WO2021047558A1 - 断裂型内含肽、使用其的重组多肽的制备方法 - Google Patents

断裂型内含肽、使用其的重组多肽的制备方法 Download PDF

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WO2021047558A1
WO2021047558A1 PCT/CN2020/114271 CN2020114271W WO2021047558A1 WO 2021047558 A1 WO2021047558 A1 WO 2021047558A1 CN 2020114271 W CN2020114271 W CN 2020114271W WO 2021047558 A1 WO2021047558 A1 WO 2021047558A1
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flanking sequence
sequence
intein
flanking
fragmented
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French (fr)
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张敬
罗芳
龚承
王鑫
方丽娟
周鹏飞
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武汉友芝友生物制药有限公司
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Priority to CN202080063341.7A priority Critical patent/CN114450292A/zh
Priority to US17/641,431 priority patent/US20220332757A1/en
Publication of WO2021047558A1 publication Critical patent/WO2021047558A1/zh

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Definitions

  • the present invention relates to a fragmented intein containing a novel pair of flanking sequences and a recombinant polypeptide using the same, and the application of the intein in preparing antibodies, especially bispecific antibodies.
  • the present invention also relates to a method for screening the fragmented intein containing the novel pair of flanking sequences.
  • Protein trans-splicing refers to a protein splicing reaction mediated by a fragmented intein.
  • the N-terminal fragment or N-terminal protein splicing region (In) of the fragmented intein and the C-terminal fragment or C-terminal protein splicing region (Ic) recognize each other and are non-covalently bonded.
  • the fragmented intein that reconstructs the active center completes the protein splicing reaction according to the typical protein splicing pathway, and connects the exon peptides on both sides (Saleh.L., Chemical Record.6( 2006)183-193).
  • the gene expressing the precursor protein can be split into two open reading frames, and the N-terminal protein splicing region (N'fragment of intein, abbreviated as In) and the C-terminal protein splicing region (In) can be used by splitting the gene expressing the precursor protein into two open reading frames.
  • C'fragment of intein, abbreviated as Ic) The two-part split intein (split intein) catalyzes the trans-splicing reaction of the protein, thereby combining the two separate exopeptides (En, Ec) that constitute the precursor protein. Connected by peptide bonds to obtain a recombinant protein (Ozawa.T., Nat Biotechbol. 21 (2003) 287-93).
  • Bispecific antibody refers to an antibody molecule that can recognize two antigens or two epitopes at the same time.
  • bispecific or multispecific antibodies that can bind more than two antigens are known in the art and can pass through cells. Fusion method, chemical modification method, gene recombination and other methods are obtained in eukaryotic expression system or prokaryotic expression system.
  • bispecific antibody formats have been developed. For example, by fusing a tetravalent bispecific antibody such as an IgG antibody format and a single chain domain (see, for example, Coloma, MJ, et al., Nature Biotech. 15 (1997) 159-163; WO 2001077342; and Morrison, S., L ., Nature Biotech. 25 (2007) 1233-1234).
  • a tetravalent bispecific antibody such as an IgG antibody format and a single chain domain
  • small molecule antibodies such as minibodies, several single-chain forms (scFv double-scFv) and so on.
  • the central structure of the antibody (IgA, IgD, IgE, IgG or IgM) is no longer maintained (Holliger, P., etc., Nature Biotech. 23 (2005) 1126-1136; Fischer, N, and Leger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., J. Immunol. Methods. 318 (2007) 65-74; Wu, C., et al., Nature Biotech. 25 ( 2007)1290-1297).
  • these foreign proteins may cause an immune response against the connecting peptide itself or the protein containing the connecting peptide, and even cause an immune storm.
  • these connecting peptides due to the flexible nature of these connecting peptides, they are prone to protein degradation, which easily leads to poor antibody stability, easy aggregation, shortened half-life and further enhancement of immunogenicity.
  • Amgen's Bonatumomab (Blinatumomab) has a half-life in the blood of only 1.25 hours, resulting in a 24-hour continuous administration through a syringe pump, which greatly limits its application (Bargou, R and Leo.E ., Science. 321 (2008) 974-7).
  • the Fc fragment effector function of the antibody can be retained: for example, CDC (complement-dependent cytotoxicity), or ADCC (cytotoxicity), and make the antibody interact with the vascular wall FcRn (Fc The half-life of receptor) binding is prolonged. These functions must be mediated by the Fc region. Therefore, it is necessary to retain the Fc region in the modified bispecific antibody.
  • bispecific antibodies with structures that are extremely similar to those of naturally occurring antibodies (such as IgA, IgD, IgE, IgG, IgM), and further, humanized bispecific antibodies with minimal differences from human antibody sequences are needed. And fully human bispecific antibodies.
  • NpuDnaE NpuDnaE
  • bispecific antibody has a great risk of misfolding and instability, and there are also problems with splicing efficiency (Han L, Zong H, etc., Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies, Methods, .Vol 154 , 2019 Feb 1; 154: 32-37).
  • the protein splicing efficiency mediated by the fragmented intein is directly related to the intein sequence and flanking sequences of the intein.
  • NpuDnaE In the NEB database (http://inteins.com/), more than 600 fragmented inteins are listed, the more commonly used ones are NpuDnaE and SspDnaE.
  • the In flanking sequence of NpuDnaE is AEY (En-AEY-In)
  • the Ic flanking sequence is CFNGT (Ic-CFNGT-Ec)
  • the In flanking sequence of SspDnaE is AEY (En- AEY-In) and Ic flanking sequence is CFNKS (Ic-CFNKS-Ec)
  • En-AEY-In and Ic-CFNGT-Ec are the protein form of En-AEYCFNGT-Ec after splicing
  • En-AEY-In and Ic -CFNKS-Ec is the protein form of En-AEYCFNKS-Ec after splicing, and all have a cysteine residue, so that there are
  • flanking sequence pair of the existing fragmented intein In order to avoid free sulfhydryl groups in the spliced product, it is necessary to improve the flanking sequence pair of the existing fragmented intein, and a novel flanking sequence is needed that maintains the good cleavage efficiency of the intein and does not contain half. Novel flanking sequence pairs of cystine residues.
  • flanking sequence pairs of the existing fragmented intein will affect the efficiency of trans-splicing. Therefore, a screening method is needed to screen for the inclusion of new flanking sequence pairs.
  • Peptides have excellent trans-splicing efficiency and do not introduce free sulfhydryl groups at the interface into the spliced product.
  • fragmented intein suitable for preparing antibodies, especially bispecific antibodies, which has excellent trans-splicing efficiency and does not introduce free sulfhydryl groups at the interface of the spliced product containing novel flanking sequence pairs. Fragmented intein.
  • the present invention through the inventor’s diligent research, through regular amino acid mutations on the flanking sequence pairs of the existing intein, and screening the flanking sequence pairs with excellent trans-splicing efficiency, thereby obtaining a new type of flanking sequence.
  • the fragmented intein of the sequence pair has flanking sequences without cysteine residues, and does not introduce free sulfhydryl groups at the interface in the cleavage product.
  • the efficiency of trans-splicing is excellent, and it is especially suitable for preparing antibodies (especially Bispecific antibodies).
  • polypeptide fragments from different proteins can be spliced together with high shear efficiency under relatively mild conditions (such as normal temperature, physiological salt concentration, neutral pH, etc.) to form a recombinant fusion Peptide protein.
  • the present inventors established a method for preparing recombinant polypeptides, especially bispecific antibodies, using the fragmented intein based on the screening of the above-mentioned fragmented intein.
  • the bispecific antibody prepared according to the method for preparing bispecific antibodies of the present invention does not have unnatural domains, and its structure is very similar to that of natural antibodies (IgA, IgD, IgE, IgG or IgM), and has Fc Structural domain.
  • the bispecific antibody has good structural integrity and stability, and can retain or remove CDC (complement-dependent cytotoxicity) or ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent cellular phagocytosis) according to different IgG subtypes ) Or FcRn (Fc receptor) binding activity.
  • the bispecific antibody prepared by the method of the present invention has the following advantages: the bispecific antibody has a long in vivo half-life and low immunogenicity; no linker is introduced in any form, and the stability of the antibody molecule is improved. The immune response is reduced.
  • the bispecific antibody prepared by the method of the present invention can be prepared by the mammalian cell expression system, so that it has the same glycosylation modification as the wild-type IgG, obtains better biological functions, is more stable, and has a long half-life in vivo;
  • the in vitro splicing method by intein can completely avoid the problems of heavy chain mismatch and light chain mismatch that are very easy to occur in traditional methods.
  • the method for preparing bispecific antibodies of the present invention can also be used to produce humanized bispecific antibodies and bispecific antibodies with fully human sequences.
  • the sequence of such an antibody prepared by the method of the present invention is closer to that of a human antibody, and can effectively reduce the occurrence of immune response.
  • the method for preparing bispecific antibodies of the present invention is a method for constructing universal bispecific antibodies, which is not restricted by antibody subtypes (IgG, IgA, IgM, IgD, IgE, and light chain ⁇ and ⁇ types), and does not require Different mutations are designed according to the specific target, which can be used to construct any bispecific antibody.
  • antibody subtypes IgG, IgA, IgM, IgD, IgE, and light chain ⁇ and ⁇ types
  • the present invention provides the following technical solutions.
  • the flanking sequence pair includes: a flanking sequence a and a flanking sequence b; the flanking sequence a is located at the N-terminus of the fragmented intein N-terminal protein splicing region (In), and between the N-terminal exon (En) and Between In; the flanking sequence b is located at the C-terminus of the fragmented intein C-terminal protein splicing region (Ic), and between Ic and the C-terminal exopeptide (Ec);
  • the fragmented intein is selected from: SspDnaE, SspDnaB, MxeGyrA, MjaTFIIB, PhoVMA, TvoVMA, Gp41-1, Gp41-8, IMPDH-1 or PhoRadA,
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or deletion, or preferably G or D;
  • a -2 is X or deletion, or preferably G or K;
  • a -1 is selected from G or T;
  • B 1 is S;
  • B 2 is I or T or S;
  • B 3 is X or missing;
  • flanking sequence a is G, XG, XGG, DKG or DKT and the flanking sequence b is SI, ST, SS, SIX, STX or SSX;
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or missing;
  • a -2 is selected from N or D;
  • a -1 is selected from R or K;
  • flanking sequence a is NR, XNR, DK, XDK, DR or XDR and the flanking sequence b is SA or SAX;
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or missing;
  • a -2 is selected from S or D;
  • a -1 is selected from G or K;
  • flanking sequence a is SG, XSG, DK, XDK, and the flanking sequence b is SI or SIX;
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where
  • a -3 is X or missing;
  • a -2 is selected from T or D;
  • a -1 is selected from Y;
  • B 1 is T;
  • B 2 is I or H;
  • B 3 is X or missing, or preferably H or T;
  • flanking sequence a is TY, DY, XTY or XDY
  • flanking sequence b is TI, TIX, TH or THX
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or missing;
  • a -2 is selected from G or D;
  • a -1 is selected from K;
  • flanking sequence a is GK, XGK, DK or XDK, and the flanking sequence b is TQ, TH, TQX or THX;
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or missing;
  • a -2 is selected from G or D;
  • a -1 is K;
  • flanking sequence a is GK, XGK, DK or XDK
  • flanking sequence b is TV, TH, TVX or THX;
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or missing;
  • a -2 is selected from R or D;
  • a -1 is selected from Y, K or T;
  • flanking sequence a is RY, XRY, DK or XDK
  • flanking sequence b is TE, TH, TEX or THX
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or missing;
  • a -2 is selected from G or D;
  • a -1 is selected from K;
  • flanking sequence a is GK, XGK, DK or XDK
  • flanking sequence b is TV, TH, TVX or THX;
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or missing;
  • a -2 is selected from G or D;
  • a -1 is selected from Y or K;
  • B 1 is S or T;
  • B 2 is S or H;
  • B 3 is X or missing, or preferably S or T;
  • flanking sequence a is GY, XGY, DK or XDK
  • flanking sequence b is SS, SH, SSX or SHX;
  • flanking sequence a is A -3 A -2 A -1 and the flanking sequence b is B 1 B 2 B 3 , where:
  • a -3 is X or missing;
  • a -2 is selected from G or D;
  • a -1 is selected from G, S or K;
  • B 1 is T or S;
  • B 2 is E or H;
  • B 3 is X or missing, or preferably T;
  • flanking sequence a is GG, XGG, GK, XGK, DK or XDK, and the flanking sequence b is SE, TH, SEX or THX;
  • said X is selected from: any of G, A, V, L, M, I, S, T, P, N, Q, F, Y, W, K, R, H, D, E, C An amino acid.
  • the SspDnaE is composed of In with the sequence of SEQ ID NO: 31 and Ic with the sequence of SEQ ID NO: 32,
  • the SspDnaB is composed of In with the sequence of SEQ ID NO: 33 and Ic with the sequence of SEQ ID NO: 34,
  • the MxeGyrA is composed of In with the sequence of SEQ ID NO: 35 and Ic with the sequence of SEQ ID NO: 36,
  • the MjaTFHB is composed of In with the sequence of SEQ ID NO: 37 and Ic with the sequence of SEQ ID NO: 38,
  • the PhoVMA is composed of In with the sequence of SEQ ID NO: 39 and Ic with the sequence of SEQ ID NO: 40,
  • the TvoVMA is composed of In with the sequence of SEQ ID NO: 41 and Ic with the sequence of SEQ ID NO: 42,
  • the Gp41-1 is composed of In with the sequence of SEQ ID NO: 43 and Ic with the sequence of SEQ ID NO: 44,
  • the Gp41-8 is composed of In with the sequence of SEQ ID NO: 45 and Ic with the sequence of SEQ ID NO: 46,
  • the IMPDH-1 is composed of In with the sequence of SEQ ID NO: 47 and Ic with the sequence of SEQ ID NO: 48,
  • the PhoRadA is composed of In with the sequence of SEQ ID NO: 49 and Ic with the sequence of SEQ ID NO: 50,
  • 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;
  • 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;
  • flanking sequence a is SG and the flanking sequence b is SIE;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • said X is selected from: any of G, A, V, L, M, I, S, T, P, N, Q, F, Y, W, K, R, H, D, E, C An amino acid.
  • Recombinant polypeptide which is obtained by trans-splicing the flanking sequence pair for the fragmented intein described in 1 or 2 above.
  • the N-terminus of the flanking sequence a is connected to the C-terminus of En, and the C-terminus of the flanking sequence a is connected to the In, and optionally a tag protein is attached to the C-terminus of In;
  • the C-terminus of the flanking sequence b is connected to the N-terminus of Ec, and the N-terminus of the flanking sequence b is connected to the Ic, and optionally a tag protein is connected to the N-terminus of Ic;
  • the coding sequences of En and Ec are respectively derived from the N-terminal part and the C-terminal part of the same protein
  • the tag protein is selected from SEQ ID NO: 24, 25, 26, 27, 28, 29 or 30.
  • the N-terminus of the flanking sequence a is connected to the C-terminus of En, and the C-terminus of the flanking sequence a is connected to the In, and optionally a tag protein is attached to the C-terminus of In;
  • the C-terminus of the flanking sequence b is connected to the N-terminus of Ec, and the N-terminus of the flanking sequence b is connected to the Ic, and optionally a tag protein is connected to the N-terminus of Ic;
  • En and Ec are from different proteins.
  • the recombinant polypeptide according to 4 or 5 above which is a fluorescent protein, a protease, a signal peptide, an antibacterial peptide, an antibody, or a polypeptide with biological toxicity.
  • the antibody is a full-length antibody or a functional fragment of an antibody.
  • antibody heavy chain variable region VH antibody heavy chain variable region VL
  • antibody heavy chain constant region fragment Fc antibody heavy chain One or more of 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.
  • the antigen A includes: 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- ⁇ , Her2, IL-10, Siglec-15 , Ras, C-myc, the epitope A is an immunogenic epitope of the antigen A.
  • the antigen B includes: 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- ⁇ , Her2, IL-10, Siglec-15 , Ras, C-myc, the epitope B is an immunogenic epitope of the antigen B.
  • the recombinant polypeptide according to 11 above which is a bispecific antibody that can simultaneously bind to antigens or epitopes A and B, preferably a humanized bispecific antibody or a bispecific antibody of fully human sequence.
  • the component A includes: the light chain of the antibody, the VH+CH1 chain of the antibody with In fused to the C-terminus, or the variable region VHHa of the single domain antibody with In fused to the C-terminus, optionally with a tag attached to the C-terminus of In protein,
  • the component B includes: the light chain of the antibody, the complete heavy chain of the antibody, and the Fc chain with Ic fused to the N-terminus, or the single domain antibody variable region VHHb with Ic fused to the N-terminus, optionally at the N-terminus of Ic A tag protein is attached, and the VHHa and VHHb may be the same or different.
  • the tag protein is selected from: Fc, His-tag, Strep-tag, Flag, HA or maltose binding protein MBP.
  • composition comprising the recombinant polypeptide described in any one of 3 to 14 above.
  • composition comprising a carrier in addition to the recombinant polypeptide described in any one of 3 to 14 above.
  • composition according to 16 above which is a pharmaceutical composition
  • the carrier is a pharmaceutically acceptable carrier.
  • a kit comprising the recombinant polypeptide described in any one of 3 to 14 above, which is used to detect the presence of antigen or epitope A and/or antigen or epitope B in a sample, wherein preferably the recombinant polypeptide is
  • the state stored in the liquid or the freeze-dried powder may optionally exist alone or in the state of being connected, complexed, associated, chelated and fixed to the carrier.
  • a method for preparing a recombinant polypeptide which includes:
  • component A includes flanking sequence a, N-terminal exo peptide En and In, the N-terminus of said flanking sequence a and the C of said N-terminal exo peptide En
  • the C-terminus of the flanking sequence a is connected to the In, and optionally a tag protein is also connected to the C-terminus of the In;
  • the component B includes a flanking sequence b, C-terminal exoneptides Ec and Ic, the C-terminus of the flanking sequence b is connected to the N-terminus of the C-terminal exoneptide Ec, and the N-terminus of the flanking sequence b is connected to the Said Ic is connected, optionally with a tag protein connected to the N-terminus of Ic;
  • flanking sequence a and the flanking sequence b are as described in 1 or 2 above, and the coding sequences of the N-terminal exoneptide En and the C-terminal exoneptide Ec are derived from the same protein or different proteins;
  • step (1) it includes allowing cells containing the nucleic acid sequences encoding component A and component B to express said component A and component B; preferably, the N-terminal exoneptide En and the C-terminal exogenous peptide
  • the peptide Ec can be different domains of the antibody.
  • the chromatography method in the first purification step is selected from proteinA, proteinG, nickel column, Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography or cross-linked starch affinity Chromatography, and
  • the chromatography method in the second purification step is selected from affinity chromatography methods corresponding to the tag protein to remove unspliced components, or remove unspliced components through ion exchange, hydrophobicity, and molecular sieve.
  • Component A includes flanking sequence a, En P and In, the N-terminus of the flanking sequence a is connected to the C-terminus of En P , and the C-terminus of the flanking sequence a is connected to the In, optionally in In
  • the C-terminus of the flanking sequence is also connected to a tag protein
  • component B includes flanking sequences b, Ec P and Ic, the C-terminus of the flanking sequence b is connected to the N-terminus of Ec P , and the N-terminus of the flanking sequence b is connected to the Ic connection, optionally with a tag protein connected to the N-terminus of Ic;
  • Component A' includes flanking sequence a, En R and In, the N-terminus of the flanking sequence a is connected to the C-terminus of Ra, and the C-terminus of the flanking sequence a is connected to the In, optionally in In
  • the C-terminus of the flanking sequence is also connected to a tag protein
  • component B'in includes flanking sequences b, Ec R and Ic, the C-terminus of the flanking sequence b is connected to the N-terminus of Ec R , and the N-terminus of the flanking sequence b is connected to the Said Ic is connected, optionally with a tag protein connected to the N-terminus of Ic;
  • a method for screening flanking sequence pairs of a fragmented intein comprising:
  • flanking sequence a is an independently designed combination of 2 to 3 amino acids, which is denoted as the flanking sequence a1 to an
  • flanking sequence b is an independently designed combination of 2 to 3 amino acids, which is denoted as the flanking sequence b1 to bn
  • the amino acid is any amino acid selected from G, A, V, L, M, I, S, T, P, N, Q, F, Y, W, K, R, H, D, E, C;
  • flanking sequences a1 to an and b1-bn designed in 2) to design the expression sequences of components A1 to An and components B1 to Bn that contain the sequence of protein P split;
  • flanking sequence pair selected in 5 Analyze the flanking sequence pair selected in 5), and eliminate the flanking sequence that can cause free sulfhydryl groups after splicing in the flanking sequence to optimize the flanking sequence pair selected in 5);
  • n 2 or 3
  • m is a positive integer
  • flanking sequence pair 1 ⁇ m design the expression sequences of component A’1 ⁇ A’m and component B’1 ⁇ B’m;
  • a method for preparing a recombinant polypeptide characterized in that the flanking sequence pair for fragmented intein described in 1 or 2 is used for trans-splicing.
  • flanking sequence pair for the fragmented intein as described in 1 or 2 above, characterized in that it is used for preparing a recombinant polypeptide, preferably for trans-splicing together with the fragmented intein.
  • flanking sequence for the fragmented intein of the present invention to mediate splicing of recombinant polypeptides (such as bispecific antibodies), including (1) no free sulfhydryl groups; (2) high-throughput and high-efficiency preparation ; (3) The target product and impurities are easy to distinguish and identify.
  • non-specific number of entities shall refer to one or more (species) of such entities; for example, "bispecific antibodies” shall be understood to mean one or more (species) of bispecific antibodies.
  • bispecific antibodies shall be understood to mean one or more (species) of bispecific antibodies.
  • the terms "one or more” and “at least one” that are not explicitly quantitatively limited are used interchangeably herein.
  • polypeptide as used herein includes a singular "polypeptide” and a plural “polypeptide”, and also refers to a molecule composed of monomers (amino acids) linearly connected by amide bonds (also called peptide bonds).
  • Polypeptides can be derived from natural biological sources or produced by recombinant technology, not necessarily translated from a specified nucleic acid sequence, and can be produced in any way, including chemical synthesis.
  • the term "recombinant” refers to a form of polypeptide or polynucleotide that does not exist in the natural state when referring to a polypeptide or polynucleotide. Combine acids or peptides to achieve this.
  • “Homology” or “identity” or “similarity” refers to the degree of sequence similarity between two peptide chain molecules or between two nucleic acid molecules. When there is the same base or amino acid at a position in the sequence being compared, the molecules at that position are homologous. The degree of homology between multiple sequences is a function of the number of pairs or homologous sites shared by these sequences.
  • the “irrelevant” or “non-homologous” sequence has less than 40% homology with one of the sequences of the invention, but preferably less than 25% homology.
  • a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) "sequence identity" means that the percentage of bases (or amino acids) is the same when the two sequences are compared during the alignment.
  • a biologically equivalent polynucleotide is a polynucleotide that has the above-mentioned specific percentage of homology and encodes the same or similar biologically active polypeptide.
  • fragment intein refers to two parts consisting of the N-terminal protein splicing region or N-terminal fragment (In, N'fragment of intein) and the C-terminal protein splicing region or C-terminal fragment (Ic, C fragment of intein) It is composed of a split intein, the gene expressing the precursor protein is split into two open reading frames, and the break site is inside the intein sequence.
  • N-terminal precursor protein refers to the fusion protein formed by translation of the gene of the N-terminal exon (En) and the fragmented N-terminal fragment (In) of the intein.
  • C-terminal precursor protein refers to a fusion protein produced after translation of a fragmented C-terminal fragment of intein (Ic) and C-terminal exonin (Ec) expression gene to form a fusion gene.
  • the N-terminal fragment (In) or C-terminal fragment (Ic) of the split-type intein alone does not have the function of protein splicing.
  • the In in the N-terminal precursor protein and the Ic of the C-terminal precursor protein bind to each other through non-covalent bonds to form a functional intein, which can catalyze the protein trans-splicing reaction, thereby using peptides. Bonding two separate protein exons (N-terminal protein exon or N-terminal exon is called En, C-terminal protein exon or C-terminal exon is called Ec) (Ozawa.T.Nat Biotechbol .21 (2003) 28793).
  • Protein trans-splicing refers to a protein splicing reaction mediated by a fragmented intein.
  • the N-terminal fragment (In) and the C-terminal fragment (Ic) of the fragmented intein recognize each other and bind with non-covalent bonds. Once combined, its structure is folded correctly. At this time, the broken intein has a reconstructed active center. Then, the protein splicing reaction is completed according to the typical protein splicing pathway, thereby connecting the exopeptides on both sides.
  • N-terminal part of a single split-type intein refers to the N-terminal part of a single split-type intein, and is also referred to herein as the N-terminal fragment of the split-type intein or the N-terminal protein splicing region.
  • Ic refers to the C-terminal part of a single split-type intein, and is also referred to herein as the C-terminal fragment of the split-type intein or the C-terminal protein splicing region.
  • the flanking sequence a is an amino acid sequence flanking the N-terminus of In and flanking the C-terminus of En, connecting In and En.
  • the first amino acid immediately adjacent to the N-terminus of In is defined as position -1
  • the second amino acid residue to the N-terminus is position-2
  • the third amino acid residue is the-th position. 3 digits, and so on until En.
  • the core sequence of the flanking sequence a is position -1 and position -2, which are directly related to splicing efficiency.
  • the flanking sequence b is an amino acid sequence flanking the C-terminus of Ic and flanking the N-terminus of Ec, connecting Ic and Ec.
  • the first amino acid residue immediately adjacent to the C-terminus of Ic is defined as position +1
  • the second amino acid residue to the C-terminus is position +2
  • the third amino acid residue is Position +3, and so on until Ec.
  • the core sequence of the flanking sequence b is the +1 and +2 positions, which are directly related to the splicing efficiency.
  • Fragmented intein-mediated trans-splicing for example, as shown in Figure 5, In is separated from the flanking sequence a, Ic is separated from the flanking sequence b, and the flanking sequence a and the flanking sequence b are connected. En and Ec are connected so that the amino acid residue at position -1 of the flanking sequence a is directly connected to the amino acid residue at position +1 of the flanking sequence b, and the amino acid at position -1 is located at the N-terminus of the amino acid at position +1.
  • the present invention uses 20 common amino acids for flanking sequence screening (hereinafter referred to as 20 amino acids), referring to: glycine (G), alanine (A), valine (V), leucine (L), methionine Acid (M), Isoleucine (I), Serine (S), Threonine (T), Proline (P), Asparagine (N), Glutamine (Q), Phenylalanine Acid (F), Tyrosine (Y), Tryptophan (W), Lysine (K), Arginine (R), Histidine (H), Aspartic acid (D), Glutamine Acid (E) and Cysteine (C).
  • G glycine
  • A alanine
  • V valine
  • L leucine
  • M methionine Acid
  • Serine Serine
  • T Threonine
  • P Proline
  • Asparagine N
  • Glutamine Phenylalanine Acid
  • F Phenylalanine Acid
  • antibody or “antigen-binding polypeptide” refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen or an immunogenic epitope.
  • the antibody can be a whole antibody or any antigen-binding fragment or single chain thereof.
  • the term "antibody” therefore includes any protein or peptide containing a specific molecule containing at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen or immunogenic epitope. Examples of this situation include, but are not limited to, the complementarity determining region (CDR) of the heavy chain or light chain or its ligand binding portion, the variable region of the heavy chain or light chain, the constant region of the heavy chain or light chain, and the framework (FR) Region or any part thereof, or at least part of a binding protein.
  • CDR complementarity determining region
  • FR framework
  • antibody fragment or "antigen-binding fragment” as used herein is a part of an antibody.
  • antibody fragment includes aptamers, aptamer enantiomers (aptamer enantiomers (spiegelmers) and diabodies, as well as any synthetic Or genetically modified proteins, which, like antibodies, can bind to specific antigens or immunogenic epitopes to form complexes.
  • Single chain variable fragment or “scFv” refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin.
  • antibody includes a wide variety of polypeptides that can be biochemically recognized. Those skilled in the art should understand that heavy chains are classified into ⁇ , ⁇ , ⁇ , ⁇ , ⁇ and have some subclasses (for example, ⁇ 1-4). The nature of this chain determines the "class" of the antibody, such as IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and functionally specific. Those skilled in the art can easily identify each modified form of these classes and isotypes with reference to this application, and therefore, these forms are within the scope of this application.
  • a standard immunoglobulin molecule contains two identical light chain polypeptides (molecular weight approximately 23,000 Daltons) linked together in a "Y" type via disulfide bonds, and two identical heavy chain polypeptides (molecular weight approximately 53,000-70,000 Daltons).
  • the antibodies, antigen-binding polypeptides, and their variants or derivatives of the present application include but are not limited to: polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized ( primatized) antibody, or chimeric antibody, single chain antibody, epitope binding fragment, for example, Fab, Fab' and F(ab') 2 , Fd, Fvs, single chain Fvs (scFv), single chain antibody, disulfide Bonded Fvs (sdFv), fragments containing VL domains or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies.
  • polyclonal antibodies monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized ( primatized) antibody, or chimeric antibody, single chain antibody, epitope binding fragment, for example, Fab, Fab' and F(ab') 2 , Fd, Fvs, single chain Fv
  • the immunoglobulin molecules or antibody molecules of the present application can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any type of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 ) Or subcategory.
  • immunoglobulins derived from camelid species or engineered based on camelid immunoglobulins may consist of only heavy chains without light chains. See, for example, Hamers-Casterman et al., Nature. 363:446-448 (1993).
  • Both the light chain and the heavy chain are divided into structural regions and functionally homologous regions.
  • the terms "constant” and “variable” are used functionally.
  • the light chain variable domain (VL) and the heavy chain variable domain (VH) simultaneously determine antigen recognition and specificity.
  • the number of constant region domains increases with distance from the antigen-binding site or amino-terminal end position of the antibody.
  • the N-terminal part is the variable region
  • the C-terminal part is the constant region; the CH3 and CL domains actually contain the carboxyl ends of the heavy chain and light chain, respectively.
  • the antigen binding site refers to: for any given heavy chain or light chain variable region, those skilled in the art can easily identify the amino acids including the CDR and framework regions, respectively, because they have been clearly defined (see, “Sequences of Proteins of Immunological Interest, "Kabat, E., etc., U.S. Department of Health and Human Services (USDepartment of Health and Human Services,), (1983); Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987), which is hereby incorporated by reference in its entirety).
  • CDR complementarity determining region
  • Kabat number used in this article refers to the numbering system described by Kabat et al., and its content is recorded in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983).
  • heavy chain constant region includes amino acid sequences derived from immunoglobulin heavy chains.
  • a polypeptide comprising a heavy chain constant region includes at least one of the following: CH1 domain, hinge (for example, upper hinge region, middle hinge region, and/or lower hinge region) domain, CH2 domain, CH3 domain, or a variant thereof Body or fragment.
  • the antigen-binding polypeptide used in the present application may include a polypeptide chain having a CH1 domain; a polypeptide having a CH1 domain, at least a part of a hinge domain, and a CH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; A polypeptide chain having a CH1 domain, at least a part of a hinge domain, and a CH3 domain, or a polypeptide chain having a CH1 domain, at least a part of a hinge structure, a CH2 domain, and a CH3 domain.
  • the polypeptide of the present application includes a polypeptide chain having a CH3 domain.
  • the antibodies used in this application may lack at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain).
  • the heavy chain constant regions may be modified so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.
  • the heavy chain constant regions of the antibodies disclosed herein can be derived from different immunoglobulin molecules.
  • the heavy chain constant region of a polypeptide may include the CH1 domain from an IgG1 molecule and the hinge region from an IgG3 molecule.
  • the heavy chain constant region may include a hinge region that is partly derived from an IgG1 molecule and partly derived from an IgG3 molecule.
  • the heavy chain portion may comprise a chimeric hinge that is partly derived from an IgG1 molecule and partly derived from an IgG4 molecule.
  • light chain constant region includes the amino acid sequence from the light chain of an antibody.
  • the light chain constant region includes at least one of a constant kappa domain and a constant lambda domain.
  • VH domain includes the amino terminal variable domain of an immunoglobulin heavy chain
  • CH1 domain includes the first (mostly amino terminal) constant region of an immunoglobulin heavy chain.
  • the CH1 domain is adjacent to the VH domain and is the amino terminus of the hinge region of an immunoglobulin heavy chain molecule.
  • CH2 domain includes a portion of a heavy chain molecule that ranges, for example, from about residue 244 to residue 360 of an antibody, using a conventional numbering scheme (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest” (1983).
  • the CH2 domain is unique because it does not pair closely with another domain. On the contrary. , Two N-linked branched sugar chains are inserted between the two CH2 domains of the intact natural IgG molecule. According to the literature, the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains About 108 residues.
  • telomere binding usually means that when an antibody binds to the epitope, the binding via the antigen-binding domain is compared to binding to a random, unrelated epitope Easier.
  • specificity is used herein to determine the affinity of a certain antibody to bind to a specific epitope.
  • treat refers to therapeutic treatment and preventive or preventive measures, in which the subject is prevented or slowed down (reduced) undesirable physiological changes or diseases, such as cancer development of.
  • beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the degree of disease, stabilizing (such as not worsening) the disease state, delaying or slowing the development of the disease, improving or alleviating the disease state, and alleviating (whether it is Part or all), regardless of whether it can be detected.
  • Treatment can also mean prolonging survival compared to expected survival when not receiving treatment.
  • any of the aforementioned antibodies or polypeptides may also include additional polypeptides, for example, the encoded polypeptide as described herein, a signal peptide at the N-terminus of the antibody for directing secretion, or other heterologous polypeptides as described herein.
  • polypeptides of the present application may contain conservative amino acid substitutions.
  • Constant amino acid substitution refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain.
  • a family of amino acid residues with similar side chains has been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid) ), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine , Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chains (for example, threonine, valine, isoleucine) Acid) and aromatic side chains (e.g.
  • non-essential amino acid residues of immunoglobulin polypeptides are preferably replaced by other amino acid residues from the same side chain family.
  • a string of amino acids can be replaced by a structurally similar string of amino acids, the latter being different in order and/or in the composition of the side chain family.
  • Transient transfection is one of the ways to introduce DNA into eukaryotic cells.
  • recombinant DNA is introduced into a highly infectious cell line to obtain temporary but high-level expression of the target gene.
  • the transfected DNA does not have to be integrated into the host chromosome, the transfected cells can be harvested in a shorter time than stable transfection, and the target product in the expression supernatant can be detected.
  • Figure 1 is a schematic diagram (A) of the splicing of homologous polypeptide fragments mediated by a fragmented intein and a schematic diagram of the primary protein structure of each component (B).
  • Figure 2 is a schematic diagram (A) of the splicing of heterologous polypeptide fragments mediated by a fragmented intein and a schematic diagram of the primary protein structure of each component (B).
  • Fig. 3 is a schematic diagram of in vitro splicing of antibodies mediated by a fragmented intein (A) and a schematic diagram of the primary protein structure of each component (B).
  • the splicing product is a bispecific antibody.
  • C is an exemplary schematic diagram of the amino acid sequence near the antibody splicing site mediated by the fragmented intein. "X" indicates that the amino acid at this position is any amino acid or is missing.
  • Figure 4 is a schematic diagram of the construction of the component A expression plasmid of the bispecific antibody (A) and the schematic diagram of the construction of the component B expression plasmid (B).
  • Figure 5 is a schematic diagram of flanking sequence numbers.
  • Figure 6 shows the reduced SDS-PAGE and Coomassie brilliant blue staining detection results of the expression supernatant of 293E cells co-transfected with expression plasmids corresponding to different intein and different flanking sequences after proteinA affinity purification.
  • (A) ⁇ (E) are the cell supernatants of fraction A and fraction B co-transfected with different intein peptides based on different flanking sequences, and the detection results after purification.
  • Fig. 7 shows the non-reducing SDS-PAGE and Coomassie brilliant blue staining detection results of the purified products of component A and component B'with different intein peptides respectively expressed by 293E cells.
  • A Detection results of purified products of Fab5, Fab9 and Fab11
  • B Detection results of purified products of HAb5, HAb9 and HAb11.
  • Figure 8 shows the non-reducing SDS-PAGE and Coomassie brilliant blue staining detection of the splicing products of component A and component B'of different intein, in which (A) the intein is IMPDH-1, and the flanking sequence a is GGG , The flanking sequence b is SI; (B) the intein is PhoRadA, the flanking sequence a is GK, and the flanking sequence b is THT.
  • Splicing product 1 means adding DTT before mixing components A and B
  • splicing product 2 means adding DTT after mixing components A and B'
  • reducing means adding DTT
  • non-reducing means not adding DTT
  • not splicing Indicates that the mixed components A and B'do not add DTT.
  • C The intein is PhoRadA, the flanking sequence a is GK, the flanking sequence b is THT, "splicing 1" and "no splicing 1" are component A and component B.
  • the concentrations are 5 ⁇ M and 4 ⁇ M, respectively, and the reaction system Contains 2mM DTT, "spliced 2" and “no spliced 2" are component A and component B'concentrations are 10uM and 1uM, respectively, and the reaction system contains 2mM DTT, "spliced 3" and “no spliced 3” are components
  • concentrations of A and component B' are 5uM and 1uM respectively, and the reaction system contains 2mM DTT, in which "splice 1" to "splice 3" are incubated overnight at 37°C, and "no splice 1" to "no splice 3" are all Incubate overnight at 4°C; the control band is Fab11 (non-reduced) for component A, HAb11 (non-reduced) for component B', and monoclonal antibody.
  • Figure 9 shows the results of the double antigen sandwich ELISA detection of the spliced product with the intein being IMPDH-1, the flanking sequence a being GGG, and the flanking sequence b being SI.
  • the coating antigen is CD38
  • the detection antigen is horseradish peroxidase (HRP) labeled PD-L1.
  • Fig. 10 is a base peak ion (BPI) map after enzymatic hydrolysis of Fab5+HAb5 (splicing product 1).
  • BPI base peak ion map after enzymatic hydrolysis of Fab5+HAb5 (splicing product 1).
  • A BPI pattern of Fab5+HAb5 (spliced product 1) after digestion with trypsin;
  • B BPI pattern of Fab5+HAb5 (spliced product 1) after chymotrypsin digestion;
  • C Fab5+HAb5 (Spliced product 1) BPI map after digestion with Glu-C enzyme.
  • Figure 11 shows the intein peptide PhoRadA and IMPDH-1 applied to the co-transfection expression and affinity purification of component A and component B of human IgG2, IgG3 or IgG4 subtype by SDS-PAGE and Coomassie brilliant blue staining detection.
  • the present invention relates to a preparation method for obtaining bispecific antibodies, which includes: splitting the corresponding DNA sequence of the target antibody, constructing a mammalian cell expression vector through total gene synthesis, purifying the vector, and transiently transfecting or transfecting the purified vector respectively.
  • Stable transfection of mammalian cells such as HEK293 or CHO. Collect the fermentation broth separately, and purify component A by proteinA, proteinL, nickel column, Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography or cross-linked starch affinity chromatography, etc.
  • component B the purified component A and component B are subjected to in vitro trans-splicing, and the spliced product is subjected to affinity chromatography corresponding to the tag protein such as a nickel column to obtain a high-purity bispecific antibody.
  • the process flow is as follows Shown in Figure 3A.
  • the antibodies described herein can be from any animal source, including birds and mammals.
  • the antibody is a human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibody.
  • the variable region may be derived from a condricthoid (e.g., from a shark).
  • the antibody may be combined with: therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • the antibody may be linked or fused to a therapeutic agent, which may include a detectable label, such as a radiolabel, immunomodulator, hormone, enzyme, oligonucleotide, photoactive therapeutic or diagnostic agent, cytotoxicity Agents, which can be: drugs or toxins, ultrasound enhancers, non-radioactive markers, combinations thereof, and other such components known in the art.
  • a detectable label such as a radiolabel, immunomodulator, hormone, enzyme, oligonucleotide, photoactive therapeutic or diagnostic agent, cytotoxicity Agents, which can be: drugs or toxins, ultrasound enhancers, non-radioactive markers, combinations thereof, and other such components known in the art.
  • the antibody By coupling it to a chemiluminescent compound, the antibody is detectably labeled. Then, the presence of the antigen-binding polypeptide labeled with the chemiluminescent substance is determined by detecting the luminescence produced during the chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • the antibody can also be detectably labeled with fluorescent light-emitting metals such as 152Eu, or other lanthanide labels. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPN) or ethylenediaminetetraacetic acid (EDTA).
  • DTPN diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the binding specificity of the antigen-binding polypeptide of the present application can be measured by in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).
  • in vitro experiments such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).
  • the cell line used to produce the recombinant polypeptide can be selected and cultured using techniques well known to those skilled in the art.
  • the variants 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, encoding 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 Replacement, less than 5 amino acid replacements, less than 4 amino acid replacements, less than 3 amino acid replacements, or less than 2 amino acid replacements.
  • mutations can be randomly introduced along all or part of the coding sequence, for example, by saturation mutagenesis, the resulting mutants can be screened for biological activity to determine mutations that retain activity.
  • the tag protein used in the present invention can be Fc, oligohistidine (His-tag), Strep-tag, Flag, HA, or maltose binding protein (MBP).
  • the transfection used in the present invention can be transient transfection or stable transfection.
  • Mammalian cells such as HEK293 or CHO are used in the present invention, but are not limited thereto.
  • Liquids containing expression products from mammalian cells can be used proteinA, proteinG, nickel column, Strep-Tactin affinity chromatography, anti-Flag antibody affinity chromatography, anti-HA antibody affinity chromatography Or cross-linked starch affinity chromatography and other methods for purification.
  • the spliced product can be subjected to affinity chromatography corresponding to the tag protein to remove unspliced components.
  • the gene fragment used for constructing the vector of the present invention can be constructed by whole gene synthesis, but is not limited to this.
  • the vector used in the present invention is pcDNA3.1 or pCHO1.0, but it is not limited thereto.
  • restriction endonuclease used in the present invention may include, for example, NotI, NruI, or BamHI-HF, but it is not limited thereto.
  • BLAST is a comparison program that uses default parameters. Specifically, the programs are BLASTN and BLASTP. Detailed information on these procedures can be obtained at the following Internet address: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi .
  • the component A expression plasmid (pPa-FSa-In-Tag) and the component B expression plasmid (pTag-Ic-FSb-Pb) can be constructed.
  • the Pa-HIn and Pa-L can be constructed into the same plasmid by molecular cloning methods such as restriction digestion and enzyme ligation, that is, component A Expression plasmid (pBi-Pa-FSa-In-Tag); or construct pB'-L, pB'-H and pB'-FcIc into the same plasmid, namely component B'expression plasmid (pBi-Tag-Ic- FSb-Rb).
  • component A Expression plasmid pBi-Pa-FSa-In-Tag
  • pB'-FcIc pB'-L, pB'-H and pB'-FcIc into the same plasmid, namely component B'expression plasmid (pBi-Tag-Ic- FSb-Rb).
  • the component B expression plasmid may include three types of expression plasmids pB-L, pB-H, and pB-FcIc.
  • Pa is also used to indicate the N-terminal protein exon or N-terminal exon of protein P, which is also expressed as Enp;
  • Pb is also used to indicate the C-terminal protein exon or C-terminal exon of protein P.
  • Peptide also expressed as Ecp.
  • Ra is also used to indicate the N-terminal protein exon or N-terminal exon of protein R, also expressed as En R ;
  • Rb is also used to indicate the C-terminal protein exon or C-terminal exon of protein R, and also indicates It is Ec R.
  • serial number serial number Flanking sequence a amino acid sequence 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 20 amino acids (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, C) any amino acid.
  • the DNA sequences in the examples of the present invention are all obtained by reverse translation based on amino acid sequences and synthesized by Wuhan Jinkairui.
  • the recombinant polypeptides involved in the examples were prepared by the following method: under the action of recombinase, the DNA sequence was ligated with the vector pcDNA3.1 digested with restriction enzyme EcoRI at 37°C for 30 minutes, and then Trans10 competent cells were transformed by heat shock method. After verification by sequencing (Wuhan Jinkairui Company), 293E cells (purchased from Thermo Fisher Company) were transiently transfected. Purify after expression.
  • the plasmids Pa-HIn and Pa-L need to be co-transfected into 293E cells for expression, or single transfected plasmid pBi-Pa-FSa-In-Tag for expression; expression Figure 3
  • the indicated component B' requires the plasmids pB'-L, pB'-H and pB'-FcIc to be co-transfected into 293E cells for expression, or a single transfection plasmid pBi-Tag-Ic-FSb-Rb for expression.
  • the ratio of the number of moles of the two plasmids can be 1:1, or any other ratio. If the three plasmids are co-transfected and expressed, the ratio of the number of moles of the three plasmids can be 1:1:1, or any other ratio.
  • tag protein is Fc
  • affinity chromatography is used, and MabSelect SuRe (GE, article number 17-5438-01), 18ml column is used.
  • tag protein is Strep-tag, Flag, HA or MBP, etc.
  • Strep-Tactin affinity chromatography anti-Flag antibody affinity chromatography
  • anti-HA antibody affinity chromatography anti-HA antibody affinity chromatography
  • cross-linked starch affinity chromatography respectively Corresponding fillers and buffers are sufficient for chromatography.
  • Ion exchange chromatography when component A (A') or component B (B') does not have a tagged protein, ion exchange chromatography can be used to separate spliced products according to the difference in isoelectric point, and the layer used
  • the analytical packing can be a cation exchange chromatography packing or an anion exchange chromatography packing, such as Hitrap SP-HP (GE Company).
  • hydrophobic chromatography when component A (A') or component B (B') does not have a tagged protein, hydrophobic chromatography can be used to separate the spliced products according to the difference in hydrophobicity.
  • the chromatography packing used is such as Capto phenyl ImpRes filler (GE Company).
  • molecular sieve chromatography when component A (A') or component B (B') does not have tagged protein, molecular sieve chromatography can be used to separate the spliced products according to the difference in molecular weight.
  • the chromatography packing used is HiLoad Superdex 200pg (GE Company).
  • pA-HIn(20) ⁇ pA-HIn(21) corresponding to A-Fab20 and A-Fab21, and pTag-Ic-FSb corresponding to plasmid B-FcIc20 and B-FcIc21 were constructed -(B-FcIc20), pTag-Ic-FSb-(B-FcIc21).
  • plasmids pA-HIn(30) ⁇ pA-HIn(31) corresponding to A-Fab30 and A-Fab31, and plasmids pTag-Ic-FSb- corresponding to B-FcIc30 and B-FcIc31 were constructed.
  • plasmids pA-HIn(40) ⁇ pA-HIn(41) corresponding to A-Fab40 and A-Fab41, and plasmids pTag-Ic-FSb- corresponding to B-FcIc40 and B-FcIc41 were constructed.
  • B-FcIc40 pTag-Ic-FSb-(B-FcIc41).
  • plasmids pA-HIn(50) ⁇ pA-HIn(51) corresponding to A-Fab50 and A-Fab51 were constructed.
  • B-FcIc50 pTag-Ic-FSb-(B-FcIc51).
  • plasmids pA-HIn(60) ⁇ pA-HIn(61) corresponding to A-Fab60 and A-Fab61, and plasmids pTag-Ic-FSb- corresponding to B-FcIc60 and B-FcIc61 were constructed.
  • plasmids pA-HIn(70) ⁇ pA-HIn(71) corresponding to A-Fab70 and A-Fab71, and plasmids pTag-Ic- corresponding to B-FcIc70 and B-FcIc71 were constructed.
  • plasmids pA-HIn(80) ⁇ pA-HIn(81) corresponding to A-Fab80 and A-Fab81, and plasmids pTag-Ic- corresponding to B-FcIc80 and B-FcIc81 were constructed.
  • plasmids pA-HIn(90) ⁇ pA-HIn(92) corresponding to A-Fab90, A-Fab91, and A-Fab92, and plasmids corresponding to B-FcIc90 ⁇ B-FcIc92 were constructed pTag-Ic-FSb-(B-FcIc90) ⁇ pTag-Ic-FSb-(B-FcIc92).
  • plasmids pA-HIn(100) ⁇ pA-HIn(101) corresponding to A-Fab100 and A-Fab101, and plasmids pTag-Ic-FSb- corresponding to B-FcIc100 and B-FcIc101 were constructed.
  • the plasmids expressing component A used in this example include: pA-HIn(20) ⁇ (21), (30) ⁇ (31), (40) ⁇ (41), (50) ⁇ (51), ( 60) to (61), (70) to (71), (80) to (81), (90) to (91), (100) to (101), and pA-L.
  • the plasmids expressing component B used in this example include: pTag-Ic-FSb-(B-FcIc20-21), (30)-(31), (40)-(41), (50)-(51) ), (60) to (61), (70) to (71), (80) to (81), (90) to (91), (100) to (101).
  • Transfection was performed according to the pairing in Table 34.
  • the transfected cells were cultured for 5 days and the supernatant was taken. Protein A affinity chromatography was performed on the protein in the supernatant. After protein A affinity chromatography, the protein in the supernatant was detected by Coomassie brilliant blue staining by SDS-PAGE (with reducing agent). The results are shown in FIGS. 6A to 6D. According to the results, it can be seen that significant splicing occurred in groups A22, A27, A31, A45, A49, A52, A53, A55, and A56.
  • 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
  • flanking sequence a is GGG, flanking sequence b is SI; or flanking sequence a is DKG, flanking The sequence b is ST; or the flanking sequence a is DKG, and the flanking sequence b is SI.
  • flanking sequence pairs with excellent splicing efficiency are: when flanking sequence a is NR, flanking sequence b is SAV; or when flanking sequence a is DK, flanking sequence b is SAV.
  • flanking sequence a is SG
  • flanking sequence b is SIE
  • flanking sequence pairs with excellent splicing efficiency are: when flanking sequence a is TY, flanking sequence b is TIH; or when flanking sequence a is TY, flanking sequence b is THT.
  • flanking sequence a is GK
  • flanking sequence b is TQL or THT
  • flanking sequence a is DK
  • flanking sequence b is TQL
  • pcDNA3.1 was used to construct the intein SspDnaB, MxeGyrA, MjaTFIIB, PhoVMA, TvoVMA, Gp41-1, Gp41-8, IMPDH according to the composition shown in Tables 31 and 33, respectively.
  • -1 The expression plasmid of PhoRadA components.
  • the above-mentioned component expression plasmids are divided into B'-L expression plasmid (pB'-L), B'-H expression plasmid (pB'-H) and B'-FcIc expression plasmid (pB '-FcIc) three kinds.
  • the pB'-L and B'-H expression plasmids are common among the components B'.
  • plasmids pB'-FcIc(20) to B'-FcIc(21) corresponding to B'-HAb20 to B'-HAb21 were constructed.
  • plasmids pB'-FcIc(30) to B'-FcIc(31) corresponding to B'-HAb30 to B'-HAb31 were constructed.
  • plasmids pB'-FcIc(40) to B'-FcIc(41) corresponding to B'-HAb40 to B'-HAb41 were constructed.
  • plasmids pB'-FcIc(50) to B'-FcIc(51) corresponding to B'-HAb50 to B'-HAb51 were constructed.
  • plasmids pB'-FcIc(60) to B'-FcIc(61) corresponding to B'-HAb60 to B'-HAb61 were constructed.
  • plasmids pB'-FcIc(70) to B'-FcIc(71) corresponding to B'-HAb70 to B'-HAb71 were constructed.
  • plasmids pB'-FcIc(80) to B'-FcIc(81) corresponding to B'-HAb80 to B'-HAb81 were constructed.
  • plasmids pB'-FcIc(90) to B'-FcIc(92) corresponding to B'-HAb90 to B'-HAb92 were constructed.
  • plasmids pB'-FcIc(100) to B'-FcIc(101) corresponding to B'-HAb100 to B'-HAb101 were constructed.
  • the plasmids expressing component A used in this example include: pA-HIn(90), pA-HIn(80), pA-HIn(81), pA-HIn(61), pA-HIn(20), pA -HIn(40), pA-HIn(100) and pA-L.
  • the plasmids expressing component B'used in this example include: pB'-FcIc(90), pB'-FcIc(80), pB'-FcIc(61), pB'-FcIc(20), pB'- FcIc (41), pB'-FcIc (101) and pB'-L, pB'-H.
  • the supernatant was purified by nickel column chromatography (Jiangsu Qianchun, Item No.: A41002-06) to obtain a purified polypeptide fragment of component A.
  • Plasmid pB'-L, plasmid pB'-H and each plasmid pB'-FcIc were co-transfected into 293E cells and cultured at 37°C.
  • the obtained polypeptide fragments of component A and component B' are referred to as Fab5 to Fab11 and HAb5 to HAb11, respectively.
  • E1, E2, E3 represent the elution components with different concentrations of imidazole in the process of nickel column chromatography. It can be seen from Figure 7A that both Fab5 and Fab11 have obtained higher expression levels. In addition, in the Fab5 and Fab11 groups, the peptides with higher purity can be obtained by using nickel column chromatography to purify the peptides. It can be seen from Fig. 7B that HAb5, HAb9 and HAb11 all have higher expression levels, and HAb5, HAb9 and HAb11 can obtain higher purity polypeptides by nickel column chromatography.
  • the purified polypeptide fragments Fab5, Fab11, HAb5 and HAb11 of component A and component B' were respectively dialyzed into a buffer solution at 4°C with a 3kD dialysis bag (purchased from Sigma).
  • the protein concentration of the component 1 ⁇ 10 micromolar.
  • the buffer solution includes: 10-50mM Tris/HCl (pH7.0-8.0), 100-500mM NaCl, and 0-0.5mM EDTA.
  • component A and component B'from the same source of intein were mixed according to the corresponding serial numbers (for example, Fab5 and HAb5, etc.) at a molar ratio of 1:5 to 5:1, and DTT was added to 0.5 to 5mM. Incubate overnight at 37°C.
  • splice 1" and “no splicing 1" are components A and B'at concentrations of 5 ⁇ M and 4 ⁇ M, and the reaction system contains 2 mM DTT, and "splice 2" and “no splicing 2" are components The concentrations of A and B'are 10 ⁇ M and 1 ⁇ M, and the reaction system contains 2mM DTT, “spliced 3" and “no splicing 3" are components A and B'at 5 ⁇ M and 1 ⁇ M, and the reaction system contains 2mM DTT.
  • control band is component A and Fab11 (non-reduced ), component B'is HAb11 (non-reduced), and monoclonal antibodies.
  • the two fragmented intein IMPDH-1 and PhoRadA with the novel flanking sequence pair of the present invention can be spliced efficiently and effectively in vitro, thereby obtaining in vitro spliced recombinant polypeptides of polypeptide fragments from different proteins.
  • the splicing products Fab5+HAb5 and Fab11+HAb11 were obtained respectively. These spliced products are the same as the monoclonal antibody control, and their band size is 150kD, which proves that the theoretical molecular weight of the product is consistent with the natural IgG monoclonal antibody.
  • Fab5+HAb5 the biological activity detection based on double antigen sandwich ELISA was carried out.
  • Antigen preparation For the protein PD-L1 and CD38, the construction was carried out by selecting only the extracellular domain, and an expression plasmid with a His tag was constructed. The vector used was pcDNA3.1.
  • 293E cells were used for transient transfection, and expression purification including nickel column purification and molecular sieve purification was performed. After purification, an antigen protein with a purity of not less than 95% detected by SDS-PAGE was obtained.
  • the PD-L1 protein was labeled with horseradish peroxidase (HRP).
  • the first antigen coating adjust the CD38 protein concentration to 2 ⁇ g/ml, use the liquid containing CD38 protein to coat the ELISA plate at 100 ⁇ l/well, overnight at 4°C; discard the supernatant, and add 250 ⁇ l blocking solution to each well (PBS containing 3% BSA);
  • Antibody addition According to the experimental design, operate at room temperature, and dilute the antibody in a gradient with 1% BSA in PBS. For example, the initial concentration of antibody dilution is 20 ⁇ g/ml, and it is diluted by 2 times and diluted in 5 concentration gradients. Add 200 ⁇ l of diluted antibody per well to the wells of the microtiter plate, incubate at room temperature for 2h, and then discard the supernatant;
  • Second antigen incubation Add the diluted second antigen (PD-L1 protein labeled with HRP), and use the second antigen after dilution 1:1000.
  • the diluent is 1% BSA in PBS with a volume of 100 ⁇ l/well, and incubate at room temperature for 1h;
  • Fab5+HAb5 (splicing 1) has the activity of simultaneously binding CD38 and PD-L1 antigens.
  • the results can prove that the Fab5+HAb5 (splicing 1) spliced product obtained by using the intein of the present invention and the novel flanking sequence pair contained therein has good bispecific antibody activity.
  • Peptide coverage refers to the ratio of the number of amino acids in the detected peptide to the total number of amino acids in the protein.
  • the detection of peptide coverage of protein test products is of great significance for the confirmation of the primary amino acid sequence of protein drugs, ensuring the formation of high-level structure of protein drugs and maintaining the properties of protein drugs.
  • the detection of protein peptide coverage is carried out by mass spectrometry in accordance with the requirements of drug declaration. The detection of peptide coverage can be completed quickly, accurately and efficiently.
  • the peptide coverage of the protein Fab5+HAb5 was analyzed. Trypsin, chymotrypsin, and Glu-C enzymes were used to enzymatically digest the protein Fab5+HAb5 (splicing product 1). Then use LC-MS/MS (XevoG2-XS QTof, waters) to analyze the digested peptide samples. UNIFI (1.8.2, Waters) software was used to analyze the LC-MS/MS data, and the peptide coverage of Fab5+HAb5 (splicing product 1) was determined according to the results of the algorithm.
  • Chymotrypsin Chymotrypsin, Sigma
  • Trypsin, chymotrypsin, and Glu-C enzymatic digestion Take appropriate amount of Fab5+HAb5 (splicing 1) and add trypsin, chymotrypsin, and Glu-C enzymes after appropriate pretreatment, and digest at 37°C for 20 hour.
  • Fab5+HAb5 (splicing product 1) is subjected to enzymatic hydrolysis and then separated by the ultra-high performance liquid system Acquity UPLC I-Class.
  • Liquid phase A is a 0.1% FA aqueous solution
  • B is a 0.1% FA acetonitrile solution.
  • Fab5+HAb5 (spliced product 1) was loaded onto the Column by an autosampler, and then separated by a chromatographic column. The column temperature was 55°C, the flow rate was 300 ⁇ l/min, and the wavelength of the TUV detector was 214nm.
  • the relevant liquid phase gradients are shown in Table 37.
  • Mass spectrometry identification Fab5+HAb5 (spliced product 1) was desalted and separated by ultra-high performance liquid chromatography and then analyzed by mass spectrometry with XevoG2-XS QTof mass spectrometer (Waters). Analysis time: 63min, detection method: positive ion, MS, scanning range (m/z): 300-2000.
  • the coverage rate after chymotrypsin hydrolysis is 100%
  • the digested samples were analyzed by LC-MS/MS and the library search results were integrated. Finally, the peptide coverage of the Fab5+HAb5 (splicing 1) was 100.00%. Based on the splicing principle of intein, according to the molecular weight of the splicing product obtained in the present invention, the double antigen sandwich ELISA and peptide map coverage test results, it can be inferred that the present invention obtains an effective and natural IgG-like bispecific antibody. The test results Confirm that the structure of the bispecific antibody is a heterodimer IgG structure composed of two different heavy chains and two different light chains, rather than a homodimer composed of two identical heavy chains and two light chains. A mixture of IgG structures.
  • transfection was carried out in the same manner as in Example 2.
  • the positive control monoclonal antibody was set up in the same way as above.
  • the transfected cells were cultured for 5 days and the supernatant was taken. Protein A affinity chromatography was performed on the protein in the supernatant. After protein A affinity chromatography, the protein in the supernatant was detected by Coomassie brilliant blue staining by SDS-PAGE (with reducing agent). The results are shown in Figure 11.
  • A102 is the intein PhoRadA applied to the intracellular expression of component A and component B of human IgG2 subtype, which can be spliced intracellularly to form a complete IgG2 monoclonal antibody
  • A103 is the intein PhoRadA applied to human IgG3 subtype The intracellular expression of component A and component B can be spliced intracellularly to form a complete IgG3 monoclonal antibody
  • A104 is the intein IMPDH-1 applied to the cells of component A and component B of human IgG4 subtype Intracellular expression, splicing can occur in the cell to form a complete IgG4 monoclonal antibody.
  • the green fluorescent protein is EGFP (source: UniProtKB-A0A076FL24), and its full-length amino acid sequence is SEQ ID No: 23, with a total of 239 amino acid residues.
  • the sequence is divided into component A and component B, among which (1) component A is the fusion of amino acids 1-158 of EGFP with intein, and the corresponding coding DNA is constructed into the eukaryotic expression vector pcDNA3.1.
  • the C-terminus adds flanking sequence a, intein N-terminus, and stop codon (TAA, TGA or TAG).
  • the constructed expression plasmid is named in Table 42; (2) Component B is EGFP amino acids 159-239 and contains The peptide fusion, the corresponding coding DNA was constructed into the eukaryotic expression vector pcDNA3.1, and the start codon ATG, the intein C-terminus and flanking sequence b were added to the N-terminus, and the termination codon (TAA, TGA or TAG), the constructed expression plasmid is named in Table 43
  • the EGFP full-length protein encoding DNA was constructed to pcDNA3.1 (with stop codon), and the plasmid was designated as pEGFP.
  • the plasmid pEGFP-A and pEGFP were transfected separately or co-transfected into 293 cells or CHO cells with a co-transfection ratio of 1:1. Refer to Example 1 for the transfection method.
  • pEGFP was separately transfected into 293 or CHO cells As a positive control. The concentration of each plasmid for single or co-transformation remains the same. After 48 hours of transfection, the green fluorescence expression of the cells was detected by flow cytometry. See Table 44 for specific statistics.
  • pGFP-N3 177 0 pGFP-C3 133 0 pGFP-N4+pGFP-C4 7 ⁇ 10 ⁇ 4 88% pGFP-N4 321 0.2% pGFP-C4 152 0 pGFP-N5+pGFP-C5 8 ⁇ 10 ⁇ 4 95% pGFP-N5 274 0.1% pGFP-C5 106 0 Blank control 139 0
  • the present invention provides a method for preparing recombinant polypeptides, especially bispecific antibodies, by using fragmented inteins with novel pairs of flanking sequences.
  • the fragmented intein with a novel pair of flanking sequences according to the present invention can be widely used in the preparation of recombinant polypeptides in the fields of medicine and bioengineering, especially the field of antibodies, especially the preparation of bispecific antibodies.
  • the bispecific antibody prepared by using the fragmented intein with novel flanking sequence pairs of the present invention has no unnatural domains and its structure is very similar to that of natural antibodies (IgA, IgD, IgE, IgG or IgM) , And has an Fc domain.
  • the bispecific antibody has good structural integrity and stability, and can retain or remove CDC (complement-dependent cytotoxicity) or ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent cellular phagocytosis) according to different IGG subtypes ) Or FcRn (Fc receptor) binding activity.
  • the bispecific antibody prepared by the method of the present invention has a long in vivo half-life and low immunogenicity; without introducing any form of connecting peptide, the stability of the antibody molecule is improved, and the immune response in the body is reduced.
  • the bispecific antibody prepared by the method of the present invention has the same glycosylation modification as the wild-type IgG, obtains better biological functions, is more stable, and has a long in vivo half-life; using the in vitro splicing method by intein, It can completely avoid the problems of heavy chain mismatch and light chain mismatch that are extremely easy to occur in traditional methods.
  • the method for preparing bispecific antibodies of the present invention can be used to produce humanized bispecific antibodies and bispecific antibodies with fully human sequences.
  • the sequence of such an antibody prepared by the method of the present invention is closer to that of a human antibody, and can effectively reduce the occurrence of immune response.
  • the method for preparing bispecific antibodies of the present invention can construct any bispecific antibody without being restricted by antibody subtypes (IgG, IgA, IgM, IgD, IgE, and light chain K and ⁇ types).

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Abstract

涉及了用于SspDnaE、SspDnaB、MxeGyrA、MjaTFIIB、PhoVMA、TvoVMA、Gp41-1、Gp41-8、IMPDH-1或PhoRadA断裂型内含肽的侧翼序列对。所述侧翼序列对包括：侧翼序列a和侧翼序列b；所述侧翼序列a位于断裂型内含肽N端蛋白质剪接区域(In)的N端，且介于N端外显肽(En)和In之间；所述侧翼序列b位于断裂型内含肽C端蛋白质剪接区域(Ic)的C端，且介于Ic和C端外显肽(Ec)之间。

Description

断裂型内含肽、使用其的重组多肽的制备方法 技术领域
本发明涉及含有新型侧翼序列对的断裂型内含肽、使用其的重组多肽，所述内含肽在制备抗体，特别是双特异性抗体中的应用。本发明还涉及所述含有新型侧翼序列对的断裂型内含肽的筛选方法。
背景技术
蛋白质反式剪接(Protein trans-splicing)是指由断裂型内含肽介导的蛋白质剪接反应。在这种类型的剪接过程中，首先是断裂型内含肽的N端片段或N端蛋白质剪接区域(In)和C端片段或C端蛋白质剪接区域(Ic)相互识别并以非共价键结合，一旦结合后正确折叠其结构，则重建活性中心的断裂型内含肽按照典型的蛋白质剪接途径完成蛋白质剪接反应，将两侧的外显肽连接(Saleh.L.，Chemical Record.6(2006)183-193)。
在制备重组蛋白的技术中，可以通过将表达前体蛋白质的基因***在两个开放阅读框中，利用包括N端蛋白质剪接区域(N’fragment of intein，简称In)和C端蛋白质剪接区域(C’fragment of intein，简称Ic)两部分的断裂型内含肽(split intein)催化蛋白质反式剪接反应进行，从而将构成所述前体蛋白质的两个分离的外显肽(En、Ec)以肽键连接，得到重组蛋白(Ozawa.T.，Nat Biotechbol.21(2003)287-93)。
双特异性抗体是指可以同时识别两个抗原或者两个表位的一个抗体分子，诸如能够结合两种以上抗原的双特异性或者多特异型抗体在本领域中是已知的，可以通过细胞融合法、化学修饰法、基因重组等方法，在真核表达***或者在原核表达***中获得。
目前，已经开发了广泛多样的重组双特异性抗体形式。例如，通过融合例如IgG抗体形式和单链结构域的四价双特异性抗体(参见例如Coloma，M.J.，等，Nature Biotech.15(1997)159-163；WO 2001077342；和Morrison，S.，L.，Nature Biotech.25(2007)1233-1234)。但这样的抗体由于与天然抗体结构相差大，进入体内之后会引起强烈的免疫反应以及较短的半衰期。
此外，也开发了能够结合两种以上抗原的若干其他新型形式，例如：小分子抗体如微型抗体(minibodies)、若干单链形式(scFv双-scFv)等。在这些小分子抗体中，抗体中心结构(IgA、IgD、IgE、IgG或IgM)不再保持(Holliger，P.，等，Nature Biotech.23(2005)1126-1136；Fischer，N，和Leger，O.，Pathobiology(病理学)74(2007)3-14；Shen，J.，等，J.Immunol.Methods.318(2007)65-74；Wu，C.，等.，Nature Biotech.25(2007)1290-1297)。
这种将抗体的核心结合区域通过连接肽(linker)与其它抗体核心结合区相连接的改造，虽然相对于双特异性抗体有明显优势，但是，在作为药物应用中也存在问题，大大限制了其成药。
实际上，就免疫原性而言，这些外源蛋白可能引起针对连接肽本身、或含有连接肽的蛋白质的免疫反应，甚至引起免疫风暴。此外，由于这些连接肽灵活的本质，使其倾向于发生蛋白质的降解，容易导致抗体的稳定性差，易于聚集，半衰期缩短以及使免疫原性进一步增强。例如，安进公司的博纳吐单抗(Blinatumomab)，在血液中的半衰期仅为1.25小时，导致需通过注射泵24小时持续给药，这大大限制了其应用(Bargou，R和Leo.E.，Science.321(2008)974-7)。
此外，希望在对双特异性抗体的改造中，能够保留抗体的Fc片段效应功能：例如，CDC(补体依赖的细胞毒性)、或ADCC(细胞毒作用)，并使抗体与血管内壁FcRn(Fc受体)结合的半衰期延长。而这些功能必须通过Fc区来介导，因此，需要在改造后的双特异性抗体中保留Fc区。
因此，需要开发结构与天然存在的抗体(如IgA、IgD、IgE、IgG、IgM)的结构极其相似的双特异性抗体，进一步，需要与人抗体序列具有最小差异的人源化双特异性抗体以及全人源的双特异性抗体。
目前，已经尝试了使用Npu-PCC73102 DnaE(简称NpuDnaE)内含肽的反式剪接机制来制备双特异性抗体。使用内含肽的反式剪接机制制备双特异性抗体，在剪接产物中不具有连接肽，但存在以下问题：在这样得到的双特异性抗体中，无法避免Ic侧翼序列所引入的自由巯基，导致该类双特异性抗体存在很大的错误折叠和不稳定风险，剪接效率也存在问题(Han L，Zong H，等，Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies，Methods，.Vol 154，2019 Feb 1；154：32-37)。
断裂型内含肽介导的蛋白质剪接效率，与所述内含肽的内含肽序列以及侧翼序列(flanking sequences)直接相关。
在NEB的数据库里(http：//inteins.com/)列举了超过600种断裂型内含肽，较为常用的例如：NpuDnaE及SspDnaE。但是，根据这些内含肽的侧翼序列，如NpuDnaE的In侧翼序列为AEY(En-AEY-In)，Ic侧翼序列为CFNGT(Ic-CFNGT-Ec)，SspDnaE的In侧翼序列为AEY(En-AEY-In)、Ic侧翼序列为CFNKS(Ic-CFNKS-Ec)可知，En-AEY-In和Ic-CFNGT-Ec在剪接后为En-AEYCFNGT-Ec的蛋白形式，En-AEY-In和Ic-CFNKS-Ec剪接后为En-AEYCFNKS-Ec的蛋白形式，均残留有一个半胱氨酸，从而在剪接产物中具有自由巯基，使产物的错误折叠和不稳定性风险大大增加。
为了避免在剪接产物中具有自由巯基，需要对现有的断裂型内含肽的侧翼序列对进行改良，需要这样的新型侧翼序列：其保持了内含肽的良好剪切效率，且不含有半胱氨酸残基的新型的侧翼序列对。
已有文献报道，一些断裂型内含肽，它们本身的Ic侧翼序列中就没有半胱氨酸而具有丝氨酸或苏氨酸，使用这些内含肽则可以让剪接产物的连接处不产生自由巯基。例如，SspDnaB、TvoVMA、MxeGyrA、PhoRadA、Gp41-1、Gp41-8、Nrdj-1、IMPDH-1等(Bareket Dassa，等，Nucleic Acids Res.2009 May；37(8)：2560-2573)。但对于使用这些内含肽进行双特异性抗体的制备，尚没有报道。
另外，对已有的断裂型内含肽的侧翼序列对进行氨基酸突变后，会对其进行反式剪接的效率造成影响，因此，需要一种筛选方法，来筛选含有新型侧翼序列对的内含肽，其反式剪接的效率优秀、且不在剪接产物中引入接口处的自由巯基。进一步，需要一种适用于制备抗体，特别是双特异性抗体的断裂型内含肽，其反式剪接的效率优秀、且不在剪切产物中的接口处引入游离巯基的含有新型侧翼序列对的断裂型内含肽。
发明内容
本发明通过发明人的努力研究，通过对已有的内含肽的侧翼序列对进行有规律的氨基酸突变，并筛选其中反式剪接的效率优秀的侧翼序列对，从而获得了一类具有新型侧翼序列对的断裂型内含肽，其具有无半胱氨酸残基的侧翼序列，且不在剪切产物中的接口处引入游离巯基，反式剪接的效率优秀，特别适用于制备 抗体(特别是双特异性抗体)。
使用本发明的断裂型内含肽，可以在相对温和的条件(如常温、生理盐浓度、中性pH等)下，以高剪切效率将来自不同蛋白的多肽片段剪接在一起，形成重组融合多肽蛋白。
此外，本发明人在基于对上述断裂型内含肽的筛选基础上，建立了一种利用断裂型内含肽制备重组多肽，特别是双特异性抗体的方法。根据本发明的制备双特异性抗体的方法制备的双特异性抗体，不存在非天然的结构域，其结构与天然抗体(IgA、IgD、IgE、IgG或IgM)的结构极其相似，并具有Fc结构域。所述双特异性抗体的结构完整稳定性好，可以根据不同的IgG亚型保留或去除CDC(补体依赖的细胞毒性)或者ADCC(抗体依赖的细胞毒作用)或者ADCP(抗体依赖的细胞吞噬作用)或者FcRn(Fc受体)结合活性。
利用本发明的方法制备的双特异性抗体具有以下优点：所述双特异性抗体的体内半衰期长，免疫原性低；不引入任何形式的连接肽(linker)，抗体分子稳定性提高，在体内的免疫反应降低。
利用本发明的方法制备的双特异性抗体可以利用哺乳动物细胞表达***制备，从而具有与野生型IgG一致的糖基化修饰，得到更好的生物学功能，并且更加稳定，体内半衰期长；利用由内含肽进行的体外剪接方法，可以完全避免传统方法中极易出现的重链错配、轻链错配的问题。
本发明的制备双特异性抗体的方法，还可以用于生产人源化的双特异性抗体，以及全人序列的双特异性抗体。利用本发明的方法制备的这样的抗体的序列与人源抗体更接近，可以有效降低免疫反应的发生。
本发明的制备双特异性抗体的方法是通用型双特异性抗体的构建方法，不受抗体亚型(IgG、IgA、IgM、IgD、IgE，以及轻链κ和λ型)的限制，不需要根据具体的靶点设计不同的突变，可以用于构建任何双特异性的抗体。
本发明提供以下技术方案。
1.用于断裂型内含肽的侧翼序列对，其中，
所述侧翼序列对包括：侧翼序列a和侧翼序列b；所述侧翼序列a位于断裂型内含肽N端蛋白质剪接区域(In)的N端，且介于N端外显肽(En)和In之间；所述侧翼序列b位于断裂型内含肽C端蛋白质剪接区域(Ic)的C端，且介于Ic和C端外显肽(Ec)之间；
所述断裂型内含肽选自：SspDnaE、SspDnaB、MxeGyrA、MjaTFIIB、PhoVMA、TvoVMA、Gp41-1、Gp41-8、IMPDH-1或PhoRadA，
(1)所述断裂型内含肽为IMPDH-1时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失，或优选为G或D；A -2为X或缺失，或优选为G或K；A -1选自G或T；
B 1为S；B 2为I或T或S；B 3为X或缺失；
优选地，
侧翼序列a为G、XG、XGG、DKG或DKT且侧翼序列b为SI、ST、SS、SIX、STX或SSX；
(2)所述断裂型内含肽为Gp41-8时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失；A -2选自N或D；A -1选自R或K；
B 1为S或T；B 2为A或H；B 3为X或缺失，或优选为V、Y或T，
优选地，
侧翼序列a为NR、XNR、DK、XDK、DR或XDR且侧翼序列b为SA或SAX；
(3)所述断裂型内含肽为SspDnaB时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失；A -2选自S或D；A -1选自G或K；
B 1为S；B 2为I；B 3为X或缺失，或优选为E或T，
优选地，
侧翼序列a为SG、XSG、DK、XDK，侧翼序列b为SI或SIX；
(4)所述内含肽为MjaTFIIB时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中
A -3为X或缺失；A -2选自T或D；A -1选自Y；
B 1为T；B 2为I或H；B 3为X或缺失，或优选为H或T；
优选地，
侧翼序列a为TY、DY、XTY或XDY，侧翼序列b为TI、TIX、TH或THX；
(5)所述断裂型内含肽为PhoRadA时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失；A -2选自G或D；A -1选自K；
B 1为T；B 2为Q或H；B 3为X或缺失，或优选为L或T，
优选地，
侧翼序列a为GK、XGK、DK或XDK，侧翼序列b为TQ、TH、TQX或THX；
(6)所述断裂型内含肽为TvoVMA时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失；A -2选自G或D；A -1为K；
B 1为T；B 2为V或H；B 3为X或缺失，或优选为I或T，
优选地，
侧翼序列a为GK、XGK、DK或XDK，侧翼序列b为TV、TH、TVX或THX；
(7)所述断裂型内含肽为MxeGyrA时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失；A -2选自R或D；A -1选自Y、K或T；
B 1为T；B 2为E或H；B 3为X或缺失，或优选为A或T，
优选地，
侧翼序列a为RY、XRY、DK或XDK，侧翼序列b为TE、TH、TEX或THX；
(8)所述断裂型内含肽为PhoVMA时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失；A -2选自G或D；A -1选自K；
B 1为T；B 2为V或H；B 3为X或缺失，或优选为I或T，
优选地，
侧翼序列a为GK、XGK、DK或XDK，侧翼序列b为TV、TH、TVX或THX；
(9)所述断裂型内含肽为Gp41-1时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失；A -2选自G或D；A -1选自Y或K；
B 1为S或T；B 2为S或H；B 3为X或缺失，或优选为S或T；
优选地，
侧翼序列a为GY、XGY、DK或XDK，侧翼序列b为SS、SH、SSX或SHX；
(10)所述断裂型内含肽为SspDnaE时，
侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
A -3为X或缺失；A -2选自G或D；A -1选自G、S或K；
B 1为T或S；B 2为E或H；B 3为X或缺失，或优选为T；
优选地，
侧翼序列a为GG、XGG、GK、XGK、DK或XDK，侧翼序列b为SE、TH、SEX或THX；
其中所述X为选自：G、A、V、L、M、I、S、T、P、N、Q、F、Y、W、K、R、H、D、E、C中的任一种氨基酸。
2.根据上述1所述的用于断裂型内含肽的侧翼序列对，其中，所述断裂型内含肽与所述侧翼序列对共同使用用于进行反式剪接，
其中，
所述SspDnaE由序列为SEQ ID NO：31的In和序列为SEQ ID NO：32的Ic组成，
所述SspDnaB由序列为SEQ ID NO：33的In和序列为SEQ ID NO：34的Ic组成，
所述MxeGyrA由序列为SEQ ID NO：35的In和序列为SEQ ID NO：36的Ic组成，
所述MjaTFHB由序列为SEQ ID NO：37的In和序列为SEQ ID NO：38的Ic组成，
所述PhoVMA由序列为SEQ ID NO：39的In和序列为SEQ ID NO：40的Ic组成，
所述TvoVMA由序列为SEQ ID NO：41的In和序列为SEQ ID NO：42的Ic组成，
所述Gp41-1由序列为SEQ ID NO：43的In和序列为SEQ ID NO：44的Ic组成，
所述Gp41-8由序列为SEQ ID NO：45的In和序列为SEQ ID NO：46的Ic组成，
所述IMPDH-1由序列为SEQ ID NO：47的In和序列为SEQ ID NO：48的Ic组成，
所述PhoRadA由序列为SEQ ID NO：49的In和序列为SEQ ID NO：50的Ic组成，
优选地，
(1)当所述断裂型内含肽为IMPDH-1时，所述侧翼序列a为XGG且侧翼序列b为SI、ST、SS；或侧翼序列a为DKG且侧翼序列b为SI、ST、SS；或侧翼序列a为DKT且侧翼序列b为SI、ST、SS；
(2)当所述断裂型内含肽为Gp41-8时，所述侧翼序列a为NR且侧翼序列b为SAV；或侧翼序列a为DK且侧翼序列b为SAV；侧翼序列a为NR且侧翼序列b为SAT；或侧翼序列a为DK且侧翼序列b为SAT；
(3)当所述断裂型内含肽为SspDnaB时，所述侧翼序列a为SG且侧翼序列b为SIE；
(4)当所述断裂型内含肽为PhoRadA时，所述侧翼序列a为GK且侧翼序列b为TQL或THT；或侧翼序列a为DK且侧翼序列b为TQL或THT；
(5)当所述断裂型内含肽为TvoVMA时，所述侧翼序列a为GK且侧翼序列b为TVI或THT；或侧翼序列a为DK且侧翼序列b为TVI或THT；
(6)当所述断裂型内含肽为MxeGyrA时，所述侧翼序列a为RY且侧翼序列b为TEA或THT；或侧翼序列a为DK且侧翼序列b为TEA或THT；
(7)当所述断裂型内含肽为MjaTFIIB时，所述侧翼序列a为TY且侧翼序列b为TIH；或侧翼序列a为TY且侧翼序列b为THT；
(8)当所述断裂型内含肽为PhoVMA时，所述侧翼序列a为GK且侧翼序列b为TVI或THT；或侧翼序列a为DK且侧翼序列b为TVI或THT；
(9)当所述断裂型内含肽为Gp41-1时，所述侧翼序列a为GY且侧翼序列b为SSS或SHT；或侧翼序列a为DK且侧翼序列b为SSS或SHT；
(10)当所述断裂型内含肽为SspDnaE时，所述侧翼序列a为GG且侧翼序列b为SET或THT；或侧翼序列a为GK且侧翼序列b为SET或THT；或侧翼序列a为DK且侧翼序列b为SET或THT；
其中所述X为选自：G、A、V、L、M、I、S、T、P、N、Q、F、Y、W、K、R、H、D、E、C中的任一种氨基酸。
3.重组多肽，其由利用上述1或2所述的用于断裂型内含肽的侧翼序列对进行反式剪接而获得。
4.根据上述3所述的重组多肽，其中，所述重组多肽由组分A与组分B经过反式剪接得到；
在组分A中，所述侧翼序列a的N端与En的C端连接、且所述侧翼序列a的C端与所述In连接，任选在In的C端连接有标签蛋白；
在组分B中，所述侧翼序列b的C端与Ec的N端连接、且所述侧翼序列b的N端与所述Ic连接，任选在Ic的N端连接有标签蛋白；
其中，所述En与Ec的编码序列分别来自同一蛋白的N端部分和C端部分，
优选地，所述标签蛋白选自SEQ ID NO：24、25、26、27、28、29或30。
5.根据上述3所述的重组多肽，其中，所述重组多肽由组分A与组分B经过反式剪接得到；
在组分A中，所述侧翼序列a的N端与En的C端连接、且所述侧翼序列a的C端与所述In连接，任选在In的C端连接有标签蛋白；
在组分B中，所述侧翼序列b的C端与Ec的N端连接、且所述侧翼序列b的N端与所述Ic连接，任选在Ic的N端连接有标签蛋白；
其中，所述En与Ec的编码序列来自不同蛋白。
6.根据上述4或5所述的重组多肽，其为荧光蛋白、蛋白酶、信号肽、抗菌肽、抗体、或具备生物毒性的多肽。
7.根据上述4或5所述的重组多肽，其中，所述同一蛋白、或所述不同蛋白中的一种以上为抗体。
8.根据上述7所述的重组多肽，其中，所述抗体为天然免疫球蛋白IgG、IgM、IgA、IgD、或IgE类，或免疫球蛋白亚类：IgG1、IgG2、IgG3、IgG4、IgG5，或不同类的轻链：κ、λ；或单域抗体；或
所述抗体为全长抗体或抗体的功能片段。
9.根据上述8所述的重组多肽，其中，所述抗体的功能片段为选自：抗体重链可变区VH、抗体轻链可变区VL、抗体重链恒定区片段Fc、抗体重链恒定区1 CH1、抗体重链恒定区2 CH2、抗体重链恒定区3 CH3、抗体轻链恒定区CL、或单域抗体可变区VHH中的一种或多种。
10.根据上述7所述的重组多肽，其中，所述同一蛋白、或所述不同蛋白中的一种以上针对抗原或表位A具有特异性，
所述抗原A包括：肿瘤细胞表面抗原、免疫细胞表面抗原、细胞因子、细胞因子受体、转录因子、膜蛋白、肌动蛋白、病毒、细菌、内毒素、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，所述表位A为所述抗原A的免疫原性表位。
11.根据上述10所述的重组多肽，其中，所述同一蛋白、或所述不同蛋白中的一种以上针对与抗原或表位A不同的抗原或表位B具有特异性，
所述抗原B包括：肿瘤细胞表面抗原、免疫细胞表面抗原、细胞因子、细胞因子受体、转录因子、膜蛋白、肌动蛋白、病毒、细菌、内毒素、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，所述表位B为所述抗原B的免疫原性表位。
12.根据上述11所述的重组多肽，其为双特异性抗体，可同时结合抗原或表位A和B，优选为人源化的双特异性抗体或全人序列的双特异性抗体。
13.根据上述7～11任一项所述的重组多肽，其中，
所述组分A包含：抗体的轻链、C端融合有In的抗体的VH+CH1链，或C端融合有In的单域抗体可变区VHHa，任选在In的C端连接有标签蛋白，
所述组分B包含：抗体的轻链、抗体的完整重链、以及N端融合有Ic的Fc链，或N端融合有Ic的单域抗体可变区VHHb，任选在Ic的N端连接有标签蛋白，所述VHHa和VHHb可以相同或不同。
14.根据上述3～13中任一项所述的重组多肽，其中，
所述标签蛋白选自：Fc、His-tag、Strep-tag、Flag、HA或麦芽糖结合蛋白MBP。
15.组合物，其包含上述3～14中任一项所述的重组多肽。
16.组合物，其中，除了上述3～14中任一项所述的重组多肽以外，还包含载体。
17.根据上述16所述的组合物，其为药物组合物，所述载体为药学上可接受的载体。
18.载体，其连接有上述3～14中任一项所述的重组多肽，优选用于包括层析的纯化用途。
19.试剂盒，其包含上述3～14中任一项所述的重组多肽，其用于检测样品中是否存在抗原或表位A和/或抗原或表位B，其中优选所述重组多肽为保存于液体中的状态或冻干粉，任选为单独存在或为被连接、络合、缔合、螯合而固定于载体的状态。
20.表达载体，其用于制备上述3～14中任一项所述的重组多肽的表达载体。
21.重组多肽的制备方法，其包括：
(1)提供组分A和组分B，所述组分A包括侧翼序列a、N端外显肽En和In，所述侧翼序列a的N端与所述N端外显肽En的C端连接、且所述侧翼序列a的C端与所述In连接，任选在In的C端还连接有标签蛋白；
所述组分B包括侧翼序列b、C端外显肽Ec和Ic，所述侧翼序列b的C端与C端外显肽Ec的N端连接、且所述侧翼序列b的N端与所述Ic连接，任选在Ic的N端连接有标签蛋白；
其中，所述的侧翼序列a和侧翼序列b如上述1或2中所述，所述N端外显肽En与C端外显肽Ec的编码序列来自同一蛋白或不同蛋白；和
(2)将所述组分A和组分B进行体外反式剪接，得到重组多肽；
优选地，在步骤(1)中，包括使含有编码组分A和组分B的核酸序列的细胞表达所述组分A和组分B；优选地，N端外显肽En和C端外显肽Ec可以为抗体的不同结构域。
22.上述21所述的重组多肽的制备方法，其还包括：
对进行反式剪接前的组分A、组分B进行层析的第一纯化步骤；
对反式剪接得到的重组多肽进行层析的第二纯化步骤；
优选所述第一纯化步骤中的层析方法选自proteinA、proteinG、镍柱、Strep-Tactin亲和层析、抗Flag抗体亲和层析、抗HA抗体亲和层析或交联淀粉亲和层析，和
优选所述第二纯化步骤中的层析方法为选自与标签蛋白对应的亲和层析方法，以去除未剪接组分，或通过离子交换、疏水、分子筛去除未剪接组分。
23.根据上述21所述的重组多肽的制备方法，其中，所述重组多肽为双特异性抗体，其中所述双特异性抗体的编码序列分别属于两种不同的抗体P和抗体R；
1)对于抗体P进行拆分为En P和Ec P，设计组分A和组分B的序列；对于抗体R进行拆分为En R和Ec R，设计组分A’和组分B’；其中，
组分A包括侧翼序列a、En P和In，所述侧翼序列a的N端与所述En P的C端连接、且所述侧翼序列a的C端与所述In连接，任选在In的C端还连接有标签蛋白；组分B包括侧翼序列b、Ec P和Ic，所述侧翼序列b的C端与Ec P的N端连接、且所述侧翼序列b的N端与所述Ic连接，任选在Ic的N端连接有标签蛋白；
组分A’包括侧翼序列a、En R和In，所述侧翼序列a的N端与所述Ra的C端连接、且所述侧翼序列a的C端与所述In连接，任选在In的C端还连接有标签蛋白；组分B’包括侧翼序列b、Ec R和Ic，所述侧翼序列b的C端与Ec R的N端连接、且所述侧翼序列b的N端与所述Ic连接，任选在Ic的N端连接有标签蛋白；
2)将所述组分A与组分B’，和/或将组分A’与组分B进行反式剪接，得到所述双特异性抗体。
24.用于断裂型内含肽的侧翼序列对的筛选方法，所述方法包括：
1)将蛋白P的氨基酸序列拆分；
2)侧翼序列a为独立设计的2～3个氨基酸组合，记为侧翼序列a1～an，侧翼序列b为独立设计的2～3个氨基酸组合，记为侧翼序列b1～bn；其中，所述氨基酸为选自G、A、V、L、M、I、S、T、P、N、Q、F、Y、W、K、R、H、D、E、C中的任一种氨基酸；
3)针对断裂型内含肽，使用2)中设计的侧翼序列a1～an和b1-bn，设计含有蛋白P拆分而成序列的组分A1～An和组分B1～Bn的表达序列；
4)将所述表达序列分别与载体连接，进行组分A和B一一对应的共转染及细胞内反式剪接，得到剪接产物F1～Fn；
5)检测剪接产物F1～Fn，选择剪接效率超过20％的侧翼序列对；
6)对5)中选择的侧翼序列对进行分析，淘汰该侧翼序列中的能够导致剪接后产生自由巯基的侧翼序列，以优化5)中选择的侧翼序列对；
7)重复所述步骤1)～5)，选择剪接效率在所有候选序列对中处于前20％，且作为剪接产物的重组多肽中不存在自由巯基的侧翼序列对1～m，
其中n为2或3，m为正整数。
25.根据上述24所述的断裂型内含肽的侧翼序列对的筛选方法，所述方法还包括：
1)将与蛋白P不同的蛋白R拆分；
2)利用侧翼序列对1～m，设计组分A’1～A’m和组分B’1～B’m的表达序列；
3)将所述表达序列与载体连接，进行转染、表达和纯化，得到组分A’1～A’m、组分B’1～B’m，
4)分别将与利用侧翼序列对1～m得到的组分A1～Am与组分B’1～B’m，和/或组分A’1～A’m与组分B1～Bm一一对应进行体外反式剪接，检测剪接产物蛋白，选择剪接效率超过50％的多个侧翼序列对。
26.制备重组多肽的方法，其特征在于利用上述1或2所述的用于断裂型内含肽的侧翼序列对进行反式剪接。
27.上述1或2所述的用于断裂型内含肽的侧翼序列对的用途，其特征在于，用于制备重组多肽，优选地用于与断裂型内含肽共同进行反式剪接。
利用本发明所述的用于断裂型内含肽的侧翼序列对介导剪接的重组多肽(如双特异性抗体)的优势，包括(1)无自由巯基；(2)高通量高效率制备；(3)目的产物和杂质易于区分和鉴别。
定义
应注意，非明确数量的实体限定应指一或多个(种)该实体；例如，“双特异性抗体”应理解为表示一或多个(种)双特异性抗体。同样地，非明确数量限定的、术语“一个或多个”和“至少一个”本文中可互换使用。
本文使用的术语“多肽”包括单数的“多肽”以及复数的“多肽”，并且也指通过酰胺键(也称为肽键)线性连接的单体(氨基酸)组成的分子。多肽可衍生自天然的生物源或通过重组技术生产，不一定由指定核酸序列翻译而成，可以任何方式产生，包括化学合成方式。
本文使用的术语“重组”，在涉及多肽或多核苷酸时指自然状态下不存在的多肽或多核苷酸的形式，其中一个非限制性的例子，可以通过将通常不会一起出现的多核苷酸或多肽组合在一起来实现。
“同源性”或“同一性”或“相似性”，是指两个肽链分子之间或两个核酸分子之间的序列相似程度。当在被比较的序列中的位置上有相同的碱基或氨基酸时，在该位置上的分子为同源的。多个序列之间的同源度为这些序列共有的配对或同源位点数量的函数。“无关的”或“非同源的”序列与本发明的序列之一之间具有少于40％的同源性，但优选小于25％的同源性。
多核苷酸或多核苷酸区域(或多肽或多肽区域)与另一序列具有一定的百分比(例如，60％、65％、70％、75％、80％、85％、90％、95％、98％或99％)的“序列同一性”是指，比对时在这两个序列比较时该百分比的碱基(或氨基酸)相同。
生物学等效的多核苷酸是具有上面提到的特定百分比的同源性，并编码具有相同或相似的生物活性多肽的多核苷酸。
术语“断裂型内含肽”，是指由N端蛋白质剪接区域或N端片段(In，N’fragment of intein)和C端蛋白质剪接区域或C端片段(Ic，C fragment of intein)两部分组成的断裂型内含肽(split intein)，表达前体蛋白质的基因被***在两个开放阅读框中，断裂位点在内含肽序列的内部。
“N端前体蛋白质”是指N端外显肽(En)与断裂型内含肽的N端片段(In)的基因形成融合基因，翻译形成的融合蛋白。
“C端前体蛋白质”是指断裂型内含肽的C端片段(Ic)与C端外显肽(Ec)的表达基因形成融合基因翻译后产生的融合蛋白。
单独的断裂型内含肽的N端片段(In)或C端片段(Ic)不具有蛋白质剪接功能。在蛋白质翻译以后，N端前体蛋白质中的In与C端前体蛋白质的Ic通过互相识 别以非共价键结合，形成有功能的内含肽，能够催化蛋白质反式剪接反应，从而以肽键连接两个分离的蛋白质外显子(N端蛋白质外显子或N端外显肽称为En、C端蛋白质外显子或C端外显肽称为Ec)(Ozawa.T.Nat Biotechbol.21(2003)28793)。
蛋白质反式剪接(protein trans-splicing)，是指由断裂型内含肽介导的蛋白质剪接反应。在反式剪接过程中，首先，断裂型内含肽的N端片段(In)和C端片段(Ic)相互识别，并以非共价键结合。在一旦结合后，其结构发生正确折叠，此时的断裂型内含肽具有重建的活性中心，然后，按照典型的蛋白质剪接途径完成蛋白质剪接反应，从而将两侧的外显肽连接。
In是指单独的断裂型内含肽的N端部分，在本文中也称为断裂型内含肽的N端片段或N端蛋白质剪接区域。
Ic是指单独的断裂型内含肽的C端部分，在本文中也称为断裂型内含肽的C端片段或C端蛋白质剪接区域。
侧翼序列a是侧接In的N端并且侧接En的C端的氨基酸序列，连接In和En。这里，如图5所示，将紧邻In的N端第一个氨基酸定义为第-1位，再往N端的第二个氨基酸残基为第-2位，第三个氨基酸残基为第-3位，依此类推直至En为止。一般而言，侧翼序列a的核心序列为第-1位和第-2位，与剪接效率直接相关。
侧翼序列b是侧接Ic的C端并且侧接Ec的N端的氨基酸序列，连接Ic和Ec。这里，如图5所示，将紧邻Ic的C端第一个氨基酸残基定义为第+1位，再往C端的第二个氨基酸残基为第+2位，第三个氨基酸残基为第+3位，依此类推直至Ec为止。一般而言，侧翼序列b的核心序列为第+1位和第+2位，与剪接效率直接相关。
断裂型内含肽介导的反式剪接，例如如图5所示，In与侧翼序列a分开，Ic与侧翼序列b分开，侧翼序列a和侧翼序列b连接，由此将与侧翼序列相连的En和Ec连接，使得侧翼序列a的第-1位氨基酸残基与侧翼序列b的第+1位氨基酸残基直接肽键相连，且第-1位氨基酸位于第+1位氨基酸的N端。
本发明使用进行侧翼序列筛选的20种常见氨基酸(下文简称20种氨基酸)，指：甘氨酸(G)、丙氨酸(A)、缬氨酸(V)、亮氨酸(L)、甲硫氨酸(M)、异亮氨酸(I)、丝氨酸(S)、苏氨酸(T)、脯氨酸(P)、天冬酰胺(N)、谷氨酰胺(Q)、苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)、赖氨酸(K)、精氨酸(R)、组氨酸(H)、天冬氨酸(D)、谷氨酸(E)和半胱氨酸(C)。
本文使用的“抗体”或者“抗原结合多肽”指特定识别并结合抗原或免疫原性表位的多肽或多肽复合体。
抗体可为完整抗体也可为任何抗原结合片段或者其单链。因此术语“抗体”包括含有特定分子的任何蛋白或肽，该特定分子含有至少一部分的免疫球蛋白分子，该免疫球蛋白分子具有结合至抗原或免疫原性表位的生物活性。此种情况的实例包括但不限于，重链或轻链或其配体结合部分的互补决定区(CDR)、重链或轻链可变区、重链或轻链恒定区、框架(FR)区或其任何部分，或结合蛋白的至少一部分。
本文使用的术语“抗体片段”或“抗原结合片段”为抗体的一部分，术语“抗体片段”包括适配体、适配体对映体(spiegelmers)和双体(diabodies)，也包括任何合成的或基因改造的蛋白，它们与抗体一样可结合至特定的抗原或免疫原性表位以形成复合体。
“单链可变片段”或“scFv”指免疫球蛋白的重链(VH)和轻链(VL)的可变区域的融合蛋白。
术语“抗体”包括多种可被生化识别的宽泛类别的多肽。本领域技术人员应理解，重链分为γ、μ、α、δ、ε并具有一些亚类(例如，γ1-4)。该链的性质决定了抗体的“类”，如IgG、IgM、IgA、IgD或IgE。免疫球蛋白亚类(同型)例如IgG1、IgG2、IgG3、IgG4、IgG5等被很好表征并在功能上具有特异性。本领域技术人员参考本申请可容易识别这些类和同型的每一种修饰形式，因此，这些形式在本申请范围内。
所有免疫球蛋白类明确地在本申请的范围内，下列讨论通常将针对免疫球蛋白分子的IgG类。
关于IgG，标准免疫球蛋白分子包含经二硫键以“Y”型连接在一起的两个相同的轻链多肽(分子量约为23,000道尔顿)、和两个相同的重链多肽(分子量约为53,000-70,000道尔顿)。
本申请的抗体、抗原结合多肽、它们的变体或衍生物，包括但不限于：多克隆抗体、单克隆抗体、多特异性抗体、人抗体、人源化的抗体、灵长类化的(primatized)抗体、或嵌合抗体、单链抗体、抗原表位结合片段，例如，Fab、Fab′和F(ab′) 2、Fd、Fvs、单链Fvs(scFv)、单链抗体、二硫键连接的Fvs(sdFv)、包含VL结构域或VH结构域的片段、由Fab表达库产生的片段、和抗独特型(Anti-idiotypic)(抗-Id)抗体。本申请的免疫球蛋白分子或抗体分子可为任何类型的(例如IgG、IgE、IgM、IgD、IgA和IgY)、免疫球蛋白分子的任何类(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。
在一些实例中，例如，某些衍生自骆驼种或基于骆驼免疫球蛋白改造的免疫球蛋白，完整的免疫球蛋白分子可仅由重链组成，而没有轻链。参见，例如，Hamers-Casterman等，Nature.363：446-448(1993)。
轻链和重链两者都分成结构区和功能同源区。术语“恒定”和“可变的”为功能上的使用。在此，应认识到轻链可变结构域(VL)和重链可变结构域(VH)同时决定了抗原识别和特异性。通常恒定区结构域的数量随着远离抗体的抗原结合位点或氨基端的末端位置而增加。N末端部分为可变区，而在C末端部分为恒定区；CH3和CL结构域实际上分别包含重链和轻链的羧基末端。
抗原结合位点是指：针对任何给定的重链或轻链可变区，本领域技术人员可容易地识别分别包括CDR和框架区的氨基酸，因为它们已经被明确定义(参见，“Sequences of Proteins of Immunological Interest，”Kabat，E.，等，美国卫生和公共服务部(U.S.Department of Health and Human Services，)，(1983)；Chothia和Lesk，J.MoI.Biol.，196：901-917(1987)，其在此通过引用以全文形式结合至本文)。
在本技术领域内使用和/或可接受的情况下，一个术语有两个或两个以上定义时，本文使用的术语的定义用于包括所有的含义，除非明确说明与此相反。
术语“互补决定区”(“CDR”)描述在重链和轻链多肽的可变区中都存在的非连续的抗原结合位点。这种具体区域由Kabat等描述于美国卫生和公共服务部，“Sequences of Proteins of Immunological Interest”(1983)和由Chothia等描述与J.MoI.Biol.196：901-917(1987)中，其通过全文引用结合至本文。给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定哪些残基包含特定CDR。
本文使用的“Kabat编号”是指Kabat等描述的编号***，其内容记载于美国卫生和公共服务部，“Sequence of Proteins of Immunological Interest”(1983)。
本文使用的术语“重链恒定区”包括来自免疫球蛋白重链的氨基酸序列。包含 重链恒定区的多肽至少包含以下一种：CH1结构域、铰链(例如，上部铰链区、中间铰链区，和/或下部铰链区)结构域、CH2结构域、CH3结构域，或其变体或片段。例如，本申请中使用的抗原结合多肽可包含具有CH1结构域的多肽链；具有CH1结构域、至少一部分的铰链结构域和CH2结构域的多肽；具有CH1结构域和CH3结构域的多肽链；具有CH1结构域、至少一部分铰链结构域和CH3结构域的多肽链，或者具有CH1结构域，至少一部分铰链结构，CH2结构域，和CH3结构域的多肽链。在另一个实施例中，本申请的多肽包括具有CH3结构域的多肽链。另外，在本申请中使用的抗体可能缺少至少一部分CH2结构域(例如，所有的或一部分的CH2结构域)。如上文所述，但本技术领域的普通技术人员应理解，重链恒定区可能会被修改，使得它们在氨基酸序列上与天然存在的免疫球蛋白分子不同。
本文所公开的抗体的重链恒定区可以来自于不同的免疫球蛋白分子。例如，多肽的重链恒定区可以包含来自IgG1分子的CH1结构域和来自IgG3的分子的铰链区。在另一例子中，重链恒定区可以包含铰链区，该铰链区部分来自IgG1分子，且部分地来自IgG3分子。在另一例子中，重链部分可包含嵌合铰链，该嵌合铰链一部分来自IgG1分子，并且一部分来自IgG4分子。
术语“轻链恒定区”包括来自抗体轻链的氨基酸序列。优选地，所述轻链恒定区包括恒定kappa结构域和恒定lambda结构域中的至少一个。
术语“VH结构域”包括免疫球蛋白重链的氨基末端可变结构域，而术语“CH1结构域”包括免疫球蛋白重链的第一(多数为氨基末端)恒定区。CH1结构域邻近VH结构域并且是免疫球蛋白重链分子的铰链区的氨基末端。
术语“CH2结构域”包括一部分的重链分子，该部分范围，例如，从抗体的约残基244到残基360，使用常规的编号方案(残基244至360，Kabat编号***；和残基231-340，EU编号***；见Kabat等，美国卫生和公共服务部，“Sequences of Proteins of Immunological Interest”(1983)。CH2结构域是独特的，因为它与另一个结构域配对不紧密。相反，两个N-连接的支链的糖链***至完整的天然IgG分子的两个CH2结构域之间。有文献记载，CH3结构域从CH2结构域延伸至IgG分子的C-末端，并包含约108个残基。
所谓“特异性结合”或“对...有特异性”，通常意味着抗体结合到该抗原表位时，经抗原结合结构域的结合相比于结合至随机的、不相关的抗原表位更容易。本文中使用术语“特异性”以确定某一抗体结合至特定抗原表位的亲和力。
本文使用的术语“治疗”(“treat”或“treatment”)是指治疗性治疗和预防或防治措施，其中对于受试者进行防止或减慢(减轻)不良的生理变化或疾病，如癌症的发展。有益的或所需的临床结果包括但不限于，减轻症状，降低疾病的程度、稳定(例如使其不恶化)疾病的状态，延迟或减缓疾病发展，改善或缓和疾病状态，并缓解(无论是部分或全部)，无论可否被检测到。“治疗”也可指与不接受治疗时的预计生存期相比能延长生存期。
任何上述的抗体或多肽还可包括额外的多肽，例如，如本文所述的编码的多肽，抗体N端的用于指导分泌的信号肽，或如本文所述的其他异源多肽。
在其它实施例中，本申请的多肽可包含保守的氨基酸替换。
“保守氨基酸替换”是指氨基酸残基被具有类似侧链的氨基酸残基替换。具有类似侧链的氨基酸残基家族已在本领域中定义，其包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)，酸性侧链(例如天冬氨酸，谷氨酸)，不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)，非 极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)，β-支链的侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，免疫球蛋白多肽的非必需氨基酸残基优选被来自相同侧链家族的其他氨基酸残基替换。在另一实施例中，一串氨基酸可被结构上类似的氨基酸串替换，后者在顺序上和/或侧链家族的组成上不同。
瞬时转染：瞬时转染是将DNA导入真核细胞的方式之一。在瞬时转染中，重组DNA导入感染性强的细胞系以获得目的基因暂时但高水平的表达。转染的DNA不必整合到宿主染色体，可在比稳定转染较短时间内收获转染的细胞，并对表达上清中的目的产物进行检测。
附图说明
图1为断裂型内含肽介导的同源多肽片段剪接示意图(A)和各组分的蛋白质一级结构示意图(B)。
图2为断裂型内含肽介导的异源多肽片段剪接示意图(A)和各组分的蛋白质一级结构示意图(B)。
图3为断裂型内含肽介导的抗体体外剪接示意图(A)和各组分的蛋白质一级结构示意图(B)，该剪接产物为双特异性抗体。(C)为断裂型内含肽介导的抗体剪接处附近的氨基酸序列的示例性示意图，“X”表示该位置氨基酸为任意氨基酸或者缺失。
图4为双特异性抗体的组分A表达质粒构建示意图(A)，和组分B表达质粒构建示意图(B)。
图5为侧翼序列编号示意图。
图6为不同内含肽及不同侧翼序列相对应的表达质粒共转染293E细胞的表达上清经proteinA亲和纯化后的还原SDS-PAGE和考马斯亮蓝染色检测结果。(A)～(E)分别为不同内含肽基于不同侧翼序列共转染组分A和组分B的细胞上清，纯化后检测结果。
图7为293E细胞分别表达的具有不同内含肽的组分A和组分B’的纯化产物的非还原SDS-PAGE和考马斯亮蓝染色检测结果。(A)Fab5，Fab9和Fab11纯化产物检测结果；(B)HAb5，HAb9和HAb11纯化产物检测结果。
图8为不同内含肽的组分A和组分B’的剪接产物的非还原SDS-PAGE和考马斯亮蓝染色检测，其中，(A)内含肽为IMPDH-1，侧翼序列a为GGG，侧翼序列b为SI；(B)内含肽为PhoRadA，侧翼序列a为GK，侧翼序列b为THT。(A)和(B)剪接产物1表示组分A和B混合前加DTT，剪接产物2表示组分A和B’混合后加DTT，还原表示加DTT，非还原表示未加DTT，未剪接表示混合组分A和B’不加DTT。(C)内含肽为PhoRadA，侧翼序列a为GK，侧翼序列b为THT，“剪接1”和“无剪接1”为组分A与组分B’浓度分别为5μM和4μM且反应体系中含2mM DTT，“剪接2”和“无剪接2”为组分A和组分B’浓度分别为10uM和1uM且反应体系中含2mM DTT，“剪接3”和“无剪接3”为组分A和组分B’浓度分别为5uM和1uM且反应体系中含2mM DTT，其中“剪接1”～“剪接3”均为37℃孵育过夜，“无剪接1”～“无剪接3”均为4℃孵育过夜；对照条带为组分A为Fab11(非还原)，组分B’为HAb11(非还原)，以及单抗。
图9为内含肽为IMPDH-1，侧翼序列a为GGG，侧翼序列b为SI的剪接产 物的双抗原夹心ELISA检测结果。其中，包被抗原为CD38，检测抗原为辣根过氧化物酶(HRP)标记的PD-L1。
图10为Fab5+HAb5(剪接产物1)的酶解后的基峰离子(Base peak ion，BPI)图谱。(A)Fab5+HAb5(剪接产物1)经胰蛋白酶酶解后的BPI图谱；(B)Fab5+HAb5(剪接产物1)经胰凝乳蛋白酶酶解后的BPI图谱；(C)Fab5+HAb5(剪接产物1)经Glu-C酶酶解后的BPI图谱。
图11为内含肽PhoRadA和IMPDH-1应用于人IgG2，IgG3或IgG4亚型的组分A和组分B的共转染表达和亲和纯化后SDS-PAGE及考马斯亮蓝染色检测。
具体实施方式
本发明涉及得到双特异性抗体的制备方法，其包括：将目标的抗体相应的DNA序列进行拆分，经过全基因合成构建哺乳动物细胞表达载体，纯化载体，纯化后的载体分别瞬时转染或稳定转染HEK293或CHO等哺乳动物细胞。分别收集发酵液，通过proteinA、proteinL、镍柱、Strep-Tactin亲和层析、抗Flag抗体亲和层析、抗HA抗体亲和层析或交联淀粉亲和层析等方法纯化组分A和组分B，将纯化所得组分A和组分B进行体外反式剪接，对剪接所得产物进行镍柱等标签蛋白对应的亲和层析，得到高纯度的双特异性抗体，工艺流程如图3A所示。
本文描述的抗体可来自任何动物源，包括鸟和哺乳动物。优选地，抗体为人、鼠、驴、兔、山羊、豚鼠、骆驼、驼马、马或鸡的抗体。在另一个实施例中，可变区可来自于软骨鱼(condricthoid)(例如，来自鲨鱼)。
在一些实施例中，所述抗体可以结合：治疗剂、药物前体、肽、蛋白、酶、病毒、脂质、生物反应调节剂、药剂或PEG。
该抗体可以连接至或融合至治疗剂上，该治疗剂可包括可检测标记物，如放射性标记物、免疫调节剂、激素、酶、寡核苷酸、光活性治疗剂或诊断剂、细胞毒性剂，其可为：药物或毒素、超声增强剂、非放射性标记物，它们的组合和其他这类本领域已知的成分。
通过将其偶联化学发光化合物，该抗体被可检测地标记。然后，通过检测化学反应过程中产生的发光来确定化学发光物标记的抗原结合多肽的存在。特别有用的化学发光物标记化合物的例子有鲁米诺、异鲁米诺、热性(theromatic)吖啶鎓酯、咪唑、吖啶鎓盐和草酸酯。
该抗体也可使用荧光发光金属如152Eu，或其他的镧系标记可检测地标记。这些金属可使用金属螯合基团如二亚乙基三胺五乙酸(DTPN)或乙二胺四乙酸(EDTA)连接到抗体上。
本申请的抗原结合多肽的结合特异性可通过体外实验，例如：免疫沉淀、放射免疫分析法(RIA)或酶联免疫吸附法(ELISA)测得。
用于产生重组多肽的细胞系可使用本领域技术人员熟知的技术选择和培养。
对在编码本申请抗体的核苷酸序列中引入突变，可使用本领域技术人员公知的标准技术，包括但不限于：产生氨基酸替换的定点诱变和PCR介导的突变。优选地，所述变体(包括衍生物)，相对于参考可变重链区，CDR-H1、CDR-H2、CDR-H3、轻链可变区、CDR-L1、CDR-L2或CDR-L3，编码少于50个氨基酸替换、少于40个氨基酸替换、少于30个氨基酸替换、少于25个氨基酸替换、少于20个氨基酸替换、少于15个氨基酸替换、少于10个氨基酸替换、少于5个氨基酸替换、少于4个氨基酸替换、少于3个氨基酸替换、或少于2个氨基酸 替换。或者，可沿着全部或部分的编码序列随机引入突变，例如通过饱和诱变可对所得到的突变体针对生物活性筛选，以确定保留有活性的突变。
本发明中使用的标签蛋白可以是Fc、寡聚组氨酸(His-tag)、Strep-tag、Flag、HA或麦芽糖结合蛋白(MBP)等。
本发明中使用的转染可以是瞬时转染或稳定转染。
本发明中使用了HEK293或CHO等哺乳动物细胞，但不限于此。
来自哺乳细胞的包含表达产物的液体例如发酵液、培养基上清，可以采用proteinA、proteinG、镍柱、Strep-Tactin亲和层析、抗Flag抗体亲和层析、抗HA抗体亲和层析或交联淀粉亲和层析等方法纯化。
剪接所得产物可以进行标签蛋白对应的亲和层析，去除未剪接的成分。
本发明用于构建载体的基因片段，可以通过全基因合成构建，但不限于此。
本发明所用的载体为pcDNA3.1或pCHO1.0，但不限于此。
本发明所用的限制性内切酶，可列举例如：NotI、NruI或BamHI-HF等，但不限于此。
BLAST是一种比对程序，使用默认参数。具体地，程序为BLASTN和BLASTP。这些程序的详细信息，可于以下互联网地址获得： http：//www.ncbi.nlm.nih.gov/blast/Blast.cgi。
在本发明的一个具体实施方式中，如图1、2、3所示，可以构建组分A表达质粒(pPa-FSa-In-Tag)和组分B表达质粒(pTag-Ic-FSb-Pb)，或组分A’表达质粒(pRa-FSa-In-Tag)和组分B’表达质粒(pTag-Ic-FSb-Rb)。
在本发明的另一个具体实施方式中，如图4A和B所示，可以通过酶切、酶连等分子克隆方法，将Pa-HIn和Pa-L构建到同一个质粒中，即组分A表达质粒(pBi-Pa-FSa-In-Tag)；或将pB’-L、pB’-H和pB’-FcIc构建到同一个质粒，即组分B’表达质粒(pBi-Tag-Ic-FSb-Rb)。
在本发明的另一个具体实施方式中，组分B表达质粒可以包括pB-L、pB-H、pB-FcIc三类表达质粒。
在本发明中，Pa也用来表示蛋白P的N端蛋白质外显子或N端外显肽，也表示为Enp；Pb也用来表示蛋白P的C端蛋白质外显子或C端外显肽，也表示为Ecp。Ra也用来表示蛋白R的N端蛋白质外显子或N端外显肽，也表示为En R；Rb也用来表示蛋白R的C端蛋白质外显子或C端外显肽，也表示为Ec R。
Figure PCTCN2020114271-appb-000001
Figure PCTCN2020114271-appb-000002
Figure PCTCN2020114271-appb-000003
Figure PCTCN2020114271-appb-000004
Figure PCTCN2020114271-appb-000005
表4 部分断裂型内含肽的侧翼序列a
序列号 编号 侧翼序列a氨基酸序列
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代表选自本发明定义的20种氨基酸(A、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、C)中的任意氨基酸。
Figure PCTCN2020114271-appb-000006
Figure PCTCN2020114271-appb-000007
Figure PCTCN2020114271-appb-000008
Figure PCTCN2020114271-appb-000009
Figure PCTCN2020114271-appb-000010
Figure PCTCN2020114271-appb-000011
Figure PCTCN2020114271-appb-000012
Figure PCTCN2020114271-appb-000013
Figure PCTCN2020114271-appb-000014
Figure PCTCN2020114271-appb-000015
Figure PCTCN2020114271-appb-000016
Figure PCTCN2020114271-appb-000017
Figure PCTCN2020114271-appb-000018
Figure PCTCN2020114271-appb-000019
Figure PCTCN2020114271-appb-000020
Figure PCTCN2020114271-appb-000021
Figure PCTCN2020114271-appb-000022
Figure PCTCN2020114271-appb-000023
Figure PCTCN2020114271-appb-000024
Figure PCTCN2020114271-appb-000025
Figure PCTCN2020114271-appb-000026
Figure PCTCN2020114271-appb-000027
Figure PCTCN2020114271-appb-000028
Figure PCTCN2020114271-appb-000029
Figure PCTCN2020114271-appb-000030
Figure PCTCN2020114271-appb-000031
Figure PCTCN2020114271-appb-000032
Figure PCTCN2020114271-appb-000033
Figure PCTCN2020114271-appb-000034
Figure PCTCN2020114271-appb-000035
Figure PCTCN2020114271-appb-000036
Figure PCTCN2020114271-appb-000037
Figure PCTCN2020114271-appb-000038
Figure PCTCN2020114271-appb-000039
Figure PCTCN2020114271-appb-000040
Figure PCTCN2020114271-appb-000041
Figure PCTCN2020114271-appb-000042
Figure PCTCN2020114271-appb-000043
实施例1
试验方法
1.重组多肽的制备
本发明的实施例中的DNA序列，均根据氨基酸序列进行逆向翻译获得，并由武汉金开瑞合成。
实施例中涉及的重组多肽制备均是通过下述方法制备：将DNA序列在重组酶作用下，与经限制性内切酶EcoRI酶切处理后的载体pcDNA3.1于37℃连接30分钟，然后通过热激法转化Trans10感受态细胞。通过测序(武汉金开瑞公司)验证正确后瞬时转染293E细胞(购自Thermo Fisher公司)。表达后进行纯化。
2.实施例中涉及的共转染的质粒DNA具体如下：
1)表达图1所示的组分A和组分B，需要质粒pPa-FSa-In-Tag和pTag-Ic-FSb-Pb分别转染或共转染至293E细胞进行表达；。
2)表达图2所示的组分A和组分B’，需要质粒pPa-FSa-In-Tag和pTag-Ic-FSb-Rb分别转染或共转染至293E细胞进行表达；
3)表达图3所示的组分A，需要质粒Pa-HIn和Pa-L共转染至293E细胞进行表达，或者单转染质粒pBi-Pa-FSa-In-Tag进行表达；表达图3所示的组分B’，需要质粒pB’-L、pB’-H和pB’-FcIc共转染至293E细胞进行表达，或者单转染质粒pBi-Tag-Ic-FSb-Rb进行表达。
一般情况下，如是两种质粒共转染表达，则两种质粒的摩尔数之比可以为1∶1，也可以为其他任意比例。如是三种质粒共转染表达，则三种质粒的摩尔数之比可为1∶1∶1，也可以为其他任意比例。
3.具有标签蛋白的多肽的纯化
(1)当标签蛋白为Fc时，采用亲和层析，使用MabSelect SuRe(GE，货号17-5438-01)，18ml柱。
(2)当标签蛋白为His-tag时，采用亲和层析，使用Ni-NTA(江苏千纯，货号：A41002-06)。
(3)当标签蛋白为Strep-tag，Flag、HA或MBP等，分别选择Strep-Tactin亲和层析、抗Flag抗体亲和层析、抗HA抗体亲和层析、或交联淀粉亲和层析相应的填料和缓冲液即可。
(4)离子交换层析，当组分A(A’)或组分B(B’)不带标签蛋白时，可根据等电点的差异使用离子交换层析方法分离剪接产物，使用的层析填料可以是阳离子交换层析填料或阴离子交换层析填料，如Hitrap SP-HP(GE公司)。
(5)疏水层析，当组分A(A’)或组分B(B’)不带标签蛋白时，可根据疏水性的差异使用疏水层析方法分离剪接产物，使用的层析填料如Capto phenyl ImpRes填料(GE公司)。
(6)分子筛，当组分A(A’)或组分B(B’)不带标签蛋白时，可根据分子量的差异使用分子筛层析方法分离剪接产物，使用的层析填料如HiLoad Superdex 200pg(GE公司)。
实施例2内含肽SspDnaB、MxeGyrA、MjaTFIIB、PhoVMA、TvoVMA、Gp41-1、Gp41-8、IMPDH-1、PhoRadA的侧翼序列对的筛选
●表达质粒A-Hln、pA-L，质粒(pTag-Ic-FSb-Pb)的构建
使用“重组多肽的制备”中的条件，如图4A、4B所示，使用pcDNA3.1质粒载体按照表31、表32中所示的构成分别构建了内含肽SspDnaB、MxeGyrA、MjaTFIIB、PhoVMA、TvoVMA、Gp41-1、Gp41-8、IMPDH-1、PhoRadA的组分表达质粒。pA-L质粒使用了与实施例1中相同的pA-L质粒。
其中，对于内含肽SspDnaB，构建了与A-Fab20、A-Fab21对应的pA-HIn(20)～pA-HIn(21)，以及质粒B-FcIc20、B-FcIc21对应的pTag-Ic-FSb-(B-FcIc20)、pTag-Ic-FSb-(B-FcIc21)。
对于内含肽MxeGyrA，构建了与A-Fab30、A-Fab31对应的质粒pA-HIn(30)～pA-HIn(31)，以及B-FcIc30、B-FcIc31对应的质粒pTag-Ic-FSb-(B-FcIc30)、pTag-Ic-FSb-(B-FcIc31)。
对于内含肽MjaTFIIB，构建了与A-Fab40、A-Fab41对应的质粒pA-HIn(40)～pA-HIn(41)，以及B-FcIc40、B-FcIc41对应的质粒pTag-Ic-FSb-(B-FcIc40)、pTag-Ic-FSb-(B-FcIc41)。
对于内含肽PhoVMA，构建了与A-Fab50、A-Fab51对应的质粒pA-HIn(50)～pA-HIn(51)，以及B-FcIc50、B-FcIc51对应的质粒pTag-Ic-FSb-(B-FcIc50)、pTag-Ic-FSb-(B-FcIc51)。
对于内含肽TvoVMA，构建了与A-Fab60、A-Fab61对应的质粒pA-HIn(60)～pA-HIn(61)，以及B-FcIc60、B-FcIc61对应的质粒pTag-Ic-FSb-(B-FcIc60)、pTag-Ic-FSb-(B-FcIc61)。
对于内含肽Gp41-1，构建了与A-Fab70、A-Fab71对应的质粒pA-HIn(70)～pA-HIn(71)，以及B-FcIc70、B-FcIc71对应的质粒pTag-Ic-FSb-(B-FcIc70)、pTag-Ic-FSb-(B-FcIc71)。
对于内含肽Gp41-8，构建了与A-Fab80、A-Fab81对应的质粒pA-HIn(80)～pA-HIn(81)，以及B-FcIc80、B-FcIc81对应的质粒pTag-Ic-FSb-(B-FcIc80)、pTag-Ic-FSb-(B-FcIc81)。
对于内含肽IMPDH-1，构建了与A-Fab90、A-Fab91、A-Fab92对应的质粒pA-HIn(90)～pA-HIn(92)，以及B-FcIc90～B-FcIc92对应的质粒pTag-Ic-FSb-(B-FcIc90)～pTag-Ic-FSb-(B-FcIc92)。
对于内含肽PhoRadA，构建了与A-Fab100、A-Fab101对应的质粒pA-HIn(100)～pA-HIn(101)，以及B-FcIc100、B-FcIc101对应的质粒pTag-Ic-FSb-(B-FcIc100)、pTag-Ic-FSb-(B-FcIc101)。
本实施例中使用的表达组分A的质粒包括：pA-HIn(20)～(21)、(30)～(31)、(40)～(41)、(50)～(51)、(60)～(61)、(70)～(71)、(80)～(81)、(90)～(91)、(100)～(101)、以及pA-L。
本实施例中使用的表达组分B的质粒包括：pTag-Ic-FSb-(B-FcIc20～21)、(30)～(31)、(40)～(41)、(50)～(51)、(60)～(61)、(70)～(71)、(80)～(81)、(90)～(91)、(100)～(101)。
表34 内含肽的共转染配对表
Figure PCTCN2020114271-appb-000044
Figure PCTCN2020114271-appb-000045
按表34中的配对进行了转染。转染条件为：质粒摩尔数比例为pTag-Ic-FSb(XX或XXX)-(B-FcIc)∶pA-HIn(XX或XXX)∶pA-L＝3∶1∶1。并设置了阳性对照为单克隆抗体的瞬时转染。
经转染的细胞培养5天后取上清。对上清中的蛋白进行proteinA亲和层析，proteinA亲和层析后，通过SDS-PAGE法(加还原剂)进行考马斯亮蓝染色检测上清中的蛋白。结果示于图6A～D，根据结果可知，在组A22、A27、A31、A45、 A49、A52、A53、A55、A56中发生了显著剪接。
图6E，结果显示，组A58、A59发生显著剪接。
组A22、A27、A31、A45、A49、A52、A53、A55、A56、A58、A59对应的内含肽及侧翼序列示于表35中。
表35 不同的内含肽及相应的有效侧翼序列对
内含肽 编号 侧翼序列a 侧翼序列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
综上所述，结果显示，对于内含肽IMPDH-1，其对应的具有优秀剪接效率的侧翼序列对为：侧翼序列a为GGG时，侧翼序列b为SI；或者侧翼序列a为DKG，侧翼序列b为ST；或者侧翼序列a为DKG，侧翼序列b为SI。
对于内含肽Gp41-8，其对应的具有优秀剪接效率的侧翼序列对为：侧翼序列a为NR时，侧翼序列b为SAV；或侧翼序列a为DK时，侧翼序列b为SAV。
对于内含肽SSpDnaB，其对应的具有优秀剪接效率的侧翼序列对为：侧翼序列a为SG时，侧翼序列b为SIE。
对于内含肽MjaTFIIB，其对应的具有优秀剪接效率的侧翼序列对为：侧翼序列a为TY时，侧翼序列b为TIH；或侧翼序列a为TY时，侧翼序列b为THT。
对于内含肽PhoRadA，其对应的具有优秀剪接效率的侧翼序列对为：侧翼序列a为GK时，侧翼序列b为TQL或THT；或侧翼序列a为DK时，侧翼序列b为TQL。
实施例3不同蛋白来源的多肽片段的内含肽介导的体外剪接
●载体构建及多肽的表达
使用与实施例1中相同的条件，使用pcDNA3.1按照表31和33中所示的构成分别构建了经由内含肽SspDnaB、MxeGyrA、MjaTFIIB、PhoVMA、TvoVMA、Gp41-1、Gp41-8、IMPDH-1、PhoRadA的组分表达质粒。
对于同一种组分B’，上述组分表达质粒均分为B’-L表达质粒(pB’-L)、B’-H表达质粒(pB’-H)和B’-FcIc表达质粒(pB’-FcIc)三种。其中，pB’-L、B’-H表达质粒在各组分B’之间是通用的。
对于内含肽SspDnaB，构建了与B’-HAb20～B’-HAb21对应的质粒pB’-FcIc(20)～B’-FcIc(21)。
对于内含肽MxeGyrA，构建了与B’-HAb30～B’-HAb31对应的质粒pB’-FcIc(30)～B’-FcIc(31)。
对于内含肽MjaTFIIB，构建了与B’-HAb40～B’-HAb41对应的质粒pB’-FcIc(40)～B’-FcIc(41)。
对于内含肽PhoVMA，构建了与B’-HAb50～B’-HAb51对应的质粒pB’-FcIc(50)～B’-FcIc(51)。
对于内含肽TvoVMA，构建了与B’-HAb60～B’-HAb61对应的质粒pB’-FcIc(60)～B’-FcIc(61)。
对于内含肽Gp41-1，构建了与B’-HAb70～B’-HAb71对应的质粒pB’-FcIc(70)～B’-FcIc(71)。
对于内含肽Gp41-8，构建了与B’-HAb80～B’-HAb81对应的质粒pB’-FcIc(80)～B’-FcIc(81)。
对于内含肽IMPDH-1，构建了与B’-HAb90～B’-HAb92对应的质粒pB’-FcIc(90)～B’-FcIc(92)。
对于内含肽PhoRadA，构建了与B’-HAb100～B’-HAb101对应的质粒pB’-FcIc(100)～B’-FcIc(101)。
本实施例中使用的表达组分A的质粒包括：pA-HIn(90)、pA-HIn(80)、pA-HIn(81)、pA-HIn(61)、pA-HIn(20)、pA-HIn(40)、pA-HIn(100)以及pA-L。
本实施例中使用的表达组分B’的质粒包括：pB’-FcIc(90)、pB’-FcIc(80)、pB’-FcIc(61)、pB’-FcIc(20)、pB’-FcIc(41)、pB’-FcIc(101)以及pB’-L、pB’-H。
组分A的表达和纯化：
将各质粒pA-HIn和质粒pA-L共转染至CHO细胞，37℃培养，质粒摩尔数比例为pA-HIn∶pA-L＝1∶1，转染后10天收获细胞上清。用镍柱层析(江苏千纯，货号：A41002-06)纯化上清液，获得经纯化的组分A的多肽片段。
组分B’的表达和纯化：
将质粒pB’-L，质粒pB’-H和各质粒pB’-FcIc共转染至293E细胞，37℃培养，质粒摩尔数比例为pB’-L∶pB’-H∶pB’-FcIc＝1∶1∶3，转染后10天收获细胞上清。用镍柱层析纯化上清液，获得经纯化的组分B’的多肽片段。
如表36所示，将获得的组分A及组分B’的多肽片段分别称为Fab5～Fab11，HAb5～HAb11。
表36 获得的组分A及组分B’的多肽片段
Figure PCTCN2020114271-appb-000046
Figure PCTCN2020114271-appb-000047
将获得的经纯化的组分A和组分B’的多肽片段进行非还原SDS-PAGE和考马斯亮蓝染色，结果示于图7A～B。
E1、E2、E3表示镍柱层析过程中咪唑浓度从低到高不同的洗脱组分。由图7A可知，Fab5和Fab11均得到了较高的表达水平。并且，在Fab5和Fab11组中，通过采用镍柱层析对多肽进行纯化可获得纯度较高的多肽。由图7B可知，HAb5、HAb9和HAb11均得到了较高的表达水平，且HAb5、HAb9和HAb11经镍柱层析可获得纯度较高的多肽。
●体外剪接
将获得的纯化后的组分A和组分B’的多肽片段Fab5、Fab11、HAb5、HAb11分别用3kD的透析袋(购自Sigma公司)4℃透析至缓冲液中，组分的蛋白的浓度1～10微摩尔。所述缓冲液包含：10～50mM Tris/HCl(pH7.0～8.0)、100～500mM NaCl、0～0.5mM EDTA。再将具有同一内含肽来源的组分A和组分B’分别按对应序号(例如，Fab5与HAb5等)进行摩尔比1∶5～5∶1混合，补加DTT至0.5～5mM，于37℃孵育过夜。
对得到的剪接产物多肽进行SDS-PAGE和考马斯亮蓝染色，结果示于图8A～C。
在图8A～B中，“剪接1”为先混合组分A和组分B’，再加入2mM DTT；“剪接2”为先分别在组分A和组分B’里加入2mM DTT，再将两者混合；“还原”表示该组分含2mM DTT，非还原表示不含DTT；“无剪接”表示溶液中不加DTT；单抗为Herceptin(购自罗氏)。
图8C中，“剪接1”和“无剪接1”为组分A与组分B’浓度分别为5μM和4μM且反应体系中含2mM DTT，“剪接2”和“无剪接2”为组分A和组分B’浓度分别为10μM和1μM且反应体系中含2mM DTT，“剪接3”和“无剪接3”为组分A和组分B’浓度分别为5μM和1μM且反应体系中含2mM DTT，其中“剪接1”～“剪接3”均为37℃孵育过夜，“无剪接1”～“无剪接3”均为4℃孵育过夜；对照条带为组分A为Fab11(非还原)，组分B’为HAb11(非还原)，以及单抗。
由图8可知，具有本发明的新型侧翼序列对的两种断裂型内含肽IMPDH-1和PhoRadA均能在体外发生高效率的有效剪接，从而得到来自不同蛋白的多肽片段的体外剪接重组多肽，分别得到了剪接产物Fab5+HAb5及Fab11+HAb11。这些剪接产物与单抗对照，其条带大小一致，为150kD，证明该产物其理论分子量与天然IgG单抗一致。
●剪接产物的生物学活性检测
针对重组多肽Fab5+HAb5(剪接1)进行了基于双抗原夹心ELISA的生物学活性检测。1)抗原准备：针对蛋白PD-L1和CD38，以只选择胞外结构域的方式进行构建，构建了带His标签的表达质粒，使用载体为pcDNA3.1。
构建后，使用293E细胞进行瞬时转染，进行了包括镍柱纯化和分子筛纯化两步的表达纯化。纯化后，获得了经SDS-PAGE检测纯度不小于95％的抗原蛋白。
用辣根过氧化物酶(HRP)标记了PD-L1蛋白。
2)第一抗原包被：调整使得CD38蛋白浓度为2μg/ml，使用所述含有CD38蛋白的液体以100μl/孔包被酶标板，4度过夜；弃上清，每孔加入250μl封闭液(含3％BSA的PBS)；
3)抗体添加：按照实验设计，室温操作，梯度稀释抗体，稀释液为1％BSA的PBS。比如抗体稀释的初始浓度为20μg/ml，2倍稀释，稀释5个浓度梯度。将稀释好的抗体以每孔200μl加入到酶标板孔中，室温下静置孵育2h，然后弃上清；
4)洗涤：用200μl/孔PBST(PBS含0.1％Tween20)洗涤3次；
5)第二抗原孵育：加入稀释好的第二抗原(经HRP标记的PD-L1蛋白)，第二抗原以1∶1000稀释后进行使用，
稀释液为1％BSA的PBS，体积为100μl/孔，室温孵育1h；
6)洗涤：用200μl/孔PBST洗涤5次；
7)显色：加TMB显色液(配制A，B显色液，购自武汉博士德公司；按A∶B＝1∶1混匀，即用即配)100μl/孔，37℃显色5min。
8)加2M HCl终止液100μl/孔，终止液加入后，在30min内进行酶标仪450nm读数。
对Fab5、HAb5多肽片段、两者未剪接的混合物及两者经由内含肽体外剪接后的多肽片段Fab5+HAb5的ELISA检测结果示于图9。
由图9可知，Fab5+HAb5(剪接1)具有同时结合CD38和PD-L1两种抗原的活性。而经体外未剪接的混合物，以及单独的组分A(Fab5)和组分B(HAb5)不具有同时结合两种抗原的活性。
结果可以证明，利用本发明的内含肽及其含有的新型侧翼序列对进行剪接，得到的所述Fab5+HAb5(剪接1)剪接产物具备良好的双特异性抗体活性。
●剪接产物的肽图覆盖检测
肽段覆盖率是指检测到的肽段的氨基酸数量占该蛋白质总氨基酸数量的比例。
蛋白质供试品肽段覆盖率的检测，对于蛋白质类药物的一级氨基酸序列的确证，保证蛋白质类药物的高级结构的形成及维持蛋白质类药物性质都具有重要的意义。目前，对蛋白质肽段覆盖率的检测根据药物申报要求的规定，均采用质谱法进行检测。可以快速、准确、高效的完成肽段覆盖率的检测。
本实施例对蛋白质Fab5+HAb5(剪接产物1)的肽段覆盖率进行了分析，使用胰蛋白酶、胰凝乳蛋白酶和Glu-C酶分别对蛋白质Fab5+HAb5(剪接产物1)进行酶解，然后使用LC-MS/MS(XevoG2-XS QTof，waters)对酶解后的肽段样品进行分析。并使用UNIFI(1.8.2，Waters)软件对LC-MS/MS数据进行分析，根据算法结果确定了Fab5+HAb5(剪接产物1)的肽段覆盖率。
实验仪器：
1)高分辨质谱仪：XevoG2-XS QTof(Waters公司)
2)超高效液相色谱：UPLC(Acquity UPLC I-Class)(Waters公司)
材料和试剂：
1)Guanidine HCl(Sigma)
2)Urea(Bio-Rad)
3)Tris-base(Bio-Rad)
4)DTT(Bio-Rad)
5)IAM(Sigma)
6)Zeba Spin column(Pierce)
7)ACQUITY UPLC CSH C18 Column，
Figure PCTCN2020114271-appb-000048
1.7μm，2.1mm X 150mm(Waters)
8)UNIFI(Waters)
9)胰蛋白酶(Trypsin，Promega)
10)胰凝乳蛋白酶(Chymotrypsin，Sigma)
11)Glu-C酶(Wako)
实验方法
1)胰蛋白酶、胰凝乳蛋白酶、Glu-C酶解：取适量Fab5+HAb5(剪接1)经适当前处理后分别加入胰蛋白酶、胰凝乳蛋白酶、Glu-C酶，37℃酶切20小时。
2)高效液相色谱：Fab5+HAb5(剪接产物1)经酶解处理后采超高效液相***Acquity UPLC I-Class进行分离。液相A液为0.1％FA水溶液，B液为0.1％FA乙腈溶液。Fab5+HAb5(剪接产物1)由自动进样器上样到Column，再经色谱柱分离，柱温为55℃，流速为300μl/min，TUV检测器波长为214nm。相关液相梯度如表37。
表37 高效液相色谱A、B溶液比例
  时间/min A液比例(％) B液比例(％)
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
3)质谱鉴定：Fab5+HAb5(剪接产物1)经超高效液相色谱脱盐及分离后用XevoG2-XS QTof质谱仪(Waters)进行质谱检测分析。分析时长：63min，检测方式：正离子，MS，扫描范围(m/z)：300-2000。
4)质谱数据处理：原始使用UNIFI(1.8.2，Waters)软件查库，主要参数如(表38)：
表38 质谱数据处理主要参数列表
Figure PCTCN2020114271-appb-000049
Figure PCTCN2020114271-appb-000050
实验结果和分析
在使用胰蛋白酶、胰凝乳蛋白酶、Glu-C酶分别对Fab5+HAb5(剪接产物1)进行溶液内酶解后得到的肽段样品，经过LC-MS/MS设备的分析，经过UNIFI软件对得到的原始数据进行了查库。所使用的数据库为客户提供的 Fab5+HAb5(剪接产物1)理论序列。
1)Fab5+HAb5(剪接产物1)的酶解后的BPI图谱见图10A～C。
2)胰蛋白酶、胰凝乳蛋白酶、Glu-C酶等酶解覆盖率分别为：
胰蛋白酶酶解后覆盖率100％，
胰凝乳蛋白酶酶解后覆盖率100％，
Glu-C酶酶解后覆盖率100％，
酶解后的样品经过LC-MS/MS分析后查库结果进行整合，最终得到该Fab5+HAb5(剪接1)的肽段覆盖率为100.00％。基于内含肽的剪接原理，根据本发明中获得的剪接产物分子量、双抗原夹心ELISA及肽图覆盖检测结果，可推测通过本发明获得了有效且类天然IgG结构的双特异性抗体，试验结果确认该双特异性抗体的结构是两条不同重链和两条不同轻链组合成的异二聚体IgG结构，而不是两条相同重链两条和相同轻链组合成的同二聚体IgG结构的混合物。
实施例4内含肽介导的不同IgG亚型的体外剪接
(1)组分A的序列
如表39所示，三种不同IgG亚型的组分A对应的序列为：
表39 人的IgG2，IgG3和IgG4的组分A对应的序列
Figure PCTCN2020114271-appb-000051
如表40所示，三种不同IgG亚型的组分B对应的序列为：
表40.人的IgG2，IgG3和IgG4的组分B对应的序列
Figure PCTCN2020114271-appb-000052
按表41中的配对与实施例2同样地进行了转染。转染条件为：质粒摩尔数比例为pTag-Ic-FSb-(B-FcIcxxx)∶pA-HIn(xxx)∶pA-L(1)＝3∶1∶1。并与上述同样地设置了阳性对照单抗。
表41 内含肽的共转染表达不同IgG亚型的配对表
Figure PCTCN2020114271-appb-000053
经转染的细胞培养5天后取上清。对上清中的蛋白进行proteinA亲和层析，proteinA亲和层析后，通过SDS-PAGE法(加还原剂)进行考马斯亮蓝染色检测上清中的蛋白。结果示于图11。
根据结果可知，应用所述的内含肽在人IgG2，IgG3和IgG4亚型中均发生了显著剪接。A102为内含肽PhoRadA应用于人IgG2亚型的组分A和组分B的胞内表达，可在胞内发生剪接形成完整的IgG2单抗；A103为内含肽PhoRadA应用于人IgG3亚型的组分A和组分B的胞内表达，可在胞内发生剪接形成完整的IgG3单抗；A104为内含肽IMPDH-1应用于人IgG4亚型的组分A和组分 B的胞内表达，可在胞内发生剪接形成完整的IgG4单抗。
实施例5内含肽介导的绿色荧光蛋白的体外剪接
绿色荧光蛋白为EGFP(来源：UniProtKB-A0A076FL24)，其全长氨基酸序列为SEQ ID No：23，共239个氨基酸残基。将其序列分为组分A和组分B，其中(1)组分A为EGFP第1～158位氨基酸与内含肽的融合，对应的编码DNA构建至真核表达载体pcDNA3.1中，C端增加侧翼序列a、内含肽N端和终止密码子(TAA，TGA或TAG)，构建的表达质粒命名见表42；(2)组分B为EGFP第159～239位氨基酸与内含肽的融合，对应的编码DNA构建至真核表达载体pcDNA3.1中，并在其N端增加起始密码子ATG、内含肽C端和侧翼序列b，C端增加终止密码子(TAA，TGA或TAG)，构建的表达质粒命名见表43。另外构建EGFP全长蛋白编码DNA至pcDNA3.1(含终止密码子)，该质粒记命名为pEGFP。
表42 EGFP的组分A表达质粒命名
Figure PCTCN2020114271-appb-000054
表43 EGFP的组分B表达质粒命名
Figure PCTCN2020114271-appb-000055
将质粒pEGFP-A和pEGFP单独转染或共转染至293细胞或CHO细胞中，共转染比例为1∶1，转染方式参考实施例1，另外单独转染pEGFP至293或CHO细胞中作为阳性对照。单转或共转的每个质粒浓度均保持相同。转染48小时用流式细胞仪检测细胞的绿色荧光表达情况，具体统计见表44。
表44 转染48小时的293细胞的绿色荧光表达情况统计
转染质粒 平均荧光强度 荧光细胞比例
pEGFP 1×10^5 99％
pGFP-N1+pGFP-C1 3×10^4 57％
pGFP-N1 221 0.1％
pGFP-C1 105 0
pGFP-N2+pGFP-C2 9.9×10^4 99％
pGFP-N2 277 0.1％
pGFP-C2 146 0
pGFP-N3+pGFP-C3 1×10^4 47％
pGFP-N3 177 0
pGFP-C3 133 0
pGFP-N4+pGFP-C4 7×10^4 88％
pGFP-N4 321 0.2％
pGFP-C4 152 0
pGFP-N5+pGFP-C5 8×10^4 95％
pGFP-N5 274 0.1％
pGFP-C5 106 0
空白对照 139 0
根据以上结果可知，不同内含肽及侧翼序列均可在细胞内有效剪接绿色荧光蛋白，并形成与原绿色荧光蛋白非常相似的结构，从而产生绿色荧光。组分A或组分B单独表达均不能发出绿色荧光。
工业实用性
本发明提供了利用具备新型侧翼序列对的断裂型内含肽制备重组多肽，特别是双特异性抗体的方法。根据本发明的具备新型侧翼序列对的断裂型内含肽，可以广泛用于在医药、生物工程领域制备重组多肽，特别是抗体领域，尤其是双特异性抗体的制备。使用本发明的具备新型侧翼序列对的断裂型内含肽制备的双特异性抗体，不存在非天然的结构域，其结构与天然抗体(IgA、IgD、IgE、IgG或IgM)的结构极其相似，并具有Fc结构域。所述双特异性抗体的结构完整稳定性好，可以根据不同的IGG亚型保留或去除CDC(补体依赖的细胞毒性)或者ADCC(抗体依赖的细胞毒作用)或者ADCP(抗体依赖的细胞吞噬作用)或者FcRn(Fc受体)结合活性。
利用本发明的方法制备的双特异性抗体的体内半衰期长，免疫原性低；不引入任何形式的连接肽，抗体分子稳定性提高，在体内的免疫反应降低。利用本发明的方法制备的双特异性抗体具有与野生型IgG一致的糖基化修饰，得到更好的生物学功能，并且更加稳定，体内半衰期长；利用由内含肽进行的体外剪接方法，可以完全避免传统方法中极易出现的重链错配、轻链错配的问题。
本发明的制备双特异性抗体的方法，可以用于生产人源化的双特异性抗体，以及全人序列的双特异性抗体。利用本发明的方法制备的这样的抗体的序列与人源抗体更接近，可以有效降低免疫反应的发生。本发明的制备双特异性抗体的方法可以不受抗体亚型(IgG、IgA、IgM、IgD、IgE，以及轻链K和λ型)的限制地构建任何双特异性的抗体。

Claims (12)

  1. 用于断裂型内含肽的侧翼序列对，其中，
    所述侧翼序列对包括：侧翼序列a和侧翼序列b；所述侧翼序列a位于断裂型内含肽N端蛋白质剪接区域(In)的N端，且介于N端外显肽(En)和In之间；所述侧翼序列b位于断裂型内含肽C端蛋白质剪接区域(Ic)的C端，且介于Ic和C端外显肽(Ec)之间；
    所述断裂型内含肽选自：SspDnaE、SspDnaB、MxeGyrA、MjaTFIIB、PhoVMA、TvoVMA、Gp41-1、Gp41-8、IMPDH-1或PhoRadA，
    (1)所述断裂型内含肽为IMPDH-1时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失，或优选为G或D；A -2为X或缺失，或优选为G或K；A -1选自G或T；
    B 1为S；B 2为I或T或S；B 3为X或缺失；
    优选地，
    侧翼序列a为G、XG、XGG、DKG或DKT且侧翼序列b为SI、ST、SS、SIX、STX或SSX；
    (2)所述断裂型内含肽为Gp41-8时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失；A -2选自N或D；A -1选自R或K；
    B 1为S或T；B 2为A或H；B 3为X或缺失，或优选为V、Y或T，
    优选地，
    侧翼序列a为NR、XNR、DK、XDK、DR或XDR且侧翼序列b为SA或SAX；
    (3)所述断裂型内含肽为SspDnaB时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失；A -2选自S或D；A -1选自G或K；
    B 1为S；B 2为I；B 3为X或缺失，或优选为E或T，
    优选地，
    侧翼序列a为SG、XSG、DK、XDK，侧翼序列b为SI或SIX；
    (4)所述内含肽为MjaTFIIB时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中
    A -3为X或缺失；A -2选自T或D；A -1选自Y；
    B 1为T；B 2为I或H；B 3为X或缺失，或优选为H或T；
    优选地，
    侧翼序列a为TY、DY、XTY或XDY，侧翼序列b为TI、TIX、TH或THX；
    (5)所述断裂型内含肽为PhoRadA时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失；A -2选自G或D；A -1选自K；
    B 1为T；B 2为Q或H；B 3为X或缺失，或优选为L或T，
    优选地，
    侧翼序列a为GK、XGK、DK或XDK，侧翼序列b为TQ、TH、TQX或THX；
    (6)所述断裂型内含肽为TvoVMA时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失；A -2选自G或D；A -1选自K；
    B 1为T；B 2为V或H；B 3为X或缺失，或优选为I或T，
    优选地，
    侧翼序列a为GK、XGK、DK或XDK，侧翼序列b为TV、TH、TVX或THX；
    (7)所述断裂型内含肽为MxeGyrA时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失；A -2选自R或D；A -1选自Y、K或T；
    B 1为T；B 2为E或H；B 3为X或缺失，或优选为A或T，
    优选地，
    侧翼序列a为RY、XRY、DK或XDK，侧翼序列b为TE、TH、TEX或THX；
    (8)所述断裂型内含肽为PhoVMA时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失；A -2选自G或D；A -1为K；
    B 1为T；B 2为V或H；B 3为X或缺失，或优选为I或T，
    优选地，
    侧翼序列a为GK、XGK、DK或XDK，侧翼序列b为TV、TH、TVX或THX；
    (9)所述断裂型内含肽为Gp41-1时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失；A -2选自G或D；A -1选自Y或K；
    B 1为S或T；B 2为S或H；B 3为X或缺失，或优选为S或T；
    优选地，
    侧翼序列a为GY、XGY、DK或XDK，侧翼序列b为SS、SH、SSX或SHX；
    (10)所述断裂型内含肽为SspDnaE时，
    侧翼序列a为A -3A -2A -1，侧翼序列b为B 1B 2B 3，其中：
    A -3为X或缺失；A -2选自G或D；A -1选自G、S或K；
    B 1为T或S；B 2为E或H；B 3为X或缺失，或优选为T；
    优选地，
    侧翼序列a为GG、XGG、GK、XGK、DK或XDK，侧翼序列b为SE、TH、SEX或THX；
    其中所述X为选自：G、A、V、L、M、I、S、T、P、N、Q、F、Y、W、K、R、H、D、E、C中的任一种氨基酸。
  2. 根据权利要求1所述的用于断裂型内含肽的侧翼序列对，其中，所述断裂型内含肽与所述侧翼序列对共同使用用于进行反式剪接，
    其中，
    所述SspDnaE由序列为SEQ ID NO：31的In和序列为SEQ ID NO：32的Ic组成，
    所述SspDnaB由序列为SEQ ID NO：33的In和序列为SEQ ID NO：34的Ic组成，
    所述MxeGyrA由序列为SEQ ID NO：35的In和序列为SEQ ID NO：36的Ic组成，
    所述MjaTFIIB由序列为SEQ ID NO：37的In和序列为SEQ ID NO：38的Ic组成，
    所述PhoVMA由序列为SEQ ID NO：39的In和序列为SEQ ID NO：40的Ic组成，
    所述TvoVMA由序列为SEQ ID NO：41的In和序列为SEQ ID NO：42的Ic组成，
    所述Gp41-1由序列为SEQ ID NO：43的In和序列为SEQ ID NO：44的Ic组成，
    所述Gp41-8由序列为SEQ ID NO：45的In和序列为SEQ ID NO：46的Ic组成，
    所述IMPDH-1由序列为SEQ ID NO：47的In和序列为SEQ ID NO：48的Ic组成，
    所述PhoRadA由序列为SEQ ID NO：49的In和序列为SEQ ID NO：50的Ic组成，
    优选地，
    (1)当所述断裂型内含肽为IMPDH-1时，所述侧翼序列a为XGG且侧翼序列b为SI、ST、SS；或侧翼序列a为DKG且侧翼序列b为SI、ST、SS；或侧翼序列a为DKT且侧翼序列b为SI、ST、SS；
    (2)当所述断裂型内含肽为Gp41-8时，所述侧翼序列a为NR且侧翼序列b为SAV；或侧翼序列a为DK且侧翼序列b为SAV；侧翼序列a为NR且侧翼序列b为SAT；或侧翼序列a为DK且侧翼序列b为SAT；
    (3)当所述断裂型内含肽为SspDnaB时，所述侧翼序列a为SG且侧翼序列b为SIE；
    (4)当所述断裂型内含肽为PhoRadA时，所述侧翼序列a为GK且侧翼序列b为TQL或THT；或侧翼序列a为DK且侧翼序列b为TQL或THT；
    (5)当所述断裂型内含肽为TvoVMA时，所述侧翼序列a为GK且侧翼序列b为TVI或THT；或侧翼序列a为DK且侧翼序列b为TVI或THT；
    (6)当所述断裂型内含肽为MxeGyrA时，所述侧翼序列a为RY且侧翼序列b为TEA或THT；或侧翼序列a为DK且侧翼序列b为TEA或THT；
    (7)当所述断裂型内含肽为MjaTFIIB时，所述侧翼序列a为TY且侧翼序列b为TIH；或侧翼序列a为TY且侧翼序列b为THT；
    (8)当所述断裂型内含肽为PhoVMA时，所述侧翼序列a为GK且侧翼序列b为TVI或THT；或侧翼序列a为DK且侧翼序列b为TVI或THT；
    (9)当所述断裂型内含肽为Gp41-1时，所述侧翼序列a为GY且侧翼序列b为SSS或SHT；或侧翼序列a为DK且侧翼序列b为SSS或SHT；
    (10)当所述断裂型内含肽为SspDnaE时，所述侧翼序列a为GG且侧翼序列b为SET或THT；或侧翼序列a为GK且侧翼序列b为SET或THT；或侧翼序列a为DK且侧翼序列b为SET或THT；
    其中所述X为选自：G、A、V、L、M、I、S、T、P、N、Q、F、Y、W、K、R、H、D、E、C中的任一种氨基酸。
  3. 重组多肽，其由利用权利要求1或2所述的用于断裂型内含肽的侧翼序列对进行反式剪接而获得。
  4. 根据权利要求3所述的重组多肽，其中，所述重组多肽由组分A与组分B经过反式剪接得到；
    在组分A中，所述侧翼序列a的N端与En的C端连接、且所述侧翼序列a的C端与所述In连接，任选在In的C端连接有标签蛋白；
    在组分B中，所述侧翼序列b的C端与Ec的N端连接、且所述侧翼序列b的N端与所述Ic连接，任选在Ic的N端连接有标签蛋白；
    其中，所述En与Ec的编码序列分别来自同一蛋白的N端部分和C端部分，
    优选地，所述标签蛋白选自SEQ ID NO：24、25、26、27、28、29或30。
  5. 根据权利要求3所述的重组多肽，其中，所述重组多肽由组分A与组分B经过反式剪接得到；
    在组分A中，所述侧翼序列a的N端与En的C端连接、且所述侧翼序列a的C端与所述In连接，任选在In的C端连接有标签蛋白；
    在组分B中，所述侧翼序列b的C端与Ec的N端连接、且所述侧翼序列b的N端与所述Ic连接，任选在Ic的N端连接有标签蛋白；
    其中，所述En与Ec的编码序列来自不同蛋白。
  6. 根据权利要求4或5所述的重组多肽，其为荧光蛋白、蛋白酶、信号肽、抗菌肽、抗体、或具备生物毒性的多肽。
  7. 根据权利要求4或5所述的重组多肽，其中，所述同一蛋白、或所述不同蛋白中 的一种以上为抗体。
  8. 根据权利要求7所述的重组多肽，其中，所述抗体为天然免疫球蛋白IgG、IgM、IgA、IgD、或IgE类，或免疫球蛋白亚类：IgG1、IgG2、IgG3、IgG4、IgG5，或不同类的轻链：κ、λ；或单域抗体；或
    所述抗体为全长抗体或抗体的功能片段。
  9. 根据权利要求8所述的重组多肽，其中，所述抗体的功能片段为选自：抗体重链可变区VH、抗体轻链可变区VL、抗体重链恒定区片段Fc、抗体重链恒定区1 CH1、抗体重链恒定区2 CH2、抗体重链恒定区3 CH3、抗体轻链恒定区CL、或单域抗体可变区VHH中的一种或多种。
  10. 根据权利要求7所述的重组多肽，其中，所述同一蛋白、或所述不同蛋白中的一种以上针对抗原或表位A具有特异性，
    所述抗原A包括：肿瘤细胞表面抗原、免疫细胞表面抗原、细胞因子、细胞因子受体、转录因子、膜蛋白、肌动蛋白、病毒、细菌、内毒素、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，所述表位A为所述抗原A的免疫原性表位。
  11. 根据权利要求10所述的重组多肽，其中，所述同一蛋白、或所述不同蛋白中的一种以上针对与抗原或表位A不同的抗原或表位B具有特异性，
    所述抗原B包括：肿瘤细胞表面抗原、免疫细胞表面抗原、细胞因子、细胞因子受体、转录因子、膜蛋白、肌动蛋白、病毒、细菌、内毒素、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，所述表位B为所述抗原B的免疫原性表位。
  12. 根据权利要求11所述的重组多肽，其为双特异性抗体，可同时结合抗原或表位A和B，优选为人源化的双特异性抗体或全人序列的双特异性抗体。
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