu | pro | Gin 230 | val | Tyr | Thr | Leu | pro 235 | Pro | Ser | Arg | Glu | Glu 240 |
40 | Met | Thr | Lys | Asn | Gin 245 | val | Ser | Leu | Thr | Cys 250 | Leu | Val | Lys | Gly | Phe 255 | Tyr |
45 | pro | ser | Asp | île 260 | Al a | val | Glu | Trp | G1 u 265 | ser | Asn | Gly | Gin | Pro 270 | Glu | Asn |
Asn | Tyr | Lys 275 | Thr | Thr | Pro | Pro | Val 280 | Leu | Asp | Ser | Asp | Gly 285 | Ser | Phe | Phe | |
50 | Leu | Tyr 290 | ser | Lys | Leu | Thr | val 295 | Asp | Lys | ser | Arg | Trp 300 | Gin | Gin | Gly | Asn |
55 | val 305 | Phe | ser | Cys | ser | val 310 | Met | Hl s | Glu | Ala | Leu 315 | His | Asn | His | Tyr | Thr 320 |
60 | G1 n | Lys | ser | Leu | Ser 325 | Leu | Ser | pro | Gly |
<210> 29 <211> 106
105 <212> PRT <213> Artificial <220>
<223> GM285 Kappa chain <400> 29
Thr 1 | val | Ala | Ala | Pro 5 | Ser | val | Phe | Ile | Phe 10 | Pro | Pro | Ser | Asp | Glu 15 | Gin |
Leu | Lys | Ser | G1 y 20 | Thr | Al a | Ser | val | val 25 | Cys | Leu | Leu | Asn | Asn 30 | Phe | Tyr |
pro | Arg | Glu 35 | Al a | Lys | val | Gin | Trp 40 | Lys | val | Asp | Asn | Ala 45 | Leu | Gin | Ser |
Gly | Asn 50 | Ser | G1 n | Glu | ser | val 55 | Thr | Glu | G1 n | Asp | Ser 60 | Lys | ASp | ser | Thr |
Tyr 65 | ser | Leu | Ser | Ser | Thr 70 | Leu | Thr | Leu | Ser | Lys 75 | Ala | Asp | Tyr | Glu | Lys 80 |
Hl Ξ | Lys | Val | Tyr | Ala 85 | Cys | G1 u | val | Thr | His 90 | Gin | Gly | Leu | Ser | ser 95 | Pro |
val | Thr | Lys | Ser 100 | Phe | Asn | Arg | Gly | Glu 105 | Cys | ||||||
<210> <211> <212> <213> . | 30 116 PRT Arti fi ci al | ||||||||||||||
<220> <223> i | GM37 | variant | 1 heavy | chain | |||||||||||
<400> | 30 | ||||||||||||||
Glu 1 | val | Gin | Leu | Leu 5 | Glu | Ser | Gly | Gly | Gly 10 | Leu | Val | Gin | Thr | Gly 15 | Gly |
ser | Leu | Arg | Leu 20 | Ser | cys | Al a | Ala | Ser 25 | Gly | Phe | Thr | Phe | Ser 30 | Ser | Tyr |
Ala | Met | Thr 35 | Trp | val | Arg | G1 n | Ala 40 | Pro | Gly | Lys | Gly | Leu 45 | Glu | Trp | val |
Ser | Al a 50 | Ile | Arg | Ser | Ser | Gly 55 | Asp | Arg | Thr | ASp | Tyr 60 | Ala | Asp | ser | val |
Lys 65 | Gly | Arg | Phe | Thr | Ile 70 | Ser | Arg | Asp | Asn | Ser 75 | Gin | Asn | Thr | Leu | Tyr 80 |
106
Leu Gin Met Asn Ser Leu Arg Ala 85 | Glu Asp 90 | Thr Ala | Val | Tyr | Tyr Cys 95 |
Ala Lys Asn Trp Ala Pro Phe Asp | ser Trp | Gly Gin | Gly | Thr | Leu val |
100 105 110
Thr val Ser Ser
115
<210> | 31 |
<211> | 116 |
<212> | PRT |
<213> | Artificial |
<220> <223> | GM 37 variant 2 heavy chain |
<400> | 31 |
Glu 1 | val | Gin | Leu | Leu 5 | Glu | Ser | Gly | Gly | Gly 10 | Leu | Val | Gin | Thr | Gly Gly 15 | |
Ser | Leu | Arg | Leu 20 | ser | Cys | Ala | Al a | Ser 25 | Gly | Phe | Thr | Phe | Ser 30 | Ser | Tyr |
Al a | Met | Thr 35 | Trp | val | Arg | Gin | Ala 40 | Pro | Gly | Lys | Gly | Leu 45 | Glu | Trp | Val |
Ser | Ala 50 | île | Arg | Ser | Gin | Gly 55 | Asp | Arg | Thr | Asp | Tyr 60 | Ala | Asp | Ser | Val |
Lvs 65 | Gly | Arg | Phe | Thr | île 70 | ser | Arg | Asp | Asn | Ser 75 | Gin | Asn | Thr | Leu | Tyr 80 |
Leu | G1 n | Met | Asn | Ser 85 | Leu | Arg | Ala | Glu | Asp 90 | Thr | Ala | val | Tyr | Tyr 95 | Cys |
Ala | Lys | Asn | Trp 100 | Ala | Pro | Phe | Asp | Ser 105 | Trp | Gly | Gin | Gly | Thr 110 | Leu | val |
Thr Val ser ser
115 <210> 32 <211> 116 <212> PRT <213> Artificial <22O>
<223> gm 37 variant 3 heavy chain
107 <400> 32
5 | Glu 1 | val | Gin | Leu | Leu 5 | Glu | Ser | Gly | Gly | Gly 10 | Leu | val | Gin | Thr | Gly 15 | Gly |
Ser | Leu | Arg | Leu 20 | ser | cys | Ala | Ala | Ser 25 | Gly | Phe | Thr | Phe | Ser 30 | Ser | Tyr | |
10 | Ala | Met | Thr 35 | T rp | val | Arg | G1 n | Ala 40 | Pro | Gly | Lys | Gly | Leu 45 | Glu | T rp | val |
15 | Ser | Al a 50 | île | Arg | ser | His | Gly 55 | Asp | Arg | Thr | ASp | Tyr 60 | Ala | ASp | Ser | val |
20 | Lys 65 | Gly | Arg | Phe | Thr | île 70 | ser | Arg | Asp | Asn | ser 75 | Gin | Asn | Thr | Leu | Tyr 80 |
25 | Leu | Gin | Met | Asn | Ser 85 | Leu | Arg | Ala | Glu | Asp 90 | Thr | Al a | val | Tyr | Tyr 95 | cys |
Ala | Lys | Asn | Trp 100 | Ala | Pro | Phe | Asp | Ser 105 | Trp | Gly | Gin | Gly | Thr 110 | Leu | val |
Thr Val ser Ser | |
115 | |
35 40 45 | <210> 33 <211> 17 <212> PRT <213> Artificial <220> <223> GM37 variant 1 heavy chain CDR 2 <400> 33 Ala Ile Arg Ser Ser Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val Lys |
50 55 60 | 1 5 10 15 Gly <210> 34 <211> 17 <212> PRT <213> Artificial <220> <223> GM37 variant 2 CDR 2 heavy chain <400> 34 Ala île Arg Ser Gin Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val Lys |
15 10 15 |
108
Gly <210> 35 <211> 17 <212> PRT <213> Artificial <22O>
<223> GM37 variant 3 CDR 2 heavy chain <400> 35
Ala Ile Arg Ser. His Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val Lys
10 15
Gly <210> 36 <211> 5 <212> PRT <213> Artificial <220>
<223> 9E4 binding epitope <400> 36
Asn Glu Ala Tyr Glu 1 5 <210> 37 <211> 134 <212> PRT <213> Artificial <220>
<223> HUMAN Beta-synuclein <400> 37
Met Asp val Phe Met Lys Gly Leu Ser Met Ala Lys Glu Gly val val
1015
Ala Ala Ala Glu Lys Thr Lys Gin Gly val Thr Glu Ala Ala Glu Lys
2530
Thr Lys Glu Gly val Leu Tyr val Gly ser Lys Thr Arg Glu Gly Val
4045
Val Gin Gly Val Ala Ser Val Ala Glu Lys Thr Lys Glu Gin Ala ser
5560
109
Hi s 65 | Leu Gly | Gly | Ala val 70 | phe Ser | G1 y Al a | Gly Asn 75 | Ile | Ala | Ala | Ala 80 |
Thr | Gly Leu | val | Lys Arg 85 | Glu Glu | Phe Pro 90 | Thr Asp | Leu | Lys | Pro 95 | Glu |
Glu | val Ala | Gin 100 | Glu Ala | Ala Glu | Glu Pro 105 | Leu île | Glu | pro 110 | Leu | Met |
Glu Pro Glu Gly Glu Ser Tyr Glu 115 120 Glu Tyr Glu Pro Glu Ala 130 <210> 38 <211> 127 <212> PRT <213> Artificial <220> <223> HUMAN Gamma-synuclein <400> 38 | Asp Pro | pro Gin | Glu 125 | Glu | Tyr | Gin | ||||
Met 1 | Asp Val | Phe | Lys Lys 5 | Gly Phe | Ser Ile 10 | Ala Lys | Glu | Gly | Val 15 | Val |
Gly | Ala val | Glu 20 | Lys Thr | Lys Gin | Gly val 25 | Thr Glu | Ala | Ala 30 | Glu | Lys |
Thr | Lys Glu 35 | Gly | Val Met | Tyr Val 40 | Gly Ala | Lys Thr | Lys 45 | Glu | Asn | Val |
Val | Gin Ser 50 | Val | Thr Ser | val Ala 55 | Glu Lys | Thr Lys 60 | G1 u | G1 n | Ala | Asn |
Al a 65 | Val Ser | Glu | Ala Val 70 | Val Ser | Ser Val | Asn Thr 75 | val | Ala | Thr | Lys 80 |
Thr | val Glu | Glu | Ala Glu 85 | Asn Ile | Ala Val 90 | Thr ser | Gly | val | val 95 | Arg |
Lys | Glu ASp | Leu 100 | Arg Pro | ser Ala | Pro Gin 105 | Gin Glu | Gly | Glu 110 | Ala | Ser |
Lys | Glu Lys 115 | Glu | Glu Val | Ala Glu 120 | Glu Ala | Gin Ser | Gly Gly 125 | Asp |
<210> 39 <211> 140
110 <212> PRT <213> Artificial <220>
<223> alpha-synuclein ortholog for Cynomolgus monkey <400> 39
10 | Met ASp 1 | val | Phe Met Lys 5 | Gly | Leu | Ser Lys Ala 10 | Lys Glu | Gly | val 15 | val | |||||
15 | Ala Ala | Ala | Glu 20 | Lys | Thr | Lys | Gin | Gly 25 | val | Ala | Glu | Ala | Ala 30 | Gly | Lys |
Thr Lys | Glu 35 | Gly | val | Leu | Tyr | val 40 | Gly | Ser | Lys | Thr | Lys 45 | Glu | Gly | val | |
20 | Val His 50 | Gly | Val | Al a | Thr | Val 55 | Al a | Glu | Lys | Thr | Lys 60 | G1 u | Gin | Val | Thr |
25 | Asn Val 65 | Gly | Gly | Ala | Val 70 | val | Thr | Gly | val | Thr 75 | Ala | Val | Ala | Gin | Lys 80 |
30 | Thr val | Glu | Gly | Al a 85 | Gly | Ser | Ile | Ala | Al a 90 | Ala | Thr | Gly | phe | Ile 95 | Lys |
35 | Lys Asp | Gin | Leu 100 | Gly | Lys | Asn | Glu | Glu 105 | Gly | Ala | pro | G1 n | Glu 110 | Gly | île |
Leu Gin | Asp 115 | Met | Pro | Val | Asp | Pro 120 | ASp | Asn | Glu | Al a | Tyr 125 | Glu | Met | Pro | |
40 | Ser Glu 130 | Glu | Gly | Tyr | Gin | Asp 135 | Tyr | Glu | Pro | Glu | Ala 140 | ||||
45 | <210> <211> <212> <213> . | 40 140 PRT Arti fi ci al | |||||||||||||
50 | <220> <223> . | alpha-synuclein ortholog | for | Rat | |||||||||||
<400> | 40 | ||||||||||||||
55 | Met Asp 1 | Val | Phe | Met 5 | Lys | Gly | Leu | Ser | Lys 10 | Ala | Lys | Glu | Gly | val 15 | Val |
60 | Ala Ala | Ala | Glu 20 | Lys | Thr | Lys | G1 n | Gly 25 | Val | Al a | Glu | Al a | Al a 30 | Gly | Lys |
Thr Lys | Glu 35 | Gly | Val | Leu | Tyr | val 40 | Gly | Ser | Lys | Thr | Lys 45 | Glu | Gly | val |
111
Val | HIS 50 | Gly val Thr | Thr | val Ala 55 | Glu Lys | Thr | Lys 60 | Glu Gin | val | Thr | |||||
Asn 65 | val | Gly | Gly | Ala | val 70 | val | Thr | Gly | Val | Thr 75 | Al a | Val | Ala | Gin | Lys 80 |
Thr | val | Glu | Gly | Ala 85 | Gly | Asn | île | Al a | Al a 90 | Ala | Thr | Gly | Phe | Val 95 | Lys |
Lys | Asp | Gin | Met 100 | Gly | Lys | Gly | Glu | Glu 105 | Gly | Tyr | Pro | Gin | Glu 110 | Gly | île |
Leu | Glu | Asp 1.15 | Met | Pro | Val | Asp | Pro 120 | Ser | Ser | Glu | Ala | Tyr 125 | Glu | Met | Pro |
Ser | G1U | Glu | Gly | Tyr | Gin | Asp | Tyr | Glu | Pro | Glu | Al a |
130 135 140 <210> 41 <211> 140 <212> PRT <213> Artificial <220>
<223> | alpha-synuclein i | ortholog | for | Mouse | |||||||||||
<400> | 41 | ||||||||||||||
Met 1 | Asp | Val | Phe | Met 5 | Lys | Gly | Leu | Ser | Lys 10 | Al a | Lys | G1 u | Gly | val 15 | Val |
Ala | Al a | Ala | Glu 20 | Lys | Thr | Lys | Gin | Gly 25 | val | Ala | Glu | Ala | Ala 30 | Gly | Lys |
Thr | Lys | Glu 35 | Gly | val | Leu | Tyr | Val 40 | Gly | Ser | Lys | Thr | Lys 45 | Glu | Gly | Val |
val | Hi s 50 | Gly | Val | Thr | Thr | Val 55 | Ala | Glu | Lys | Thr | Lys 60 | Glu | Gin | val | Thr |
Asn 65 | val | Gly | Gly | Ala | val 70 | val | Thr | Gly | val | Thr 75 | Ala | val | Ala | Gin | Lys 80 |
Thr | Val | Glu | Gly | Ala 85 | Gly | Asn | Ile | Ala | Ala 90 | Ala | Thr | Gly | Phe | Val 95 | Lys |
Lys | Asp | Gin | Met 100 | Gly | Lys | Gly | Glu | Glu 105 | Gly | Tyr | Pro | Gin | Glu 110 | Gly | île |
112
Leu Glu Asp Met Pro Val Asp Pro
115 120
Gly ser Glu Ala Tyr Glu Met Pro
125 ser Glu Glu Gly Tyr Gin Asp Tyr
130 135 <210> 42 <211> 446 <212> PRT <213> artificial <220>
<223> 9E4 HC <400> 42
Glu val Gin Leu val Glu Ser Gly
5
Glu Pro Glu Ala
140
Gly Gly Leu val Gin Pro Gly Gly
15
Ser Leu Arg Leu Ser Cys Ala Ala 20
Ser Gly Phe Thr Phe Ser Asn Tyr
30
Gly Met Ser Trp val Arg Gin Ala
40
Pro Gly Lys Gly Leu Glu Trp Val
Ala Ser île Ser ser Gly Gly Gly
55
Ser Thr Tyr Tyr Pro Asp Asn val
Lys Gly Arg Phe Thr Ile ser Arg
70
Asp Asn Ala Lys Asn Ser Leu Tyr
80
Leu Gin Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala val Tyr Tyr cys
95
Ala Arg Gly Gly Ala Gly Ile Asp
100
Tyr Trp Gly Gin Gly Thr Leu Val
105 110
Thr Val ser ser Ala ser Thr Lys
115 120
Gly Pro Ser Val Phe Pro Leu Ala
125
Pro ser Ser Lys Ser Thr Ser Gly
130 135
Gly Thr Ala Ala Leu Gly Cys Leu
140
Val Lys Asp Tyr Phe Pro Glu Pro
145 150 val Thr Val ser Trp Asn Ser Gly
155 160
Ala Leu Thr ser Gly val His Thr
165
Phe Pro Ala val Leu Gin ser ser
170 175
Gly Leu Tyr ser Leu Ser Ser Val
180
Val Thr val Pro Ser Ser Ser Leu
185 190
113
5 | Gly | Thr | Gin 195 | Thr | Tyr | Ile | cys | Asn 200 | Val | Asn | His | Lys | Pro 205 | Ser | Asn | Thr |
Lys | val 210 | Asp | Lys | Arg | val | G1 u 215 | Pro | Lys | Ser | cys | Asp 220 | Lys | Thr | His | Thr | |
10 | cys 225 | Pro | Pro | cys | Pro | Ala 230 | Pro | Glu | Leu | Leu | Gly 235 | Gly | Pro | Ser | val | Phe 240 |
15 | Leu | Phe | Pro | pro | Lys 245 | Pro | Lys | ASp | Thr | Leu 250 | Met | Ile | Ser | Arg | Thr 255 | Pro |
20 | Glu | Val | Thr | cys 260 | Val | val | Val | ASp | val 265 | Ser | Hi s | Glu | Asp | ΡΓΟ 270 | Glu | val |
25 | Lys | Phe | Asn 275 | Trp | Tyr | val | Asp | Gly 280 | val | Glu | val | His | Asn 285 | Ala | Lys | Thr |
Lys | Pro 290 | Arg | Glu | G1 u | Gin | Tyr 295 | Asn | Ser | Thr | Tyr | Arg 300 | val | val | ser | val | |
30 | Leu 305 | Thr | val | Leu | His | Gin 310 | Asp | Trp | Leu | Asn | Gly 315 | Lys | Glu | Tyr | Lys | Cys 320 |
35 | Lys | val | ser | Asn | LVS 325 | Ala | Leu | Pro | Ala | Pro 330 | île | Glu | Lys | Thr | Ile 335 | Ser |
40 | Lys | Al a | Lys | Gly 340 | Gin | Pro | Arg | Glu | Pro 345 | Gin | val | Tyr | Thr | Leu 350 | Pro | Pro |
45 | Ser | Arg | Glu 355 | Glu | Met | Thr | Lys | Asn 360 | G1 n | Val | Ser | Leu | Thr 365 | Cys | Leu | val |
Lys | G1 y 370 | Phe | Tyr | Pro | ser | ASp 375 | île | Al a | val | Glu | Trp 380 | Glu | ser | Asn | Gly | |
50 | Gin 385 | Pro | Glu | Asn | Asn | Tyr 390 | Lys | Thr | Thr | Pro | Pro 395 | Val | Leu | Asp | ser | Asp 400 |
55 | Gly | Ser | Phe | Phe | Leu 405 | Tyr | Ser | Lys | Leu | Thr 410 | Val | Asp | Lys | Ser | Arg 415 | Trp |
60 | Gin | G1 n | Gly | Asn 420 | val | Phe | Ser | Cys | Ser 425 | Val | Met | Hi s | Glu | Ala 430 | Leu | His |
Asn | Hi s | Tyr 435 | Thr | Gin | Lys | Ser | Leu 440 | ser | Leu | ser | Pro | Gly 445 | Lys |
114 <210> 43 <211> 220 <212> PRT <213> artificial <220>
<223> 9E4 LC <400> 43
Asp île Gin Met Thr Gin Ser Pro
5
Ser Ser Leu Ser Ala Ser Val Gly
15
Asp Arg Val Thr Ile Thr Cys Lys ser île Gin Thr Leu Leu Tyr ser
30
Ser Asn Gin Lys Asn Tyr Leu Ala
40
Trp Phe Gin Gin Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Trp
55
Ala Ser Ile Arg Lys Ser Gly Val 60
Pro Ser Arg Phe Ser Gly Ser Gly
70
Ser Gly Thr Asp Phe Thr Leu Thr
80 île ser Ser Leu Gin Pro Glu Asp
Leu Ala Thr Tyr Tyr Cys Gin Gin
95
Tyr Tyr Ser Tyr Pro Leu Thr Phe
100
Gly Gly Gly Thr Lys Leu Glu île
105 110
Lys Arg Thr Val Ala Ala Pro ser
115 120 val Phe île Phe Pro Pro Ser Asp
125
Glu Gin Leu Lys ser Gly Thr Ala
130 135
Ser val val Cys Leu Leu Asn Asn
140
Phe Tyr pro Arg Glu Ala Lys val
145 150
Gin Trp Lys val Asp Asn Ala Leu
155 160
Gin ser Gly Asn ser Gin Glu ser
165 val Thr Glu Gin Asp ser Lys Asp
170 175
Ser Thr Tyr Ser Leu Ser Ser Thr
180
Leu Thr Leu Ser Lys Ala Asp Tyr
185 190
Glu Lys His Lys Val Tyr Ala Cys
195 200
Glu Val Thr His Gin Gly Leu Ser
205
115
18689“
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
116
3500
Claims (14)
- A monoclonal antibody capable of specifically binding to human alpha-synuclein,1- , .wherein said antibody binds an epitope within amino acids 112-117 (SEQ ID NO:9 (ILEDMP)) of human alpha-synuclein (SEQ ID NO:10), or antigen-binding fragment thereof that binds said epitope.5 2 The monoclonal antibody, or antigen-binding fragment thereof, according to daim 1, wherein said antibody is capable of competing with an antibody comprising the light chain variable domain of SEQ ID NO:8 and the heavy chain variable domain of SEQ ID NO:7, 30, 31 or 32 for binding to said epitope.3v A monoclonal antibody according to claim 1 capable of specifically binding to an epitope -tq τ within amino acids 112-115 (SEQ ID NO:19 (ILED) of human alpha-synuclein (SEQ IDNO:10)), or antigen-binding fragment thereof that binds said epitope.4* The monoclonal antibody, or antigen-binding fragment thereof, according to daims 1 or 3, wherein said antibody is capable of competing with an antibody comprising the heavy chain variable domain of SEQ ID NO:26 and the light chain variable domain of SEQ ID15 NO:27 for binding to said epitope5, A monoclonal antibody or the monoclonal antibody according to any one of daims 1-2, or a fragment thereof, comprising:(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:1;(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO;2;
- 2CX (c) a Heavy Chain CDR
- 3 having the amino acid sequence of SEQ ID NO:3;(d) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:
- 4;(e) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:6.6. The monodonai antibody according to daim 10, comprising the heavy chain variable25 domain of SEQ ID NO:7 or the light chain variable domain of SEQ ID NO:8.y The monodonai antibody according to daim 6, comprising a heavy chain consisting of a variable domain of SEQ ID NO:7 and a light chain consisting of a variable domain of SEQ ID NO:8.w ··.#. · ·' ·; Λ: 117 g A monoclonal antibody or the monoclonal antibody according to any one of daims 1-2, or a fragment thereof, comprising:(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:1;(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:33;
- 5 (c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:3;(d) a Light Chain CDR1 having the amino add sequence of SEQ ID NO:4;(e) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a Light Chain CDR3 having the amino add sequence of SEQ ID NO:6.g, The monoclonal antibody according to daim 8, comprising a heavy chain consisting of a <o variable domain of SEQ ID NO:30 and a light chain consisting of a variable domain ofSEQ ID NO:8.
- 10- A monoclonal antibody or the monoclonal antibody according to any one of daims 1-2 and 5-9, or a fragment thereof, comprising:(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:1;(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:34;(c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:3;(d) a Light Chain CDR1 having the amino acid sequence of SEQ IDNO:4;(e) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a Light Chain CDR3 having the amino acid sequence of SEQ IDNO:6.
- 11 The monodonal antibody according to daim 10, comprising a heavy chain consisting of a variable domain of SEQ ID NO:31 and a variable domain of SEQ ID NO:8..^2 A monoclonal antibody orthe monodonal antibody according to any one of claims 1-2, or a fragment thereof, comprising:(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:1;(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:35;(c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:3;(d) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:4;(e) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and ' (f) a Light Chain CDR3 having the amino add sequence of SEQ ID NO:6.
- 13- The monoclonal antibody according to claim 12, comprising a heavy chain consisting of > a variable domain of SEQ ID NO:32 and a variable domain of SEQ ID NO:8.118
- 14. A monoclonal antibody or the monoclonal antibody according to any one of claims 1, 3, or a fragment thereof, comprising:(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21;5 (c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22;(d) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:23;(e) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:24; and (f) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:25.
- 15- The monoclonal antibody according to claim 14, comprising a heavy chain consisting-of-rc ‘ “ a variable domain of SEQ ID NO:26 and a variable domain of SEQ ID NO:27.
- 16- A pharmaceutical composition comprising the monoclonal antibody according to any one ofthe claims 1-15, and a pharmaceutical acceptable carrier.
- 17., A nucleic acid encoding the antibody or the fragment according to any one of claims 5-15.; ή g The monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1-15 for use in therapy.
- 19. The monoclonal antibody, or antigen-binding fragment thereof, according to any one of —* claims 1-15 for use in treating a synucleinopathy.
- 20- The monoclonal antibody, or antigen-binding fragment thereof, according to claim 19 for use in treating Parkinson's disease, idiopathic and inherited forms of Parkinson's2C disease, Gauchers Disease (GD), Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Combîned Alzheimer's and Parkinson disease, pure autonomie failure or multiple system atrophy.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1512203.9 | 2015-07-13 |
Publications (1)
Publication Number | Publication Date |
---|---|
OA18689A true OA18689A (en) | 2019-05-17 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11421024B2 (en) | Agents, uses and methods for the treatment of synucleinopathy | |
US20210236634A1 (en) | Agents, uses and methods for the treatment of synucleinopathy | |
EP3555127A1 (en) | Agents, uses and methods | |
OA18689A (en) | Agents, Uses and Methods for the Treatment of Synucleinopathy |