CN107698684A - The fusion proteins of GLP 1 of Fc portion of immunoglobulin comprising mutation - Google Patents
The fusion proteins of GLP 1 of Fc portion of immunoglobulin comprising mutation Download PDFInfo
- Publication number
- CN107698684A CN107698684A CN201710630742.6A CN201710630742A CN107698684A CN 107698684 A CN107698684 A CN 107698684A CN 201710630742 A CN201710630742 A CN 201710630742A CN 107698684 A CN107698684 A CN 107698684A
- Authority
- CN
- China
- Prior art keywords
- ser
- val
- glu
- amino acid
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 83
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 83
- 108060003951 Immunoglobulin Proteins 0.000 title claims abstract description 43
- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 43
- 230000035772 mutation Effects 0.000 title abstract description 21
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 title abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 43
- 239000003814 drug Substances 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 20
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims description 94
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 claims description 69
- 235000001014 amino acid Nutrition 0.000 claims description 66
- 150000001413 amino acids Chemical class 0.000 claims description 54
- 229940024606 amino acid Drugs 0.000 claims description 45
- 210000004027 cell Anatomy 0.000 claims description 28
- 238000006467 substitution reaction Methods 0.000 claims description 21
- 239000012634 fragment Substances 0.000 claims description 12
- 102000040430 polynucleotide Human genes 0.000 claims description 12
- 108091033319 polynucleotide Proteins 0.000 claims description 12
- 239000002157 polynucleotide Substances 0.000 claims description 12
- 210000004899 c-terminal region Anatomy 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 10
- 125000000539 amino acid group Chemical group 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 5
- 235000004279 alanine Nutrition 0.000 claims description 5
- 206010012601 diabetes mellitus Diseases 0.000 claims description 5
- 150000004665 fatty acids Chemical class 0.000 claims description 5
- 239000000710 homodimer Substances 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 102200069889 rs104893964 Human genes 0.000 claims description 5
- 102200077973 rs113068438 Human genes 0.000 claims description 5
- 102220074722 rs796053167 Human genes 0.000 claims description 5
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 claims description 4
- 241000699802 Cricetulus griseus Species 0.000 claims description 4
- 208000008589 Obesity Diseases 0.000 claims description 4
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 4
- 229930195729 fatty acid Natural products 0.000 claims description 4
- 239000000194 fatty acid Substances 0.000 claims description 4
- 230000002209 hydrophobic effect Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 235000020824 obesity Nutrition 0.000 claims description 4
- 210000001672 ovary Anatomy 0.000 claims description 4
- 239000000539 dimer Substances 0.000 claims description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 2
- 229960000310 isoleucine Drugs 0.000 claims description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 2
- 229930182817 methionine Natural products 0.000 claims description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 2
- 239000004474 valine Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 102100025101 GATA-type zinc finger protein 1 Human genes 0.000 claims 8
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims 1
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 description 65
- 125000003275 alpha amino acid group Chemical group 0.000 description 43
- 108010001064 glycyl-glycyl-glycyl-glycine Proteins 0.000 description 27
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 19
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 19
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 18
- 102000004196 processed proteins & peptides Human genes 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 15
- 229940079593 drug Drugs 0.000 description 13
- 231100000350 mutagenesis Toxicity 0.000 description 13
- 238000002703 mutagenesis Methods 0.000 description 12
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 11
- LLWQVJNHMYBLLK-CDMKHQONSA-N Gly-Thr-Phe Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LLWQVJNHMYBLLK-CDMKHQONSA-N 0.000 description 10
- HAPWZEVRQYGLSG-IUCAKERBSA-N His-Gly-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O HAPWZEVRQYGLSG-IUCAKERBSA-N 0.000 description 10
- IVDFVBVIVLJJHR-LKXGYXEUSA-N Thr-Ser-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IVDFVBVIVLJJHR-LKXGYXEUSA-N 0.000 description 10
- KSCVLGXNQXKUAR-JYJNAYRXSA-N Tyr-Leu-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O KSCVLGXNQXKUAR-JYJNAYRXSA-N 0.000 description 10
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 10
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 10
- 108010045383 histidyl-glycyl-glutamic acid Proteins 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- QMOQBVOBWVNSNO-UHFFFAOYSA-N 2-[[2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]acetyl]amino]acetate Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(O)=O QMOQBVOBWVNSNO-UHFFFAOYSA-N 0.000 description 9
- DPNZTBKGAUAZQU-DLOVCJGASA-N Ala-Leu-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N DPNZTBKGAUAZQU-DLOVCJGASA-N 0.000 description 9
- OINVDEKBKBCPLX-JXUBOQSCSA-N Ala-Lys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OINVDEKBKBCPLX-JXUBOQSCSA-N 0.000 description 9
- BVLIJXXSXBUGEC-SRVKXCTJSA-N Asn-Asn-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BVLIJXXSXBUGEC-SRVKXCTJSA-N 0.000 description 9
- NYGILGUOUOXGMJ-YUMQZZPRSA-N Asn-Lys-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O NYGILGUOUOXGMJ-YUMQZZPRSA-N 0.000 description 9
- HNXWVVHIGTZTBO-LKXGYXEUSA-N Asn-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O HNXWVVHIGTZTBO-LKXGYXEUSA-N 0.000 description 9
- KBQOUDLMWYWXNP-YDHLFZDLSA-N Asn-Val-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC(=O)N)N KBQOUDLMWYWXNP-YDHLFZDLSA-N 0.000 description 9
- WSGVTKZFVJSJOG-RCOVLWMOSA-N Asp-Gly-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O WSGVTKZFVJSJOG-RCOVLWMOSA-N 0.000 description 9
- CUQDCPXNZPDYFQ-ZLUOBGJFSA-N Asp-Ser-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O CUQDCPXNZPDYFQ-ZLUOBGJFSA-N 0.000 description 9
- OHLLDUNVMPPUMD-DCAQKATOSA-N Cys-Leu-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N OHLLDUNVMPPUMD-DCAQKATOSA-N 0.000 description 9
- SMEYEQDCCBHTEF-FXQIFTODSA-N Cys-Pro-Ala Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O SMEYEQDCCBHTEF-FXQIFTODSA-N 0.000 description 9
- KSMSFCBQBQPFAD-GUBZILKMSA-N Cys-Pro-Pro Chemical compound SC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 KSMSFCBQBQPFAD-GUBZILKMSA-N 0.000 description 9
- ALTQTAKGRFLRLR-GUBZILKMSA-N Cys-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N ALTQTAKGRFLRLR-GUBZILKMSA-N 0.000 description 9
- YJIUYQKQBBQYHZ-ACZMJKKPSA-N Gln-Ala-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YJIUYQKQBBQYHZ-ACZMJKKPSA-N 0.000 description 9
- CGVWDTRDPLOMHZ-FXQIFTODSA-N Gln-Glu-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O CGVWDTRDPLOMHZ-FXQIFTODSA-N 0.000 description 9
- KCJJFESQRXGTGC-BQBZGAKWSA-N Gln-Glu-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O KCJJFESQRXGTGC-BQBZGAKWSA-N 0.000 description 9
- AQPZYBSRDRZBAG-AVGNSLFASA-N Gln-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N AQPZYBSRDRZBAG-AVGNSLFASA-N 0.000 description 9
- HMIXCETWRYDVMO-GUBZILKMSA-N Gln-Pro-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O HMIXCETWRYDVMO-GUBZILKMSA-N 0.000 description 9
- RFDHKPSHTXZKLL-IHRRRGAJSA-N Glu-Gln-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)O)N RFDHKPSHTXZKLL-IHRRRGAJSA-N 0.000 description 9
- YHOJJFFTSMWVGR-HJGDQZAQSA-N Glu-Met-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YHOJJFFTSMWVGR-HJGDQZAQSA-N 0.000 description 9
- AAJHGGDRKHYSDH-GUBZILKMSA-N Glu-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O AAJHGGDRKHYSDH-GUBZILKMSA-N 0.000 description 9
- WGYHAAXZWPEBDQ-IFFSRLJSSA-N Glu-Val-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WGYHAAXZWPEBDQ-IFFSRLJSSA-N 0.000 description 9
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 9
- GMTXWRIDLGTVFC-IUCAKERBSA-N Gly-Lys-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMTXWRIDLGTVFC-IUCAKERBSA-N 0.000 description 9
- OOCFXNOVSLSHAB-IUCAKERBSA-N Gly-Pro-Pro Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 OOCFXNOVSLSHAB-IUCAKERBSA-N 0.000 description 9
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 description 9
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 9
- JSLVAHYTAJJEQH-QWRGUYRKSA-N Gly-Ser-Phe Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JSLVAHYTAJJEQH-QWRGUYRKSA-N 0.000 description 9
- WZPIKDWQVRTATP-SYWGBEHUSA-N Ile-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)[C@@H](C)CC)C(O)=O)=CNC2=C1 WZPIKDWQVRTATP-SYWGBEHUSA-N 0.000 description 9
- DFJJAVZIHDFOGQ-MNXVOIDGSA-N Ile-Glu-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N DFJJAVZIHDFOGQ-MNXVOIDGSA-N 0.000 description 9
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 9
- 241000880493 Leptailurus serval Species 0.000 description 9
- UHNQRAFSEBGZFZ-YESZJQIVSA-N Leu-Phe-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N UHNQRAFSEBGZFZ-YESZJQIVSA-N 0.000 description 9
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 description 9
- CHJKEDSZNSONPS-DCAQKATOSA-N Leu-Pro-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O CHJKEDSZNSONPS-DCAQKATOSA-N 0.000 description 9
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 9
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 9
- YKIRNDPUWONXQN-GUBZILKMSA-N Lys-Asn-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YKIRNDPUWONXQN-GUBZILKMSA-N 0.000 description 9
- KWUKZRFFKPLUPE-HJGDQZAQSA-N Lys-Asp-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWUKZRFFKPLUPE-HJGDQZAQSA-N 0.000 description 9
- DUTMKEAPLLUGNO-JYJNAYRXSA-N Lys-Glu-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DUTMKEAPLLUGNO-JYJNAYRXSA-N 0.000 description 9
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 9
- GILLQRYAWOMHED-DCAQKATOSA-N Lys-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN GILLQRYAWOMHED-DCAQKATOSA-N 0.000 description 9
- JDMKQHSHKJHAHR-UHFFFAOYSA-N Phe-Phe-Leu-Tyr Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)CC1=CC=CC=C1 JDMKQHSHKJHAHR-UHFFFAOYSA-N 0.000 description 9
- SJRQWEDYTKYHHL-SLFFLAALSA-N Phe-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CC=CC=C3)N)C(=O)O SJRQWEDYTKYHHL-SLFFLAALSA-N 0.000 description 9
- IHCXPSYCHXFXKT-DCAQKATOSA-N Pro-Arg-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O IHCXPSYCHXFXKT-DCAQKATOSA-N 0.000 description 9
- LHALYDBUDCWMDY-CIUDSAMLSA-N Pro-Glu-Ala Chemical compound C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1)C(O)=O LHALYDBUDCWMDY-CIUDSAMLSA-N 0.000 description 9
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 9
- ULWBBFKQBDNGOY-RWMBFGLXSA-N Pro-Lys-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N2CCC[C@@H]2C(=O)O ULWBBFKQBDNGOY-RWMBFGLXSA-N 0.000 description 9
- KHRLUIPIMIQFGT-AVGNSLFASA-N Pro-Val-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O KHRLUIPIMIQFGT-AVGNSLFASA-N 0.000 description 9
- UEJYSALTSUZXFV-SRVKXCTJSA-N Rigin Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O UEJYSALTSUZXFV-SRVKXCTJSA-N 0.000 description 9
- WDXYVIIVDIDOSX-DCAQKATOSA-N Ser-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CCCN=C(N)N WDXYVIIVDIDOSX-DCAQKATOSA-N 0.000 description 9
- HZWAHWQZPSXNCB-BPUTZDHNSA-N Ser-Arg-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O HZWAHWQZPSXNCB-BPUTZDHNSA-N 0.000 description 9
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 9
- QPFJSHSJFIYDJZ-GHCJXIJMSA-N Ser-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CO QPFJSHSJFIYDJZ-GHCJXIJMSA-N 0.000 description 9
- MOVJSUIKUNCVMG-ZLUOBGJFSA-N Ser-Cys-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N)O MOVJSUIKUNCVMG-ZLUOBGJFSA-N 0.000 description 9
- ZOHGLPQGEHSLPD-FXQIFTODSA-N Ser-Gln-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZOHGLPQGEHSLPD-FXQIFTODSA-N 0.000 description 9
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 9
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 9
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 9
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 9
- QJKPECIAWNNKIT-KKUMJFAQSA-N Ser-Lys-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QJKPECIAWNNKIT-KKUMJFAQSA-N 0.000 description 9
- HNDMFDBQXYZSRM-IHRRRGAJSA-N Ser-Val-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HNDMFDBQXYZSRM-IHRRRGAJSA-N 0.000 description 9
- LAFLAXHTDVNVEL-WDCWCFNPSA-N Thr-Gln-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O LAFLAXHTDVNVEL-WDCWCFNPSA-N 0.000 description 9
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 9
- SSSDKJMQMZTMJP-BVSLBCMMSA-N Trp-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CC=C(O)C=C1 SSSDKJMQMZTMJP-BVSLBCMMSA-N 0.000 description 9
- GZUIDWDVMWZSMI-KKUMJFAQSA-N Tyr-Lys-Cys Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CS)C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 GZUIDWDVMWZSMI-KKUMJFAQSA-N 0.000 description 9
- BMGOFDMKDVVGJG-NHCYSSNCSA-N Val-Asp-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BMGOFDMKDVVGJG-NHCYSSNCSA-N 0.000 description 9
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 9
- OACSGBOREVRSME-NHCYSSNCSA-N Val-His-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(N)=O)C(O)=O OACSGBOREVRSME-NHCYSSNCSA-N 0.000 description 9
- BGTDGENDNWGMDQ-KJEVXHAQSA-N Val-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N)O BGTDGENDNWGMDQ-KJEVXHAQSA-N 0.000 description 9
- 108010005233 alanylglutamic acid Proteins 0.000 description 9
- 108010087924 alanylproline Proteins 0.000 description 9
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 9
- 108010047857 aspartylglycine Proteins 0.000 description 9
- 230000004927 fusion Effects 0.000 description 9
- 108010013768 glutamyl-aspartyl-proline Proteins 0.000 description 9
- 108010049041 glutamylalanine Proteins 0.000 description 9
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 9
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 9
- 108010010147 glycylglutamine Proteins 0.000 description 9
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 9
- 108010064235 lysylglycine Proteins 0.000 description 9
- 108010073101 phenylalanylleucine Proteins 0.000 description 9
- 108010051242 phenylalanylserine Proteins 0.000 description 9
- 108010077112 prolyl-proline Proteins 0.000 description 9
- 108010031719 prolyl-serine Proteins 0.000 description 9
- 108010070643 prolylglutamic acid Proteins 0.000 description 9
- 108010071097 threonyl-lysyl-proline Proteins 0.000 description 9
- 108010080629 tryptophan-leucine Proteins 0.000 description 9
- 108010051110 tyrosyl-lysine Proteins 0.000 description 9
- 108010052774 valyl-lysyl-glycyl-phenylalanyl-tyrosine Proteins 0.000 description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 8
- YUELDQUPTAYEGM-XIRDDKMYSA-N Asp-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC(=O)O)N YUELDQUPTAYEGM-XIRDDKMYSA-N 0.000 description 8
- 108091006020 Fc-tagged proteins Proteins 0.000 description 8
- BPCLDCNZBUYGOD-BPUTZDHNSA-N Glu-Trp-Glu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 BPCLDCNZBUYGOD-BPUTZDHNSA-N 0.000 description 8
- BKTXKJMNTSMJDQ-AVGNSLFASA-N Leu-His-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N BKTXKJMNTSMJDQ-AVGNSLFASA-N 0.000 description 8
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 8
- QYSBJAUCUKHSLU-JYJNAYRXSA-N Tyr-Arg-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O QYSBJAUCUKHSLU-JYJNAYRXSA-N 0.000 description 8
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 108010011459 Exenatide Proteins 0.000 description 7
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 7
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 7
- RKIIYGUHIQJCBW-SRVKXCTJSA-N Met-His-Glu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O RKIIYGUHIQJCBW-SRVKXCTJSA-N 0.000 description 7
- OGWAVGNOAMXIIM-UHFFFAOYSA-N albiglutide Chemical compound O=C(O)C(NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)CNC(=O)C(NC(=O)CNC(=O)C(N)CC=1(N=CNC=1))CCC(=O)O)C(O)C)CC2(=CC=CC=C2))C(O)C)CO)CC(=O)O)C(C)C)CO)CO)CC3(=CC=C(O)C=C3))CC(C)C)CCC(=O)O)CCC(=O)N)C)C)CCCCN)CCC(=O)O)CC4(=CC=CC=C4))C(CC)C)C)CC=6(C5(=C(C=CC=C5)NC=6)))CC(C)C)C(C)C)CCCCN)CCCNC(=N)N OGWAVGNOAMXIIM-UHFFFAOYSA-N 0.000 description 7
- 229960001519 exenatide Drugs 0.000 description 7
- 108700027806 rGLP-1 Proteins 0.000 description 7
- HTQBXNHDCUEHJF-XWLPCZSASA-N Exenatide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 HTQBXNHDCUEHJF-XWLPCZSASA-N 0.000 description 6
- 108010019598 Liraglutide Proteins 0.000 description 6
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 description 6
- 241000700159 Rattus Species 0.000 description 6
- 229960004733 albiglutide Drugs 0.000 description 6
- 229960002701 liraglutide Drugs 0.000 description 6
- HPSVTWMFWCHKFN-GARJFASQSA-N Arg-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O HPSVTWMFWCHKFN-GARJFASQSA-N 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- LBFXVAXPDOBRKU-LKTVYLICSA-N Ala-His-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LBFXVAXPDOBRKU-LKTVYLICSA-N 0.000 description 4
- ZJBUILVYSXQNSW-YTWAJWBKSA-N Arg-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ZJBUILVYSXQNSW-YTWAJWBKSA-N 0.000 description 4
- 108010067722 Dipeptidyl Peptidase 4 Proteins 0.000 description 4
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 4
- XVVOERDUTLJJHN-UHFFFAOYSA-N Lixisenatide Chemical compound C=1NC2=CC=CC=C2C=1CC(C(=O)NC(CC(C)C)C(=O)NC(CCCCN)C(=O)NC(CC(N)=O)C(=O)NCC(=O)NCC(=O)N1C(CCC1)C(=O)NC(CO)C(=O)NC(CO)C(=O)NCC(=O)NC(C)C(=O)N1C(CCC1)C(=O)N1C(CCC1)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)CC)NC(=O)C(NC(=O)C(CC(C)C)NC(=O)C(CCCNC(N)=N)NC(=O)C(NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(CCC(O)=O)NC(=O)C(CCC(O)=O)NC(=O)C(CCSC)NC(=O)C(CCC(N)=O)NC(=O)C(CCCCN)NC(=O)C(CO)NC(=O)C(CC(C)C)NC(=O)C(CC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(CC=1C=CC=CC=1)NC(=O)C(NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)CNC(=O)C(N)CC=1NC=NC=1)C(C)O)C(C)O)C(C)C)CC1=CC=CC=C1 XVVOERDUTLJJHN-UHFFFAOYSA-N 0.000 description 4
- FWAHLGXNBLWIKB-NAKRPEOUSA-N Met-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCSC FWAHLGXNBLWIKB-NAKRPEOUSA-N 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 108010005794 dulaglutide Proteins 0.000 description 4
- 229960005175 dulaglutide Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 108010040030 histidinoalanine Proteins 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 229960001093 lixisenatide Drugs 0.000 description 4
- 108010004367 lixisenatide Proteins 0.000 description 4
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical group C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- HKZAAJSTFUZYTO-LURJTMIESA-N (2s)-2-[[2-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoic acid Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O HKZAAJSTFUZYTO-LURJTMIESA-N 0.000 description 3
- WQLJRNRLHWJIRW-KKUMJFAQSA-N Asn-His-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC(=O)N)N)O WQLJRNRLHWJIRW-KKUMJFAQSA-N 0.000 description 3
- 102000016622 Dipeptidyl Peptidase 4 Human genes 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 101000930822 Giardia intestinalis Dipeptidyl-peptidase 4 Proteins 0.000 description 3
- 108010086246 Glucagon-Like Peptide-1 Receptor Proteins 0.000 description 3
- 102100032882 Glucagon-like peptide 1 receptor Human genes 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 108700005078 Synthetic Genes Proteins 0.000 description 3
- QARCDOCCDOLJSF-HJPIBITLSA-N Tyr-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N QARCDOCCDOLJSF-HJPIBITLSA-N 0.000 description 3
- 125000001931 aliphatic group Chemical group 0.000 description 3
- 239000012930 cell culture fluid Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229940126534 drug product Drugs 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 230000002218 hypoglycaemic effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000000825 pharmaceutical preparation Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 229940081330 tena Drugs 0.000 description 3
- 230000003442 weekly effect Effects 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 101800004266 Glucagon-like peptide 1(7-37) Proteins 0.000 description 2
- LYCVKHSJGDMDLM-LURJTMIESA-N His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 LYCVKHSJGDMDLM-LURJTMIESA-N 0.000 description 2
- KXODZBLFVFSLAI-AVGNSLFASA-N Leu-His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CN=CN1 KXODZBLFVFSLAI-AVGNSLFASA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- WEQAYODCJHZSJZ-KKUMJFAQSA-N Ser-His-Tyr Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 WEQAYODCJHZSJZ-KKUMJFAQSA-N 0.000 description 2
- BXPOOVDVGWEXDU-WZLNRYEVSA-N Tyr-Ile-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BXPOOVDVGWEXDU-WZLNRYEVSA-N 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 108010036413 histidylglycine Proteins 0.000 description 2
- 108010018006 histidylserine Proteins 0.000 description 2
- 125000001165 hydrophobic group Chemical group 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000002708 random mutagenesis Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- NGJOFQZEYQGZMB-KTKZVXAJSA-N (4S)-5-[[2-[[(2S,3R)-1-[[(2S)-1-[[(2S,3R)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[2-[[(1S)-4-carbamimidamido-1-carboxybutyl]amino]-2-oxoethyl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-2-oxoethyl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-2-oxoethyl]amino]-4-[[(2S)-2-[[(2S)-2-amino-3-(1H-imidazol-4-yl)propanoyl]amino]propanoyl]amino]-5-oxopentanoic acid Chemical group C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 NGJOFQZEYQGZMB-KTKZVXAJSA-N 0.000 description 1
- PXAFZDXYEIIUTF-LKTVYLICSA-N Ala-Trp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(O)=O PXAFZDXYEIIUTF-LKTVYLICSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108020004414 DNA Proteins 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
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 1
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 1
- 241000270322 Lepidosauria Species 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 230000002862 amidating effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- JUFFVKRROAPVBI-PVOYSMBESA-N chembl1210015 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N[C@H]1[C@@H]([C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@]3(O[C@@H](C[C@H](O)[C@H](O)CO)[C@H](NC(C)=O)[C@@H](O)C3)C(O)=O)O2)O)[C@@H](CO)O1)NC(C)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 JUFFVKRROAPVBI-PVOYSMBESA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000003890 endocrine cell Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000030136 gastric emptying Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 150000002885 octadecanoids Chemical class 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Abstract
The present invention relates to the fusion protein comprising GLP 1 or its analog, the Fc portion of immunoglobulin of peptide linker and mutation, and it has increased half-life period.The present invention also provides the application of the method and the fusion protein that produce the fusion protein in medicine is prepared.
Description
Technical field
The present invention relates to glucagon-like-peptide-1 (glucagon-like peptide-1, GLP-1) fusion protein,
It includes the Fc portion of immunoglobulin of mutation, thus with half-life period inside extending.The fusion protein can be used for treatment sugar
Urine disease, fat and other relevant diseases or illness.
Background technology
Glucagon-like-peptide-1 (glucagon-like peptide-1, GLP-1) is separated from intestinal mucosa
Extract, be a kind of braingut petide of ileum endocrine cells secrete, at present mainly as the pharmaceutically-active target of diabetes B
Point.Because GLP-1 can suppress gastric emptying, enterocinesia is reduced, therefore contributes to control to ingest, is lost weight.
Initially caused GLP-1 is 37 peptides to enteron aisle, is inactive peptide chain, need to digest the excision peptide of N-terminal 6, turn into life
The GLP-1 (7~37) of thing activity, its C-terminal glycine can be so, naturally-produced in enteron aisle as the substrate of amidating enzyme
GLP-1 to have 80% or so be GLP-1 (7~36) acid amides, its sequence all same in the mammal studied at present.C
It is terminus amidated to add stability inside GLP-1.
Natural GLP-1 (7-37) amino acid sequence is:
HisAlaGluGlyThrPheThrSerAspValSerSerTyrLeuGluGlyGlnAlaAlaLysGluPheIleAlaTrpLe
uValLysGlyArgGly
GLP-1 has N-terminal and C-terminal, and N-terminal is relevant with its physiologically active, and C-terminal is combined relevant with acceptor.Dipeptidyl peptidase Ⅳ
(dipeptidyl peptidase- IV, DPP IV) can catalyzing hydrolysis GLP-1N the 2nd, ends alanine, formation GLP-1 (9~
36)NH2Lose activity, be natural agonist inside GLP-1R.GLP-1 biological half-life is shorter, is 1~1.5min, very
Degraded soon by dipeptidyl peptidase Ⅳ, be difficult clinically the concentration for detecting it in blood therefore.Therefore structure is carried out to GLP-1 to repair
Decorations, the GLP-1 analogs with same pharmacological activity are formed, and cover the binding site of DPP-IV, it is such to extend half-life period
The major subjects of medicament research and development.
In the past few years, gift comes, Novo Nordisk, and GSK etc. competitively transforms the albumen, to obtain long-acting GLP-1 class
Hypoglycemic medicine.
Exenatide (exenatide) is the biologically active peptide extracted from lizard salivary gland, its amino acid sequence with
GLP-1 has 53% homology.Research shows that its dosage period can extend to twice daily.The amino acid sequence of Exenatide is such as
Shown in lower:
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-
Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
Pro-Pro-Pro-Ser-NH2
Because its N-terminal second by Gly instead of Ala in GLP-1, do not degraded by DPP-IV, and partly declined with longer
Phase and stronger bioactivity.
Liraglutide (liraglutide) is the medicine that fatty acid chain modification is carried out to GLP-1 albumen, and dosage period extends
To once a day.Liraglutide is that 34 Lys are substituted by Arg on GLP-1 (7-37) chain, and access is through 16 on the Lys of 26
The glutamine of alkanoic acid modification.GLP-1 adds the affinity between albumin after aliphatic chain is modified, so as to reduce
By the hydrolysis rate and renal clearance of DPP-IV, extend biological half-life.
Li Sina peptides (Lixisenatide) (trade name:Lyxumia it is) by French Sanofi Aventis and Zealand
Company's joint development.Li Sina peptide amino acid sequences are as follows:
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-
Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH2
From structure, Li Sina peptides are the Pro that Exendin-4 removes 38, and meet 6 Lys in the Ser of 39.Through
Structural modification is crossed, Lixisenatide half-life period has extended with respect to Exenatide, can be subcutaneously injected once a day.
Albiglutide (Albiglutide) (trade name:Eperzan) researched and developed on every Mondays by GlaxoSmithKline
Secondary hypodermic long-acting GLP-1 analog.From structure, Albiglutide is by GLP-1 (7-36) chain 8
Ala has been substituted for Gly, then two GLP-1 peptide chains fused in tandem through modified are white in the serum containing 585 residues
On albumen, half-life period so substantially prolongs.
Du Lalu peptides (Dulaglutide) are the weekly hypodermic long-acting GLP-1 classes researched and developed by Lily companies
Like thing.From structure, Dulaglutide is that the Ala of 8 on GLP-1 (7-37) chain has been substituted for into Gly, and the Gly of 22 is replaced
Glu is changed into, the Arg of 36 has been substituted for Gly, then is fused to restructuring IgG4 by " GGGGSGGGGSGGGGSA " coupling bridge and exempts from
On the glutamic acid of 216 of epidemic disease albumin (containing 227 amino acid Fc fragments), average organism long half time was up to 90 hours.
Suo Malu peptides (Semeglutide) are by the weekly hypodermic long-acting of Nove Nordisk companies research and development
GLP-1 analogs.Semeglutide is that the Ala of 8 on GLP-1 (7-37) chain is substituted for into Aib from structure, 34
Lys is substituted for Arg, and the Lys of 26 connects octadecanoid acid aliphatic chain.Compared with Liraglutide, Suo Malu peptide aliphatic chains are longer, dredge
Water-based increase, but Suo Malu peptides are modified by the PEG of short chain, hydrophily greatly enhances.Not only can be with white egg after PEG modifications
Combine closely in vain, cover DPP-4 enzyme hydrolysis site, moreover it is possible to reduce renal excretion, half-life period can be extended, reach macrocyclic effect.
GLP-1 class medicines the most long-acting in the market, its frequency of injection is weekly.The developmental function time is more excellent
Long-acting GLP-1 class medicine, advantageously reduce frequently drug injection, improve the compliance of patient.
The content of the invention
In one aspect of the invention, there is provided a kind of fusion protein, it includes or consisted of:GLP-1 or its class
Like thing;Peptide linker;And Fc portion of immunoglobulin, wherein the Fc portion of immunoglobulin (is numbered according to EU in 434 sites and is
Unified editing number) amino acid N replaced by weak hydrophobic amino acid, wherein the hydrophobic amino acid is selected from alanine, valine, bright
Propylhomoserin, isoleucine, proline, phenylalanine, tryptophan and methionine, preferably alanine.Thus have in animal (preferably
Mammal, such as mouse, more preferably people) internal increased half-life period.
In an embodiment of the fusion protein of the present invention, the C-terminal of the GLP-1 or its analog passes through described
Peptide linker merges with the N-terminal of the Fc parts, and/or wherein described Fc portion of immunoglobulin from IgG 1, IgG2,
IgG3 or IgG4.
In another embodiment of the fusion protein of the present invention, the Fc portion of immunoglobulin also comprising one or
The amino acid substitution that multiple (for example, 1,2,3,4,5,6,7 or 8) are selected from the group consisted of (is compiled according to EU numbering systems
Number):S228P, F234A, L235A, M252Y, T256E, T307A, E380A and M428L, S228P, F234A are preferably comprised,
L235A and optionally one or more (such as 1,2,3,4 or 5) be selected from the group consisted of amino acid substitution (according to
EU numbering systems are numbered):M252Y, T256E, T307A, E380A and M428L, more preferably comprising S228P, F234A, L235A and
The amino acid substitution that optionally one or more (such as 1,2,3,4 or 5) is selected from the group consisted of (is numbered according to EU and is
Unified editing number):T307A, E380A and M428L.
In another embodiment of the fusion protein of the present invention, the Fc portion of immunoglobulin is included selected from following
Amino acid substitution (according to EU numbering systems number) combination:
1)S228P+F234A+L235A+N434A;
2)S228P+F234A+L235A+M428L+N434A;
3)S228P+F234A+L235A+T307A+E380A+N434A;With
4)S228P+F234A+L235A+M252Y+T256E+N434A。
In another embodiment of the fusion protein of the present invention, the fusion protein have it is one or more be selected from
Under feature:
1) there are one or more (2 or 3) to be selected from following amino acid substitution (root for the GLP-1 or its analog
Numbered according to EU numbering systems):A8G, G22E and R36G;
2) GLP-1 or its analog have on 1-5 (such as 1,2,3,4 or 5) amino acid residues
(C12-24, such as C12、C14、C16Or C18Long-chain) fatty acid modifying;With
3) the 27th amino acids and Fc portion of immunoglobulin of the GLP-1 of the fusion protein or its analog part
216th amino acids (being numbered according to EU numbering systems) carry negative electrical charge, and GLP-1 or the 34th ammonia of its analog part
218th amino acids site of base acid and Fc portion of immunoglobulin (being numbered according to EU numbering systems) carries positive charge, and
And GLP-1 or its analog and Fc portion of immunoglobulin tundish are containing (10-30 amino acid residue) continuous polarity ammonia
Base acid residue segment.
In another embodiment of the fusion protein of the present invention, the amino acid sequence of the fusion protein is selected from SEQ
ID NO.1, SEQ ID NO.2, the group of SEQ ID NO.3 and SEQ ID NO.4 compositions;And/or wherein described GLP-1 or its class
Like the amino acid sequence of thing as shown in SEQ ID NO.6;And/or the amino acid sequence of wherein described peptide linker such as SEQ ID
Shown in NO.7.
In another embodiment of the fusion protein of the present invention, in the form of the fusion protein is in the form of dimer
In the presence of the preferably presence in the form of homodimer.
In another aspect of the present invention, there is provided polynucleotides, it encodes the fusion protein of the present invention.
In another aspect of the present invention, carrier is additionally provided, it includes the polynucleotides according to the present invention.
In another aspect of the present invention, host cell is additionally provided, it includes support according to the present invention, preferably described
Host cell is Chinese hamster ovary celI.
In another aspect of the present invention, a kind of method for producing the fusion protein according to the present invention is additionally provided, it is described
Method, which is included in host cell, expresses support according to the present invention.
In another aspect of the present invention, application of the fusion protein of the present invention in medicine is prepared is additionally provided, preferably
The medicine is used to treat diabetes or obesity.
Brief description of the drawings
Fig. 1 the present invention comprising mutation Fc portion of immunoglobulin GLP-1/mTf schematic diagram (from a left side to
It is right:N-terminal is to C-terminal);
Fig. 2 include the amino acid sequence (SEQ ID NO.1) of the fusion protein (Na) of Fc mutant 1;
Fig. 3 include the amino acid sequence (SEQ ID NO.2) of the fusion protein (MNa) of Fc mutant 2;
Fig. 4 include the amino acid sequence (SEQ ID NO.3) of the fusion protein (TENa) of Fc mutant 3;
Fig. 5 include the amino acid sequence (SEQ ID NO.4) of the fusion protein (MTNa) of Fc mutant 4;
The amino acid sequence (SEQ ID NO.5) of Fig. 6 Du Lalu peptides;
Fig. 7 .YES amino acid sequence (S228P, F234A, L235A, M252Y, T256E, N434S) (SEQ ID
NO.8);
Fig. 8 .YTE amino acid sequence (S228P, F234A, L235A, M252Y, S254T, T256E) (SEQ ID
NO.9);
Fig. 9 .YTELS amino acid sequence (S228P, F234A, L235A, M252Y, S254T, T256E, M428L,
N434S)(SEQ ID NO.10);
Figure 10 .YE amino acid sequence (S228P, F234A, L235A, M252Y, T256E) (SEQ ID NO.11);
The active determination in vitro of Figure 11 fusion proteins of the present invention;With
The design diagram of Figure 12 homodimer fusion proteins of the embodiment of the present invention, wherein Fc fusion proteins include anti-
Body Fc regions and unique drug fusion fragment (GLP-1 or its analog), unique drug fusion fragment contain one section about
5nm flexible region, the E27 sites of GLP-1 albumen and the E216 sites (being numbered according to EU numbering systems) of Fc albumen carry
Negative electrical charge, and the K34 sites of GLP-1 albumen and the K218 sites (being numbered according to EU numbering systems) of Fc albumen carry positive electricity
Lotus, it is latent that the structure make it that the drug fusion fragment that the about 5nm grows exists with the charged residues and polar residues having in Fc fragments
Interaction, further influence medicine fragment spatiality.
Embodiment
Fc fusion proteins medicines are using technologies such as genetic engineerings, and functional protein is mutually melted with immunoglobulin Fc segments
The New function recombinant protein of conjunction.Fc fusion proteins and antibody belong to different type albumen.Its essential distinction is:Antibody includes two
Individual heavy chain and two light chains, Fc fragments are located at the constant region of heavy chain;And Fc fusion proteins include functional protein and Fc fragments.Fc melts
This feature of hop protein also causes it to remain the biological activity of functional protein, and also has the antibody such as long-acting half-life period
Property.
By carrying out structure of modification to the Fc regions of such Fc fusion protein, obtain medicine has substantially for half-life period for this research
The depot drug product of advantage.
More specifically, the present invention provides a kind of GLP-1 class Fc fusion proteins with long-acting hypoglycemic ability, the albumen by
GLP-1 analogs are obtained with Fc mutant by being connected peptide, and wherein Fc mutant at least has a mutation:The quilt of amino acid N 434
A434 substitutes (numbering is according to EU indexes).
According to the specific embodiment of the present invention, there is provided a kind of fusion protein, it includes or consisted of:
GLP-1 or its analog;Peptide linker;And Fc portion of immunoglobulin, wherein the Fc portion of immunoglobulin is in 434 sites
The amino acid N of (being numbered according to EU numbering systems) is replaced by weak hydrophobic amino acid, preferably described Fc portion of immunoglobulin bag
Include amino acid substitution N434A and (note (is numbered) according to EU numbering systems:Herein, the amino acid of Fc portion of immunoglobulin replaces
Change and use following name:Initial, position (are numbered) according to EU numbering systems, replace amino acid.Separated with plus sige (+) more
Individual mutation).
Natural GLP-1 is in vivo by processing, and its preceding 6 amino acid is removed, therefore this area is generally by GLP-1 ammonia
Cardinal extremity (N-terminal) is appointed as 7, and c-terminus (C-terminal) is 37.
Other GLP-1 analogs for keeping GLP-1 natural bioactives be well known to a person skilled in the art or can be with
Determined by normal experiment.
Preferably, heretofore described GLP-1 analogs be included in natural GLP-1 sequences 1-10 (such as 1,
2nd, 3,4,5,6,7,8,9 or 10) amino acid replacement (such as conservative amino acid replacement), missing or insertion, so as to extend GLP-1
Inside half-life period, while retain GLP-1 natural bioactive.Preferably, such as disclosed in CN1802386A
GLP-1 analogs, the SEQ ID NOs.1-6 particularly disclosed in CN1802386A.
Preferably, heretofore described GLP-1 analogs be included in natural GLP-1 sequences 1-5 (such as 1,2,3,
4 or 5) (C on amino acid residue12-24, such as C12、C14、C16Or C18Long-chain) fatty acid modifying, so as to partly be declined in extension body
Phase.
In a preferred embodiment of the invention, heretofore described GLP-1 analogs are selected from by Exenatide
(Exenatide), Liraglutide (Liraglutide), Li Sina peptide (Lixisenatide), albiglutide
(Albiglutide), the group of Du Lalu peptides (Dulaglutide) and Suo Malu peptides (Semeglutide) composition.Wherein Ah
Must Shandong peptide (Albiglutide) and Du Lalu peptides (Dulaglutide) refer to GLP-1 parts in its structure.
One representative series of heretofore described GLP-1 analogs are (from left to right:N-terminal is to C-terminal) it is as follows:
HisGly8GluGlyThrPheThrSerAspValSerSerTyrLeuGluGlu22GlnAlaAlaLysGlu
PheIleAlaTrpLeuValLysGlyGly36Gly(SEQ ID NO:6)
Compared with natural human activity in vivo GLP-1 (7-37), there are three amino acid to be replaced:A8G, G22E, R36G,
Purpose is to reduce degraded of the endogenous enzyme to the analog, reduces the potentiality of molecule aggregation and/or reduce immunogenicity.At this
In sequence, the 27th amino acids E27 carries negative electrical charge, and the 34th amino acids K34 carries positive charge.The distribution exhibition of these residues
The peculiar charge distribution characteristics of GLP-1 activated protein residues are shown.
Heretofore described GLP-1/mTf has following characteristics:The amino acids (such as E27) of GLP-1 albumen the 27th
Negative electrical charge is carried with the 216th amino acids of immunoglobulin Fc (being numbered according to EU numbering systems, such as E216), and GLP-1 eggs
218 amino acids of white 34th amino acids (such as K34) and immunoglobulin Fc (number, such as K218) band according to EU numbering systems
There is a positive charge, among GLP-1 albumen and immunoglobulin Fc for one section of continuous polar residues fragment (such as
GGGGSGGGGSGGGGS)。
Peptide linker in the fusion protein of the present invention can select peptide linker well known in the art (such as in CN1802386A
Disclosed peptide linker, particularly wherein disclosed SEQ ID NOs.8,19 and 21), as long as it is for GLP-1 in fusion protein
Activity and/or the stability of fusion protein not adversely affect.For the representational preferred peptide in the fusion protein of the present invention
The amino acid sequence of joint adds A to form by (GGGGS) repetitive sequence, represent sequence as:
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAla(SEQ ID NO:7)
According to the specific embodiment of the present invention, the C-terminal of the GLP-1 or its analog pass through the peptide linker
Merged with the N-terminal of the Fc parts.
The Fc parts of the present invention can come from IgG 1, IgG2, IgG3 or IgG4.
It is preferred that the Fc parts of the present invention by mutation so that effector function to be minimized to (such as L235A, F234A, according to EU
Numbering system is numbered, referring to Kabat, E.A. et al., (1991), Sequences of Proteins of Immunological
Interest, the 5th edition, U.S.Dept.of Health and Human Services, Bethesda, MD, NIH publication 91-
3242).Additionally, it is preferred that the Fc parts of the present invention are mutated (such as S228P) so as to form stable dimeric structure.
After transformation research of the present inventor by carrying out system to the residue in fusion protein F c sections, it has further been found that
The mutation in N434A sites, the medicines of GLP-1/Fc in blood can be dramatically increased for half-life period, be expected to develop into a kind of more long
The GLP-1 class hypoglycemic medicines of effect.
It is described in fusion protein of the present invention in addition to N434A is replaced according to the specific embodiment of the present invention
Fc portion of immunoglobulin is also comprising one or more (for example, 1,2,3,4,5,6,7 or 8) selected from the group consisted of
Amino acid substitution (is numbered) according to EU numbering systems:S228P, F234A, L235A, M252Y, T256E, T307A, E380A and
M428L, preferably comprises S228P, and F234A, L235A and optionally other one or more (such as 1,2,3,4 or 5) are selected from
The amino acid substitution (being numbered according to EU numbering systems) of the group consisted of:M252Y, T256E, T307A, E380A and
M428L, more preferably comprising S228P, F234A, L235A and optionally one or more (such as 1,2,3,4 or 5) be selected from by with
The amino acid substitution (being numbered according to EU numbering systems) of the group of lower composition:T307A, E380A and M428L.
In a preferred embodiment of the invention, the Fc portion of immunoglobulin, which includes, is selected from what is consisted of
The combination of the amino acid substitution (being numbered according to EU numbering systems) of group:
1)S228P+F234A+L235A+N434A;
2)S228P+F234A+L235A+M428L+N434A;
3)S228P+F234A+L235A+T307A+E380A+N434A;With
4)S228P+F234A+L235A+M252Y+T256E+N434A。
In a preferred embodiment of the invention, there is the fusion protein one or more to be selected from following spy
Sign:
1) there are one or more (2 or 3) to be selected from following amino acid substitution (root for the GLP-1 or its analog
Numbered according to EU numbering systems):A8G, G22E and R36G, to improve GLP-1 or its analog biological half-life;
2) GLP-1 or its analog have the (C12- on 1-5 (such as 1,2,3,4 or 5) amino acid residues
24, such as C12、C14、C16Or C18Long-chain) fatty acid modifying, to improve GLP-1 or its analog biological half-life;With
3) the 27th amino acids and Fc portion of immunoglobulin of the GLP-1 of the fusion protein or its analog part
216th amino acids (being numbered according to EU numbering systems) carry negative electrical charge, and GLP-1 or the 34th ammonia of its analog part
218th amino acids of base acid and Fc portion of immunoglobulin (being numbered according to EU numbering systems) carry positive charge, and
Between GLP-1 or its analog and Fc portion of immunoglobulin comprising (for example, 10,11,12,13,14,15,16,17,18,19,
20th, 21,22,23,24,25,26,27,28,29 or 30 amino acid residues) continuous polar amino acid residues or mainly by
Its soft segment formed.
In a preferred embodiment of the invention, described fusion protein exists in the form of dimer;In this hair
In a bright particularly preferred embodiment, described fusion protein exists in the form of homodimer.
In the particularly preferred embodiment of the present invention, the amino acid sequence of the Fc portion of immunoglobulin is selected from
SEQ ID NO.1, SEQ ID NO.2, the group of SEQ ID NO.3 and SEQ ID NO.4 compositions;And/or wherein described GLP-1 or
The amino acid sequence of its analog is as shown in SEQ ID NO.6;And/or the amino acid sequence of wherein described peptide linker such as SEQ ID
Shown in NO.7.
The mutant of the Fc portion of immunoglobulin of the present invention can be come using any method of mutagenesis as known in the art
Prepare, such as direct mutagenesis, synthetic gene structure, semi-synthetic gene constructed, random mutagenesis, reorganization etc..
Direct mutagenesis is wherein to limit site in one or more of polynucleotides of coding parent to manufacture one or more
The technology of individual (several) mutation.
Direct mutagenesis can be completed by PCR in vitro, and the PCR is related to be made comprising the Oligonucleolide primers for it is expected mutagenesis
With.Direct mutagenesis can also be carried out by box mutagenesis in vitro, and the box mutagenesis is related to Restriction Enzyme comprising the more of coding parent
The oligonucleotides that mutation is included in polynucleotides is cracked and is then attached on a site in the plasmid of nucleotides.Generally
The Restriction Enzyme of digested plasmid and oligonucleotides is identical so that the cohesive end and insetion sequence of plasmid are connected to each other.It is fixed
Point mutagenesis can also be completed in vivo by methods known in the art.
The present invention can use any direct mutagenesis program.There are many available commercial kits to can be used in preparing
Variant.
Mutagenesis/Shuffling Method can combine with high-throughout, automatic screening technique, to detect gram of host cell expression
The activity of grand, mutagenic treatment polypeptide.The mutagenized dna molecule of encoding active polypeptide can reclaim from host cell and use this
The standard method in field is quickly sequenced.These methods allow the quick determination of single amino acids residue importance in polypeptide.
It is semi-synthetic it is gene constructed by be applied in combination synthetic gene structure, and/or direct mutagenesis, and/or random mutagenesis,
And/or reorganize to complete.The typical case of semi-synthetic structure is the method for polynucleotide passage and the group of round pcr using synthesis
Close.Therefore can de novo formation limit gene region, but other regions can be used site-specific mutagenesis primer expanded,
And other regions can be expanded by fallibility PCR or non-fallibilities PCR.Then polynucleotides subsequence can be reorganized.
Present invention also offers the polynucleotides of the fusion protein of the coding present invention, the carrier comprising the polynucleotides is (special
It is not expression vector), the host cell (preferably Chinese hamster ovary celI) of the included carrier.Term " polynucleotides ", " carrier " and
" host cell " has implication well known in the art, except as otherwise noted.
Present invention also offers the method for producing fusion protein of the present invention, methods described is included in host cell
The carrier of the polynucleotides of fusion protein of the expression comprising the coding present invention.This method can be according to as well known to those skilled in the art
Recombinant-protein expression carry out.
In addition, the present invention also provides application of the fusion protein of the present invention in medicine is prepared, preferably described medicine is used for
Treat diabetes or obesity.
The invention further relates to pharmaceutical composition, and it includes the fusion protein of the present invention, and optionally, at least one medicinal load
Body, diluent or excipient.
The present invention also provides a kind of method for treating diabetes or obesity, and methods described is included to its subject of needs
(such as mammal, preferably any primate, but people in particular) using the fusion of the invention of therapeutically effective amount
Albumen.
Below by way of following non-limitative experiment part and the mode of accompanying drawing, the present invention is further described.Technology path is general
State:
1. designing the mutational site of Fc portion of immunoglobulin, GLP-1 analogs are mutated by peptide linker and IgG-Fc
Body merges;The mutant fusion protein includes three domains, as shown in figure 1, wherein, GLP-1 is GLP-1 class activated proteins
Sequence, represent sequence as:
HisGly8GluGlyThrPheThrSerAspValSerSerTyrLeuGluGlu22GlnAlaAlaLys
GluPheIleAlaTrpLeuValLysGlyGly36Gly
Compared with natural human activity in vivo GLP-1, there are three amino acid to be replaced:A8G, G22E, R36G,
Peptide linker sequence adds A to form by (GGGGS) repetitive sequence, represent sequence as:
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAla
Fc represents immunoglobulin Fc mutant.
The mutant fusion protein represents sequence as SEQ ID NOs.1-4, wherein:
EU numberings are E216-G446 corresponding to Fc parts in SEQ ID NO.1, including following amino acid substitution:S228P,
F234A,L235A,N434A;
EU numberings are E216-G446 corresponding to Fc parts in SEQ ID NO.2, including following amino acid substitution:S228P,
F234A,L235A,M428L,N434A;
EU numberings are E216-G446 corresponding to Fc parts in SEQ ID NO.3, including following amino acid substitution:S228P,
F234A,L235A,T307A,E380A,N434A;
EU numberings are E216-G446 corresponding to Fc parts in SEQ ID NO.4, including following amino acid substitution:S228P,
F234A,L235A,M252Y,T256E,N434A;
2. a mutant fusion protein for pair design carries out gene design, and carries out full genome synthesis (trust money this auspicious);
3. pair fully synthetic gene uses molecular cloning method, be building up to carrier for expression of eukaryon (pcDNA3.3,
Invitrogen in), expression construct (Plasmid-X) is obtained;
4. expression construct (Plasmid-X) uses electric method for transformation transfection host cell (Chinese hamster ovary celI), and with 800 μ g/
ML G418 (Geneticin, Geneticin) carries out pressurization screening;
5. mix the Fc mutant fusion proteins expression of clone strain;
6. pair Fc mutant fusion proteins carry out affinitive layer purification using Protein A fillers;
7. pair albumen collected carries out Mass Spectrometric Identification, the correctness of product is confirmed, while carry out SDS-PAGE researchs, it is determined that
The purity of purifying protein;
8. the fusion protein of pair purifying is diluted to concentration 0.1mg/ml with 10mM PBS, by the injection of 0.1mg/kg rats
Amount, every rat about inject 0.03-0.04mg albumen.And sampled in 0h, 2h, 5h, 8h, 24h, 48h, 72h, 120h, 168h, adopt
Detected with GLP-1 kits, finally calculate medicine for half-life period.
The structure of the carrier of embodiment 1
Based on above-mentioned technology path, using the method for molecular cloning, construct following (from the N-terminal to C-terminal) of the present invention
Various expression vectors:
GLP-1 analogs (amino acid sequence is as shown in SEQ ID NO.6)+peptide linker (amino acid sequence such as SEQ ID
Shown in NO.7)+mutation Fc portion of immunoglobulin.
The amino acid sequence for the fusion protein that structure obtains is respectively as shown in SEQ ID NOs.1-4,8-11.
As control, the carrier of structure coding following (from N-terminal to C-terminal):
Du Lalu peptides (WT), amino acid sequence is as shown in SEQ ID NO.5.
The Fc portion of immunoglobulin of the mutation of the present invention is summarized as follows:
Table 1
The nucleotide sequence for encoding WT fusion proteins (Du Lalu peptides) and IgG4-Fc mutant fusion proteins is trust money
Si Rui bio tech ltd (Nanjing of China) is obtained based on its amino acid sequence encoded by chemical synthesis.Obtained
Composition sequence by double digestion after, be inserted between the identical restriction enzyme site of carrier for expression of eukaryon, build Plasmid-GLP-1-
Fc and a series of carriers of mutant.Then a series of correct tables of empirical tests are extracted using Invitrogen plasmid extraction kits
Up to carrier, and recovery, -20 DEG C of preservations are purified after being linearized with restriction enzyme.
The carrier of embodiment 2 is transfected and expressed in cell
After CHO host cells are cultivated with the recovery of CHO culture mediums, as cell density about 8x105Collected during individual cell/mL thin
Born of the same parents are transfected.Transfectional cell about 1x107Individual cell, the μ g of plasmid about 40, (Bio-Rad, Gene are transfected by electric-shocking method
pulser Xcell).Cell is cultivated in 20mL CHO culture mediums after electric shock.Culture second day, is collected by centrifugation cell, and adding
Enter and culture is resuspended in G418 (Geneticin, Geneticin) to the μ g/mL of final concentration 800 20mL CHO culture mediums.When cell is close
Spend about 0.6x106During individual cell/mL, the mixing clone strain of acquisition is passed on CHO culture mediums, passage cell density is about
0.2x106Individual cell/mL.When cell survival rate about 90%, cell culture fluid is collected.
The purified fusion protein from animal cell culture fluid of embodiment 3
Detection in translation skill is carried out to 1 a series of fusion protein of embodiment.Using Protein A fillers to a small amount of
Cell culture fluid is enriched with, and collects fusion protein, obtained fusion protein be by disulfide bond and it is a variety of it is non-covalent mutually
The homodimer formed is acted on, carries out Mass Spectrometer Method, Mass Spectrometer Method molecular weight about 62KD is consistent with theoretical molecular.To receiving
The fusion protein of collection is purified using Protein A chromatographic columns.The sample of collection passes through 10%SDS-PAGE electricity after carrying out reduction
Swimming detection, electrophoresis pattern show single band, about 36KD.Purification of samples is saturating at 4 DEG C using pH 7.2 10mM PBSs
Analysis is overnight.
The pharmacokinetics of the fusion protein of embodiment 4 in rats
Concentration 0.1mg/mL is diluted to 10mM PBS to a series of fusion proteins of purifying.To big by the SD of body weight
Mouse (0.3-0.4kg) randomly chooses, and the fusion protein that 0.1mg/kg is injected by every SD rat carries out calculating administration, each fusion
3 rats are subcutaneously injected in albumen.
Before administration, about 200 μ l blood is taken out from the jugular vein of every rat respectively, is suppressed with EDTA-K2 and DPP-4
Agent carries out anti-freezing processing, in -20 DEG C of preservations.2h, 5h, 8h after every animal administration, 24h, 48h, 72h, 120h, 168h are adopted respectively
Blood, same -20 DEG C of preservations after carrying out anti-freezing processing.
Each blood sample drug residue amount is detected using GLP-1ELISA detection kits (Millipore), and carries out medicine generation
Data calculate, and as a result see the table below:
Table 2
The active determination in vitro of the fusion protein of embodiment 5
Fusion protein after purification is quantified with BCA methods, then with Assay buffer (DMEM20ml, FBS 200
μ l of μ l, IBMX 20) carry out three times gradient dilution.Stimulated with cAMP detection kits (producer cisbio) measure fusion protein
The cAMP contents of intracellular after GLP-1R/HEK293 cells, i.e., 5 μ l dilute sample liquid are added in a 384 shallow bore hole plates, it is rear to add
5 μ l cell suspensions (cell density is 100/μ l), the warm bath 30min in CO2gas incubator, it is anti-to add reaction reagent
Should be after the fluorescent value that 665nm, 620nm are detected in multi-function microplate reader.According to the concentration of cAMP standard items and its corresponding fluorescence
The ratio of value draws standard curve, calculates the quantity that test sample under various concentrations stimulates GLP-1R/HEK293 cells to produce cAMP.
Using the logarithm value of test sample concentration as abscissa, curve is made by ordinate of cAMP nM values.As a result show, pierced through test sample
The cAMP contents curve of intracellular is in typical " S " type curve after swashing, and EC is calculated according to these curves50Value.As a result such as following table and Figure 11:
Table 3
Fusion protein | WT | Na | TENa | MNa |
EC50It is worth (nM) | 10.350 | 6.363 | 3.652 | 3.784 |
Embodiment 6 is summarized and discussed
Crystal structure (the PDB of antibody Fc region and its acceptor:1FRT, 4N0U) show amino of the Fc regions by its flank
Acid identification FcRn, the amino acid of these flanks include:M252, S254, T256, M428, N434 etc. (Figure 12).By to these sides
The rite-directed mutagenesis of wing amino acid can change Fc and its acceptor adhesion, if any seminar by the way that M252, S254, T256 are dashed forward
It is changed into Y252, T254, E256, the adhesion of mutant and acceptor can be increased, the mutation introducesNegative electrical charge residueE256 andPolar amino acidT254.N434 site mutations are S434 etc. by Ye You seminar, are introducedPolar amino acidCome increase mutant with
The combination of acceptor.
The Fc fusion proteins that the present invention designs include antibody Fc region and unique drug fusion fragment (GLP-1 or its
Analog) (Figure 12).Wherein unique drug fusion fragment contains one section of about 5nm flexible region.In Fc fusion proteins,
The E27 sites of GLP-1 albumen, the E216 sites (being numbered according to EU numbering systems) with Fc albumen, with negative electrical charge;And GLP-
The K34 sites of 1 albumen, the K218 sites (being numbered according to EU numbering systems) with Fc albumen, with positive charge.Also, GLP-1
It is that one section of continuous polar residues fragment is the peptide linker (G among albumen and immunoglobulin38GGGSGGGGSGGGGS52)。
This distinctive architectural feature make it that the drug fusion fragment that the 5nm grows and the charged residues and polarity that are newly introduced in Fc fragments are residual
There is potential interaction in base, further influence the spatiality of medicine fragment.
The mutation of the present invention and medicine generation experiment also demonstrate that this viewpoint (experimental method and the step embodiment with before).Remove
Introduced after the above-mentioned fusion protein of the present invention, in Fc regions of the inventor also in fusion protein medicine corresponding to the present invention negative
Charged residues E256, (as shown in SEQ ID NO.8, Fc contains in region amino acid sequence built-up four drug candidate YES
Mutation
S228P+F234A+L235A+M252Y+T256E+N434S), YTE (amino acid sequence as shown in SEQ ID NO.9,
Fc contains in region mutation
S228P+F234A+L235A+M252Y+S254T+T256E), YTELS (amino acid sequence such as SEQ ID NO.10 institutes
Show, Fc contains in region mutation
) and YE (amino acid sequence such as SEQ S228P+F234A+L235A+M252Y+S254T+T256E+M428L+N434S
Shown in ID NO.11, Fc contains in region mutation
S228P+F234A+L235A+M252Y+T256E), its CmaxValue is reduced, t1/2Time advance, and without potential
Depot drug product druggability (with the comparative result of fusion protein of the present invention referring to table 2).As can be seen that it is residual to introduce polarity in Fc regions
Base S434, t can be caused1/2Time advance, without potential depot drug product druggability.In medicine in experimentation, we sieve
Choosing obtains, and introduces weak hydrophobic residue A434 in Fc regions, can dramatically increase t1/2, its CmaxAlso show drug for excellent etc. index
Gesture.Therefore, present inventors have surprisingly discovered that:Weak hydrophobic residue (such as A434) is being introduced in medicine Fc fragments
(and be not polarity or electrically charged residue), structure between drug fusion fragment and Fc fragments can be reduced from pressing down
System, so as to increase medicine for effect, so as to complete the present invention.
Although in order to be clearly understood from, foregoing invention is described in some details by means of drawings and examples,
But description and embodiments are not construed as limiting the scope of the present invention.All patents referred to herein and scientific literature
Disclosure by quote intactly clearly be incorporated to.
Sequence table
<110>Guangdong Dongyang Guang Pharmaceutical Co., Ltd
<120>The GLP-1/mTf of Fc portion of immunoglobulin comprising mutation
<130> IB178011
<150> CN 201610633073.3
<151> 2016-08-03
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>Include the fusion protein of Fc mutant 1(Na)Amino acid sequence
<400> 1
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 2
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>Include the fusion protein of Fc mutant 2(MNa)Amino acid sequence
<400> 2
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Leu His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 3
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>Include the fusion protein of Fc mutant 3(TENa)Amino acid sequence
<400> 3
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Ala Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Ala Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 4
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>Include the fusion protein of Fc mutant 4(MTNa)Amino acid sequence
<400> 4
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Ser Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 5
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>Du Lalu peptides
<400> 5
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 6
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223>Representative GLP-1 analogs
<400> 6
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly
20 25 30
<210> 7
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223>Joint
<400> 7
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
1 5 10 15
<210> 8
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>YES amino acid sequence
<400> 8
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Ser Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 9
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>YTE amino acid sequence
<400> 9
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 10
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>YTELS amino acid sequence
<400> 10
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 11
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>YE amino acid sequence
<400> 11
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Ser Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
Claims (12)
1. fusion protein, it includes or consisted of:GLP-1 or its analog;Peptide linker;And Fc portion of immunoglobulin,
Wherein described Fc portion of immunoglobulin is dredged in the amino acid (such as amino acid N) of 434 sites (being numbered according to EU numbering systems)
Water-based amino acid substitution, wherein the hydrophobic amino acid be selected from alanine, valine, leucine, isoleucine, proline,
Phenylalanine, tryptophan and methionine, preferably alanine, most preferably described Fc portion of immunoglobulin include amino acid substitution
N434A。
2. fusion protein according to claim 1, wherein the C-terminal of the GLP-1 or its analog is connect by the peptide
Head merges with the N-terminal of the Fc parts, and/or wherein described Fc portion of immunoglobulin is from IgG 1, IgG2, IgG3
Or IgG4.
3. fusion protein according to claim 1 or 2, wherein the Fc portion of immunoglobulin is also comprising one or more
(for example, 1,2,3,4,5,6,7 or 8) is selected from the amino acid substitution (being numbered according to EU numbering systems) of the group consisted of:
S228P, F234A, L235A, M252Y, T256E, T307A, E380A and M428L, preferably comprise S228P, F234A, L235A and
The amino acid substitution that optionally one or more (such as 1,2,3,4 or 5) is selected from the group consisted of (is numbered according to EU and is
Unified editing number):M252Y, T256E, T307A, E380A and M428L, more preferably comprising S228P, F234A, L235A and optionally one
Individual or multiple (such as 1,2,3,4 or 5) is selected from the amino acid substitution (being numbered according to EU numbering systems) of the group consisted of:
T307A, E380A and M428L.
4. fusion protein according to claim 3, wherein the Fc portion of immunoglobulin, which includes, is selected from following amino
The combination that acid is replaced and (numbered according to EU numbering systems):
1)S228P+F234A+L235A+N434A;
2)S228P+F234A+L235A+M428L+N434A;
3)S228P+F234A+L235A+T307A+E380A+N434A;With
4)S228P+F234A+L235A+M252Y+T256E+N434A。
5. fusion protein according to claim 1, wherein there is the fusion protein one or more to be selected from following spy
Sign:
1) there are one or more (2 or 3) to be selected from following amino acid substitution (according to EU for the GLP-1 or its analog
Numbering system is numbered):A8G, G22E and R36G;
2) GLP-1 or its analog have the (C on 1-5 (such as 1,2,3,4 or 5) amino acid residues12-24, such as
C12、C14、C16Or C18Long-chain) fatty acid modifying;With
3) the of the 27th amino acids and Fc portion of immunoglobulin of the GLP-1 of the fusion protein or its analog part
216 amino acids (being numbered according to EU numbering systems) carry negative electrical charge, and GLP-1 or the 34th bit amino of its analog part
218th amino acids of acid and Fc portion of immunoglobulin (being numbered according to EU numbering systems) carry positive charge, and GLP-1
Or its analog and Fc portion of immunoglobulin tundish are containing (10-30 amino acid residue) continuous polar amino acid residues
Fragment.
6. the fusion protein according to claim 4 or 5, wherein the amino acid sequence of the fusion protein is selected from SEQ ID
NO.1, SEQ ID NO.2, the group of SEQ ID NO.3 and SEQ ID NO.4 compositions;And/or wherein described GLP-1 or its is similar
The amino acid sequence of thing is as shown in SEQ ID NO.6;And/or the amino acid sequence of wherein described peptide linker such as SEQ ID NO.7
It is shown.
7. fusion protein according to claim 1, it exists in the form of dimer, preferably with the shape of homodimer
Formula is present.
8. polynucleotides, it encodes the fusion protein according to any one of claim 1-7.
9. carrier, it includes polynucleotides according to claim 8.
10. host cell, it includes carrier according to claim 9, and preferably described host cell is Chinese hamster ovary celI.
11. a kind of method for producing the fusion protein according to any one of claim 1-7, methods described are included in host
Carrier according to claim 9 is expressed in cell.
12. application of the fusion protein according to any one of claim 1-7 in medicine is prepared, preferably described medicine is used
In treatment diabetes or obesity.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2016106330733 | 2016-08-03 | ||
CN201610633073 | 2016-08-03 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107698684A true CN107698684A (en) | 2018-02-16 |
CN107698684B CN107698684B (en) | 2021-09-28 |
Family
ID=61072912
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710630742.6A Active CN107698684B (en) | 2016-08-03 | 2017-07-28 | GLP-1 fusion proteins comprising a mutated immunoglobulin Fc portion |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN107698684B (en) |
WO (1) | WO2018024162A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110878127A (en) * | 2018-09-06 | 2020-03-13 | 浙江柏拉阿图医药科技有限公司 | Long-acting recombinant GLP1-Fc-CD47 protein and preparation and application thereof |
CN112424233A (en) * | 2018-06-18 | 2021-02-26 | 戴纳立制药公司 | Fusion proteins comprising a progranulin |
CN117143242A (en) * | 2023-10-30 | 2023-12-01 | 南京佰抗生物科技有限公司 | Monoclonal antibody composition for resisting Galectin-3 protein and application thereof |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110964116A (en) * | 2018-09-26 | 2020-04-07 | 北京辅仁瑞辉生物医药研究院有限公司 | GLP1-Fc fusion proteins and conjugates thereof |
MX2021012997A (en) * | 2019-04-24 | 2022-03-04 | Univ Pennsylvania | Bi-functional humanized anti-c5 antibodies and factor h fusion proteins and uses thereof. |
EP3990476A1 (en) * | 2019-06-25 | 2022-05-04 | Gilead Sciences, Inc. | Flt3l-fc fusion proteins and methods of use |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104327187A (en) * | 2014-10-11 | 2015-02-04 | 上海兴迪金生物技术有限公司 | Recombinant human GLP-1-Fc fusion protein |
WO2016106302A1 (en) * | 2014-12-23 | 2016-06-30 | Bristol-Myers Squibb Company | Antibodies to tigit |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100166741A1 (en) * | 2006-07-13 | 2010-07-01 | Genentech , Inc. | Altered br-3 binding polypeptides |
AU2008275180A1 (en) * | 2007-07-10 | 2009-01-15 | Eli Lilly And Company | GLP-1-Fc fusion protein formulation |
SI2808343T1 (en) * | 2007-12-26 | 2019-10-30 | Xencor Inc | Fc variants with altered binding to FcRn |
CA2960797A1 (en) * | 2014-11-06 | 2016-05-12 | F. Hoffmann-La Roche Ag | Fc-region variants with modified fcrn-binding and methods of use |
RS60631B1 (en) * | 2014-11-21 | 2020-09-30 | Bristol Myers Squibb Co | Antibodies against cd73 and uses thereof |
-
2017
- 2017-07-28 WO PCT/CN2017/094916 patent/WO2018024162A1/en active Application Filing
- 2017-07-28 CN CN201710630742.6A patent/CN107698684B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104327187A (en) * | 2014-10-11 | 2015-02-04 | 上海兴迪金生物技术有限公司 | Recombinant human GLP-1-Fc fusion protein |
WO2016106302A1 (en) * | 2014-12-23 | 2016-06-30 | Bristol-Myers Squibb Company | Antibodies to tigit |
Non-Patent Citations (2)
Title |
---|
M KUMAR等: "Gene therapy of diabetes using a novel GLP-1/IgG1-Fc fusion construct normalizes glucose levels in db/db mice", 《GENE THERAPY》 * |
YANG Y等: "Expression and Characterization of a Potent Long-Acting GLP-1 Receptor Agonist, GLP-1-IgG2σ-Fc", 《PLOS ONE》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112424233A (en) * | 2018-06-18 | 2021-02-26 | 戴纳立制药公司 | Fusion proteins comprising a progranulin |
CN110878127A (en) * | 2018-09-06 | 2020-03-13 | 浙江柏拉阿图医药科技有限公司 | Long-acting recombinant GLP1-Fc-CD47 protein and preparation and application thereof |
CN110878127B (en) * | 2018-09-06 | 2022-06-28 | 浙江柏拉阿图医药科技有限公司 | Long-acting recombinant GLP1-Fc-CD47 protein and preparation and application thereof |
CN117143242A (en) * | 2023-10-30 | 2023-12-01 | 南京佰抗生物科技有限公司 | Monoclonal antibody composition for resisting Galectin-3 protein and application thereof |
Also Published As
Publication number | Publication date |
---|---|
CN107698684B (en) | 2021-09-28 |
WO2018024162A1 (en) | 2018-02-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107698684A (en) | The fusion proteins of GLP 1 of Fc portion of immunoglobulin comprising mutation | |
CN104271588B (en) | The engineered polypeptide of immunogenicity with enhanced acting duration and reduction | |
CN103370083B (en) | There is the engineered polypeptide of the acting duration of enhancing | |
JP5827218B2 (en) | Peptides for improving in vivo stability, such as physiologically active substances, and physiologically active substances with improved in vivo stability | |
CN104582736A (en) | Incretin receptor ligand polypeptide Fc-region fusion polypeptides and conjugates with altered Fc-effector function | |
BRPI1008061B1 (en) | Extended recombinant polypeptides (xten), isolated fusion protein comprising the same and method of improving a property of a biologically active protein | |
JP2016183180A (en) | Engineered polypeptide having enhanced duration of action and reduced immunogenicity | |
JP6519929B2 (en) | GLP-1 analog fusion protein, method for producing the same and use thereof | |
US11078250B2 (en) | High-activity long-acting hypoglycemic fusion protein as well as preparation method and medical application thereof | |
CN106029087A (en) | Lipidated incretin receptor ligand human immunoglobulin fc-region fusion polypeptides | |
CN102690352A (en) | Fusion protein containing GLP-1, and pharmaceutical compositions and applications thereof | |
CN105254763B (en) | A kind of Exendin-4 fusion protein, preparation method and applications | |
CN115175920A (en) | Barcoded XTEN polypeptides and compositions thereof, and methods of making and using the same | |
CN113105561B (en) | Preparation method and application of double-target fusion protein | |
JP6612360B2 (en) | Fusion protein complex and fusion protein having medicinal action | |
CN113968911A (en) | insulin-Fc fusion protein and application thereof | |
CN110172103B (en) | GLP-1 analogue-Fc fusion protein, and preparation method and application thereof | |
US10017555B2 (en) | Long-acting blood sugar decreasing fusion protein | |
WO2023280133A1 (en) | Fusion protein and application thereof | |
CN104086655A (en) | Amylin-type fusion protein and its coding gene and use | |
CN106146667A (en) | A kind of Exendin-4 fusion protein and preparation method thereof and purposes | |
US20180344813A1 (en) | Engineered polypeptides having enhanced duration of action with reduced immunogenicity |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CP03 | Change of name, title or address | ||
CP03 | Change of name, title or address |
Address after: 523808 No.1, Gongye North Road, Songshanhu Park, Dongguan City, Guangdong Province Patentee after: Guangdong Dongyangguang Pharmaceutical Co.,Ltd. Address before: 523808 No.1 Gongye North Road, Songshan Hubei Industrial Park, Dongguan City, Guangdong Province Patentee before: SUNSHINE LAKE PHARMA Co.,Ltd. |