SPLIT INTEIN AND PREPARATION METHOD FOR RECOMBINANT POLYPEPTIDE USING THE SAME

Abstract

The present disclosure relates to a pair of flanking sequences for a split intein, wherein the pair of flanking sequences includes: 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 selected from the group consisting of SspDnaE, SspDnaB, MxeGyrA, MjaTFIIB, PhoVMA, TVoVMA, Gp41-1, Gp41-8, IMPDH-1 and PhoRadA.

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 selected from the group consisting of SspDnaE, SspDnaB, MxeGyrA, MjaTFIIB, PhoVMA, TVoVMA, Gp41-1, Gp41-8, IMPDH-1 or PhoRadA, (1) when the split intein is IMPDH-1, 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, or preferably G or D; A.sub.−2 is X or deletion, or preferably G or K; A.sub.−1 is selected from G or T; B.sub.1 is S; B.sub.2 is I or T or S; B.sub.3 is X or deletion; preferably, the flanking sequence a is G, XG, XGG, DKG or DKT, and the flanking sequence b is SI, ST, SS, SIX, STX or SSX; (2) when the split intein is Gp41-8, 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 N or D; A.sub.−1 is selected from R or K; B.sub.1 is S or T; B.sub.2 is A or H; B.sub.3 is X or deletion, or preferably V, Y or T, preferably, the flanking sequence a is NR, XNR, DK, XDK, DR or XDR, and the flanking sequence b is SA or SAX; (3) when the split intein is SspDnaB, 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 S or D; A.sub.−1 is selected from G or K; B.sub.1 is S; B.sub.2 is I; B.sub.3 is X or deletion, or preferably E or T, preferably, the flanking sequence a is SG, XSG, DK, XDK, and the flanking sequence b is SI or SIX; (4) when the intein is MjaTFIIB, 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 T or D; A.sub.−1 is selected from Y; B.sub.1 is T; B.sub.2 is I or H; B.sub.3 is X or deletion, or preferably H or T; preferably, the flanking sequence a is TY, DY, XTY or XDY, and the flanking sequence b is TI, TIX, TH or THX; (5) when the split intein is PhoRadA, 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 G or D; A.sub.−1 is selected from K; B.sub.1 is T; B.sub.2 is Q or H; B.sub.3 is X or deletion, or preferably L or T, preferably, the flanking sequence a is GK, XGK, DK or XDK, and the flanking sequence b is TQ, TH, TQX or THX; (6) when the split intein is TVoVMA, 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.−3is X or deletion; A.sub.−2 is selected from G or D; A.sub.−1 is K; B.sub.1 is T; B.sub.2 is V or H; B.sub.3 is X or deletion, or preferably I or T, preferably, the flanking sequence a is GK, XGK, DK or XDK, and the flanking sequence b is TV, TH, TVX or THX; (7) when the split intein is MxeGyrA, 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.−3is X or deletion; A.sub.−2 is selected from R or D; A.sub.−1 is selected from Y, K or T; B.sub.1 is T; B.sub.2 is E or H; B.sub.3 is X or deletion, or preferably A or T, preferably, the flanking sequence a is RY, XRY, DK or XDK, and the flanking sequence b is TE, TH, TEX or THX; (8) when the split intein is PhoVMA, 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 G or D; A.sub.−1 is selected from K; B.sub.1 is T; B.sub.2 is V or H; B.sub.3 is X or deletion, or preferably I or T, preferably, the flanking sequence a is GK, XGK, DK or XDK, and the flanking sequence b is TV, TH, TVX or THX; (9) when the split intein is Gp41-1, 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 G or D; A.sub.−1 is selected from Y or K; B.sub.1 is S or T; B.sub.2 is S or H; B.sub.3 is X or deletion, or preferably S or T; preferably, the flanking sequence a is GY, XGY, DK or XDK, and the flanking sequence b is SS, SH, SSX or SHX; (10) when the split intein is SspDnaE, 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.−3is X or deletion; A.sub.−2 is selected from G or D; A.sub.−1 is selected from G, S or K; B.sub.1 is T or S; B.sub.2 is E or H; B.sub.3 is X or deletion, or preferably T; preferably, the flanking sequence a is GG, XGG, GK, XGK, DK or XDK, and the flanking sequence b is SE, TH, SEX or THX; 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 SspDnaE is composed of the In of sequence as SEQ ID NO:31 and the Ic of sequence as SEQ ID NO:32, the SspDnaB is composed of the In of sequence as SEQ ID NO:33 and the Ic of sequence as SEQ ID NO:34, the MxeGyrA is composed of the In of sequence as SEQ ID NO:35 and the Ic of sequence as SEQ ID NO:36, the MjaTFIIB is composed of the In of sequence as SEQ ID NO:37 and the Ic of sequence as SEQ ID NO:38, the PhoVMA is composed of the In of sequence as SEQ ID NO:39 and the Ic of sequence as SEQ ID NO:40, the TvoVMA is composed of the In of sequence as SEQ ID NO:41 and the Ic of sequence as SEQ ID NO:42, the Gp41-1 is composed of the In of sequence as SEQ ID NO:43 and the Ic of sequence as SEQ ID NO:44, the Gp41-8 is composed of the In of sequence as SEQ ID NO:45 and the Ic of sequence as SEQ ID NO:46, the IMPDH-1 is composed of the In of sequence as SEQ ID NO:47 and the Ic of sequence as SEQ ID NO:48, the PhoRadA is composed of the In of sequence as SEQ ID NO:49 and the Ic of sequence as SEQ ID NO:50.

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 an 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 2, wherein: (1) when the split intein is IMPDH-1, the flanking sequence a is XGG and the flanking sequence b is SI, ST, SS; or the flanking sequence a is DKG and the flanking sequence b is SI, ST, SS; or the flanking sequence a is DKT and the flanking sequence b is SI, ST, SS; (2) when the split intein is Gp41-8, the flanking sequence a is NR and the flanking sequence b is SAV; or the flanking sequence a is DK and the flanking sequence b is SAV; the flanking sequence a is NR and the flanking sequence b is SAT; or the flanking sequence a is DK and the flanking sequence b is SAT; (3) when the split intein is SspDnaB, the flanking sequence a is SG and the flanking sequence b is SIE; (4) when the split intein is PhoRadA, the flanking sequence a is GK and the flanking sequence b is TQL or THT; or the flanking sequence a is DK and the flanking sequence b is TQL or THT; (5) when the split intein is TVoVMA, the flanking sequence a is GK and the flanking sequence b is TVI or THT; or the flanking sequence a is DK and the flanking sequence b is TVI or THT; (6) when the split intein is MxeGyrA, the flanking sequence a is RY and the flanking sequence b is TEA or THT; or the flanking sequence a is DK and the flanking sequence b is TEA or THT; (7) when the split intein is MjaTFIIB, the flanking sequence a is TY and the flanking sequence b is TIH; or the flanking sequence a is TY and the flanking sequence b is THT; (8) when the split intein is PhoVMA, the flanking sequence a is GK and the flanking sequence b is TVI or THT; or the flanking sequence a is DK and the flanking sequence b is TVI or THT; (9) when the split intein is Gp41-1, the flanking sequence a is GY and the flanking sequence b is SSS or SHT; or the flanking sequence a is DK and the flanking sequence b is SSS or SHT; (10) when the split intein is SspDnaE, the flanking sequence a is GG and the flanking sequence b is SET or THT; or the flanking sequence a is GK and the flanking sequence b is SET or THT; or the flanking sequence a is DK and the flanking sequence b is SET or THT; 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, C.

14. 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.

15. 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

[0220] FIG. 1 is a schematic diagram (A) of split intein-mediated splicing of homologous polypeptide fragments and a schematic diagram (B) of the protein primary structure of each component. (Pa, N-terminal fragment of the split protein P; In, N-terminal fragment of the split intein; Pb, C-terminal fragment of the split protein P; Ic, C-terminal fragment of the split intein; TAG, tag protein; FS, flanking sequence).

[0221] FIG. 2 is a schematic diagram (A) of split intein-mediated splicing of heterologous polypeptide fragments and a schematic diagram (B) of the protein primary structure of each component. (Pa, N-terminal fragment of the split protein P; Ra, N-terminal fragment of the split protein R; In, N-terminal fragment of the split intein; Pb, C-terminal fragment of the split protein P; Rb, C-terminal fragment of the split protein R; TAG, tag protein; Ic, C-terminal fragment of the split intein; FS, flanking sequence).

[0222] FIG. 3 is a schematic diagram (A) of split intein-mediated antibody splicing in vitro and a schematic diagram (B) of the protein primary structure of each component, wherein the spliced product is a bispecific antibody. (C) is an exemplary schematic diagram of the amino acid sequence near the split intein-mediated antibody splicing site, “X” indicates that the amino acid at that position is any amino acid or deletion. (LC, light chain; HC, heavy chain; TAG, tag protein; FS, flanking sequence).

[0223] FIG. 4 is a schematic diagram (A) for the construction of an expression plasmid for the component A of bispecific antibody, and a schematic diagram (B) for the construction of an expression plasmid for the component B.

[0224] FIG. 5 is a schematic diagram of flanking sequence numbering. (TAG, tag protein; FS, flanking sequence).

[0225] FIG. 6 shows the detection results of reducing SDS-PAGE and coomassie brilliant blue staining of the expression supernatants of 293E cells after proteinA affinity purification, wherein the 293E cells are co-transfected with expression plasmids corresponding to different inteins and different flanking sequences. FIG. 6 (A) to (E) shows the detection results after purification of cell supernatants of component A and component B co-transfected with different inteins based on different flanking sequences, respectively.(MW, molecular weight)

[0226] FIG. 7 shows the results of non-reducing SDS-PAGE and coomassie brilliant blue staining of the purified products of component A and component B′ with different inteins expressed by 293E cells, respectively. (A) Detection results of purified products of Fab5, Fab9 and Fab11; (B) Detection results of purified products of HAb5, HAb9 and HAb11.

[0227] FIG. 8 shows the detection of non-reducing SDS-PAGE and coomassie brilliant blue staining of the spliced products of component A and component B′ with different inteins, wherein (A) the intein is IMPDH-1, the flanking sequence a is GGG, and the flanking sequence b is SI; (B) the intein is PhoRadA, the flanking sequence a is GK, and the flanking sequence b is THT. In FIGS. 8(A) and (B), the spliced product 1 means that the DTT is added before mixing the components A and B; the spliced product 2 means that the DTT is added after mixing the components A and B′; the reduced (ie., RD) means that the DTT is added; the non-reduced (ie., NON-RD) means that no DTT is added; the “non-splicing” indicates that components A and B′ are mixed without adding DTT. In FIG. 8(C), the intein is PhoRadA, the flanking sequence a is GK, the flanking sequence b is THT. “SPLICING 1” and “NON-SPLICING 1” refer to reaction systems containing the component A and component B′ at concentrations of 5 μM and 4 μM, respectively, as well as 2 mM DTT; “SPLICING 2” and “NON-SPLICING 2” refer to reaction systems containing the component A and component B′ with concentrations of 10 μM and 1 μM, respectively, as well as 2 mM DTT; “SPLICING 3” and “NON-SPLICING 3” refer to reaction systems containing the component A and component B′ with concentrations of 5 μM and 1 μM, respectively, as well as 2 mM DTT; wherein “SPLICING 1” to “SPLICING 3” are incubated overnight at 37° C., and “NON-SPLICING 1” to “NON-SPLICING 3” are incubated at 4° C. overnight; the control bands are Fab11 (non-reduced) for component A, and HAb11 (non-reduced) for component B′, and mAb. (RD, reduced; MW, molecular weight; mAb, monoclonal antibody)

[0228] FIG. 9 shows the detection result of spliced product by double antigen sandwich ELISA in which the intein is IMPDH-1, the flanking sequence a is GGG, and the flanking sequence b is SI; wherein, the coating antigen is CD38, and the detection antigen is horseradish peroxidase (HRP)-labeled PD-L1.

[0229] FIG. 10 shows the base peak ion (BPI) map of Fab5+HAb5 (spliced product 1) after digestion. (A) BPI map of Fab5+HAb5 (spliced product 1) after trypsin digestion; (B) BPI map of Fab5+HAb5 (spliced product 1) after chymotrypsin digestion; (C) BPI map of Fab5+HAb5 (spliced product 1) after Glu-C digestion.

[0230] FIG. 11 shows the SDS-PAGE and coomassie staining detection after co-transfection expression and affinity purification of component A and component B by applying intein PhoRadA and IMPDH-1 to human IgG2, IgG3 or IgG4 subclasses. (RD, reduced; MW, molecular weight)

DETAILED DESCRIPTION OF THE INVENTION

[0231] 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 FIG. 3A.

[0232] 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).

[0233] In some embodiments, the antibody may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

[0234] 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.

[0235] 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.

[0236] 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).

[0237] 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).

[0238] Cell lines for production of recombinant polypeptides can be selected and cultured by using techniques well known to those skilled in the art.

[0239] 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.

[0240] 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.

[0241] The transfection used in the present disclosure may be transient transfection or stable transfection.

[0242] Mammalian cells such as HEK293 or CHO are used in the present disclosure, but are not limited thereto.

[0243] 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.

[0244] The spliced product can be subjected to affinity chromatography for the tag protein to remove unspliced components.

[0245] The gene fragment used for constructing the vector of the present disclosure can be constructed by whole gene synthesis, but is not limited thereto.

[0246] The vector used in the present disclosure is pcDNA3.1 or pCHO1.0, but is not limited thereto.

[0247] The restriction enzymes used in the present disclosure include, but are not limited to, NotI, NruI, or BamHI-HF, for example.

[0248] 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.

[0249] In a specific embodiment of the present disclosure, as shown in FIGS. 1, 2, and 3, a component A expression plasmid (pPa-FSa-In-Tag) and a component B expression plasmid (pTag-Ic-FSb-Pb) or component A′ expression plasmid (pRa-FSa-In-Tag) and component B′ expression plasmid (pTag-Ic-FSb-Rb) can be constructed.

[0250] In another specific embodiment of the present disclosure, as shown in FIGS. 4A and 4B, the Pa-HIn and Pa-L can be constructed into the same plasmid, namely component A expression plasmid (pBi-Pa-FSa-In-Tag); or the pB′-L, pB′-H and pB′-FcIc can be constructed into the same plasmid, namely component B′ expression plasmid (pBi-Tag-Ic-FSb-Rb) by molecular cloning methods such as enzyme cleavage and enzyme ligation.

[0251] 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.

[0252] 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 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRND (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 (Source: UniProtKB- MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIIS P11836) GSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGIL SVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPI ENDSSP 18 Human Claudin18.2 MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVS (Source: UniProtKB-P56856) IFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCI ACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV 19 Human FIXa (Source: UniProtKB- YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQ P00740) FCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPW QVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIA DKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEE CAMKGKYGIYTKVSRYVNWIKEKTKLT 20 Human FX (Source: UniProtKB- ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCELFTRKLCSLDNGDCD P00742) QFCHEEQNSVVCSCARGYTLADNGKACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTR IVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTP ITFRMNVAPACLPERDWAESTLMTQKTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGPHVTRFK DTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSSPLK 21 Human HER2 (Source: UniProtKB- TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDN P04626) GDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLT RTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTL VCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYL YISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGE GLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGV KPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT 22 Human IL-10R HGTELPSPPSVWFEAEFFHHILHWTPIPNQSESTCYEVALLRYGIESWNSISNCSQTLSYDLTAVTLDLYHSNGYRARVRAVDGSRHSNWTVTNTRF (Source: UniProtKB-Q13651) SVDEVTLTVGSVNLEIHNGFILGKIQLPRPKMAPANDTYESIFSHFREYEIAIRKVPGNFTFTHKKVKHENFSLLTSGEVGEFCVQVKPSVASRSNK GMWSKEECISLTRQYFTVTN 23 EGFP (Source: UniProtKB- MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQER A0A076FL24) TIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPV LLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE-US-00002 TABLE 2 Amino acid sequences of some tag proteins SEQ ID NO Tag protein name Amino acid sequence 24 His-tag HHHHHHH (Oligo-histidine) 25 Flag DYKDDDDK 26 HA YPYDVPDYA 27 C-MYC EQKLISEEDL 28 St rep-tag WSHPQFEK 29 Avi-tag GLNDIFEAQKIEWHE 30 Fc PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE-US-00003 TABLE 3 In and 1c sequences of some split inteins SEQ SEQ ID Intein ID Intein NO name In NO name Ic 31 SspDnaE CLSFGTEILTVEYGPLPIGKIVSEEINCSVYSV 32 SspDnaE MVKVIGRRSLGVQRIFDIGLPQDHNFLLANGAIAAN DPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVI RATSDHRFLTTDYQLLAIEEIFARQLDLLTLEN IKQTEEALDNHRLPFPLLDAGTIK 33 SspDnaB CISGDSLISLASTGKRVSIKDLLDEKDFEIWAI 34 SspDnaB SPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVA NEQTMKLESAKVSRVFCTGKKLVYILKTRLGRT NDIIVHN IKATANHRFLTIDGWKRLDELSLKEHIALPRKL ESSSLQ 35 MxeGyrA CITGDALVALPEGESVRIADIVPGARPNSDNAI 36 MxeGyrA GKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGR DLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG FYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHN LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKP GDYAVIQRSAFSVDCAGFAR 37 MjaTFIIB SVDYNEPIIIKENGEIKVVKIGELIDKIIENSE 38 MjaTFIIB NSDFIFLKIKEINKVEPTSGYAYDLTVPNAENFVAGFGGFV NIRREGILEIAKCKGIEVIAFNSNYKFKFMPVS LHN EVSRHPVSEMFEIVVEGNKKVRVTRSHSVFTIR DNEVVPIRVDELKVGDILVLAK 39 PhoVMA CVSGDTPVLLDAGERRIGDLFMEAIRPKERGEI 40 PhoVMA MHISGVFDVYDLMVPDYGYNFIGGNGLIVLHN GQNEEIVRLHDSWRIYSMVGSEIVETVSHAIYH GKSNAIVNVRTENGREVRVTPVHKLFVKIGNSV IERPASEVNEGDEIAWPSVSENGDSQTVTTTLV LTFDRVVSKE 41 TvoVMA CVSGETPVYLA 42 TvoVMA DGKTIKIKDLYSSERKKEDNIVEAGSGEEIIHLKDPIQIYS YVDGTIVRSRSRLLYKGKSSYLVRIETIGGRSVSVTPVHKL FVLTEKGIEEVMASNLKVGDMIAAVAESESEARDCGMSEEC VMEAEVYTSLEATFDRVKSIAYEKGDFDVYDLSVPEYGRNF IGGEGLLVLHN 43 Gp41-1 CLDLKTQVQTPQGMKEISNIQVGDLVLSNTGYN 44 Gp41-1 MMLKKILKIEELDERELIDIEVSGNHLFYANDILTHN EVLNVFPKSKKKSYKITLEDGKEIICSEEHLFP TQTGEMNISGGLKEGMCLYVKE 45 Gp41-8 CLSLDTMVVTNGKAIEIRDVKVGDWLESECGPV 46 Gp41-8 MCEIFENEIDWDEIASIEYVGVEETIDINVTNDRLFFANGI QVTEVLPIIKQPVFEIVLKSGKKIRVSANHKFP LTHN TKDGLKTINSGLKVGDFLRSRAK 47 IMPDH-1 CFVPGTLVNTENGLKKIEEIKVGDKVFSHTGKLQ 48 IMPDH-1 MKFKLKEITSIETKHYKGKVHDLTVNQDHSYNVRGTVVHN EVVDTLIFDRDEEIISINGIDCTKNHEFYVIDKE NANRVNEDNIHLFARWVHAEELDMKKHLLIELE 49 PhoRadA CFARDTEVYYENDTVPHMESIEEMYSKYASMNGE 50 PhoRadA NGYAVPLDNVFVYTLDIASGEIKKTRASYIYREKVEKLIEI LPFD KLSSGYSLKVTPSHPVLLFRDGLQWVPAAEVKPGDVVVGVR EEVLRRRIISKGELEFHEVSSVRIIDYNNWVYDLVIPETHN FIAPNGLVLHN

TABLE-US-00004 TABLE 4 Flanking sequences a of some split inteins SEQ Amino acid sequences of ID NO No. flanking sequence a 51 FSa1 AEY 52 FSa2 SG 53 FSa3 GS 54 FSa4 MGG 55 FSa5 RY 56 FSa6 TY 57 FSa7 GK 58 FSa8 NR 59 FSa9 GGG 60 FSa10 DK 61 FSa11 GY 62 FSa12 XX* 63 FSa13 XXX* 202 FSa14 DKG 203 FSa15 DKT *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 Flanking sequences b of some split inteins SEQ Amino acid sequences of ID NO No. flanking sequence b 64 FSb1 CFN 65 FSb2 SVY 66 FSb3 SIE 67 FSb4 TEA 68 FSb5 TIH 69 FSb6 TVI 70 FSb7 SSS 71 FSb8 SAV 72 FSb9 SI 73 FSb10 TQL 74 FSb11 SEI 75 FSb12 SEH 76 FSb13 SET 77 FSb14 THT 78 FSb15 XX* 79 FSb16 XXX* 204 FSb17 ST *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 Some amino acid sequences and sequence No. of the En domains involved in the construction of component A or A′ SEQ ID NO Domain Code Amino acid sequences 168 Hinge Hin1 DKTHT 169 Hinge Hin2 EKCCVE 170 Hinge Hin3 GDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPR 171 Hinge Hin4 YGPP 172 CL Lc1 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 173 CL Lc2 GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTVAPTECS 174 CL Lc3 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS 175 CL Lc4 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPAKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS 176 CL Lc5 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPAECS 177 CL Lc6 GQPKAAPTVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADSSPAKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS 178 CL Lc7 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 179 CH1 G1CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSC 180 CH1 G2CH1 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ TYTCNVDHKPSNTKV 181 CH1 G3CH1 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYTCNVNHKPSNTKVDKRVE 182 CH1 G4CH1 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKR 198 Pa CD38-Pa VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDL AHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKN STF 200 Pa GFP-Pa MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAM PEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIED

TABLE-US-00007 TABLE 7 Some amino acid sequences and sequence numbers of the Ec domains involved in the construction of component B or B′ SEQ ID NO Domain Code Amino acid sequences 183 CH2 G1CH2 CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 184 CH2 G2CH2 CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK 185 CH2 G2DCH2 CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK 186 CH2 G3CH2 CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK 187 CH2 G4CH2 CPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK 188 CH3 G1CH3 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 189 CH3 G2CH3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 190 CH3 G3CH3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK 191 CH3 G4CH3 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 192 CH3 G1CH3- GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR CW WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 193 CH3 G1CH3- GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR CSAV WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 194 CH3 G1CH3-W GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 195 CH3 G1CH3- GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR SAV WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 196 CH3 G1CH3-V GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 197 CH3 G1CH3-RF GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 199 Pb CD38-Pb EVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI 201 Pb EGFP-Pb VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE-US-00008 TABLE 8 Variable region sequences of anti-CD3 antibody Amino acid sequences 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWV 80 QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANW 81 RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR VQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAA DDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSW LTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK FAYWGQGTLVTVSS 2j5a QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWV 82 QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANW 83 RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR FQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAA DDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSW LTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK AAYWGQGTLVTVSS

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 84 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLH 85 (Cancer Research VRQRPEQGLEWIGWIFPGDGSTQYNEKFKGKATLTT WYQQKSHESPRLLIKYASQSISGIPSRFSGSGSG 61, 4048-4054, DTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYW SDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGT May 15, 2001) GQGTLVTVSS KLELK BRCA69D QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQW 86 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLN 87 (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 88 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW 89 (US9040050) APGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNT YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD LYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTL FTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVE VTVSS IK MOR QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQ 90 DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWY 91 (US8088896) APGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNT QQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTA LYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVT TLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLT VSS VLGQ 2F5 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQ 92 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAW 93 (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 94 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQ 95 (US20100310463 WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI QKPGQAPRLIIYGASTTASGIPARFSASGSGTDFTLT TADESTSTAYMELSSLRSEDTAVYYCARGLLWNYW ISSLQSEDFAVYYCQQYNNWPPAYTFGQGTKLEIK GQGTLVTVSS 2-6 EVQLVESGPELKKPGETVKISCKASGYTFTDYSMHW 96 DIQMTQSPSSLSASLGERVSLTCRASQEISVSLSWLQ 97 (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  98 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHSNGNTYLH  99 (US9598500) WVRQAPGQGLEWMGWIYFASGNSEYNQKFTGRVTM WYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTL TRDTSINTAYMELSSLTSEDTAVYFCASLYDYDWY KISRVEAEDVGIYYCSQSSIYPWTFGQGTKLEIK FDVWGQGTMVTVSS B140153 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS 100 LPVLTQPPSASGTPGQRVTISCSGRSSNIGSNSVNWYRQ 101 WVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTI LPGAAPKLLIYSNNQRPPGVPVRFSGSKSGTSASLAISG (WO2016090320A1) TADKSTSTAYMELSSLRSEDTAVYYCARGGYYSHD LQSEDEATYYCATWDDNLNVHYVFGTGTKVTVLG MWSEDWGQGTLVTVSS B69 QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYF 102 SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPP 103 WGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVT GQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVE (US2017051068A1) ISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVA AGDEAVYYCQVWDSSSDHVVFGGGTKLTVL 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 104 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLA 105 (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 106 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGV 107 (US20090258013A1) WVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTI KENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRF SRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNT TGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPL FDSWGQGTLVTVSS TFGDGTKLEIK MAB10 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYWG 108 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLA 109 (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 110 EIVLTQSPATLSLSPGERATLSCRASQSISSYLA 111 (US9505839B2) WIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLS WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG LDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYN TDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGT WFDPWGQGTLVTVSS NLEIK L3E3 EVQLLESGAEVKKPGASVKVSCKASGYTFTSYYMH 112 QSVLTQPASASGSPGQSITISCTGTSSDVGGYNY 113 (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 114 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA 115 (WO2006121168) WVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTI WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG SRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQ TDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGT GTLVTVSS KVEIK H409A11 QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMY 116 EIVLTQSPATLSLSPGERATLSCRASKGVSTSGY 117 (WO2008156712A1) WVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTL SYLHWYQQKPGQAPRLLIYLASYLESGVPARFSG TTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDM SGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTF GFDYWGQGTTVTVSS GGGTKVEIK

TABLE-US-00017 TABLE 17 variable region sequences of anti-PD-Ll 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 S70 EVQLVESGGGLVQPGGSLRLSCAASGFT 118 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAP 119 (WO2010077634A1) FSDSWIHWVRQAPGKGLEWVAWISPYGG KLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC STYYADSVKGRFTISADTSKNTAYLQMN QQYLYHPATFGQGTKVEIK SLRAEDTAVYYCARRHWPGGFDYWGQGT LVTVSS 12A4 QVQLVQSGAEVKKPGSSVKVSCKTSGDT 120 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAP 121 (US7943743B2) FSTYAISWVRQAPGQGLEWMGGIIPIFG RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC KAHYAQKFQGRVTITADESTSTAYMELS QQRSNWPTFGQGTKVEIK MSLRSEDTAVYFCARKFHFVSGSPFGDV WGQGTTVTVSS

TABLE-US-00018 TABLE 18 Variable region sequences of anti-CD16 antibody Amino acid sequences of anti-CD16 antibody variable region Antibody (Bold and underlined amino acids are CDR regions) code SEQ SEQ (sequence ID ID source) VH NO VL NO NM3E2 EVQLVESGGGVVRPGGSLRLSCAASGFT 122 SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYG 123 FDDYGMSWVRQAPGKGLEWVSGINWNGG KNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHV STGYADSVKGRFTISRDNAKNSLYLQMN VFGGGTKLTVL SLRAEDTAVYYCARGRSLLFDYWGQGTL VTVSR

TABLE-US-00019 TABLE 19 Variable region sequences of anti-SLAMF7 antibody Amino acid sequences 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 EVQLVESGGGLVQPGGSLRLSCAASGFD 124 DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVP 125 (WO2004100898A2) FSRYWMSWVRQAPGKGLEWIGEINPDSS KLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYC TINYAPSLKDKFIISRDNAKNSLYLQMN QQYSSYPYTFGQGTKVEIK SLRAEDTAVYYCARPDGNYWYFDVWGQG TLVTVSS

TABLE-US-00020 TABLE 20 Variable region sequences of anti-CEA antibody Amino acid sequences 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 QVQLVQSGSELKKPGASVKVSCKASGYT 126 DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLI 127 Immunol FTVFGMNWVRQAPGQGLEWMGWINTKTG YSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYYTYPL Immunother EATYVEEFKGRFVFSLDTSVSTAYLQIS FTFGQGTKVEIK (1999) 47: SLKADDTAVYYCARWDFYDYVEAMDYWG 299-306) QGTTVTVSS

TABLE-US-00021 TABLE 21 Variable region sequences of anti-VEGF antibody Amino acid sequences of anti-VEGF antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO Avastin EVQLVESGGGLVQPGGSLRLSCAASGYT 128 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAP 129 FTNYGMNWVRQAPGKGLEWVGWINTYTG KVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC EPTYAADFKRRFTFSLDTSKSTAYLQMN QQYSTVPWTFGQGTKVEIK SLRAEDTAVYYCAKYPHYYGSSHWYFDV WGQGTLVTVSS B2041 EVQLVESGGGLVQPGGSLRLSCAASGFS 130 DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQQKPGKAP 131 (WO2005012359A2) INGSWIFWVRQAPGKGLEWVGAIWPFGG KLLIYAASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC YTHYADSVKGRFTISADTSKNTAYLQMN QQSNTSPLTFGQGTKVEIK SLRAEDTAVYYCARWGHSTSPWAMDYWG QGTLVTVSS G631 EVQLVESGGGLVQPGGSLRLSCAASGFT 132 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAP 133 (WO2005012359A2) ISDYWIHWVRQAPGKGLEWVAGITPAGG KLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC YTYYADSVKGRFTISADTSKNTAYLQMN QQGYGNPFTFGQGTKVEIK SLRAEDTAVYYCARFVFFLPYAMDYWGQ GTLVTVSS

TABLE-US-00022 TABLE 22 Anti-TGF-beta antibody variable regions Amino acid sequences of anti-TGF-beta antibody variable region Antibody (Bold and underlined amino acids are CDR regions) code SEQ SEQ (sequence ID ID source) VH NO VL NO 3G12 QVQLVQSGAEVKKPGSSVKVSCKASGYT 134 ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLL 135 FSSNVISWVRQAPGQGLEWMGGVIPIVD IYGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSP IANYAQRFKGRVTITADESTSTTYMELS ITFGQGTRLEIK SLRSEDTAVYYCASTLGLVLDAMDYWGQ GTLVTVSS 4B9 QVQLVQSGAEVKKPGSSVKVSCKASGYT 136 ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLL 137 FSSNVISWVRQAPGQGLEWMGGVIPIVD IYGASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSP IANYAQRFKGRVTITADESTSTTYMELS ITFGQGTRLEIK SLRSEDTAVYYCALPRAFVLDAMDYWGQ GTLVTVSS

TABLE-US-00023 TABLE 23 Anti-IL-10 antibody variable regions Amino acid sequences of anti-IL-10 antibody variable region Antibody (Bold and underlined amino acids are CDR regions) code SEQ SEQ (sequence ID ID source) VH NO VL NO B-N10 QVQLKQSGPGLLQPSQSLSISCTVSGFS 138 DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQS 139 LATYGVHWVRQSPGKGLEWLGVIWRGGS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRLEAEDLGVYYCFQG TDYSAAFMSRLSITKDNSKSQVFFKMNS SHVPWTFGGGTKLEIK LQADDTAIYFCAKQAYGHYMDYWGQGTS VTVSS BT-063 EVQLVESGGGLVQPGGSLRLSCAASGFS 140 DVVMTQSPLSLPVTLGQPASISCRSSQNIVHSNGNTYLEWYLQRPGQS 141 FATYGVHWVRQSPGKGLEWLGVIWRGGS PRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG TDYSAAFMSRLTISKDNSKNTVYLQMNS SHVPWTFGQGTKVEIK LRAEDTAVYFCAKQAYGHYMDYWGQGTS VTVSS

TABLE-US-00024 TABLE 24 Variable region sequences of anti-CD20 antibody Amino acid sequences 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 QVQLVQSGAEVKKPGSSVKVSCKASGYA 142 DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQS 143 (WO2005044859) FSYSWINWVRQAPGQGLEWMGRIFPGDG PQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQN DTDYNGKFKGRVTITADKSTSTAYMELS LELPYTFGGGTKVEIK SLRSEDTAVYYCARNVFDGYWLVYWGQG TLVTVSS

TABLE-US-00025 TABLE 25 Variable region sequences of anti-Claudinl8.2 antibody Amino acid sequences of anti-Claudinl8.2 antibody variable region (Bold and underlined amino acids are CDR regions) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO IMAB362 QVQLKQSGPGLLQPSQSLSISCTVSGFS 144 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQ 145 (US20090169547A1) LATYGVHWVRQSPGKGLEWLGVIWRGGS KPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAED TDYSAAFMSRLSITKDNSKSQVFFKMNS LAVYYCQNDYSYPFTFGSGTKLEIK LQADDTAIYFCAKQAYGHYMDYWGQGTS VTVSS

TABLE-US-00026 TABLE 26 Variable region sequences of anti-FIXa antibody Amino acid sequences of anti-FIXa antibody variable region (Bold and underlined amino acids are CDR region) Antibody code SEQ SEQ (sequence ID ID source) VH NO VL NO A44 QVQLQQSGAELAKPGASVKLSCKASGYT 146 DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLI 147 (US8062635B2) FTSSWMHWIKQRPGQGLEWLGYINPSSG YWASTRHTGVPDRFTGSRYGTDFTLTISNVQSEDLADYLCQQYSNYIT YTKYNRKFRDKATLTADKSSSTAYMQLT FGGGTKLELK SLTYEDSAVYYCARGGNGYYFDYWGQGT TLTVSS A50 QVQLQQSGAELAKPGASVKLSCKASGYT 148 DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGLSPKLLI 149 (US8062635B2) FTTYWMHWVKQRPGQGLEWIGYINPSSG YWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYLT YTKYNQKFKVKATLTADKSSSTAYMQLS FGAGTKLEIK SLTDEDSAVYYCANGNLGYFFDYWGQGT TLTVSS A69 EVQLQQSGAELVKPGASVKLSCTASGFN 150 DIQMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLI 151 (US8062635B2) IKDYYMHWIKQRPGQGLEWLGYINPSSG YWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYLCQQYSNYIT YTKYNRKFRDKATLTADKSSSTAYMQLT FGAGTKLELK SLTYEDSAVYYCARGGNGYYLDYWGQGT TLTVSS XB12 EVQLQQSGPGLVKPTQSLSLTCSVTGYS 152 DIVLTQSPAIMSASLGEKVTMSCRATSSVNYIYWYQQKSDASPKLWIF 153 (US8062635B2) ITSGYYWTWIRQFPGNNLEWIGYISFDG YTSNLAPGVPPRFSGSGSGNSYSLTISSMEAEDAATYYCQQFSSSPWT TNDYNPSLKNRISITRDTSENQFFLKLN FGGGTKLEIK SVTTEDTATYYCARGPPCTYWGQGTLVT VSA

TABLE-US-00027 TABLE 27 Variable region sequences of anti-FX antibody Amino acid sequences 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 QVQLQQSGPELVKPGASVKMSCKASGYT 154 DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQ 155 (US8062635B2) FTHFVLHWVKQNPGQGLEWIGYIIPYND KPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAED GTKYNEKFKGKATLTSDKSSSTAYMELS LAVYLCQQYYRFPYTFGGGTKLEIK SLTSEDSAVYYCARGNRYDVGSYAMDYW GQGTSVTVSS B26 QVQLQQSGPELVKPGASVKISCKASGYT 156 DIVLTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQKPGQSP 157 (US8062635B2) FTDNNMDWVKQSHGKGLEWIGDINTKSG KALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC GSIYNQKFKGKATLTIDKSSSTAYMELR QQYNSYPLTFGAGTKLEIK SLTSEDTAVYYCARRRSYGYYFDYWGQG TTLTVSS

TABLE-US-00028 TABLE 28 Variable region sequences of anti-HER2 antibody Amino acid sequences of anti-HER2 antibody variable region Antibody (Bold and underlined amino acids are CDR regions) code SEQ SEQ (sequence ID ID source) VH NO VL NO Herceptin EVQLVESGGGLVQPGGSLRLSCAASGFN 158 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI 159 IKDTYIHWVRQAPGKGLEWVARIYPTNG YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP YTRYADSVKGRFTISADTSKNTAYLQMN TFGQGTKVEIK SLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSS Perjeta EVQLVESGGGLVQPGGSLRLSCAASGFT 160 DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLI 161 FTDYTMDWVRQAPGKGLEWVADVNPNSG YSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPY GSIYNQRFKGRFTLSVDRSKNTLYLQMN TFGQGTKVEIK SLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSS

TABLE-US-00029 TABLE 29 Variable region sequences of anti-Siglec-15 antibody Amino acid sequences of anti-Siglec-15 antibody variable region Antibody  Bold and underlined amino acids are CDR regions) code VH SEQ VL SEQ (sequence ID ID source) NO NO 34A1 EVQILETGGGLVKPGGSLRLSCATSGFN 162 DIVLTQSPALAVSLGQRATISCRASQSVTISGYSFIHWYQQKPGQQPR 163 FNDYFMNWVRQAPEKGLEWVAQIRNKIY LLIYRASNLASGIPARFSGSGSGTDFTLTINPVQADDIATYFCQQSRK TYATFYAESLEGRVTISRDDSESSVYLQ SPWTFAGGTKLELR VSSLRAEDTAIYYCTRSLTGGDYFDYWG QGVMVTVSS H34A1 EVQLVESGGGLVQPGGSLRLSCAASGFN 164 EILMTQSPATLSLSPGERATLSCRASQSVTISGYSFIHWYQQKPGQAP 165 FNDYFMNWVRQAPGKGLEWVAQIRNKIY RLLIYRASNLASGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQSR TYATFYAASVKGRFTISRDNAKNSLYLQ KSPWTFGQGTKVEIK MNSLRAEDTAVYYCARSLTGGDYFDYWG QGTLVTVSS

TABLE-US-00030 TABLE 30 Variable region sequences of anti-luciferase antibody Amino acid sequences of anti-luciferase antibody variable region Antibody (Bold and underlined amino acids are CDR regions) code SEQ SEQ (sequence ID ID source) VH NO VL NO 4420 EVKLDETGGGLVQPGRPMKLSCVASGFT 166 DWMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQS 167 FSDYWMNWVRQSPEKGLEWVAQIRNKPY PKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQS NYETYYSDSVKGRFTISRDDSKSSVYLQ THVPWTFGGGTKLEIK MNNLRVEDMGIYYCTGSYYGMDYWGQGT SVTVSS

TABLE-US-00031 TABLE 31 Amino acid sequences of some components A Expression Corresponding SEQ Code Peptide plasmid name Domain code ID NO A-Fab10 A-HIn pA-HIn(10) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa1 51 In SspDnaE 31 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab11 A-HIn pA-HIn(11) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In SspDnaE 31 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab20 A-HIn pA-HIn(20) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa2 52 In SspDnaB 33 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab21 A-HIn pA-HIn(21) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In SspDnaB 33 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab30 A-HIn pA-HIn(30) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa5 55 In MxeGyrA 35 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab31 A-HIn pA-HIn(31) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In MxeGyrA 35 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab40 A-HIn pA-HIn(40) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa6 56 In MjaTFIIB 37 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab41 A-HIn pA-HIn(41) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In MjaTFIIB 37 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab50 A-HIn pA-HIn(50) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa7 57 In PhoVMA 39 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab51 A-HIn pA-HIn(51) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In PhoVMA 39 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab60 A-HIn pA-HIn(60) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa7 57 In TvoVMA 41 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab61 A-HIn pA-HIn(61) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In TvoVMA 41 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab70 A-HIn pA-HIn(70) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa11 61 In Gp41-1 43 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab71 A-HIn pA-HIn(71) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In Gp41-1 43 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab80 A-HIn pA-HIn(80) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa8 58 In Gp41-8 45 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab81 A-HIn pA-HIn(81) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In Gp41-8 45 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab90 A-HIn pA-HIn(90) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa9 59 In IMPDH-1 47 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab91 A-HIn pA-HIn(91) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In IMPDH-1 47 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab92 A-HIn pA-HIn(92) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa14 202 In IMPDH-1 47 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab100 A-HIn pA-HIn(100) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa7 57 In PhoRadA 49 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 A-Fab101 A-HIn pA-HIn(101) VHa S70 118 CH1 G1CH1 179 flanking sequence a FSa10 60 In PhoRadA 49 tag protein His-tag 24 Strep-tag 28 A-L pA-L VLa S70 119 CL Lc1 172 Note: The sequences of domains such as VHa, CH1, flanking sequence a, tag protein, VLa and CL 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 different inteins Expression Corresponding SEQ Code Peptide plasmid name Domain code ID NO B-FcIc10 component B pTag-Ic-FSb-(B-FcIc10) tag protein Strep-tag 28 His-tag 24 Ic SspDnaE 32 flanking sequence b FSb1 64 Pb G1CH2 183 G1CH3 188 B-FcIc11 component B pTag-Ic-FSb-(B-FcIc11) tag protein Strep-tag 28 His-tag 24 Ic SspDnaE 32 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc20 component B pTag-Ic-FSb-(B-FcIc20) tag protein Strep-tag 28 His-tag 24 Ic SspDnaB 34 flanking sequence b FSb3 64 Pb G1CH2 183 G1CH3 188 B-FcIc21 component B pTag-Ic-FSb-(B-FcIc21) tag protein Strep-tag 28 His-tag 24 Ic SspDnaB 34 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc30 component B pTag-Ic-FSb-(B-FcIc30) tag protein Strep-tag 28 His-tag 24 Ic MxeGyrA 36 flanking sequence b FSb4 67 Pb G1CH2 183 G1CH3 188 B-FcIc31 component B pTag-Ic-FSb-(B-FcIc31) tag protein Strep-tag 28 His-tag 24 Ic MxeGyrA 36 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc40 component B pTag-Ic-FSb-(B-FcIc40) tag protein Strep-tag 28 His-tag 24 Ic MjaTFIIB 38 flanking sequence b FSb5 68 Pb G1CH2 183 G1CH3 188 B-FcIc41 component B pTag-Ic-FSb-(B-FcIc41) tag protein Strep-tag 28 His-tag 24 Ic MjaTFIIB 38 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc50 component B pTag-Ic-FSb-(B-FcIc50) tag protein Strep-tag 28 His-tag 24 Ic PhoVMA 40 flanking sequence b FSb6 69 Pb G1CH2 183 G1CH3 188 B-FcIc51 component B pTag-Ic-FSb-(B-FcIc51) tag protein Strep-tag 28 His-tag 24 Ic PhoVMA 40 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc60 component B pTag-Ic-FSb-(B-FcIc60) tag protein Strep-tag 28 His-tag 24 Ic TvoVMA 42 flanking sequence b FS6 69 Pb G1CH2 183 G1CH3 188 B-FcIc61 component B pTag-Ic-FSb-(B-FcIc61) tag protein Strep-tag 28 His-tag 24 Ic TvoVMA 42 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc70 component B pTag-Ic-FSb-(B-FcIc70) tag protein Strep-tag 28 His-tag 24 Ic Gp41-1 44 flanking sequence b FSb7 70 Pb G1CH2 183 G1CH3 188 B-FcIc71 component B pTag-Ic-FSb-(B-FcIc71) tag protein Strep-tag 28 His-tag 24 Ic Gp41-1 44 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc80 component B pTag-Ic-FSb-(B-FcIc80) tag protein Strep-tag 28 His-tag 24 Ic Gp41-8 46 flanking sequence b FSb8 71 Pb G1CH2 183 G1CH3 188 B-FcIc81 component B pTag-Ic-FSb-(B-FcIc81) tag protein Strep-tag 28 His-tag 24 Ic Gp41-8 46 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc90 component B pTag-Ic-FSb-(B-FcIc90) tag protein Strep-tag 28 His-tag 24 Ic IMPDH-1 48 flanking sequence b FSb9 72 Pb G1CH2 183 G1CH3 188 B-FcIc91 component B pTag-Ic-FSb-(B-FcIc91) tag protein Strep-tag 28 His-tag 24 Ic IMPDH-1 48 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188 B-FcIc92 component B pTag-Ic-FSb-(B-FcIc92) tag protein Strep-tag 28 His-tag 24 Ic IMPDH-1 48 flanking sequence b FSb17 204 Pb G1CH2 183 G1CH3 188 B-FcIc100 component B pTag-Ic-FSb-(B-FcIc100) tag protein Strep-tag 28 His-tag 24 Ic PhoRadA 50 flanking sequence b FSb10 73 Pb G1CH2 183 G1CH3 188 B-FcIc101 component B pTag-Ic-FSb-(B-FcIc101) tag protein Strep-tag 28 His-tag 24 Ic PhoRadA 50 flanking sequence b FSb14 77 Pb G1CH2 183 G1CH3 188

TABLE-US-00033 TABLE 33 Component B′ including different inteins Expression Corresponding SEQ Code Polypeptide plasmid name Domain code ID NO Types of inteins SspDnaE B′-HAb10 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(10) tag protein Strep-tag 28 His-tag 24 Ic SspDnaE 32 flanking sequence b FSb1 64 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb11 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(11) tag protein Strep-tag 28 His-tag 24 Ic SspDnaE 32 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb20 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(20) tag protein Strep-tag 28 His-tag 24 Ic SspDnaB 34 flanking sequence b FSb3 66 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb21 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(21) tag protein Strep-tag 28 His-tag 24 Ic SspDnaB 34 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 Types of inteins MxeGyrA B′-HAb30 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(30) tag protein Strep-tag 28 His-tag 24 Ic MxeGyrA 36 flanking sequence b FSb4 67 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb31 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(31) tag protein Strep-tag 28 His-tag 24 Ic MxeGyrA 36 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 Types of inteins MjaTFIIB B′-HAb40 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(40) tag protein Strep-tag 28 His-tag 24 Ic MjaTFIIB 38 flanking sequence b FSb5 68 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb41 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(41) tag protein Strep-tag 28 His-tag 24 Ic MjaTFIIB 38 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 Types of inteins PhoVMA B′-HAb50 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(50) tag protein Strep-tag 28 His-tag 24 Ic PhoVMA 40 flanking sequence b FSb6 69 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb51 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(51) tag protein Strep-tag 28 His-tag 24 Ic PhoVMA 40 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 Types of inteins TvoVMA B′-HAb60 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(60) tag protein Strep-tag 28 His-tag 24 Ic TvoVMA 42 flanking sequence b FS6 69 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb61 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(61) tag protein Strep-tag 28 His-tag 24 Ic TvoVMA 42 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 Types of inteins Gp41-1 B′-HAb70 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(70) tag protein Strep-tag 28 His-tag 24 Ic Gp41-1 44 flanking sequence b FSb7 70 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb71 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(71) tag protein Strep-tag 28 His-tag 24 Ic Gp41-1 44 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 Types of inteins Gp41-8 B′-HAb80 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(80) tag protein Strep-tag 28 His-tag 24 Ic Gp41-8 46 flanking sequence b FSb8 71 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb81 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(81) tag protein Strep-tag 28 His-tag 24 Ic Gp41-8 46 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 Types of inteins IMPDH-1 B′-HAb90 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(90) tag protein Strep-tag 28 His-tag 24 Ic IMPDH-1 48 flanking sequence b FSb9 72 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb91 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(91) tag protein Strep-tag 28 His-tag 24 Ic IMPDH-1 48 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb92 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(92) tag protein Strep-tag 28 His-tag 24 Ic IMPDH-1 48 flanking sequence b FSb17 204 CH2 G1CH2 183 CH3 G1CH3 188 Types of inteins PhoRadA B′-HAb100 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(100) tag protein Strep-tag 28 His-tag 24 Ic PhoRadA 50 flanking sequence b FSb10 73 CH2 G1CH2 183 CH3 G1CH3 188 B′-HAb101 B′-L pB′-L VLb Dara 89 CL Lc1 172 B′-H pB′-H VHb Dara 88 CH1 G1CH1 179 hinge Hin1 168 CH2 G1CH2 183 CH3 G1CH3 188 B′-FcIc pB′-FcIc(101) tag protein Strep-tag 28 His-tag 24 Ic PhoRadA 50 flanking sequence b FSb14 77 CH2 G1CH2 183 CH3 G1CH3 188 Note The sequences of domains such as VLb, CL, VHb, CH1, hinge, CH2, CH3, and tag protein in the table can be replaced with the protein sequences of other corresponding domains mentioned in the present specification.

EXAMPLE 1

Experimental Method

[0253] 1. Preparation of Recombinant Polypeptides

[0254] 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.

[0255] 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.

[0256] 2. The Co-Transfected Plasmids Involved in the Examples were Shown as Follows:

[0257] 1) To express the component A and component B shown in FIG. 1, the plasmids pPa-FSa-In-Tag and pTag-Ic-FSb-Pb were required to be respectively transfected or co-transfected into 293E cells;

[0258] 2) To express the component A and component B′ shown in FIG. 2, the plasmids pPa-FSa-In-Tag and pTag-Ic-FSb-Rb were required to be respectively transfected or co-transfected into 293E cells;

[0259] 3) To express the component A shown in FIG. 3, co-transfection of plasmids Pa-HIn and Pa-L or separate transfection of plasmid pBi-Pa-FSa-In-Tag into 293E cells was required; to express the component B′ shown in FIG. 3, co-transfection of plasmids pB′-L, pB′-H and pB′-FcIc or separate transfection of plasmid pBi-Tag-Ic-FSb-Rb into 293E cells was required.

[0260] 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.

[0261] 3. Purification of Polypeptides with Tag Proteins

[0262] (1) When the tag protein was Fc, the polypeptide was purified by affinity chromatography, for example, MabSelect SuRe (GE, Cat. No. 17-5438-01), 18 ml column.

[0263] (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).

[0264] (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.

[0265] (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).

[0266] (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).

[0267] (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 2

Screening of Flanking Sequence Pairs of Inteins such as SspDnaB, MxeGyrA, MjaTFIIB, PhoVMA, TVoVMA, Gp41-1, Gp41-8, IMPDH-1, PhoRadA

[0268] Construction of Expression Plasmids A-HIn, pA-L, and Plasmid (pTag-Ic-FSb-Pb)

[0269] Under the conditions as described in “Preparation of recombinant polypeptides” of Example 1, as shown in FIGS. 4A and 4B, component expression plasmids for the inteins SspDnaB, MxeGyrA, MjaTFIIB, PhoVMA, TvoVMA, GP41-1, GP41-8, IMPDH-1 and PhoRadA were respectively constructed by pcDNA3.1 plasmid vector based on the structure as shown in Table 31 and Table 32. The pA-L plasmid was the same as that in Example 1.

[0270] For the intein SspDnaB, the following plasmids were constructed: plasmids pA-HIn(20)˜pA-HIn(21) corresponding to A-Fab20 and A-Fab21, and plasmids pTag-Ic-FSb-(B-FcIc20) and pTag-Ic-FSb-(B-FcIc21) corresponding to B-FcIc20 and B-FcIc21.

[0271] For the intein MxeGyrA, the following plasmids were constructed: plasmids pA-HIn(30)˜pA-HIn(31) corresponding to A-Fab30 and A-Fab31, and plasmids pTag-Ic-FSb-(B-FcIc30) and pTag-Ic-FSb-(B-FcIc31) corresponding to B-FcIc30 and B-FcIc31.

[0272] For the intein MjaTFIIB, the following plasmids were constructed: plasmids pA-HIn(40)˜pA-HIn(41) corresponding to A-Fab40 and A-Fab41, and plasmids pTag-Ic-FSb-(B-FcIc40) and pTag-Ic-FSb-(B-FcIc41) corresponding to B-FcIc40 and B-FcIc41.

[0273] For the intein PhoVMA, the following plasmids were constructed: plasmids pA-HIn(50)˜pA-HIn(51) corresponding to A-Fab50 and A-Fab51, and plasmids pTag-Ic-FSb-(B-FcIc50) and pTag-Ic-FSb-(B-FcIc51) corresponding to B-FcIc50 and B-FcIc51.

[0274] For the intein TVoVMA, the following plasmids were constructed: plasmids pA-HIn(60)˜pA-HIn(61) corresponding to A-Fab60 and A-Fab61, and plasmids pTag-Ic-FSb-(B-FcIc60) and pTag-Ic-FSb-(B-FcIc61) corresponding to B-FcIc60 and B-FcIc61.

[0275] For the intein Gp41-1, the following plasmids were constructed: plasmids pA-HIn(70)˜pA-HIn(71) corresponding to A-Fab70 and A-Fab71, and plasmids pTag-Ic-FSb-(B-FcIc70) and pTag-Ic-FSb-(B-FcIc71) corresponding to B-FcIc70 and B-FcIc71.

[0276] For the intein Gp41-8, the following plasmids were constructed: plasmids pA-HIn(80)˜pA-HIn(81) corresponding to A-Fab80 and A-Fab81, and plasmids pTag-Ic-FSb-(B-FcIc80) and pTag-Ic-FSb-(B-FcIc81) corresponding to B-FcIc80 and B-FcIc81.

[0277] For the intein IMPDH-1, the following plasmids were constructed: plasmids pA-HIn(90)˜pA-HIn(92) corresponding to A-Fab90, A-Fab91 and A-Fab92, and plasmids pTag-Ic-FSb-(B-FcIc90)˜pTag-Ic-FSb-(B-FcIc92) corresponding to B-FcIc90˜B-FcIc92.

[0278] For the intein PhoRadA, the following plasmids were constructed: plasmids pA-HIn(100)˜pA-HIn(101) corresponding to A-Fab100 and A-Fab101, and plasmids pTag-Ic-FSb-(B-FcIc100) and pTag-Ic-FSb-(B-FcIc101) corresponding to B-FcIc100 and B-FcIc101.

[0279] The plasmids used in this Example to express the component A included: pA-HIn(20)˜(21), (30)˜(31), (40)˜(41), (50)˜(51), (60)˜(61), (70)˜(71), (80)˜(81), (90)˜(91), (100)˜(101), and pA-L.

[0280] The plasmids used in this Example to express the component B included: pTag-Ic-FSb-(B-FcIc20)˜21), (30)˜(31), (40)˜(41), (50)˜(51), (60)˜(61), (70)˜(71), (80)˜(81), (90)˜(91), (100)˜(101).

TABLE-US-00034 TABLE 34 Co-transfection pairings for inteins Number Component A Component B A21 pA-HIn(81) pA-L pTag-Ic-FSb-(B-FcIc81) A22 pA-HIn(90) pA-L pTag-Ic-FSb-(B-FcIc90) A23 pA-HIn(50) pA-L pTag-Ic-FSb-(B-FcIc50) A24 pA-HIn(51) pA-L pTag-Ic-FSb-(B-FcIc51) A25 pA-HIn(70) pA-L pTag-Ic-FSb-(B-FcIc70) A26 pA-HIn(71) pA-L pTag-Ic-FSb-(B-FcIc71) A27 pA-HIn(80) pA-L pTag-Ic-FSb-(B-FcIc80) A28 pA-HIn(91) pA-L pTag-Ic-FSb-(B-FcIc91) A29 pA-HIn(51) pA-L pTag-Ic-FSb-(B-FcIc50) A30 pA-HIn(71) pA-L pTag-Ic-FSb-(B-FcIc70) A31 pA-HIn(81) pA-L pTag-Ic-FSb-(B-FcIc80) A32 pA-HIn(90) pA-L pTag-Ic-FSb-(B-FcIc91) A33 pA-HIn(50) pA-L pTag-Ic-FSb-(B-FcIc51) A34 pA-HIn(70) pA-L pTag-Ic-FSb-(B-FcIc71) A35 pA-HIn(80) pA-L pTag-Ic-FSb-(B-FcIc81) A36 pA-HIn(91) pA-L pTag-Ic-FSb-(B-FcIc90) A37 pA-HIn(30) pA-L pTag-Ic-FSb-(B-FcIc30) A38 pA-HIn(31) pA-L pTag-Ic-FSb-(B-FcIc31) A39 pA-HIn(31) pA-L pTag-Ic-FSb-(B-FcIc30) A40 pA-HIn(30) pA-L pTag-Ic-FSb-(B-FcIc31) A41 pA-HIn(60) pA-L pTag-Ic-FSb-(B-FcIc60) A42 pA-HIn(61) pA-L pTag-Ic-FSb-(B-FcIc61) A43 pA-HIn(61) pA-L pTag-Ic-FSb-(B-FcIc60) A44 pA-HIn(60) pA-L pTag-Ic-FSb-(B-FcIc61) A45 pA-HIn(20) pA-L pTag-Ic-FSb-(B-FcIc20) A46 pA-HIn(21) pA-L pTag-Ic-FSb-(B-FcIc21) A47 pA-HIn(21) pA-L pTag-Ic-FSb-(B-FcIc20) A48 pA-HIn(20) pA-L pTag-Ic-FSb-(B-FcIc21) A49 pA-HIn(40) pA-L pTag-Ic-FSb-(B-FcIc40) A50 pA-HIn(41) pA-L pTag-Ic-FSb-(B-FcIc41) A51 pA-HIn(41) pA-L pTag-Ic-FSb-(B-FcIc40) A52 pA-HIn(40) pA-L pTag-Ic-FSb-(B-FcIc41) A53 pA-HIn(100) pA-L pTag-Ic-FSb-(B-FcIc100) A54 pA-HIn(101) pA-L pTag-Ic-FSb-(B-FcIc101) A55 pA-HIn(101) pA-L pTag-Ic-FSb-(B-FcIc100) A56 pA-HIn(100) pA-L pTag-Ic-FSb-(B-FcIc101) A58 pA-HIn(92) pA-L pTag-Ic-FSb-(B-FcIc90) A59 pA-HIn(92) pA-L pTag-Ic-FSb-(B-FcIc92)

[0281] Transfections were performed based on the pairs of Table 34. The transfection conditions were as follows: the molar ratio of plasmids was pTag-Ic-FSb(XX or XXX)-(B-FcIc): pA-HIn(XX or XXX): pA-L=3:1:1. And the transient transfection of monoclonal antibody was set as a positive control.

[0282] The transfected cells were cultured for 5 days and the supernatant was taken. Protein A affinity chromatography was performed on the proteins in the supernatant, and then a coomassie brilliant blue staining was performed by SDS-PAGE method (adding a reducing agent) to detect the proteins in the supernatant. The results were shown in FIGS. 6A to 6D, As can be seen from the result, groups A22, A27, A31, A45, A49, A52, A53, A55, and A56 show a significant splicing.

[0283] As can be seen from the result of FIG. 6E, groups A58 and A59 show a significant splicing.

[0284] The inteins and flanking sequences corresponding to groups A22, A27, A31, A45, A49, A52, A53, A55, A56, A58 and A59 are shown in Table 35.

TABLE-US-00035 TABLE 35 Different inteins and corresponding effective flanking sequence pairs Intein Number Flanking sequence a Flanking sequence b IMPDH-1 A22 GGG SI IMPDH-1 A58 DKG SI IMPDH-1 A59 DKG ST Gp41-8 A27 NR SAV Gp41-8 A31 DK SAV SSpDnaB A45 SG SIE MjaTFIIB A49 TY TIH MjaTFIIB A52 TY THT PhoRadA A53 GK TQL PhoRadA A55 GK THT PhoRadA A56 DK TQL

[0285] In summary, the results show that for the intein IMPDH-1, the corresponding flanking sequence pair with excellent splicing efficiency is: when the flanking sequence a is GGG, the flanking sequence b is SI; or when the flanking sequence a is DKG, the flanking sequence b is ST; or when the flanking sequence a is DKG, the flanking sequence b is SI.

[0286] For the intein Gp41-8, the corresponding flanking sequence pair with excellent splicing efficiency is: when the flanking sequence a is NR, the flanking sequence b is SAV; or when the flanking sequence a is DK, the flanking sequence b is SAV.

[0287] For the intein SSpDnaB, the corresponding flanking sequence pair with excellent splicing efficiency is: when the flanking sequence a is SG, the flanking sequence b is SIE.

[0288] For the intein MjaTFIIB, the corresponding flanking sequence pair with excellent splicing efficiency is: when the flanking sequence a is TY, the flanking sequence b is TIH; or when the flanking sequence a is TY, the flanking sequence b is THT.

[0289] For the intein PhoRadA, the corresponding flanking sequence pair with excellent splicing efficiency is: when the flanking sequence a is GK, the flanking sequence b is TQL or THT; or when the flanking sequence a is DK, the flanking sequence b is TQL.

EXAMPLE 3

Intein-Mediated In Vitro Splicing of Polypeptide Fragments from Different Protein Sources

[0290] Construction of Vectors and Expression of Polypeptides

[0291] Under the same condition as that in Example 1, component expression plasmids of inteins SspDnaB, MxeGyrA, MjaTFIIB, PhoVMA, TVoVMA, Gp41-1, Gp41-8, IMPDH-1, PhoRadA were respectively constructed by pcDNA3.1 based on the structure as shown in Tables 31 and 33.

[0292] For the same component B′, the above component expression plasmids were averagely divided into three types: B′-L expression plasmid (pB′-L), B′-H expression plasmid (pB′-H) and B′-FcIc expression plasmid (pB′-FcIc); wherein, each component B′ shared the same pB′-L and B′-H expression plasmids.

[0293] For the intein SspDnaB, plasmids pB′-FcIc(20)˜B′-FcIc(21) corresponding to B′-HAb20˜B′-HAb21 were constructed.

[0294] For the intein MxeGyrA, plasmids pB′-FcIc(30)˜B′-FcIc(31) corresponding to B′-HAb30˜B′-HAb31 were constructed.

[0295] For the intein MjaTFIIB, plasmids pB′-FcIc(40)˜B′-FcIc(41) corresponding to B′-HAb40˜B′-HAb41 were constructed.

[0296] For the intein PhoVMA, plasmids pB′-FcIc(50)˜B′-FcIc(51) corresponding to B′-HAb50˜B′-HAb51 were constructed.

[0297] For the intein TVoVMA, plasmids pB′-FcIc(60)˜B′-FcIc(61) corresponding to B′-HAb60˜B′-HAb61 were constructed.

[0298] For the intein Gp41-1, plasmids pB′-FcIc(70)˜B′-FcIc(71) corresponding to B′-HAb70˜B′-HAb71 were constructed.

[0299] For the intein Gp41-8, plasmids pB′-FcIc(80)˜B′-FcIc(81) corresponding to B′-HAb80˜B′-HAb81 were constructed.

[0300] For the intein IMPDH-1, plasmids pB′-FcIc(90)˜B′-FcIc(92) corresponding to B′-HAb90˜B′-HAb92 were constructed.

[0301] For the intein PhoRadA, plasmids pB′-FcIc(100)˜B′-FcIc(101) corresponding to B′-HAb100˜B′-HAb101 were constructed.

[0302] The plasmids used in this Example to express the component A included: pA-HIn(90), pA-HIn(80), pA-HIn(81), pA-HIn(61), pA-HIn(20), pA-HIn(40), pA-HIn(100) and pA-L.

[0303] The plasmids used in this Example to express the component B′ included: pB′-FcIc(90), pB′-FcIc(80), pB′-FcIc(61), pB′-FcIc(20), pB′-FcIc(41), pB′-FcIc(101) and pB′-L, pB′-H.

[0304] Expression and purification of component A:

[0305] Each plasmid pA-HIn 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.

[0306] Expression and purification of component B′:

[0307] 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′.

[0308] As shown in Table 36, the obtained polypeptide fragments of component A and component B′ were referred to as Fab5˜Fab 11 and HAb5˜HAb11, respectively.

TABLE-US-00036 TABLE 36 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′ Fab5 pA-HIn(90) HAb5 pB′-L pA-L pB′-H pB′-FcIc(90) Fab6 pA-HIn(80) HAb6 pB′-L pA-L pB′-H pB′-FcIc(80) Fab7 pA-HIn(81) HAb7 pB′-L pA-L pB′-H pB′-FcIc(80) Fab8 pA-HIn(61) HAb8 pB′-L pA-L pB′-H pB′-FcIc(61) Fab9 pA-HIn(20) HAb9 pB′-L pA-L pB′-H pB′-FcIc(20) Fab10 pA-HIn(40) HAb10 pB′-L pA-L pB′-H pB′-FcIc(41) Fab11 pA-HIn(100) HAb11 pB′-L pA-L pB′-H pB′-FcIc(101)

[0309] 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 FIGS. 7A to 7B.

[0310] 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 FIG. 7A that both Fab5 and Fab11 are expressed at a high level. Moreover, in the Fab5 and Fab11 groups, polypeptides with a high purity can be obtained by purifying with nickel column chromatography. It can be seen from FIG. 7B that HAb5, HAb9 and HAb11 are all expressed at a high level, and polypeptides with a higher purity can be obtained after HAb5, HAb9 and Hab11 being subjected to nickel column chromatography.

[0311] In Vitro Splicing

[0312] The obtained purified polypeptide fragments of component A and component B′, Fab5, Fab11, HAb5 and HAb11, were dialyzed into a buffer at 4° C. with a 31(D dialysis bag (purchased from Sigma), with a concentration of 1 to 10 micromolar. The buffer contained 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 and B′ with the same intein source were respectively mixed according to corresponding serial numbers thereof (for example, Fab5 and HAb5, etc.) at a molar ratio of 1:5 to 5:1, and DTT was added to be 0.5 to 5 mM, then the mixture was incubated overnight at 37° C.

[0313] The obtained spliced product polypeptides were subjected to SDS-PAGE and coomassie brilliant blue staining, and the results were shown in FIGS. 8A to 8C.

[0314] In FIGS. 8A to 8B, “SPLICING 1” shows the result of a reaction system obtained by mixing component A and component B′ firstly, and then adding 2 mM DTT; “SPLICING 2” shows the result of a reaction system obtained by adding 2 mM DTT to component A and component B′ respectively, and then mixing the two; “reduced (i.e., RD)” means that the component contains 2 mM DTT, “non-reduced (i.e., NON-RD)” means that the component does not contain DTT; “NON-SPLICING ” means no DTT is added to the solution; the monoclonal antibody is Herceptin (purchased from Roche).

[0315] In FIG. 8C, “SPLICING 1” and “NON-SPLICING 1” show the results of reaction systems containing the component A and component B′ at concentrations of 5 μM and 4 μM, respectively, as well as 2 mM DTT; “SPLICING 2” and “NON-SPLICING 2” show the results of reaction systems containing the component A and component B′ with concentrations of 10 μM and 1 μM, respectively, as well as 2 mM DTT; “SPLICING 3” and “NON-SPLICING 3” show the results of reaction systems containing the component A and component B′ with concentrations of 5 μM and 1 μM, respectively, as well as 2 mM DTT; wherein “SPLICING 1” to “SPLICING 3” are incubated overnight at 37° C., and “NON-SPLICING 1” to “NON-SPLICING 3” are incubated at 4° C. overnight; the control bands are Fab11 (non-reduced, i.e., NON-RD) for component A, and HAb 11 (non-reduced, i.e., NON-RD) for component B′, and mAb.

[0316] It can be seen from FIG. 8 that the two split inteins IMPDH-1 and PhoRadA with the novel flanking sequence pair of the present disclosure have a high efficiency in effective splicing in vitro, thereby obtaining in vitro spliced recombinant polypeptides derived from polypeptide fragments of different proteins (i.e., spliced products Fab5+HAb5 and Fab11+HAb11, respectively). The band size of these spliced products are the same as that of the monoclonal antibody control (150 kD), demonstrating that the theoretical molecular weight of the product is consistent with that of natural IgG monoclonal antibody.

[0317] Biological Activity Detection of Spliced Product

[0318] The biological activity detection based on double antigen sandwich ELISA was performed for the recombinant polypeptide Fab5+HAb5 (SPLICING 1). The steps were as follows: 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.

[0319] 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.

[0320] PD-L1 protein was labeled with horseradish peroxidase (HRP).

[0321] 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;

[0322] 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;

[0323] 4) washing: the plate was washed by 200 μl/well PBST (PBS containing 0.1% Tween20) for 3 times;

[0324] 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;

[0325] 6) washing: the plate was washed with 200 μl/well PBST for 5 times;

[0326] 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;

[0327] 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.

[0328] FIG. 9 shows the ELISA results of Fab5 polypeptide fragment, HAb5 polypeptide fragment, unspliced mixture of Fab5 and HAb5, and Fab5+HAb5 polypeptide fragment obtained by splicing Fab5 and HAb5 via the intein in vitro.

[0329] It can be seen from FIG. 9 that the Fab5+HAb5 (SPLICING 1) has the activity of simultaneously binding to both CD38 and PD-L1 antigens. The in vitro unspliced mixture, and the component A (Fab5) and component B (HAb5) alone, does not have the activity of simultaneously binding to both antigens.

[0330] The results prove that the spliced product Fab5+HAb5 (SPLICING 1) obtained by using the intein and the novel flanking sequence pair contained therein of the present disclosure has a good bispecific antibody activity.

[0331] Peptide Map Overlay Detection of Spliced Products

[0332] Peptide coverage refers to the ratio of the number of detected peptide amino acids to the total number of protein amino acids.

[0333] The detection of peptide coverage of a protein product is of great significance for confirming the primary amino acid sequence of protein drugs, ensuring the formation of higher-order structures of protein drugs and maintaining the properties of protein drugs. At present, the detection of protein peptide coverage is carried out by mass spectrometry according to the requirements of drug declaration. The detection of peptide coverage can be completed quickly, accurately and efficiently.

[0334] The peptide coverage of the protein Fab5+HAb5 (spliced product 1) was analyzed in this Example. The protein Fab5+HAb5 (spliced product 1) was digested by trypsin, chymotrypsin and Glu-C enzyme respectively, and the digested peptide samples were then analyzed by LC-MS/MS (XevoG2-XS QTof, waters). The LC-MS/MS data was analyzed by UNIFI (1.8.2, Waters) software, and the peptide coverage of Fab5+HAb5 (spliced product 1) was determined according to the algorithm results.

[0335] Experimental Apparatus:

[0336] 1) High resolution mass spectrometer: XevoG2-XS QTof (Waters)

[0337] 2) Ultra-high performance liquid chromatography: UPLC (Acquity UPLC I-Class) (Waters)

[0338] Materials and Reagents:

[0339] 1) Guanidine HCl (Sigma)

[0340] 2) Urea (Bio-Rad)

[0341] 3) Tris-base (Bio-Rad)

[0342] 4) DTT (Bio-Rad)

[0343] 5) IAM (Sigma)

[0344] 6) Zeba Spin column (Pierce)

[0345] 7) ACQUITY UPLC CSH C18 Column, 130 Å, 1.7 μm, 2.1 mm×150 mm (Waters)

[0346] 8) UNIFI (Waters)

[0347] 9) Trypsin (Promega)

[0348] 10) Chymotrypsin (Sigma)

[0349] 11) Glu-C enzyme (Wako)

[0350] Experimental Method

[0351] 1) Digestion with trypsin, chymotrypsin and Glu-C enzyme: the trypsin, chymotrypsin and Glu-C enzyme were added respectively to an appropriate amount of Fab5+HAb5 (splicing 1) after appropriate pretreatment and then digested at 37° C. for 20 hours.

[0352] 2) High performance liquid chromatography: after digestion, the Fab5+HAb5 (spliced product 1) was separated by a ultra-high performance liquid chromatography system, Acquity UPLC I-Class, with a liquid phase A solution of 0.1% FA aqueous solution and a liquid phase B solution of 0.1% FA acetonitrile solution. The Fab5+HAb5 (spliced product 1) was loaded into the column by a autosampler, and then separated by the chromatographic column, with a column temperature of 55° C., a flow rate of 300 μl/min, and a 214 nm wavelength of TUV detector. The relevant liquid phase gradients were shown in Table 37.

TABLE-US-00037 TABLE 37 The ratio of solutions A and B in high performance liquid chromatography Solution A Solution B Time/min percentage (%) percentage (%) 1 3 98 2 2 63 60 40 3 63.1 2 98 4 66 2 98 5 66.1 98 2 6 75 98 2

[0353] 3) Mass spectrometry identification: the Fab5+HAb5 (spliced product 1) was detected and analyzed by XevoG2-XS QTof mass spectrometer (Waters) after being desalted and separated by the ultra-high performance liquid chromatography. Analysis time: 63 minutes; detection mode: positive ion, MS, scanning range (m/z): 300-2000.

[0354] 4) Mass spectrometry data processing: the raw data were checked against the database by UNIFI (1.8.2, Waters) software, and the main parameters were as follows (Table 38):

TABLE-US-00038 TABLE 38 List of main parameters for mass spectrometry data processing Item Specific situation Protease Trypsin, chymotrypsin and Glu-C enzyme protein Glycosylated O-GN-G ST, Glycosylated O-GN-G-SA ST, modification Glycosylated O-G-SA ST, G0(N), G0F(N), G1F(N), G2F(N), Carbamidomethyl (C), Deamidated (NQ), Oxidation(M), Protein Terminal Acetyl (N-terminal) M/Z ±15 ppm tolerance Fragment ±20 ppm tolerance theoretical Light chain 1: sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS Fab5 + HAb5 RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSD (spliced EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL product 1) SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 205) Heavy chain 1: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGS TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGGSICPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLP PSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 206) Heavy chain 2: EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGG TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 207) Light chain 2: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:208) Filter Minimum fragmentions: 3

[0355] Experimental Results and Analysis

[0356] The peptide samples obtained after digesting Fab5+HAb5 (spliced product 1) with trypsin, chymotrypsin and Glu-C enzyme respectively were analyzed by LC-MS/MS. The obtained raw data were checked against the database by UNIFI software. The database used was the theoretical sequence of Fab5+HAb5 (spliced product 1) provided by the customer.

[0357] 1) The BPI map after digestion of Fab5+HAb5 (spliced product 1) was shown in FIGS. 10A to 10C.

[0358] 2) The coverage percentage after digestion by trypsin, chymotrypsin, and Glu-C enzyme were:

[0359] after trypsin digestion, the coverage percentage was 100%;

[0360] after chymotrypsin digestion, the coverage percentage was 100%;

[0361] after Glu-C enzyme digestion, the coverage percentage was 100%.

[0362] The digested samples were analyzed by LC-MS/MS and the database search results were integrated, finally obtaining a 100.00% peptide coverage for the Fab5+HAb5 (splicing 1). 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 and peptide map coverage, 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.

EXAMPLE 4

Intein-Mediated In Vitro Splicing of Different IgG Subclasses (1) Sequence of Component A

[0363] As shown in Table 39, the sequences corresponding to component A of three different IgG subclasses were as follows:

TABLE-US-00039 TABLE 39 Sequences corresponding to component A of human IgG2, IgG3 and IgG4 Expression Corre- Human IgG Poly- plasmid sponding SEQ subclass peptide name Domain code ID NO IgG2 Fab102 pA-HIn(102) VHa 10A7 106 component CH1 G2CH1 180 A flanking FSa7 57 sequence a In PhoRadA 49 tag protein His-tag 24 Strep-tag 28 PA-L(1) VLa 10A7 107 CL Lc2 173 IgG3 Fab103 pA-HIn(103) VHa 10A7 106 component CH1 G3CH1 181 A flanking FSa7 57 sequence a In PhoRadA 49 tag protein His-tag 24 Strep-tag 28 PA-L(1) VLa 10A7 107 CL Lc2 173 IgG4 Fab104 pA-HIn(104) VHa 10A7 106 component CH1 G4CH1 182 A flanking FSa9 59 sequence a In IMPDH-1 47 tag protein His-tag 24 Strep-tag 28 PA-L(1) VLa 10A7 107 CL Lc2 173

[0364] As shown in Table 40, the sequences corresponding to component B of the three different IgG subclasses were as follows:

TABLE-US-00040 TABLE 40 Sequences corresponding to component B of human IgG2, IgG3 and IgG4 Expression Corre- Human IgG Poly- plasmid sponding SEQ subclass peptide name Domain code ID NO IgG2 FcIc102 pTag-Ic- tag protein Strep-tag 28 component FSb-(B- His-tag 24 B FcIc102) Ic PhoRadA 50 flanking FSb10 73 sequence b hinge Hin2 169 Pb G2CH2 184 G2CH3 189 IgG3 FcIc103 pTag-Ic- tag protein Strep-tag 28 component FSb-(B- His-tag 24 B FcIc103) Ic PhoRadA 50 flanking FSb10 73 sequence b hinge Hin3 170 Pb G3CH2 186 G3CH3 190 IgG4 FcIc104 pTag-Ic- tag protein Strep-tag 28 component FSb-(B- His-tag 24 B FcIc104) Ic IMPDH-1 48 flanking FSb9 72 sequence b hinge Hin4 171 Pb G4CH2 187 G4CH3 191

[0365] Transfections were performed based on the pairs of Table 41 in the same manner as that in Example 2. The transfection conditions were as follows: the molar ratio of plasmids was pTag-Ic-FSb-(B-FcIcxxx):pA-HIn(xxx):pA-L(1)=3:1:1. A positive control monoclonal antibody was also set as described above.

TABLE-US-00041 TABLE 41 Co-transfection pairing table for inteins for expression of different IgG subclasses Number Component A Component B A102 pA-HIn(102) PA-L(1) pTag-Ic-FSb-(B-FcIc102) A103 pA-HIn(103) PA-L(1) pTag-Ic-FSb-(B-FcIc103) A104 pA-HIn(104) PA-L(1) pTag-Ic-FSb-(B-FcIc104)

[0366] The transfected cells were cultured for 5 days and the supernatant was taken. Protein A affinity chromatography was performed on the proteins in the supernatant, and then a coomassie brilliant blue staining was performed by SDS-PAGE method (adding a reducing agent) to detect the proteins in the supernatant. The results were shown in FIG. 11.

[0367] As can be seen from the results, there was a significant splicing in human IgG2, IgG3 and IgG4 subclasses by using the intein; wherein, the A102 showed the intracellular expression by applying the intein PhoRadA to the component A and component B of human IgG2 subclass, indicating that an intact IgG2 mAb was formed by intracellular splicing; A103 showed the intracellular expression by applying the intein PhoRadA to the component A and component B of human IgG3 subclass, indicating that an intact IgG3 mAb was formed by intracellular splicing; A104 showed the intracellular expression by applying the intein IMPDH-1 to the component A and component B of human IgG4 subclass, indicating that an intact IgG4 mAb was formed by intracellular splicing.

EXAMPLE 5

Intein-Mediated In Vitro Splicing of Green Fluorescent Protein

[0368] The green fluorescent protein was EGFP (source: UniProtKB—A0A076FL24), and its full-length amino acid sequence was SEQ ID No: 23, with a total of 239 amino acid residues. The sequence was split into component A and component B; wherein (1) the component A was a fusion of amino acids at positions 1-158 of EGFP and an intein, and the corresponding coding DNA was constructed into an eukaryotic expression vector pcDNA3.1, with the flanking sequence a, the N′ fragment of intein (In) and a stop codon (TAA, TGA or TAG) added to the C-terminus, and the names of the constructed expression plasmids were shown in Table 42; (2) the component B was a fusion of amino acids at positions 159-239 of EGFP and an intein, and the corresponding coding DNA was constructed into an eukaryotic expression vector pcDNA3.1, with a start codon ATG, the C′fragment of intein (Ic) and the flanking sequence b added to its N-terminus, as well as with a stop codon (TAA, TGA or TAG) added to the C-terminus, and the names of the constructed expression plasmids were shown in Table 43. In addition, the EGFP full-length protein-encoding DNA was constructed into pcDNA3.1 (with a stop codon), and the plasmid was referred to as pEGFP.

TABLE-US-00042 TABLE 42 Names of the expression plasmid for component A of EGFP Plasmid name Pa Flanking sequence a In pGFP-N1 N-terminus DK (SEQ ID No: 60) Gp41-8 (SEQ ID No: 45) pGFP-N2 of EGFP DKG (SEQ ID No: 202) IMPDH-1 (SEQ ID No: 47) pGFP-N3 (amino acids GK (SEQ ID No: 57) TvoVMA (SEQ ID No: 41) pGFP-N4 at positions SG (SEQ ID No: 52) SpDnaB (SEQ ID No: 33) pGFP-N5 1-158 of SEQ GK (SEQ ID No: 57) PhoRadA (SEQ ID No: 49) ID No: 23)

TABLE-US-00043 TABLE 43 Names of the expression plasmid for component B of EGFP Plasmid name Ic Flanking sequence b Pb pGFP-C1 Gp41-8 (SEQ ID No: 46) SAV (SEQ ID No: 71) C-terminus pGFP-C2 IMPDH-1 (SEQ ID No: 48) SI (SEQ ID No: 72) of EGFP pGFP-C3 TvoVMA (SEQ ID No: 42) THT (SEQ ID No: 77) (amino acids pGFP-C4 SpDnaB (SEQ ID No: 34) SIE (SEQ ID No: 66) at positions pGFP-C5 PhoRadA (SEQ ID No: 50) THT (SEQ ID No: 77) 159 to 239 of SEQ ID No: 23)

[0369] Based on the method of Example 1, the plasmids pEGFP-A and pEGFP were separately transfected or co-transfected into 293 cells or CHO cells with a co-transfection ratio of 1:1. In addition, the pEGFP was separately transfected into 293 or CHO cells as a positive control. The concentration of each plasmid was the same for separate transfection or co-transfection. 48 hours after transfection, the green fluorescence expression of cells was detected by flow cytometer. and the results were shown in Table 44.

TABLE-US-00044 TABLE 44 Green fluorescence expression results in 293 cells 48 hours after transfection Mean fluorescence Fluorescent cell Transfected plasmid intensity percentage pEGFP 1 × 10{circumflex over ( )}5 99% pGFP-N1 + pGFP-C1 3 × 10{circumflex over ( )}4 57% pGFP-N1 221 0.1%  pGFP-C1 105 0 pGFP-N2 + pGFP-C2 9.9 × 10{circumflex over ( )}4.sup.  99% pGFP-N2 277 0.1%  pGFP-C2 146 0 pGFP-N3 + pGFP-C3 1 × 10{circumflex over ( )}4 47% pGFP-N3 177 0 pGFP-C3 133 0 pGFP-N4 + pGFP-C4 7 × 10{circumflex over ( )}4 88% pGFP-N4 321 0.2%  pGFP-C4 152 0 pGFP-N5 + pGFP-C5 8 × 10{circumflex over ( )}4 95% pGFP-N5 274 0.1%  pGFP-C5 106 0 Blank control 139 0

[0370] As can be seen from the above results, different inteins and flanking sequences can effectively splice the green fluorescent protein in cells and form a structure very similar to that of the original green fluorescent protein, thereby generating the green fluorescence. Separate expression of component A or component B cannot generate the green fluorescence.

INDUSTRIAL APPLICABILITY

[0371] 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.

[0372] 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.

[0373] 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.