Anti-human IL-17RC monoclonal antibody and application thereof
阅读说明：本技术 一种抗人il-17rc的单克隆抗体及其应用 (Anti-human IL-17RC monoclonal antibody and application thereof ) 是由 季鸣 于 2021-09-30 设计创作，主要内容包括：本发明提供了一种抗人IL-17RC的单克隆抗体及其应用。所述抗人IL-17RC的单克隆抗体包括重链和轻链,所述重链的CDR3包括如SEQ ID NO.26～36任意一项所示的氨基酸序列。本发明中所述的抗人IL-17RC的单克隆抗体特异性识别并结合人、食蟹猴和小鼠的IL-17RC。本发明中提供的抗人IL-17RC的单克隆抗体能够特异性与人、食蟹猴和小鼠的IL-17RC抗原结合,有效阻断IL-17A和IL-17F与IL-17RC的结合,抑制和/或阻断由IL-17RC介导的IL-17A和IL-17F的信号传导,能用于治疗IL-17通路相关的疾病和病症。(The invention provides a monoclonal antibody of anti-human IL-17RC and application thereof. The monoclonal antibody of anti-human IL-17RC comprises a heavy chain and a light chain, wherein a CDR3 of the heavy chain comprises an amino acid sequence shown in any one of SEQ ID NO. 26-36. The monoclonal antibody of anti-human IL-17RC specifically recognizes and binds to IL-17RC of human, cynomolgus monkey and mouse. The monoclonal antibody of anti-human IL-17RC provided by the invention can be specifically combined with IL-17RC antigen of human, cynomolgus monkey and mouse, effectively block the combination of IL-17A and IL-17F and IL-17RC, inhibit and/or block IL-17A and IL-17F signal transduction mediated by IL-17RC, and can be used for treating IL-17 pathway related diseases and symptoms.)
1. The monoclonal antibody for resisting human IL-17RC is characterized by comprising a heavy chain and a light chain, wherein a CDR3 of the heavy chain comprises an amino acid sequence shown in any one of SEQ ID NO. 26-36.
2. The monoclonal antibody against human IL-17RC of claim 1, wherein the CDR1 of the heavy chain comprises an amino acid sequence shown in any one of SEQ ID nos. 1 to 11;
preferably, the CDR2 of said heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID No.12 to 25.
3. The monoclonal antibody against human IL-17RC of claim 1 or 2, wherein the CDR3 of the light chain has an amino acid sequence as set forth in any one of SEQ ID nos. 63-72.
4. The monoclonal antibody against human IL-17RC as claimed in any one of claims 1 to 3, wherein CDR1 of the light chain comprises an amino acid sequence as set forth in any one of SEQ ID nos. 37 to 50;
preferably, the CDR2 of the light chain comprises an amino acid sequence as set forth in any one of SEQ ID nos. 51-62.
5. The monoclonal antibody against human IL-17RC as claimed in any one of claims 1 to 4, wherein the heavy chain variable region of the monoclonal antibody against human IL-17RC comprises an amino acid sequence as shown in SEQ ID No.73, SEQ ID No.75, SEQ ID No.77, SEQ ID No.79, SEQ ID No.81, SEQ ID No.83, SEQ ID No.85, SEQ ID No.87, SEQ ID No.89, SEQ ID No.91, SEQ ID No.93, SEQ ID No.95, SEQ ID No.97, SEQ ID No.99 or SEQ ID No. 101.
6. The monoclonal antibody against human IL-17RC as claimed in any one of claims 1 to 5, wherein the light chain variable region of the monoclonal antibody against human IL-17RC comprises an amino acid sequence as shown in SEQ ID NO.74, SEQ ID NO.76, SEQ ID NO.78, SEQ ID NO.80, SEQ ID NO.82, SEQ ID NO.84, SEQ ID NO.86, SEQ ID NO.88, SEQ ID NO.90, SEQ ID NO.92, SEQ ID NO.94, SEQ ID NO.96, SEQ ID NO.98, SEQ ID NO.100 or SEQ ID NO. 102.
7. A nucleic acid fragment comprising a nucleotide sequence encoding the anti-human IL-17RC monoclonal antibody according to any one of claims 1 to 6.
8. An expression vector comprising at least one copy of the nucleic acid fragment of claim 7.
9. A recombinant host cell comprising the nucleic acid fragment of claim 7 or the expression vector of claim 8.
10. Use of any one or a combination of at least two of the anti-human IL-17RC monoclonal antibody according to any one of claims 1 to 6, the nucleic acid fragment according to claim 7, the expression vector according to claim 8 or the recombinant host cell according to claim 9 for the preparation of a medicament for the treatment or prevention of autoimmune diseases and/or tumors;
preferably, the autoimmune disease and/or tumor is an IL-17 mediated autoimmune disease and/or tumor;
preferably, the autoimmune disease comprises any one of psoriasis, rheumatoid arthritis or ankylosing spondylitis;
preferably, the tumor comprises any one of colon cancer, lung cancer or breast cancer.
Technical Field
The invention belongs to the technical field of antibody engineering, and particularly relates to an anti-human IL-17RC monoclonal antibody and application thereof.
Background
The interleukin-17 (IL-17) cytokine family consists of 6 homologs, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, respectively. Wherein IL-17A and IL-17F have the highest homology and perform similar functions. Overexpression and abnormal expression of IL-17A and IL-17F are associated with a variety of diseases. Including rheumatoid arthritis, psoriasis, multiple sclerosis, tracheal hypersensitivity (including asthma), cutaneous hypersensitivity (including atopic dermatitis) and inflammatory bowel disease (including ulcerative enteritis and crohn's disease). The existing literature indicates that IL-17 has important influence on the pathogenesis and treatment process of colorectal cancer, breast cancer, liver cancer, gastric cancer, lung cancer, prostatic cancer, pancreatic cancer, skin cancer and the like.
IL-17A and IL-17F may form homodimers, respectively, or may combine with each other to form heterodimers. IL-17A and IL-17F bind to heterodimeric receptors consisting of IL-17RA and IL-17RC, signaling. Animal experiments show that the inflammatory reaction caused by IL-17A and IL-17F can be effectively reduced by blocking IL-17RC mediated signals of IL-17A and IL-17F.
Research shows that in an experimental autoimmune encephalomyelitis model of a mouse, the knock-out of IL-17RC obviously relieves inflammation symptoms; in a mouse-simulated psoriasis model, knock-out of IL-17RC can eliminate most inflammatory symptoms; IL-17RC knockout mice also exhibit mild symptoms during the onset of prostate cancer. Taken together, IL-17RC is the major receptor for IL-17A and IL-17F. The blocking of IL-17RC has the effect of effectively reducing the inflammatory response caused by IL-17A and IL-17F.
CN102276727A discloses a human interleukin receptor C (IL-17RC) fusion protein (IL-17RC-hFc fusion protein), which comprises a key region for binding human IL-17RC with interleukin 17A (IL-17A) and interleukin 17F (IL-17F) and a human IgG1-Fc segment, wherein the IL-17RC-hFc fusion protein can inhibit the stimulation of IL-17A or IL-17F on inflammatory cytokines, reduce the release of IL-6 and inhibit the expression of RANKL molecules, is used for treating autoimmune diseases such as IL-17-related rheumatoid arthritis and the like, and plays roles of inhibiting inflammation and antagonizing bone destruction.
CN111100211A discloses an Fc fusion protein for simultaneously blocking TNF and IL-17, wherein the first antigen binding domain of the Fc fusion protein comprises TNF alpha R2 and IL-17RA, and the second antigen binding domain comprises TNF alpha R2 and IL-17 RC. The TNF alpha R2 can be combined with TNF alpha molecules, IL-17RA and IL-17RC can be combined with IL-17, and the main function of the TNF alpha R2 is to block partial inflammatory reaction of autoimmune diseases so as to achieve the effect of improving symptoms.
Existing antagonists for the IL-17 pathway are primarily monoclonal antibodies targeting IL-17A and IL-17 RA. Firstly, the monoclonal antibody is prepared to be combined with IL-17A and compete with a receptor of IL-17 for a substrate to inhibit inflammatory reaction; and secondly, the monoclonal antibody is prepared to be combined with IL-17RA, so that the combination of IL-17A and IL-17RA is reduced, and the effect of inhibiting inflammatory reaction is achieved. The existing monoclonal antibody medicament obtains better curative effect on psoriasis, ankylosing spondylitis, psoriatic arthritis and other indications.
To date, there are no monoclonal antibody antagonists developed to target IL-17RC for the treatment of IL-17 pathway related diseases and disorders. Wherein IL-17RA is mainly expressed in hematopoietic cells, IL-17RC is expressed in non-hematopoietic tissues and can be combined with IL-17A and IL-17F, and inflammatory cytokines IL-17A and IL-17F are closely related to the progression of inflammation and autoimmune diseases. Therefore, the development of a monoclonal antibody antagonist targeting IL-17RC plays an important role in treating diseases caused by IL-17 family cytokines.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an anti-human IL-17RC monoclonal antibody and application thereof. The anti-human IL-17RC monoclonal antibody is suitable for treating diseases and symptoms caused by IL-17 family cytokines, and can be combined with IL-17RC to reduce the combination of IL-17A and IL-17RA, so that the effect of inhibiting inflammatory reaction is achieved.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an anti-human IL-17RC monoclonal antibody, which comprises a heavy chain and a light chain, wherein the CDR3 of the heavy chain comprises an amino acid sequence shown in any one of SEQ ID nos. 26 to 36.
Preferably, the CDR1 of the heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID No. 1-11.
Preferably, the CDR2 of said heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID No.12 to 25.
Preferably, the CDR3 of the light chain comprises an amino acid sequence as set forth in any one of SEQ ID nos. 63-72.
Preferably, the CDR1 of the light chain comprises an amino acid sequence as set forth in any one of SEQ ID No. 37-50.
Preferably, the CDR2 of the light chain comprises an amino acid sequence as set forth in any one of SEQ ID nos. 51-62.
The amino acid sequences of the anti-human IL-17RC monoclonal antibodies including CDR1, CDR2 and CDR3 of the heavy and light chains are shown in Table 1.
TABLE 1
The monoclonal antibody of anti-human IL-17RC provided by the invention is a fully human monoclonal antibody, can be specifically combined with IL-17RC antigen, effectively blocks the combination of IL-17A and IL-17F and IL-17RC, and inhibits and/or blocks the signal transduction of IL-17A and IL-17F mediated by IL-17 RC.
Preferably, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.73, SEQ ID NO.75, SEQ ID NO.77, SEQ ID NO.79, SEQ ID NO.81, SEQ ID NO.83, SEQ ID NO.85, SEQ ID NO.87, SEQ ID NO.89, SEQ ID NO.91, SEQ ID NO.93, SEQ ID NO.95, SEQ ID NO.97, SEQ ID NO.99 or SEQ ID NO. 101.
Preferably, the light chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.74, SEQ ID NO.76, SEQ ID NO.78, SEQ ID NO.80, SEQ ID NO.82, SEQ ID NO.84, SEQ ID NO.86, SEQ ID NO.88, SEQ ID NO.90, SEQ ID NO.92, SEQ ID NO.94, SEQ ID NO.96, SEQ ID NO.98, SEQ ID NO.100 or SEQ ID NO. 102.
As a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.12 or SEQ ID NO. 26; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.37, SEQ ID NO.51 or SEQ ID NO.63 and is marked as clone 10.
Wherein the sequence of the heavy chain variable region of clone 10 (SEQ ID NO.73) is:
MGWIWIFLLLLSGTAGVHSEVQLQQSGPELVKTGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGYISCYNGATNYKQKFRGKATFTVDTSSSTAYMQFNSLTSEDSAVYYCARSYGYPNTGAMDYWGQGTSVTVSS。
wherein the sequence of the light chain variable region of clone 10 (SEQ ID NO.74) is:
MESQTQVFLSLLLWVSGTCGNILMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQFLSSLTFGAGTKLELK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.2, SEQ ID NO.13 or SEQ ID NO. 27; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.38, SEQ ID NO.51 or SEQ ID NO.64, and is marked as clone 26.
Wherein the sequence of the heavy chain variable region of clone 26 (SEQ ID NO.75) is:
MGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLRTSGMGAGWIRQPSGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSSNQVFLKIASVDTADTATYYCARIANRYFDVWGAGTTVTVSS。
wherein the sequence of the light chain variable region of clone 26 (SEQ ID NO.76) is:
MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVTLSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGFGTDFTLTISSVKAEDLAVYYCQQYYSFPYTFGGGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.3, SEQ ID NO.14 or SEQ ID NO. 28; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.39, SEQ ID NO.52 or SEQ ID NO.65, and is marked as clone 38.
Wherein the sequence of the heavy chain variable region of clone38 (SEQ ID NO.77) is:
MGWSWIFLFLLSGTGGVLSEVLLQQSGPELVKPGASVMMSCKASGYTFTDYYIKWVKQSHGKSLEWIGDINPNNGDIFYNQNFKGKVTLTVDKSSSTAYMKINSLTSEDSAVYYCAKGWFAYWGQGTLVTVSA。
wherein the sequence of the light chain variable region of clone38 (SEQ ID NO.78) is:
MRTPAQFLGILLLWFPGIKCDIKMTQSPSSMFASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLMDGVPSGFSGSGSGQDYSLTISSLDYEDMGIYYCLQYDEFPFTFGGGTTLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.15 or SEQ ID NO. 29; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.40, SEQ ID NO.51 or SEQ ID NO.63 and is marked as clone 50.
Wherein the sequence of the heavy chain variable region of clone 50 (SEQ ID NO.79) is:
MGWIWIFLFLLSGTAGVHSEVQLQQSGPELLKTGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGYINCYNGATGYNQKFKGKATFTVDTSSSTAYMQFDSLTSEDSAIYYCARSYGYPITGAMDYWGQGTSVTVSS。
wherein the sequence of the light chain variable region of clone 50 (SEQ ID NO.80) is:
MESQTQVFLSLLLWVSGTCGNILMTQSPSSLAVSAGEKVTMSCKSSQTVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCHQFLSSLTFGAGTKLELK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.4, SEQ ID NO.16 or SEQ ID NO. 30; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.41, SEQ ID NO.53 or SEQ ID NO.65, and is marked as clone 52.
Wherein the sequence of the heavy chain variable region of clone 52 (SEQ ID NO.81) is:
MNFGFSLIFLVLVLKGVQCEVNLVESGGGLVRPGGSLKLSCAASGFTFNTYAMSWVRQTPEKRLEWVASITSGGIPYYPDNMKGRFTVSRDNARNILYLQMSSLRSEDTAMYYCASLHYYGGPSYAMDYWGQGTSVTVSS。
wherein the sequence of the light chain variable region of clone 52 (SEQ ID NO.82) is:
MRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTITCKASQDLNRYLSWLQQKPGKSPKTLIYRANSLVDGVPSRFSGSGSGQDYSLTISSLEYEDVGFYYCLQYDEFPFTLGSGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.5, SEQ ID NO.17 or SEQ ID NO. 31; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.42, SEQ ID NO.54 or SEQ ID NO.66 and is marked as clone 53.
Wherein the sequence of the heavy chain variable region of clone 53 (SEQ ID NO.83) is:
MECNWILPFILSVISGVYSEVQLQQSGTVLARPGASVKMSCKASGYSFTDYWLHWLKQRPGQGLEWIGAMNPGKSDIRYNQRFKGKAKLTAVTSASTAYMELSSLTNEDSAVYYCTRWGDSIDNWGQGTTLIVSS。
wherein the sequence of the light chain variable region of clone 53 (SEQ ID NO.84) is:
MEKDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSSGISFMNWFQQKPGQAPKLIIYAASKQGSGVPARYSGSGSGTDFSLNIQPMKEDDSAMYFCQQSKELPYTFGGGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.18 or SEQ ID NO. 32; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown in SEQ ID NO.43, SE ID NO.55 or SEQ ID NO.67 and is marked as clone 68.
Wherein the sequence of the heavy chain variable region of clone 68 (SEQ ID NO.85) is:
MGWIWIFLFLLSGTAGVHSEVQLQQSGPELVKTGASMKISCKASGYSFTGYYMHWVKQSHGKSLEWIGYISCYNGATTYTQKFKGKATFTVDTSSSTAYMQFNSLTSEDSAVYYCARSYGYPITNSMDYWGQGTSVTVSS。
wherein the sequence of the light chain variable region of clone 68 (SEQ ID NO.86) is:
MESQTQVFLSLLLWVSGTCGNIMMTQSPSSLAVSAGEKVTMSCKSSQSVLSSSNQKNYLAWYQQKPGQSPKLLIYWASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLTVYYCHQYLSSLTFGAGTKLELK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.6, SEQ ID NO.19 or SEQ ID NO. 33; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown in SEQ ID NO.38, SEQ ID NO.56 or SEQ ID NO.68, and is marked as clone 83.
Wherein the sequence of the heavy chain variable region of clone83 (SEQ ID NO.87) is:
MGWSSIILFLVATATGVHSQGQLQQPGAELVRPGAAVKLSCKASGYSFTNYWMNWVKQRPGQGLEWIGMIHPSDSATRLNPKFKDKATLTVDRSSSTAYMQLSSPTSEDSAVYYCARDGYWGQGTTLTVSS。
wherein the sequence of the light chain variable region of clone83 (SEQ ID NO.88) is:
MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLTVSVGEKVTMSCKSSQSLLYSSNQKNYLAWFQQKPGQSPKLLIYWTSTRESGVPDRFTGSGSGTDFTLTISSVMAEDLAVYYCQQYYSIPYTFGGGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.12 or SEQ ID NO. 29; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.44, SEQ ID NO.57 or SEQ ID NO.69 and is marked as clone 91.
Wherein the sequence of the heavy chain variable region of clone 91 (SEQ ID NO.89) is:
MGWIWIFLLLLSGTAGVHSEVQLQQSGPELVKTGASVKISCKASGYSFTGYYMHWVKQSHGRSLEWIGYISCYNGATNYKQKFRGKATFTVDTSSSTAYMQFNSLTSEDSAVYYCARSYGYPITGAMDYWGQGTSVTVSS。
wherein the sequence of the light chain variable region of clone 91 (SEQ ID NO.90) is:
MSSAQFLGLLLLCFQGTRCDIQMTQTPSSLSASLGDRVTISCRASQDINNYFNWYQQRPDGTVKHLIYYSSRLHSGVPSRFSGSGSGADYSLTISNLEQEDIATYFCQQGYTLPWTFGRGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.7, SEQ ID NO.20 or SEQ ID NO. 30; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.45, SEQ ID NO.58 or SEQ ID NO.65, and is marked as clone 102.
Wherein the sequence of the heavy chain variable region of clone 102 (SEQ ID NO.91) is:
MNFGFSLIFLVLVLKGVQCEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASITSGGITYYPDSVKGRITVSRDNARNILYLQMSSLRSEDTAMYYCASLHYYGGPSYAMDYWGQGSSVTVSS。
wherein the sequence of the light chain variable region of clone 102 (SEQ ID NO.92) is:
MRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTITCKASQDINRYLSWLQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPFTFGSGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.7, SEQ ID NO.21 or SEQ ID NO. 30; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.46, SEQ ID NO.59 or SEQ ID NO.65, and is marked as clone 104.
Wherein the sequence of the heavy chain variable region of clone 104 (SEQ ID NO.93) is:
MNFGFSLIFLVLVLKGVQCEVKLVESGGGLVKPGGSLKLSCAASGFIFSSYAMSWVRQSPEKRLEWVASITSGGIPYYVDNMKGRFTVSRDNARNILYLQMSSLRSEDTAMYFCASLHYYGGPSYAMDYWGQGTSVTVSS。
wherein the sequence of the light chain variable region of clone 104 (SEQ ID NO.94) is:
MRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTITCKASRDLNRYLSWLQQKPGKSPKTLIYRASNLVDGVPSRFSGSGSGQDYSLTISSLEYEDVGIYFCLQYDEFPFTLGSGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.8, SEQ ID NO.22 or SEQ ID NO. 34; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.47, SEQ ID NO.60 or SEQ ID NO.70, and is marked as clone 113.
Wherein the sequence of the heavy chain variable region of clone 113 (SEQ ID NO.95) is:
MAVLGLLLCLVTFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQPPGQGLEWLGVIWGGGPTYYNSALKSRLRFTKDNSKNQVFLKMNSLQTDDTAMYFCAKHENFYGGSSAMDYWGQGTSVTVSS。
wherein the sequence of the light chain variable region of clone 113 (SEQ ID NO.96) is:
MSPAQFLFLLVLWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.9, SEQ ID NO.23 or SEQ ID NO. 31; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.48, SEQ ID NO.54 or SEQ ID NO.66 and is marked as clone 115.
Wherein the sequence of the heavy chain variable region of clone 115 (SEQ ID NO.97) is:
MECNWILPFILSVISGVYSEVQLQQSGTVLARPGASVKMSCEASGYTFTNYWMHWLKQRPGQGLEWIGAMYPGKSDIRYNQKFKGKAKLTAVTSASTAYMELSSLTNEDSAVYYCTRWGDSIDNWGQGTTLTVSS。
wherein the sequence of the light chain variable region of clone 115 (SEQ ID NO.98) is:
MEKDTLLLWVLLLWVPGSTGDIILTQSPASLAVSLGQRATISCRATESVDSSGISFMNWFQQKPGQPPKLLIYAASKQGSGVPARYSGSGSGTDFSLNIQPMEEDDSAMYFCQQSKELPYTFGGGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.10, SEQ ID NO.24 or SEQ ID NO. 35; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.49, SEQ ID NO.61 or SEQ ID NO.71 and is marked as clone 129.
Wherein the sequence of the heavy chain variable region of clone 129 (SEQ ID NO.99) is:
MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGNTFTTYVMHWVKQKPGQGLEWIGYINPYNDGSKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAGYDKTVDWFAYWGQGTLVTVSA。
wherein the sequence of the light chain variable region of clone 129 (SEQ ID NO.100) is:
MSPAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLFSNGKTYLNWLLQRPGQSPKRLFYLVSQLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPWTFGGGTKLEIK。
as a preferred technical scheme of the invention, the heavy chain variable region of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.11, SEQ ID NO.25 or SEQ ID NO. 36; the variable region of the light chain of the anti-human IL-17RC monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO.50, SEQ ID NO.62 or SEQ ID NO.72, and is marked as clone 143.
Wherein the sequence of the heavy chain variable region of clone 143 (SEQ ID NO.101) is:
MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTIHWMKQSHGKSLEWIGGINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGNYVGYYAMDYWGQGTSVTVSS。
wherein the sequence of the light chain variable region of clone 143 (SEQ ID NO.102) is:
MSVLTQVLALLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASGNIHNYLVWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPPTFGGGTKLEIK。
the pharmacological activity of the anti-human IL-17RC monoclonal antibody is as follows:
in the presence of the anti-human IL-17RC monoclonal antibody, HDF cells or HT29 cells are stimulated with recombinant IL-17A, respectively. Supernatants were removed 48 hours after stimulation and assayed for secretion of IL-6 or GRO-. alpha.by ELISA. The results show that the IC50 value of the monoclonal antibody clone 26 of anti-human IL-17RC for inhibiting GRO-alpha secretion is 0.071 mu g/mL, the IC50 value of clone83 for inhibiting GRO-alpha secretion is 0.059 mu g/mL, and the IC50 value of clone38 for inhibiting GRO-alpha secretion is 0.439 mu g/mL. The IC50 value of the monoclonal antibody clone 26 of anti-human IL-17RC for inhibiting IL-6 secretion is 0.338 mu g/mL, the IC50 value of clone83 for inhibiting IL-6 secretion is 0.238 mu g/mL, and the IC50 value of clone38 for inhibiting IL-6 secretion is 2.632 mu g/mL.
The anti-human IL-17RC monoclonal antibody can be simultaneously combined with human IL-17RC and cynomolgus monkey IL-17RC, and is not combined with mouse IL-17 RC. Wherein the antibody heavy chain constant region is of the IgG4 subtype and the light chain constant region comprises the kappa subtype.
The monoclonal antibody of the invention against human IL-17RC can:
(1) specifically recognizing and binding IL-17RC (including human IL-17RC, cynomolgus monkey IL-17RC, and murine IL-17 RC).
(2) Binds to IL-17RC on the surface of HEK-Blue IL-17 cells overexpressing the IL-17RC antigen; binds to IL-17RC on the cell surface of HFDa.
(3) Inhibit GRO-alpha secretion from IL-17A-stimulated HT-29 cells.
(4) Inhibit IL-17A-stimulated secretion of IL-6 by HDF cells.
In a second aspect, the present invention also provides a nucleic acid fragment comprising a nucleotide sequence encoding the anti-human IL-17RC monoclonal antibody according to the first aspect.
In a third aspect, the present invention also provides an expression vector comprising at least one copy of a nucleic acid fragment according to the second aspect.
In a fourth aspect, the present invention also provides a recombinant host cell comprising a nucleic acid fragment according to the second aspect or an expression vector according to the third aspect.
In a fifth aspect, the present invention also provides a use of any one or a combination of at least two of the anti-human IL-17RC monoclonal antibody according to the first aspect, the nucleic acid fragment according to the second aspect, the expression vector according to the third aspect, or the recombinant host cell according to the fourth aspect, in the preparation of a medicament for treating or preventing an autoimmune disease and/or tumor.
Preferably, the autoimmune disease and/or tumor is an IL-17 mediated autoimmune disease and/or tumor.
Preferably, the autoimmune disease comprises any one of psoriasis, rheumatoid arthritis or ankylosing spondylitis.
Preferably, the tumor comprises any one of colon cancer, lung cancer or breast cancer.
It is to be noted that scientific and technical terms and abbreviations thereof used in the present invention have meanings commonly understood by those skilled in the art. Some of the terms and abbreviations used in the present invention are listed below:
heavy chain: heavy chain, HC; light chain: light chain, LC.
Heavy chain variable region: a variable region of heavy chain, VH.
Light chain variable region: variable region of kappa chain, VK.
Complementarity determining region: complementary determining region, CDR, refers to the antigen complementary binding region of an antibody.
Interleukin-17 cytokine family: interleukin 17, IL-17, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F.
Interleukin-17 receptor: interleukin 17Receptor A, IL-17 RA.
Interleukin-17 receptor: interleukin 17Receptor C, IL-17RC, IL-17RC is a type I membrane protein similar to IL-17 RA.
Interleukin-6: interleukin-6, IL-6.
Nuclear factor kappa-B ligand receptor activators: receptor activator of nuclear factor kappa-B ligand, RANKL.
Tumor necrosis factor: tumor necrosis factor, TNF.
TNF α type ii receptors: TNF α R2.
Human growth regulating oncogene- α: growth-related oncogene-alpha, GRO-alpha.
Horse radish peroxidase: horseradish peroxidase, HRP.
3,3',5,5' -tetramethylbenzidine: 3,3',5,5' -Tetramethylbenzidine, TMB.
Enzyme-linked immunosorbent assay: enzyme linked immunological assay, ELISA.
Surface plasmon resonance: surface plasma resonance, SPR.
In the present invention, "EC 50" means the concentration of antibody at which 50% of maximum effect is caused, i.e., the concentration for 50% of maximum effect.
The "IC 50" in the present invention is the half inhibitory concentration (the half inhibitory concentration).
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention separates, analyzes and screens B cells at a single cell level by a single B cell antibody screening technology, obtains antibody nucleotide information from positive single B cells, screens and obtains antibody sequences capable of simultaneously combining human and cynomolgus monkey IL-17RC, constructs an expression vector of the anti-human IL-17RC monoclonal antibody, expresses and purifies the anti-human IL-17RC monoclonal antibody by a mammalian cell expression system, and the mammalian cell expression system can provide post-translational modification which is closest to a natural state for the monoclonal antibody, so that the obtained protein is closer to a natural protein and has better biological activity.
(2) The pharmacological activity of the anti-human IL-17RC monoclonal antibody is as follows: the monoclonal antibody of anti-human IL-17RC can inhibit GRO-alpha secretion of HT-29 cells and IL-6 secretion of HDF cells. The results show that the IC50 value of the monoclonal antibody clone 26 of anti-human IL-17RC for inhibiting GRO-alpha secretion is 0.071 mu g/mL, the IC50 value of clone83 for inhibiting GRO-alpha secretion is 0.059 mu g/mL, and the IC50 value of clone38 for inhibiting GRO-alpha secretion is 0.439 mu g/mL. The IC50 value of the monoclonal antibody clone 26 of anti-human IL-17RC for inhibiting IL-6 secretion is 0.338 mu g/mL, the IC50 value of clone83 for inhibiting IL-6 secretion is 0.238 mu g/mL, and the IC50 value of clone38 for inhibiting IL-6 secretion is 2.632 mu g/mL.
(3) The anti-human IL-17RC monoclonal antibody can specifically recognize and specifically combine human IL-17RC and cynomolgus monkey IL-17 RC; effectively block the combination of IL-17A and IL-17F and IL-17RC, inhibit and/or block the signal transduction of IL-17A and IL-17F mediated by IL-17 RC; the monoclonal antibody of the anti-human IL-17RC can be combined with HEK-Blue IL-17 cell surface IL-17RC over-expressing IL-17RC antigen; the anti-human IL-17RC monoclonal antibody developed by taking IL-17RC as a target point can be used for treating IL-17 pathway related diseases.
Drawings
FIG. 1 is a graph showing the detection of the binding ability of the monoclonal antibody against human IL-17RC and human IL-17RC in example 4.
FIG. 2 is a graph showing the detection of the binding ability of the monoclonal antibody against human IL-17RC and cynomolgus monkey IL-17RC in example 5.
FIG. 3 is a graph showing the detection of the binding ability of the monoclonal antibody against human IL-17RC and mouse IL-17RC in example 5
FIG. 4 is a graph showing the binding of the anti-human IL-17RC monoclonal antibody in example 6 to HEK-Blue IL-17 cell surface IL-17RC, including clone 10, clone 26, clone38, clone 50, clone 52, clone 53, clone 68 and clone 83.
FIG. 5 is a graph showing the binding of the anti-human IL-17RC monoclonal antibody of example 6 to IL-17RC on the cell surface of HEK-Blue IL-17, including clone 91, clone 102, clone 104, clone 115, clone 129 and clone 143.
FIG. 6 is a graph showing the measurement of GRO-. alpha.secretion amount of IL-17A-stimulated HT-29 cells treated with clone 26, clone83 and clone38 in the anti-human IL-17RC monoclonal antibody of example 8.
FIG. 7 is a graph showing the measurement of IL-6 secretion amount after treating IL-17A-stimulated HDF cells with clone 26, clone83 and clone38 in the anti-human IL-17RC monoclonal antibody of example 8.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 Single B cell antibody screening
The single B cell antibody screening technology is based on a microfluidic technology, and directly performs single cell level separation, analysis and screening on B cells, so that the B cells secreting target antibody molecules are accurately and efficiently screened, and then a single cell sequencing technology is combined, so that a target antibody sequence can be obtained. This technique is one of the most mature antibody library techniques at present, and has been successfully applied to the preparation of human monoclonal antibody drugs.
The specific method comprises the following steps:
(1) single B cell isolation and Beacon screening: immunizing splenocytes of mice with human IL-17RC recombinant protein, separating splenocytes from animals meeting the titer requirement, enriching B cells by using CD138B cell separation kit on the same day, and controlling the cell density at 1.5 × 107cells/mL, CD138B cell isolation kit was purchased from Meitian whirlpool Biotechnology, Inc., Germany, and isolated according to the instructions. B cells were loaded onto Beacon chips and 20000B cells were expected to be loaded per round of experiment. Antibody affinity-based screening of all B cells was performed against human/cynomolgus monkey target proteins. And (4) leading out positive single B cells meeting the screening requirement to a 96-well plate, and storing the positive single B cells in a special lysate, and freezing and storing the positive single B cells at-80 ℃ for sequencing.
(2) Single B cell sequencing: extracting RNA of positive single B cells, purifying the RNA of the positive single B cells, and carrying out RNA reverse transcription and cDNA amplification. Antibody heavy chain variable region (VH) and light chain variable region (VK) amplifications were then performed, and the amplification products were subjected to VH and VK sequencing and analysis.
By this example, which describes strategies and methods for screening monoclonal antibodies against human IL-17RC using a mature single B-cell screening method, antibody sequences that bind both human IL-17RC and cynomolgus IL-17RC are obtained. The nucleotide sequences of the variable regions of the heavy chain (VH) and light chain (VK) of the antibodies are shown in Table 2.
TABLE 2
Antibody numbering
Heavy chain variable region nucleotide sequence (VH)
Light chain variable region nucleotide sequence (VK)
clone 10
SEQ ID NO.73
SEQ ID NO.74
clone 26
SEQ ID NO.75
SEQ ID NO.76
clone 38
SEQ ID NO.77
SEQ ID NO.78
clone 50
SEQ ID NO.79
SEQ ID NO.80
clone 52
SEQ ID NO.81
SEQ ID NO.82
clone 53
SEQ ID NO.83
SEQ ID NO.84
clone 68
SEQ ID NO.85
SEQ ID NO.86
clone 83
SEQ ID NO.87
SEQ ID NO.88
clone 91
SEQ ID NO.89
SEQ ID NO.90
clone 102
SEQ ID NO.91
SEQ ID NO.92
clone 104
SEQ ID NO.93
SEQ ID NO.94
clone 113
SEQ ID NO.95
SEQ ID NO.96
clone 115
SEQ ID NO.97
SEQ ID NO.98
clone 129
SEQ ID NO.99
SEQ ID NO.100
clone 143
SEQ ID NO.101
SEQ ID NO.102
Example 2 expression and purification of monoclonal antibodies against human IL-17RC
The heavy chain and light chain genes of the anti-human IL-17RC monoclonal antibody are respectively cloned to a eukaryotic expression vector pcDNA3.1, wherein the heavy chain constant region of the antibody is of an IgG4 subtype, and the light chain constant region of the antibody is of a kappa subtype.
24h before transfection, cells of Expi293F were cultured normally in Expi293 expression medium, cells of Expi293F were purchased from Nanjing Kinshire Biotech, Inc., and cultured according to the instructions and then cultured at 37 ℃ under 8% CO2In a rocking bed according to (1). Cell suspensions were taken for observation of contamination and for counting. Preheated Opti-MEM medium was added to each of the two tubes. The heavy chain plasmid and the light chain plasmid were added to one of the centrifuge tubes, and the transfection reagent was added to the other centrifuge tube, and incubated at room temperature for 5 min. The plasmid and transfection reagent were then mixed to give a complex, and the complex was incubated at room temperature for 20 min.
The complexes were added to Expi293F cells and the flasks were returned to 37 ℃ with 8% CO2The cultivation was continued for 16h in the shaker. Adding enhancer1 into the shake flask&enhancer2, and then the flask was returned to the shaker for further cultivation. The cells were harvested and centrifuged, and the supernatant collected and purified. Using AmMagTMProtein A Magnetic Beads (Kinsley, L00695) purified cell supernatants.
The cell supernatant was filtered through a 0.22 μm filter. The Protein A/G Resin affinity column was removed from the refrigerator and 12 column volumes were equilibrated with Binding buffer. The filtered supernatant was applied to a Protein A/G Resin affinity column at 4 ℃ and a flow rate of 3 mL/min. The Protein A/G Resin affinity column was equilibrated to 12 column volumes with Binding buffer until A280 reached baseline. Eluting the Protein A/G Resin affinity column by using an Elution buffer and collecting according to A280 of a nucleic acid Protein detector to obtain the anti-human IL-17RC monoclonal antibody. The Protein A/G Resin affinity column was equilibrated with Binding buffer for 12 column volumes and stored at 4 ℃ for future use.
The eluted and neutralized monoclonal antibody against human IL-17RC was packed in a 0.01 μm Dialysis bag, dialyzed at room temperature against a Dialysis buffer for 2 hours, and replaced with fresh buffer for 12 hours at 4 ℃. The anti-human IL-17RC monoclonal antibody was transferred from the dialysis bag to a 50mL tube and the volume was recorded. The concentration was measured by Nanodrop 2000 and the extinction coefficient was 1.43 (IgG).
Example 3 preliminary functional analysis of monoclonal antibody against human IL-17RC
Human IL-17RC, cynomolgus monkey IL-17RC and mouse IL-17RC recombinant proteins were coated with Phosphate Buffered Saline (PBS) at pH7.4, 100. mu.L of 1.0. mu.g/mL recombinant protein was added to each well, and the mixture was coated at 4 ℃ for 12 hours. PBST (0.05% Tween20 in neutral PBS) was washed three times and blocked for 1h at 37 ℃ for PBST-4% mil. The monoclonal antibody against human IL-17RC was diluted, and 100. mu.L of the detection stock solution was added to each well of the ELISA plate and incubated at 37 ℃ for 1 hour. PBST washing ELISA plate, adding HRP-Anti-Human IgG, HRP-Anti-Monkey IgG and HRP-Anti-Mouse IgG secondary antibody 1:5000 dilution, each well adding 100 u L, 37 degrees C placed for 1 h. Developing with TMB developing solution for 15min, 1M H2SO4The development was stopped at 50. mu.L/well, and the optical density was measured at OD450 wavelength with a microplate reader, the results of which are shown in Table 3.
TABLE 3
Example 4 binding experiment of monoclonal antibody against human IL-17RC and human IL-17RC
The ability of the monoclonal antibody against human IL-17RC to bind to human IL-17RC-His was analyzed by ELISA. Human IL-17RC-his was coated (1. mu.g/mL, 100. mu.L/well, 12h at 4 ℃) at an initial concentration of 1000ng/mL for each monoclonal antibody and a 3-fold gradient dilution was performed, setting 11 dilutions for each monoclonal antibody sample. The ability of monoclonal antibodies of various dilutions to bind to human IL-17RC was tested using HRP-coat-anti-human IgG and the results are shown in FIG. 1.
It can be seen from FIG. 1 that 14 monoclonal antibodies, clone 10, clone 26, clone38, clone 50, clone 52, clone 53, clone 68, clone83, clone 91, clone 102, clone 104, clone 115, clone 129 and clone 143, are capable of specifically binding to human IL-17 RC.
Example 5 binding of monoclonal antibodies against human IL-17RC to IL-17RC of different species
IL-17RC recombinant proteins (cynomolgus monkey IL-17RC, mouse IL-17RC) were coated in 96-well ELISA plates (1. mu.g/mL, 100. mu.L/well, coating at 4 ℃ for 12h), respectively. Adding different anti-IL-17 RC monoclonal antibodies, binding at 37 deg.C for 1h, washing with PBST for 4 times, adding different HRP-labeled IgG (secondary antibody), binding at 37 deg.C for 1h, washing with PBST for 4 times, adding TMB substrate developing solution, and washing with 1M H after 10min2SO4The color development was stopped and the absorbance value was measured at OD 450.
As shown in FIG. 2, 14 of clone 10, clone 26, clone38, clone 50, clone 52, clone 53, clone 68, clone83, clone 91, clone 102, clone 104, clone 115, clone 129 and clone 143 in the anti-human IL-17RC monoclonal antibody provided by the present invention can specifically bind to cynomolgus monkey IL-17RC, as shown in FIG. 3, wherein clone38 can also bind to mouse IL-17 RC.
Example 6 binding ability of monoclonal antibody against human IL-17RC to cell surface IL-17RC
While functional anti-human IL-17RC monoclonal antibodies should block binding between IL-17RC and IL-17 cytokines at the protein level, this example analyzes the binding ability of 14 different anti-human IL-17RC monoclonal antibodies (including clone 10, clone 26, clone38, clone 50, clone 52, clone 53, clone 68, clone83, clone 91, clone 102, clone 104, clone 115, clone 129 and clone 143) to cell surface IL-17 RC.
HFDa cells were purchased from ATCC and cultured according to the cell's product specifications. Detecting activity by flow cytometry, collecting HFDa cell, incubating with antibody to be detected, and incubating at 1 × 10/tube5The concentration of the monoclonal antibody to be tested against human IL-17RC was 45. mu.g/mL per cell (50. mu.L). After incubation at 4 ℃ for 1h, wash 2 times with PBS. Alexa was added at 1. mu.g/mL per tube647 Affinipure Goat Anti-Human IgG secondary antibody, incubation at 4 deg.C for 20min, washing with PBS 2 timesAnd detecting in a flow cytometer. The binding ability of the monoclonal antibody against human IL-17RC to cell surface IL-17RC is shown in Table 4, and the larger the MFI value is, the stronger the monoclonal antibody is bound to cell surface antigen, wherein the binding ability of clone 26 and clone83 is the strongest.
TABLE 4
Antibody numbering
Fluorescence signal value (MFI)
Antibody numbering
Fluorescence signal value (MFI)
clone 10
128
clone 83
1458
clone 26
1327
clone 91
119
clone 38
72
clone 102
111
clone 50
64
clone 104
89
clone 52
52
clone 113
80
clone 53
59
clone 115
628
clone 68
861
clone 129
50
Negative control IgG1
27
-
-
While functional anti-human IL-17RC monoclonal antibodies should block binding between IL-17RC and IL-17 cytokines at the protein level, this example analyzes the binding ability of 14 different anti-human IL-17RC monoclonal antibodies (including clone 10, clone 26, clone38, clone 50, clone 52, clone 53, clone 68, clone83, clone 91, clone 102, clone 104, clone 115, clone 129 and clone 143) to cell surface IL-17 RC.
HEK-Blue IL-17 cells were purchased from Invivogen and cultured according to the cell's product instructions. Performing activity detection by flow cytometry, collecting HEK-Blue IL-17 cells, incubating with the antibody to be detected, and incubating at 1 × 10 per tube5Each cell (50. mu.L) was assayed at an initial concentration of 45. mu.g/mL of anti-human IL-17RC monoclonal antibody, 3-fold diluted, 11 concentrations. After incubation at 4 ℃ for 1h, wash 2 times with PBS. Alexa was added at 1. mu.g/mL per tube647 Affinipure Goat Anti-Human IgG secondary antibody, incubated at 4 ℃ for 20min, washed 2 times with PBS, and detected by flow cytometry. The binding ability of various anti-human IL-17RC monoclonal antibodies to cell surface IL-17RC is shown in FIGS. 4 and 5, wherein the binding ability of clone 26 and clone83 is strongest, and EC50 is 0.00063. mu.g/mL and 0.00055. mu.g/mL, respectively.
Example 7 affinity of clone38 for mouse IL-17RC protein in monoclonal antibody against human IL-17RC
On the basis of determining the binding capacity of different monoclonal antibodies against human IL-17RC and cell surface IL-17RC, the affinity of the candidate clone38 and mouse IL-17RC protein is also detected by using an SPR method.
The affinity of clone38 was tested using a Biacore instrument at 25 ℃. Different concentrations of clone38 were coupled on the chip by Fc capture method and the mouse IL-17RC recombinant protein was detected as mobile phase. The affinity of clone38 for the mouse IL-17RC recombinant protein is shown in Table 5.
TABLE 5
Ligands
Analyte
ka(1/Ms)
kd(1/s)
KD(M)
Rmax(RU)
Chi2(RU2)
clone 38
Mouse IL-17RC
1.80E+04
2.47E-02
1.37E-06
30.54
0.016
EXAMPLE 8 in vitro evaluation of the pharmacological effects of clone38, clone 26 and clone83 in monoclonal antibodies against human IL-17RC
(1) IL-17A stimulates HT-29 cells to secrete GRO-alpha
IL-17A was used to stimulate GRO-alpha secretion from HT-29 cells and the ability of clone 26, clone83 and clone38 to inhibit GRO-alpha secretion was examined. HT-29 cells were purchased from the cell resource center of the institute of basic medicine, academy of Chinese medical sciences and cultured according to the product instructions for the cells.
HT-29 cell digestion inoculation 96-well plate, 3X 104100 μ L/well, 12h, the next day, discard old medium, add 150 μ L complete medium containing antibody + IL-17A (200ng/mL) to each well, incubate for 48h, and then take 100 μ L supernatant for ELISA detection. As shown in FIG. 4, under the conditions, the IC50 value of clone 26 of anti-human IL-17RC for inhibiting GRO-alpha secretion is 0.071 ug/mL, the IC50 value of clone83 for inhibiting GRO-alpha secretion is 0.059 ug/mL, and the IC50 value of clone38 for inhibiting GRO-alpha secretion is 0.439 ug/mL.
(2) IL-17A stimulation of IL-6 secretion by HDF cells
IL-17A was used to stimulate IL-6 secretion from HDF cells, and the ability of clone 26, clone83 and clone38 to inhibit IL-6 secretion was examined. HDF cells were purchased from ScienCell, inc and cultured according to the cell's product instructions.
HDF cell digestion inoculation 96-well plate, 2X 104100 μ L/well, culturing for 12h, discarding the old medium, adding 150 μ L complete medium containing antibody + IL-17A (200ng/mL) + TNF- α (10ng/mL) to each well, incubating for 48h,then 100. mu.L of the supernatant was used for ELISA detection. As shown in FIG. 5, under the conditions, the IC50 value of the monoclonal antibody clone 26 of anti-human IL-17RC for inhibiting IL-6 secretion is 0.338 μ g/mL, the IC50 value of clone83 for inhibiting IL-6 secretion is 0.238 μ g/mL, and the IC50 value of clone38 for inhibiting IL-6 secretion is 2.632 μ g/mL.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Harbour grass biology (Suzhou) Ltd
<120> anti-human IL-17RC monoclonal antibody and application thereof
<130> 2021
<160> 102
<170> PatentIn version 3.3
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<213> artificially synthesized sequence
<400> 42
Arg Ala Ser Glu Ser Val Asp Ser Ser Gly Ile Ser Phe Met Asn
1 5 10 15
<210> 43
<211> 17
<212> PRT
<213> artificially synthesized sequence
<400> 43
Lys Ser Ser Gln Ser Val Leu Ser Ser Ser Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 44
<211> 11
<212> PRT
<213> artificially synthesized sequence
<400> 44
Arg Ala Ser Gln Asp Ile Asn Asn Tyr Phe Asn
1 5 10
<210> 45
<211> 11
<212> PRT
<213> artificially synthesized sequence
<400> 45
Lys Ala Ser Gln Asp Ile Asn Arg Tyr Leu Ser
1 5 10
<210> 46
<211> 11
<212> PRT
<213> artificially synthesized sequence
<400> 46
Lys Ala Ser Arg Asp Leu Asn Arg Tyr Leu Ser
1 5 10
<210> 47
<211> 16
<212> PRT
<213> artificially synthesized sequence
<400> 47
Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn
1 5 10 15
<210> 48
<211> 15
<212> PRT
<213> artificially synthesized sequence
<400> 48
Arg Ala Thr Glu Ser Val Asp Ser Ser Gly Ile Ser Phe Met Asn
1 5 10 15
<210> 49
<211> 16
<212> PRT
<213> artificially synthesized sequence
<400> 49
Lys Ser Ser Gln Ser Leu Leu Phe Ser Asn Gly Lys Thr Tyr Leu Asn
1 5 10 15
<210> 50
<211> 11
<212> PRT
<213> artificially synthesized sequence
<400> 50
Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Val
1 5 10
<210> 51
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 51
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 52
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 52
Arg Ala Asn Arg Leu Met Asp
1 5
<210> 53
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 53
Arg Ala Asn Ser Leu Val Asp
1 5
<210> 54
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 54
Ala Ala Ser Lys Gln Gly Ser
1 5
<210> 55
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 55
Trp Ala Ser Thr Arg Ala Ser
1 5
<210> 56
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 56
Trp Thr Ser Thr Arg Glu Ser
1 5
<210> 57
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 57
Tyr Ser Ser Arg Leu His Ser
1 5
<210> 58
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 58
Arg Ala Asn Arg Leu Val Asp
1 5
<210> 59
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 59
Arg Ala Ser Asn Leu Val Asp
1 5
<210> 60
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 60
Leu Val Ser Lys Leu Asp Ser
1 5
<210> 61
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 61
Leu Val Ser Gln Leu Asp Ser
1 5
<210> 62
<211> 7
<212> PRT
<213> artificially synthesized sequence
<400> 62
Asn Ala Lys Thr Leu Ala Asp
1 5
<210> 63
<211> 8
<212> PRT
<213> artificially synthesized sequence
<400> 63
His Gln Phe Leu Ser Ser Leu Thr
1 5
<210> 64
<211> 9
<212> PRT
<213> artificially synthesized sequence
<400> 64
Gln Gln Tyr Tyr Ser Phe Pro Tyr Thr
1 5
<210> 65
<211> 9
<212> PRT
<213> artificially synthesized sequence
<400> 65
Leu Gln Tyr Asp Glu Phe Pro Phe Thr
1 5
<210> 66
<211> 9
<212> PRT
<213> artificially synthesized sequence
<400> 66
Gln Gln Ser Lys Glu Leu Pro Tyr Thr
1 5
<210> 67
<211> 8
<212> PRT
<213> artificially synthesized sequence
<400> 67
His Gln Tyr Leu Ser Ser Leu Thr
1 5
<210> 68
<211> 9
<212> PRT
<213> artificially synthesized sequence
<400> 68
Gln Gln Tyr Tyr Ser Ile Pro Tyr Thr
1 5
<210> 69
<211> 9
<212> PRT
<213> artificially synthesized sequence
<400> 69
Gln Gln Gly Tyr Thr Leu Pro Trp Thr
1 5
<210> 70
<211> 9
<212> PRT
<213> artificially synthesized sequence
<400> 70
Trp Gln Gly Thr His Phe Pro Arg Thr
1 5
<210> 71
<211> 9
<212> PRT
<213> artificially synthesized sequence
<400> 71
Val Gln Gly Thr His Phe Pro Trp Thr
1 5
<210> 72
<211> 9
<212> PRT
<213> artificially synthesized sequence
<400> 72
Gln His Phe Trp Ser Thr Pro Pro Thr
1 5
<210> 73
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 73
Met Gly Trp Ile Trp Ile Phe Leu Leu Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
Thr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Lys
65 70 75 80
Gln Lys Phe Arg Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
85 90 95
Thr Ala Tyr Met Gln Phe Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Ser Tyr Gly Tyr Pro Asn Thr Gly Ala Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
<210> 74
<211> 132
<212> PRT
<213> artificially synthesized sequence
<400> 74
Met Glu Ser Gln Thr Gln Val Phe Leu Ser Leu Leu Leu Trp Val Ser
1 5 10 15
Gly Thr Cys Gly Asn Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Ala
20 25 30
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
35 40 45
Val Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr
100 105 110
Tyr Cys His Gln Phe Leu Ser Ser Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Leu Lys
130
<210> 75
<211> 137
<212> PRT
<213> artificially synthesized sequence
<400> 75
Met Gly Arg Leu Thr Ser Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr
1 5 10 15
Val Leu Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln
20 25 30
Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu
35 40 45
Arg Thr Ser Gly Met Gly Ala Gly Trp Ile Arg Gln Pro Ser Gly Lys
50 55 60
Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
65 70 75 80
Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser
85 90 95
Asn Gln Val Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala
100 105 110
Thr Tyr Tyr Cys Ala Arg Ile Ala Asn Arg Tyr Phe Asp Val Trp Gly
115 120 125
Ala Gly Thr Thr Val Thr Val Ser Ser
130 135
<210> 76
<211> 133
<212> PRT
<213> artificially synthesized sequence
<400> 76
Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser
1 5 10 15
Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala
20 25 30
Val Ser Val Gly Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser
35 40 45
Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr
100 105 110
Tyr Cys Gln Gln Tyr Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly Thr
115 120 125
Lys Leu Glu Ile Lys
130
<210> 77
<211> 133
<212> PRT
<213> artificially synthesized sequence
<400> 77
Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Gly Gly
1 5 10 15
Val Leu Ser Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Met Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Asp Tyr Tyr Ile Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Asp Ile Asn Pro Asn Asn Gly Asp Ile Phe Tyr Asn
65 70 75 80
Gln Asn Phe Lys Gly Lys Val Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Lys Ile Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Lys Gly Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
115 120 125
Val Thr Val Ser Ala
130
<210> 78
<211> 127
<212> PRT
<213> artificially synthesized sequence
<400> 78
Met Arg Thr Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ile Lys Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Phe
20 25 30
Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp
35 40 45
Ile Asn Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro
50 55 60
Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Met Asp Gly Val Pro Ser
65 70 75 80
Gly Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser
85 90 95
Ser Leu Asp Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp
100 105 110
Glu Phe Pro Phe Thr Phe Gly Gly Gly Thr Thr Leu Glu Ile Lys
115 120 125
<210> 79
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 79
Met Gly Trp Ile Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Leu Lys
20 25 30
Thr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Tyr Ile Asn Cys Tyr Asn Gly Ala Thr Gly Tyr Asn
65 70 75 80
Gln Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
85 90 95
Thr Ala Tyr Met Gln Phe Asp Ser Leu Thr Ser Glu Asp Ser Ala Ile
100 105 110
Tyr Tyr Cys Ala Arg Ser Tyr Gly Tyr Pro Ile Thr Gly Ala Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
<210> 80
<211> 132
<212> PRT
<213> artificially synthesized sequence
<400> 80
Met Glu Ser Gln Thr Gln Val Phe Leu Ser Leu Leu Leu Trp Val Ser
1 5 10 15
Gly Thr Cys Gly Asn Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Ala
20 25 30
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Thr
35 40 45
Val Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr
100 105 110
Tyr Cys His Gln Phe Leu Ser Ser Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Leu Lys
130
<210> 81
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 81
Met Asn Phe Gly Phe Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Arg
20 25 30
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Asn Thr Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu
50 55 60
Glu Trp Val Ala Ser Ile Thr Ser Gly Gly Ile Pro Tyr Tyr Pro Asp
65 70 75 80
Asn Met Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Asn Ile
85 90 95
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr
100 105 110
Tyr Cys Ala Ser Leu His Tyr Tyr Gly Gly Pro Ser Tyr Ala Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
<210> 82
<211> 127
<212> PRT
<213> artificially synthesized sequence
<400> 82
Met Arg Thr Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ile Lys Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr
20 25 30
Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp
35 40 45
Leu Asn Arg Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Ser Pro
50 55 60
Lys Thr Leu Ile Tyr Arg Ala Asn Ser Leu Val Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser
85 90 95
Ser Leu Glu Tyr Glu Asp Val Gly Phe Tyr Tyr Cys Leu Gln Tyr Asp
100 105 110
Glu Phe Pro Phe Thr Leu Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 83
<211> 135
<212> PRT
<213> artificially synthesized sequence
<400> 83
Met Glu Cys Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Ile Ser Gly
1 5 10 15
Val Tyr Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg
20 25 30
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Asp Tyr Trp Leu His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Gly Ala Met Asn Pro Gly Lys Ser Asp Ile Arg Tyr Asn
65 70 75 80
Gln Arg Phe Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Thr Arg Trp Gly Asp Ser Ile Asp Asn Trp Gly Gln Gly
115 120 125
Thr Thr Leu Ile Val Ser Ser
130 135
<210> 84
<211> 131
<212> PRT
<213> artificially synthesized sequence
<400> 84
Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser
35 40 45
Val Asp Ser Ser Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro
50 55 60
Gly Gln Ala Pro Lys Leu Ile Ile Tyr Ala Ala Ser Lys Gln Gly Ser
65 70 75 80
Gly Val Pro Ala Arg Tyr Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser
85 90 95
Leu Asn Ile Gln Pro Met Lys Glu Asp Asp Ser Ala Met Tyr Phe Cys
100 105 110
Gln Gln Ser Lys Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys
130
<210> 85
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 85
Met Gly Trp Ile Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
Thr Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Thr Tyr Thr
65 70 75 80
Gln Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
85 90 95
Thr Ala Tyr Met Gln Phe Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Ser Tyr Gly Tyr Pro Ile Thr Asn Ser Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
<210> 86
<211> 132
<212> PRT
<213> artificially synthesized sequence
<400> 86
Met Glu Ser Gln Thr Gln Val Phe Leu Ser Leu Leu Leu Trp Val Ser
1 5 10 15
Gly Thr Cys Gly Asn Ile Met Met Thr Gln Ser Pro Ser Ser Leu Ala
20 25 30
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
35 40 45
Val Leu Ser Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
65 70 75 80
Ala Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Thr Val Tyr
100 105 110
Tyr Cys His Gln Tyr Leu Ser Ser Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Leu Lys
130
<210> 87
<211> 131
<212> PRT
<213> artificially synthesized sequence
<400> 87
Met Gly Trp Ser Ser Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Gln Gly Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg
20 25 30
Pro Gly Ala Ala Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Asn Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Gly Met Ile His Pro Ser Asp Ser Ala Thr Arg Leu Asn
65 70 75 80
Pro Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser
85 90 95
Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Asp Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr
115 120 125
Val Ser Ser
130
<210> 88
<211> 133
<212> PRT
<213> artificially synthesized sequence
<400> 88
Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser
1 5 10 15
Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Thr
20 25 30
Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
35 40 45
Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Phe Gln Gln
50 55 60
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg
65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Val Met Ala Glu Asp Leu Ala Val Tyr
100 105 110
Tyr Cys Gln Gln Tyr Tyr Ser Ile Pro Tyr Thr Phe Gly Gly Gly Thr
115 120 125
Lys Leu Glu Ile Lys
130
<210> 89
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 89
Met Gly Trp Ile Trp Ile Phe Leu Leu Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
Thr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Arg Ser Leu
50 55 60
Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Lys
65 70 75 80
Gln Lys Phe Arg Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
85 90 95
Thr Ala Tyr Met Gln Phe Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Ser Tyr Gly Tyr Pro Ile Thr Gly Ala Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
<210> 90
<211> 126
<212> PRT
<213> artificially synthesized sequence
<400> 90
Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln Gly
1 5 10 15
Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Pro Ser Ser Leu Ser Ala
20 25 30
Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile
35 40 45
Asn Asn Tyr Phe Asn Trp Tyr Gln Gln Arg Pro Asp Gly Thr Val Lys
50 55 60
His Leu Ile Tyr Tyr Ser Ser Arg Leu His Ser Gly Val Pro Ser Arg
65 70 75 80
Phe Ser Gly Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Asn
85 90 95
Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr
100 105 110
Leu Pro Trp Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 91
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 91
Met Asn Phe Gly Phe Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu
50 55 60
Glu Trp Val Ala Ser Ile Thr Ser Gly Gly Ile Thr Tyr Tyr Pro Asp
65 70 75 80
Ser Val Lys Gly Arg Ile Thr Val Ser Arg Asp Asn Ala Arg Asn Ile
85 90 95
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr
100 105 110
Tyr Cys Ala Ser Leu His Tyr Tyr Gly Gly Pro Ser Tyr Ala Met Asp
115 120 125
Tyr Trp Gly Gln Gly Ser Ser Val Thr Val Ser Ser
130 135 140
<210> 92
<211> 127
<212> PRT
<213> artificially synthesized sequence
<400> 92
Met Arg Thr Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ile Lys Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr
20 25 30
Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp
35 40 45
Ile Asn Arg Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Ser Pro
50 55 60
Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser
85 90 95
Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp
100 105 110
Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 93
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 93
Met Asn Phe Gly Phe Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe
35 40 45
Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu
50 55 60
Glu Trp Val Ala Ser Ile Thr Ser Gly Gly Ile Pro Tyr Tyr Val Asp
65 70 75 80
Asn Met Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Asn Ile
85 90 95
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr
100 105 110
Phe Cys Ala Ser Leu His Tyr Tyr Gly Gly Pro Ser Tyr Ala Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
<210> 94
<211> 127
<212> PRT
<213> artificially synthesized sequence
<400> 94
Met Arg Thr Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ile Lys Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr
20 25 30
Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asp
35 40 45
Leu Asn Arg Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Ser Pro
50 55 60
Lys Thr Leu Ile Tyr Arg Ala Ser Asn Leu Val Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser
85 90 95
Ser Leu Glu Tyr Glu Asp Val Gly Ile Tyr Phe Cys Leu Gln Tyr Asp
100 105 110
Glu Phe Pro Phe Thr Leu Gly Ser Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 95
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 95
Met Ala Val Leu Gly Leu Leu Leu Cys Leu Val Thr Phe Pro Ser Cys
1 5 10 15
Val Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala
20 25 30
Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Thr Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu
50 55 60
Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Pro Thr Tyr Tyr Asn Ser
65 70 75 80
Ala Leu Lys Ser Arg Leu Arg Phe Thr Lys Asp Asn Ser Lys Asn Gln
85 90 95
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr
100 105 110
Phe Cys Ala Lys His Glu Asn Phe Tyr Gly Gly Ser Ser Ala Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
<210> 96
<211> 131
<212> PRT
<213> artificially synthesized sequence
<400> 96
Met Ser Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg Glu
1 5 10 15
Thr Asn Gly Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val
20 25 30
Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu
35 40 45
Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro
50 55 60
Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
100 105 110
Trp Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys
130
<210> 97
<211> 135
<212> PRT
<213> artificially synthesized sequence
<400> 97
Met Glu Cys Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Ile Ser Gly
1 5 10 15
Val Tyr Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg
20 25 30
Pro Gly Ala Ser Val Lys Met Ser Cys Glu Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Asn Tyr Trp Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Gly Ala Met Tyr Pro Gly Lys Ser Asp Ile Arg Tyr Asn
65 70 75 80
Gln Lys Phe Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Thr Arg Trp Gly Asp Ser Ile Asp Asn Trp Gly Gln Gly
115 120 125
Thr Thr Leu Thr Val Ser Ser
130 135
<210> 98
<211> 131
<212> PRT
<213> artificially synthesized sequence
<400> 98
Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Ile Leu Thr Gln Ser Pro Ala Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Thr Glu Ser
35 40 45
Val Asp Ser Ser Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Lys Gln Gly Ser
65 70 75 80
Gly Val Pro Ala Arg Tyr Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser
85 90 95
Leu Asn Ile Gln Pro Met Glu Glu Asp Asp Ser Ala Met Tyr Phe Cys
100 105 110
Gln Gln Ser Lys Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys
130
<210> 99
<211> 138
<212> PRT
<213> artificially synthesized sequence
<400> 99
Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Asn Thr Phe
35 40 45
Thr Thr Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn
65 70 75 80
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Gly Tyr Asp Lys Thr Val Asp Trp Phe Ala Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ala
130 135
<210> 100
<211> 131
<212> PRT
<213> artificially synthesized sequence
<400> 100
Met Ser Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Gln Glu
1 5 10 15
Thr Asn Gly Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val
20 25 30
Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu
35 40 45
Leu Phe Ser Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro
50 55 60
Gly Gln Ser Pro Lys Arg Leu Phe Tyr Leu Val Ser Gln Leu Asp Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
100 105 110
Val Gln Gly Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys
130
<210> 101
<211> 140
<212> PRT
<213> artificially synthesized sequence
<400> 101
Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe
35 40 45
Thr Glu Tyr Thr Ile His Trp Met Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn
65 70 75 80
Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Arg Gly Asn Tyr Val Gly Tyr Tyr Ala Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
<210> 102
<211> 127
<212> PRT
<213> artificially synthesized sequence
<400> 102
Met Ser Val Leu Thr Gln Val Leu Ala Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser
20 25 30
Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn
35 40 45
Ile His Asn Tyr Leu Val Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
50 55 60
Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
85 90 95
Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp
100 105 110
Ser Thr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
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