CN108264557A

CN108264557A - A kind of combination CD3 and T cell bear bifunctional molecule and its application of costimulatory molecules

Info

Publication number: CN108264557A
Application number: CN201611256643.8A
Authority: CN
Inventors: 朱化星; 陈帅; 廖远平
Original assignee: SINOBIO BIOTECH CO
Current assignee: Cytocares (shanghai) Inc
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2018-07-10
Anticipated expiration: 2036-12-30
Also published as: CN108264557B

Abstract

The invention belongs to biomedicine technical fields, and in particular to a kind of combination CD3 and T cell bear bifunctional molecule and its application of costimulatory molecules.The present invention will combine and activate the first functional domain of T cell surface C D3 molecules and can combine and blocking t cell bears the second functional domain of costimulatory molecules and is blended in same protein peptide chain and forms bifunctional molecule, using eukaryotic cell expression system production, expression product structure is single, purifying process is easy, protein yield is high, and preparation process and product are stablized；With AntiCD3 McAb and anti-T cell just（It is negative）The combination of costimulatory molecules full length antibody is compared, and bifunctional molecule of the present invention is more excellent to the amplification in vitro effect of T cell, and albumen dosage is less, and using simplicity, can directly be added by solution form, without optimizing the relative scale of two kinds of full length antibodies.

Description

A kind of combination CD3 and T cell bear bifunctional molecule and its application of costimulatory molecules

Technical field

The invention belongs to biomedicine technical fields, and in particular to a kind of combination CD3 and T cell bear the double of costimulatory molecules Functional molecular and its application.

Background technology

T lymphocytes (T lymphocyte) claim T cell from thymus gland (Thymus).Mature T cells are present in outer The thymus dependent area of all immune organs occupies core status in adaptability cellullar immunologic response, while resists in thymus-dependent Important booster action is also played in the humoral immune response of original induction.According to the difference of function, T cell can be divided into cytotoxicity T cell (Cytotoxic T lymphocyte, CTL), T helper cell (Helper T cell, Th) and regulatory T cells (Regulatory T cell, Treg).Wherein CTL expresses CD8, is the main effects cell of adaptability cellular immunity, main Function is endogenous antigen peptide/MHC I MHC molecule complex of specific recognition target cell surface, can secrete and wear after autoactivation The substances direct killing target cell (tumour such as Kong Su (Peforin), granzyme (Granzyme), particle cytolysin (Granulysin) The cell of cell or parasitic pathogenic infection), it can also pass through Fas/FasL signal pathway inducing target cell apoptosis；And Th is expressed CD4 by secreting different types of cell factor and directly interacting between other cells, adjusts the cell of CTL Activity participates in cellular immunity indirectly；In addition, Treg can inhibit target cell activation and secretion IL-10, TGF β by being in direct contact Etc. cell factors to cellullar immunologic response carry out negative regulation, it is more in immune tolerance, autoimmunity disease, infectious diseases and tumour etc. It plays an important role in kind disease.

Complete activation and the efficient amplification of CD8 positive T cells are that it effectively kills the basis of target cell, dependent on dual signal The effect of pipeline：The MHC I/ on wherein antigen presenting cell (Antigen presenting cell, APC) surface are endogenous Property the expression of antigenic peptide complexes specific recognition T cell TCR/CD3 compounds, lead to the cytoplasm section phase of CD3 and co-receptor CD8 Interaction activates the protein tyrosine kinase being connected with cytoplasm segment trailer, swashs CD3 cytoplasmic region immunity receptor tyrosine kinase Tyrosine phosphorylation in die body (Immunoreceptor tyrosine-based activation motif, ITAM) living, Enabling signal transduction molecule cascade reaction, activating transcription factor so that T cell primary activation, this is the first letter of T cell activation Number；Meanwhile T cell surface costimulatory molecules (Co-stimulatory molecule, for example, CD28,4-1BB, ICOS, OX40, GITR, CD40L, CD27, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT and BTLA etc.) it can be with APC cell surface phases Answer costimulatory molecules ligand (such as CD80, CD86,4-1BBL, B7RP-1, OX40L, GITRL, CD40, CD70, PD-L1, PD-L2, Galectin-9, HVEM etc.) it interacts, generate the second signal (costimulatory signal) of T cell activation：Wherein CD28,4-1BB, ICOS, OX40, GITR, CD40L and CD27 etc. belong to positive costimulatory molecules, with respective ligand (CD80, CD86, 4-1BBL, B7RP-1, OX40L, GITRL, CD40, CD70 etc.) second signal (positive costimulatory signal) caused by interaction It can lead to the complete activation of T cell；And CTLA-4, PD-1, LAG-3, TIM-3, TIGIT and BTLA etc. belong to negative costimulation point Son, with respective ligand (CD80, CD86, PD-L1, PD-L2, Galectin-9, HVEM etc.) interaction caused by second letter Number (negative costimulatory signal) can descend to reconcile the activation for terminating T cell.

Currently for the first signal pathway of T cell activation, appear in the newspapers and a system is designed and constructed by genetic engineering Row CD 3-resisting monoclonal full length antibody (Beverley PC et al., Eur J Immunol, 11:329-334,1981； Lanzavecchia A et al., Eur J Immunol, 17:105-111,1987；Yannelli JR et al., J Immunol Methods,130:91-100,1990).Existing experimental data shows that such monoclonal antibody being capable of specific recognition T cell The CD3 molecules on surface generate the first signal of T cell activation.However, only not only the first signaling pathways cannot effectively swash T cell living can cause T cell disability even to generate T cell death (the Activation induced of activation-inducing instead Cell death, AICD).In order to overcome this shortcoming of CD3 monoclonal full length antibodies, people design and construct anti-CD28, Activated form monoclonal full length antibody (the US Patent of the positive costimulatory molecules such as anti-4-1BB and anti-ICOS 20100168400A1；US Patent 20100183621A1；US Patent 009193789B2) or it is anti-PD-1, anti- Blocking-up type monoclonal full length antibody (the World Patent of the negative costimulatory molecules such as CTLA-4 and anti-lag-3 2013173223Al；US Patent 007452535B2；US Patent 2015116539A1), by resisting with AntiCD3 McAb overall length Body is used in combination, and complete dual signal activated channel can be provided for T cell.Make however, two kinds of monoclonal full length antibodies are combined There are still some shortcomings in concrete application for mode, such as significantly increase workload and the life of recombinant antibodies expression and purification Cost is produced, is applied to T cell Activated in Vitro with the relative scale of two kinds of full length antibodies must be optimized during amplification.In addition, two When a full length antibody is used in combination, to promote receptor activation, needing to add the antibody-solutions of higher concentration or arriving antibody coating To enhance its activation effect to receptor on culture plate or microballoon.

Invention content

In order to overcome the problems of in the prior art, the purpose of the present invention is to provide one kind in combination with CD3 and T Cell bears bifunctional molecule and its application of costimulatory molecules.

To achieve these goals and other related purposes, the present invention adopt the following technical scheme that：

The first aspect of the present invention, provides a kind of bifunctional molecule, and structure includes to combine and activating T cell table First functional domain of face CD3 molecules and the second functional domain that costimulatory molecules can be born with reference to simultaneously blocking t cell.

Preferably, the bifunctional molecule can be combined and be blocked while combining and activating T cell surface C D3 molecules T cell bears costimulatory molecules, so as to generate the first signal and the second signal needed for T cell activation.

Preferably, first functional domain is the antibody of AntiCD3 McAb, and second functional domain bears costimulation point for anti-T cell The antibody of son.

Preferably, the antibody is small molecular antibody.

Preferably, the antibody is selected from Fab antibody, Fv antibody or single-chain antibody (scFv).

Preferably, first functional domain is connected with second functional domain by junction fragment.The junction fragment Amino acid quantity can be >=2.

Preferably, the junction fragment is selected from the hinge area piece of the junction fragment or Immunoglobulin IgD as unit of G4S Section.

The G4S is specially GGGGS.The junction fragment as unit of G4S includes one or more G4S units.Example Such as, it is one, two, three or more than four G4S units that can include.In some embodiments of the present invention, one is listed In the bifunctional molecule of monomeric form, connected between the first functional domain and the second functional domain by the junction fragment as unit of G4S It connects, junction fragment G4S units containing there are three, the amino acid sequence of the junction fragment is as shown in SEQ ID NO.1.

The hinge area segment of the Immunoglobulin IgD can be the hinge Ala90-Val170 of Immunoglobulin IgD.This In some embodiments of invention, in the bifunctional molecule for listing a dimeric forms, the first functional domain and the second functional domain it Between connected by the hinge area segment of Immunoglobulin IgD, the hinge area segment of the Immunoglobulin IgD is immunoglobulin The hinge Ala90-Val170 of IgD, the amino acid sequence such as SEQ ID NO.3 of the hinge area segment of the Immunoglobulin IgD It is shown.The junction fragment can be interconnected to form dimer by disulfide bond.

Preferably, the C-terminal of first functional domain is connect with the N-terminal of second structural domain.

Preferably, first functional domain is the single-chain antibody of AntiCD3 McAb, and second functional domain bears common thorn for anti-T cell Swash the single-chain antibody of molecule, the single-chain antibody includes heavy chain variable region and light chain variable region.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.36. The amino acid sequence of the light chain variable region of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.37.

Preferably, the single-chain antibody that the anti-T cell bears costimulatory molecules can be the single-chain antibody of anti-PD-1, resist The single-chain antibody of CTLA-4, the single-chain antibody of anti-lag-3, the single-chain antibody of anti-TIM-3, anti-TIGIT single-chain antibody or anti- The single-chain antibody of BTLA it is any.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-PD-1 such as SEQ ID NO.39 institutes Show.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-PD-1 is as shown in SEQ ID NO.40.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CTLA-4 such as SEQ ID NO.42 institutes Show.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-CTLA-4 is as shown in SEQ ID NO.43.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-lag-3 such as SEQ ID NO.45 institutes Show.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-lag-3 is as shown in SEQ ID NO.46.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-TIM-3 such as SEQ ID NO.48 institutes Show.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-TIM-3 is as shown in SEQ ID NO.49.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-TIGIT such as SEQ ID NO.51 institutes Show.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-TIGIT is as shown in SEQ ID NO.52.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-BTLA such as SEQ ID NO.54 institutes Show.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-BTLA is as shown in SEQ ID NO.55.

In some embodiments of the invention, the amino acid sequence such as SEQ ID of the single-chain antibody of the AntiCD3 McAb are listed Shown in NO.35.The amino acid sequence of the single-chain antibody of the anti-PD-1 is as shown in SEQ ID NO.38.The list of the anti-CTLA-4 The amino acid sequence of chain antibody is as shown in SEQ ID NO.41.The amino acid sequence of the single-chain antibody of the anti-lag-3 such as SEQ Shown in ID NO.44.The amino acid sequence of the single-chain antibody of the anti-TIM-3 is as shown in SEQ ID NO.47.The anti-TIGIT Single-chain antibody amino acid sequence as shown in SEQ ID NO.50.The amino acid sequence of the single-chain antibody of the anti-BTLA is such as Shown in SEQ ID NO.53.

In the preferable case of this case, amino acid sequence such as SEQ ID NO.11, the SEQ of the bifunctional molecule of monomeric form ID NO.15, SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27 or SEQ ID NO.31 it is any shown in.Dimerization The amino acid sequence of the bifunctional molecule of body form such as SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.29 or SEQ ID NO.33 it is any shown in.

The second aspect of the present invention provides a kind of polynucleotides, encodes aforementioned bifunctional molecule.

The third aspect of the present invention provides a kind of expression vector, contains foregoing polynucleotides.

The fourth aspect of the present invention provides a kind of host cell, is converted by foregoing expression vectors.

The fifth aspect of the present invention provides a kind of method for preparing aforementioned bifunctional molecule, including：Structure is containing difunctional Then the expression vector of the gene order containing bifunctional molecule is converted into host cell and lured by the expression vector of molecular gene sequence Expression is led, is detached from expression product and obtains the bifunctional molecule.

In the preferable case of the present invention, the expression vector uses pcDNA3.1.The host cell uses Chinese hamster Gonad cell (Chinese hamster ovary ce1l, CHO).

The sixth aspect of the present invention provides the purposes that aforementioned bifunctional molecule is used to prepare T cell amplification in vitro agent.

The seventh aspect of the present invention provides a kind of T cell amplification in vitro agent, contains aforementioned bifunctional molecule.

The eighth aspect of the present invention discloses a kind of method of amplification in vitro T cell, including aforementioned bifunctional molecule is made For T cell.The method can be non-treatment purpose.

Compared with prior art, the present invention has the advantages that：

(1) present invention will combine and activate the first functional domain of T cell surface C D3 molecules and can combine and block The second functional domain that T cell bears costimulatory molecules is blended in same protein peptide chain formation bifunctional molecule, using eukaryocyte table Up to system production, expression product structure is single, and purifying process is easy, and protein yield is high, and preparation process and product are stablized；And resist CD3 monoclonals full length antibody is just being total to (negative) with anti-T cell if stimulation molecule monoclonal full length antibody is used in combination, two antibody Expression and purification need to be distinguished, preparation process is more complicated, and workload and production cost dramatically increase.

(2) bifunctional molecule of the present invention is single albumen, relative to AntiCD3 McAb full length antibody and anti-T cell just (negative) costimulatory molecules full length antibody is used in combination, more excellent to the Activated in Vitro and expanding effect of T cell, and albumen dosage is less, And it using simplicity, can directly be added by solution form, without optimizing the relative scale of two kinds of full length antibodies.

Description of the drawings

Fig. 1：A. monomeric form AntiCD3 McAb/anti- T cell bears the structure chart of costimulatory molecules bispecific antibody；B. dimer Form AntiCD3 McAb/anti- T cell bears the structure chart of costimulatory molecules bispecific antibody.

Fig. 2：A. the CD3-PD-1 BsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight protein Marker；Swimming Road 2：Reproducibility CD3-PD-1 BsAb_M；Swimming lane 3：Irreducibility CD3-PD-1 BsAb_M；B. the CD3-PD-1 purified BsAb_D SDS-PAGE analysis charts, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：Reproducibility CD3-PD-1 BsAb_D；Swimming lane 3：Irreducibility CD3-PD-1 BsAb_D.

Fig. 3 A：The ELISA qualification results of CD3-PD-1 BsAb_M, the curve in figure represent three kinds of testing results respectively：■ 1 μ g/ml recombinant antigen CD3-hFc are coated with,It is coated with 1 μ g/ml recombinant antigens PD-1-hFc；▲ it is not coated with the measure of any antigen As a result.

Fig. 3 B：The ELISA qualification results of CD3-PD-1 BsAb_D, the curve in figure represent three kinds of testing results respectively：■ It is coated with 1 μ g/ml recombinant antigens CD3-hFc；It is coated with 1 μ g/ml recombinant antigens PD-1-hFc；▲ it is not coated with the measure of any antigen As a result.

Fig. 4：CIK cell amplification times curve using peripheral blood PBMC as experimental cell, adds CD3-PD-1 BsAb_ respectively M, (Anti-CD3/Anti-CD28) is used in combination in CD3-PD-1 BsAb_D or AntiCD3 McAb/anti- CD28 monoclonal full length antibodies, always Meter culture 30 days, with the cell quantity counted every time divided by the cell quantity of the 1st day, cells expanded is compared in counting, wherein Control group：5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 coated cell culture plates；Experimental group 1：It is added under solution state 10ng/ml CD3-PD-1 BsAb_M；Experimental group 2：10ng/ml CD3-PD-1 BsAb_D are added under solution state.

Fig. 5：The CIK cell IFN-γ secretion of CD3-PD-1 bispecific antibodies mediation.Control group：It is compareed in Example 4 Group (Anti-CD3/Anti-CD28) cultivates the CIK cell 2 × 10 of 25 days⁵A, centrifuging and taking supernatant is detected by ELISA Kit The IFN-γ quantity of cell secretion, is defined as 1；Experimental group 1 and experimental group 2 are respectively to add CD3-PD-1 under solution state BsAb_M and CD3-PD-1 BsAb_D cultivate the CIK cell of 25 days, take the cell of quantity identical with control group, centrifuging and taking supernatant, The IFN-γ quantity divided by control group of detection cell secretion are the opposite secretory volume of IFN-γ.

Fig. 6：A. the CD3-CTLA-4 BsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight protein Marker； Swimming lane 2：Reproducibility CD3-CTLA-4 BsAb_M；Swimming lane 3：Irreducibility CD3-CTLA-4 BsAb_M；B. the CD3- purified CTLA-4 BsAb_D SDS-PAGE analysis charts, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：Reproducibility CD3-CTLA-4 BsAb_D；Swimming lane 3：Irreducibility CD3-CTLA-4 BsAb_D.

Fig. 7 A：The ELISA qualification results of CD3-CTLA-4 BsAb_M, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigen CD3-hFc,It is coated with 1 μ g/ml recombinant antigens CTLA-4-hFc；▲ it is not coated with any antigen Measurement result.

Fig. 7 B：The ELISA qualification results of CD3-CTLA-4 BsAb_D, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigens CD3-hFc；It is coated with 1 μ g/ml recombinant antigens CTLA-4-hFc；▲ it is not coated with any antigen Measurement result.

Fig. 8：CIK cell amplification times curve, using peripheral blood PBMC as experimental cell, adds CD3-CTLA-4 respectively (Anti-CD3/Anti- is used in combination in BsAb_M, CD3-CTLA-4 BsAb_D or AntiCD3 McAb/anti- CD28 monoclonal full length antibodies CD28), culture 30 days is amounted to, with the cell quantity counted every time divided by the cell quantity of the 1st day, cell amplification times is compared in counting Number, wherein control group：5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 coated cell culture plates；Experimental group 1：Solution shape 10ng/ml CD3-CTLA-4 BsAb_M are added under state；Experimental group 2：10ng/ml CD3-CTLA-4 are added under solution state BsAb_D。

Fig. 9：The CIK cell IFN-γ secretion of CD3-CTLA-4 bispecific antibodies mediation.Control group：It is right in Example 9 The CIK cell 2 × 10 of 25 days is cultivated according to group (Anti-CD3/Anti-CD28)⁵A, centrifuging and taking supernatant is examined by ELISA Kit The IFN-γ quantity of cell secretion is surveyed, is defined as 1；Experimental group 1 and experimental group 2 are respectively to add CD3- under solution state CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D cultivate the CIK cell of 25 days, take the cell of quantity identical with control group, from The heart takes supernatant, and the IFN-γ quantity divided by control group of detection cell secretion are the opposite secretory volume of IFN-γ.

Figure 10：A. the CD3-LAG-3 BsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight protein Marker； Swimming lane 2：Reproducibility CD3-LAG-3 BsAb_M；Swimming lane 3：Irreducibility CD3-LAG-3 BsAb_M；B. the CD3-LAG-3 purified BsAb_D SDS-PAGE analysis charts, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：Reproducibility CD3-LAG-3 BsAb_D；Swimming Road 3：Irreducibility CD3-LAG-3 BsAb_D.

Figure 11 A：The ELISA qualification results of CD3-LAG-3 BsAb_M, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigen CD3-hFc,It is coated with 1 μ g/ml recombinant antigens LAG-3-hFc；▲ it is not coated with any antigen Measurement result.

Figure 11 B：The ELISA qualification results of CD3-LAG-3 BsAb_D, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigens CD3-hFc；It is coated with 1 μ g/ml recombinant antigens LAG-3-hFc；▲ it is not coated with any antigen Measurement result.

Figure 12：CIK cell amplification times curve, using peripheral blood PBMC as experimental cell, adds CD3-LAG-3 respectively (Anti-CD3/Anti- is used in combination in BsAb_M, CD3-LAG-3 BsAb_D or AntiCD3 McAb/anti- CD28 monoclonal full length antibodies CD28), culture 30 days is amounted to, with the cell quantity counted every time divided by the cell quantity of the 1st day, cell amplification times is compared in counting Number, wherein control group：5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 coated cell culture plates；Experimental group 1：Solution shape 10ng/ml CD3-LAG-3 BsAb_M are added under state；Experimental group 2：10ng/ml CD3-LAG-3 are added under solution state BsAb_D。

Figure 13：The CIK cell IFN-γ secretion of CD3-LAG-3 bispecific antibodies mediation.Control group：It is right in Example 14 The CIK cell 2 × 10 of 25 days is cultivated according to group (Anti-CD3/Anti-CD28)⁵A, centrifuging and taking supernatant is examined by ELISA Kit The IFN-γ quantity of cell secretion is surveyed, is defined as 1；Experimental group 1 and experimental group 2 are respectively to add CD3- under solution state LAG-3 BsAb_M and CD3-LAG-3 BsAb_D cultivate the CIK cell of 25 days, take the cell of quantity identical with control group, centrifugation Supernatant is taken, the IFN-γ quantity divided by control group of detection cell secretion are the opposite secretory volume of IFN-γ.

Figure 14：A. the CD3-TIM-3 BsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight protein Marker； Swimming lane 2：Reproducibility CD3-TIM-3 BsAb_M；Swimming lane 3：Irreducibility CD3-TIM-3 BsAb_M；B. the CD3-TIM-3 purified BsAb_D SDS-PAGE analysis charts, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：Reproducibility CD3-TIM-3 BsAb_D；Swimming Road 3：Irreducibility CD3-TIM-3 BsAb_D.

Figure 15 A：The ELISA qualification results of CD3-TIM-3 BsAb_M, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigen CD3-hFc,It is coated with 1 μ g/ml recombinant antigens TIM-3-hFc；▲ it is not coated with any antigen Measurement result.

Figure 15 B：The ELISA qualification results of CD3-TIM-3 BsAb_D, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigens CD3-hFc；It is coated with 1 μ g/ml recombinant antigens TIM-3-hFc；▲ it is not coated with any antigen Measurement result.

Figure 16：CIK cell amplification times curve, using peripheral blood PBMC as experimental cell, adds CD3-TIM-3 respectively (Anti-CD3/Anti- is used in combination in BsAb_M, CD3-TIM-3 BsAb_D or AntiCD3 McAb/anti- CD28 monoclonal full length antibodies CD28), culture 30 days is amounted to, with the cell quantity counted every time divided by the cell quantity of the 1st day, cell amplification times is compared in counting Number.Wherein, control group：5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 wrapper sheets；Experimental group 1：It is added under solution state 10ng/ml CD3-TIM-3 BsAb_M；Experimental group 2：10ng/ml CD3-TIM-3 BsAb_D are added under solution state.

Figure 17：The CIK cell IFN-γ secretion of CD3-TIM-3 bispecific antibodies mediation.Control group：It is right in Example 19 The CIK cell 2 × 10 of 25 days is cultivated according to group (Anti-CD3/Anti-CD28)⁵A, centrifuging and taking supernatant is examined by ELISA Kit The IFN-γ quantity of cell secretion is surveyed, is defined as 1；Experimental group 1 and experimental group 2 are respectively to add CD3- under solution state TIM-3 BsAb_M and CD3-TIM-3 BsAb_D cultivate the CIK cell of 25 days, take the cell of quantity identical with control group, centrifugation Supernatant is taken, the IFN-γ quantity divided by control group of detection cell secretion are the opposite secretory volume of IFN-γ.

Figure 18：A. the CD3-TIGIT BsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight protein Marker； Swimming lane 2：Reproducibility CD3-TIGIT BsAb_M；Swimming lane 3：Irreducibility CD3-TIGIT BsAb_M；B. the CD3-TIGIT purified BsAb_D SDS-PAGE analysis charts, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：Reproducibility CD3-TIGIT BsAb_D；Swimming Road 3：Irreducibility CD3-TIGIT BsAb_D.

Figure 19 A：The ELISA qualification results of CD3-TIGIT BsAb_M, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigen CD3-hFc,It is coated with 1 μ g/ml recombinant antigens TIGIT-hFc；▲ it is not coated with any antigen Measurement result.

Figure 19 B：The ELISA qualification results of CD3-TIGIT BsAb_D, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigens CD3-hFc；It is coated with 1 μ g/ml recombinant antigens TIGIT-hFc；▲ it is not coated with any antigen Measurement result.

Figure 20：CIK cell amplification times curve, using peripheral blood PBMC as experimental cell, adds CD3-TIGIT respectively (Anti-CD3/Anti- is used in combination in BsAb_M, CD3-TIGIT BsAb_D or AntiCD3 McAb/anti- CD28 monoclonal full length antibodies CD28), culture 30 days is amounted to, with the cell quantity counted every time divided by the cell quantity of the 1st day, cell amplification times is compared in counting Number.Wherein, control group：5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 wrapper sheets；Experimental group 1：It is added under solution state 10ng/ml CD3-TIGIT BsAb_M；Experimental group 2：10ng/ml CD3-TIGIT BsAb_D are added under solution state.

Figure 21：The CIK cell IFN-γ secretion of CD3-TIGIT bispecific antibodies mediation.Control group：It is right in Example 24 The CIK cell 2 × 10 of 25 days is cultivated according to group (Anti-CD3/Anti-CD28)⁵A, centrifuging and taking supernatant is examined by ELISA Kit The IFN-γ quantity of cell secretion is surveyed, is defined as 1；Experimental group 1 and experimental group 2 are respectively to add CD3- under solution state TIGIT BsAb_M and CD3-TIGIT BsAb_D cultivate the CIK cell of 25 days, take the cell of quantity identical with control group, centrifugation Supernatant is taken, the IFN-γ quantity divided by control group of detection cell secretion are the opposite secretory volume of IFN-γ.

Figure 22：A. the CD3-BTLA BsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight protein Marker；Swimming Road 2：Reproducibility CD3-BTLA BsAb_M；Swimming lane 3：Irreducibility CD3-BTLA BsAb_M；B. the CD3-BTLA purified BsAb_D SDS-PAGE analysis charts, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：Irreducibility CD3-BTLA BsAb_D；Swimming Road 3：Reproducibility CD3-BTLA BsAb_D.

Figure 23 A：The ELISA qualification results of CD3-BTLA BsAb_M, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigen CD3-hFc,It is coated with 1 μ g/ml Recombinant antigen Bs TLA-hFc；▲ it is not coated with the survey of any antigen Determine result.

Figure 23 B：The ELISA qualification results of CD3-BTLA BsAb_D, the curve in figure represent three kinds of testing results respectively： ■ is coated with 1 μ g/ml recombinant antigens CD3-hFc；It is coated with 1 μ g/ml Recombinant antigen Bs TLA-hFc；▲ it is not coated with the survey of any antigen Determine result.

Figure 24：CIK cell amplification times curve, using peripheral blood PBMC as experimental cell, adds CD3-BTLA respectively (Anti-CD3/Anti- is used in combination in BsAb_M, CD3-BTLA BsAb_D or AntiCD3 McAb/anti- CD28 monoclonal full length antibodies CD28), culture 30 days is amounted to, with the cell quantity counted every time divided by the cell quantity of the 1st day, cell amplification times is compared in counting Number.Wherein, control group：5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 wrapper sheets；Experimental group 1：It is added under solution state 10ng/ml CD3-BTLA BsAb_M；Experimental group 2：10ng/ml CD3-BTLA BsAb_D are added under solution state.

Figure 25：The CIK cell IFN-γ secretion of CD3-BTLA bispecific antibodies mediation.Control group：It is right in Example 29 The CIK cell 2 × 10 of 25 days is cultivated according to group (Anti-CD3/Anti-CD28)⁵A, centrifuging and taking supernatant is examined by ELISA Kit The IFN-γ quantity of cell secretion is surveyed, is defined as 1；Experimental group 1 and experimental group 2 are respectively to add CD3- under solution state BTLA BsAb_M and CD3-BTLA BsAb_D cultivate the CIK cell of 25 days, take the cell of quantity identical with control group, centrifuging and taking Supernatant, the IFN-γ quantity divided by control group of detection cell secretion are the opposite secretory volume of IFN-γ.

Specific embodiment

First, term and abbreviation：

BsAb：Bispecific antibody (Bi-specific Antibody)

scFv：Single chain variable fragment (Single-chain variable fragment), also known as single-chain antibody

Fab：Antigen-binding fragment (Fragment of antigen binding)

Fv：Variable region fragment (Variable fragment)

V_H：Heavy chain variable region (Heavy chain variable region)

V_L：Light chain variable region (Light chain variable region)

Linker：Junction fragment

Extracellular domain：Extracellular region

Co-stimulatory molecule：Costimulatory molecules

CD3-PD-1 BsAb_M:The AntiCD3 McAb of monomeric form/anti- PD-1 bispecific antibodies

CD3-PD-1 BsAb_D:The AntiCD3 McAb of dimeric forms/anti- PD-1 bispecific antibodies

CD3-CTLA-4 BsAb_M:The AntiCD3 McAb of monomeric form/anti- CTLA-4 bispecific antibodies

CD3-CTLA-4 BsAb_D:The AntiCD3 McAb of dimeric forms/anti- CTLA-4 bispecific antibodies

CD3-LAG-3 BsAb_M:The AntiCD3 McAb of monomeric form/anti-lag-3 bispecific antibody

CD3-LAG-3 BsAb_D:The AntiCD3 McAb of dimeric forms/anti-lag-3 bispecific antibody

CD3-TIM-3 BsAb_M:The AntiCD3 McAb of monomeric form/anti- TIM-3 bispecific antibodies

CD3-TIM-3 BsAb_D:The AntiCD3 McAb of dimeric forms/anti- TIM-3 bispecific antibodies

CD3-TIGIT BsAb_M:The AntiCD3 McAb of monomeric form/anti- TIGIT bispecific antibodies

CD3-TIGIT BsAb_D:The AntiCD3 McAb of dimeric forms/anti- TIGIT bispecific antibodies

CD3-BTLA BsAb_M:The AntiCD3 McAb of monomeric form/anti- BTLA bispecific antibodies

CD3-BTLA BsAb_D:The AntiCD3 McAb of dimeric forms/anti- BTLA bispecific antibodies

2nd, bifunctional molecule

Bifunctional molecule of the present invention, structure include to combine and activate the of T cell surface C D3 molecules One functional domain and the second functional domain that costimulatory molecules can be born with reference to simultaneously blocking t cell.

Further, the bifunctional molecule can be combined and be hindered while combining and activating T cell surface C D3 molecules Disconnected T cell bears costimulatory molecules, so as to generate the first signal and the second signal needed for T cell activation.The T cell bears common thorn Sharp molecule includes but not limited to the mankind PD-1, CTLA-4, LAG-3, TIM-3, TIGIT and BTLA etc..

The present invention the first functional domain and the second functional domain are had no it is specifically limited, as long as can combine and activate T thin It is combined while cellular surface CD3 molecules and blocking t cell bears costimulatory molecules, so as to generate the first letter needed for T cell activation Number and second signal.For example, first functional domain can be the antibody of AntiCD3 McAb, second functional domain can be anti-T Cell bears the antibody of costimulatory molecules.The antibody can be arbitrary form.But the either antibody of which kind of form, antigen knot It closes position and contains heavy chain variable region and light chain variable region.The antibody preferably can be small molecular antibody.The small molecule Antibody is the smaller antibody fragment of molecular weight, and antigen-binding site includes heavy chain variable region and light chain variable region.Described small point Though the molecular weight of sub- antibody is small but maintains the affinity of parent's monoclonal antibody, the specificity for having parent's monoclonal antibody the same.Described small point The type of sub- antibody mainly includes Fab antibody, Fv antibody, single-chain antibody (scFv) etc..Fab antibody is (variable by complete light chain Area V_LWith constant region C_L) and Fd sections of (variable region V of heavy chain_HWith the first constant region C_H1) it connects to be formed by disulfide bond.Fv antibody is only It is connected by light chain with the variable region of heavy chain by non-covalent bond, is the minimum function that antibody molecule retains intact antigen binding site Segment.Single-chain antibody (scFv) is the single protein peptide chain that heavy chain variable region and light chain variable region are formed by connecting by junction fragment Molecule.

First functional domain is connected with second functional domain by junction fragment.The present invention does not have the order of connection Particular/special requirement, as long as not limiting the purpose of the present invention.For example, the C-terminal of first functional domain can be with described second The N-terminal connection of structural domain.The amino acid quantity of the junction fragment preferably >=2.The present invention does not have junction fragment yet Special limitation, as long as do not limit the purpose of the present invention.

Further, the junction fragment is selected from the hinge area of the junction fragment or Immunoglobulin IgD as unit of G4S Segment.

In the preferred embodiment, the structure diagram of the bifunctional molecule is as shown in Figure 1, for bispecific Antibody.The bifunctional molecule can be that monomeric form can also be dimeric forms.The present invention monomeric form it is difunctional The structure diagram of molecule contains that there are one the with CD3 antigen bindings as shown in A in Fig. 1, in the structure of the bifunctional molecule One functional domain and second functional domain that costimulatory molecules antigen binding is born with any T cell, first functional domain be with The single-chain antibody (scFv) of CD3 antigen bindings, second functional domain are to bear costimulatory molecules extracellular region with T cell The single-chain antibody (scFv) that (Extracellular domain) is combined.The knot of the bifunctional molecule of the dimeric forms of the present invention Structure schematic diagram is as shown in B in Fig. 1, containing there are two the first functional domains with CD3 antigen bindings in the structure of the bifunctional molecule The second functional domain for bearing with any T cell costimulatory molecules antigen binding with two, first functional domain are and CD3 antigen knots The single-chain antibody (scFv) of conjunction, second functional domain are to bear costimulatory molecules extracellular region (Extracellular with T cell Domain) the single-chain antibody (scFv) combined.The antigen binding potency of the bifunctional molecule of the dimeric forms of the present invention is single Two times or more of body form, the effect of amplification in vitro T cell is more excellent.

It can be the mankind PD-1, CTLA-4, LAG-3, TIM-3, TIGIT or BTLA etc. that the T cell, which bears costimulatory molecules,.

T cell bears costimulatory molecules mankind PD-1 (Uniprot ID：Q15116) the amino acid sequence of extracellular region such as SEQ Shown in ID NO.5, specially：

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV。

T cell bears costimulatory molecules mankind CTLA-4 (Uniprot ID：P16410) the amino acid sequence of extracellular region such as SEQ Shown in ID NO.6, specially：

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQV NLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD。

T cell bears costimulatory molecules mankind LAG-3 (Uniprot ID：P18627) the amino acid sequence of extracellular region such as SEQ Shown in ID NO.7, specially：

VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVG PGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRAS DWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTV LGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIH LQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQ LYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHL。

T cell bears costimulatory molecules mankind TIM-3 (Uniprot ID：Q8TDQ0) the amino acid sequence of extracellular region such as SEQ Shown in ID NO.8, specially：

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIE NVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQ ISTLANELRDSRLANDLRDSGATIRIG。

T cell bears costimulatory molecules mankind TIGIT (Uniprot ID：Q495A1) the amino acid sequence of extracellular region such as SEQ Shown in ID NO.9, specially：

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSL TVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP。

T cell bears costimulatory molecules mankind BTLA (Uniprot ID：Q7Z6A9) the amino acid sequence of extracellular region such as SEQ Shown in ID NO.10, specially：

KESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLP NDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPSKDEMASRPWLLYR。

Specifically, first functional domain is the single-chain antibody of AntiCD3 McAb.The single-chain antibody of the AntiCD3 McAb can including heavy chain Become area and light chain variable region.The amino acid sequence of the heavy chain variable region of the single-chain antibody of the AntiCD3 McAb such as SEQ ID NO.36 institutes Show.The amino acid sequence of the light chain variable region of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.37.Further, institute The amino acid sequence of the single-chain antibody of AntiCD3 McAb is stated as shown in SEQ ID NO.35.

Second functional domain bears the single-chain antibody of costimulatory molecules for anti-T cell.The anti-T cell bears costimulatory molecules Single-chain antibody include heavy chain variable region and light chain variable region.

The single-chain antibody that the anti-T cell bears costimulatory molecules can be the list of the single-chain antibody of anti-PD-1, anti-CTLA-4 Chain antibody, the single-chain antibody of anti-lag-3, the single-chain antibody of anti-TIM-3, the single-chain antibody of anti-TIGIT or the single-stranded of anti-BTLA resist Body it is any.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-PD-1 is as shown in SEQ ID NO.39.It is described anti- The amino acid sequence of the light chain variable region of the single-chain antibody of PD-1 is as shown in SEQ ID NO.40.The single-chain antibody of the anti-PD-1 Amino acid sequence as shown in SEQ ID NO.38.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CTLA-4 is as shown in SEQ ID NO.42.It is described The amino acid sequence of the light chain variable region of the single-chain antibody of anti-CTLA-4 is as shown in SEQ ID NO.43.The list of the anti-CTLA-4 The amino acid sequence of chain antibody is as shown in SEQ ID NO.41.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-lag-3 is as shown in SEQ ID NO.45.It is described The amino acid sequence of the light chain variable region of the single-chain antibody of anti-lag-3 is as shown in SEQ ID NO.46.The anti-lag-3 it is single-stranded The amino acid sequence of antibody is as shown in SEQ ID NO.44.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-TIM-3 is as shown in SEQ ID NO.48.It is described The amino acid sequence of the light chain variable region of the single-chain antibody of anti-TIM-3 is as shown in SEQ ID NO.49.The anti-TIM-3's is single-stranded The amino acid sequence of antibody is as shown in SEQ ID NO.47.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-TIGIT is as shown in SEQ ID NO.51.It is described The amino acid sequence of the light chain variable region of the single-chain antibody of anti-TIGIT is as shown in SEQ ID NO.52.The anti-TIGIT's is single-stranded The amino acid sequence of antibody is as shown in SEQ ID NO.50.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-BTLA is as shown in SEQ ID NO.54.It is described anti- The amino acid sequence of the light chain variable region of the single-chain antibody of BTLA is as shown in SEQ ID NO.55.The single-chain antibody of the anti-BTLA Amino acid sequence as shown in SEQ ID NO.53.

In the preferable case of this case, amino acid sequence such as SEQ ID NO.11, the SEQ of the bifunctional molecule of monomeric form ID NO.15, SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27 or SEQ ID NO.31 it is any shown in.Dimerization The amino acid sequence of the bifunctional molecule of body form such as SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.29 or SEQ ID NO.33 it is any shown in.But it is not limited to tool cited in preferably case of the invention Body form.

3rd, the polynucleotides of bifunctional molecule are encoded

The polynucleotides of the coding bifunctional molecule of the present invention, can be DNA form or rna form.DNA form packet Include cDNA, genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.

The polynucleotides of the coding bifunctional molecule of the present invention, can be by well known to those skilled in the art any It is prepared by appropriate technology.The technology sees the general description of this field, such as《Molecular Cloning:A Laboratory guide》(J. Pehanorm Brookers Deng, Science Press, 1995).Including but not limited to recombinant DNA technology, chemical synthesis the methods of；For example, by using overlap-extension PCR PCR methods.

In the preferred embodiment, the nucleotides sequence of the heavy chain variable region of the single-chain antibody of the AntiCD3 McAb is encoded Row are as shown in SEQ ID NO.57.Encode the nucleotide sequence such as SEQ ID of the light chain variable region of the single-chain antibody of the AntiCD3 McAb Shown in NO.58.The nucleotide sequence of the single-chain antibody of the AntiCD3 McAb is encoded as shown in SEQ ID NO.56.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-PD-1 is encoded as shown in SEQ ID NO.60.It compiles The nucleotide sequence of the light chain variable region of the single-chain antibody of the code anti-PD-1 is as shown in SEQ ID NO.61.It encodes described anti- The nucleotide sequence of the single-chain antibody of PD-1 is as shown in SEQ ID NO.59.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-CTLA-4 is encoded as shown in SEQ ID NO.63. The nucleotide for encoding the light chain variable region of the single-chain antibody of the anti-CTLA-4 is arranged as shown in SEQ ID NO.64.It encodes described anti- The nucleotide sequence of the single-chain antibody of CTLA-4 is as shown in SEQ ID NO.62.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-lag-3 is encoded as shown in SEQ ID NO.66. The nucleotide sequence of the light chain variable region of the single-chain antibody of the anti-lag-3 is encoded as shown in SEQ ID NO.67.Described in coding The nucleotide sequence of the single-chain antibody of anti-lag-3 is as shown in SEQ ID NO.65.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-TIM-3 is encoded as shown in SEQ ID NO.69. The nucleotide sequence of the light chain variable region of the single-chain antibody of the anti-TIM-3 is encoded as shown in SEQ ID NO.70.Described in coding The nucleotide sequence of the single-chain antibody of anti-TIM-3 is as shown in SEQ ID NO.68.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-TIGIT is encoded as shown in SEQ ID NO.72. The nucleotide sequence of the light chain variable region of the single-chain antibody of the anti-TIGIT is encoded as shown in SEQ ID NO.73.Described in coding The nucleotide sequence of the single-chain antibody of anti-TIGIT is as shown in SEQ ID NO.71.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-BTLA is encoded as shown in SEQ ID NO.75.It compiles The nucleotide sequence of the light chain variable region of the single-chain antibody of the code anti-BTLA is as shown in SEQ ID NO.76.It encodes described anti- The nucleotide sequence of the single-chain antibody of BTLA is as shown in SEQ ID NO.74.

The nucleotide sequence of junction fragment of the encoding amino acid sequence as shown in SEQ ID NO.1 such as SEQ ID NO.2 institutes Show.

The nucleotide sequence of junction fragment of the encoding amino acid sequence as shown in SEQ ID NO.3 such as SEQ ID NO.4 institutes Show.

Further, the nucleotide sequence of the bifunctional molecule of coded cell form such as SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28 or SEQ ID NO.32 it is any shown in.Encode dimerization The nucleotide sequence of the bifunctional molecule of body form is as such as SEQ ID NO.14, SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.26, SEQ ID NO.30 or SEQ ID NO.34 it is any shown in.

4th, expression vector

The expression vector of the present invention contains the polynucleotides for encoding the bifunctional molecule.Those skilled in the art Well known method can be used to build the expression vector.These methods include recombinant DNA technology, DNA synthetic technologys etc..It can will compile The DNA of the code fusion protein is effectively connected in the multiple cloning sites in carrier, mRNA to be instructed to synthesize and then expresses albumen, Or for homologous recombination.In the preferable case of the present invention, the expression vector uses pcDNA3.1.The host cell uses Chinese hamster ovary cell (Chinese hamster ovary ce1l, CHO).

5th, the method for preparing bifunctional molecule

The method for preparing aforementioned bifunctional molecule of the present invention, including：Build the table containing bifunctional molecule gene order Up to carrier, then the expression vector of the gene order containing bifunctional molecule is converted to induced expression in host cell, is produced from expression Separation obtains the bifunctional molecule in object.In the preferable case of the present invention, the expression vector uses pcDNA3.1.It is described Host cell uses Chinese hamster ovary cell (Chinese hamster ovary ce1l, CHO).

6th, the purposes of bifunctional molecule

The bifunctional molecule of the present invention can be used for preparing T cell amplification in vitro agent.In present pre-ferred embodiments, pass through examination It verifies that the bifunctional molecule of bright monomer and dimeric forms is respectively provided with and bears costimulatory molecules recombinant antigen to CD3 and corresponding T cell It is external combine activity, can be applied to T cell Activated in Vitro and amplification, wherein dimer has better effect compared with monomer.

7th, the method for amplification in vitro T cell

The method of amplification in vitro T cell of the present invention, including aforementioned bifunctional molecule is acted on T cell.It is described Method can be non-treatment purpose.In present pre-ferred embodiments, monomer and double work(of dimeric forms are proved by experiment Energy molecule is respectively provided with the external combination activity that costimulatory molecules recombinant antigen is born to CD3 and corresponding T cell, can be applied to T cell Activated in Vitro and amplification, wherein dimer have better effect compared with monomer.

The present invention is led to for AntiCD3 McAb and the shortcoming of anti-T cell just (negative) costimulatory molecules full length antibody use in conjunction Crossing the method structure of genetic engineering and antibody engineering can identify and activate CD3 and identify and any one T cell is blocked to bear altogether The bifunctional molecule of stimulation molecule, which not only has the characteristic that above-mentioned double antibody is used in combination, while is preparing There is apparent advantage in terms of technique and practical application, can reach when adding in the form of a solution even better than two it is antibody combined Addition or the effect of coating culture plate substantially increase the effect of Activated in Vitro is with amplification T cell, increase the convenience used Property.

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention.The test method of actual conditions is not specified in the following example, Usually according to normal condition or the condition proposed by according to each manufacturer.

When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, according to record of the those skilled in the art to the grasp of the prior art and the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

Embodiment 1：The structure of CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D carrier for expression of eukaryon

In the present invention, costimulatory molecules PD-1 albumen is born as target spot using T cell surface mankind CD3 albumen and T cell Bispecific antibody is named as CD3-PD-1 BsAb.

First, CD3-PD-1 BsAb_M and the design of CD3-PD-1 BsAb_D constructing plans

The specific constructing plans of CD3-PD-1 BsAb_M of monomeric form are：AntiCD3 McAb scFv and anti-PD-1 scFv sequences it Between pass through (GGGGS)₃Linker is connected.

The specific constructing plans of CD3-PD-1 BsAb_D of dimeric forms are：AntiCD3 McAb scFv and anti-PD-1 scFv sequences Between Linker be used as by IgD hinge areas be connected.

For bispecific antibody is made to be expressed in mammalian cell, for AntiCD3 McAb scFv, anti-PD-1 scFv and connection The sequence of segment (Linker) has carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.57, specially：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGG CTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCA ACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGC ACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCA CTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

Specifically, the nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.58, specially：

GACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAG CAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCA AGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATG GAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCT GGAGCTGAAG。

Specifically, the nucleotide sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.56, specially：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGG CTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCA ACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGC ACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCA CTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCG GCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAG AAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCC CAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCA GCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCC CTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

Specifically, the nucleotide sequence of the heavy chain variable region of anti-PD-1 scFv is as shown in SEQ ID NO.60, specially：

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGGACTGCAAGGCCAGCGG CATCACCTTCAGCAACAGCGGCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCT GGTACGACGGCAGCAAGCGCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAAC ACCCTGTTCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCACCAACGACGACTACTG GGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

Specifically, the nucleotide sequence of the light chain variable region of anti-PD-1 scFv is as shown in SEQ ID NO.61, specially：

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAG CCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCA GCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGCACCTTCGGCCAGGGCACCAA GGTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of anti-PD-1 scFv is as shown in SEQ ID NO.59, specially：

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGGACTGCAAGGCCAGCGG CATCACCTTCAGCAACAGCGGCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCT GGTACGACGGCAGCAAGCGCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAAC ACCCTGTTCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCACCAACGACGACTACTG GGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG AGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGC CAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAG CAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCC TGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGCACCTTCGGCCAGGGCACCAAG GTGGAGATCAAGCGC。

The nucleotide sequence of the CD3-PD-1 BsAb_M junction fragments of monomeric form is as shown in SEQ ID NO.2, specifically For：

GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC。

The nucleotide sequence of the CD3-PD-1 BsAb_D junction fragments of dimeric forms is as shown in SEQ ID NO.4, specifically For：

GCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCA GCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGG AGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAG CCCCTGGGCGTG。

For bispecific antibody is made to be expressed in CHO-S cells and in successful secretion to culture medium, has selected antibody-secreting The signal peptide of type expression is used for this embodiment.

The amino acid sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.77, specially：

MTRLTVLALLAGLLASSRA。

The nucleotide sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.78, specially：

ATGACCCGCCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCAGCAGCCGCGCC。

2nd, CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D construction of eukaryotic expression vector

The construction and expression of bispecific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1 (being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the bispecific antibody of dimeric forms, separately design Primer as shown in table 1, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplification Zhou Hongxun Science and Technology Ltd.s synthesize.

It builds for the clone of CD3-PD-1 BsAb_M, is amplified first using primer pcDNA3.1-Sig-F and Sig-R Then signal peptide fragment is utilized respectively primer Sig-CD3-F and CD3-R, CD3- (GGGGS)₃- PD-1-F and pcDNA3.1-PD- 1-R amplifies AntiCD3 McAb scFv, (GGGGS)₃The gene order of Linker, anti-PD-1 scFv；For CD3-PD-1 BsAb_D Clone structure, equally amplify signal peptide fragment using primer pcDNA3.1-Sig-F and Sig-R first, be then utilized respectively Primer Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-PD-1-F and pcDNA3.1-PD-1-R amplify AntiCD3 McAb The gene order of scFv, IgD hinge area, anti-PD-1 scFv.After amplification, utilizeMono- step directed clonings of PCR Kit (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.) splices monomer respectively and dimeric forms bispecific antibody is complete Long gene order simultaneously seamless is cloned on the pcDNA3.1 expression vectors through EcoRI and HindIII linearization process.Purpose carrier Bacillus coli DH 5 alpha is converted, positive clone identification is carried out using bacterium colony PCR, positive recon (recombinant plasmid) is accredited as and carries out Sequencing identification.Correct recon (recombinant plasmid) will then be sequenced to arrange to take out in plasmid, for the transfection of CHO-S cells.

Know through sequencing, the CD3-PD-1 BsAb_M of monomeric form and the CD3-PD-1 BsAb_D's of dimeric forms is complete Long gene order is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD3-PD-1 BsAb_M of monomeric form is as shown in SEQ ID NO.12, specifically For：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGGACTG CAAGGCCAGCGGCATCACCTTCAGCAACAGCGGCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGG TGGCCGTGATCTGGTACGACGGCAGCAAGCGCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAC AACAGCAAGAACACCCTGTTCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCACCAA CGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGC TGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGAT CTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGCACCTTCGGC CAGGGCACCAAGGTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of the CD3-PD-1 BsAb_D of dimeric forms is as shown in SEQ ID NO.14, specifically For：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGG CCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCAC CACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGG AGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGGAGAGC GGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGGACTGCAAGGCCAGCGGCATCACCTTCAGCAACAGCGG CATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAAGCGCT ACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTTCCTGCAGATGAAC AGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTGGTGAC CGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCC CCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTG GCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCC CGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCG TGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

The primer used in table 1.CD3-PD-1 bispecific antibody gene clonings

Embodiment 2：The expression and purification of CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D

First, the expression of CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D

1.1.CHO-S 1 day passage density is 0.5 before cell (being purchased from Thermo Fisher Scientific companies) transfection ~0.6 × 10⁶/ml；

1.2. the transfection same day carries out cell density statistics, when density is 1~1.4 × 10⁶/ ml, vigor>When 90%, it can use In plasmid transfection；

1.3. transfection composite is prepared：Each project (CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D) need to prepare two A centrifuge tube/culture bottle, by taking 20ml as an example, is placed respectively, prepared recombinant plasmid in Example 1：

Manage 1. 600 μ l PBS of middle addition, 20 μ g recombinant plasmids, mixing；

Manage 2. 600 μ l PBS, 20ul FreeStyle of middle addition^TMMAX Transfection Reagent (are purchased from Thermo Fisher Scientific companies), mixing；

1.4. it by the transfection reagent after dilution, adds in the recombinant plasmid to after diluting, is uniformly mixed, it is multiple to be configured to transfection Close object；

1.5. after transfection composite stands 15~20min, single drop is at the uniform velocity added in cell culture；

1.6. in 37 DEG C, CO₂Concentration 8% carries out Transfected cells culture under the conditions of shaking speed 130rpm, is received after 5 days Collect culture supernatant and carry out destination protein detection of expression.

2nd, the purifying of CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D

2.1 sample pretreatment

Above-mentioned Transfected cells culture supernatant 20ml is taken, buffer solution 20mM PB, 200mM NaCl is added in and adjusts pH to 7.5；

2.2Protein L affinity chromatography column purifications

Protein purification chromatographic column：Protein L affinity columns (purchased from GE Healthcare companies, column volume 1.0ml)

Buffer solution A (Buffer A)：PBS, pH7.4

Buffer solution B (Buffer B)：0.1M Glycine,pH3.0

Buffer solution C (Buffer C)：0.1M Glycine,pH2.7

Purification process：Using 100 type protein purification systems of AKTA explorer (be purchased from GE Healthcare companies), Protein L affinity columns are pre-processed with Buffer A, take culture supernatant loading, collect efflux.After loading, with extremely Few 1.5ml Buffer A balance chromatographic column, are eluted respectively with Buffer B and Buffer C after balance, collect destination protein and wash (collecting pipe of eluent needs to be previously added 1% 1M Tris, pH8.0 to neutralize eluent pH value, Tris final concentrations de- liquid About 10mM), in finally concentration dialysis to buffer solution PBS.

CD3-PD-1 BsAb_M and CD3-PD-1 the BsAb_D recombinant proteins finally purified are analyzed through SDS-PAGE, reduction It is as shown in Figure 2 with electrophoretogram under non reducing conditions.It can be seen from the figure that through Protein L affinity columns after purification, CD3- The purity of PD-1 BsAb_M and CD3-PD-1 BsAb_D recombinant proteins is equal>95%；Wherein CD3-PD-1 BsAb_M recombinant proteins Theoretical molecular weight for 52.5kDa, single electrophoretic band, molecular weight and monomer is presented in the albumen under reduction and non reducing conditions Unanimously, therefore the bispecific antibody is monomeric form (Fig. 2A)；The theoretical molecular weight of CD3-PD-1 BsAb_D recombinant proteins is 60.4kDa, the protein electrophoresis band, non reducing conditions under the electrophoretic band institute consistent with monomer that be presented molecular weight under reducing condition It is consistent with dimer (Fig. 2 B) that molecular weight is presented, illustrates that two protein moleculars can be connected with each other by disulfide bond, therefore this pair is special Heterogenetic antibody is dimeric forms.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sample Equal frame is errorless, consistent with theoretical N/C terminal amino acid sequences, and mass spectral analysis further confirms that CD3-PD-1 BsAb_M are single Body form, CD3-PD-1 BsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence of the CD3-PD-1 BsAb_M of monomeric form as shown in SEQ ID NO.11, Specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSG GGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR。

The amino acid sequence of the CD3-PD-1 BsAb_D of dimeric forms is as shown in SEQ ID NO.13, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEK EKEEQEERETKTPECPSHTQPLGVQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWY DGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSEI VLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLE PEDFAVYYCQQSSNWPRTFGQGTKVEIKR。

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.35, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELK。

The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.36, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS。

The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.37, specially：

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTS YSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。

The amino acid sequence of anti-PD-1 scFv is as shown in SEQ ID NO.38, specially：

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFT ISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGER ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIKR。

The amino acid sequence of the heavy chain variable region of anti-PD-1 scFv is as shown in SEQ ID NO.39, specially：

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFT ISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS。

The amino acid sequence of the light chain variable region of anti-PD-1 scFv is as shown in SEQ ID NO.40, specially：

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR。

The amino acid sequence of junction fragment is as shown in SEQ ID NO.1 in the CD3-PD-1 BsAb_M of monomeric form, specifically For：

GGGGSGGGGSGGGGS。

The amino acid sequence of junction fragment is as shown in SEQ ID NO.3 in the CD3-PD-1 BsAb_D of dimeric forms, tool Body is：

ASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKT PECPSHTQPLGV。

Embodiment 3：The antigen-binding activity of ELISA detection CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD3-hFc (is purchased from Wujiang offshore protein section with mankind PD-1-hFc fusion proteins Skill Co., Ltd) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes, and coating condition is 37 DEG C 1 Hour or 4 DEG C overnight, coating buffer solution (PBS) formula be：3.58g Na₂HPO4,0.24g NaH₂PO4,0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing to 1L；

2. closing：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes, 37 DEG C of closings are added in 1 hour；

3. sample-adding：After PBS board-washings 4 times, the bispecific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1 Hour, sample gradient preparation method：Using CD3-PD-1 BsAb_M or the CD3-PD-1 BsAb_D that 10 μ g/ml are purified as starting Concentration, carries out 6 gradients of doubling dilution, and each gradient sets 2 multiple holes；

4. colour developing：It is dilute by 1/5000 with confining liquid PBSA after PBST (PBS+0.05%Tween-20 (V/V)) board-washing 4 times The colour developing antibody (purchased from Abcam companies) of HRP labels is released, is added in by 100 μ l/ holes, 37 DEG C are incubated 1 hour.After PBS board-washings 4 times, Developing solution TMB (being purchased from KPL companies) is added, 100 μ l/ holes, room temperature is protected from light colour developing 5~10 minutes；

5. it terminates reaction to measure with result：Add terminate liquid (1M HCl), 100 μ l/ holes, the 450nm wavelength in microplate reader Lower reading light absorption value.

ELISA results are as shown in Figure 3A and Figure 3B：Fig. 3 A illustrate CD3-PD-1 BsAb_M and recombinant antigen CD3-hFc and PD-1-hFc is respectively provided with external combination activity, and wherein PD-1 combines activity and combines activity higher compared with CD3；Fig. 3 B illustrate CD3-PD-1 BsAb_D is similary with recombinant antigen CD3-hFc and PD-1-hFc to have external combination activity, and wherein PD-1 combines active higher.

Embodiment 4：CIK (Cytokine induced killer) cell Proliferation of CD3-PD-1 bispecific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC) Material, with bispecific antibody CD3-PD-1 BsAb_M of the above-mentioned monomeric form prepared by the present invention, dimeric forms it is double special Antibody CD3-PD-1 BsAb_D and AntiCD3 McAb/anti- CD28 monoclonals full length antibody combination (Anti-CD3/Anti-CD28) point The people blood PBMC of same donor source is not acted on, is counted after cell culture, compares amplification times.

The separation of 1.PBMC：Anticoagulation is taken, adds in isometric medical saline, edge centrifugation tube wall is slowly added to and blood The isometric lymphocyte separation medium of liquid (is purchased from GE Healthcare companies), keeps liquid level layering apparent, 2000rpm centrifugations 20min draws the cellular layer of intermediate white haze shape in new centrifuge tube, adds in the PBS buffer solution washing of 2 times or more volume, 1100rpm centrifuges 10min, and repeated washing is primary, (public purchased from Lonza with 15 serum free mediums of X-vivo being pre-chilled on a small quantity Department) it is resuspended, cell count is for use；

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) are resuspended, It is 1 × 10 to adjust cell density⁶/ ml separately designs following 3 experimental groups：Control group (Anti-CD3 5ug/ml and Anti- CD285ug/ml coated cells culture plate)；Experimental group 1 (adds bispecific antibody CD3-PD-1 BsAb_ under solution state M10ng/ml)；Experimental group 2 (adds bispecific antibody CD3-PD-1 BsAb_D 10ng/ml) under solution state.In addition, 3 groups Experimental cell adds cell factor IFN-γ (200ng/ml, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) and IL-1 simultaneously β (2ng/ml, purchased from Wujiang Alongshore Protein Technology Co., Ltd.), is placed in incubator, in saturated humidity, 37 DEG C, 5.0%CO₂ Under conditions of cultivated.After overnight incubation, add the IL-2 of 500U/ml (purchased from Wujiang Alongshore Protein Technology Co., Ltd.) Continue to cultivate, be counted per 2-3 days and press 1 × 10 with the CIK basal mediums of addition 500U/ml IL-2⁶The density of/ml carries out Cell passes on.Method culture 30 days like this, the final amplification times for counting cell, draw growth curve；

Testing result as shown in figure 4, the CD3-PD-1 bispecific antibody single uses of monomer and dimeric forms to CIK The cultivation effect of cell is superior to AntiCD3 McAb/anti- CD28 monoclonal full length antibodies and is used in combination, after cultivating 18 days, Anti-CD3/ There are a large amount of cell deaths in Anti-CD28 combinations, and cells expanded is remarkably decreased；And add the CD3-PD-1 of monomeric form BsAb_M or the CD3-PD-1 BsAb_D of dimeric forms do not occur cell death, and only cell amplification rate is opposite slows down. Therefore the CD3-PD-1 bispecific antibodies of two kinds of forms that prepared by the present invention can effectively expand and extend the existence of CIK cell Phase, wherein dimeric forms effect are more preferable.

Embodiment 5：The CIK cell IFN-γ secretion of CD3-PD-1 bispecific antibodies induction

Operating procedure：

1st, after being cultivated 25 days in Example 4 CIK cell supernatant (be adjusted to same cell density, cell number for 2 × 10⁵It is a) 100 μ l, 37 DEG C of incubation 45min, it is detected by Human IFN-γ ELISA Kit (purchased from doctor's moral biology), Three groups of every group of experiments take three samples to repeat；

2nd, it is cleaned three times with PBS, the IFN-γ antibody of addition HRP labels, 37 DEG C of incubation 45min；

3rd, it is cleaned three times with PBS, addition TMB 100 μ l colour developings, color development at room temperature 5-10min；

4th, addition terminate liquid HCl (1M) is terminated, and light absorption value is read under 450nm wavelength.

The results are shown in Figure 5：Wherein secreted by the CIK cell of Anti-CD3/Anti-CD28 full length antibodies combination culture IFN-γ quantity is defined as 1, and the CIK cell IFN-γ of the CD3-PD-1 BsAb_M cultures of monomeric form is added under solution state It is 2.45 with respect to secretory volume, the CIK cell IFN-γ of the CD3-PD-1 BsAb_D cultures of addition dimeric forms under solution state It is 4.12 with respect to secretory volume, therefore the CD3-PD-1 bispecific antibodies of two kinds of forms prepared by the present invention are more advantageous to activating CIK cell and the secretion for inducing IFN-γ, wherein dimeric forms are better.

Embodiment 6：The structure of CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D carrier for expression of eukaryon

In the present invention, costimulatory molecules CTLA-4 albumen is born as target spot using T cell surface mankind CD3 albumen and T cell Bispecific antibody be named as CD3-CTLA-4 BsAb.

First, CD3-CTLA-4 BsAb_M and the design of CD3-CTLA-4 BsAb_D constructing plans

The specific constructing plans of CD3-CTLA-4 BsAb_M of monomeric form are：AntiCD3 McAb scFv and anti-CTLA-4 scFv sequences Pass through (GGGGS) between row₃Linker is connected.

The specific constructing plans of CD3-CTLA-4 BsAb_D of dimeric forms are：AntiCD3 McAb scFv and anti-CTLA-4 scFv It is used as Linker by IgD hinge areas between sequence to be connected.

For bispecific antibody is made to be expressed in mammalian cell, for AntiCD3 McAb scFv, anti-CTLA-4 scFv and company The sequence of tab segments (Linker) has carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.57.

Specifically, the nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.58.

Specifically, the nucleotide sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.56.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-CTLA-4 scFv is as shown in SEQ ID NO.63, specifically For：

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCC GCCAGCGGCTTCACCTTCAGCAGCTACACCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAC CTTCATCAGCTACGACGGCAACAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACA GCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCATCTACTACTGCGCCCGCACCGGC TGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

Specifically, the nucleotide sequence of the light chain variable region of anti-CTLA-4 scFv is as shown in SEQ ID NO.64, specifically For：

GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGC CGCGCCAGCCAGAGCGTGGGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGAT CTACGGCGCCTTCAGCCGCGCCACCGGCATCCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCCGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCTGGACCTTCGGC CAGGGCACCAAGGTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of anti-CTLA-4 scFv is as shown in SEQ ID NO.62, specially：

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCC GCCAGCGGCTTCACCTTCAGCAGCTACACCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAC CTTCATCAGCTACGACGGCAACAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACA GCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCATCTACTACTGCGCCCGCACCGGC TGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGCGCG CCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGGGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCC CCCCGCCTGCTGATCTACGGCGCCTTCAGCCGCGCCACCGGCATCCCCGACCGCTTCAGCGGCAGCGGCAGCGGCAC CGACTTCACCCTGACCATCAGCCGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCC CCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence of the CD3-CTLA-4 BsAb_M junction fragments of monomeric form is as shown in SEQ ID NO.2.

The nucleotide sequence of the CD3-CTLA-4 BsAb_D junction fragments of dimeric forms is as shown in SEQ ID NO.4.

The amino acid sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.77.

The nucleotide sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.78.

2nd, CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D construction of eukaryotic expression vector

The construction and expression of bispecific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1 (being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the bispecific antibody of dimeric forms, separately design Primer as shown in table 2, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplification Zhou Hongxun Science and Technology Ltd.s synthesize.

It builds for the clone of CD3-CTLA-4 BsAb_M, is expanded first using primer pcDNA3.1-Sig-F and Sig-R Go out signal peptide fragment, be then utilized respectively primer Sig-CD3-F and CD3-R, CD3- (GGGGS)₃- CTLA-4-F and PcDNA3.1-CTLA-4-R amplifies AntiCD3 McAb scFv, (GGGGS)₃The gene order of Linker, anti-CTLA-4 scFv；For Clone's structure of CD3-CTLA-4 BsAb_D, equally amplifies signal peptide using primer pcDNA3.1-Sig-F and Sig-R first Segment, be then utilized respectively primer Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CTLA-4-F and PcDNA3.1-CTLA-4-R amplifies the gene order of AntiCD3 McAb scFv, IgD hinge area, anti-CTLA-4 scFv.Amplification finishes Afterwards, it utilizesMono- step directed cloning kits of PCR (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.) splice respectively Monomer and dimeric forms bispecific antibody full-length gene order simultaneously seamless are cloned into through at EcoRI and HindIII linearisations On the pcDNA3.1 expression vectors of reason.Purpose carrier converts bacillus coli DH 5 alpha, and positive clone identification is carried out using bacterium colony PCR, It is accredited as positive recon (recombinant plasmid) and carries out sequencing identification.Correct recon (recombinant plasmid) arrangement will then be sequenced It is taken out in plasmid, for the transfection of CHO-S cells.

Know through sequencing, the CD3-CTLA-4 BsAb_M of monomeric form and the CD3-CTLA-4 BsAb_D of dimeric forms Full-length gene order it is correct, with expection be consistent.

Specifically, the nucleotide sequence of the CD3-CTLA-4 BsAb_M of monomeric form is as shown in SEQ ID NO.16, specifically For：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTG CGCCGCCAGCGGCTTCACCTTCAGCAGCTACACCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGG TGACCTTCATCAGCTACGACGGCAACAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAC AACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCATCTACTACTGCGCCCGCAC CGGCTGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAG CGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGGGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCA GGCCCCCCGCCTGCTGATCTACGGCGCCTTCAGCCGCGCCACCGGCATCCCCGACCGCTTCAGCGGCAGCGGCAGCG GCACCGACTTCACCCTGACCATCAGCCGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGC AGCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of the CD3-CTLA-4 BsAb_D of dimeric forms is as shown in SEQ ID NO.18, tool Body is：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGG CCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCAC CACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGG AGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGGAGAGC GGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAC CATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGACCTTCATCAGCTACGACGGCAACAACAAGT ACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAAC AGCCTGCGCGCCGAGGACACCGCCATCTACTACTGCGCCCGCACCGGCTGGCTGGGCCCCTTCGACTACTGGGGCCA GGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCG TGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGC GTGGGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGGCGCCTTCAG CCGCGCCACCGGCATCCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCCGCCTGG AGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCTGGACCTTCGGCCAGGGCACCAAGGTG GAGATCAAGCGC。

The primer used in table 2.CD3-CTLA-4 bispecific antibody gene clonings

Embodiment 7：The expression and purification of CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D

First, the expression of CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D

1.3. transfection composite is prepared：Each project (CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D) needs standard Standby two centrifuge tube/culture bottles, by taking 20ml as an example, are placed, prepared recombinant plasmid in Example 6 respectively：

2nd, the purifying of CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D

2.1 sample pretreatment

2.2Protein L affinity chromatography column purifications

Buffer solution A (Buffer A)：PBS, pH7.4

Buffer solution B (Buffer B)：0.1M Glycine,pH3.0

Buffer solution C (Buffer C)：0.1M Glycine,pH2.7

CD3-CTLA-4 BsAb_M and CD3-CTLA-4 the BsAb_D recombinant proteins finally purified are analyzed through SDS-PAGE, Electrophoretogram is as shown in Figure 6 under reduction and non reducing conditions.It can be seen from the figure that through Protein L affinity columns after purification, The purity of CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D recombinant proteins is equal>95%；Wherein CD3-CTLA-4 The theoretical molecular weight of BsAb_M recombinant proteins is 53.2kDa, and single electrophoresis strip is presented in the albumen under reduction and non reducing conditions Band, molecular weight is consistent with monomer, therefore the bispecific antibody is monomeric form (Fig. 6 A)；CD3-CTLA-4 BsAb_D are recombinated The theoretical molecular weight of albumen is 61.2kDa, and it is consistent with monomer that molecular weight is presented in the protein electrophoresis band under reducing condition, it is non-and also It is consistent with dimer (Fig. 6 B) that molecular weight is presented in electrophoretic band under old terms, illustrates that two protein moleculars can be by disulfide bond phase It connects, therefore the bispecific antibody is dimeric forms.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sample Equal frame is errorless, and consistent with theoretical N/C terminal amino acid sequences, mass spectral analysis further confirms that CD3-CTLA-4 BsAb_M are Monomeric form, CD3-CTLA-4 BsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence such as SEQ ID NO.15 institutes of the CD3-CTLA-4 BsAb_M of monomeric form Show, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPG KGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSG GGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKR。

The amino acid sequence of the CD3-CTLA-4 BsAb_D of dimeric forms is as shown in SEQ ID NO.17, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEK EKEEQEERETKTPECPSHTQPLGVQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISY DGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSGGGGSGGGGSGG GGSEIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKR。

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.35.

The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.36.

The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.37.

The amino acid sequence of anti-CTLA-4 scFv is as shown in SEQ ID NO.41, specially：

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSL SPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYGSSPWTFGQGTKVEIKR。

The amino acid sequence of the heavy chain variable region of anti-CTLA-4 scFv is as shown in SEQ ID NO.42, specially：

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS。

The amino acid sequence of the light chain variable region of anti-CTLA-4 scFv is as shown in SEQ ID NO.43, specially：

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKR。

The amino acid sequence of junction fragment is as shown in SEQ ID NO.1 in the CD3-CTLA-4 BsAb_M of monomeric form.

The amino acid sequence of junction fragment is as shown in SEQ ID NO.3 in the CD3-CTLA-4 BsAb_D of dimeric forms.

Embodiment 8：The antigen-binding activity of ELISA detection CD3-CTLA-4 BsAb_M and CD3-CTLA-4 BsAb_D

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD3-hFc (is purchased from Wujiang offshore protein with mankind CTLA-4-hFc fusion proteins Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes, and coating condition is 37 DEG C 1 hour or 4 DEG C overnight, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO4,0.24g NaH₂PO4,0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing to 1L；

2. closing：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes are added in.37 DEG C of closings 1 hour；

3. sample-adding：After PBS board-washings 4 times, the bispecific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1 Hour, sample gradient preparation method：Using 10 μ g/ml purify CD3-CTLA-4 BsAb_M or CD3-CTLA-4 BsAb_D as Initial concentration, carries out 6 gradients of doubling dilution, and each gradient sets 2 multiple holes；

ELISA results are as shown in figs. 7 a-b：Fig. 7 A illustrate CD3-CTLA-4 BsAb_M and recombinant antigen CD3-hFc and CTLA-4-hFc is respectively provided with external combination activity, and wherein CTLA-4 combines activity and combines activity higher compared with CD3；Fig. 7 B illustrate CD3- CTLA-4 BsAb_D are similary with recombinant antigen CD3-hFc and CTLA-4-hFc to have external combination activity, and wherein CTLA-4 is combined Active higher.

Embodiment 9：The CIK cell proliferation of CD3-CTLA-4 bispecific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC) Material, with bispecific antibody CD3-CTLA-4 BsAb_M of the above-mentioned monomeric form prepared by the present invention, double spies of dimeric forms (Anti-CD3/Anti- is used in combination in xenoantibody CD3-CTLA-4 BsAb_D and AntiCD3 McAb/anti- CD28 monoclonal full length antibodies CD28 the people blood PBMC of same donor source) is respectively acting on, is counted after cell culture, compares amplification times.

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) are resuspended, It is 1 × 10 to adjust cell density⁶/ ml separately designs following 3 experimental groups：Control group (Anti-CD3 5ug/ml and Anti- CD285ug/ml coated cells culture plate)；Experimental group 1 (adds bispecific antibody CD3-CTLA-4 BsAb_ under solution state M10ng/ml)；Experimental group 2 (adds bispecific antibody CD3-CTLA-4 BsAb_D 10ng/ml) under solution state.In addition, 3 Group experimental cell add simultaneously cell factor IFN-γ (200ng/ml, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) and IL-1 β (2ng/ml, purchased from Wujiang Alongshore Protein Technology Co., Ltd.), are placed in incubator, saturated humidity, 37 DEG C, 5.0% CO₂Under conditions of cultivated.After overnight incubation, the IL-2 of 500U/ml is added (purchased from the limited public affairs of Wujiang offshore protein science and technology Department) continue to cultivate, it was counted per 2-3 days and presses 1 × 10 with the CIK basal mediums of addition 500U/ml IL-2⁶The density of/ml into Row cell passes on.Method culture 30 days like this, the final amplification times for counting cell, draw growth curve；

Testing result is as shown in figure 8, the CD3-CTLA-4 bispecific antibody single uses pair of monomer and dimeric forms The cultivation effect of CIK cell is superior to AntiCD3 McAb/anti- CD28 monoclonal full length antibodies and is used in combination, after cultivating 18 days, Anti- There are a large amount of cell deaths in CD3/Anti-CD28 combinations, and cells expanded is remarkably decreased；And add the CD3- of monomeric form CTLA-4 BsAb_M or the CD3-CTLA-4 BsAb_D of dimeric forms do not occur cell death, only cell amplification rate It is opposite to slow down.Therefore the CD3-CTLA-4 bispecific antibodies of two kinds of forms that prepared by the present invention effectively can be expanded and be extended The life cycle of CIK cell, wherein dimeric forms effect are more preferable.

Embodiment 10：The CIK cell IFN-γ secretion of CD3-CTLA-4 bispecific antibodies induction

Operating procedure：

1st, after being cultivated 25 days in Example 9 CIK cell supernatant (be adjusted to same cell density, cell number for 2 × 10⁵It is a) 100 μ l, 37 DEG C of incubation 45min, it is detected by Human IFN-γ ELISA Kit (purchased from doctor's moral biology), Three groups of every group of experiments take three samples to repeat；

The results are shown in Figure 9：Wherein secreted by the CIK cell of Anti-CD3/Anti-CD28 full length antibodies combination culture IFN-γ quantity is defined as 1, and the CIK cell IFN- of the CD3-CTLA-4 BsAb_M cultures of monomeric form is added under solution state γ is 1.94 with respect to secretory volume, the CIK cell of the CD3-CTLA-4 BsAb_D cultures of addition dimeric forms under solution state IFN-γ is 2.85 with respect to secretory volume, therefore the CD3-CTLA-4 bispecific antibodies of two kinds of forms prepared by the present invention are more advantageous In activation CIK cell, the secretion of IFN-γ is induced, wherein dimeric forms are better.

Embodiment 11：The structure of CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D carrier for expression of eukaryon

In the present invention, costimulatory molecules LAG-3 albumen is born as target spot using T cell surface mankind CD3 albumen and T cell Bispecific antibody is named as CD3-LAG-3 BsAb.

First, CD3-LAG-3 BsAb_M and the design of CD3-LAG-3 BsAb_D constructing plans

The specific constructing plans of CD3-LAG-3 BsAb_M of monomeric form are：AntiCD3 McAb scFv and anti-lag-3 scFv sequences Between pass through (GGGGS)₃Linker is connected.

The specific constructing plans of CD3-LAG-3 BsAb_D of dimeric forms are：AntiCD3 McAb scFv and anti-lag-3 scFv sequences It is used as Linker by IgD hinge areas between row to be connected.

For bispecific antibody is made to be expressed in mammalian cell, for AntiCD3 McAb scFv, anti-lag-3 scFv and company The sequence of tab segments (Linker) has carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-lag-3 scFv is as shown in SEQ ID NO.66, specially：

CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCC GTGTACGGCGGCAGCTTCAGCGACTACTACTGGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGG CGAGATCAACCACCGCGGCAGCACCAACAGCAACCCCAGCCTGAAGAGCCGCGTGACCCTGAGCCTGGACACCAGCA AGAACCAGTTCAGCCTGAAGCTGCGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCTTCGGCTACAGC GACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

Specifically, the nucleotide sequence of the light chain variable region of anti-lag-3 scFv is as shown in SEQ ID NO.67, specially：

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGC CGCGCCAGCCAGAGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTA CGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCA TCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCTGACCTTCGGCCAG GGCACCAACCTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of anti-lag-3 scFv is as shown in SEQ ID NO.65, specially：

CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCC GTGTACGGCGGCAGCTTCAGCGACTACTACTGGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGG CGAGATCAACCACCGCGGCAGCACCAACAGCAACCCCAGCCTGAAGAGCCGCGTGACCCTGAGCCTGGACACCAGCA AGAACCAGTTCAGCCTGAAGCTGCGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCTTCGGCTACAGC GACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCG AGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAG GCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGG CACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACT GGCCCCTGACCTTCGGCCAGGGCACCAACCTGGAGATCAAGCGC。

The nucleotide sequence of the CD3-LAG-3 BsAb_M junction fragments of monomeric form is as shown in SEQ ID NO.2.

The nucleotide sequence of the CD3-LAG-3 BsAb_D junction fragments of dimeric forms is as shown in SEQ ID NO.4.

2nd, CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D construction of eukaryotic expression vector

The construction and expression of bispecific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1 (being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the bispecific antibody of dimeric forms, separately design Primer as shown in table 3, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplification Zhou Hongxun Science and Technology Ltd.s synthesize.

It builds for the clone of CD3-LAG-3 BsAb_M, is expanded first using primer pcDNA3.1-Sig-F and Sig-R Go out signal peptide fragment, be then utilized respectively primer Sig-CD3-F and CD3-R, CD3- (GGGGS)₃- LAG-3-F and pcDNA3.1- LAG-3-R amplifies AntiCD3 McAb scFv, (GGGGS)₃The gene order of Linker, anti-lag-3 scFv；For CD3-LAG-3 Clone's structure of BsAb_D, equally amplifies signal peptide fragment, Ran Houfen using primer pcDNA3.1-Sig-F and Sig-R first Not Li Yong primer Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-LAG-3-F and pcDNA3.1-LAG-3-R expand Go out the gene order of AntiCD3 McAb scFv, IgD hinge area, anti-lag-3 scFv.After amplification, utilizePCR mono- It walks directed cloning kit (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.) and splices monomer and the double spies of dimeric forms respectively Heterogenetic antibody full-length gene order simultaneously seamless is cloned into the pcDNA3.1 expression vectors through EcoRI and HindIII linearization process On.Purpose carrier converts bacillus coli DH 5 alpha, and positive clone identification is carried out using bacterium colony PCR, is accredited as positive recon (weight Group plasmid) carry out sequencing identification.Correct recon (recombinant plasmid) will then be sequenced to arrange to take out in plasmid, it is thin for CHO-S The transfection of born of the same parents.

Know through sequencing, the CD3-LAG-3 BsAb_M of monomeric form and the CD3-LAG-3 BsAb_D's of dimeric forms Full-length gene order is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD3-LAG-3 BsAb_M of monomeric form is as shown in SEQ ID NO.20, specifically For：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTG CGCCGTGTACGGCGGCAGCTTCAGCGACTACTACTGGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGA TCGGCGAGATCAACCACCGCGGCAGCACCAACAGCAACCCCAGCCTGAAGAGCCGCGTGACCCTGAGCCTGGACACC AGCAAGAACCAGTTCAGCCTGAAGCTGCGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCTTCGGCTA CAGCGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCA GCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCC GGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGG CCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCA GCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGC AACTGGCCCCTGACCTTCGGCCAGGGCACCAACCTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of the CD3-LAG-3 BsAb_D of dimeric forms is as shown in SEQ ID NO.22, tool Body is：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGG CCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCAC CACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGG AGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGCAGCAGTGG GGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGACTACTA CTGGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCACCGCGGCAGCACCAACA GCAACCCCAGCCTGAAGAGCCGCGTGACCCTGAGCCTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGCGCAGC GTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCTTCGGCTACAGCGACTACGAGTACAACTGGTTCGACCCCTG GGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG AGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGC CAGAGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAG CAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCC TGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCTGACCTTCGGCCAGGGCACCAAC CTGGAGATCAAGCGC。

The primer used in table 3.CD3-LAG-3 bispecific antibody gene clonings

Embodiment 12：The expression and purification of CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D

First, the expression of CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D

1.3. transfection composite is prepared：Each project (CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D) needs to prepare Two centrifuge tube/culture bottles, by taking 20ml as an example, are placed respectively, prepared recombinant plasmid in Example 11：

2nd, the purifying of CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D

2.1 sample pretreatment

2.2Protein L affinity chromatography column purifications

Buffer solution A (Buffer A)：PBS, pH7.4

Buffer solution B (Buffer B)：0.1M Glycine,pH3.0

Buffer solution C (Buffer C)：0.1M Glycine,pH2.7

CD3-LAG-3 BsAb_M and CD3-LAG-3 the BsAb_D recombinant proteins finally purified are analyzed through SDS-PAGE, also Electrophoretogram is as shown in Figure 10 under former and non reducing conditions.It can be seen from the figure that through Protein L affinity columns after purification, The purity of CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D recombinant proteins is equal>95%；Wherein CD3-LAG-3BsAb_M weights The theoretical molecular weight of histone is 53.5kDa, and single electrophoretic band, molecular weight is presented in the albumen under reduction and non reducing conditions It is consistent with monomer, therefore the bispecific antibody is monomeric form (Figure 10 A)；The theory of CD3-LAG-3 BsAb_D recombinant proteins Molecular weight is 61.4kDa, and it is consistent with monomer that molecular weight is presented in the protein electrophoresis band under reducing condition, electric under non reducing conditions It is consistent with dimer (Figure 10 B) that swimming band is presented molecular weight, illustrates that two protein moleculars can be connected with each other by disulfide bond, because This bispecific antibody is dimeric forms.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sample Equal frame is errorless, consistent with theoretical N/C terminal amino acid sequences, and mass spectral analysis further confirms that CD3-LAG-3 BsAb_M are single Body form, CD3-LAG-3 BsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence of the CD3-LAG-3 BsAb_M of monomeric form as shown in SEQ ID NO.19, Specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPG KGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVS SGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKR。

The amino acid sequence of the CD3-LAG-3 BsAb_D of dimeric forms is as shown in SEQ ID NO.21, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEK EKEEQEERETKTPECPSHTQPLGVQVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINH RGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSGGGGSGGGGS GGGGSEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKR。

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.35.

The amino acid sequence of anti-lag-3 scFv is as shown in SEQ ID NO.44, specially：

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTL SLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATL SLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPLTFGQGTNLEIKR。

The amino acid sequence of the heavy chain variable region of anti-lag-3 scFv is as shown in SEQ ID NO.45, specially：

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTL SLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSS。

The amino acid sequence of the light chain variable region of anti-lag-3 scFv is as shown in SEQ ID NO.46, specially：

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKR。

The amino acid sequence of junction fragment is as shown in SEQ ID NO.1 in the CD3-LAG-3 BsAb_M of monomeric form.

The amino acid sequence of junction fragment is as shown in SEQ ID NO.3 in the CD3-LAG-3 BsAb_D of dimeric forms.

Embodiment 13：The antigen-binding activity of ELISA detection CD3-LAG-3 BsAb_M and CD3-LAG-3 BsAb_D

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD3-hFc (is purchased from Wujiang offshore protein with mankind LAG-3-hFc fusion proteins Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes, and coating condition is 37 DEG C 1 hour or 4 DEG C overnight, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO4,0.24g NaH₂PO4,0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing to 1L；

3. sample-adding：After PBS board-washings 4 times, the bispecific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1 Hour, sample gradient preparation method：Using 10 μ g/ml purify CD3-LAG-3 BsAb_M or CD3-LAG-3 BsAb_D as Beginning concentration, carries out 6 gradients of doubling dilution, and each gradient sets 2 multiple holes；

ELISA results are as seen in figs. 11a and 11b：Figure 11 A illustrate CD3-LAG-3 BsAb_M and recombinant antigen CD3- HFc and LAG-3-hFc is respectively provided with external combination activity, and wherein LAG-3 combines activity and combines activity higher compared with CD3；Figure 11 B explanations CD3-LAG-3 BsAb_D are similary with recombinant antigen CD3-hFc and LAG-3-hFc to have external combination activity, wherein LAG-3 knots Close active higher.

Embodiment 14：The CIK cell proliferation of CD3-LAG-3 bispecific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC) Material, with bispecific antibody CD3-LAG-3 BsAb_M of the above-mentioned monomeric form prepared by the present invention, double spies of dimeric forms Xenoantibody CD3-LAG-3 BsAb_D and AntiCD3 McAb/anti- CD28 monoclonals full length antibody combination (Anti-CD3/Anti-CD28) The people blood PBMC of same donor source is respectively acting on, is counted after cell culture, compares amplification times.

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) are resuspended, It is 1 × 10 to adjust cell density⁶/ ml separately designs following 3 experimental groups：Control group (Anti-CD3 5ug/ml and Anti- CD285ug/ml coated cell culture plates, full length antibody are purchased from Wujiang Alongshore Protein Technology Co., Ltd.)；Experimental group 1 is (molten Bispecific antibody CD3-LAG-3 BsAb_M 10ng/ml are added under liquid status)；Experimental group 2 (is added double special under solution state Heterogenetic antibody CD3-LAG-3 BsAb_D 10ng/ml).In addition, 3 groups of experimental cells add cell factor IFN-γ simultaneously (200ng/ml, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) and IL-1 β (2ng/ml, purchased from Wujiang offshore protein section Skill Co., Ltd), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, add The IL-2 (purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of 500U/ml is added to continue to cultivate, was counted per 2-3 days and with adding The CIK basal mediums of 500U/ml IL-2 press 1 × 10⁶The density of/ml carries out cell passage.Method culture 30 days like this, most The amplification times of finish-unification meter cell draw growth curve.

Testing result is as shown in figure 12, the CD3-LAG-3 bispecific antibody single uses pair of monomer and dimeric forms The cultivation effect of CIK cell is superior to AntiCD3 McAb/anti- CD28 monoclonal full length antibodies and is used in combination, after cultivating 18 days, Anti- There are a large amount of cell deaths in CD3/Anti-CD28 combinations, and cells expanded is remarkably decreased；And add the CD3- of monomeric form LAG-3 BsAb_M or the CD3-LAG-3 BsAb_D of dimeric forms do not occur cell death, only cell amplification rate phase To slowing down.Therefore the CD3-LAG-3 bispecific antibodies of two kinds of forms that prepare of the present invention can be expanded effectively and to extend CIK thin The life cycle of born of the same parents, wherein dimeric forms effect are more preferable.

Embodiment 15：The CIK cell IFN-γ secretion of CD3-LAG-3 bispecific antibodies induction

Operating procedure：

1st, the CIK cell supernatant after being cultivated 25 days in Example 14 (is adjusted to same cell density, cell number 2 ×10⁵It is a) 100 μ l, 37 DEG C of incubation 45min, are examined by Human IFN-γ ELISA Kit (purchased from doctor's moral biology) It surveys, three groups of every group of experiments take three samples to repeat；

As a result as shown in figure 13：Wherein secreted by the CIK cell of Anti-CD3/Anti-CD28 full length antibodies combination culture IFN-γ quantity be defined as 1, the CIK cell IFN- of the CD3-LAG-3 BsAb_M cultures of addition monomeric form under solution state γ is 2.25 with respect to secretory volume, the CIK cell of the CD3-LAG-3 BsAb_D cultures of addition dimeric forms under solution state IFN-γ is 3.37 with respect to secretory volume, therefore the CD3-LAG-3 bispecific antibodies of two kinds of forms prepared by the present invention are more advantageous In activation CIK cell, the secretion of IFN-γ is induced, wherein dimeric forms are better.

Embodiment 16：The structure of CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D carrier for expression of eukaryon

In the present invention, costimulatory molecules TIM-3 albumen is born as target spot using T cell surface mankind CD3 albumen and T cell Bispecific antibody is named as CD3-TIM-3 BsAb.

First, CD3-TIM-3 BsAb_M and the design of CD3-TIM-3 BsAb_D constructing plans

The specific constructing plans of CD3-TIM-3 BsAb_M of monomeric form are：AntiCD3 McAb scFv and anti-TIM-3 scFv sequences Between pass through (GGGGS)₃Linker is connected.

The specific constructing plans of CD3-TIM-3 BsAb_D of dimeric forms are：AntiCD3 McAb scFv and anti-TIM-3 scFv sequences It is used as Linker by IgD hinge areas between row to be connected.

For bispecific antibody is made to be expressed in mammalian cell, for AntiCD3 McAb scFv, anti-TIM-3 scFv and company The sequence of tab segments (Linker) has carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-TIM-3 scFv is as shown in SEQ ID NO.69, specially：

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAG GCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGG CGACATCTACCCCGGCCAGGGCGACACCAGCTACAACCAGAAGTTCAAGGGCCGCGCCACCATGACCGCCGACAAGA GCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGTGGGC GGCGCCTTCCCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

Specifically, the nucleotide sequence of the light chain variable region of anti-TIM-3 scFv is as shown in SEQ ID NO.70, specially：

GACATCGTGCTGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTGC CGCGCCAGCGAGAGCGTGGAGTACTACGGCACCAGCCTGATGCAGTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAA GCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACT TCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCCGCAAGGACCCCAGC ACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of anti-TIM-3 scFv is as shown in SEQ ID NO.68, specially：

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAG GCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGG CGACATCTACCCCGGCCAGGGCGACACCAGCTACAACCAGAAGTTCAAGGGCCGCGCCACCATGACCGCCGACAAGA GCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGTGGGC GGCGCCTTCCCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGGCGGCGGCGGCAGCGACATCGTGCTGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCG CCACCATCAACTGCCGCGCCAGCGAGAGCGTGGAGTACTACGGCACCAGCCTGATGCAGTGGTACCAGCAGAAGCCC GGCCAGCCCCCCAAGCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGG CAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCC GCAAGGACCCCAGCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence of the CD3-TIM-3 BsAb_M junction fragments of monomeric form is as shown in SEQ ID NO.2.

The nucleotide sequence of the CD3-TIM-3 BsAb_D junction fragments of dimeric forms is as shown in SEQ ID NO.4.

2nd, CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D construction of eukaryotic expression vector

The construction and expression of bispecific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1 (being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the bispecific antibody of dimeric forms, separately design Primer as shown in table 4, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplification Zhou Hongxun Science and Technology Ltd.s synthesize.

It builds for the clone of CD3-TIM-3 BsAb_M, is expanded first using primer pcDNA3.1-Sig-F and Sig-R Go out signal peptide fragment, be then utilized respectively primer Sig-CD3-F and CD3-R, CD3- (GGGGS)₃- TIM-3-F and pcDNA3.1- TIM-3-R amplifies AntiCD3 McAb scFv, (GGGGS)₃The gene order of Linker, anti-TIM-3 scFv；For CD3-TIM-3 Clone's structure of BsAb_D, equally amplifies signal peptide fragment, Ran Houfen using primer pcDNA3.1-Sig-F and Sig-R first Not Li Yong primer Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-TIM-3-F and pcDNA3.1-TIM-3-R expand Go out the gene order of AntiCD3 McAb scFv, IgD hinge area, anti-TIM-3 scFv.After amplification, utilizeMono- steps of PCR Directed cloning kit (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.) splices monomer respectively and dimeric forms are double special Property antibody full-length gene order simultaneously seamless is cloned on the pcDNA3.1 expression vectors through EcoRI and HindIII linearization process. Purpose carrier converts bacillus coli DH 5 alpha, and positive clone identification is carried out using bacterium colony PCR, is accredited as (the recombination of positive recon Plasmid) carry out sequencing identification.Correct recon (recombinant plasmid) will then be sequenced to arrange to take out in plasmid, for CHO-S cells Transfection.

Know through sequencing, the CD3-TIM-3 BsAb_M of monomeric form and the CD3-TIM-3 BsAb_D's of dimeric forms Full-length gene order is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD3-TIM-3 BsAb_M of monomeric form is as shown in SEQ ID NO.24, specifically For：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTG CAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGA TCGGCGACATCTACCCCGGCCAGGGCGACACCAGCTACAACCAGAAGTTCAAGGGCCGCGCCACCATGACCGCCGAC AAGAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGT GGGCGGCGCCTTCCCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGCTGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAG CGCGCCACCATCAACTGCCGCGCCAGCGAGAGCGTGGAGTACTACGGCACCAGCCTGATGCAGTGGTACCAGCAGAA GCCCGGCCAGCCCCCCAAGCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCCGACCGCTTCAGCGGCA GCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAG AGCCGCAAGGACCCCAGCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of the CD3-TIM-3 BsAb_D of dimeric forms is as shown in SEQ ID NO.26.Tool Body is：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGG CCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCAC CACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGG AGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGCAGAGC GGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAA CATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGACATCTACCCCGGCCAGGGCGACACCA GCTACAACCAGAAGTTCAAGGGCCGCGCCACCATGACCGCCGACAAGAGCACCAGCACCGTGTACATGGAGCTGAGC AGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGTGGGCGGCGCCTTCCCCATGGACTACTGGGGCCA GGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCG TGCTGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTGCCGCGCCAGCGAGAGC GTGGAGTACTACGGCACCAGCCTGATGCAGTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGC CGCCAGCAACGTGGAGAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCA GCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCCGCAAGGACCCCAGCACCTTCGGCGGCGGC ACCAAGGTGGAGATCAAGCGC。

The primer used in table 4.CD3-TIM-3 bispecific antibody gene clonings

Embodiment 17：The expression and purification of CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D

First, the expression of CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D

1.3. transfection composite is prepared：Each project (CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D) needs to prepare Two centrifuge tube/culture bottles, by taking 20ml as an example, are placed respectively, prepared recombinant plasmid in Example 16：

2nd, the purifying of CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D

2.1 sample pretreatment

2.2Protein L affinity chromatography column purifications

Buffer solution A (Buffer A)：PBS, pH7.4

Buffer solution B (Buffer B)：0.1M Glycine,pH3.0

Buffer solution C (Buffer C)：0.1M Glycine,pH2.7

CD3-TIM-3 BsAb_M and CD3-TIM-3 the BsAb_D recombinant proteins finally purified are analyzed through SDS-PAGE, also Electrophoretogram is as shown in figure 14 under former and non reducing conditions.It can be seen from the figure that through Protein L affinity columns after purification, The purity of CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D recombinant proteins is equal>95%；Wherein CD3-TIM-3 BsAb_M The theoretical molecular weight of recombinant protein is 53.2kDa, and single electrophoretic band, molecule is presented in the albumen under reduction and non reducing conditions Amount is consistent with monomer, therefore the bispecific antibody is monomeric form (Figure 14 A)；The reason of CD3-TIM-3 BsAb_D recombinant proteins It is 61.1kDa by molecular weight, it is consistent with monomer that molecular weight is presented in the protein electrophoresis band under reducing condition, under non reducing conditions It is consistent with dimer (Figure 14 B) that electrophoretic band is presented molecular weight, illustrates that two protein moleculars can be connected with each other by disulfide bond, Therefore the bispecific antibody is dimeric forms.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sample Equal frame is errorless, consistent with theoretical N/C terminal amino acid sequences, and mass spectral analysis further confirms that CD3-TIM-3 BsAb_M are single Body form, CD3-TIM-3 BsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence of the CD3-TIM-3 BsAb_M of monomeric form as shown in SEQ ID NO.23, Specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPG QGLEWIGDIYPGQGDTSYNQKFKGRATMTADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATINCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIKR。

The amino acid sequence of the CD3-TIM-3 BsAb_D of dimeric forms is as shown in SEQ ID NO.25, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEK EKEEQEERETKTPECPSHTQPLGVQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGDIYP GQGDTSYNQKFKGRATMTADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLVTVSSGGGGSGGGGSGG GGSDIVLTQSPDSLAVSLGERATINCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTD FTLTISSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIKR。

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.35.

The amino acid sequence of anti-TIM-3 scFv is as shown in SEQ ID NO.47, specially：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGDIYPGQGDTSYNQKFKGRAT MTADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAV SLGERATINCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQSRKDPSTFGGGTKVEIKR。

The amino acid sequence of the heavy chain variable region of anti-TIM-3 scFv is as shown in SEQ ID NO.48, specially：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGDIYPGQGDTSYNQKFKGRAT MTADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLVTVSS。

The amino acid sequence of the light chain variable region of anti-TIM-3 scFv is as shown in SEQ ID NO.49, specially：

DIVLTQSPDSLAVSLGERATINCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGS GSGTDFTLTISSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIKR。

The amino acid sequence of junction fragment is as shown in SEQ ID NO.1 in the CD3-TIM-3 BsAb_M of monomeric form.

The amino acid sequence of junction fragment is as shown in SEQ ID NO.3 in the CD3-TIM-3 BsAb_D of dimeric forms.

Embodiment 18：The antigen-binding activity of ELISA detection CD3-TIM-3 BsAb_M and CD3-TIM-3 BsAb_D

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD3-hFc (is purchased from Wujiang offshore protein with mankind TIM-3-hFc fusion proteins Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes, and coating condition is 37 DEG C 1 hour or 4 DEG C overnight, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO4,0.24g NaH₂PO4,0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing to 1L；

3. sample-adding：After PBS board-washings 4 times, the bispecific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1 Hour, sample gradient preparation method：Using 10 μ g/ml purify CD3-TIM-3 BsAb_M or CD3-TIM-3 BsAb_D as Beginning concentration, carries out 6 gradients of doubling dilution, and each gradient sets 2 multiple holes；

ELISA results are as shown in fig. 15 a and fig. 15b：Figure 15 A illustrate CD3-TIM-3 BsAb_M and recombinant antigen CD3- HFc and TIM-3-hFc is respectively provided with external combination activity, and wherein TIM-3 combines activity and combines activity higher compared with CD3；Figure 15 B explanations CD3-TIM-3 BsAb_D are similary with recombinant antigen CD3-hFc and TIM-3-hFc to have external combination activity, wherein TIM-3 knots Close active higher.

Embodiment 19：The CIK cell proliferation of CD3-TIM-3 bispecific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC) Material, with bispecific antibody CD3-TIM-3 BsAb_M of the above-mentioned monomeric form prepared by the present invention, double spies of dimeric forms Xenoantibody CD3-TIM-3 BsAb_D and AntiCD3 McAb/anti- CD28 monoclonals full length antibody combination (Anti-CD3/Anti-CD28) The people blood PBMC of same donor source is respectively acting on, is counted after cell culture, compares amplification times.

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) are resuspended, It is 1 × 10 to adjust cell density⁶/ ml separately designs following 3 experimental groups：Control group (Anti-CD3 5ug/ml and Anti- CD285ug/ml coated cell culture plates, full length antibody are purchased from Wujiang Alongshore Protein Technology Co., Ltd.)；Experimental group 1 is (molten Bispecific antibody CD3-TIM-3 BsAb_M 10ng/ml are added under liquid status)；Experimental group 2 (is added double special under solution state Heterogenetic antibody CD3-TIM-3 BsAb_D 10ng/ml).In addition, 3 groups of experimental cells add cell factor IFN-γ simultaneously (200ng/ml, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) and IL-1 β (2ng/ml, purchased from Wujiang offshore protein section Skill Co., Ltd), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, add The IL-2 (purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of 500U/ml is added to continue to cultivate, was counted per 2-3 days and with adding The CIK basal mediums of 500U/ml IL-2 press 1 × 10⁶The density of/ml carries out cell passage.Method culture 30 days like this, most The amplification times of finish-unification meter cell draw growth curve；

Testing result is as shown in figure 16, the CD3-TIM-3 bispecific antibody single uses pair of monomer and dimeric forms The cultivation effect of CIK cell is superior to AntiCD3 McAb/anti- CD28 monoclonal full length antibodies and is used in combination, after cultivating 18 days, Anti- There are a large amount of cell deaths in CD3/Anti-CD28 combinations, and cells expanded is remarkably decreased；And add the CD3- of monomeric form TIM-3 BsAb_M or the CD3-TIM-3 BsAb_D of dimeric forms do not occur cell death, only cell amplification rate phase To slowing down.Therefore the CD3-TIM-3 bispecific antibodies of two kinds of forms that prepare of the present invention can be expanded effectively and to extend CIK thin The life cycle of born of the same parents, wherein dimeric forms effect are more preferable.

Embodiment 20：The CIK cell IFN-γ secretion of CD3-TIM-3 bispecific antibodies induction

Operating procedure：

1st, the CIK cell supernatant after being cultivated 25 days in Example 19 (is adjusted to same cell density, cell number 2 ×10⁵It is a) 100 μ l, 37 DEG C of incubation 45min, are examined by Human IFN-γ ELISA Kit (purchased from doctor's moral biology) It surveys, three groups of every group of experiments take three samples to repeat；

As a result as shown in figure 17：Wherein secreted by the CIK cell of Anti-CD3/Anti-CD28 full length antibodies combination culture IFN-γ quantity be defined as 1, the CIK cell IFN- of the CD3-TIM-3 BsAb_M cultures of addition monomeric form under solution state γ is 2.07 with respect to secretory volume, the CIK cell of the CD3-TIM-3 BsAb_D cultures of addition dimeric forms under solution state IFN-γ is 3.04 with respect to secretory volume, therefore the CD3-TIM-3 bispecific antibodies of two kinds of forms prepared by the present invention are more advantageous In activation CIK cell, the secretion of IFN-γ is induced, wherein dimeric forms are better.

Embodiment 21：The structure of CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D carrier for expression of eukaryon

In the present invention, costimulatory molecules TIGIT albumen is born as target spot using T cell surface mankind CD3 albumen and T cell Bispecific antibody is named as CD3-TIGIT BsAb.

First, CD3-TIGIT BsAb_M and the design of CD3-TIGIT BsAb_D constructing plans

The specific constructing plans of CD3-TIGIT BsAb_M of monomeric form are：AntiCD3 McAb scFv and anti-TIGIT scFv sequences Between pass through (GGGGS)₃Linker is connected.

The specific constructing plans of CD3-TIGIT BsAb_D of dimeric forms are：AntiCD3 McAb scFv and anti-TIGIT scFv sequences It is used as Linker by IgD hinge areas between row to be connected.

For bispecific antibody is made to be expressed in mammalian cell, for AntiCD3 McAb scFv, anti-TIGIT scFv and company The sequence of tab segments (Linker) has carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-TIGIT scFv is as shown in SEQ ID NO.72, specially：

GAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTGCAGC GTGACCGGCAGCAGCATCGCCAGCGACTACTGGGGCTGGATCCGCAAGTTCCCCGGCAACAAGATGGAGTGGATGGG CTTCATCACCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGAGCCGCATCAGCATCACCCGCGACACCAGCA AGAACCAGTTCTTCCTGCAGCTGCACAGCGTGACCACCGACGACACCGCCACCTACAGCTGCGCCCGCATGCCCAGC TTCATCACCCTGGCCAGCCTGAGCACCTGGGAGGGCTACTTCGACTTCTGGGGCCCCGGCACCATGGTGACCGTGAG CAGC。

Specifically, the nucleotide sequence of the light chain variable region of anti-TIGIT scFv is as shown in SEQ ID NO.73, specially：

GACATCCAGATGACCCAGAGCCCCAGCCTGCTGAGCGCCAGCGTGGGCGACCGCGTGACCCTGAACTGC AAGGCCAGCCAGAGCATCCACAAGAACCTGGCCTGGTACCAGCAGAAGCTGGGCGAGGCCCCCAAGTTCCTGATCTA CTACGCCAACAGCCTGCAGACCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCA TCAGCGGCCTGCAGCCCGAGGACGTGGCCACCTACTTCTGCCAGCAGTACTACAGCGGCTGGACCTTCGGCGGCGGC ACCAAGGTGGAGCTGAAGCGC。

Specifically, the nucleotide sequence of anti-TIGIT scFv is as shown in SEQ ID NO.71, specially：

GAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTGCAGC GTGACCGGCAGCAGCATCGCCAGCGACTACTGGGGCTGGATCCGCAAGTTCCCCGGCAACAAGATGGAGTGGATGGG CTTCATCACCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGAGCCGCATCAGCATCACCCGCGACACCAGCA AGAACCAGTTCTTCCTGCAGCTGCACAGCGTGACCACCGACGACACCGCCACCTACAGCTGCGCCCGCATGCCCAGC TTCATCACCCTGGCCAGCCTGAGCACCTGGGAGGGCTACTTCGACTTCTGGGGCCCCGGCACCATGGTGACCGTGAG CAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCC TGCTGAGCGCCAGCGTGGGCGACCGCGTGACCCTGAACTGCAAGGCCAGCCAGAGCATCCACAAGAACCTGGCCTGG TACCAGCAGAAGCTGGGCGAGGCCCCCAAGTTCCTGATCTACTACGCCAACAGCCTGCAGACCGGCATCCCCAGCCG CTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCGGCCTGCAGCCCGAGGACGTGGCCACCTACT TCTGCCAGCAGTACTACAGCGGCTGGACCTTCGGCGGCGGCACCAAGGTGGAGCTGAAGCGC。

The nucleotide sequence of the CD3-TIGIT BsAb_M junction fragments of monomeric form is as shown in SEQ ID NO.2.

The nucleotide sequence of the CD3-TIGIT BsAb_D junction fragments of dimeric forms is as shown in SEQ ID NO.4.

2nd, CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D construction of eukaryotic expression vector

The construction and expression of bispecific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1 (being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the bispecific antibody of dimeric forms, separately design Primer as shown in table 5, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplification Zhou Hongxun Science and Technology Ltd.s synthesize.

It builds for the clone of CD3-TIGIT BsAb_M, is expanded first using primer pcDNA3.1-Sig-F and Sig-R Go out signal peptide fragment, be then utilized respectively primer Sig-CD3-F and CD3-R, CD3- (GGGGS)₃- TIGIT-F and pcDNA3.1- TIGIT-R amplifies AntiCD3 McAb scFv, (GGGGS)₃The gene order of Linker, anti-TIGIT scFv；For CD3-TIGIT Clone's structure of BsAb_D, equally amplifies signal peptide fragment, Ran Houfen using primer pcDNA3.1-Sig-F and Sig-R first Not Li Yong primer Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-TIGIT-F and pcDNA3.1-TIGIT-R expand Go out the gene order of AntiCD3 McAb scFv, IgD hinge area, anti-TIGIT scFv.After amplification, utilizeMono- steps of PCR Directed cloning kit (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.) splices monomer respectively and dimeric forms are double special Property antibody full-length gene order simultaneously seamless is cloned on the pcDNA3.1 expression vectors through EcoRI and HindIII linearization process. Purpose carrier converts bacillus coli DH 5 alpha, and positive clone identification is carried out using bacterium colony PCR, is accredited as (the recombination of positive recon Plasmid) carry out sequencing identification.Correct recon (recombinant plasmid) will then be sequenced to arrange to take out in plasmid, for CHO-S cells Transfection.

Know through sequencing, the CD3-TIGIT BsAb_M of monomeric form and the CD3-TIGIT BsAb_D's of dimeric forms Full-length gene order is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD3-TIGIT BsAb_M of monomeric form is as shown in SEQ ID NO.28, specifically For：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTG CAGCGTGACCGGCAGCAGCATCGCCAGCGACTACTGGGGCTGGATCCGCAAGTTCCCCGGCAACAAGATGGAGTGGA TGGGCTTCATCACCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGAGCCGCATCAGCATCACCCGCGACACC AGCAAGAACCAGTTCTTCCTGCAGCTGCACAGCGTGACCACCGACGACACCGCCACCTACAGCTGCGCCCGCATGCC CAGCTTCATCACCCTGGCCAGCCTGAGCACCTGGGAGGGCTACTTCGACTTCTGGGGCCCCGGCACCATGGTGACCG TGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCC AGCCTGCTGAGCGCCAGCGTGGGCGACCGCGTGACCCTGAACTGCAAGGCCAGCCAGAGCATCCACAAGAACCTGGC CTGGTACCAGCAGAAGCTGGGCGAGGCCCCCAAGTTCCTGATCTACTACGCCAACAGCCTGCAGACCGGCATCCCCA GCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCGGCCTGCAGCCCGAGGACGTGGCCACC TACTTCTGCCAGCAGTACTACAGCGGCTGGACCTTCGGCGGCGGCACCAAGGTGGAGCTGAAGCGC。

Specifically, the nucleotide sequence of the CD3-TIGIT BsAb_D of dimeric forms is as shown in SEQ ID NO.30, tool Body is：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGG CCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCAC CACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGG AGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGGAGGTGCAGCTGCAGGAGAGC GGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTGCAGCGTGACCGGCAGCAGCATCGCCAGCGACTA CTGGGGCTGGATCCGCAAGTTCCCCGGCAACAAGATGGAGTGGATGGGCTTCATCACCTACAGCGGCAGCACCAGCT ACAACCCCAGCCTGAAGAGCCGCATCAGCATCACCCGCGACACCAGCAAGAACCAGTTCTTCCTGCAGCTGCACAGC GTGACCACCGACGACACCGCCACCTACAGCTGCGCCCGCATGCCCAGCTTCATCACCCTGGCCAGCCTGAGCACCTG GGAGGGCTACTTCGACTTCTGGGGCCCCGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCG GCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCCTGCTGAGCGCCAGCGTGGGCGACCGCGTG ACCCTGAACTGCAAGGCCAGCCAGAGCATCCACAAGAACCTGGCCTGGTACCAGCAGAAGCTGGGCGAGGCCCCCAA GTTCCTGATCTACTACGCCAACAGCCTGCAGACCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACT TCACCCTGACCATCAGCGGCCTGCAGCCCGAGGACGTGGCCACCTACTTCTGCCAGCAGTACTACAGCGGCTGGACC TTCGGCGGCGGCACCAAGGTGGAGCTGAAGCGC。

The primer used in table 5.CD3-TIGIT bispecific antibody gene clonings

Embodiment 22：The expression and purification of CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D

First, the expression of CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D

1.3. transfection composite is prepared：Each project (CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D) needs to prepare Two centrifuge tube/culture bottles, by taking 20ml as an example, are placed respectively, prepared recombinant plasmid in Example 21：

2nd, the purifying of CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D

2.1 sample pretreatment

2.2Protein L affinity chromatography column purifications

Buffer solution A (Buffer A)：PBS, pH7.4

Buffer solution B (Buffer B)：0.1M Glycine,pH3.0

Buffer solution C (Buffer C)：0.1M Glycine,pH2.7

CD3-TIGIT BsAb_M and CD3-TIGIT the BsAb_D recombinant proteins finally purified are analyzed through SDS-PAGE, also Electrophoretogram is as shown in figure 18 under former and non reducing conditions.It can be seen from the figure that through Protein L affinity columns after purification, The purity of CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D recombinant proteins is equal>95%；Wherein CD3-TIGIT BsAb_M The theoretical molecular weight of recombinant protein is 54.0kDa, and single electrophoretic band, molecule is presented in the albumen under reduction and non reducing conditions Amount is consistent with monomer, therefore the bispecific antibody is monomeric form (Figure 18 A)；The reason of CD3-TIGIT BsAb_D recombinant proteins It is 61.9kDa by molecular weight, it is consistent with monomer that molecular weight is presented in the protein electrophoresis band under reducing condition, under non reducing conditions It is consistent with dimer (Figure 18 B) that electrophoretic band is presented molecular weight, illustrates that two protein moleculars can be connected with each other by disulfide bond, Therefore the bispecific antibody is dimeric forms.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sample Equal frame is errorless, consistent with theoretical N/C terminal amino acid sequences, and mass spectral analysis further confirms that CD3-TIGIT BsAb_M are single Body form, CD3-TIGIT BsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence of the CD3-TIGIT BsAb_M of monomeric form as shown in SEQ ID NO.27, Specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPG NKMEWMGFITYSGSTSYNPSLKSRISITRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGP GTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYANSL QTGIPSRFSGSGSGTDFTLTISGLQPEDVATYFCQQYYSGWTFGGGTKVELKR。

The amino acid sequence of the CD3-TIGIT BsAb_D of dimeric forms is as shown in SEQ ID NO.29, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEK EKEEQEERETKTPECPSHTQPLGVEVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFITY SGSTSYNPSLKSRISITRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGPGTMVTVSSGGG GSGGGGSGGGGSDIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYANSLQTGIPSRFSGS GSGTDFTLTISGLQPEDVATYFCQQYYSGWTFGGGTKVELKR。

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.35.

The amino acid sequence of anti-TIGIT scFv is as shown in SEQ ID NO.50, specially：

EVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFITYSGSTSYNPSLKSRISI TRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGPGTMVTVSSGGGGSGGGGSGGGGSDIQM TQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYANSLQTGIPSRFSGSGSGTDFTLTISGLQPE DVATYFCQQYYSGWTFGGGTKVELKR。

The amino acid sequence of the heavy chain variable region of anti-TIGIT scFv is as shown in SEQ ID NO.51, specially：

EVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFITYSGSTSYNPSLKSRISI TRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGPGTMVTVSS。

The amino acid sequence of the light chain variable region of anti-TIGIT scFv is as shown in SEQ ID NO.52, specially：

DIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYANSLQTGIPSRFSGSGSGT DFTLTISGLQPEDVATYFCQQYYSGWTFGGGTKVELKR。

The amino acid sequence of junction fragment is as shown in SEQ ID NO.1 in the CD3-TIGIT BsAb_M of monomeric form.

The amino acid sequence of junction fragment is as shown in SEQ ID NO.3 in the CD3-TIGIT BsAb_D of dimeric forms.

Embodiment 23：The antigen-binding activity of ELISA detection CD3-TIGIT BsAb_M and CD3-TIGIT BsAb_D

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD3-hFc (is purchased from Wujiang offshore protein with mankind TIGIT-hFc fusion proteins Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes, and coating condition is 37 DEG C 1 hour or 4 DEG C overnight, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO4,0.24g NaH₂PO4,0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing to 1L；

3. sample-adding：After PBS board-washings 4 times, the bispecific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1 Hour, sample gradient preparation method：Using 10 μ g/ml purify CD3-TIGIT BsAb_M or CD3-TIGIT BsAb_D as Beginning concentration, carries out 6 gradients of doubling dilution, and each gradient sets 2 multiple holes；

ELISA results are as shown in Figure 19A and Figure 19B：Figure 19 A illustrate CD3-TIGIT BsAb_M and recombinant antigen CD3- HFc and TIGIT-hFc is respectively provided with external combination activity, and wherein TIGIT combines activity and combines activity higher compared with CD3；Figure 19 B explanations CD3-TIGIT BsAb_D are similary with recombinant antigen CD3-hFc and TIGIT-hFc to have external combination activity, wherein TIGIT knots Close active higher.

Embodiment 24：The CIK cell proliferation of CD3-TIGIT bispecific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC) Material, with bispecific antibody CD3-TIGIT BsAb_M of the above-mentioned monomeric form prepared by the present invention, double spies of dimeric forms Xenoantibody CD3-TIGIT BsAb_D and AntiCD3 McAb/anti- CD28 monoclonals full length antibody combination (Anti-CD3/Anti-CD28) The people blood PBMC of same donor source is respectively acting on, is counted after cell culture, compares amplification times.

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) are resuspended, It is 1 × 10 to adjust cell density⁶/ ml separately designs following 3 experimental groups：Control group (Anti-CD3 5ug/ml and Anti- CD285ug/ml coated cell culture plates, full length antibody are purchased from Wujiang Alongshore Protein Technology Co., Ltd.)；Experimental group 1 is (molten Bispecific antibody CD3-TIGIT BsAb_M 10ng/ml are added under liquid status)；Experimental group 2 (is added double special under solution state Heterogenetic antibody CD3-TIGIT BsAb_D 10ng/ml).In addition, 3 groups of experimental cells add cell factor IFN-γ simultaneously (200ng/ml, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) and IL-1 β (2ng/ml, purchased from Wujiang offshore protein section Skill Co., Ltd), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, add The IL-2 (purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of 500U/ml is added to continue to cultivate, was counted per 2-3 days and with adding The CIK basal mediums of 500U/ml IL-2 press 1 × 10⁶The density of/ml carries out cell passage.Method culture 30 days like this, most The amplification times of finish-unification meter cell draw growth curve；

Testing result is as shown in figure 20, the CD3-TIGIT bispecific antibody single uses pair of monomer and dimeric forms The cultivation effect of CIK cell is superior to AntiCD3 McAb/anti- CD28 monoclonal full length antibodies and is used in combination, after cultivating 18 days, Anti- There are a large amount of cell deaths in CD3/Anti-CD28 combinations, and cells expanded is remarkably decreased；And add the CD3- of monomeric form TIGIT BsAb_M or the CD3-TIGIT BsAb_D of dimeric forms do not occur cell death, only cell amplification rate phase To slowing down.Therefore the CD3-TIGIT bispecific antibodies of two kinds of forms that prepare of the present invention can be expanded effectively and to extend CIK thin The life cycle of born of the same parents, wherein dimeric forms effect are more preferable.

Embodiment 25：The CIK cell IFN-γ secretion of CD3-TIGIT bispecific antibodies induction

Operating procedure：

1st, the CIK cell supernatant after being cultivated 25 days in Example 24 (is adjusted to same cell density, cell number 2 ×10⁵It is a) 100 μ l, 37 DEG C of incubation 45min, are examined by Human IFN-γ ELISA Kit (purchased from doctor's moral biology) It surveys, three groups of every group of experiments take three samples to repeat；

As a result as shown in figure 21：Wherein secreted by the CIK cell of Anti-CD3/Anti-CD28 full length antibodies combination culture IFN-γ quantity be defined as 1, the CIK cell IFN- of the CD3-TIGIT BsAb_M cultures of addition monomeric form under solution state γ is 1.66 with respect to secretory volume, the CIK cell of the CD3-TIGIT BsAb_D cultures of addition dimeric forms under solution state IFN-γ is 2.30 with respect to secretory volume, therefore the CD3-TIGIT bispecific antibodies of two kinds of forms prepared by the present invention are more advantageous In activation CIK cell, the secretion of IFN-γ is induced, wherein dimeric forms are better.

Embodiment 26：The structure of CD3-BTLA BsAb_M and CD3-BTLA BsAb_D carrier for expression of eukaryon

In the present invention, costimulatory molecules BTLA albumen is born as target spot using T cell surface mankind CD3 albumen and T cell Bispecific antibody is named as CD3-BTLA BsAb.

First, CD3-BTLA BsAb_M and the design of CD3-BTLA BsAb_D constructing plans

The specific constructing plans of CD3-BTLA BsAb_M of monomeric form are：AntiCD3 McAb scFv and anti-BTLA scFv sequences it Between pass through (GGGGS)₃Linker is connected.

The specific constructing plans of CD3-BTLA BsAb_D of dimeric forms are：AntiCD3 McAb scFv and anti-BTLA scFv sequences Between Linker be used as by IgD hinge areas be connected.

For bispecific antibody is made to be expressed in mammalian cell, for AntiCD3 McAb scFv, anti-BTLA scFv and connection The sequence of segment (Linker) has carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-BTLA scFv is as shown in SEQ ID NO.75, specially：

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCC GCCAGCGGCTTCACCATCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCACCGGCAAGGGCCTGGAGTGGGTGAG CGTGATCGGCCCCGCCGGCGACACCTACTACCCCGGCAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAGAACGCCA AGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGGCGACACCGCCGTGTACTACTGCGCCCGCGAGGGCATG GCCGCCCACAACTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

Specifically, the nucleotide sequence of the light chain variable region of anti-BTLA scFv is as shown in SEQ ID NO.76, specially：

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGC CGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTA CGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCA TCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCATCACCTTCGGC CAGGGCACCCGCCTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of anti-BTLA scFv is as shown in SEQ ID NO.74, specially：

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCC GCCAGCGGCTTCACCATCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCACCGGCAAGGGCCTGGAGTGGGTGAG CGTGATCGGCCCCGCCGGCGACACCTACTACCCCGGCAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAGAACGCCA AGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGGCGACACCGCCGTGTACTACTGCGCCCGCGAGGGCATG GCCGCCCACAACTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAG CGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCG GCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGC CAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAG CGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCA ACTGGCCCCCCATCACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

The nucleotide sequence of the CD3-BTLA BsAb_M junction fragments of monomeric form is as shown in SEQ ID NO.2.

The nucleotide sequence of the CD3-BTLA BsAb_D junction fragments of dimeric forms is as shown in SEQ ID NO.4.

2nd, CD3-BTLA BsAb_M and CD3-BTLA BsAb_D construction of eukaryotic expression vector

The construction and expression of bispecific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1 (being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the bispecific antibody of dimeric forms, separately design Primer as shown in table 6, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplification Zhou Hongxun Science and Technology Ltd.s synthesize.

It builds for the clone of CD3-BTLA BsAb_M, is amplified first using primer pcDNA3.1-Sig-F and Sig-R Then signal peptide fragment is utilized respectively primer Sig-CD3-F and CD3-R, CD3- (GGGGS)₃- BTLA-F and pcDNA3.1- BTLA-R amplifies AntiCD3 McAb scFv, (GGGGS)₃The gene order of Linker, anti-BTLA scFv；For CD3-BTLA Clone's structure of BsAb_D, equally amplifies signal peptide fragment, Ran Houfen using primer pcDNA3.1-Sig-F and Sig-R first It is not amplified using primer Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-BTLA-F and pcDNA3.1-BTLA-R The gene order of AntiCD3 McAb scFv, IgD hinge area, anti-BTLA scFv.After amplification, utilizeMono- steps of PCR are determined Splice monomer and dimeric forms bispecific respectively to Cloning Kit (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.) Antibody full-length gene order simultaneously seamless is cloned on the pcDNA3.1 expression vectors through EcoRI and HindIII linearization process.Mesh Carrier conversion bacillus coli DH 5 alpha, carry out positive clone identification using bacterium colony PCR, be accredited as positive recon (recombination matter Grain) carry out sequencing identification.Correct recon (recombinant plasmid) will then be sequenced to arrange to take out in plasmid, for CHO-S cells Transfection.

Know through sequencing, the CD3-BTLA BsAb_M of monomeric form and the CD3-BTLA BsAb_D's of dimeric forms is complete Long gene order is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD3-BTLA BsAb_M of monomeric form is as shown in SEQ ID NO.32, specifically For：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTG CGCCGCCAGCGGCTTCACCATCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCACCGGCAAGGGCCTGGAGTGGG TGAGCGTGATCGGCCCCGCCGGCGACACCTACTACCCCGGCAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAGAAC GCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGGCGACACCGCCGTGTACTACTGCGCCCGCGAGGG CATGGCCGCCCACAACTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCG GCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGC CCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCC CGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCG GCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGC AGCAACTGGCCCCCCATCACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

Specifically, the nucleotide sequence of the CD3-BTLA BsAb_D of dimeric forms is as shown in SEQ ID NO.34, specifically For：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAG ACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGG CTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGA GCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTAC GACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGG CAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGC ACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAG CGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCA GCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGG CCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCAC CACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGG AGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGGAGGTGCAGCTGGTGGAGAGC GGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCATCAGCAGCTACGA CATGCACTGGGTGCGCCAGGCCACCGGCAAGGGCCTGGAGTGGGTGAGCGTGATCGGCCCCGCCGGCGACACCTACT ACCCCGGCAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGC CTGCGCGCCGGCGACACCGCCGTGTACTACTGCGCCCGCGAGGGCATGGCCGCCCACAACTACTACGGCATGGACGT GTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCA GCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCC AGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGC CAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCA GCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCATCACCTTCGGCCAGGGC ACCCGCCTGGAGATCAAGCGC。

The primer used in table 6.CD3-BTLA bispecific antibody gene clonings

Embodiment 27：The expression and purification of CD3-BTLA BsAb_M and CD3-BTLA BsAb_D

First, the expression of CD3-BTLA BsAb_M and CD3-BTLA BsAb_D

1.3. transfection composite is prepared：Each project (CD3-BTLA BsAb_M and CD3-BTLA BsAb_D) need to prepare two A centrifuge tube/culture bottle, by taking 20ml as an example, is placed respectively, prepared recombinant plasmid in Example 26：

2nd, the purifying of CD3-BTLA BsAb_M and CD3-BTLA BsAb_D

2.1 sample pretreatment

2.2Protein L affinity chromatography column purifications

Buffer solution A (Buffer A)：PBS, pH7.4

Buffer solution B (Buffer B)：0.1M Glycine,pH3.0

Buffer solution C (Buffer C)：0.1M Glycine,pH2.7

CD3-BTLA BsAb_M and CD3-BTLA the BsAb_D recombinant proteins finally purified are analyzed through SDS-PAGE, reduction It is as shown in figure 22 with electrophoretogram under non reducing conditions.It can be seen from the figure that through Protein L affinity columns after purification, The purity of CD3-BTLA BsAb_M and CD3-BTLA BsAb_D recombinant proteins is equal>95%；Wherein CD3-BTLA BsAb_M are recombinated The theoretical molecular weight of albumen is 53.1kDa, and the albumen is presented single electrophoretic band under reduction and non reducing conditions, molecular weight with Monomer is consistent, therefore the bispecific antibody is monomeric form (Figure 22 A)；The theory of CD3-BTLA BsAb_D recombinant proteins point Son amount is 61.0kDa, the protein electrophoresis band, non reducing conditions under the electrophoresis consistent with monomer that be presented molecular weight under reducing condition It is consistent with dimer (Figure 22 B) that band is presented molecular weight, illustrates that two protein moleculars can be connected with each other by disulfide bond, therefore The bispecific antibody is dimeric forms.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sample Equal frame is errorless, consistent with theoretical N/C terminal amino acid sequences, and mass spectral analysis further confirms that CD3-BTLA BsAb_M are single Body form, CD3-BTLA BsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence of the CD3-BTLA BsAb_M of monomeric form as shown in SEQ ID NO.31, Specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTISSYDMHWVRQATG KGLEWVSVIGPAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAREGMAAHNYYGMDVWGQGTTVTV SSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIKR。

The amino acid sequence of the CD3-BTLA BsAb_D of dimeric forms is as shown in SEQ ID NO.33, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEK EKEEQEERETKTPECPSHTQPLGVEVQLVESGGGLVQPGGSLRLSCAASGFTISSYDMHWVRQATGKGLEWVSVIGP AGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAREGMAAHNYYGMDVWGQGTTVTVSSGGGGSGGGG SGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDF TLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIKR。

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.35.

The amino acid sequence of anti-BTLA scFv is as shown in SEQ ID NO.53, specially：

EVQLVESGGGLVQPGGSLRLSCAASGFTISSYDMHWVRQATGKGLEWVSVIGPAGDTYYPGSVKGRFTI SRENAKNSLYLQMNSLRAGDTAVYYCAREGMAAHNYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPAT LSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYY CQQRSNWPPITFGQGTRLEIKR。

The amino acid sequence of the heavy chain variable region of anti-BTLA scFv is as shown in SEQ ID NO.54, specially：

EVQLVESGGGLVQPGGSLRLSCAASGFTISSYDMHWVRQATGKGLEWVSVIGPAGDTYYPGSVKGRFTI SRENAKNSLYLQMNSLRAGDTAVYYCAREGMAAHNYYGMDVWGQGTTVTVSS。

The amino acid sequence of the light chain variable region of anti-BTLA scFv is as shown in SEQ ID NO.55, specially：

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIKR。

The amino acid sequence of junction fragment is as shown in SEQ ID NO.1 in the CD3-BTLA BsAb_M of monomeric form.

The amino acid sequence of junction fragment is as shown in SEQ ID NO.3 in the CD3-BTLA BsAb_D of dimeric forms.

Embodiment 28：The antigen-binding activity of ELISA detection CD3-BTLA BsAb_M and CD3-BTLA BsAb_D

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD3-hFc (is purchased from Wujiang offshore protein section with mankind BTLA-hFc fusion proteins Skill Co., Ltd) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes, and coating condition is 37 DEG C 1 Hour or 4 DEG C overnight, coating buffer solution (PBS) formula be：3.58g Na₂HPO4,0.24g NaH₂PO4,0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing to 1L；

3. sample-adding：After PBS board-washings 4 times, the bispecific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1 Hour, sample gradient preparation method：Using CD3-BTLA BsAb_M or the CD3-BTLA BsAb_D that 10 μ g/ml are purified as starting Concentration, carries out 6 gradients of doubling dilution, and each gradient sets 2 multiple holes；

ELISA results are as shown in Figure 23 A and Figure 23 B：Figure 23 A illustrate CD3-BTLA BsAb_M and recombinant antigen CD3-hFc External combination activity is respectively provided with BTLA-hFc, wherein BTLA combines activity and combines activity higher compared with CD3；Figure 23 B illustrate CD3- BTLA BsAb_D are similary with recombinant antigen CD3-hFc and BTLA-hFc to have external combination activity, and wherein BTLA combines activity more It is high.

Embodiment 29：The CIK cell proliferation of CD3-BTLA bispecific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC) Material, with bispecific antibody CD3-BTLA BsAb_M of the above-mentioned monomeric form prepared by the present invention, dimeric forms it is double special Antibody CD3-BTLA BsAb_D and AntiCD3 McAb/anti- CD28 monoclonals full length antibody combination (Anti-CD3/Anti-CD28) point The people blood PBMC of same donor source is not acted on, is counted after cell culture, compares amplification times.

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) are resuspended, It is 1 × 10 to adjust cell density⁶/ ml separately designs following 3 experimental groups：Control group (Anti-CD3 5ug/ml and Anti- CD285ug/ml coated cell culture plates, full length antibody are purchased from Wujiang Alongshore Protein Technology Co., Ltd.)；Experimental group 1 is (molten Bispecific antibody CD3-BTLA BsAb_M 10ng/ml are added under liquid status)；Experimental group 2 (is added double special under solution state Property antibody CD3-BTLA BsAb_D 10ng/ml).In addition, three groups of experimental cells add cell factor IFN-γ (200ng/ simultaneously Ml, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) and IL-1 β (2ng/ml, purchased from the limited public affairs of Wujiang offshore protein science and technology Department), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, 500U/ is added The IL-2 (be purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of ml continues to cultivate, and is counted per 2-3 days and with adding 500U/ml The CIK basal mediums of IL-2 press 1 × 10⁶The density of/ml carries out cell passage.Method culture 30 days like this, final statistics are thin The amplification times of born of the same parents draw growth curve；

Testing result is as shown in figure 24, the CD3-BTLA bispecific antibody single uses pair of monomer and dimeric forms The cultivation effect of CIK cell is superior to AntiCD3 McAb/anti- CD28 monoclonal full length antibodies and is used in combination, after cultivating 18 days, Anti- There are a large amount of cell deaths in CD3/Anti-CD28 combinations, and cells expanded is remarkably decreased；And add the CD3- of monomeric form BTLA BsAb_M or the CD3-BTLA BsAb_D of dimeric forms do not occur cell death, and only cell amplification rate is opposite Slow down.Therefore the CD3-BTLA bispecific antibodies of two kinds of forms that prepared by the present invention can effectively expand and extend CIK cell Life cycle, wherein dimeric forms effect is more preferable.

Embodiment 30：The CIK cell IFN-γ secretion of CD3-BTLA bispecific antibodies induction

Operating procedure：

1st, the CIK cell supernatant after being cultivated 25 days in Example 29 (is adjusted to same cell density, cell number 2 ×10⁵It is a) 100 μ l, 37 DEG C of incubation 45min, are examined by Human IFN-γ ELISA Kit (purchased from doctor's moral biology) It surveys, three groups of every group of experiments take three samples to repeat；

As a result as shown in figure 25：Wherein secreted by the CIK cell of Anti-CD3/Anti-CD28 full length antibodies combination culture IFN-γ quantity be defined as 1, the CIK cell IFN- of the CD3-BTLA BsAb_M cultures of addition monomeric form under solution state γ is 1.54 with respect to secretory volume, the CIK cell IFN- of the CD3-BTLA BsAb_D cultures of addition dimeric forms under solution state γ is 2.24 with respect to secretory volume, therefore the CD3-BTLA bispecific antibodies of two kinds of forms prepared by the present invention are more advantageous to activating CIK cell, induces the secretion of IFN-γ, and wherein dimeric forms are better.

The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation, It should be pointed out that for those skilled in the art, under the premise of the method for the present invention is not departed from, can also make Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more Dynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile all substantial technologicals pair according to the present invention The variation, modification and evolution of any equivalent variations that above-described embodiment is made still fall within the range of technical scheme of the present invention It is interior.

SEQUENCE LISTING

<110>Shanghai Xinbainuo Biology Science Co., Ltd

<120>A kind of combination CD3 and T cell bear bifunctional molecule and its application of costimulatory molecules

<130> 164635

<160> 102

<170> PatentIn version 3.3

<210> 1

<211> 15

<212> PRT

<213> Artificial

<220>

<223>The AntiCD3 McAb of monomeric form/anti- T cell bears the amino acid sequence of junction fragment in costimulatory molecules bispecific antibody

Row

<400> 1

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 2

<211> 45

<212> DNA

<213> Artificial

<220>

<223>The AntiCD3 McAb of monomeric form/anti- T cell bears the nucleotides sequence of junction fragment in costimulatory molecules bispecific antibody

Row

<400> 2

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45

<210> 3

<211> 81

<212> PRT

<213> Artificial

<220>

<223>The AntiCD3 McAb of dimeric forms/anti- T cell bears the amino of junction fragment in costimulatory molecules bispecific antibody

Acid sequence

<400> 3

Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu Ser Pro Lys

1 5 10 15

Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala Glu Gly Ser

20 25 30

Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg

35 40 45

Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu Glu

50 55 60

Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly

65 70 75 80

Val

<210> 4

<211> 243

<212> DNA

<213> Artificial

<220>

<223>The AntiCD3 McAb of dimeric forms/anti- T cell bears the nucleosides of junction fragment in costimulatory molecules bispecific antibody

Acid sequence

<400> 4

gccagcaaga gcaagaagga gatcttccgc tggcccgaga gccccaaggc ccaggccagc 60

agcgtgccca ccgcccagcc ccaggccgag ggcagcctgg ccaaggccac caccgccccc 120

gccaccaccc gcaacaccgg ccgcggcggc gaggagaaga agaaggagaa ggagaaggag 180

gagcaggagg agcgcgagac caagaccccc gagtgcccca gccacaccca gcccctgggc 240

gtg 243

<210> 5

<211> 150

<212> PRT

<213> Artificial

<220>

<223>T cell bears the amino acid sequence of costimulatory molecules mankind's PD-1 extracellular regions

<400> 5

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 6

<211> 126

<212> PRT

<213> Artificial

<220>

<223>T cell bears the amino acid sequence of costimulatory molecules mankind's CTLA-4 extracellular regions

<400> 6

Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg

1 5 10 15

Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp

50 55 60

Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

115 120 125

<210> 7

<211> 422

<212> PRT

<213> Artificial

<220>

<223>T cell bears the amino acid sequence of costimulatory molecules mankind's LAG-3 extracellular regions

<400> 7

Val Pro Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys

1 5 10 15

Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly

20 25 30

Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro

35 40 45

Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp

50 55 60

Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly

65 70 75 80

Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu

85 90 95

Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg

100 105 110

Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg

115 120 125

Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr

130 135 140

Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn

145 150 155 160

Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg

165 170 175

Asn Arg Gly Gln Gly Arg Val Pro Val Arg Glu Ser Pro His His His

180 185 190

Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser

195 200 205

Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser

210 215 220

Ile Met Tyr Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu

225 230 235 240

Thr Val Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu

245 250 255

Pro Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro

260 265 270

Pro Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe

275 280 285

Thr Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr

290 295 300

Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu

305 310 315 320

Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu

325 330 335

Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe

340 345 350

Val Trp Ser Ser Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro

355 360 365

Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys

370 375 380

Gln Leu Tyr Gln Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr

385 390 395 400

Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala

405 410 415

Leu Pro Ala Gly His Leu

420

<210> 8

<211> 181

<212> PRT

<213> Artificial

<220>

<223>T cell bears the amino acid sequence of costimulatory molecules mankind's TIM-3 extracellular regions

<400> 8

Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln Asn Ala Tyr Leu Pro

1 5 10 15

Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu Val Pro Val Cys Trp

20 25 30

Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly Asn Val Val Leu Arg

35 40 45

Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser Arg Tyr Trp Leu Asn

50 55 60

Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr Ile Glu Asn Val Thr

65 70 75 80

Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile Gln Ile Pro Gly Ile

85 90 95

Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val Ile Lys Pro Ala Lys

100 105 110

Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe Thr Ala Ala Phe Pro

115 120 125

Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala Glu Thr Gln Thr Leu

130 135 140

Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile Ser Thr Leu Ala Asn

145 150 155 160

Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu Arg Asp Ser Gly Ala

165 170 175

Thr Ile Arg Ile Gly

180

<210> 9

<211> 120

<212> PRT

<213> Artificial

<220>

<223>T cell bears the amino acid sequence of costimulatory molecules mankind's TIGIT extracellular regions

<400> 9

Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys

1 5 10 15

Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln

20 25 30

Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys

35 40 45

Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val

50 55 60

Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn

65 70 75 80

Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr

85 90 95

Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu

100 105 110

His Gly Ala Arg Phe Gln Ile Pro

115 120

<210> 10

<211> 127

<212> PRT

<213> Artificial

<220>

<223>T cell bears the amino acid sequence of costimulatory molecules mankind's BTLA extracellular regions

<400> 10

Lys Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg Gln Ser Glu His

1 5 10 15

Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys Pro Val Lys Tyr

20 25 30

Cys Ala Asn Arg Pro His Val Thr Trp Cys Lys Leu Asn Gly Thr Thr

35 40 45

Cys Val Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys Glu Glu Lys Asn

50 55 60

Ile Ser Phe Phe Ile Leu His Phe Glu Pro Val Leu Pro Asn Asp Asn

65 70 75 80

Gly Ser Tyr Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu Ser

85 90 95

His Ser Thr Thr Leu Tyr Val Thr Asp Val Lys Ser Ala Ser Glu Arg

100 105 110

Pro Ser Lys Asp Glu Met Ala Ser Arg Pro Trp Leu Leu Tyr Arg

115 120 125

<210> 11

<211> 494

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-PD-1 BsAb_M of monomeric form

<400> 11

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

260 265 270

Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser

275 280 285

Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

290 295 300

Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp

305 310 315 320

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

325 330 335

Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

340 345 350

Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

355 360 365

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

370 375 380

Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser

385 390 395 400

Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser

405 410 415

Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

420 425 430

Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe

435 440 445

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

450 455 460

Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp

465 470 475 480

Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

485 490

<210> 12

<211> 1482

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-PD-1 BsAb_M of monomeric form

<400> 12

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccaggtg 780

cagctggtgg agagcggcgg cggcgtggtg cagcccggcc gcagcctgcg cctggactgc 840

aaggccagcg gcatcacctt cagcaacagc ggcatgcact gggtgcgcca ggcccccggc 900

aagggcctgg agtgggtggc cgtgatctgg tacgacggca gcaagcgcta ctacgccgac 960

agcgtgaagg gccgcttcac catcagccgc gacaacagca agaacaccct gttcctgcag 1020

atgaacagcc tgcgcgccga ggacaccgcc gtgtactact gcgccaccaa cgacgactac 1080

tggggccagg gcaccctggt gaccgtgagc agcggcggcg gcggcagcgg cggcggcggc 1140

agcggcggcg gcggcagcga gatcgtgctg acccagagcc ccgccaccct gagcctgagc 1200

cccggcgagc gcgccaccct gagctgccgc gccagccaga gcgtgagcag ctacctggcc 1260

tggtaccagc agaagcccgg ccaggccccc cgcctgctga tctacgacgc cagcaaccgc 1320

gccaccggca tccccgcccg cttcagcggc agcggcagcg gcaccgactt caccctgacc 1380

atcagcagcc tggagcccga ggacttcgcc gtgtactact gccagcagag cagcaactgg 1440

ccccgcacct tcggccaggg caccaaggtg gagatcaagc gc 1482

<210> 13

<211> 560

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-PD-1 BsAb_D of dimeric forms

<400> 13

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu

245 250 255

Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala

260 265 270

Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn

275 280 285

Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu

290 295 300

Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln

305 310 315 320

Pro Leu Gly Val Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

325 330 335

Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr

340 345 350

Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

355 360 365

Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr

370 375 380

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

385 390 395 400

Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

405 410 415

Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu

420 425 430

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

450 455 460

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

465 470 475 480

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

500 505 510

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

515 520 525

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser

530 535 540

Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

545 550 555 560

<210> 14

<211> 1680

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-PD-1 BsAb_D of dimeric forms

<400> 14

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780

caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840

accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900

gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960

cccctgggcg tgcaggtgca gctggtggag agcggcggcg gcgtggtgca gcccggccgc 1020

agcctgcgcc tggactgcaa ggccagcggc atcaccttca gcaacagcgg catgcactgg 1080

gtgcgccagg cccccggcaa gggcctggag tgggtggccg tgatctggta cgacggcagc 1140

aagcgctact acgccgacag cgtgaagggc cgcttcacca tcagccgcga caacagcaag 1200

aacaccctgt tcctgcagat gaacagcctg cgcgccgagg acaccgccgt gtactactgc 1260

gccaccaacg acgactactg gggccagggc accctggtga ccgtgagcag cggcggcggc 1320

ggcagcggcg gcggcggcag cggcggcggc ggcagcgaga tcgtgctgac ccagagcccc 1380

gccaccctga gcctgagccc cggcgagcgc gccaccctga gctgccgcgc cagccagagc 1440

gtgagcagct acctggcctg gtaccagcag aagcccggcc aggccccccg cctgctgatc 1500

tacgacgcca gcaaccgcgc caccggcatc cccgcccgct tcagcggcag cggcagcggc 1560

accgacttca ccctgaccat cagcagcctg gagcccgagg acttcgccgt gtactactgc 1620

cagcagagca gcaactggcc ccgcaccttc ggccagggca ccaaggtgga gatcaagcgc 1680

<210> 15

<211> 500

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-CTLA-4 BsAb_M of monomeric form

<400> 15

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

260 265 270

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

275 280 285

Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

290 295 300

Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp

305 310 315 320

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

325 330 335

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr

340 345 350

Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln

355 360 365

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

370 375 380

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly

385 390 395 400

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

405 410 415

Ser Gln Ser Val Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro

420 425 430

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr

435 440 445

Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

450 455 460

Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

465 470 475 480

Gln Gln Tyr Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

485 490 495

Glu Ile Lys Arg

500

<210> 16

<211> 1500

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-CTLA-4 BsAb_M of monomeric form

<400> 16

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccaggtg 780

cagctggtgg agagcggcgg cggcgtggtg cagcccggcc gcagcctgcg cctgagctgc 840

gccgccagcg gcttcacctt cagcagctac accatgcact gggtgcgcca ggcccccggc 900

aagggcctgg agtgggtgac cttcatcagc tacgacggca acaacaagta ctacgccgac 960

agcgtgaagg gccgcttcac catcagccgc gacaacagca agaacaccct gtacctgcag 1020

atgaacagcc tgcgcgccga ggacaccgcc atctactact gcgcccgcac cggctggctg 1080

ggccccttcg actactgggg ccagggcacc ctggtgaccg tgagcagcgg cggcggcggc 1140

agcggcggcg gcggcagcgg cggcggcggc agcgagatcg tgctgaccca gagccccggc 1200

accctgagcc tgagccccgg cgagcgcgcc accctgagct gccgcgccag ccagagcgtg 1260

ggcagcagct acctggcctg gtaccagcag aagcccggcc aggccccccg cctgctgatc 1320

tacggcgcct tcagccgcgc caccggcatc cccgaccgct tcagcggcag cggcagcggc 1380

accgacttca ccctgaccat cagccgcctg gagcccgagg acttcgccgt gtactactgc 1440

cagcagtacg gcagcagccc ctggaccttc ggccagggca ccaaggtgga gatcaagcgc 1500

<210> 17

<211> 566

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-CTLA-4 BsAb_D of dimeric forms

<400> 17

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu

245 250 255

Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala

260 265 270

Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn

275 280 285

Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu

290 295 300

Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln

305 310 315 320

Pro Leu Gly Val Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

325 330 335

Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

340 345 350

Phe Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

355 360 365

Leu Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr

370 375 380

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

385 390 395 400

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

405 410 415

Ile Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp

420 425 430

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

450 455 460

Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

465 470 475 480

Arg Ala Ser Gln Ser Val Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln

485 490 495

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Phe Ser Arg

500 505 510

Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

515 520 525

Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr

530 535 540

Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr

545 550 555 560

Lys Val Glu Ile Lys Arg

565

<210> 18

<211> 1698

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-CTLA-4 BsAb_D of dimeric forms

<400> 18

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780

caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840

accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900

gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960

cccctgggcg tgcaggtgca gctggtggag agcggcggcg gcgtggtgca gcccggccgc 1020

agcctgcgcc tgagctgcgc cgccagcggc ttcaccttca gcagctacac catgcactgg 1080

gtgcgccagg cccccggcaa gggcctggag tgggtgacct tcatcagcta cgacggcaac 1140

aacaagtact acgccgacag cgtgaagggc cgcttcacca tcagccgcga caacagcaag 1200

aacaccctgt acctgcagat gaacagcctg cgcgccgagg acaccgccat ctactactgc 1260

gcccgcaccg gctggctggg ccccttcgac tactggggcc agggcaccct ggtgaccgtg 1320

agcagcggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag cgagatcgtg 1380

ctgacccaga gccccggcac cctgagcctg agccccggcg agcgcgccac cctgagctgc 1440

cgcgccagcc agagcgtggg cagcagctac ctggcctggt accagcagaa gcccggccag 1500

gccccccgcc tgctgatcta cggcgccttc agccgcgcca ccggcatccc cgaccgcttc 1560

agcggcagcg gcagcggcac cgacttcacc ctgaccatca gccgcctgga gcccgaggac 1620

ttcgccgtgt actactgcca gcagtacggc agcagcccct ggaccttcgg ccagggcacc 1680

aaggtggaga tcaagcgc 1698

<210> 19

<211> 501

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-LAG-3 BsAb_M of monomeric form

<400> 19

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

260 265 270

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser

275 280 285

Asp Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

290 295 300

Trp Ile Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser

305 310 315 320

Leu Lys Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe

325 330 335

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp

355 360 365

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

370 375 380

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

385 390 395 400

Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

405 410 415

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys

420 425 430

Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala

435 440 445

Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

450 455 460

Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr

465 470 475 480

Cys Gln Gln Arg Ser Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Asn

485 490 495

Leu Glu Ile Lys Arg

500

<210> 20

<211> 1503

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-LAG-3 BsAb_M of monomeric form

<400> 20

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccaggtg 780

cagctgcagc agtggggcgc cggcctgctg aagcccagcg agaccctgag cctgacctgc 840

gccgtgtacg gcggcagctt cagcgactac tactggaact ggatccgcca gccccccggc 900

aagggcctgg agtggatcgg cgagatcaac caccgcggca gcaccaacag caaccccagc 960

ctgaagagcc gcgtgaccct gagcctggac accagcaaga accagttcag cctgaagctg 1020

cgcagcgtga ccgccgccga caccgccgtg tactactgcg ccttcggcta cagcgactac 1080

gagtacaact ggttcgaccc ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 1140

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg agatcgtgct gacccagagc 1200

cccgccaccc tgagcctgag ccccggcgag cgcgccaccc tgagctgccg cgccagccag 1260

agcatcagca gctacctggc ctggtaccag cagaagcccg gccaggcccc ccgcctgctg 1320

atctacgacg ccagcaaccg cgccaccggc atccccgccc gcttcagcgg cagcggcagc 1380

ggcaccgact tcaccctgac catcagcagc ctggagcccg aggacttcgc cgtgtactac 1440

tgccagcagc gcagcaactg gcccctgacc ttcggccagg gcaccaacct ggagatcaag 1500

cgc 1503

<210> 21

<211> 567

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-LAG-3 BsAb_D of dimeric forms

<400> 21

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu

245 250 255

Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala

260 265 270

Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn

275 280 285

Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu

290 295 300

Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln

305 310 315 320

Pro Leu Gly Val Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

325 330 335

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser

340 345 350

Phe Ser Asp Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly

355 360 365

Leu Glu Trp Ile Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn

370 375 380

Pro Ser Leu Lys Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn

385 390 395 400

Gln Phe Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val

405 410 415

Tyr Tyr Cys Ala Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp

420 425 430

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr

450 455 460

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

465 470 475 480

Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala Trp Tyr Gln

485 490 495

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn

500 505 510

Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

515 520 525

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

530 535 540

Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu Thr Phe Gly Gln Gly

545 550 555 560

Thr Asn Leu Glu Ile Lys Arg

565

<210> 22

<211> 1701

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-LAG-3 BsAb_D of dimeric forms

<400> 22

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780

caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840

accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900

gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960

cccctgggcg tgcaggtgca gctgcagcag tggggcgccg gcctgctgaa gcccagcgag 1020

accctgagcc tgacctgcgc cgtgtacggc ggcagcttca gcgactacta ctggaactgg 1080

atccgccagc cccccggcaa gggcctggag tggatcggcg agatcaacca ccgcggcagc 1140

accaacagca accccagcct gaagagccgc gtgaccctga gcctggacac cagcaagaac 1200

cagttcagcc tgaagctgcg cagcgtgacc gccgccgaca ccgccgtgta ctactgcgcc 1260

ttcggctaca gcgactacga gtacaactgg ttcgacccct ggggccaggg caccctggtg 1320

accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgag 1380

atcgtgctga cccagagccc cgccaccctg agcctgagcc ccggcgagcg cgccaccctg 1440

agctgccgcg ccagccagag catcagcagc tacctggcct ggtaccagca gaagcccggc 1500

caggcccccc gcctgctgat ctacgacgcc agcaaccgcg ccaccggcat ccccgcccgc 1560

ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcagcct ggagcccgag 1620

gacttcgccg tgtactactg ccagcagcgc agcaactggc ccctgacctt cggccagggc 1680

accaacctgg agatcaagcg c 1701

<210> 23

<211> 503

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-TIM-3 BsAb_M of monomeric form

<400> 23

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

260 265 270

Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

275 280 285

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

290 295 300

Trp Ile Gly Asp Ile Tyr Pro Gly Gln Gly Asp Thr Ser Tyr Asn Gln

305 310 315 320

Lys Phe Lys Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Thr Ser Thr

325 330 335

Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

340 345 350

Tyr Cys Ala Arg Val Gly Gly Ala Phe Pro Met Asp Tyr Trp Gly Gln

355 360 365

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

370 375 380

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Asp

385 390 395 400

Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala

405 410 415

Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln Trp Tyr Gln

420 425 430

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn

435 440 445

Val Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

450 455 460

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

465 470 475 480

Tyr Tyr Cys Gln Gln Ser Arg Lys Asp Pro Ser Thr Phe Gly Gly Gly

485 490 495

Thr Lys Val Glu Ile Lys Arg

500

<210> 24

<211> 1509

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-TIM-3 BsAb_M of monomeric form

<400> 24

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagccaggtg 780

cagctggtgc agagcggcgc cgaggtgaag aagcccggcg ccagcgtgaa ggtgagctgc 840

aaggccagcg gctacacctt caccagctac aacatgcact gggtgcgcca ggcccccggc 900

cagggcctgg agtggatcgg cgacatctac cccggccagg gcgacaccag ctacaaccag 960

aagttcaagg gccgcgccac catgaccgcc gacaagagca ccagcaccgt gtacatggag 1020

ctgagcagcc tgcgcagcga ggacaccgcc gtgtactact gcgcccgcgt gggcggcgcc 1080

ttccccatgg actactgggg ccagggcacc ctggtgaccg tgagcagcgg cggcggcggc 1140

agcggcggcg gcggcagcgg cggcggcggc agcgacatcg tgctgaccca gagccccgac 1200

agcctggccg tgagcctggg cgagcgcgcc accatcaact gccgcgccag cgagagcgtg 1260

gagtactacg gcaccagcct gatgcagtgg taccagcaga agcccggcca gccccccaag 1320

ctgctgatct acgccgccag caacgtggag agcggcgtgc ccgaccgctt cagcggcagc 1380

ggcagcggca ccgacttcac cctgaccatc agcagcctgc aggccgagga cgtggccgtg 1440

tactactgcc agcagagccg caaggacccc agcaccttcg gcggcggcac caaggtggag 1500

atcaagcgc 1509

<210> 25

<211> 569

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-TIM-3 BsAb_D of dimeric forms

<400> 25

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu

245 250 255

Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala

260 265 270

Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn

275 280 285

Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu

290 295 300

Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln

305 310 315 320

Pro Leu Gly Val Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

325 330 335

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

340 345 350

Phe Thr Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

355 360 365

Leu Glu Trp Ile Gly Asp Ile Tyr Pro Gly Gln Gly Asp Thr Ser Tyr

370 375 380

Asn Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Thr

385 390 395 400

Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

405 410 415

Val Tyr Tyr Cys Ala Arg Val Gly Gly Ala Phe Pro Met Asp Tyr Trp

420 425 430

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser

450 455 460

Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys

465 470 475 480

Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln Trp

485 490 495

Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala

500 505 510

Ser Asn Val Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

515 520 525

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val

530 535 540

Ala Val Tyr Tyr Cys Gln Gln Ser Arg Lys Asp Pro Ser Thr Phe Gly

545 550 555 560

Gly Gly Thr Lys Val Glu Ile Lys Arg

565

<210> 26

<211> 1707

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-TIM-3 BsAb_D of dimeric forms

<400> 26

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780

caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840

accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900

gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960

cccctgggcg tgcaggtgca gctggtgcag agcggcgccg aggtgaagaa gcccggcgcc 1020

agcgtgaagg tgagctgcaa ggccagcggc tacaccttca ccagctacaa catgcactgg 1080

gtgcgccagg cccccggcca gggcctggag tggatcggcg acatctaccc cggccagggc 1140

gacaccagct acaaccagaa gttcaagggc cgcgccacca tgaccgccga caagagcacc 1200

agcaccgtgt acatggagct gagcagcctg cgcagcgagg acaccgccgt gtactactgc 1260

gcccgcgtgg gcggcgcctt ccccatggac tactggggcc agggcaccct ggtgaccgtg 1320

agcagcggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag cgacatcgtg 1380

ctgacccaga gccccgacag cctggccgtg agcctgggcg agcgcgccac catcaactgc 1440

cgcgccagcg agagcgtgga gtactacggc accagcctga tgcagtggta ccagcagaag 1500

cccggccagc cccccaagct gctgatctac gccgccagca acgtggagag cggcgtgccc 1560

gaccgcttca gcggcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctgcag 1620

gccgaggacg tggccgtgta ctactgccag cagagccgca aggaccccag caccttcggc 1680

ggcggcacca aggtggagat caagcgc 1707

<210> 27

<211> 507

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-TIGIT BsAb_M of monomeric form

<400> 27

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

260 265 270

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala

275 280 285

Ser Asp Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu

290 295 300

Trp Met Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser

305 310 315 320

Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe

325 330 335

Phe Leu Gln Leu His Ser Val Thr Thr Asp Asp Thr Ala Thr Tyr Ser

340 345 350

Cys Ala Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp

355 360 365

Glu Gly Tyr Phe Asp Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly

405 410 415

Asp Arg Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Ile His Lys Asn

420 425 430

Leu Ala Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Phe Leu Ile

435 440 445

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

450 455 460

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

465 470 475 480

Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

485 490 495

Phe Gly Gly Gly Thr Lys Val Glu Leu Lys Arg

500 505

<210> 28

<211> 1521

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-TIGIT BsAb_M of monomeric form

<400> 28

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagcgaggtg 780

cagctgcagg agagcggccc cggcctggtg aagcccagcc agagcctgag cctgacctgc 840

agcgtgaccg gcagcagcat cgccagcgac tactggggct ggatccgcaa gttccccggc 900

aacaagatgg agtggatggg cttcatcacc tacagcggca gcaccagcta caaccccagc 960

ctgaagagcc gcatcagcat cacccgcgac accagcaaga accagttctt cctgcagctg 1020

cacagcgtga ccaccgacga caccgccacc tacagctgcg cccgcatgcc cagcttcatc 1080

accctggcca gcctgagcac ctgggagggc tacttcgact tctggggccc cggcaccatg 1140

gtgaccgtga gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 1200

gacatccaga tgacccagag ccccagcctg ctgagcgcca gcgtgggcga ccgcgtgacc 1260

ctgaactgca aggccagcca gagcatccac aagaacctgg cctggtacca gcagaagctg 1320

ggcgaggccc ccaagttcct gatctactac gccaacagcc tgcagaccgg catccccagc 1380

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcgg cctgcagccc 1440

gaggacgtgg ccacctactt ctgccagcag tactacagcg gctggacctt cggcggcggc 1500

accaaggtgg agctgaagcg c 1521

<210> 29

<211> 573

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-TIGIT BsAb_D of dimeric forms

<400> 29

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu

245 250 255

Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala

260 265 270

Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn

275 280 285

Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu

290 295 300

Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln

305 310 315 320

Pro Leu Gly Val Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

325 330 335

Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser

340 345 350

Ile Ala Ser Asp Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys

355 360 365

Met Glu Trp Met Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn

370 375 380

Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn

385 390 395 400

Gln Phe Phe Leu Gln Leu His Ser Val Thr Thr Asp Asp Thr Ala Thr

405 410 415

Tyr Ser Cys Ala Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser

420 425 430

Thr Trp Glu Gly Tyr Phe Asp Phe Trp Gly Pro Gly Thr Met Val Thr

435 440 445

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser

465 470 475 480

Val Gly Asp Arg Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Ile His

485 490 495

Lys Asn Leu Ala Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Phe

500 505 510

Leu Ile Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe

515 520 525

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu

530 535 540

Gln Pro Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly

545 550 555 560

Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Leu Lys Arg

565 570

<210> 30

<211> 1719

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-TIGIT BsAb_D of dimeric forms

<400> 30

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780

caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840

accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900

gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960

cccctgggcg tggaggtgca gctgcaggag agcggccccg gcctggtgaa gcccagccag 1020

agcctgagcc tgacctgcag cgtgaccggc agcagcatcg ccagcgacta ctggggctgg 1080

atccgcaagt tccccggcaa caagatggag tggatgggct tcatcaccta cagcggcagc 1140

accagctaca accccagcct gaagagccgc atcagcatca cccgcgacac cagcaagaac 1200

cagttcttcc tgcagctgca cagcgtgacc accgacgaca ccgccaccta cagctgcgcc 1260

cgcatgccca gcttcatcac cctggccagc ctgagcacct gggagggcta cttcgacttc 1320

tggggccccg gcaccatggt gaccgtgagc agcggcggcg gcggcagcgg cggcggcggc 1380

agcggcggcg gcggcagcga catccagatg acccagagcc ccagcctgct gagcgccagc 1440

gtgggcgacc gcgtgaccct gaactgcaag gccagccaga gcatccacaa gaacctggcc 1500

tggtaccagc agaagctggg cgaggccccc aagttcctga tctactacgc caacagcctg 1560

cagaccggca tccccagccg cttcagcggc agcggcagcg gcaccgactt caccctgacc 1620

atcagcggcc tgcagcccga ggacgtggcc acctacttct gccagcagta ctacagcggc 1680

tggaccttcg gcggcggcac caaggtggag ctgaagcgc 1719

<210> 31

<211> 503

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-BTLA BsAb_M of monomeric form

<400> 31

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

260 265 270

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser

275 280 285

Ser Tyr Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu

290 295 300

Trp Val Ser Val Ile Gly Pro Ala Gly Asp Thr Tyr Tyr Pro Gly Ser

305 310 315 320

Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu

325 330 335

Tyr Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Glu Gly Met Ala Ala His Asn Tyr Tyr Gly Met Asp Val

355 360 365

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln

385 390 395 400

Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser

405 410 415

Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln

420 425 430

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg

435 440 445

Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

450 455 460

Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr

465 470 475 480

Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Ile Thr Phe Gly Gln Gly

485 490 495

Thr Arg Leu Glu Ile Lys Arg

500

<210> 32

<211> 1509

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD3-BTLA BsAb_M of monomeric form

<400> 32

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagcgaggtg 780

cagctggtgg agagcggcgg cggcctggtg cagcccggcg gcagcctgcg cctgagctgc 840

gccgccagcg gcttcaccat cagcagctac gacatgcact gggtgcgcca ggccaccggc 900

aagggcctgg agtgggtgag cgtgatcggc cccgccggcg acacctacta ccccggcagc 960

gtgaagggcc gcttcaccat cagccgcgag aacgccaaga acagcctgta cctgcagatg 1020

aacagcctgc gcgccggcga caccgccgtg tactactgcg cccgcgaggg catggccgcc 1080

cacaactact acggcatgga cgtgtggggc cagggcacca ccgtgaccgt gagcagcggc 1140

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgagatcgt gctgacccag 1200

agccccgcca ccctgagcct gagccccggc gagcgcgcca ccctgagctg ccgcgccagc 1260

cagagcgtga gcagctacct ggcctggtac cagcagaagc ccggccaggc cccccgcctg 1320

ctgatctacg acgccagcaa ccgcgccacc ggcatccccg cccgcttcag cggcagcggc 1380

agcggcaccg acttcaccct gaccatcagc agcctggagc ccgaggactt cgccgtgtac 1440

tactgccagc agcgcagcaa ctggcccccc atcaccttcg gccagggcac ccgcctggag 1500

atcaagcgc 1509

<210> 33

<211> 569

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD3-BTLA BsAb_D of dimeric forms

<400> 33

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu

245 250 255

Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala

260 265 270

Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn

275 280 285

Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu

290 295 300

Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln

305 310 315 320

Pro Leu Gly Val Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

325 330 335

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

340 345 350

Ile Ser Ser Tyr Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly

355 360 365

Leu Glu Trp Val Ser Val Ile Gly Pro Ala Gly Asp Thr Tyr Tyr Pro

370 375 380

Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn

385 390 395 400

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val

405 410 415

Tyr Tyr Cys Ala Arg Glu Gly Met Ala Ala His Asn Tyr Tyr Gly Met

420 425 430

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu

450 455 460

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

465 470 475 480

Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr

485 490 495

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser

500 505 510

Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

515 520 525

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala

530 535 540

Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Ile Thr Phe Gly

545 550 555 560

Gln Gly Thr Arg Leu Glu Ile Lys Arg

565

<210> 34

<211> 1707

<212> DNA

<213> Artificial

<220>

<223>The nucleotide acid sequence of the CD3-BTLA BsAb_D of dimeric forms

<400> 34

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaagg ccagcaagag caagaaggag atcttccgct ggcccgagag ccccaaggcc 780

caggccagca gcgtgcccac cgcccagccc caggccgagg gcagcctggc caaggccacc 840

accgcccccg ccaccacccg caacaccggc cgcggcggcg aggagaagaa gaaggagaag 900

gagaaggagg agcaggagga gcgcgagacc aagacccccg agtgccccag ccacacccag 960

cccctgggcg tggaggtgca gctggtggag agcggcggcg gcctggtgca gcccggcggc 1020

agcctgcgcc tgagctgcgc cgccagcggc ttcaccatca gcagctacga catgcactgg 1080

gtgcgccagg ccaccggcaa gggcctggag tgggtgagcg tgatcggccc cgccggcgac 1140

acctactacc ccggcagcgt gaagggccgc ttcaccatca gccgcgagaa cgccaagaac 1200

agcctgtacc tgcagatgaa cagcctgcgc gccggcgaca ccgccgtgta ctactgcgcc 1260

cgcgagggca tggccgccca caactactac ggcatggacg tgtggggcca gggcaccacc 1320

gtgaccgtga gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 1380

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 1440

ctgagctgcc gcgccagcca gagcgtgagc agctacctgg cctggtacca gcagaagccc 1500

ggccaggccc cccgcctgct gatctacgac gccagcaacc gcgccaccgg catccccgcc 1560

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc 1620

gaggacttcg ccgtgtacta ctgccagcag cgcagcaact ggccccccat caccttcggc 1680

cagggcaccc gcctggagat caagcgc 1707

<210> 35

<211> 243

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of AntiCD3 McAb scFv

<400> 35

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys

<210> 36

<211> 119

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv

<400> 36

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 37

<211> 106

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv

<400> 37

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 38

<211> 236

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-PD-1 scFv

<400> 38

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

130 135 140

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

145 150 155 160

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

165 170 175

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

180 185 190

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

195 200 205

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

210 215 220

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

225 230 235

<210> 39

<211> 113

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-PD-1 scFv

<400> 39

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 40

<211> 108

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-PD-1 scFv

<400> 40

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 41

<211> 242

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-CTLA-4 scFv

<400> 41

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu

130 135 140

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

145 150 155 160

Ser Val Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile

180 185 190

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln

210 215 220

Tyr Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

225 230 235 240

Lys Arg

<210> 42

<211> 118

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-CTLA-4 scFv

<400> 42

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 43

<211> 109

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-CTLA-4 scFv

<400> 43

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 44

<211> 243

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-lag-3 scFv

<400> 44

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Ser Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

210 215 220

Gln Arg Ser Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Asn Leu Glu

225 230 235 240

Ile Lys Arg

<210> 45

<211> 120

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-lag-3 scFv

<400> 45

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 46

<211> 108

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-lag-3 scFv

<400> 46

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg

100 105

<210> 47

<211> 245

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-TIM-3 scFv

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Gly Gln Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Gly Ala Phe Pro Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu

130 135 140

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu

145 150 155 160

Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu

180 185 190

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

210 215 220

Cys Gln Gln Ser Arg Lys Asp Pro Ser Thr Phe Gly Gly Gly Thr Lys

225 230 235 240

Val Glu Ile Lys Arg

245

<210> 48

<211> 118

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-TIM-3 scFv

<400> 48

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Gly Gln Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Gly Ala Phe Pro Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 49

<211> 112

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-TIM-3 scFv

<400> 49

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Asp Pro Ser Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

<210> 50

<211> 249

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-TIGIT scFv

<400> 50

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met

35 40 45

Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu

65 70 75 80

Gln Leu His Ser Val Thr Thr Asp Asp Thr Ala Thr Tyr Ser Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

130 135 140

Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly Asp Arg

145 150 155 160

Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Ile His Lys Asn Leu Ala

165 170 175

Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Phe Leu Ile Tyr Tyr

180 185 190

Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly

195 200 205

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro Glu Asp

210 215 220

Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Val Glu Leu Lys Arg

245

<210> 51

<211> 127

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-TIGIT scFv

<400> 51

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met

35 40 45

Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu

65 70 75 80

Gln Leu His Ser Val Thr Thr Asp Asp Thr Ala Thr Tyr Ser Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 52

<211> 107

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-TIGIT scFv

<400> 52

Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Leu Lys Arg

100 105

<210> 53

<211> 245

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-BTLA scFv

<400> 53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Gly Pro Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Gly Met Ala Ala His Asn Tyr Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro

130 135 140

Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

145 150 155 160

Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro

165 170 175

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr

180 185 190

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

195 200 205

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

210 215 220

Gln Gln Arg Ser Asn Trp Pro Pro Ile Thr Phe Gly Gln Gly Thr Arg

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 54

<211> 121

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-BTLA scFv

<400> 54

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Gly Pro Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Gly Met Ala Ala His Asn Tyr Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 55

<211> 109

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-BTLA scFv

<400> 55

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

100 105

<210> 56

<211> 729

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of AntiCD3 McAb scfv

<400> 56

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaag 729

<210> 57

<211> 357

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv

<400> 57

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagc 357

<210> 58

<211> 318

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv

<400> 58

gacatccagc tgacccagag ccccgccatc atgagcgcca gccccggcga gaaggtgacc 60

atgacctgcc gcgccagcag cagcgtgagc tacatgaact ggtaccagca gaagagcggc 120

accagcccca agcgctggat ctacgacacc agcaaggtgg ccagcggcgt gccctaccgc 180

ttcagcggca gcggcagcgg caccagctac agcctgacca tcagcagcat ggaggccgag 240

gacgccgcca cctactactg ccagcagtgg agcagcaacc ccctgacctt cggcgccggc 300

accaagctgg agctgaag 318

<210> 59

<211> 708

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-PD-1 scFv

<400> 59

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

gactgcaagg ccagcggcat caccttcagc aacagcggca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa gcgctactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgttc 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caccaacgac 300

gactactggg gccagggcac cctggtgacc gtgagcagcg gcggcggcgg cagcggcggc 360

ggcggcagcg gcggcggcgg cagcgagatc gtgctgaccc agagccccgc caccctgagc 420

ctgagccccg gcgagcgcgc caccctgagc tgccgcgcca gccagagcgt gagcagctac 480

ctggcctggt accagcagaa gcccggccag gccccccgcc tgctgatcta cgacgccagc 540

aaccgcgcca ccggcatccc cgcccgcttc agcggcagcg gcagcggcac cgacttcacc 600

ctgaccatca gcagcctgga gcccgaggac ttcgccgtgt actactgcca gcagagcagc 660

aactggcccc gcaccttcgg ccagggcacc aaggtggaga tcaagcgc 708

<210> 60

<211> 339

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-PD-1 scFv

<400> 60

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

gactgcaagg ccagcggcat caccttcagc aacagcggca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa gcgctactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgttc 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caccaacgac 300

gactactggg gccagggcac cctggtgacc gtgagcagc 339

<210> 61

<211> 324

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-PD-1 scFv

<400> 61

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcca gagcgtgagc agctacctgg cctggtacca gcagaagccc 120

ggccaggccc cccgcctgct gatctacgac gccagcaacc gcgccaccgg catccccgcc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc 240

gaggacttcg ccgtgtacta ctgccagcag agcagcaact ggccccgcac cttcggccag 300

ggcaccaagg tggagatcaa gcgc 324

<210> 62

<211> 726

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-CTLA-4 scFv

<400> 62

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacacca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtgaccttc atcagctacg acggcaacaa caagtactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccatct actactgcgc ccgcaccggc 300

tggctgggcc ccttcgacta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 360

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg agatcgtgct gacccagagc 420

cccggcaccc tgagcctgag ccccggcgag cgcgccaccc tgagctgccg cgccagccag 480

agcgtgggca gcagctacct ggcctggtac cagcagaagc ccggccaggc cccccgcctg 540

ctgatctacg gcgccttcag ccgcgccacc ggcatccccg accgcttcag cggcagcggc 600

agcggcaccg acttcaccct gaccatcagc cgcctggagc ccgaggactt cgccgtgtac 660

tactgccagc agtacggcag cagcccctgg accttcggcc agggcaccaa ggtggagatc 720

aagcgc 726

<210> 63

<211> 354

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-CTLA-4 scFv

<400> 63

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacacca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtgaccttc atcagctacg acggcaacaa caagtactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccatct actactgcgc ccgcaccggc 300

tggctgggcc ccttcgacta ctggggccag ggcaccctgg tgaccgtgag cagc 354

<210> 64

<211> 327

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-CTLA-4 scFv

<400> 64

gagatcgtgc tgacccagag ccccggcacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcca gagcgtgggc agcagctacc tggcctggta ccagcagaag 120

cccggccagg ccccccgcct gctgatctac ggcgccttca gccgcgccac cggcatcccc 180

gaccgcttca gcggcagcgg cagcggcacc gacttcaccc tgaccatcag ccgcctggag 240

cccgaggact tcgccgtgta ctactgccag cagtacggca gcagcccctg gaccttcggc 300

cagggcacca aggtggagat caagcgc 327

<210> 65

<211> 729

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-lag-3 scFv

<400> 65

caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60

acctgcgccg tgtacggcgg cagcttcagc gactactact ggaactggat ccgccagccc 120

cccggcaagg gcctggagtg gatcggcgag atcaaccacc gcggcagcac caacagcaac 180

cccagcctga agagccgcgt gaccctgagc ctggacacca gcaagaacca gttcagcctg 240

aagctgcgca gcgtgaccgc cgccgacacc gccgtgtact actgcgcctt cggctacagc 300

gactacgagt acaactggtt cgacccctgg ggccagggca ccctggtgac cgtgagcagc 360

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgagat cgtgctgacc 420

cagagccccg ccaccctgag cctgagcccc ggcgagcgcg ccaccctgag ctgccgcgcc 480

agccagagca tcagcagcta cctggcctgg taccagcaga agcccggcca ggccccccgc 540

ctgctgatct acgacgccag caaccgcgcc accggcatcc ccgcccgctt cagcggcagc 600

ggcagcggca ccgacttcac cctgaccatc agcagcctgg agcccgagga cttcgccgtg 660

tactactgcc agcagcgcag caactggccc ctgaccttcg gccagggcac caacctggag 720

atcaagcgc 729

<210> 66

<211> 360

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-lag-3 scFv

<400> 66

caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60

acctgcgccg tgtacggcgg cagcttcagc gactactact ggaactggat ccgccagccc 120

cccggcaagg gcctggagtg gatcggcgag atcaaccacc gcggcagcac caacagcaac 180

cccagcctga agagccgcgt gaccctgagc ctggacacca gcaagaacca gttcagcctg 240

aagctgcgca gcgtgaccgc cgccgacacc gccgtgtact actgcgcctt cggctacagc 300

gactacgagt acaactggtt cgacccctgg ggccagggca ccctggtgac cgtgagcagc 360

<210> 67

<211> 324

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-lag-3 scFv

<400> 67

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcca gagcatcagc agctacctgg cctggtacca gcagaagccc 120

ggccaggccc cccgcctgct gatctacgac gccagcaacc gcgccaccgg catccccgcc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc 240

gaggacttcg ccgtgtacta ctgccagcag cgcagcaact ggcccctgac cttcggccag 300

ggcaccaacc tggagatcaa gcgc 324

<210> 68

<211> 735

<212> DNA

<213> Artificial

<220>

<223>The nucleotide acid sequence of anti-TIM-3 scFv

<400> 68

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctacaaca tgcactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatcggcgac atctaccccg gccagggcga caccagctac 180

aaccagaagt tcaagggccg cgccaccatg accgccgaca agagcaccag caccgtgtac 240

atggagctga gcagcctgcg cagcgaggac accgccgtgt actactgcgc ccgcgtgggc 300

ggcgccttcc ccatggacta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 360

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatcgtgct gacccagagc 420

cccgacagcc tggccgtgag cctgggcgag cgcgccacca tcaactgccg cgccagcgag 480

agcgtggagt actacggcac cagcctgatg cagtggtacc agcagaagcc cggccagccc 540

cccaagctgc tgatctacgc cgccagcaac gtggagagcg gcgtgcccga ccgcttcagc 600

ggcagcggca gcggcaccga cttcaccctg accatcagca gcctgcaggc cgaggacgtg 660

gccgtgtact actgccagca gagccgcaag gaccccagca ccttcggcgg cggcaccaag 720

gtggagatca agcgc 735

<210> 69

<211> 354

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-TIM-3 scFv

<400> 69

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctacaaca tgcactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatcggcgac atctaccccg gccagggcga caccagctac 180

aaccagaagt tcaagggccg cgccaccatg accgccgaca agagcaccag caccgtgtac 240

atggagctga gcagcctgcg cagcgaggac accgccgtgt actactgcgc ccgcgtgggc 300

ggcgccttcc ccatggacta ctggggccag ggcaccctgg tgaccgtgag cagc 354

<210> 70

<211> 336

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-TIM-3 scFv

<400> 70

gacatcgtgc tgacccagag ccccgacagc ctggccgtga gcctgggcga gcgcgccacc 60

atcaactgcc gcgccagcga gagcgtggag tactacggca ccagcctgat gcagtggtac 120

cagcagaagc ccggccagcc ccccaagctg ctgatctacg ccgccagcaa cgtggagagc 180

ggcgtgcccg accgcttcag cggcagcggc agcggcaccg acttcaccct gaccatcagc 240

agcctgcagg ccgaggacgt ggccgtgtac tactgccagc agagccgcaa ggaccccagc 300

accttcggcg gcggcaccaa ggtggagatc aagcgc 336

<210> 71

<211> 747

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-TIGIT scFv

<400> 71

gaggtgcagc tgcaggagag cggccccggc ctggtgaagc ccagccagag cctgagcctg 60

acctgcagcg tgaccggcag cagcatcgcc agcgactact ggggctggat ccgcaagttc 120

cccggcaaca agatggagtg gatgggcttc atcacctaca gcggcagcac cagctacaac 180

cccagcctga agagccgcat cagcatcacc cgcgacacca gcaagaacca gttcttcctg 240

cagctgcaca gcgtgaccac cgacgacacc gccacctaca gctgcgcccg catgcccagc 300

ttcatcaccc tggccagcct gagcacctgg gagggctact tcgacttctg gggccccggc 360

accatggtga ccgtgagcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 420

ggcagcgaca tccagatgac ccagagcccc agcctgctga gcgccagcgt gggcgaccgc 480

gtgaccctga actgcaaggc cagccagagc atccacaaga acctggcctg gtaccagcag 540

aagctgggcg aggcccccaa gttcctgatc tactacgcca acagcctgca gaccggcatc 600

cccagccgct tcagcggcag cggcagcggc accgacttca ccctgaccat cagcggcctg 660

cagcccgagg acgtggccac ctacttctgc cagcagtact acagcggctg gaccttcggc 720

ggcggcacca aggtggagct gaagcgc 747

<210> 72

<211> 381

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-TIGIT scFv

<400> 72

gaggtgcagc tgcaggagag cggccccggc ctggtgaagc ccagccagag cctgagcctg 60

acctgcagcg tgaccggcag cagcatcgcc agcgactact ggggctggat ccgcaagttc 120

cccggcaaca agatggagtg gatgggcttc atcacctaca gcggcagcac cagctacaac 180

cccagcctga agagccgcat cagcatcacc cgcgacacca gcaagaacca gttcttcctg 240

cagctgcaca gcgtgaccac cgacgacacc gccacctaca gctgcgcccg catgcccagc 300

ttcatcaccc tggccagcct gagcacctgg gagggctact tcgacttctg gggccccggc 360

accatggtga ccgtgagcag c 381

<210> 73

<211> 321

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-TIGIT scFv

<400> 73

gacatccaga tgacccagag ccccagcctg ctgagcgcca gcgtgggcga ccgcgtgacc 60

ctgaactgca aggccagcca gagcatccac aagaacctgg cctggtacca gcagaagctg 120

ggcgaggccc ccaagttcct gatctactac gccaacagcc tgcagaccgg catccccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcgg cctgcagccc 240

gaggacgtgg ccacctactt ctgccagcag tactacagcg gctggacctt cggcggcggc 300

accaaggtgg agctgaagcg c 321

<210> 74

<211> 735

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-BTLA scFv

<400> 74

gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60

agctgcgccg ccagcggctt caccatcagc agctacgaca tgcactgggt gcgccaggcc 120

accggcaagg gcctggagtg ggtgagcgtg atcggccccg ccggcgacac ctactacccc 180

ggcagcgtga agggccgctt caccatcagc cgcgagaacg ccaagaacag cctgtacctg 240

cagatgaaca gcctgcgcgc cggcgacacc gccgtgtact actgcgcccg cgagggcatg 300

gccgcccaca actactacgg catggacgtg tggggccagg gcaccaccgt gaccgtgagc 360

agcggcggcg gcggcagcgg cggcggcggc agcggcggcg gcggcagcga gatcgtgctg 420

acccagagcc ccgccaccct gagcctgagc cccggcgagc gcgccaccct gagctgccgc 480

gccagccaga gcgtgagcag ctacctggcc tggtaccagc agaagcccgg ccaggccccc 540

cgcctgctga tctacgacgc cagcaaccgc gccaccggca tccccgcccg cttcagcggc 600

agcggcagcg gcaccgactt caccctgacc atcagcagcc tggagcccga ggacttcgcc 660

gtgtactact gccagcagcg cagcaactgg ccccccatca ccttcggcca gggcacccgc 720

ctggagatca agcgc 735

<210> 75

<211> 363

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-BTLA scFv

<400> 75

gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60

agctgcgccg ccagcggctt caccatcagc agctacgaca tgcactgggt gcgccaggcc 120

accggcaagg gcctggagtg ggtgagcgtg atcggccccg ccggcgacac ctactacccc 180

ggcagcgtga agggccgctt caccatcagc cgcgagaacg ccaagaacag cctgtacctg 240

cagatgaaca gcctgcgcgc cggcgacacc gccgtgtact actgcgcccg cgagggcatg 300

gccgcccaca actactacgg catggacgtg tggggccagg gcaccaccgt gaccgtgagc 360

agc 363

<210> 76

<211> 327

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-BTLA scFv

<400> 76

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcca gagcgtgagc agctacctgg cctggtacca gcagaagccc 120

ggccaggccc cccgcctgct gatctacgac gccagcaacc gcgccaccgg catccccgcc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc 240

gaggacttcg ccgtgtacta ctgccagcag cgcagcaact ggccccccat caccttcggc 300

cagggcaccc gcctggagat caagcgc 327

<210> 77

<211> 19

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of secreting, expressing signal peptide

<400> 77

Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser

1 5 10 15

Ser Arg Ala

<210> 78

<211> 57

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of secreting, expressing signal peptide

<400> 78

atgacccgcc tgaccgtgct ggccctgctg gccggcctgc tggccagcag ccgcgcc 57

<210> 79

<211> 59

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-Sig-F

<400> 79

gtgctggata tctgcagaat tcgccgccac catgacccgg ctgaccgtgc tggccctgc 59

<210> 80

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Sig-R

<400> 80

ggccctggag gaggccagca ggccggccag cagggccagc acggtcagc 49

<210> 81

<211> 41

<212> DNA

<213> Artificial

<220>

<223> Sig-CD3-F

<400> 81

gctggcctcc tccagggccg acatcaagct gcagcagagc g 41

<210> 82

<211> 20

<212> DNA

<213> Artificial

<220>

<223> CD3-R

<400> 82

cttcagctcc agcttggtgc 20

<210> 83

<211> 91

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-PD-1-F

<400> 83

ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60

ggcagccagg tgcagctggt ggagagcggc g 91

<210> 84

<211> 49

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-PD-1-R

<400> 84

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttgg 49

<210> 85

<211> 41

<212> DNA

<213> Artificial

<220>

<223> CD3-IgD-F

<400> 85

gcaccaagct ggagctgaag gccagcaaga gcaagaagga g 41

<210> 86

<211> 21

<212> DNA

<213> Artificial

<220>

<223> IgD-R

<400> 86

cacgcccagg ggctgggtgt g 21

<210> 87

<211> 43

<212> DNA

<213> Artificial

<220>

<223> IgD-PD-1-F

<400> 87

cacacccagc ccctgggcgt gcaggtgcag ctggtggaga gcg 43

<210> 88

<211> 87

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-CTLA-4-F

<400> 88

ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60

ggcagccagg tgcagctggt ggagagc 87

<210> 89

<211> 49

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-CTLA-4-R

<400> 89

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttgg 49

<210> 90

<211> 40

<212> DNA

<213> Artificial

<220>

<223> IgD-CTLA-4-F

<400> 90

acacccagcc cctgggcgtg ccaaggtgga gatcaagcgc 40

<210> 91

<211> 87

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-LAG-3-F

<400> 91

ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60

ggcagccagg tgcagctgca gcagtgg 87

<210> 92

<211> 49

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-LAG-3-R

<400> 92

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaggttgg 49

<210> 93

<211> 40

<212> DNA

<213> Artificial

<220>

<223> IgD-LAG-3-F

<400> 93

acacccagcc cctgggcgtg ccaacctgga gatcaagcgc 40

<210> 94

<211> 87

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-TIM-3-F

<400> 94

ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60

ggcagccagg tgcagctggt gcagagc 87

<210> 95

<211> 50

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-TIM-3-R

<400> 95

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt 50

<210> 96

<211> 40

<212> DNA

<213> Artificial

<220>

<223> IgD-TIM-3-F

<400> 96

acacccagcc cctgggcgtg ccaaggtgga gatcaagcgc 40

<210> 97

<211> 87

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-TIGIT-F

<400> 97

ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60

ggcagcgagg tgcagctgca ggagagc 87

<210> 98

<211> 49

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-TIGIT-R

<400> 98

ctgatcagcg gtttaaactt aagctttcag cgcttcagct ccaccttgg 49

<210> 99

<211> 40

<212> DNA

<213> Artificial

<220>

<223> IgD-TIGIT-F

<400> 99

acacccagcc cctgggcgtg ccaaggtgga gctgaagcgc 40

<210> 100

<211> 87

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-BTLA-F

<400> 100

ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60

ggcagcgagg tgcagctggt ggagagc 87

<210> 101

<211> 49

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-BTLA-R

<400> 101

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaggcggg 49

<210> 102

<211> 42

<212> DNA

<213> Artificial

<220>

<223> IgD-BTLA-F

<400> 102

cacacccagc ccctgggcgt ggaggtgcag ctggtggaga gc 42

Claims

1. a kind of bifunctional molecule, structure include to combine and activate T cell surface C D3 molecules the first functional domain and The second functional domain that simultaneously blocking t cell bears costimulatory molecules can be combined.

2. bifunctional molecule according to claim 1, which is characterized in that the bifunctional molecule can combined and activated It is combined while T cell surface C D3 molecules and blocking t cell bears costimulatory molecules, so as to generate first needed for T cell activation Signal and second signal.

3. bifunctional molecule according to claim 1, which is characterized in that antibody of first functional domain for AntiCD3 McAb, institute State the antibody that the second functional domain bears costimulatory molecules for anti-T cell.

4. bifunctional molecule according to claim 3, which is characterized in that the antibody is selected from Fab antibody, Fv antibody or list Chain antibody.

5. bifunctional molecule according to claim 1, which is characterized in that first functional domain and second functional domain It is connected by junction fragment.

6. bifunctional molecule according to claim 5, which is characterized in that the junction fragment is selected from as unit of G4S The hinge area segment of junction fragment or Immunoglobulin IgD.

7. bifunctional molecule according to claim 6, which is characterized in that the amino acid of the junction fragment as unit of G4S Sequence is as shown in SEQ ID NO.1；The amino acid sequence of the hinge area segment of Immunoglobulin IgD is as shown in SEQ ID NO.3.

8. bifunctional molecule according to claim 1, which is characterized in that first functional domain is the single-stranded anti-of AntiCD3 McAb Body, second functional domain bear the single-chain antibodies of costimulatory molecules for anti-T cell, the single-chain antibody include heavy chain variable region and Light chain variable region.

9. bifunctional molecule according to claim 8, which is characterized in that the weight chain variable of the single-chain antibody of the AntiCD3 McAb The amino acid sequence in area is as shown in SEQ ID NO.36；The amino acid sequence of the light chain variable region of the single-chain antibody of the AntiCD3 McAb As shown in SEQ ID NO.37；The single-chain antibody that the anti-T cell bears costimulatory molecules is selected from the single-chain antibody of anti-PD-1, resists The single-chain antibody of CTLA-4, the single-chain antibody of anti-lag-3, the single-chain antibody of anti-TIM-3, anti-TIGIT single-chain antibody or anti- The single-chain antibody of BTLA it is any；The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-PD-1 such as SEQ ID Shown in NO.39；The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-PD-1 is as shown in SEQ ID NO.40；It is described The amino acid sequence of the heavy chain variable region of the single-chain antibody of anti-CTLA-4 is as shown in SEQ ID NO.42；The list of the anti-CTLA-4 The amino acid sequence of the light chain variable region of chain antibody is as shown in SEQ ID NO.43；The heavy chain of the single-chain antibody of the anti-lag-3 The amino acid sequence of variable region is as shown in SEQ ID NO.45；The amino of the light chain variable region of the single-chain antibody of the anti-lag-3 Acid sequence is as shown in SEQ ID NO.46；The amino acid sequence such as SEQ of the heavy chain variable region of the single-chain antibody of the anti-TIM-3 Shown in ID NO.48；The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-TIM-3 is as shown in SEQ ID NO.49； The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-TIGIT is as shown in SEQ ID NO.51；The anti-TIGIT's The amino acid sequence of the light chain variable region of single-chain antibody is as shown in SEQ ID NO.52；The heavy chain of the single-chain antibody of the anti-BTLA The amino acid sequence of variable region is as shown in SEQ ID NO.54；The amino acid of the light chain variable region of the single-chain antibody of the anti-BTLA Sequence is as shown in SEQ ID NO.55.

10. bifunctional molecule according to claim 9, which is characterized in that the amino acid sequence of the single-chain antibody of the AntiCD3 McAb Row are as shown in SEQ ID NO.35；The amino acid sequence of the single-chain antibody of the anti-PD-1 is as shown in SEQ ID NO.38；It is described The amino acid sequence of the single-chain antibody of anti-CTLA-4 is as shown in SEQ ID NO.41；The amino of the single-chain antibody of the anti-lag-3 Acid sequence is as shown in SEQ ID NO.44；The amino acid sequence of the single-chain antibody of the anti-TIM-3 is as shown in SEQ ID NO.47； The amino acid sequence of the single-chain antibody of the anti-TIGIT is as shown in SEQ ID NO.50；The ammonia of the single-chain antibody of the anti-BTLA Base acid sequence is as shown in SEQ ID NO.53.

11. bifunctional molecule according to claim 1, which is characterized in that the amino acid sequence of the bifunctional molecule is such as SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31 or SEQ ID NO.33's is any shown.

12. a kind of polynucleotides, coding bifunctional molecule as described in any one of claim 1~11.

13. a kind of expression vector contains polynucleotides as claimed in claim 12.

14. a kind of host cell is converted by expression vector as claimed in claim 13.

15. the preparation method of bifunctional molecule as described in any one of claim 1~11, including：Structure contains bifunctional molecule Then the expression vector of gene order converts the expression vector of the gene order containing bifunctional molecule to induction table in host cell It reaches, is detached from expression product and obtain the bifunctional molecule.

16. the bifunctional molecule as described in any one of claim 1~11 is used to prepare the purposes of T cell amplification in vitro agent.

17. a kind of method of amplification in vitro T cell, including bifunctional molecule will be acted on as described in any one of claim 1~11 In T cell.