SPLIT INTEIN AND PREPARATION METHOD FOR RECOMBINANT POLYPEPTIDE USING THE SAME
20220340677 · 2022-10-27
Inventors
- Jing Zhang (Wuhan, CN)
- Fang Luo (Wuhan, CN)
- Cheng Gong (Wuhan, CN)
- Xin WANG (Wuhan, CN)
- Lijuan Fang (Wuhan, CN)
- Pengfei Zhou (Wuhan, CN)
Cpc classification
C12N9/22
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/64
CHEMISTRY; METALLURGY
C07K5/081
CHEMISTRY; METALLURGY
C12N15/82
CHEMISTRY; METALLURGY
C07K16/2896
CHEMISTRY; METALLURGY
C07K2317/10
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
C07K2317/62
CHEMISTRY; METALLURGY
International classification
C07K16/28
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to a pair of flanking sequences for a split intein wherein, the pair of flanking sequences comprises: a flanking sequence a and a flanking sequence b; the flanking sequence a is located at the N-terminus of the split intein N-terminal protein splicing region (In), and is between the N-terminal extein (En) and the In; the flanking sequence b is located at the C-terminus of the split intein C-terminal protein splicing region (Ic), and is between the Ic and the C-terminal extein (Ec); and the split intein is NpuDnaE.
Claims
1. A flanking sequence pair for a split intein, wherein, the flanking sequence pair comprises: a flanking sequence a and a flanking sequence b; wherein, the flanking sequence a is located at N-terminus of a split intein N-terminal protein splicing region (In), and is between a N-terminal extein (En) and the In; the flanking sequence b is located at C-terminus of a split intein C-terminal protein splicing region (Ic), and is between the Ic and a C-terminal extein (Ec); the split intein is NpuDnaE; the flanking sequence a is A.sub.-3A.sub.-2A.sub.-1 and the flanking sequence b is B.sub.1B.sub.2B.sub.3, wherein: A.sub.-3 is X or deletion; A.sub.-2 is selected from D, F, G, L, N, S or W; A.sub.-1 is selected from G, A, K, Q, R, W, T or S; B.sub.1 is S; B.sub.2 is E; B.sub.3 is X or deletion; wherein the X is any amino acid selected from the group consisting of G, A, V, L, M, I, S, T, P, N, Q, F, Y, W, K, R, H, D, E, and C.
2. The flanking sequence pair for a split intein according to claim 1, wherein the split intein together with the flanking sequence pair are used for trans-splicing, wherein, the NpuDnaE is composed of the In of sequence as SEQ ID NO:31 and the Ic of sequence as SEQ ID ID:32.
3. A recombinant polypeptide obtained by trans-splicing via the flanking sequence pair for a split intein according to claim 1.
4. The recombinant polypeptide according to claim 3, wherein the recombinant polypeptide is obtained by a component A and a component B through trans-splicing; in the component A, the N-terminus of the flanking sequence a is connected to the C-terminus of the En, and the C-terminus of the flanking sequence a is connected to the In, optionally a tag protein is connected to the C-terminus of the In; in the component B, the C-terminus of the flanking sequence b is connected to the N-terminus of the Ec, and the N-terminus of the flanking sequence b is connected to the Ic, optionally a tag protein is connected to the N-terminus of the Ic; wherein, coding sequences of the En and the Ec are respectively derived from a N-terminal part and a C-terminal part of the same protein.
5. The recombinant polypeptide according to claim 3, wherein the recombinant polypeptide is obtained by a component A and a component B through trans-splicing; in the component A, the N-terminus of the flanking sequence a is connected to the C-terminus of the En, and the C-terminus of the flanking sequence a is connected to the In, optionally a tag protein is connected to the C-terminus of the In; in the component B, the C-terminus of the flanking sequence b is connected to the N-terminus of the Ec, and the N-terminus of the flanking sequence b is connected to the Ic, optionally a tag protein is connected to the N-terminus of the Ic; wherein, coding sequences of the En and the Ec are derived from different proteins.
6. The recombinant polypeptide according to claim 4, wherein the recombinant polypeptide is a fluorescent protein, protease, signal peptide, antimicrobial peptide, antibody, or a polypeptide with biological toxicity.
7. The recombinant polypeptide according to claim 4, wherein the same protein, or one or more of the different proteins is an antibody.
8. The recombinant polypeptide according to claim 7, wherein the antibody is a natural immunoglobulin class IgG, IgM, IgA, IgD or IgE, or an immunoglobulin subclass: IgG1, IgG2, IgG3, IgG4, IgG5, or with light chains of different classes: kappa, lambda; or a single domain antibody; or the antibody is a full-length antibody or a functional fragment of an antibody.
9. The recombinant polypeptide according to claim 8, wherein the functional fragment of an antibody is selected from one or more of the group consisting of: antibody heavy chain variable region VH, antibody light chain variable region VL, antibody heavy chain constant region fragment Fc, antibody heavy chain constant region 1 CH1, antibody heavy chain constant region 2 CH2, antibody heavy chain constant region 3 CH3, antibody light chain constant region CL or single domain antibody variable region VHH.
10. The recombinant polypeptide according to claim 7, wherein, the same protein or one or more of the different proteins is specific to an antigen or epitope A, the antigen A comprises: tumor cell surface antigen, immune cell surface antigen, cytokine, cytokine receptor, transcription factor, membrane protein, actin, virus, bacteria, endotoxin, FIXa, FX, CD3, SLAMF7, CD38, BCMA, CD20, CD16, CEA, PD-L1, PD-1, CTLA-4, TIGIT, LAG-3, VEGF, B7-H3, Claudin18.2, TGF-β, Her2, IL-10, Siglec-15, Ras, C-myc, and the epitope A is an immunogenic epitope of the antigen A.
11. The recombinant polypeptide according to claim 10, wherein, the same protein or one or more of the different proteins is specific to an antigen or epitope B different from the antigen or epitope A, the antigen B comprises: tumor cell surface antigen, immune cell surface antigen, cytokine, cytokine receptor, transcription factor, membrane protein, actin, virus, bacteria, endotoxin, FIXa, FX, CD3, SLAMF7, CD38, BCMA, CD20, CD16, CEA, PD-L1, PD-1, CTLA-4, TIGIT, LAG-3, VEGF, B7-H3, Claudin18.2, TGF-β, Her2, IL-10, Siglec-15, Ras, C-myc, and the epitope B is the immunogenic epitope of the antigen B.
12. The recombinant polypeptide according to claim 11, which is a bispecific antibody that can simultaneously bind to both the antigen or epitope A and the antigen or epitope B.
13. The flanking sequence pair according to claim 1, wherein the B3 is T, I, A, D, E, F, H, L, M, S, V, W or Y.
14. The flanking sequence pair according to claim 1, wherein the flanking sequence a is GG, SG, XGG, XSG, GA, GK, GQ, GR, GW, GT, GS, XGA, XGK, XGQ, XGR, XGW, XGT, XGS, DG, FG, LG, NG, WG, XDG, XFG, XLG, XNG or XWG, and the flanking sequence b is SE or SEX.
15. The flanking sequence pair according to claim 2, wherein the flanking sequence a is GG or SG, and the flanking sequence b is SET or SEI or SES or SEH; or the flanking sequence a is GA, GK, GQ, GR, GW, GT, GS and the flanking sequence b is SET or SEI or SES or SEH; or the flanking sequence a is DG, FG, LG, NG, WG and the flanking sequence b is SET or SEI or SES or SHE.
16. The recombinant polypeptide according to claim 4, wherein the tag protein is selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 29 and 30.
17. The recombinant polypeptide according to claim 12, which is a humanized bispecific antibody or a bispecific antibody of complete human sequence.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0157] “SPLICING 2” are both incubated at 37° C. overnight, and the other groups are stored at 4° C. (MW, molecular weight; RD, reduced)
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DETAILED DESCRIPTION OF THE INVENTION
[0159] The present disclosure relates to a preparation method of a bispecific antibody, which includes: splitting the DNA sequence of the target antibody, constructing a mammalian cell expression vector through whole gene synthesis, purifying the vector, and then the purified vector can be transiently transfected or stably transfected into mammalian cells such as HEK293 or CHO, respectively. The fermentation broth is collected separately, and the component A and the component B are purified by methods such as protein A, protein L, nickel column, Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography or cross-linked starch affinity chromatography; the purified component A and component B are subjected to in vitro trans-splicing, and the spliced product is subjected to affinity chromatography for tag proteins such as nickel column to obtain a bispecific antibody with high-purity. The process flow is shown in
[0160] The antibodies described herein can be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another embodiment, the variable region may be derived from a condricthoid (e.g., from a shark).
[0161] In some embodiments, the antibody may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
[0162] The antibody may be linked or fused to a therapeutic agent, which may include detectable labels, such as radioactive labels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxicity agents, which can be drugs or toxins, ultrasound enhancers, non-radioactive labels, a combination thereof and other such components known in the art.
[0163] The antibody can be detectably labeled by coupling it to chemiluminescent compounds. Then, the presence of the chemiluminescent-labeled antigen-binding polypeptide is determined by detecting the luminescence produced during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
[0164] The antibodies can also be detectably labeled by using fluorescence emitting metals such as 152Eu, or other lanthanide labels. These metals can be attached to the antibody by using the following metal chelating groups, such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0165] The binding specificity of the antigen-binding polypeptides of the present disclosure can be measured by in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
[0166] Cell lines for production of recombinant polypeptides can be selected and cultured by using techniques well known to those skilled in the art.
[0167] Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequences encoding the antibodies of the present disclosure, including, but not limited to, site-directed mutagenesis and PCR-mediated mutations which result in amino acid substitutions. Preferably, the variants (including derivatives), relative to the reference variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2 or CDR- L3, encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, and less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions. Alternatively, mutations can be randomly introduced along all or part of the encoding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutations that retain activity.
[0168] The tag protein used in the present disclosure may be Fc, oligo-histidine (His-tag), Strep-tag, Flag, HA, or maltose-binding protein (MBP) or the like.
[0169] The transfection used in the present disclosure may be transient transfection or stable transfection.
[0170] Mammalian cells such as HEK293 or CHO are used in the present disclosure, but are not limited thereto.
[0171] Liquids containing expression products from mammalian cells, such as fermentation broth and culture medium supernatant, can be purified by methods such as protein A, protein G, nickel column, Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography or cross-linked starch affinity chromatography.
[0172] The spliced product can be subjected to affinity chromatography for the tag protein to remove unspliced components.
[0173] The gene fragment used for constructing the vector of the present disclosure can be constructed by whole gene synthesis, but is not limited thereto.
[0174] The vector used in the present disclosure is pcDNA3.1 or pCH01.0, but is not limited thereto.
[0175] The restriction enzymes used in the present disclosure include, but are not limited to, Notl, Nrul, or BamHI-HF, for example.
[0176] BLAST is an alignment program that uses default parameters. Specifically, the programs are BLASTN and BLASTP. Detailed information of these programs is available at the following Internet address:
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi.
[0177] In a specific embodiment of the present disclosure, as shown in
[0178] In another specific embodiment of the present disclosure, as shown in
[0179] In another specific embodiment of the present disclosure, the component B expression plasmids may include three types of expression plasmids, pB-L, pB-H, and pB-FcIc.
[0180] In the present disclosure, Pa also refers to the N-terminal protein exon or N-terminal extein of protein P, also referred to as Enp; Pb also refers to the C-terminal protein exon or C-terminal extein of protein P, also referred to as Ecp. Ra also refers to the N-terminal protein exon or N-terminal extein of protein R, also referred to as En.sub.R; Rb also refers to the C-terminal protein exon or C-terminal extein of protein R, also referred to as Ec.sub.R.
TABLE-US-00001 TABLE 1 Amino acid sequences of some polypeptides involved in the present disclosure SEQ ID NO. Gene name (Source) Amino acid sequence 1 Human CD38 VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQ (Source: UniProtKB-P28907) RDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQ TLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI 2 Human BCMA MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (Source: UniProtKB-Q02223) 3 Human CTLA-4 MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELM (Source: UniProtKB-P16410) YPPPYYLGIGNGTQIYVIDPEPCPDSD 4 Human LAG-3 VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDER (Source: UniProtKB-P18627) GRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLA ESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLR LEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQ GERLLGAAVYFTELSSPGAQRSGRAPGALPAGHL 5 Human TIGIT MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTY (Source: UniProtKB-Q495A1) TGRIFLEVLESSVAEHGARFQIP 6 Human PD-1 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAPPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRND (Source: UniProtKB-Q15116) SGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV 7 Human PD-L1 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCM (Source: UniProtKB-Q9NZQ7) ISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFR RLDPEENHTAELVIPELPLAHPPNER 8 Human SLAMF7 SGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNRERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQE (Source: UniProtKB-Q9NQ25) YVLHVYEHLSKPKVTMGLQSNKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICVARNPVSRNFSSPILARKLCE GAADDPDSSM 9 Human CEA KLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVN (Source: UniProtKB-P06731) EEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDS VILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKP FITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISP SYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAV AFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCH SASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSA 10 Human CD3ε DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVC (Source: UniProtKB-P07766) ENCMEMD 11 Human CD16A GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQA (Source: UniProtKB-P08637) PRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQ 12 Human TGF-β1 ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPK (Source: UniProtKB-P01137) VEQLSNMIVRSCKCS 13 Human TGF-β2 ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPK (Source: UniProtKB-P61812) IEQLSNMIVKSCKCS 14 Human TGF-β3 ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPK (Source: UniProtKB-P10600) VEQLSNMVVKSCKCS 15 Human VEGFA APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFL (Source: UniProtKB-P15692) QHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLEL NERTCRCDKPRR 16 Human IL-10 PGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENL (Source: UniProtKB-P22301) KTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 17 Human CD20 MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIIS (Source: UniProtKB-P11836) GSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGIL SVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPI ENDSSP 18 Human Claudin18.2 MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVS (Source: UniProtKB-P56856) IFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCI ACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV 19 Human FIXa YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQ (Source: UniProtKB-P00740) FCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPW QVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIA DKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEE CAMKGKYGIYTKVSRYVNWIKEKTKLT 20 Human FX ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCELFTRKLCSLDNGDCD (Source: UniProtKB-P00742) QFCHEEQNSVVCSCARGYTLADNGKACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTR IVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTP ITFRMNVAPACLPERDWAESTLMTQKTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGPHVTRFK DTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSSPLK 21 Human HER2 TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDN (Source: UniProtKB-P04626) GDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLT RTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTL VCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYL YISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGE GLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGV KPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT 22 Human IL-10R HGTELPSPPSVWFEAEFFHHILHWTPIPNQSESTCYEVALLRYGIESWNSISNCSQTLSYDLTAVTLDLYHSNGYRARVRAVDGSRHSNWTVTNTRF (Source: UniProtKB-Q13651) SVDEVTLTVGSVNLEIHNGFILGKIQLPRPKMAPANDTYESIFSHFREYEIAIRKVPGNFTFTHKKVKHENFSLLTSGEVGEFCVQVKPSVASRSNK GMWSKEECISLTRQYFTVTN 23 EGFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQER (Source: UniProtKB-A0A076FL24) TIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPV LLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
TABLE-US-00002 TABLE 2 Amino acid sequences of some tag proteins SEQ Tag ID protein NO. name Amino acid sequence 24 His-tag HHHHHHH (Oligo- histidine) 25 Flag DYKDDDDK 26 HA YPYDVPDYA 27 C-MYC EQKLISEEDL 28 Strep-tag WSHPQFEK 29 Avi-tag GLNDIFEAQKIEWHE 30 Fc PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK
TABLE-US-00003 TABLE 3 In and Ic sequences of some split inteins SEQ SEQ ID Intein ID Intein NO. name In NO. name Ic 31 NpuDnaE CLSYETEILTV 32 NpuDnaE MIKIATRKY EYGLLPIGKIV LGKQNVYDI EKRIECTVYSV GVERDHNFA DNNGNIYTQPV LKNGFIASN AQWHDRGEQEV FEYCLEDGSLI RATKDHKFMTV DGQMLPIDEIF ERELDLMRVDN LPN
TABLE-US-00004 TABLE 4 Some Flanking Sequences a of Split Inteins SEQ ID Amino acid sequence of NO. Number flanking sequence a 33 FSa1 AEY 34 FSa2 SG 35 FSa3 GS 36 FSa4 MGG 37 FSa5 RY 38 FSa6 TY 39 FSa7 GK 40 FSa8 NR 41 FSa9 GGG 42 FSa10 DK 43 FSa11 GY 44 FSa12 XX* 45 FSa13 XXX* *X represents any amino acid selected from the 20 amino acids (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, C) defined in the present disclosure.
TABLE-US-00005 TABLE 5 Some Flanking Sequences b of Split Inteins SEQ ID Amino acid sequence of NO. Number flanking sequence b 46 FSb1 CFN 47 FSb2 SVY 48 FSb3 SIE 49 FSb4 TEA 50 FSb5 TIH 51 FSb6 TVI 52 FSb7 SSS 53 FSb8 SAV 54 FSb9 SI 55 FSb10 TQL 56 FSb11 SEI 57 FSb12 SEH 58 FSb13 SET 59 FSb14 THT 60 FSb15 XX* 61 FSb16 XXX* *X represents any amino acid selected from the 20 amino acids (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, C) defined in the present disclosure.
TABLE-US-00006 TABLE 6 amino acid sequences and sequence numbers of the En domains involved in the construction of component A or A′ SEQ ID Corresponding NO. Domain code Amino acid sequence 150 Hinge Hin1 DKTHT 151 Hinge Hin2 ERKCCVE 152 Hinge Hin3 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPR 153 Hinge Hin4 ESKYGPP 154 CL Lc1 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 155 CL Lc2 GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 156 CL Lc3 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSK QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 157 CL Lc4 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPAKAGVETTTPSK QSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS 158 CL Lc5 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSK QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS 159 CL Lc6 GQPKAAPTVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADSSPAKAGVETTTPSK QSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS 160 CL Lc7 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 161 CH1 G1CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 162 CH1 G2CH1 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV 163 CH1 G3CH1 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV 164 CH1 G4CH1 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV 180 Pa CD38-Pa VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNIT EEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGE FNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTF 182 Pa GFP-Pa MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIED
TABLE-US-00007 TABLE 7 amino acid sequences and sequence numbers of the Ec domains involved in the construction of component B or B′ SEQ ID Corresponding NO. Domain code Amino acid sequence 165 CH2 G1CH2 CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 166 CH2 G2CH2 CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK 167 CH2 G2DCH2 CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK 168 CH2 G3CH2 CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK 169 CH2 G4CH2 CPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK 170 CH3 G1CH3 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 171 CH3 G2CH3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 172 CH3 G3CH3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK 173 CH3 G4CH3 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSHALYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 174 CH3 G1CH3-CW GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 175 CH3 G1CH3-CSAV GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 176 CH3 G1CH3-W GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 177 CH3 G1CH3-SAV GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 178 CH3 G1CH3-V GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 179 CH3 G1CH3-RF GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 181 Pb CD38-Pb EVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI 183 Pb EGFP-Pb VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
TABLE-US-00008 TABLE 8 Variable region sequences of anti-CD3 antibody Amino acid sequence of anti-CD3 antibody variable region (Bold and underlined amino acids are CDR regions) Anti- SEQ SEQ body ID ID code VH NO. VL NO. 2a5 QVQLVESGGGVVQPGRSLRLSCAASGFTESTYAMN 62 QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYAN 63 WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRE WVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGK TISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFG AALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEI NSYVSWFAYWGQGTLVTVSS K 2j5a QVQLVESGGGVVQPGRSLRLSCAASGFTESTYAMN 64 QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYAN 65 WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRE WFQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGK TISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFG AALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEI NSYVSWAAYWGQGTLVTVSS K
TABLE-US-00009 TABLE 9 Variable region sequences of anti-B7-H3 antibody Amino acid sequences of anti-B7-H3 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO 8H9 QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINW 66 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLH 67 (Cancer Research VRQRPEQGLEWIGWIFPGDGSTQYNEKFKGKATLTT WYQQKSHESPRLLIKYASQSISGIPSRFSGSGSG 61, 4048-4054, DTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYW SDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGT May 15, 2001) GQGTLVTVSS KLELK BRCA69D QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQW 68 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLN 68 (US20120294796A1) VKQRPGQGLEWIGTIYPGDGDTRYTQKFKGKATLTA WYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSG DKSSSTAYMQLSSLASEDSAVYYCARRGIPRLWYFD TDYSLTIDNLEQEDIATYFCQQGNTLPPTFGGGT VWGAGTTVTVSS KLEIK
TABLE-US-00010 TABLE 10 Variable region sequences of anti-CD38 antibody Amino acid sequence of anti-CD38 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO Dara EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQ 70 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW 71 (US9040050) APGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNT YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD LYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTL FTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVE VTVSS IK MOR QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQ 72 DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWY 73 (US8088896) APGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNT QQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTA LYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVT TLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLT VSS VLGQ 2F5 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQ 74 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAW 75 (US9040050) APGQGLEWMGRVIPFLGIANSAQKFQGRVTITADKSTST YQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTD AYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVT FTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVE VSS IK
TABLE-US-00011 TABLE 11 Variable region sequences of anti-EpCAM antibody Amino acid sequences of anti-EpCAM antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO 3-171 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS 76 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQ 77 (US20100310463 WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI QKPGQAPRLIIYGASTTASGIPARFSASGSGTDFTLT TADESTSTAYMELSSLRSEDTAVYYCARGLLWNYW ISSLQSEDFAVYYCQQYNNWPPAYTFGQGTKLEIK GQGTLVTVSS 2-6 EVQLVESGPELKKPGETVKISCKASGYTFTDYSMHW 78 DIQMTQSPSSLSASLGERVSLTCRASQEISVSLSWLQ 79 (TW102107344) VKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSL QEPDGTIKRLIYATSTLDSGVPKRFSGSRSGSDYSLT ETSASTAYLQINNLKNEDTATYFCARTAVYWGQGTT ISSLESEDFVDYYCLQYASYPWTFGGGTKLEIK VTVSS
TABLE-US-00012 TABLE 12 Variable region sequences of anti-BCMA antibody Amino acid sequence of anti-BCMA antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO B50 QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYIN 80 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHSNGNTYL 81 (US9598500) WVRQAPGQGLEWMGWIYFASGNSEYNQKFTGRVTM HWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDF TRDTSINTAYMELSSLTSEDTAVYFCASLYDYDWY TLKISRVEAEDVGIYYCSQSSIYPWTFGQGTKLEIK FDVWGQGTMVTVSS B140153 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS 82 LPVLTQPPSASGTPGQRVTISCSGRSSNIGSNSVNWYR 83 (WO2016090320A1) WVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTI QLPGAAPKLLIYSNNQRPPGVPVRFSGSKSGTSASLAI TADKSTSTAYMELSSLRSEDTAVYYCARGGYYSHD SGLQSEDEATYYCATWDDNLNVHYVFGTGTKVTVLG MWSEDWGQGTLVTVSS B69 QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYF 84 SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQP 85 (US2017051068A1) WGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVT PGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISR ISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVA VEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVL GLFDYWGQGTLVTVSS
TABLE-US-00013 TABLE 13 Variable region sequences of anti-CTLA-4 antibody Amino acid sequences of anti-CTLA-4 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO Yervoy QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMH 86 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLA 87 (US20020086014A1) WVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTI WYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGT GTLVTVSSSRDNSKNTLYLQMNSLRAEDTAIYYCA FTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKV RTGWLGPFDYWGQ VEIK
TABLE-US-00014 TABLE 14 Variable region sequences of anti-TIGIT antibody Amino acid sequence of anti-TIGIT antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO 10A7 EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMH 88 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGV 89 (US20090258013A1) WVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTI KENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRF SRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNT TGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPL FDSWGQGTLVTVSS TFGDGTKLEIK MAB10 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYWG 90 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLA 91 (WO2017059095A1) WIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRATISVD WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT TSKNQFSLKLSSVTAADTAVYYCARDGVLALNKRSFD DFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTK IWGQGTMVTVSS VEIK
TABLE-US-00015 TABLE 15 Variable region sequences of anti-LAG-3 antibody Amino acid sequence of anti-LAG-3 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO LAG35 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWN 92 EIVLTQSPATLSLSPGERATLSCRASQSISSYLA 93 (US9505839B2) WIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLS WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG LDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYN TDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGT WFDPWGQGTLVTVSS NLEIK L3E3 EVQLLESGAEVKKPGASVKVSCKASGYTFTSYYMH 94 QSVLTQPASASGSPGQSITISCTGTSSDVGGYNY 95 (US9902772B2) WVRQAPGQGLEWMGIINPSAGSTSYAQKFQGRVTM VSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSK TRDTSTSTVYMELSSLRSEDTAVYYCARELMATGG SGNTASLTISGLQAEDEANYYCSSYTSSSTNVFG FDYWGQGTLVTVSS TGTKVTVL
TABLE-US-00016 TABLE 16 Variable region sequences of anti-PD-1 antibody Amino acid sequences of anti-PD-1 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO 5C4 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMH 96 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA 97 (WO2006121168) WVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTI WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG SRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQ TDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGT GTLVTVSS KVEIK H409A11 QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMY 98 EIVLTQSPATLSLSPGERATLSCRASKGVSTSGY 99 (WO2008156712A1) WVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTL SYLHWYQQKPGQAPRLLIYLASYLESGVPARFSG TTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDM SGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTF GFDYWGQGTTVTVSS GGGTKVEIK
TABLE-US-00017 TABLE 17 variable region sequences of anti-PD-L1 antibody Amino acid sequence of anti-PD-L1 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. S70 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYG 100 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLI 101 (WO2010077634A1) GSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQ YSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPA GTLVTVSS TFGQGTKVEIK 12A4 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIF 102 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI 103 (US7943743B2) GKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGM YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPT DVWGQGTTVTVSS FGQGTKVEIK
TABLE-US-00018 TABLE 18 Variable region sequences of anti-CD16 antibody Amino acid sequence of anti-CD16 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. NM3E2 EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAP 104 SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQK 105 GKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQ PGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTIT MNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR GAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL
TABLE-US-00019 TABLE 19 variable region sequences of anti-SLAMF7 antibody Amino acid sequence of anti-SLAMF7 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. Elotuzumab EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDS 106 DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLI 107 (WO2004100898A2) STINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWG YWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPY QGTLVTVSS TFGQGTKVEIK
TABLE-US-00020 TABLE 20 Variable region sequences of anti-CEA antibody Amino acid sequence of anti-CEA antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. hPR1A3 (Cancer QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKT 108 DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLI 109 Immunol GEATYVEEFKGRFVFSLDTSVSTAYLQISSLKADDTAVYYCARWDFYDYVEAMDY YSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYYTYPL lmmunother WGQGTTVTVSS FTFGQGTKVEIK (1999) 47:299-306)
TABLE-US-00021 TABLE 21 Variable region sequences of anti-VEGF antibody Amino acid sequence of anti-VEGF anibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. Avastin EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYT 110 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLI 111 GEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYF YFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPW DVWGQGTLVTVSS TFGQGTKVEIK B2041 EVQLVESGGGLVQPGGSLRLSCAASGFSINGSWIFWVRQAPGKGLEWVGAIWPFG 112 DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQQKPGKAPKLLI 113 (WO2005012359A2) GYTHYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDY YAASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPL WGQGTLVTVSS TFGQGTKVEIK G631 EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGITPAG 114 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLI 115 (WO2005012359A2) GYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYW YSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYGNPF GQGTLVTVSS TFGQGTKVEIK
TABLE-US-00022 TABLE 22 Anti-TGF-beta antibody variable regions Amino acid sequence of anti-TGF-beta antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. 3G12 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIV 116 ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLL 117 DIANYAQRFKGRVTITADESTSTTYMELSSLRSEDTAVYYCASTLGLVLDAMDYW IYGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSP GQGTLVTVSS ITFGQGTRLEIK 4B9 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIV 118 ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLL 119 DIANYAQRFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYW IYGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSP GQGTLVTVSS ITFGQGTRLEIK
TABLE-US-00023 TABLE 23 Anti-IL-10 antibody variable regions Amino acid sequence of anti-IL-10 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. B-N10 QVQLKQSGPGLLQPSQSLSISCTVSGFSLATYGVHWVRQSPGKGLEWLGVIWRGG 120 DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQS 121 STDYSAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKQAYGHYMDYWGQG PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRLEAEDLGVYYCFQG TSVTVSS SHVPWTFGGGTKLEIK BT-063 EVQLVESGGGLVQPGGSLRLSCAASGFSFATYGVHWVRQSPGKGLEWLGVIWRGG 122 DVVMTQSPLSLPVTLGQPASISCRSSQNIVHSNGNTYLEWYLQRPGQS 123 STDYSAAFMSRLTISKDNSKNTVYLQMNSLRAEDTAVYFCAKQAYGHYMDYWGQG PRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG TSVTVSS SHVPWTFGQGTKVEIK
TABLE-US-00024 TABLE 24 Variable region sequences of anti-CD20 antibody Amino acid sequence of anti-CD20 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. Gazyva QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGD 124 DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQS 125 (WO2005044859) GDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWG PQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQN QGTLVTVSS LELPYTFGGGTKVEIK
TABLE-US-00025 TABLE 25 variable region sequences of anti-Claudin18.2 antibody Amino acid sequence of anti-Claudin18.2 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. IMAB362 QVQLKQSGPGLLQPSQSLSISCTVSGFSLATYGVHWVRQSPGKGLEWLGVIWRGG 126 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQ 127 (US20090169547A1) STDYSAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKQAYGHYMDYWGQG PPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQN TSVTVSS DYSYPFTFGSGTKLEIK
TABLE-US-00026 TABLE 26 Variable region sequences of anti-FIXa antibody Amino acid sequence of anti-FIXa antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. A44 QVQLQQSGAELAKPGASVKLSCKASGYTFTSSWMHWIKQRPGQGLEWLGYINPSS 128 DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLI 129 (US8062635B2) GYTKYNRKFRDKATLTADKSSSTAYMQLTSLTYEDSAVYYCARGGNGYYFDYWGQ YWASTRHTGVPDRFTGSRYGTDFTLTISNVQSEDLADYLCQQYSNYIT GTTLTVSS FGGGTKLELK A50 QVQLQQSGAELAKPGASVKLSCKASGYTFTTYWMHWVKQRPGQGLEWIGYINPSS 130 DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGLSPKLLI 131 (US8062635B2) GYTKYNQKFKVKATLTADKSSSTAYMQLSSLTDEDSAVYYCANGNLGYFFDYWGQ YWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYLT GTTLTVSS FGAGTKLEIK A69 EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWIKQRPGQGLEWLGYINPSS 132 DIQMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLI 133 (US8062635B2) GYTKYNRKFRDKATLTADKSSSTAYMQLTSLTYEDSAVYYCARGGNGYYLDYWGQ YWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYLCQQYSNYIT GTTLTVSS FGAGTKLELK XB12 EVQLQQSGPGLVKPTQSLSLTCSVTGYSITSGYYWTWIRQFPGNNLEWIGYISFD 134 DIVLTQSPAIMSASLGEKVTMSCRATSSVNYIYWYQQKSDASPKLWIF 135 (US8062635B2) GTNDYNPSLKNRISITRDTSENQFFLKLNSVTTEDTATYYCARGPPCTYWGQGTL YTSNLAPGVPPRFSGSGSGNSYSLTISSMEAEDAATYYCQQFSSSPWT VTVSA FGGGTKLEIK
TABLE-US-00027 TABLE 27 Variable region sequences of anti-FX antibody Amino acid sequence of anti-FX antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. SB04 QVQLQQSGPELVKPGASVKMSCKASGYTFTHFVLHWVKQNPGQGLEWIGYIIPYN 136 DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQ 137 (US8062635B2) DGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGNRYDVGSYAMD SPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYLCQQ YWGQGTSVTVSS YYRFPYTFGGGTKLEIK B26 QVQLQQSGPELVKPGASVKISCKASGYTFTDNNMDWVKQSHGKGLEWIGDINTKS 138 DIVLTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQKPGQSPKALI 139 (US8062635B2) GGSIYNQKFKGKATLTIDKSSSTAYMELRSLTSEDTAVYYCARRRSYGYYFDYWG YSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPL QGTTLTVSS TFGAGTKLEIK
TABLE-US-00028 TABLE 28 Variable region sequences of anti-HER2 antibody Amino acid sequence of anti-HER2 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. Herceptin EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN 140 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI 141 GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP GQGTLVTVSS TFGQGTKVEIK Perjeta EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNS 142 DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLI 143 GGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWG YSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPY QGTLVTVSS TFGQGTKVEIK
TABLE-US-00029 TABLE 29 Anti-Siglec-15 antibody variable region sequences Amino acid sequence of anti-Siglec-15 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. 34A1 EVQILETGGGLVKPGGSLRLSCATSGFNFNDYFMNWVRQAPEKGLEWVAQIRNKI 144 DIVLTQSPALAVSLGQRATISCRASQSVTISGYSFIHWYQQKPGQQPR 145 YTYATFYAESLEGRVTISRDDSESSVYLQVSSLRAEDTAIYYCTRSLTGGDYFDY LLIYRASNLASGIPARFSGSGSGTDFTLTINPVQADDIATYFCQQSRK WGQGVMVTVSS SPWTFAGGTKLELR H34A1 EVQLVESGGGLVQPGGSLRLSCAASGFNFNDYFMNWVRQAPGKGLEWVAQIRNKI 146 EILMTQSPATLSLSPGERATLSCRASQSVTISGYSFIHWYQQKPGQAP 147 YTYATFYAASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSLTGGDYFDY RLLIYRASNLASGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQSR WGQGTLVTVSSI KSPWTFGQGTKVEIK
TABLE-US-00030 TABLE 30 variable region sequences of anti-Luciferase antibody Amino acid sequence of anti-Luciferase antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (Sequence ID ID source) VH NO. VL NO. 4420 EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKP 148 DWMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQS 149 YNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQ PKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQS GTSVTVSS THVPWTFGGGTKLEIK
TABLE-US-00031 TABLE 31 Amino acid sequences of some components A including intein NpuDnaE Expression Corresponding SEQ ID Code Polypeptide plasmid name Domain Code NO. A-38 Component A pA-CD38Pa Pa CD38-Pa 180 Flanking FSa2 34 sequence a In NpuDnaE 31 Tag protein His-tag 24 A-GFP Component A pA-GFPPa Pa GFP-Pa 182 Flanking FSa2 34 sequence a In NpuDnaE 31 Tag protein His-tag 24 A-Fab A-HIn pA-HIn(XX) VHa S70 100 CH1 G1CH1 161 Flanking FSa12 44 sequence a In NpuDnaE 31 Tag protein His-tag 24 A-L pA-L VLa S70 101 CL Lc1 154 A-Fab1 A-HIn pA-HIn(1) VHa S70 100 CH1 G1CH1 161 Flanking FSa1 34 sequence a In NpuDnaE 31 Tag protein His-tag 24 A-L pA-L VLa S70 101 CL Lc1 154 A-Fab2 A-HIn pA-HIn(2) VHa S70 100 CH1 G1CH1 161 Flanking FSa7 39 sequence a In NpuDnaE 31 Tag protein His-tag 24 A-L pA-L VLa S70 101 CL Lc1 154 A-Fab3 A-HIn pA-HIn(3) VHa S70 100 CH1 G1CH1 161 Flanking FSa3 35 sequence a In NpuDnaE 31 Tag protein His-tag 24 A-L pA-L VLa S70 101 CL Lc1 154 A-Fab4 A-HIn pA-HIn(4) VHa S70 100 CH1 G1CH1 161 Flanking FSa4 36 sequence a In NpuDnaE 31 Tag protein His-tag 24 A-L pA-L VLa S70 101 CL Lc1 154 A-Fab5 A-HIn pA-HIn(5) VHa S70 100 CH1 G1CH1 161 Flanking FSa10 42 sequence a In NpuDnaE 31 Tag protein His-tag 24 A-L pA-L VLa S70 101 CL Lc1 154 A-Fab6 A-HIn pA-HIn(6) VHa S70 100 CH1 G1CH1 161 Flanking FSa2 34 sequence a In NpuDnaE 31 Tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 101 CL Lc1 154 A-Fab7 A-HIn pA-HIn(7) VHa S70 100 CH1 G1CH1 161 Flanking FSa7 39 sequence a In NpuDnaE 31 Tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 101 CL Lc1 154 Note: The variable region of antibody heavy chain in the component A is denoted as VHa; the sequences of domains such as VHa, CH1, flanking sequence a and tag protein in the table can be replaced with the protein sequences of other corresponding domains mentioned in the present specification.
TABLE-US-00032 TABLE 32 Amino acid sequences of some components B including intein NpuDnaE Corresponding SEQ ID Code Polypeptide Expression plasmid name Domain Code NO. B-38 Component B pTag-Ic-FSb-CD38Pb Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb11 56 sequence b Pb CD38-Pb 181 B-GFP Component B pTag-Ic-FSb-GFPPb Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb11 56 sequence b Pb EGFP-Pb 183 B-FcIc Component B pTag-Ic-FSb(XXX)-(B-FcIc) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb16 61 sequence b Pb G1CH2 165 G1CH3 170 B-FcIc1 Component B pTag-Ic-FSb-(B-FcIc1) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb1 46 sequence b Pb G1CH2 165 G1CH3 170 B-FcIc2 Component B pTag-Ic-FSb-(B-FcIc2) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb2 47 sequence b Pb G1CH2 165 G1CH3 170 B-FcIc3 Component B pTag-Ic-FSb-(B-FcIc3) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb11 56 sequence b Pb G1CH2 165 G1CH3 170 B-FcIc4 Component B pTag-Ic-FSb-(B-FcIc4) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb12 57 sequence b Pb G1CH2 165 G1CH3 170 B-FcIc5 Component B pTag-Ic-FSb-(B-FcIc5) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb13 58 sequence b Pb G1CH2 165 G1CH3 170 B-FcIc6 Component B pTag-Ic-FSb-(B-FcIc6) Tag protein Strep-tag 28 His-tag 24 Ic NpuDnaE 32 Flanking FSb13 58 sequence b Pb G1CH2 165 G1CH3 170 B-FcIc7 Component B pTag-Ic-FSb-(B-FcIc7) Tag protein Strep-tag 28 His-tag 24 Ic NpuDnaE 32 Flanking FSb14 59 sequence b Pb G1CH2 165 G1CH3 170 Note: The sequences of domains such as Pb, flanking sequence b, and tag protein in the table can be replaced with the protein sequences of other corresponding domains mentioned in the present specification.
TABLE-US-00033 TABLE 33 Component B′ including intein NpuDnaE Expression Corresponding SEQ ID Code Polypeptide plasmid name Domain Code NO. B′-HAb1 B′-L pB′-L VLb Dara 71 CL Lc1 154 B′-H pB′-H VHb Dara 70 CH1 G1CH1 161 Hinge Hin1 150 CH2 G1CH2 165 CH3 G1CH3 170 B′-FcIc pB′-FcIc(1) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb1 46 sequence b CH2 G1CH2 165 CH3 G1CH3 170 B′-HAb2 B′-L pB′-L VLb Dara 71 CL Lc1 154 B′-H pB′-H VHb Dara 70 CH1 G1CH1 161 Hinge Hin1 150 CH2 G1CH2 165 CH3 G1CH3 170 B′-FcIc pB′-FcIc(2) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb2 47 sequence b CH2 G1CH2 165 CH3 G1CH3 170 B′-HAb3 B′-L pB′-L VLb Dara 71 CL Lc1 154 B′-H pB′-H VHb Dara 70 CH1 G1CH1 161 Hinge Hin1 150 CH2 G1CH2 165 CH3 G1CH3 170 B′-FcIc pB′-FcIc(3) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb11 76 sequence b CH2 G1CH2 165 CH3 G1CH3 170 B′-HAb4 B′-L pB′-L VLb Dara 71 CL Lc1 154 B′-H pB′-H VHb Dara 70 CH1 G1CH1 161 Hinge Hin1 150 CH2 G1CH2 165 CH3 G1CH3 170 B′-FcIc pB′-FcIc(4) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb12 57 sequence b CH2 G1CH2 165 CH3 G1CH3 170 B′-HAb5 B′-L pB′-L VLb Dara 71 CL Lc1 154 B′-H pB′-H VHb Dara 70 CH1 G1CH1 161 Hinge Hin1 150 CH2 G1CH2 165 CH3 G1CH3 170 B′-FcIc pB′-FcIc(5) Tag protein His-tag 24 Ic NpuDnaE 32 Flanking FSb13 58 sequence b CH2 G1CH2 165 CH3 G1CH3 170 B′-HAb6 B′-L pB′-L VLb Dara 71 CL Lc1 154 B′-H pB′-H VHb Dara 70 CH1 G1CH1 161 Hinge Hin1 150 CH2 G1CH2 165 CH3 G1CH3 170 B′-FcIc pB′-FcIc(6) Tag protein Strep-tag 28 His-tag 24 Ic NpuDnaE 32 Flanking FSb13 58 sequence b CH2 G1CH2 165 CH3 G1CH3 170 B′-HAb7 B′-L pB′-L VLb Dara 71 CL Lc1 154 B′-H pB′-H VHb Dara 70 CH1 G1CH1 161 Hinge Hin1 150 CH2 G1CH2 165 CH3 G1CH3 170 B′-FcIc pB′-FcIc(7) Tag protein Strep-tag 28 His-tag 24 Ic NpuDnaE 32 Flanking FSb14 59 sequence b CH2 G1CH2 165 CH3 G1CH3 170 Note: The sequences of domains such as VHa, CH1, flanking sequence a, and tag protein in the table can be replaced with the protein sequences of other corresponding domains mentioned in the present specification.
EXAMPLES
Experimental Method
1. Preparation of Recombinant Polypeptides
[0181] The DNA sequences in the Examples of the present disclosure were all obtained by reverse translation based on the amino acid sequences, and were synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd.
[0182] The recombinant polypeptides involved in the Examples were all prepared by the following method: in the presence of recombinase, the DNA sequence and a vector pcDNA3.1 digested by a restriction enzyme EcoRI were ligated at 37° C. for 30 minutes, and then transformed into a Trans10 competent cell by heat shock method, and then transiently transfected into 293E cells (purchased from Thermo Fisher) after verified by sequencing (Wuhan GeneCreate Biological Engineering Co., Ltd.). After expression, the recombinant polypeptides were purified.
2. The Co-Transfected Plasmids Involved in the Examples were Shown as Follows:
[0183] 1) To express the component A and component B shown in
[0184] 2) To express the component A and component B′ shown in
[0185] 3) To express the component A shown in
[0186] In general, if two plasmids were co-transfected and expressed, the molar ratio of the two plasmids was 1:1 or any other ratio. If three plasmids were co-transfected and expressed, the molar ratio of the three plasmids was 1:1:1, or any other ratio.
3. Purification of Ppolypeptides with Tag Proteins
[0187] (1) When the tag protein was Fc, the polypeptide was purified by affinity chromatography, for example, MabSelect SuRe (GE, Cat. No. 17-5438-01), 18m1 column.
[0188] (2) When the tag protein was His-tag, the polypeptide was purified by affinity chromatography, for example, Ni-NTA (Jiangsu Qianchun, product number: A41002-06).
[0189] (3) When the tag protein was Strep-tag, Flag, HA or MBP, etc., the polypeptide was purified by Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography, or cross-linked starch affinity chromatography by selecting corresponding packings and buffers.
[0190] (4) When the component A (A′) or component B (B′) did not have a tag protein, the spliced product can be separated by an ion exchange chromatography based on the difference in isoelectric point. The chromatography packing can be a cation exchange chromatography packing or an anion exchange chromatography packing, such as Hitrap SP-HP (GE Company).
[0191] (5) When the component A (A′) or component B (B′) did not have a tag protein, the spliced product can be separated by a hydrophobic chromatography based on the difference in hydrophobicity by using a chromatography packing such as Capto phenyl ImpRes packing (GE Company).
[0192] (6) When the component A (A′) or component B (B′) did not have a tag protein, the spliced product can be separated by a molecular sieve chromatography based on the difference in molecular weight by using a chromatography packing such as HiLoad Superdex 200pg (GE Company).
Example 1
Screening of Flanking Sequence Pairs of Intein NpuDnaE
[0193] Construction of Expression Plasmids pA-HIn, pA-L and Plasmid (pTag-Ic-FSb-Pb)
[0194] In this Example, the amino acid selected from any one of the 19 amino acids (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y) defined in the present disclosure is denoted as X amino acid. H refers to a portion of heavy chain of the antibody and L refers to a portion of light chain of the antibody. For the flanking sequence pairs of the original NpuDnaE, each amino acid is mutated by degenerate primer design to be one of the 19 amino acids described in the present disclosure.
[0195] For the A-HIn expression plasmid (pA-HIn) with the mutated flanking sequence pair, for the sake of simplicity, a plasmid with the X amino acid at position -1 of the flanking sequence a is referred to as pA-HIn(X), and a plasmid with the X amino acids independently at positions −2 and −1 (wherein the X amino acids may be the same or different) is referred to as pA-HIn(XX).
[0196] The sequences involving X amino acid in pA-HIn(XX) are all obtained by degenerate primer design.
[0197] The expression plasmid of A-Fab 1 contains two polypeptides, A-HIn and AL, wherein the plasmid for polypeptide A-HIn is denoted as pA-HIn(1), and the plasmid for polypeptide A-L is denoted as pA-L; and so on.
[0198] The B-FcIc expression plasmid with the mutated flanking sequence pair is similarly designated as the expression plasmid pTag-Ic-FSb-(B-FcIc). Similarly, the expression plasmid pTag-Ic-FSb-(B-FcIc) with the amino acids at positions +1, +2 and +3 of the flanking sequence b being independently the same or different X amino acids is denoted as plasmid pTag-Ic-FSb(XXX)-(B-FcIc), wherein the sequences involving X amino acids are all obtained by degenerate primer design.
[0199] According to the steps and conditions described in “Preparation of Recombinant Polypeptides”, as shown in
Screening of Amino Acid at Position +1
[0200] The expression plasmid pTag-Ic-FSb-(B-FcIc) was divided into 19 groups, wherein the plasmids with the same amino acid residue at position +1 of the flanking sequence b were divided into one group. Accordingly, for each group, the amino acid X at position +1 of the flanking sequence b was definite, and the amino acids at positions +2 and +3 were X (that is, any one of the 19 amino acids).
[0201] 293E cells were co-transfected with the above 19 groups of pTag-Ic-FSb(XXX)-(B-FcIc) plasmids and the corresponding expression plasmids pA-HIn(XX) and pA-L, respectively.
[0202] The molar ratio of transfection was pTag-Ic-FSb(XXX)-(B-FcIc):pA-HIn(XX):pA-L=3:1:1.
[0203] At the same time, a positive control group (the flanking sequence a was AEY, and the flanking sequence b was CFN) was set up, and the molar ratio of positive control plasmid and co-transfected plasmids thereof was pTag-Ic-FSb-(B -FcIc1):pA-HIn(1):pA-L=3:1:1.
[0204] The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot, wherein the molecular weight marker protein was PageRuler Prestained Protein Ladder (purchased from Thermo Company, Cat. No. 26616). The results showed that at the position of 50 kD (corresponding to the complete heavy chain), there was a clear band in the positive control group (intein+natural flanking sequence pair); however, for other groups, only the group with serine (S) at position +1 of the flanking sequence b showed an obvious band.
[0205] The results show that NpuDnaE has a higher splicing efficiency when the amino acid at position +1 is S.
Screening of Amino Acid at Positions −1 and +2
[0206] The above screened expression plasmid pTag-Ic-FSb(SXX)-(B-Fcic) with Serine (S) at position +1 was further grouped into 19 subgroups, wherein the plasmids with the same amino acid residue at position +2 of the flanking sequence b were divided into one group. Accordingly, for each subgroup, the amino acid at position +1 of the flanking sequence b was S, and the amino acid at position +2 was definite.
[0207] The expression plasmid pA-HIn(XX) was divided into 19 groups, wherein the plasmids with the same amino acid residue at position −1 of the flanking sequence a were divided into one group. Accordingly, for each group, the amino acid at position +1 of the flanking sequence a was definite.
[0208] 293E cells were co-transfected with the above 19 subgroups of expression plasmids pTag-Ic-FSb(SXX)-(B-FcIc) and the 19 groups of expression plasmids pA-HIn(XX) and pA-L, respectively.
Primary Screening
[0209] In order to reduce the number of experiments in the cross-pairing test, the above 19 groups of expression plasmids pA-HIn(XX) were divided into groups Al—A6 according to Table 34 below, and the above 19 subgroups of expression plasmids pTag-Ic-FSb(SXX)-(B-Fcic) were divided into groups B1˜B6 according to Table 35 below.
TABLE-US-00034 TABLE 34 Groups of expression plasmids pA-HIn(X) Amino acid at position −1 of flanking sequence a Group A1 A2 A3 A4 A5 A6 Amino acid A F S P M R V Y T Q L K G W H N I E D
TABLE-US-00035 TABLE 35 Groups of expression plasmids pTag-Ic-FSb(SXX)-(B-FcIc) Amino acid at position +2 of flanking sequence b Group B1 B2 B3 B4 B5 B6 Amino acid A F S P M R V Y T Q L K G W H N I E D
[0210] Transfection was performed in the same manner as above based on the pairings in Table 36 with a molar ratio of pTag-Ic-FSb(SXX)-(B-FcIc):pA-HIn(XX):pA-L=3:1:1. A positive control monoclonal antibody was also set in the same manner as above, and 36 groups of transfections (i.e., groups 1-1 to groups 6-6) were obtained, respectively.
TABLE-US-00036 TABLE 36 Co-transfected groups Number B1 B2 B3 B4 B5 B6 A1 1-1 1-2 1-3 1-4 1-5 1-6 A2 2-1 2-2 2-3 2-4 2-5 2-6 A3 3-1 3-2 3-3 3-4 3-5 3-6 A4 4-1 4-2 4-3 4-4 4-5 4-6 A5 5-1 5-2 5-3 5-4 5-5 5-6 A6 6-1 6-2 6-3 6-4 6-5 6-6
[0211] The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot (SDS-PAGE plus reducing agent β-mercaptoethanol, and the detection antibody was HRP-labeled goat anti-human IGG antibody purchased from Sigma). The results were shown in
[0212] The results show that NpuDnaE has a higher splicing efficiency when the amino acid at position −1 is A, V or G, and the amino acid at position +2 is R, K, E or D.
[0213] All plasmids in groups 1-6 were selected for rescreening.
Rescreening
[0214] All plasmids in groups 1-6 in which the amino acid residue at position −1 was A, V or G and the amino acid residue at position +2 was R, K, E or D were paired and co-transfected according to Table 37.
TABLE-US-00037 TABLE 37 groups for rescreening Amino acid at position +2 Number R K E D Amino acid at A A-SR A-SK A-SE A-SD position −1 V V-SR V-SK V-SE V-SD G G-SR G-SK G-SE G-SD
[0215] Transfection was performed in the same manner as above based on the pairings in Table 37 with a molar ratio of pTag-Ic-FSb(SRX or SKX or SEX or SDX)-(B-FcIc):pA-HIn(XA or XV or XG):pA-L=3:1:1. A positive control was also set in the same manner as above.
[0216] The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot ((SDS-PAGE plus reducing agent). The results were shown in
[0217] The results show that NpuDnaE has a higher splicing efficiency when the amino acid at position +1 is S, the amino acid at position +2 is E and the amino acid at position −1 is G.
[0218] Therefore, all plasmids of the pTag-Ic-FSb(SEX)-(B-FcIc):pA-HIn(XG) group were selected for the following screening of amino acids at positions −2 and +3 .
Screening of Amino Acid at Position −2
[0219] The pA-HIn(XG) plasmids were divided into 19 groups based on the amino acid X at position −2, and these 19 groups were co-transfected with all the plasmids of the group pTag-Ic-FSb(SEX)-(B-FcIc) and the plasmid pA-L, respectively for preliminary screening.
TABLE-US-00038 TABLE 38 Primary screening and grouping of amino acid at position −2 Position −2 of flanking Number sequence a AG-SE A DG-SE D EG-SE E FG-SE F GG-SE G HG-SE H IG-SE I KG-SE K LG-SE L MG-SE M NG-SE N PG-SE P QG-SE Q RG-SE R SG-SE S TG-SE T VG-SE V WG-SE W YG-SE Y
[0220] Transfection was performed in the same manner as above according to the pairings in Table 38, and 19 transfection groups (i.e., group AG-SE to group YG-SE) were obtained, respectively. The molar ratio of plasmids for transfection was pTag-Ic-FSb(SEX)-(B-FcIc):pA-HIn(XG):pA-L=3:1:1. A positive control was also set in the same manner as above.
[0221] The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot ((SDS-PAGE plus reducing agent). The results were shown in
[0222] The results show that the NpuDnaE has a higher splicing efficiency when the amino acid at position −1 is G and the amino acid at position −2 is selected from D, F, G, L, N, G, S, W, and in particular, has the highest splicing efficiency when the amino acid at position −1 is G and the amino acid at position −2 is G or S.
[0223] Other solutions of amino acids at positions −1 and −2
[0224] Eight expression plasmids pA-HIn (GA or GE or GK or GQ or GR or GW or GT or GP) were co-transfected with expression plasmid pTag-Ic-FSb(SEX)-(B-FcIc) and plasmid pA-L respectively.
TABLE-US-00039 TABLE 39 Other screening and grouping of amino acid at position −1 Flanking sequence a Number Position −2 Position −1 GA-SE G A GE-SE G E GK-SE G K GQ-SE G Q GS-SE G S GR-SE G R GW-SE G W GT-SE G T GP-SE G P
[0225] Transfection was performed in the same manner as above based on the pairings in Table 39 with a molar ratio of pTag-Ic-FSb(SEX)-(B-FcIc):pA-HIn(GA or GE or GK or GQ or GR or GW or GT or GP):pA-L=3:1:1. A positive control was also set in the same manner as above.
[0226] The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot (SDS-PAGE plus reducing agent). The results were shown in
[0227] The results show that the NpuDnaE has a higher splicing efficiency when the amino acid at position −2 is G and the amino acid at position −1 is selected from A, K, S, Q, R, W, T.
Amino Acid at Position +3
[0228] The expression plasmids pTag-Ic-FSb(SEX)-(B-FcIc) were divided into 19 groups based on the amino acid X at position +3, and were respectively co-transfected with all the plasmids of group pA-HIn(GX) and the plasmid pA-L for primary screening.
TABLE-US-00040 TABLE 40 Primary screening and grouping of amino acid at position +3 Position +3 of flanking Number sequence b G-SEA A G-SED D G-SEE E G-SEF F G-SEG G G-SEH H G-SEI I G-SEK K G-SEL L G-SEM M G-SEN N G-SEP P G-SEQ Q G-SER R G-SES S G-SET T G-SEV V G-SEW W G-SEY Y
[0229] Transfection was performed in the same manner as above according to the pairings in Table 40, and 19 transfection groups (i.e., group G-SEA to group G-SEY) were obtained, respectively. The molar ratio of plasmids for transfection was pTag-Ic-FSb(SEA—SEY)-(B-FcIc):pA-HIn(GX):pA-L=3:1:1. A positive control was also set in the same manner as above.
[0230] The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were detected by Western blot (SDS-PAGE plus reducing agent). The results were shown in
[0231] The results show that the NpuDnaE has a higher splicing efficiency when the amino acid at position +1 is S, the amino acid at position +2 is E and the amino acid at position +3 is selected from A, D, E, F, H, I, L, M, S, T, V, W, Y, and in particular, has a very high splicing efficiency when the amino acid at position +3 is selected from H, I, S, T.
[0232] In summary, Table 41 showed the novel flanking sequence pairs of split intein NpuDnaE of the present disclosure with an efficient splicing.
TABLE-US-00041 TABLE 41 Novel flanking sequence pairs of intein NpuDnaE Flanking sequence a Flanking sequence b Position −2 Position −1 Position +1 Position +2 Position +3 Natural sequence pair E Y C F N Novel D mutated F A sequence pair G D L G E N F S H W I S E L A M K S Q T G R V W W T Y S
Example 2
Splicing Comparison between Optimal Flanking Sequence Pairs and known Flanking Sequence Pairs
[0233] Construction of expression plasmids A-Hln, pA-L, plasmid (pTag-Ic-FSb-Pb)
[0234] Under the conditions as described above in “Preparation of Recombinant Polypeptides”, as shown in
[0235] The molar ratio of plasmids for transfection was pTag-Ic-FSb-(B-FcIc6):pA-HIn(2):pA-L=3:1:1, and the expression product was A61. Positive controls Al and A10 were also set, wherein the plasmids corresponding to Al were pA-HIn(4), pTag-Ic-FSb-(B-FcIc2) and pA-L, and the plasmids corresponding to A10 were pA-HIn(3), pTag-Ic-FSb-(B-FcIcI) and pA-L, with the same molar ratio of plasmids for transfection as described above.
[0236] The transfected cells were cultured for 5 days and the supernatant was taken. Proteins in the supernatant were subjected to protein A affinity chromatography and then detected by coomassie brilliant blue staining via SDS-PAGE (with a reducing agent).
[0237] The results in
[0238] The inteins and flanking sequences corresponding to groups A1, A10 and A61 were shown in Table 42.
TABLE-US-00042 TABLE 42 Inteins and corresponding effective flanking sequence pairs of Intein Number Corresponding plasmid Flanking sequence a Flanking sequence b NpuDnaE A1 pA-HIn(4) MGG SVY pTag-Ic-FSb-(B-FcIc2) pA-L NpuDnaE A10 pA-HIn(3) GS CFN pTag-Ic-FSb-(B-FcIc1) pA-L NpuDnaE A61 pA-HIn(2) GK SET pTag-Ic-FSb-(B-FcIc6) pA-L
Example 3
Intein-Mediated In Vitro Splicing of Polypeptide Fragments from Different Protein Sources
Construction of Vectors and Expression of Polypeptides
[0239] Under the same condition as that in Example 1, component expression plasmids of intein NpuDnaE were respectively constructed by pcDNA3.1 based on the structure as shown in Tables 31 and 33.
[0240] The expression plasmids of component A in this Example were pA-L and pA-HIn(x), wherein the x represented different numbers.
[0241] The expression plasmids of component B′ in this Example were divided into three types: B′-L expression plasmid (pB′-L), B′-H expression plasmid (pB′-H) and B′-FcIc expression plasmid (pB′-FcIcx), wherein, the x represented different numbers. Each component B′ shared the same pB′-L and B′-H expression plasmids.
[0242] For the intein NpuDnaE, plasmids pB′-FcIc(1)˜B′-FcIc(7) corresponding to B′-HAb1˜B′-HAb7 were constructed.
Expression and Purification of Component A:
[0243] Each plasmid pA-HIn(x) and the plasmid pA-L were co-transfected into CHO cells and cultured at 37° C. , with a plasmid molar ratio of pA-HIn:pA-L=1:1, and the cell supernatant was harvested at 10 day after transfection. The supernatant was purified by nickel column chromatography (Jiangsu Qianchun, cat no. A41002-06) to obtain a purified polypeptide fragment of component A.
Expression and Purification of Component B′:
[0244] The plasmid pB′-L, plasmid pB′-H and each plasmid pB+-FcIc were co-transfected into 293E cells and cultured at 37° C. , with a plasmid molar ratio of pB′-L:pB′-H:pB′-FcIc=1:1:3, and the cell supernatant was harvested at 10 day after transfection. The supernatant was purified by nickel column chromatography to obtain a purified polypeptide fragment of component B′.
[0245] As shown in Table 43, the obtained polypeptide fragments of component A and component B′ were referred to as Fab4 and HAb4, respectively.
TABLE-US-00043 TABLE 43 The obtained polypeptide fragments of component A and component B′ Corresponding Corresponding Number of plasmid of Number of plasmid of component A component A component B’ component B′ Fab4 pA-HIn(6) HAb4 pB′-L pA-L pB′-H pB′-FcIc(3)
[0246] The obtained purified polypeptide fragments of component A and component B′ were subjected to non-reducing SDS-PAGE and coomassie brilliant blue staining, and the results were shown in
[0247] E1, E2, and E3 represent elution fractions eluted with different imidazole concentrations (from low to high concentration) during nickel column chromatography. It can be seen from
In Vitro Splicing
[0248] The obtained purified polypeptide fragments of component A and component B′, Fab4 and HAb4, were dialyzed into a buffer at 4° C. with a 3 kD dialysis bag (purchased from Sigma) with a concentration of 1 to 10 micromolar/L. The buffer included 10 to 50 mM Tris/HCl (pH 7.0-8.0), 100 to 500 mM NaCl, and 0 to 0.5 mM EDTA. Then, the components A (Fab4) and B′ (Hab4) were respectively mixed according to corresponding serial numbers in a molar ratio of 1:10 to 10:1, and DTT was added to be 0.5 to 5 mM, then the mixture was incubated overnight at 37° C.
[0249] The obtained spliced product polypeptides were subjected to SDS-PAGE and coomassie brilliant blue staining, and the results were shown in
[0250] In
[0251] It can be seen from
Biological Activity Detection of Spliced Product
[0252] The biological activity detection based on double antigen sandwich ELISA was performed for the recombinant polypeptide “SPLICING 2”.
[0253] 1) Preparation of antigen: for the proteins PD-L1 and CD38, only the extracellular domain was selected for construction, and an expression plasmid with His-tag was constructed by using the vector pcDNA3.1.
[0254] After construction, 293E cells were used for transient transfection, and a two-step purification including nickel column purification and molecular sieve purification was carried out. After purification, an antigen protein with a purity of no less than 95% detected by SDS-PAGE was obtained.
[0255] PD-L1 protein was labeled with horseradish peroxidase (HRP).
[0256] 2) Coating of the first antigen: the concentration of CD38 protein was adjusted to 2 μg/ml, and an microtiter plate was coated with the CD38 protein-containing liquid at 100 μl/well, 4° C. overnight; the supernatant was discarded and 250 μl blocking solution (3% BSA in PBS) was added to each well;
[0257] 3) addition of antibody: according to the experimental design, the operation was performed at room temperature. The antibody was diluted in a gradient with 1% BSA in PBS. For example, the initial concentration of antibody for dilution was 20 μg/mL, and the antibody was diluted by 2-fold with 5 gradients. The diluted antibody was added into wells of microtiter plate at 200 μl/well, incubated at room temperature for 2 hours, and then the supernatant was discarded;
[0258] 4) washing: the plate was washed by 200 μl/well PBST (PBS containing 0.1% Tween20) for 3 times;
[0259] 5) incubation of secondary antigen: a diluted secondary antigen (HRP-labeled PD-L1 protein) was added with a volume of 100 μl/well and incubated at room temperature for 1 hour, wherein the secondary antigen was diluted at 1:1000 and the diluent was 1% BSA in PBS ;
[0260] 6) washing: the plate was washed with 200 μl/well PBST for 5 times;
[0261] 7) color-developing: TMB color-developing solution (prepared from A and B color-developing solutions purchased from Wuhan Boster Company, and mixed according to A:B=1:1, ready to use) was added at 100 μl/well, and the color-developing was performed at 37° C. for 5 min;
[0262] 8) 2M HCl stopping solution was added at 100 μl/well, and then the microplate reader should be read at 450 nm within 30 minutes.
[0263]
[0264] It can be seen from
[0265] The results prove that the spliced product Fab4+HAb4 obtained by using the intein and the novel flanking sequence pair contained therein of the present disclosure has a good bispecific antibody activity.
[0266] Based on the splicing principle of intein, according to the molecular weight of spliced product obtained in the present disclosure and the results of double-antigen sandwich ELISA, it can be speculated that an effective bispecific antibody with a natural IgG-like structure was obtained in the present disclosure. The test results confirmed that the structure of the bispecific antibody was a heterodimeric IgG structure composed of two different heavy chains and two different light chains, rather than a mixture of homodimeric IgG structure composed of two identical heavy chains and two identical light chains.
INDUSTRIAL APPLICABILITY
[0267] The present disclosure provides methods for preparing recombinant polypeptides, particularly bispecific antibodies, by using split inteins with novel flanking sequence pairs. The split inteins with novel flanking sequence pairs of the present disclosure can be widely used in the preparation of recombinant polypeptides in the fields of medicine and bioengineering, especially in the field of antibodies, especially in the preparation of bispecific antibodies. The bispecific antibody prepared by using the split inteins with novel flanking sequence pairs of the present disclosure does not have a non-natural domain, has a structure closely similar to that of natural antibody (IgA, IgD, IgE, IgG or IgM), and has a Fc domain. The bispecific antibody has a complete structure and good stability, and can retain or remove CDC (complement-dependent cytotoxicity) or ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent cellular phagocytosis) or FcRn (Fc receptor)-binding activity according to different IgG subclasses.
[0268] The bispecific antibody prepared by the method of the present disclosure has the following advantages: the bispecific antibody has a long half-life in vivo and low immunogenicity, and does not introduce any form of linkers; has an improved stability, and a reduced in vivo immune response. The bispecific antibody prepared by the method of the present disclosure has the same glycosylation modification as that of wild-type IgG, has better biological function, is more stable, and has a long half-life in vivo; the in vitro splicing method by using inteins can completely avoid the problems of heavy chain mismatch and light chain mismatch commonly found in traditional methods.
[0269] The preparation method for bispecific antibodies of the present disclosure can also be used to produce humanized bispecific antibodies and bispecific antibodies with complete human sequences. The sequence of such an antibody prepared by the method of the present disclosure is more similar to that of a human antibody, which can effectively reduce the immune response. The preparation method for bispecific antibodies of the present disclosure is not limited by antibody subclasses (IgG, IgA, IgM, IgD, IgE, and light chain κ and λ types) and can be used to construct any bispecific antibody.