CN108264557B

CN108264557B - Bifunctional molecule combining CD3 and T cell negative co-stimulatory molecule and application thereof

Info

Publication number: CN108264557B
Application number: CN201611256643.8A
Authority: CN
Inventors: 朱化星; 陈帅; 廖远平
Original assignee: Huihe Biotechnology Shanghai Co ltd
Current assignee: Cytocares (shanghai) Inc
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2021-08-24
Anticipated expiration: 2036-12-30
Also published as: CN108264557A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a bifunctional molecule combining CD3 and a T cell negative co-stimulatory molecule and application thereof. The invention fuses the first functional domain which can combine and activate the CD3 molecule on the surface of the T cell and the second functional domain which can combine and block the negative co-stimulatory molecule of the T cell into the same protein peptide chain to form the double-functional molecule, adopts the eukaryotic cell expression system to produce, the expression product has single structure, the purification process is simple and convenient, the protein yield is high, the preparation process and the product are stable; compared with the combination of the anti-CD3 and the anti-T cell positive (negative) co-stimulatory molecule full-length antibody, the bifunctional molecule has better in-vitro amplification effect on T cells, less protein consumption and simple and convenient use, can be directly added in a solution form, and does not need to optimize the relative proportion of the two full-length antibodies.

Description

Bifunctional molecule combining CD3 and T cell negative co-stimulatory molecule and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a bifunctional molecule combining CD3 and a T cell negative co-stimulatory molecule and application thereof.

Background

T lymphocytes (T lymphocytes) are derived from Thymus (Thymus), and are therefore called T cells. Mature T cells exist in thymus-dependent regions of peripheral immune organs, occupy a central position in adaptive cellular immune response, and play an important auxiliary role in humoral immune response induced by thymus-dependent antigens. Depending on the function, T cells can be classified into Cytotoxic T Cells (CTL), Helper T cells (Th), and Regulatory T cells (Treg). The CTL expresses CD8, is a main effector cell of adaptive cellular immunity, has the main functions of specifically recognizing endogenous antigen peptide/MHC I molecule compound on the surface of a target cell, can secrete perforin (Peforin), Granzyme (Granzyme), Granulysin (granlysin) and other substances after self activation to directly kill the target cell (tumor cell or cell infected by parasitic pathogen), and can also induce the apoptosis of the target cell through a Fas/FasL signal pathway; th all express CD4, and regulate the cell activity of CTL via secreting different kinds of cell factors and direct interaction with other cells, so as to indirectly participate in cellular immunity; in addition, tregs can negatively regulate cellular immune responses by directly contacting and inhibiting activation of target cells and secreting cytokines such as IL-10 and TGF β, and play an important role in various diseases such as immune tolerance, autoimmune diseases, infectious diseases and tumors.

The complete activation and efficient expansion of CD8 positive T cells are the basis for their effective killing of target cells, relying on the role of dual signaling pathways: wherein MHC I/endogenous Antigen peptide complex on the surface of Antigen Presenting Cell (APC) specifically recognizes TCR/CD3 complex expressed by T cell, causes CD3 to interact with cytoplasmic phase of co-receptor CD8, activates protein tyrosine kinase connected with cytoplasmic phase tail, enables tyrosine phosphorylation in immune receptor tyrosine kinase activation motif (ITAM) of CD3 cytoplasmic region, initiates signal transduction molecular cascade reaction, activates transcription factor, and enables T cell primary activation, which is the first signal for T cell activation; meanwhile, Co-stimulatory molecules (Co-stimulatory molecules such as CD28, 4-1BB, ICOS, OX40, GITR, CD40L, CD27, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, BTLA, etc.) on the surface of T cells can interact with corresponding Co-stimulatory molecule ligands (such as CD80, CD86, 4-1BBL, B7RP-1, OX40L, GITRL, CD40, CD70, PD-L1, PD-L2, Galectin-9, HVEM, etc.) on the surface of APC cells to generate a second signal (Co-stimulatory signal) for T cell activation: wherein CD28, 4-1BB, ICOS, OX40, GITR, CD40L, CD27 and the like belong to the group of positive costimulatory molecules, and the interaction with the corresponding ligands (CD80, CD86, 4-1BBL, B7RP-1, OX40L, GITRL, CD40, CD70 and the like) generates a second signal (positive costimulatory signal) that leads to the complete activation of T cells; while CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, BTLA, etc., are negative co-stimulatory molecules, the interaction of which with the corresponding ligands (CD80, CD86, PD-L1, PD-L2, Galectin-9, HVEM, etc.) generates a second signal (negative co-stimulatory signal) that downregulates and terminates the activation of T cells.

The first signaling pathway for T cell activation has been reported to be a series of monoclonal full-length antibodies against CD3 (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). The existing experimental data show that the monoclonal antibody can specifically recognize CD3 molecules on the surface of T cells and generate a first signal for activating the T cells. However, the first signaling pathway is not, but not effective in activating T cells, but rather leads to T cell disability and even Activation-induced T cell death (AICD). In order to overcome the defect of the CD3 monoclonal full-length antibody, activated monoclonal full-length antibodies (US Patent 20100168400A 1; US Patent 20100183621A 1; US Patent 009193789B2) against CD28, 4-1BB and ICOS and other positive co-stimulatory molecules or blocked monoclonal full-length antibodies (World Patent 2013173223 Al; US Patent 007452535B 2; US Patent 2015116539A1) against PD-1, CTLA-4 and LAG-3 and other negative co-stimulatory molecules are designed and constructed, and a complete dual-signal activation pathway can be provided for T cells by combining with the anti-CD3 full-length antibody. However, the combination of two monoclonal full-length antibodies still has some disadvantages in specific applications, such as significantly increasing the workload of recombinant antibody expression and purification and the production cost, and the relative proportion of the two full-length antibodies must be optimized when the antibodies are actually applied to T cell in vitro activation and amplification. In addition, when two full-length antibodies are used in combination, in order to promote receptor activation, a higher concentration of antibody solution needs to be added or the antibody needs to be coated on a culture plate or microspheres to enhance the receptor activation effect.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a bifunctional molecule which simultaneously binds CD3 and a T cell negative co-stimulatory molecule and application thereof.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the invention, there is provided a bifunctional molecule comprising in its structure a first domain capable of binding to and activating the T cell surface CD3 molecule and a second domain capable of binding to and blocking a T cell negative co-stimulatory molecule.

Preferably, the bifunctional molecule is capable of binding to and blocking a T cell negative co-stimulatory molecule while binding to and activating a T cell surface CD3 molecule, thereby generating a first signal and a second signal required for T cell activation.

Preferably, the first domain is an antibody against CD3 and the second domain is an antibody against a negative co-stimulatory molecule of a T cell.

Preferably, the antibody is a small molecule antibody.

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

Preferably, the first domain and the second domain are linked by a linker fragment. The number of amino acids of the connecting segment can be more than or equal to 2.

Preferably, the linking fragment is selected from the group consisting of a linking fragment in G4S units or a hinge region fragment of an immunoglobulin IgD.

The G4S is specifically GGGGS. The G4S-unit ligated fragment includes one or more G4S units. For example, one, two, three, or more than four G4S units may be included. In some embodiments of the present invention, a single bifunctional molecule is illustrated, wherein the first domain and the second domain are linked via a G4S unit linker, the linker comprises three G4S units, and the amino acid sequence of the linker is shown in SEQ ID NO. 1.

The hinge region fragment of an immunoglobulin IgD may be the hinge Ala90-Val170 of an immunoglobulin IgD. In some embodiments of the invention, a dimer form of the bifunctional molecule is exemplified in which the first domain and the second domain are connected by a hinge region fragment of an immunoglobulin IgD, the hinge region fragment of an immunoglobulin IgD being the hinge Ala90-Val170 of the immunoglobulin IgD, the amino acid sequence of the hinge region fragment of an immunoglobulin IgD being represented in SEQ ID NO. 3. The linking fragments may be linked to each other by a disulfide bond to form a dimer.

Preferably, the C-terminus of the first domain is linked to the N-terminus of the second domain.

Preferably, the first domain is a single chain antibody against CD3 and the second domain is a single chain antibody against a T cell negative co-stimulatory molecule, said single chain antibody comprising a heavy chain variable region and a light chain variable region.

Preferably, the amino acid sequence of the heavy chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 36. The amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 37.

Preferably, the single-chain antibody against the T-cell negative co-stimulatory molecule may be any one of a single-chain antibody against PD-1, a single-chain antibody against CTLA-4, a single-chain antibody against LAG-3, a single-chain antibody against TIM-3, a single-chain antibody against TIGIT, or a single-chain antibody against BTLA.

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

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

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

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody against TIM-3 is shown as SEQ ID No. 48. The amino acid sequence of the variable region of the light chain of the single-chain antibody for resisting the TIM-3 is shown as SEQ ID NO. 49.

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

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody against BTLA is shown in SEQ ID No. 54. The amino acid sequence of the variable region of the light chain of the single-chain antibody against BTLA is shown in SEQ ID NO. 55.

In some embodiments of the invention, the amino acid sequence of the anti-CD3 single chain antibody is shown in SEQ ID NO. 35. The amino acid sequence of the single-chain antibody for resisting PD-1 is shown in SEQ ID NO. 38. The amino acid sequence of the single-chain antibody for resisting CTLA-4 is shown in SEQ ID NO. 41. The amino acid sequence of the single-chain antibody for resisting the LAG-3 is shown as SEQ ID NO. 44. The amino acid sequence of the single-chain antibody for resisting the TIM-3 is shown as SEQ ID NO. 47. The amino acid sequence of the single-chain antibody for resisting TIGIT is shown as SEQ ID NO. 50. The amino acid sequence of the single-chain antibody for resisting BTLA is shown in SEQ ID NO. 53.

In a preferred embodiment, the amino acid sequence of the monomeric bifunctional molecule is as shown in any one of SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27 or SEQ ID NO. 31. The amino acid sequence of the bifunctional molecule in the form of a dimer is shown in any one of 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.

In a second aspect of the invention, there is provided a polynucleotide encoding the aforementioned bifunctional molecule.

In a third aspect of the present invention, there is provided an expression vector comprising the aforementioned polynucleotide.

In a fourth aspect of the present invention, there is provided a host cell transformed with the aforementioned expression vector.

In a fifth aspect of the present invention, there is provided a method for preparing the bifunctional molecule, comprising: constructing an expression vector containing the gene sequence of the bifunctional molecule, then transforming the expression vector containing the gene sequence of the bifunctional molecule into host cells for inducing expression, and separating the expression product to obtain the bifunctional molecule.

In a preferred embodiment of the invention, pcDNA3.1 is used as the expression vector. The host cell was Chinese hamster ovary (Chinese hamster ovary ce1l, CHO).

In a sixth aspect of the invention, there is provided the use of the bifunctional molecule as described above for the preparation of an in vitro T cell expansion agent.

In a seventh aspect of the present invention, there is provided an in vitro T cell expansion agent comprising the bifunctional molecule.

In an eighth aspect of the present invention, a method for expanding T cells in vitro is disclosed, which comprises allowing the aforementioned bifunctional molecule to act on T cells. The method may be for non-therapeutic purposes.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention fuses the first functional domain which can combine and activate the CD3 molecule on the surface of the T cell and the second functional domain which can combine and block the negative co-stimulatory molecule of the T cell into the same protein peptide chain to form the double-functional molecule, adopts the eukaryotic cell expression system to produce, the expression product has single structure, the purification process is simple and convenient, the protein yield is high, the preparation process and the product are stable; if the anti-CD3 monoclonal full-length antibody and the anti-T cell positive (negative) stimulation molecule monoclonal full-length antibody are used in combination, the two antibodies need to be expressed and purified respectively, the preparation process is more complicated, and the workload and the production cost are obviously increased.

(2) Compared with the combined use of an anti-CD3 full-length antibody and an anti-T cell positive (negative) costimulatory molecule full-length antibody, the in-vitro activation and amplification effect on T cells is better, the protein dosage is less, the use is simple and convenient, the bifunctional molecule is directly added in a solution form, and the relative proportion of the two full-length antibodies is not required to be optimized.

Drawings

FIG. 1: A. a structural diagram of the monomeric form of the anti-CD 3/anti-T cell negative co-stimulatory molecule bispecific antibody; B. a structural diagram of a dimeric form of the anti-CD 3/anti-T cell negative co-stimulatory molecule bispecific antibody.

FIG. 2: A. purified CD3-PD-1 BsAb _ M SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-PD-1 BsAb _ M; lane 3: non-reducing CD3-PD-1 BsAb _ M; B. purified CD3-PD-1 BsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-PD-1 BsAb _ D; lane 3: non-reducing CD3-PD-1 BsAb _ D.

FIG. 3A: the ELISA identification result of CD3-PD-1 BsAb _ M, and the curves in the figure represent three tests respectivelyAnd (3) measuring results: ■ is coated with 1 mug/ml recombinant antigen CD3-hFc,

coating 1 mug/ml recombinant antigen PD-1-hFc; assay results without any antigen coating.

FIG. 3B: the ELISA identification result of CD3-PD-1 BsAb _ D, the curves in the figure represent three detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 3-hFc;

FIG. 4: CIK cell expansion fold curve, using peripheral blood PBMC as experimental cells, and adding CD3-PD-1 BsAb _ M, CD3-PD-1 BsAb _ D or Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) respectively, culturing for 30 days in total, counting and comparing cell expansion fold by dividing cell number counted each time by cell number on day 1, wherein control group: 5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 coated cell culture plates; experimental group 1: adding 10ng/ml of CD3-PD-1 BsAb _ M in a solution state; experimental group 2: 10ng/ml CD3-PD-1 BsAb _ D was added in solution.

FIG. 5: CD3-PD-1 bispecific antibody mediated IFN-gamma secretion from CIK cells. Control group: 2X 10 CIK cells cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of example 4 were selected⁵Centrifuging to take supernatant, detecting the amount of IFN-gamma secreted by cells by ELISA Kit, and defining the amount as 1; the experimental group 1 and the experimental group 2 are CIK cells which are added with CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D and cultured for 25 days respectively in a solution state, the cells with the same number as that of the control group are taken, the supernatant is obtained by centrifugation, the number of IFN-gamma secreted by the cells is detected, and the relative secretion of the IFN-gamma is obtained by dividing the cell by the control group.

FIG. 6: A. purified CD3-CTLA-4 BsAb _ M SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-CTLA-4 BsAb _ M; lane 3: non-reducing CD3-CTLA-4 BsAb _ M; B. purified CD3-CTLA-4 BsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-CTLA-4 BsAb _ D; lane 3: non-reducing CD3-CTLA-4 BsAb _ D.

FIG. 7A: the ELISA identification result of CD3-CTLA-4 BsAb _ M, the curves in the figure represent three detection results respectively: ■ is coated with 1 mug/ml recombinant antigen CD3-hFc,

coating 1 mu g/ml recombinant antigen CTLA-4-hFc; assay results without any antigen coating.

FIG. 7B: the ELISA identification result of CD3-CTLA-4 BsAb _ D, the curves in the figure represent three detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 3-hFc;

FIG. 8: CIK cell expansion fold curve, using peripheral blood PBMC as experimental cells, and adding CD3-CTLA-4 BsAb _ M, CD3-CTLA-4 BsAb _ D or Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28), respectively, culturing for 30 days in total, counting and comparing cell expansion fold by dividing cell number counted each time by cell number on day 1, wherein control group: 5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 coated cell culture plates; experimental group 1: adding 10ng/ml CD3-CTLA-4 BsAb _ M in the solution state; experimental group 2: 10ng/ml CD3-CTLA-4 BsAb _ D was added to the solution.

FIG. 9: CD3-CTLA-4 bispecific antibody mediated IFN-gamma secretion from CIK cells. Control group: 2X 10 days of CIK cells cultured in the control group (Anti-CD3/Anti-CD28) of example 9 were selected⁵Centrifuging to take supernatant, detecting the amount of IFN-gamma secreted by cells by ELISA Kit, and defining the amount as 1; the experimental group 1 and the experimental group 2 are CIK cells which are respectively added with CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D and cultured for 25 days in a solution state, the cells with the same number as that of the control group are taken, the supernatant is obtained by centrifugation, the number of IFN-gamma secreted by the cells is detected, and the relative secretion of the IFN-gamma is obtained by dividing the cell by the control group.

FIG. 10: A. purified CD3-LAG-3BsAb _ M SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-LAG-3BsAb _ M; lane 3: non-reducing CD3-LAG-3BsAb _ M; B. purified CD3-LAG-3BsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-LAG-3BsAb _ D; lane 3: non-reducing CD3-LAG-3BsAb _ D.

FIG. 11A: the ELISA identification result of CD3-LAG-3BsAb _ M, the curves in the figure represent three detection results respectively: ■ is coated with 1 mug/ml recombinant antigen CD3-hFc,

coating 1 mu g/ml of recombinant antigen LAG-3-hFc; assay results without any antigen coating.

FIG. 11B: the ELISA identification result of CD3-LAG-3BsAb _ D, the curves in the figure represent three detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 3-hFc;

FIG. 12: CIK cell expansion fold curve, using peripheral blood PBMC as experimental cells, and adding CD3-LAG-3BsAb _ M, CD3-LAG-3BsAb _ D or Anti-CD3/Anti-CD28 monoclonal full-length antibody in combination (Anti-CD3/Anti-CD28), respectively, for 30 days of total culture, counting and comparing cell expansion fold by dividing the number of cells counted each time by the number of cells on day 1, wherein the control group: 5ug/ml Anti-CD3 and 5ug/ml Anti-CD28 coated cell culture plates; experimental group 1: adding 10ng/ml of CD3-LAG-3BsAb _ M in a solution state; experimental group 2: 10ng/ml CD3-LAG-3BsAb _ D was added to the solution.

FIG. 13: CD3-LAG-3 bispecific antibody mediated IFN-gamma secretion from CIK cells. Control group: 2X 10 CIK cells cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of example 14 were selected⁵Centrifuging to take supernatant, detecting the amount of IFN-gamma secreted by cells by ELISA Kit, and defining the amount as 1; the experimental group 1 and the experimental group 2 are CIK cells which are respectively added with CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D and cultured for 25 days in a solution state, the cells with the same number as that of the control group are taken, the supernatant is obtained by centrifugation, the IFN-gamma number secreted by the cells is detected, and the control group is divided by the IFN-gamma relative secretion numberAmount of the compound (A).

FIG. 14: A. purified CD3-TIM-3 BsAb _ M SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-TIM-3 BsAb _ M; lane 3: non-reducing CD3-TIM-3 BsAb _ M; B. purified CD3-TIM-3 BsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-TIM-3 BsAb _ D; lane 3: non-reducing CD3-TIM-3 BsAb _ D.

FIG. 15A: the ELISA identification result of CD3-TIM-3 BsAb _ M shows that the curves in the figure represent three detection results respectively: ■ is coated with 1 mug/ml recombinant antigen CD3-hFc,

coating 1 mu g/ml recombinant antigen TIM-3-hFc; assay results without any antigen coating.

FIG. 15B: the ELISA identification result of CD3-TIM-3 BsAb _ D, the curves in the figure represent three detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 3-hFc;

FIG. 16: CIK cell expansion fold curve, peripheral blood PBMC was used as experimental cells, CD3-TIM-3 BsAb _ M, CD3-TIM-3 BsAb _ D or Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) was added, and cell expansion fold was counted and compared by dividing the number of cells counted each time by the number of cells on day 1 for 30 days in total. Wherein, the control group: 5ug/ml Anti-CD3 and 5ug/ml Anti-CD 28; experimental group 1: adding 10ng/ml CD3-TIM-3 BsAb _ M in the solution state; experimental group 2: 10ng/ml CD3-TIM-3 BsAb _ D was added to the solution.

FIG. 17: CIK cell IFN-gamma secretion mediated by CD3-TIM-3 bispecific antibody. Control group: 2X 10 CIK cells cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of example 19 were selected⁵Centrifuging to take supernatant, detecting the amount of IFN-gamma secreted by cells by ELISA Kit, and defining the amount as 1; experiment group 1 and experiment group 2 were cultured in the state of solution with addition of CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D, respectively, to 2And (3) taking the same number of cells as the control group from the CIK cells in 5 days, centrifuging to take the supernatant, and detecting the IFN-gamma number secreted by the cells, wherein the IFN-gamma relative secretion is obtained by dividing the IFN-gamma number by the control group.

FIG. 18: A. purified CD3-TIGIT BsAb _ M SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-TIGIT BsAb _ M; lane 3: non-reducing CD3-TIGIT BsAb _ M; B. purified CD3-TIGIT BsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-TIGIT BsAb _ D; lane 3: non-reducing CD3-TIGIT BsAb _ D.

FIG. 19A: the ELISA identification result of CD3-TIGIT BsAb _ M shows that the curves in the figure respectively represent three detection results: ■ is coated with 1 mug/ml recombinant antigen CD3-hFc,

coating a1 mu g/ml recombinant antigen TIGIT-hFc; assay results without any antigen coating.

FIG. 19B: the ELISA identification result of CD3-TIGIT BsAb _ D, the curves in the figure represent three detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 3-hFc;

FIG. 20: CIK cell expansion fold curve, peripheral blood PBMC was used as experimental cells, CD3-TIGIT BsAb _ M, CD3-TIGIT BsAb _ D or Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) was added, and cell expansion fold was counted and compared by dividing the number of cells counted each time by the number of cells on day 1 for 30 days in total. Wherein, the control group: 5ug/ml Anti-CD3 and 5ug/ml Anti-CD 28; experimental group 1: adding 10ng/ml of CD3-TIGIT BsAb _ M in a solution state; experimental group 2: 10ng/ml CD3-TIGIT BsAb _ D was added in the solution state.

FIG. 21: CD3-TIGIT bispecific antibody mediated IFN-gamma secretion from CIK cells. Control group: 2X 10 CIK cells cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of example 24 were selected⁵The supernatant was centrifuged and ELISA was performedKit detects the amount of IFN-gamma secreted by cells, and defines the amount as 1; the experimental group 1 and the experimental group 2 are CIK cells which are added with CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D and cultured for 25 days respectively in a solution state, the cells with the same number as that of the control group are taken, the supernatant is obtained by centrifugation, the IFN-gamma number secreted by the cells is detected, and the IFN-gamma relative secretion is obtained by dividing the IFN-gamma number by the control group.

FIG. 22: A. SDS-PAGE analysis of purified CD3-BTLA BsAb _ M, lane 1: a molecular weight protein Marker; lane 2: reducing CD3-BTLA BsAb _ M; lane 3: non-reducing CD3-BTLA BsAb _ M; B. purified CD3-BTLA BsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: non-reducing CD3-BTLA BsAb _ D; lane 3: reducing CD3-BTLA BsAb _ D.

FIG. 23A: the ELISA identification result of CD3-BTLA BsAb _ M, the curves in the figure represent three detection results respectively: ■ is coated with 1 mug/ml recombinant antigen CD3-hFc,

coating 1 mu g/ml of recombinant antigen BTLA-hFc; assay results without any antigen coating.

FIG. 23B: the ELISA identification result of CD3-BTLA BsAb _ D, and the curves in the figure represent three detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 3-hFc;

FIG. 24: CIK cell expansion fold curve, peripheral blood PBMC was used as experimental cells, CD3-BTLA BsAb _ M, CD3-BTLA BsAb _ D or Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) was added, and cell expansion fold was counted and compared by dividing the number of cells counted each time by the number of cells on day 1 for 30 days. Wherein, the control group: 5ug/ml Anti-CD3 and 5ug/ml Anti-CD 28; experimental group 1: adding 10ng/ml of CD3-BTLA BsAb _ M in a solution state; experimental group 2: 10ng/ml CD3-BTLA BsAb _ D was added in solution.

FIG. 25: CD3-BTLA bispecific antibody mediated IFN-gamma fraction from CIK cellsAnd (4) secreting. Control group: 2X 10 CIK cells cultured for 25 days in the control group (Anti-CD3/Anti-CD28) of example 29 were selected⁵Centrifuging to take supernatant, detecting the amount of IFN-gamma secreted by cells by ELISA Kit, and defining the amount as 1; the experimental group 1 and the experimental group 2 are CIK cells which are added with CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D and cultured for 25 days respectively in a solution state, the cells with the same number as that of the control group are taken, the supernatant is obtained by centrifugation, the number of IFN-gamma secreted by the cells is detected, and the IFN-gamma is divided by the control group to obtain the relative secretion of the IFN-gamma.

Detailed Description

First, terms and abbreviations:

BsAb: bispecific Antibody (Bi-specific Antibody)

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

Fab: antigen binding Fragment (Fragment of antigen binding)

Fv: variable region fragments (Variable fragment)

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

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

Linker: connecting segment

Excellar domain: extracellular region

Co-simullatory molecule: co-stimulatory molecules

CD3-PD-1 BsAb _ M anti-CD 3/anti-PD-1 bispecific antibody in monomer form

CD3-PD-1 BsAb _ D dimeric form of anti-CD 3/anti-PD-1 bispecific antibody

CD3-CTLA-4 BsAb _ M monomeric anti-CD 3/anti-CTLA-4 bispecific antibody

CD3-CTLA-4 BsAb _ D dimeric form of anti-CD 3/anti-CTLA-4 bispecific antibody

CD3-LAG-3BsAb _ M monomeric anti-CD 3/anti-LAG-3 bispecific antibody

CD3-LAG-3BsAb _ D dimeric form of anti-CD 3/anti-LAG-3 bispecific antibody

CD3-TIM-3 BsAb _ M monomeric anti-CD 3/anti-TIM-3 bispecific antibody

CD3-TIM-3 BsAb _ D dimeric form of anti-CD 3/anti-TIM-3 bispecific antibody

CD3-TIGIT BsAb _ M monomeric anti-CD 3/anti-TIGIT bispecific antibody

CD3-TIGIT BsAb _ D dimeric form of anti-CD 3/anti-TIGIT bispecific antibody

CD3-BTLA BsAb _ M monomeric anti-CD 3/anti-BTLA bispecific antibody

CD3-BTLA BsAb _ D dimeric form of anti-CD 3/anti-BTLA bispecific antibody

Bi, bi-functional molecules

The bifunctional molecule comprises a first functional domain capable of binding and activating a T cell surface CD3 molecule and a second functional domain capable of binding and blocking a T cell negative co-stimulatory molecule.

Further, the bifunctional molecule is capable of binding and blocking a T cell negative co-stimulatory molecule while binding and activating a T cell surface CD3 molecule, thereby generating a first signal and a second signal required for T cell activation. The T cell negative co-stimulatory molecules include but are not limited to human PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, BTLA and the like.

The first domain and the second domain of the present invention are not particularly limited as long as they bind to and block the T cell negative costimulatory molecule while binding to and activating the T cell surface CD3 molecule, thereby generating the first signal and the second signal required for T cell activation. For example, the first domain can be an antibody against CD3 and the second domain can be an antibody against a negative co-stimulatory molecule of a T cell. The antibody may be in any form. However, in any form of antibody, the antigen-binding site thereof contains a heavy chain variable region and a light chain variable region. The antibody may preferably be a small molecule antibody. The small molecule antibody is an antibody fragment with smaller molecular weight, and the antigen combining part of the small molecule antibody comprises a heavy chain variable region and a light chain variable region. The small molecular antibody has small molecular weight, but maintains the affinity of the parent monoclonal antibody, and has the same specificity as the parent monoclonal antibody. The types of the small molecule antibodies mainly comprise Fab antibodies, Fv antibodies, single chain antibodies (scFv) and the like. Fab antibody synthesisComplete light chain (variable region V)_LAnd constant region C_L) And heavy chain Fd segment (variable region V)_HAnd a first constant region C_H1) Formed by disulfide bonding. Fv antibodies are the smallest functional fragment of an antibody molecule that retains an intact antigen-binding site, linked by non-covalent bonds only from the variable regions of the light and heavy chains. Single chain antibodies (scFv) are single protein peptide chain molecules in which a heavy chain variable region and a light chain variable region are connected by a linker.

The first domain and the second domain are linked by a linker fragment. The present invention has no particular requirement on the order of connection as long as the object of the present invention is not limited. For example, the C-terminus of the first domain may be linked to the N-terminus of the second domain. The number of amino acids of the connecting fragment is preferably 2 or more. The present invention is not limited to the ligated fragments, as long as the present invention is not limited thereto.

Further, the connecting fragment is selected from a connecting fragment with G4S as a unit or a hinge region fragment of immunoglobulin IgD.

In a preferred embodiment of the present invention, the bifunctional molecule is shown in FIG. 1, and is a bispecific antibody. The bifunctional molecule may be in monomeric or dimeric form. The structural diagram of the bifunctional molecule of the present invention in a monomeric form is shown in fig. 1a, and the structure of the bifunctional molecule comprises a first functional domain that binds to CD3 antigen and a second functional domain that binds to any T cell negative costimulatory molecule antigen, wherein the first functional domain is a single-chain antibody (scFv) that binds to CD3 antigen, and the second functional domain is a single-chain antibody (scFv) that binds to an Extracellular region (Extracellular domain) of a T cell negative costimulatory molecule. The structural diagram of the bifunctional molecule in a dimer form according to the present invention is shown in fig. 1 as B, and the structure of the bifunctional molecule comprises two first domains which bind to CD3 antigen and two second domains which bind to any T cell negative costimulatory molecule antigen, wherein the first domain is a single chain antibody (scFv) which binds to CD3 antigen, and the second domain is a single chain antibody (scFv) which binds to an Extracellular region (Extracellular domain) of a T cell negative costimulatory molecule. The antigen binding potency of the bifunctional molecule in the form of dimer is more than twice that of the monomer form, and the effect of in vitro T cell amplification is better.

The T cell negative co-stimulatory molecule can be human PD-1, CTLA-4, LAG-3, TIM-3, TIGIT or BTLA, etc.

The amino acid sequence of the extracellular region of the T cell negative co-stimulatory molecule human PD-1(Uniprot ID: Q15116) is shown in SEQ ID NO.5, and specifically comprises the following steps:

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV。

the amino acid sequence of the extracellular region of the T cell negative co-stimulatory molecule human CTLA-4 (Unit ID: P16410) is shown as SEQ ID NO.6, and specifically comprises the following steps:

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD。

the amino acid sequence of the extracellular region of the T cell negative co-stimulatory molecule human LAG-3 (Unit ID: P18627) is shown as SEQ ID NO.7, and specifically comprises the following steps:

VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHL。

the amino acid sequence of the extracellular region of the T cell negative co-stimulatory molecule human TIM-3(Uniprot ID: Q8TDQ0) is shown in SEQ ID NO.8, and specifically comprises the following steps:

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIG。

the amino acid sequence of the extracellular region of T cell negative co-stimulatory molecule human TIGIT (Unit ID: Q495A1) is shown in SEQ ID NO.9, and specifically comprises the following steps:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP。

the amino acid sequence of the extracellular region of the T cell negative co-stimulatory molecule human BTLA (Unit ID: Q7Z6A9) is shown in SEQ ID NO.10, and specifically comprises the following steps:

KESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPSKDEMASRPWLLYR。

in particular, the first domain is a single chain antibody against CD 3. The anti-CD3 single chain antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 36. The amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 37. Further, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 35.

The second functional domain is a single-chain antibody of an anti-T cell negative co-stimulatory molecule. The single-chain antibody of the anti-T cell negative co-stimulatory molecule comprises a heavy chain variable region and a light chain variable region.

The single-chain antibody against the T cell negative co-stimulatory molecule may be any one of a single-chain antibody against PD-1, a single-chain antibody against CTLA-4, a single-chain antibody against LAG-3, a single-chain antibody against TIM-3, a single-chain antibody against TIGIT, or a single-chain antibody against BTLA.

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

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

The amino acid sequence of the heavy chain variable region of the anti-LAG-3 single-chain antibody is shown in SEQ ID NO. 45. The amino acid sequence of the variable region of the light chain of the anti-LAG-3 single-chain antibody is shown in SEQ ID NO. 46. The amino acid sequence of the single-chain antibody for resisting the LAG-3 is shown as SEQ ID NO. 44.

The amino acid sequence of the heavy chain variable region of the single-chain antibody for resisting TIM-3 is shown as SEQ ID NO. 48. The amino acid sequence of the variable region of the light chain of the single-chain antibody for resisting the TIM-3 is shown as SEQ ID NO. 49. The amino acid sequence of the single-chain antibody for resisting the TIM-3 is shown as SEQ ID NO. 47.

The amino acid sequence of the heavy chain variable region of the anti-TIGIT single-chain antibody is shown in SEQ ID NO. 51. The amino acid sequence of the light chain variable region of the anti-TIGIT single-chain antibody is shown as SEQ ID NO. 52. The amino acid sequence of the single-chain antibody for resisting TIGIT is shown as SEQ ID NO. 50.

The amino acid sequence of the heavy chain variable region of the single-chain antibody for resisting BTLA is shown in SEQ ID NO. 54. The amino acid sequence of the variable region of the light chain of the single-chain antibody against BTLA is shown in SEQ ID NO. 55. The amino acid sequence of the single-chain antibody for resisting BTLA is shown in SEQ ID NO. 53.

In a preferred embodiment, the amino acid sequence of the monomeric bifunctional molecule is as shown in any one of SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27 or SEQ ID NO. 31. The amino acid sequence of the bifunctional molecule in the form of a dimer is shown in any one of 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. But are not limited to, the specific forms set forth in the preferred embodiment of the invention.

Polynucleotides encoding bifunctional molecules

The polynucleotide of the present invention encoding the bifunctional molecule may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded.

The polynucleotides encoding the bifunctional molecules of the present invention may be prepared by any suitable technique well known to those skilled in the art. Such techniques are described generally in the art, e.g., in the molecular cloning guidelines (J. SammBruk et al, scientific Press, 1995). Including but not limited to recombinant DNA techniques, chemical synthesis, and the like; for example, overlap extension PCR is used.

In a preferred embodiment of the present invention, the nucleotide sequence of the heavy chain variable region of the single chain antibody encoding anti-CD3 is shown in SEQ ID NO. 57. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-CD3 is shown as SEQ ID NO. 58. The nucleotide sequence of the single-chain antibody for encoding the anti-CD3 is shown in SEQ ID NO. 56.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-PD-1 is shown as SEQ ID NO. 60. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-PD-1 is shown as SEQ ID NO. 61. The nucleotide sequence of the single-chain antibody for coding the anti-PD-1 is shown as SEQ ID NO. 59.

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

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-LAG-3 is shown as SEQ ID NO. 66. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-LAG-3 is shown as SEQ ID NO. 67. The nucleotide sequence of the single-chain antibody for encoding the anti-LAG-3 is shown as SEQ ID NO. 65.

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

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

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the BTLA is shown as SEQ ID NO. 75. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the BTLA is shown as SEQ ID NO. 76. The nucleotide sequence of the single-chain antibody for encoding the BTLA is shown as SEQ ID NO. 74.

The nucleotide sequence of the connecting segment with the coding amino acid sequence shown as SEQ ID NO.1 is shown as SEQ ID NO. 2.

The nucleotide sequence of the connecting segment with the coding amino acid sequence shown as SEQ ID NO.3 is shown as SEQ ID NO. 4.

Further, the nucleotide sequence encoding the bifunctional molecule in monomeric form is shown in any one of 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. The nucleotide sequence of the bifunctional molecule in the form of a code dimer is shown as any one of 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.

Fourth, expression vector

The expression vector of the invention comprises a polynucleotide encoding the bifunctional molecule. Methods well known to those skilled in the art can be used to construct the expression vector. These methods include recombinant DNA techniques, DNA synthesis techniques and the like. The DNA encoding the fusion protein may be operably linked to a multiple cloning site in a vector to direct mRNA synthesis for protein expression, or for homologous recombination. In a preferred embodiment of the invention, pcDNA3.1 is used as the expression vector. The host cell was Chinese hamster ovary (Chinese hamster ovary ce1l, CHO).

Method for preparing bifunctional molecule

The method for preparing the bifunctional molecule comprises the following steps: constructing an expression vector containing the gene sequence of the bifunctional molecule, then transforming the expression vector containing the gene sequence of the bifunctional molecule into host cells for inducing expression, and separating the expression product to obtain the bifunctional molecule. In a preferred embodiment of the invention, pcDNA3.1 is used as the expression vector. The host cell was Chinese hamster ovary (Chinese hamster ovary ce1l, CHO).

Use of hexa-bifunctional molecules

The bifunctional molecules of the present invention can be used to prepare T cell in vitro amplification agents. In the preferred embodiment of the present invention, experiments prove that the bifunctional molecules in the form of monomers and dimers both have the in vitro binding activity with the recombinant antigen of CD3 and the corresponding T cell negative co-stimulatory molecule, and can be applied to the in vitro activation and amplification of T cells, wherein the dimers have better effect than the monomers.

Method for expanding T cells in vitro

The method for in vitro expansion of T cells comprises the step of acting the bifunctional molecule on the T cells. The method may be for non-therapeutic purposes. In the preferred embodiment of the present invention, experiments prove that the bifunctional molecules in the form of monomers and dimers both have the in vitro binding activity with the recombinant antigen of CD3 and the corresponding T cell negative co-stimulatory molecule, and can be applied to the in vitro activation and amplification of T cells, wherein the dimers have better effect than the monomers.

Aiming at the defects of the combined application of anti-CD3 and anti-T cell positive (negative) co-stimulatory molecule full-length antibodies, the invention constructs the bifunctional molecule capable of identifying and activating CD3 and identifying and blocking any T cell negative co-stimulatory molecule by the methods of genetic engineering and antibody engineering, the bifunctional molecule not only has the characteristic of the combined use of the double antibodies, but also has obvious advantages in the aspects of preparation process and practical application, and can achieve the effect even superior to the effect of the combined addition of the two antibodies or the coating of a culture plate when added in a solution form, thereby greatly improving the effects of in vitro activation and T cell amplification and increasing the convenience of use.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, 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; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1: construction of CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D eukaryotic expression vectors

In the present invention, bispecific antibodies targeting a T cell surface human CD3 protein and a T cell negative costimulatory molecule PD-1 protein were designated CD3-PD-1 BsAb.

Design of construction schemes for CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D

The specific construction scheme of the monomer form of CD3-PD-1 BsAb _ M is as follows: between anti-CD3 scFv and anti-PD-1 scFv sequences through (GGGGS)₃And linking with a Linker.

The specific construction scheme of the dimer form of CD3-PD-1 BsAb _ D is as follows: the anti-CD3 scFv and the anti-PD-1 scFv are connected by an IgD hinge region as a Linker.

For expression of the bispecific antibody in mammalian cells, the sequences for anti-CD3 scFv, anti-PD-1 scFv and Linker fragment (Linker) were codon optimized for mammalian system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID No.57, specifically:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

specifically, the nucleotide sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID No.58, specifically:

GACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

specifically, the nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO.56, and specifically comprises the following steps:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

specifically, the nucleotide sequence of the heavy chain variable region of the anti-PD-1 scFv is shown as SEQ ID NO.60, and specifically comprises:

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGGACTGCAAGGCCAGCGGCATCACCTTCAGCAACAGCGGCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAAGCGCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTTCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

specifically, the nucleotide sequence of the light chain variable region of the anti-PD-1 scFv is shown as SEQ ID NO.61, and specifically comprises:

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the anti-PD-1 scFv is shown as SEQ ID NO.59, and specifically comprises the following steps:

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGGACTGCAAGGCCAGCGGCATCACCTTCAGCAACAGCGGCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAAGCGCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTTCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric CD3-PD-1 BsAb _ M connecting fragment is shown as SEQ ID NO.2, and specifically comprises the following steps:

GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC。

the nucleotide sequence of the dimer form of the CD3-PD-1 BsAb _ D connecting fragment is shown as SEQ ID NO.4, and specifically comprises the following steps:

GCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTG。

for expression and successful secretion of the bispecific antibody into the culture medium in CHO-S cells, a signal peptide for secretory expression of the antibody was selected for this example.

The amino acid sequence of the secretory expression signal peptide is shown as SEQ ID NO.77, and specifically comprises the following steps:

MTRLTVLALLAGLLASSRA。

the nucleotide sequence of the secretory expression signal peptide is shown as SEQ ID NO.78, and specifically comprises the following steps:

ATGACCCGCCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCAGCAGCCGCGCC。

II, CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D eukaryotic expression vector construction

The bispecific antibody of the invention is constructed and expressed by selecting a mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.). To construct bispecific antibodies in monomeric and dimeric forms, primers as shown in table 1 were designed, respectively, and all primers were synthesized by seuzhou jingzhi biotechnology limited, and gene templates required for amplification were synthesized by seuzhou hong jun technology limited.

Cloning construction for CD3-PD-1 BsAb _ M, signal peptide fragment was first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD3-F and CD3-R, CD3- (GGGGS)₃Amplification of anti-CD3 scFv, (GGGGS) by-PD-1-F and pcDNA3.1-PD-1-R₃Linker, gene sequence of anti-PD-1 scFv; for the cloning construction of CD3-PD-1 BsAb _ D, signal peptide fragments were first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and then the gene sequences for anti-CD3 scFv, IgD hinge region and anti-PD-1 scFv were amplified using the primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-PD-1-F and pcDNA3.1-PD-1-R, respectively. After amplification, the amplified DNA is used

The PCR one-step directional cloning kit (purchased from Wujiang near-shore protein science and technology Co., Ltd.) respectively splices full-length gene sequences of the bispecific antibody in a monomer and dimer form and seamlessly clones the full-length gene sequences to a pcDNA3.1 expression vector which is subjected to linear treatment by EcoRI and HindIII. The target vector transforms escherichia coli DH5 alpha, positive clone identification is carried out by colony PCR, and sequencing identification is carried out on recombinants (recombinant plasmids) identified as positive. The correctly sequenced recombinants (recombinant plasmids) were then mapped into plasmids and used for transfection of CHO-S cells.

Sequencing revealed that the full-length gene sequences of monomeric form of CD3-PD-1 BsAb _ M and dimeric form of CD3-PD-1 BsAb _ D were correct and consistent with the expectations.

Specifically, the nucleotide sequence of the monomeric CD3-PD-1 BsAb _ M is shown as SEQ ID NO.12, and specifically comprises:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGGACTGCAAGGCCAGCGGCATCACCTTCAGCAACAGCGGCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAAGCGCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTTCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the dimeric form of CD3-PD-1 BsAb _ D is shown in SEQ ID No.14, specifically:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGGACTGCAAGGCCAGCGGCATCACCTTCAGCAACAGCGGCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAAGCGCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTTCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

TABLE 1 primers used in the cloning of the CD3-PD-1 bispecific antibody Gene

Example 2: expression and purification of CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D

Expression of CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D

1.1 the passage density of CHO-S cells (purchased from Thermo Fisher Scientific Co.) 1 day before transfection was 0.5-0.6X 10⁶/ml；

1.2. Cell density statistics is carried out on the day of transfection, and when the density is 1-1.4 multiplied by 10⁶Activity/ml>90%, can be used for plasmid transfection;

1.3. preparation of transfection complex: for each item (CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D), two centrifuge tubes/culture flasks were prepared, each containing 20ml of the recombinant plasmid prepared in example 1:

adding 600 mu l of PBS and 20 mu g of recombinant plasmid into the tube, and uniformly mixing;

add 600. mu.l PBS, 20ul FreeStyle^TMMAX Transfection Reagent (available from Thermo Fisher Scientific Co.) and blending;

1.4. adding the diluted transfection reagent into the diluted recombinant plasmid, and uniformly mixing to prepare a transfection compound;

1.5. standing the transfection complex for 15-20 min, and adding a single drop of the transfection complex into the cell culture at a constant speed;

1.6. at 37 ℃ CO₂The concentration is 8%, the cell culture after transfection is carried out under the condition of 130rpm of the shaking table, and the culture supernatant is collected for carrying out the expression detection of the target protein after 5 days.

Purification of CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D

2.1 sample pretreatment

Taking 20ml of the transfected cell culture supernatant, adding a buffer solution of 20mM PB and 200mM NaCl to adjust the pH value to 7.5;

2.2Protein L affinity column purification

Protein purification chromatography column: protein L affinity chromatography column (available from GE Healthcare, column volume 1.0ml)

Buffer a (buffer a): PBS, pH7.4

Buffer b (buffer b): 0.1M Glycine, pH3.0

Buffer c (buffer c): 0.1M Glycine, pH2.7

And (3) purification process: the Protein L affinity chromatography column was pretreated with Buffer A using AKTA explorer 100 type Protein purification system (purchased from GE Healthcare), and the culture supernatant was sampled and the effluent was collected. After the sample loading is finished, balancing the chromatographic column by using at least 1.5ml of Buffer A, eluting by using Buffer B and Buffer C respectively after balancing, collecting target protein eluent (1% of 1M Tris needs to be added in advance into a collecting pipe of the eluent, the pH value of the eluent is neutralized by pH8.0, and the final concentration of Tris is about 10mM), and finally concentrating and dialyzing into Buffer PBS.

The final purified recombinant proteins CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D were analyzed by SDS-PAGE and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 2. As can be seen from the figure, after purification by Protein L affinity chromatography column, the purity of CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D recombinant proteins is both > 95%; wherein the theoretical molecular weight of the recombinant protein CD3-PD-1 BsAb _ M is 52.5kDa, and the protein presents a single electrophoresis band under reducing and non-reducing conditions, and the molecular weight is consistent with that of a monomer, so that the bispecific antibody is in a monomer form (FIG. 2A); the theoretical molecular weight of the recombinant CD3-PD-1 BsAb _ D protein is 60.4kDa, the electrophoretic band of the protein exhibits a molecular weight consistent with that of a monomer under reducing conditions, and the electrophoretic band exhibits a molecular weight consistent with that of a dimer under non-reducing conditions (FIG. 2B), indicating that the two protein molecules can be linked to each other through disulfide bonds, and thus the bispecific antibody is in a dimer form.

In addition, the purified recombinant protein samples are subjected to N/C terminal sequence analysis, and the results show that the reading frames of the expressed recombinant protein samples are correct and consistent with the theoretical N/C terminal amino acid sequence, and the mass spectrometry further confirms that the CD3-PD-1 BsAb _ M is in a monomer form and the CD3-PD-1 BsAb _ D is in a dimer form.

Therefore, it can be known that the amino acid sequence of the monomeric form of CD3-PD-1 BsAb _ M is shown in SEQ ID No.11, specifically:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR。

the amino acid sequence of the dimer form of CD3-PD-1 BsAb _ D is shown in SEQ ID NO.13, and specifically comprises:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR。

the amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO.35, and specifically comprises the following steps:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。

the amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID NO.36, and specifically comprises the following steps:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS。

the amino acid sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID NO.37, and specifically comprises the following steps:

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。

the amino acid sequence of the anti-PD-1 scFv is shown as SEQ ID NO.38, and specifically comprises the following steps:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR。

the amino acid sequence of the heavy chain variable region of the anti-PD-1 scFv is shown as SEQ ID NO.39, and specifically comprises the following steps:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS。

the amino acid sequence of the light chain variable region of the anti-PD-1 scFv is shown as SEQ ID NO.40, and specifically comprises the following steps:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR。

the amino acid sequence of the connecting fragment in the monomeric CD3-PD-1 BsAb _ M is shown as SEQ ID NO.1, and specifically comprises the following steps:

GGGGSGGGGSGGGGS。

the amino acid sequence of the connecting fragment in the dimer form of CD3-PD-1 BsAb _ D is shown as SEQ ID NO.3, and specifically comprises the following steps:

ASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGV。

example 3: ELISA for detecting antigen binding activity of CD3-PD-1 BsAb _ M and CD3-PD-1 BsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD3-hFc and human PD-1-hFc fusion protein (purchased from Wujiang near-shore protein technologies, Ltd.) were coated on 96-well plates, respectively, with an antigen concentration of 1. mu.g/ml and a coating volume of 100. mu.l/well, under the conditions of 1 hour at 37 ℃ or overnight at 4 ℃, and the formulation of coating buffer (PBS) was: 3.58g Na₂HPO4，0.24g NaH₂PO4，0.2g KCl，8.2g NaCl，950ml H₂O, adjusting the pH value to 7.4 by using 1mol/L HCl or 1mol/L NaOH, and supplementing water to 1L;

2. and (3) sealing: after washing the plate 4 times with PBS, blocking solution PBSA (PBS + 2% BSA (V/W)) was added at 200. mu.l/well and blocked at 37 ℃ for 1 hour;

3. sample adding: after 4 PBS washes, purified bispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation: taking 10 μ g/ml purified CD3-PD-1 BsAb _ M or CD3-PD-1 BsAb _ D as the initial concentration, performing a dilution by multiple of 6 gradients, each gradient having 2 multiple wells;

4. color development: after washing the plate 4 times with PBST (PBS + 0.05% Tween-20(V/V)), the HRP-labeled chromogenic antibody (purchased from Abcam) was diluted 1/5000 with blocking solution PBSA, added at 100. mu.l/well, and incubated at 37 ℃ for 1 hour. After washing the plate for 4 times with PBS, adding a color developing solution TMB (purchased from KPL company) with 100 mul/hole, and developing for 5-10 minutes at room temperature in a dark place;

5. termination reaction and result determination: stop solution (1M HCl) was added at 100. mu.l/well and absorbance was read on a microplate reader at a wavelength of 450 nm.

The ELISA results are shown in fig. 3A and 3B: FIG. 3A illustrates that CD3-PD-1 BsAb _ M has in vitro binding activity to both recombinant antigens CD3-hFc and PD-1-hFc, wherein the binding activity of PD-1 is higher than that of CD 3; FIG. 3B illustrates that CD3-PD-1 BsAb _ D has in vitro binding activity as well as recombinant antigens CD3-hFc and PD-1-hFc, with greater PD-1 binding activity.

Example 4: CD3-PD-1 bispecific antibody mediated CIK (cytokine induced killer) cell proliferation

The bispecific antibody CD3-PD-1 BsAb _ M in monomer form, the bispecific antibody CD3-PD-1 BsAb _ D in dimer form and the Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) prepared by the invention respectively act on human blood PBMC of the same donor source by taking human Peripheral Blood Mononuclear Cells (PBMC) as experimental materials, counting is carried out after cell culture, and amplification times are compared.

Isolation of PBMC: adding anticoagulated blood, adding medical normal saline with the same volume, slowly adding lymphocyte separation liquid (purchased from GE Healthcare company) with the same volume with blood along the wall of a centrifugal tube, keeping the liquid level obviously layered, centrifuging at 2000rpm for 20min, sucking the middle white vaporous cell layer into a new centrifugal tube, adding PBS buffer solution with more than 2 times volume for washing, centrifuging at 1100rpm for 10min, repeatedly washing once, re-suspending with a small amount of precooled X-vivo15 serum-free culture medium (purchased from Lonza company), and counting cells for later use;

CIK cell culture and expansion: PBMC were cultured in CIK basal medium (90% X-vivo 1)5+ 10% FBS) and adjusted cell density to 1X 10⁶Ml, the following 3 experimental groups were designed: control group (Anti-CD 35 ug/ml and Anti-CD285ug/ml coated cell culture plate); experimental group 1 (bispecific antibody CD3-PD-1 BsAb _ M10ng/ml is added in solution); experimental group 2 (bispecific antibody CD3-PD-1 BsAb _ D10 ng/ml was added in solution). In addition, 3 groups of experimental cells were simultaneously supplemented with the cytokines IFN-. gamma. (200ng/ml, ex WUJIANG NEIGINIAN PROTEIN SCHEME Co., Ltd.) and IL-1. beta. (2ng/ml, ex WUJIANG NEIGINIAN PROTEIN SCHEME, Inc.), placed in an incubator, and subjected to 5.0% CO treatment at a saturated humidity of 37 deg.C₂Culturing under the conditions of (1). After overnight incubation, the cultures were continued with 500U/ml IL-2 (purchased from Wujiang Korea protein technology Ltd.) and counted every 2-3 days and cultured in 1X 10 CIK basal medium supplemented with 500U/ml IL-2⁶Cell passaging was performed at a density of/ml. Culturing for 30 days by the method, finally counting the amplification multiple of the cells, and drawing a growth curve;

the detection result is shown in fig. 4, the single use of the CD3-PD-1 bispecific antibody in monomer and dimer forms has better proliferation effect on CIK cells than the combined use of the Anti-CD3/Anti-CD28 monoclonal full-length antibody, after 18 days of culture, a great amount of cell death occurs in the combination of Anti-CD3/Anti-CD28, and the cell expansion fold is obviously reduced; while the addition of CD3-PD-1 BsAb _ M in monomeric form or CD3-PD-1 BsAb _ D in dimeric form did not result in cell death, but the rate of cell expansion was relatively slow. Therefore, the two forms of the CD3-PD-1 bispecific antibody prepared by the invention can effectively expand and prolong the survival period of CIK cells, and the dimer form has better effect.

Example 5: CIK cell IFN-gamma secretion induced by CD3-PD-1 bispecific antibody

The method comprises the following operation steps:

1. the supernatant of CIK cells cultured for 25 days in example 4 (adjusted to the same cell density, cell number 2X 10)⁵One) 100 μ l, incubated at 37 ℃ for 45min, and tested by the Human IFN- γ ELISA Kit (purchased from Boshide Biotech), three samples were taken for each of the three experiments and repeated;

2. washing with PBS for three times, adding HRP-labeled IFN-gamma antibody, and incubating at 37 deg.C for 45 min;

3. washing with PBS for three times, adding TMB 100 μ l for color development, and developing at room temperature for 5-10 min;

4. the addition of stop solution HCl (1M) was stopped and the absorbance was read at a wavelength of 450 nm.

The results are shown in FIG. 5: wherein the quantity of IFN-gamma secreted by CIK cells cultured by Anti-CD3/Anti-CD28 full-length antibody in a combined mode is defined as 1, the relative secretion quantity of IFN-gamma of the CIK cells cultured by adding a monomer form of CD3-PD-1 BsAb _ M in a solution state is 2.45, and the relative secretion quantity of IFN-gamma of the CIK cells cultured by adding a dimer form of CD3-PD-1 BsAb _ D in a solution state is 4.12, so that the two forms of CD3-PD-1 bispecific antibodies prepared by the invention are both more favorable for activating the CIK cells and inducing the secretion of the IFN-gamma, and the effect of the dimer form is better.

Example 6: construction of CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D eukaryotic expression vectors

In the present invention, the bispecific antibody targeting the T cell surface human CD3 protein and the T cell negative co-stimulatory molecule CTLA-4 protein was named CD3-CTLA-4 BsAb.

First, CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD3-CTLA-4 BsAb _ M is as follows: anti-CD3 scFv and anti-CTLA-4 scFv sequences through (GGGGS)₃And linking with a Linker.

The specific construction scheme of the dimer form of CD3-CTLA-4 BsAb _ D is as follows: the anti-CD3 scFv and the anti-CTLA-4 scFv are connected by an IgD hinge region as a Linker.

For expression of the bispecific antibody in mammalian cells, the sequences for anti-CD3 scFv, anti-CTLA-4 scFv and Linker fragment (Linker) were codon optimized for mammalian system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 57.

Specifically, the nucleotide sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 58.

Specifically, the nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 56.

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

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACACCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGACCTTCATCAGCTACGACGGCAACAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCATCTACTACTGCGCCCGCACCGGCTGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

specifically, the nucleotide sequence of the variable region of the light chain of the anti-CTLA-4 scFv is shown in SEQ ID No.64, and specifically comprises the following steps:

GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGGGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGGCGCCTTCAGCCGCGCCACCGGCATCCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCCGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the anti-CTLA-4 scFv is shown as SEQ ID NO.62, and specifically comprises the following steps:

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACACCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGACCTTCATCAGCTACGACGGCAACAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCATCTACTACTGCGCCCGCACCGGCTGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGGGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGGCGCCTTCAGCCGCGCCACCGGCATCCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCCGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric CD3-CTLA-4 BsAb _ M connecting fragment is shown as SEQ ID NO. 2.

The nucleotide sequence of the dimer form of CD3-CTLA-4 BsAb _ D connecting fragment is shown in SEQ ID NO. 4.

The amino acid sequence of the secretory expression signal peptide is shown as SEQ ID NO. 77.

The nucleotide sequence of the secretory expression signal peptide is shown as SEQ ID NO. 78.

Second, CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D eukaryotic expression vector construction

The bispecific antibody of the invention is constructed and expressed by selecting a mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.). To construct bispecific antibodies in monomeric and dimeric forms, primers as shown in table 2 were designed, all of which were synthesized by seujin Zhi Biotech, Inc., and gene templates for amplification were synthesized by seujin JNJTech, Inc., respectively.

Cloning construction for CD3-CTLA-4 BsAb _ M, signal peptide fragment was first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD3-F and CD3-R, CD3- (GGGGS)₃anti-CD3 scFv (GGGGS) amplified from-CTLA-4-F and pcDNA3.1-CTLA-4-R₃Linker, gene sequence of anti-CTLA-4 scFv; cloning construction for CD3-CTLA-4 BsAb _ D was carried out by first amplifying signal peptide fragments using primers pcDNA3.1-Sig-F and Sig-R, and then amplifying gene sequences for anti-CD3 scFv, IgD hinge region and anti-CTLA-4 scFv using primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CTLA-4-F and pcDNA3.1-CTLA-4-R, respectively. After amplification, the amplified DNA is used

The PCR one-step directional cloning kit (purchased from Wujiang near-shore protein science and technology Co., Ltd.) respectively splices full-length gene sequences of the bispecific antibody in a monomer and dimer form and seamlessly clones the full-length gene sequences to a pcDNA3.1 expression vector which is subjected to linear treatment by EcoRI and HindIII. The target vector is transformed into escherichia coli DH5 alpha, positive clone identification is carried out by colony PCR, and the escherichia coli DH5 alpha is identified as positiveThe recombinants (recombinant plasmids) were sequenced and identified. The correctly sequenced recombinants (recombinant plasmids) were then mapped into plasmids and used for transfection of CHO-S cells.

Sequencing revealed that the full-length gene sequences of the monomeric form of CD3-CTLA-4 BsAb _ M and the dimeric form of CD3-CTLA-4 BsAb _ D were correct and consistent with the expectations.

Specifically, the nucleotide sequence of the monomeric CD3-CTLA-4 BsAb _ M is shown as SEQ ID NO.16, and specifically comprises the following steps:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACACCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGACCTTCATCAGCTACGACGGCAACAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCATCTACTACTGCGCCCGCACCGGCTGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGGGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGGCGCCTTCAGCCGCGCCACCGGCATCCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCCGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the dimeric form of CD3-CTLA-4 BsAb _ D is shown in SEQ ID NO.18, and specifically comprises:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACACCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGACCTTCATCAGCTACGACGGCAACAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCATCTACTACTGCGCCCGCACCGGCTGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGGGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGGCGCCTTCAGCCGCGCCACCGGCATCCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCCGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

TABLE 2 primers used in the cloning of the CD3-CTLA-4 bispecific antibody Gene

Example 7: expression and purification of CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D

Expression of CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D

1.3. preparation of transfection complex: for each item (CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D), two centrifuge tubes/culture bottles were prepared, and 20ml of each was placed, and the recombinant plasmid prepared in example 6 was taken:

Secondly, purifying CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D

2.1 sample pretreatment

2.2Protein L affinity column purification

Buffer a (buffer a): PBS, pH7.4

Buffer b (buffer b): 0.1M Glycine, pH3.0

Buffer c (buffer c): 0.1M Glycine, pH2.7

The final purified CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D recombinant proteins were analyzed by SDS-PAGE, and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 6. As can be seen from the figure, after the Protein L affinity chromatography column purification, the purity of the CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D recombinant proteins is both > 95%; wherein the theoretical molecular weight of the CD3-CTLA-4 BsAb _ M recombinant protein is 53.2kDa, and the protein presents a single electrophoresis band under reducing and non-reducing conditions, and the molecular weight is consistent with that of a monomer, so that the bispecific antibody is in a monomer form (FIG. 6A); the theoretical molecular weight of the CD3-CTLA-4 BsAb _ D recombinant protein is 61.2kDa, the electrophoretic band of the protein under reducing conditions shows a molecular weight consistent with that of a monomer, and the electrophoretic band under non-reducing conditions shows a molecular weight consistent with that of a dimer (FIG. 6B), indicating that the two protein molecules can be connected with each other through a disulfide bond, so that the bispecific antibody is in a dimer form.

In addition, the N/C terminal sequence analysis of the purified recombinant protein samples shows that the reading frames of the expressed recombinant protein samples are correct and consistent with the theoretical N/C terminal amino acid sequence, and the mass spectrometry further confirms that the CD3-CTLA-4 BsAb _ M is in a monomer form and the CD3-CTLA-4 BsAb _ D is in a dimer form.

Therefore, it can be seen that the amino acid sequence of the monomeric form of CD3-CTLA-4 BsAb _ M is shown in SEQ ID NO.15, which specifically comprises:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKR。

the amino acid sequence of the dimer form of CD3-CTLA-4 BsAb _ D is shown in SEQ ID NO.17, and specifically comprises the following components:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKR。

the amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 35.

The amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 36.

The amino acid sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 37.

The amino acid sequence of the anti-CTLA-4 scFv is shown as SEQ ID NO.41, and specifically comprises the following steps:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKR。

the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 scFv is shown as SEQ ID NO.42, and specifically comprises the following steps:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS。

the amino acid sequence of the variable region of the light chain of the anti-CTLA-4 scFv is shown as SEQ ID NO.43, and specifically comprises the following steps:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKR。

the amino acid sequence of the connecting fragment in the monomeric form of CD3-CTLA-4 BsAb _ M is shown in SEQ ID NO. 1.

The amino acid sequence of the connecting fragment in the dimer form of CD3-CTLA-4 BsAb _ D is shown in SEQ ID NO. 3.

Example 8: ELISA for detecting the antigen binding activity of CD3-CTLA-4 BsAb _ M and CD3-CTLA-4 BsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD3-hFc and human CTLA-4-hFc fusion proteins (purchased from Wujiang near-shore protein technologies, Ltd.) were coated on 96-well plates, respectively, with an antigen concentration of 1. mu.g/ml and a coating volume of 100. mu.l/well, under the conditions of 1 hour at 37 ℃ or overnight at 4 ℃, and the formulation of coating buffer (PBS) was: 3.58g Na₂HPO4，0.24g NaH₂PO4，0.2g KCl，8.2g NaCl，950ml H₂O, adjusting the pH value to 7.4 by using 1mol/L HCl or 1mol/L NaOH, and supplementing water to 1L;

2. and (3) sealing: after washing the plate 4 times with PBS, blocking solution PBSA (PBS + 2% BSA (V/W)) was added at 200. mu.l/well. Blocking at 37 ℃ for 1 hour;

3. sample adding: after 4 PBS washes, purified bispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation: taking 10 μ g/ml purified CD3-CTLA-4 BsAb _ M or CD3-CTLA-4 BsAb _ D as the initial concentration, performing double dilution for 6 gradients, each gradient having 2 multiple wells;

The ELISA results are shown in figures 7A and 7B: FIG. 7A illustrates that CD3-CTLA-4 BsAb _ M has in vitro binding activity to both recombinant antigens CD3-hFc and CTLA-4-hFc, wherein CTLA-4 binding activity is higher than CD 3; FIG. 7B shows that CD3-CTLA-4 BsAb _ D has in vitro binding activity as well as recombinant antigens CD3-hFc and CTLA-4-hFc, wherein CTLA-4 binding activity is higher.

Example 9: CD3-CTLA-4 bispecific antibody mediated CIK cell proliferation

The human Peripheral Blood Mononuclear Cells (PBMC) were used as experimental materials, and the monomeric bispecific antibody CD3-CTLA-4 BsAb _ M, the dimeric bispecific antibody CD3-CTLA-4 BsAb _ D and the Anti-CD3/Anti-CD28 monoclonal full-length antibody prepared by the present invention were used in combination with Anti-CD3/Anti-CD28 to act on human PBMC derived from the same donor, respectively, and the cells were cultured, counted, and the amplification factor was compared.

CIK cell culture and expansion: the PBMC were resuspended in CIK basal medium (90% X-vivo15+ 10% FBS) and the cell density was adjusted to 1X 10⁶Ml, the following 3 experimental groups were designed: control group (Anti-CD 35 ug/ml and Anti-CD285ug/ml coated cell culture plate); experimental group 1 (bispecific antibody CD3-CTLA-4 BsAb _ M10ng/ml was added in solution); experimental group 2 (bispecific antibody CD3-CTLA-4 BsAb _ D10 ng/ml was added in solution). In addition, 3 groups of experimental cells were supplemented with the cytokines IFN-. gamma. (200ng/ml, from Wujiang Yuan protein technology Co., Ltd.) and IL-1. beta. (2ng/ml, from Wujiang Yuan protein technology Co., Ltd.)Science and technology Co., Ltd.) in an incubator at saturated humidity, 37 ℃ and 5.0% CO₂Culturing under the conditions of (1). After overnight incubation, the cultures were continued with 500U/ml IL-2 (purchased from Wujiang Korea protein technology Ltd.) and counted every 2-3 days and cultured in 1X 10 CIK basal medium supplemented with 500U/ml IL-2⁶Cell passaging was performed at a density of/ml. Culturing for 30 days by the method, finally counting the amplification multiple of the cells, and drawing a growth curve;

the detection result is shown in fig. 8, the single use of the CD3-CTLA-4 bispecific antibody in a monomer form and a dimer form has better proliferation effect on CIK cells than the combined use of the Anti-CD3/Anti-CD28 monoclonal full-length antibody, after 18 days of culture, a great amount of cell death occurs in the combination of Anti-CD3 and Anti-CD28, and the cell expansion fold is obviously reduced; the addition of CD3-CTLA-4 BsAb _ M in monomer form or CD3-CTLA-4 BsAb _ D in dimer form did not result in cell death, but the cell expansion rate was relatively slow. Therefore, the two forms of the CD3-CTLA-4 bispecific antibody prepared by the invention can effectively expand and prolong the survival period of CIK cells, and the dimer form has better effect.

Example 10: CD3-CTLA-4 bispecific antibody induced IFN-gamma secretion from CIK cells

The method comprises the following operation steps:

1. the supernatant of CIK cells cultured for 25 days in example 9 (adjusted to the same cell density, cell number 2X 10)⁵One) 100 μ l, incubated at 37 ℃ for 45min, and tested by the Human IFN- γ ELISA Kit (purchased from Boshide Biotech), three samples were taken for each of the three experiments and repeated;

The results are shown in FIG. 9: wherein the quantity of IFN-gamma secreted by CIK cells cultured by Anti-CD3/Anti-CD28 full-length antibody in a combined way is defined as 1, the relative secretion quantity of IFN-gamma of the CIK cells cultured by adding a monomer form of CD3-CTLA-4 BsAb _ M in a solution state is 1.94, and the relative secretion quantity of IFN-gamma of the CIK cells cultured by adding a dimer form of CD3-CTLA-4 BsAb _ D in a solution state is 2.85, so that the two forms of CD3-CTLA-4 bispecific antibodies prepared by the invention are both more favorable for activating the CIK cells and inducing the secretion of the IFN-gamma, and the effect of the dimer form is better.

Example 11: construction of CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D eukaryotic expression vectors

In the present invention, bispecific antibodies targeting a T cell surface human CD3 protein and a T cell negative costimulatory molecule LAG-3 protein were designated CD3-LAG-3 BsAb.

Design of CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D construction schemes

The specific construction scheme of the monomer form of CD3-LAG-3BsAb _ M is as follows: between anti-CD3 scFv and anti-LAG-3 scFv sequences through (GGGGS)₃And linking with a Linker.

The specific construction scheme of the dimer form of CD3-LAG-3BsAb _ D is as follows: the anti-CD3 scFv and anti-LAG-3 scFv sequences are linked by an IgD hinge region as a Linker.

For expression of the bispecific antibody in mammalian cells, the sequences for anti-CD3 scFv, anti-LAG-3 scFv and Linker fragment (Linker) were codon optimized for mammalian system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of the anti-LAG-3 scFv is shown in SEQ ID No.66, specifically:

CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGACTACTACTGGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCACCGCGGCAGCACCAACAGCAACCCCAGCCTGAAGAGCCGCGTGACCCTGAGCCTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGCGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCTTCGGCTACAGCGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

specifically, the nucleotide sequence of the light chain variable region of the anti-LAG-3 scFv is shown in SEQ ID No.67, specifically:

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCTGACCTTCGGCCAGGGCACCAACCTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the anti-LAG-3 scFv is shown as SEQ ID NO.65, and specifically comprises:

CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGACTACTACTGGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCACCGCGGCAGCACCAACAGCAACCCCAGCCTGAAGAGCCGCGTGACCCTGAGCCTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGCGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCTTCGGCTACAGCGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCTGACCTTCGGCCAGGGCACCAACCTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric CD3-LAG-3BsAb _ M connecting fragment is shown as SEQ ID NO. 2.

The nucleotide sequence of the dimeric form of the CD3-LAG-3BsAb _ D junction fragment is shown in SEQ ID NO. 4.

Second, CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D eukaryotic expression vector construction

The bispecific antibody of the invention is constructed and expressed by selecting a mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.). To construct bispecific antibodies in monomeric and dimeric forms, primers as shown in table 3 were designed, respectively, and all primers were synthesized by seuzhou jingzhi biotechnology limited and gene templates for amplification were synthesized by seuzhou hong jun technology limited.

Cloning construction for CD3-LAG-3BsAb _ M, signal peptide fragment was first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD3-F and CD3-R, CD3- (GGGGS)₃Amplification of-LAG-3-F and pcDNA3.1-LAG-3-R against CD3 scFv, (GGGGS)₃Linker, gene sequence of anti-LAG-3 scFv; cloning construction for CD3-LAG-3BsAb _ D, signal peptide fragments were also first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then anti-CD3 scFv, IgD hinge region, anti-LAG-3 scFv gene sequences were amplified using primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-LAG-3-F and pcDNA3.1-LAB-3-R, respectively. After amplification, the amplified DNA is used

Sequencing revealed that the full-length gene sequences of monomeric form of CD3-LAG-3BsAb _ M and dimeric form of CD3-LAG-3BsAb _ D were correct and consistent with the expectations.

Specifically, the nucleotide sequence of the monomeric form of CD3-LAG-3BsAb _ M is shown as SEQ ID NO.20, and specifically comprises:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGACTACTACTGGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCACCGCGGCAGCACCAACAGCAACCCCAGCCTGAAGAGCCGCGTGACCCTGAGCCTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGCGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCTTCGGCTACAGCGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCTGACCTTCGGCCAGGGCACCAACCTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the dimeric form of CD3-LAG-3BsAb _ D is shown in SEQ ID NO.22, and specifically comprises:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGACTACTACTGGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCACCGCGGCAGCACCAACAGCAACCCCAGCCTGAAGAGCCGCGTGACCCTGAGCCTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGCGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCTTCGGCTACAGCGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCTGACCTTCGGCCAGGGCACCAACCTGGAGATCAAGCGC。

TABLE 3 primers used in the cloning of the CD3-LAG-3 bispecific antibody Gene

Example 12: expression and purification of CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D

Expression of CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D

1.2. Cell density statistics were performed on the day of transfection whenDensity of 1 to 1.4X 10⁶Activity/ml>90%, can be used for plasmid transfection;

1.3. preparation of transfection complex: for each item (CD3-LAG-3 BsAb _ M and CD3-LAG-3BsAb _ D), two centrifuge tubes/culture flasks were prepared, each containing 20ml of the recombinant plasmid prepared in example 11:

II, purification of CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D

2.1 sample pretreatment

2.2Protein L affinity column purification

Buffer a (buffer a): PBS, pH7.4

Buffer b (buffer b): 0.1M Glycine, pH3.0

Buffer c (buffer c): 0.1M Glycine, pH2.7

The final purified recombinant proteins CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D were analyzed by SDS-PAGE, and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 10. As can be seen from the figure, after purification by Protein L affinity chromatography column, the purity of the CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D recombinant proteins is both > 95%; wherein the theoretical molecular weight of the recombinant CD3-LAG-3BsAb _ M protein is 53.5kDa, and the protein exhibits a single electrophoretic band under both reducing and non-reducing conditions, the molecular weight is consistent with that of a monomer, so that the bispecific antibody is in a monomer form (FIG. 10A); the theoretical molecular weight of the recombinant CD3-LAG-3BsAb _ D protein is 61.4kDa, the electrophoretic band of the protein exhibits a molecular weight consistent with that of a monomer under reducing conditions, and the electrophoretic band exhibits a molecular weight consistent with that of a dimer under non-reducing conditions (FIG. 10B), indicating that two protein molecules can be linked to each other through disulfide bonds, and thus the bispecific antibody is in a dimer form.

In addition, the purified recombinant protein samples are subjected to N/C terminal sequence analysis, the result shows that the expressed recombinant protein samples have correct reading frames and are consistent with the theoretical N/C terminal amino acid sequence, and mass spectrometry further confirms that the CD3-LAG-3BsAb _ M is in a monomer form and the CD3-LAG-3BsAb _ D is in a dimer form.

Therefore, it can be seen that the amino acid sequence of the monomeric form of CD3-LAG-3BsAb _ M is shown in SEQ ID No.19, specifically:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKR。

the amino acid sequence of the dimeric form of CD3-LAG-3BsAb _ D is shown in SEQ ID NO.21, and specifically comprises:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKR。

the amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 35.

The amino acid sequence of the anti-LAG-3 scFv is shown as SEQ ID NO.44, and specifically comprises the following steps:

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKR。

the amino acid sequence of the heavy chain variable region of the anti-LAG-3 scFv is shown as SEQ ID NO.45, and specifically comprises the following steps:

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSS。

the amino acid sequence of the variable region of the light chain of the anti-LAG-3 scFv is shown as SEQ ID NO.46, and specifically comprises the following steps:

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKR。

the amino acid sequence of the connecting fragment in the monomeric form of CD3-LAG-3BsAb _ M is shown in SEQ ID NO. 1.

The amino acid sequence of the connecting fragment in the dimeric form of CD3-LAG-3BsAb _ D is shown in SEQ ID NO. 3.

Example 13: ELISA for detecting antigen binding activity of CD3-LAG-3BsAb _ M and CD3-LAG-3BsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD3-hFc and human LAG-3-hFc fusion proteins (purchased from Wujiang nearshore protein technologies, Ltd.) were coated on 96-well plates, respectively, with an antigen concentration of 1. mu.g/ml and a coating volume of 100. mu.l/well, under conditions of 37 ℃ for 1 hour or 4 ℃ overnight, and the formulation of coating buffer (PBS) was: 3.58g Na₂HPO4，0.24g NaH₂PO4，0.2g KCl，8.2g NaCl，950ml H₂O, adjusting the pH value to 7.4 by using 1mol/L HCl or 1mol/L NaOH, and supplementing water to 1L;

3. sample adding: after 4 PBS washes, purified bispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation: taking 10 μ g/ml purified CD3-LAG-3BsAb _ M or CD3-LAG-3BsAb _ D as the initial concentration, performing dilution by multiple ratio for 6 gradients, each gradient being provided with 2 duplicate wells;

The ELISA results are shown in fig. 11A and 11B: FIG. 11A illustrates that CD3-LAG-3BsAb _ M has in vitro binding activity to both recombinant antigens CD3-hFc and LAG-3-hFc, wherein LAG-3 binding activity is greater than CD3 binding activity; FIG. 11B illustrates that CD3-LAG-3BsAb _ D has in vitro binding activity as well as recombinant antigens CD3-hFc and LAG-3-hFc, wherein LAG-3 binding activity is higher.

Example 14: CD3-LAG-3 bispecific antibody mediated CIK cell proliferation

The bispecific antibody CD3-LAG-3BsAb _ M in monomer form, the bispecific antibody CD3-LAG-3BsAb _ D in dimer form and the Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) prepared by the invention respectively act on human PBMC (Peripheral blood mononuclear cell (PBMC)) of the same donor source by taking PBMC as an experimental material, counting after cell culture and comparing the amplification times.

CIK cell culture and expansion: the PBMC were resuspended in CIK basal medium (90% X-vivo15+ 10% FBS) and the cell density was adjusted to 1X 10⁶Ml, the following 3 experimental groups were designed: control groups (Anti-CD 35 ug/ml and Anti-CD285ug/ml coated cell culture plates, full length antibody purchased from Wujiang near-shore protein technology, Inc.); experimental group 1 (bispecific antibody CD3-LAG-3BsAb _ M10ng/ml added in solution); experimental group 2 (bispecific antibody CD3-LAG-3BsAb _ D10 ng/ml was added in solution). In addition, 3 groups of experimental cells were simultaneously supplemented with the cytokines IFN-. gamma. (200ng/ml, ex WUJIANG NEIGINIAN PROTEIN SCHEME Co., Ltd.) and IL-1. beta. (2ng/ml, ex WUJIANG NEIGINIAN PROTEIN SCHEME, Inc.), placed in an incubator, and subjected to 5.0% CO treatment at a saturated humidity of 37 deg.C₂Culturing under the conditions of (1). After overnight incubation, the cultures were continued with 500U/ml IL-2 (purchased from Wujiang Korea protein technology Ltd.) and counted every 2-3 days and cultured in 1X 10 CIK basal medium supplemented with 500U/ml IL-2⁶Cell passaging was performed at a density of/ml. After culturing for 30 days, the amplification multiple of the cells is finally counted, and a growth curve is drawn.

The detection result is shown in fig. 12, the single use of the CD3-LAG-3 bispecific antibody in monomer and dimer forms has better proliferation effect on CIK cells than the combined use of the Anti-CD3/Anti-CD28 monoclonal full-length antibody, after 18 days of culture, a great amount of cell death occurs in the combination of Anti-CD3 and Anti-CD28, and the cell expansion fold is obviously reduced; the addition of CD3-LAG-3BsAb _ M in monomer form or CD3-LAG-3BsAb _ D in dimer form did not result in cell death, but the rate of cell expansion was relatively slow. Therefore, the two forms of the CD3-LAG-3 bispecific antibody prepared by the invention can effectively expand and prolong the survival of CIK cells, and the dimer form has better effect.

Example 15: CIK cell IFN-gamma secretion induced by CD3-LAG-3 bispecific antibody

The method comprises the following operation steps:

1. the supernatant of CIK cells obtained after 25 days of culture in example 14 (adjusted to the same cell density, cell number 2X 10)⁵One) 100 μ l, incubated at 37 ℃ for 45min, and tested by the Human IFN- γ ELISA Kit (purchased from Boshide Biotech), three samples were taken for each of the three experiments and repeated;

The results are shown in FIG. 13: wherein the quantity of IFN-gamma secreted by CIK cells cultured by Anti-CD3/Anti-CD28 full-length antibody in a combined way is defined as 1, the relative secretion quantity of IFN-gamma of the CIK cells cultured by adding a monomer form of CD3-LAG-3BsAb _ M in a solution state is 2.25, and the relative secretion quantity of IFN-gamma of the CIK cells cultured by adding a dimer form of CD3-LAG-3BsAb _ D in a solution state is 3.37, so that the two forms of the bispecific antibody 3-LAG-3 prepared by the invention are both more favorable for activating the CIK cells and inducing the secretion of the IFN-gamma, and the effect of the dimer form is better.

Example 16: construction of CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D eukaryotic expression vectors

In the present invention, bispecific antibodies targeting a T cell surface human CD3 protein and a T cell negative costimulatory molecule TIM-3 protein were designated CD3-TIM-3 BsAb.

First, CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD3-TIM-3 BsAb _ M is as follows: between anti-CD3 scFv and anti-TIM-3 scFv sequences through (GGGGS)₃And linking with a Linker.

The specific construction scheme of the dimer form of CD3-TIM-3 BsAb _ D is as follows: the anti-CD3 scFv and anti-TIM-3 scFv sequences were linked by an IgD hinge region as Linker.

For expression of the bispecific antibody in mammalian cells, the sequences for anti-CD3 scFv, anti-TIM-3 scFv and Linker fragment (Linker) were codon optimized for mammalian system expression.

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

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGACATCTACCCCGGCCAGGGCGACACCAGCTACAACCAGAAGTTCAAGGGCCGCGCCACCATGACCGCCGACAAGAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGTGGGCGGCGCCTTCCCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

specifically, the nucleotide sequence of the variable region of the light chain of the anti-TIM-3 scFv is shown in SEQ ID No.70, specifically:

GACATCGTGCTGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTGCCGCGCCAGCGAGAGCGTGGAGTACTACGGCACCAGCCTGATGCAGTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCCGCAAGGACCCCAGCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the anti-TIM-3 scFv is shown as SEQ ID NO.68, and specifically comprises the following steps:

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGACATCTACCCCGGCCAGGGCGACACCAGCTACAACCAGAAGTTCAAGGGCCGCGCCACCATGACCGCCGACAAGAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGTGGGCGGCGCCTTCCCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGCTGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTGCCGCGCCAGCGAGAGCGTGGAGTACTACGGCACCAGCCTGATGCAGTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCCGCAAGGACCCCAGCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric CD3-TIM-3 BsAb _ M connecting fragment is shown as SEQ ID NO. 2.

The nucleotide sequence of the dimeric form of the CD3-TIM-3 BsAb _ D junction fragment is shown in SEQ ID NO. 4.

Two, CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D eukaryotic expression vector construction

The bispecific antibody of the invention is constructed and expressed by selecting a mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.). To construct bispecific antibodies in monomeric and dimeric forms, primers as shown in table 4 were designed, all of which were synthesized by seuzhou jinzhi biotechnology limited and gene templates for amplification were synthesized by seuzhou hong jun science and technology limited, respectively.

Cloning construction for CD3-TIM-3 BsAb _ M Signal peptide fragments were first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD3-F and CD3-R, CD3- (GGGGS)₃Amplification of anti-CD3 scFv, (GGGGS) from-TIM-3-F and pcDNA3.1-TIM-3-R₃Linker, gene sequence of anti-TIM-3 scFv; for the cloning construction of CD3-TIM-3 BsAb _ D, signal peptide fragments were first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and then the gene sequences for anti-CD3 scFv, IgD hinge region, anti-TIM-3 scFv were amplified using the primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-TIM-3-F and pcDNA3.1-TIM-3-R, respectively. After amplification, the amplified DNA is used

Sequencing revealed that the full-length gene sequences of the monomeric form of CD3-TIM-3 BsAb _ M and the dimeric form of CD3-TIM-3 BsAb _ D were correct and consistent with the expectation.

Specifically, the nucleotide sequence of the monomeric CD3-TIM-3 BsAb _ M is shown as SEQ ID NO.24, and specifically comprises the following steps:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGACATCTACCCCGGCCAGGGCGACACCAGCTACAACCAGAAGTTCAAGGGCCGCGCCACCATGACCGCCGACAAGAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGTGGGCGGCGCCTTCCCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGCTGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTGCCGCGCCAGCGAGAGCGTGGAGTACTACGGCACCAGCCTGATGCAGTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCCGCAAGGACCCCAGCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the dimeric form of CD3-TIM-3 BsAb _ D is shown in SEQ ID NO. 26. The method specifically comprises the following steps:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGACATCTACCCCGGCCAGGGCGACACCAGCTACAACCAGAAGTTCAAGGGCCGCGCCACCATGACCGCCGACAAGAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGTGGGCGGCGCCTTCCCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGCTGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTGCCGCGCCAGCGAGAGCGTGGAGTACTACGGCACCAGCCTGATGCAGTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCCGCAAGGACCCCAGCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

TABLE 4 primers used in the cloning of the CD3-TIM-3 bispecific antibody Gene

Example 17: expression and purification of CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D

Expression of one, CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D

1.3. preparation of transfection complex: for each item (CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D), two centrifuge tubes/culture bottles were prepared, each 20ml of each was placed, and the recombinant plasmid prepared in example 16 was taken:

Purification of CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D

2.1 sample pretreatment

2.2Protein L affinity column purification

Buffer a (buffer a): PBS, pH7.4

Buffer b (buffer b): 0.1M Glycine, pH3.0

Buffer c (buffer c): 0.1M Glycine, pH2.7

The final purified recombinant proteins CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D were analyzed by SDS-PAGE, and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 14. As can be seen from the figure, after purification by Protein L affinity chromatography column, the purity of CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D recombinant proteins is both > 95%; wherein the theoretical molecular weight of the recombinant CD3-TIM-3 BsAb _ M protein is 53.2kDa, and the protein presents a single electrophoretic band under reducing and non-reducing conditions, and the molecular weight is consistent with that of a monomer, so that the bispecific antibody is in a monomer form (FIG. 14A); the theoretical molecular weight of the CD3-TIM-3 BsAb _ D recombinant protein is 61.1kDa, the electrophoretic band of the protein under reducing conditions exhibits a molecular weight consistent with that of a monomer, and the electrophoretic band of the protein under non-reducing conditions exhibits a molecular weight consistent with that of a dimer (FIG. 14B), indicating that the two protein molecules can be connected with each other through a disulfide bond, and thus the bispecific antibody is in a dimer form.

In addition, the purified recombinant protein samples are subjected to N/C terminal sequence analysis, the result shows that the expressed recombinant protein samples have correct reading frames and are consistent with the theoretical N/C terminal amino acid sequence, and mass spectrometry further confirms that the CD3-TIM-3 BsAb _ M is in a monomer form and the CD3-TIM-3 BsAb _ D is in a dimer form.

Therefore, it can be seen that the amino acid sequence of the monomeric form of CD3-TIM-3 BsAb _ M is shown in SEQ ID NO.23, specifically:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGDIYPGQGDTSYNQKFKGRATMTADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATINCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIKR。

the amino acid sequence of the dimer form of CD3-TIM-3 BsAb _ D is shown in SEQ ID NO.25, and specifically comprises:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGDIYPGQGDTSYNQKFKGRATMTADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATINCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIKR。

the amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 35.

The amino acid sequence of the anti-TIM-3 scFv is shown as SEQ ID NO.47, and specifically comprises the following steps:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGDIYPGQGDTSYNQKFKGRATMTADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATINCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIKR。

the amino acid sequence of the heavy chain variable region of the anti-TIM-3 scFv is shown as SEQ ID NO.48, and specifically comprises the following steps:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGDIYPGQGDTSYNQKFKGRATMTADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLVTVSS。

the amino acid sequence of the variable region of the light chain of the anti-TIM-3 scFv is shown as SEQ ID NO.49, and specifically comprises the following steps:

DIVLTQSPDSLAVSLGERATINCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIKR。

the amino acid sequence of the connecting fragment in the monomeric form of CD3-TIM-3 BsAb _ M is shown in SEQ ID NO. 1.

The amino acid sequence of the connecting fragment in the dimeric form of CD3-TIM-3 BsAb _ D is shown in SEQ ID NO. 3.

Example 18: ELISA for detecting antigen binding activity of CD3-TIM-3 BsAb _ M and CD3-TIM-3 BsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD3-hFc and human TIM-3-hFc fusion proteins (available from Wujiang near-shore protein technology Co., Ltd.)Separately coating 96-well plates with an antigen concentration of 1. mu.g/ml, a coating volume of 100. mu.l/well, under conditions of 37 ℃ for 1 hour or 4 ℃ overnight, and a coating buffer (PBS) formulation: 3.58g Na₂HPO4，0.24g NaH₂PO4，0.2g KCl，8.2g NaCl，950ml H₂O, adjusting the pH value to 7.4 by using 1mol/L HCl or 1mol/L NaOH, and supplementing water to 1L;

3. sample adding: after 4 PBS washes, purified bispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation: taking 10 μ g/ml purified CD3-TIM-3 BsAb _ M or CD3-TIM-3 BsAb _ D as the initial concentration, performing a double dilution of 6 gradients, each gradient having 2 duplicate wells;

The ELISA results are shown in fig. 15A and 15B: FIG. 15A illustrates that CD3-TIM-3 BsAb _ M has in vitro binding activity to both recombinant antigens CD3-hFc and TIM-3-hFc, wherein TIM-3 binding activity is greater than CD3 binding activity; FIG. 15B illustrates that CD3-TIM-3 BsAb _ D has in vitro binding activity as well as the recombinant antigens CD3-hFc and TIM-3-hFc, where TIM-3 binding activity is higher.

Example 19: CIK cell proliferation mediated by CD3-TIM-3 bispecific antibody

The bispecific antibody CD3-TIM-3 BsAb _ M in monomer form, the bispecific antibody CD3-TIM-3 BsAb _ D in dimer form and the Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) prepared by the invention respectively act on human blood PBMC of the same donor source by taking human Peripheral Blood Mononuclear Cells (PBMC) as experimental materials, counting is carried out after the cells are cultured, and the amplification times are compared.

CIK cell culture and expansion: the PBMC were resuspended in CIK basal medium (90% X-vivo15+ 10% FBS) and the cell density was adjusted to 1X 10⁶Ml, the following 3 experimental groups were designed: control groups (Anti-CD 35 ug/ml and Anti-CD285ug/ml coated cell culture plates, full length antibody purchased from Wujiang near-shore protein technology, Inc.); experimental group 1 (bispecific antibody CD3-TIM-3 BsAb _ M10ng/ml added in solution); experimental group 2 (bispecific antibody CD3-TIM-3 BsAb _ D10 ng/ml was added in solution). In addition, 3 groups of experimental cells were simultaneously supplemented with the cytokines IFN-. gamma. (200ng/ml, ex WUJIANG NEIGINIAN PROTEIN SCHEME Co., Ltd.) and IL-1. beta. (2ng/ml, ex WUJIANG NEIGINIAN PROTEIN SCHEME, Inc.), placed in an incubator, and subjected to 5.0% CO treatment at a saturated humidity of 37 deg.C₂Culturing under the conditions of (1). After overnight incubation, the cultures were continued with 500U/ml IL-2 (purchased from Wujiang Korea protein technology Ltd.) and counted every 2-3 days and cultured in 1X 10 CIK basal medium supplemented with 500U/ml IL-2⁶Cell passaging was performed at a density of/ml. Culturing for 30 days by the method, finally counting the amplification multiple of the cells, and drawing a growth curve;

the detection result is shown in fig. 16, the single use of the CD3-TIM-3 bispecific antibody in monomer and dimer forms has better proliferation effect on CIK cells than the combined use of the Anti-CD3/Anti-CD28 monoclonal full-length antibody, after 18 days of culture, a great amount of cell death occurs in the combination of Anti-CD3/Anti-CD28, and the cell expansion fold is obviously reduced; while the addition of CD3-TIM-3 BsAb _ M in monomeric form or CD3-TIM-3 BsAb _ D in dimeric form did not result in cell death, but the cell expansion rate was relatively slow. Therefore, the two forms of the CD3-TIM-3 bispecific antibody prepared by the invention can effectively expand and prolong the survival of CIK cells, and the dimer form has better effect.

Example 20: CIK cell IFN-gamma secretion induced by CD3-TIM-3 bispecific antibody

The method comprises the following operation steps:

1. the supernatant of CIK cells cultured for 25 days in example 19 (adjusted to the same cell density, cell number 2X 10)⁵One) 100 μ l, incubated at 37 ℃ for 45min, and tested by the Human IFN- γ ELISA Kit (purchased from Boshide Biotech), three samples were taken for each of the three experiments and repeated;

The results are shown in FIG. 17: wherein the quantity of IFN-gamma secreted by CIK cells cultured by Anti-CD3/Anti-CD28 full-length antibody in a combined mode is defined as 1, the relative secretion quantity of IFN-gamma of the CIK cells cultured by adding a monomer form of CD3-TIM-3 BsAb _ M in a solution state is 2.07, and the relative secretion quantity of IFN-gamma of the CIK cells cultured by adding a dimer form of CD3-TIM-3 BsAb _ D in a solution state is 3.04, so that the two forms of CD3-TIM-3 bispecific antibodies prepared by the invention are both more favorable for activating the CIK cells and inducing the secretion of the IFN-gamma, and the effect of the dimer form is better.

Example 21: construction of CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D eukaryotic expression vectors

In the present invention, bispecific antibodies targeting T cell surface human CD3 protein and T cell negative costimulatory molecule TIGIT protein were designated CD3-TIGIT BsAb.

Design of construction schemes of CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D

The specific construction scheme of the monomer form of CD3-TIGIT BsAb _ M is as follows: passing between anti-CD3 scFv and anti-TIGIT scFv sequences (GGGGS)₃And linking with a Linker.

The specific construction scheme of the dimer form of CD3-TIGIT BsAb _ D is as follows: the sequences of the anti-CD3 scFv and the anti-TIGIT scFv are connected through an IgD hinge region as a Linker.

For expression of the bispecific antibody in mammalian cells, the sequences for anti-CD3 scFv, anti-TIGIT scFv and Linker fragment (Linker) were codon optimized for mammalian system expression.

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

GAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTGCAGCGTGACCGGCAGCAGCATCGCCAGCGACTACTGGGGCTGGATCCGCAAGTTCCCCGGCAACAAGATGGAGTGGATGGGCTTCATCACCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGAGCCGCATCAGCATCACCCGCGACACCAGCAAGAACCAGTTCTTCCTGCAGCTGCACAGCGTGACCACCGACGACACCGCCACCTACAGCTGCGCCCGCATGCCCAGCTTCATCACCCTGGCCAGCCTGAGCACCTGGGAGGGCTACTTCGACTTCTGGGGCCCCGGCACCATGGTGACCGTGAGCAGC。

specifically, the nucleotide sequence of the light chain variable region of the anti-TIGIT scFv is shown in SEQ ID No.73, specifically:

GACATCCAGATGACCCAGAGCCCCAGCCTGCTGAGCGCCAGCGTGGGCGACCGCGTGACCCTGAACTGCAAGGCCAGCCAGAGCATCCACAAGAACCTGGCCTGGTACCAGCAGAAGCTGGGCGAGGCCCCCAAGTTCCTGATCTACTACGCCAACAGCCTGCAGACCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCGGCCTGCAGCCCGAGGACGTGGCCACCTACTTCTGCCAGCAGTACTACAGCGGCTGGACCTTCGGCGGCGGCACCAAGGTGGAGCTGAAGCGC。

specifically, the nucleotide sequence of the anti-TIGIT scFv is shown in SEQ ID No.71, specifically:

GAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTGCAGCGTGACCGGCAGCAGCATCGCCAGCGACTACTGGGGCTGGATCCGCAAGTTCCCCGGCAACAAGATGGAGTGGATGGGCTTCATCACCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGAGCCGCATCAGCATCACCCGCGACACCAGCAAGAACCAGTTCTTCCTGCAGCTGCACAGCGTGACCACCGACGACACCGCCACCTACAGCTGCGCCCGCATGCCCAGCTTCATCACCCTGGCCAGCCTGAGCACCTGGGAGGGCTACTTCGACTTCTGGGGCCCCGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCCTGCTGAGCGCCAGCGTGGGCGACCGCGTGACCCTGAACTGCAAGGCCAGCCAGAGCATCCACAAGAACCTGGCCTGGTACCAGCAGAAGCTGGGCGAGGCCCCCAAGTTCCTGATCTACTACGCCAACAGCCTGCAGACCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCGGCCTGCAGCCCGAGGACGTGGCCACCTACTTCTGCCAGCAGTACTACAGCGGCTGGACCTTCGGCGGCGGCACCAAGGTGGAGCTGAAGCGC。

the nucleotide sequence of the monomeric CD3-TIGIT BsAb _ M connecting fragment is shown as SEQ ID NO. 2.

The nucleotide sequence of the dimeric form of the CD3-TIGIT BsAb _ D junction fragment is shown in SEQ ID NO. 4.

II, construction of CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D eukaryotic expression vectors

The bispecific antibody of the invention is constructed and expressed by selecting a mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.). To construct bispecific antibodies in monomeric and dimeric forms, primers as shown in table 5 were designed, respectively, and all primers were synthesized by seuzhou jingzhi biotechnology limited and gene templates for amplification were synthesized by seuzhou hong jun technology limited.

Cloning for CD3-TIGIT BsAb _ M was performed by first amplifying signal peptide fragments using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD3-F and CD3-R, CD3- (GGGGS)₃Amplifying anti-CD3 scFv, (GGGGS) by-TIGIT-F and pcDNA3.1-TIGIT-R₃Linker, gene sequence of anti-TIGIT scFv; for CD3-TIGIT BsAb _ D was constructed by first amplifying signal peptide fragments using primers pcDNA3.1-Sig-F and Sig-R, and then amplifying gene sequences of anti-CD3 scFv, IgD hinge region and anti-TIGIT scFv using primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-TIGIT-F and pcDNA3.1-TIGIT-R, respectively. After amplification, the amplified DNA is used

Sequencing revealed that the full-length gene sequences of the monomeric form of CD3-TIGIT BsAb _ M and the dimeric form of CD3-TIGIT BsAb _ D were correct and consistent with the expectation.

Specifically, the nucleotide sequence of the monomer form of CD3-TIGIT BsAb _ M is shown as SEQ ID NO.28, and specifically comprises:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTGCAGCGTGACCGGCAGCAGCATCGCCAGCGACTACTGGGGCTGGATCCGCAAGTTCCCCGGCAACAAGATGGAGTGGATGGGCTTCATCACCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGAGCCGCATCAGCATCACCCGCGACACCAGCAAGAACCAGTTCTTCCTGCAGCTGCACAGCGTGACCACCGACGACACCGCCACCTACAGCTGCGCCCGCATGCCCAGCTTCATCACCCTGGCCAGCCTGAGCACCTGGGAGGGCTACTTCGACTTCTGGGGCCCCGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCCTGCTGAGCGCCAGCGTGGGCGACCGCGTGACCCTGAACTGCAAGGCCAGCCAGAGCATCCACAAGAACCTGGCCTGGTACCAGCAGAAGCTGGGCGAGGCCCCCAAGTTCCTGATCTACTACGCCAACAGCCTGCAGACCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCGGCCTGCAGCCCGAGGACGTGGCCACCTACTTCTGCCAGCAGTACTACAGCGGCTGGACCTTCGGCGGCGGCACCAAGGTGGAGCTGAAGCGC。

specifically, the nucleotide sequence of the dimer form of CD3-TIGIT BsAb _ D is shown in SEQ ID NO.30, and specifically comprises:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGGAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTGCAGCGTGACCGGCAGCAGCATCGCCAGCGACTACTGGGGCTGGATCCGCAAGTTCCCCGGCAACAAGATGGAGTGGATGGGCTTCATCACCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGAGCCGCATCAGCATCACCCGCGACACCAGCAAGAACCAGTTCTTCCTGCAGCTGCACAGCGTGACCACCGACGACACCGCCACCTACAGCTGCGCCCGCATGCCCAGCTTCATCACCCTGGCCAGCCTGAGCACCTGGGAGGGCTACTTCGACTTCTGGGGCCCCGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCCTGCTGAGCGCCAGCGTGGGCGACCGCGTGACCCTGAACTGCAAGGCCAGCCAGAGCATCCACAAGAACCTGGCCTGGTACCAGCAGAAGCTGGGCGAGGCCCCCAAGTTCCTGATCTACTACGCCAACAGCCTGCAGACCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCGGCCTGCAGCCCGAGGACGTGGCCACCTACTTCTGCCAGCAGTACTACAGCGGCTGGACCTTCGGCGGCGGCACCAAGGTGGAGCTGAAGCGC。

TABLE 5 primers used in the cloning of the CD3-TIGIT bispecific antibody Gene

Example 22: expression and purification of CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D

Expression of CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D

1.3. preparation of transfection complex: for each project (CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D), two centrifuge tubes/culture bottles were prepared, each 20ml was placed, and the recombinant plasmid prepared in example 21 was taken:

II, purification of CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D

2.1 sample pretreatment

2.2Protein L affinity column purification

Buffer a (buffer a): PBS, pH7.4

Buffer b (buffer b): 0.1M Glycine, pH3.0

Buffer c (buffer c): 0.1M Glycine, pH2.7

The final purified recombinant proteins CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D were analyzed by SDS-PAGE and electrophoretograms under reducing and non-reducing conditions are shown in FIG. 18. As can be seen from the figure, after the Protein L affinity chromatography column purification, the purity of the CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D recombinant proteins is both more than 95%; wherein the theoretical molecular weight of the CD3-TIGIT BsAb _ M recombinant protein is 54.0kDa, and the protein presents a single electrophoresis band under reducing and non-reducing conditions, and the molecular weight is consistent with that of a monomer, so that the bispecific antibody is in a monomer form (FIG. 18A); the theoretical molecular weight of the recombinant CD3-TIGIT BsAb _ D protein is 61.9kDa, the electrophoretic band of the protein exhibits a molecular weight consistent with that of a monomer under reducing conditions, and the electrophoretic band exhibits a molecular weight consistent with that of a dimer under non-reducing conditions (FIG. 18B), indicating that the two protein molecules can be linked to each other through disulfide bonds, and thus the bispecific antibody is in a dimer form.

In addition, the N/C terminal sequence analysis of the purified recombinant protein sample shows that the reading frames of the expressed recombinant protein samples are correct and consistent with the theoretical N/C terminal amino acid sequence, and the mass spectrometry further confirms that the CD3-TIGIT BsAb _ M is in a monomer form and the CD3-TIGIT BsAb _ D is in a dimer form.

Therefore, it can be known that the amino acid sequence of the monomeric form of CD3-TIGIT BsAb _ M is shown in SEQ ID NO.27, specifically:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFITYSGSTSYNPSLKSRISITRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGPGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYANSLQTGIPSRFSGSGSGTDFTLTISGLQPEDVATYFCQQYYSGWTFGGGTKVELKR。

the amino acid sequence of the dimer form of CD3-TIGIT BsAb _ D is shown in SEQ ID NO.29, and specifically comprises the following steps:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVEVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFITYSGSTSYNPSLKSRISITRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGPGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYANSLQTGIPSRFSGSGSGTDFTLTISGLQPEDVATYFCQQYYSGWTFGGGTKVELKR。

the amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 35.

The amino acid sequence of the anti-TIGIT scFv is shown as SEQ ID NO.50, and specifically comprises the following steps:

EVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFITYSGSTSYNPSLKSRISITRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGPGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYANSLQTGIPSRFSGSGSGTDFTLTISGLQPEDVATYFCQQYYSGWTFGGGTKVELKR。

the amino acid sequence of the heavy chain variable region of the anti-TIGIT scFv is shown as SEQ ID NO.51, and specifically comprises the following steps:

EVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFITYSGSTSYNPSLKSRISITRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGPGTMVTVSS。

the amino acid sequence of the light chain variable region of the anti-TIGIT scFv is shown as SEQ ID NO.52, and specifically comprises the following steps:

DIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYANSLQTGIPSRFSGSGSGTDFTLTISGLQPEDVATYFCQQYYSGWTFGGGTKVELKR。

the amino acid sequence of the connecting fragment in the monomeric form of CD3-TIGIT BsAb _ M is shown in SEQ ID NO. 1.

The amino acid sequence of the connecting fragment in the dimer form of CD3-TIGIT BsAb _ D is shown in SEQ ID NO. 3.

Example 23: ELISA for detecting antigen binding activity of CD3-TIGIT BsAb _ M and CD3-TIGIT BsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD3-hFc and human TIGIT-hFc fusion protein (purchased from Wujiang near-shore protein technologies Co., Ltd.) were coated on 96-well plates respectively at an antigen concentration of 1. mu.g/ml and a coating volume of 100. mu.l/well under the conditions of 37 ℃ for 1 hour or 4 ℃ overnight, and the formulation of coating buffer (PBS) was: 3.58g Na₂HPO4，0.24g NaH₂PO4，0.2g KCl，8.2g NaCl，950ml H₂O, adjusting the pH value to 7.4 by using 1mol/L HCl or 1mol/L NaOH, and supplementing water to 1L;

3. sample adding: after 4 PBS washes, purified bispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation: taking 10 μ g/ml purified CD3-TIGIT BsAb _ M or CD3-TIGIT BsAb _ D as the initial concentration, performing multiple dilution for 6 gradients, and setting 2 multiple wells for each gradient;

The ELISA results are shown in fig. 19A and 19B: FIG. 19A illustrates that CD3-TIGIT BsAb _ M has in vitro binding activity to both recombinant antigens CD3-hFc and TIGIT-hFc, wherein TIGIT binding activity is higher than CD3 binding activity; FIG. 19B illustrates that CD3-TIGIT BsAb _ D has in vitro binding activity as well as recombinant antigens CD3-hFc and TIGIT-hFc, with TIGIT binding activity being higher.

Example 24: CD3-TIGIT bispecific antibody mediated CIK cell proliferation

The bispecific antibody CD3-TIGIT BsAb _ M in monomer form, the bispecific antibody CD3-TIGIT BsAb _ D in dimer form and the Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) prepared by the invention respectively act on human PBMC of the same donor source by taking PBMC of human Peripheral Blood Mononuclear Cells (PBMC), and counting and comparing the amplification times after cell culture.

CIK cell culture and expansion: the PBMC were resuspended in CIK basal medium (90% X-vivo15+ 10% FBS) and the cell density was adjusted to 1X 10⁶Ml, the following 3 experimental groups were designed: control groups (Anti-CD 35 ug/ml and Anti-CD285ug/ml coated cell culture plates, full length antibody purchased from Wujiang near-shore protein technology, Inc.); experimental group 1 (bispecific antibody CD3-TIGIT BsAb _ M10ng/ml added in solution state); experimental group 2 (bispecific antibody CD3-TIGIT BsAb _ D10 ng/ml was added in solution). In addition, 3 groups of experimental cells were simultaneously supplemented with the cytokines IFN-. gamma. (200ng/ml, ex WUJIANG NEIGINIAN PROTEIN SCHEME Co., Ltd.) and IL-1. beta. (2ng/ml, ex WUJIANG NEIGINIAN PROTEIN SCHEME, Inc.), placed in an incubator, and subjected to 5.0% CO treatment at a saturated humidity of 37 deg.C₂Culturing under the conditions of (1). After overnight incubation, the cultures were continued with 500U/ml IL-2 (purchased from Wujiang Korea protein technology Ltd.) and counted every 2-3 days and cultured in 1X 10 CIK basal medium supplemented with 500U/ml IL-2⁶Cell passaging was performed at a density of/ml. Culturing for 30 days by the method, finally counting the amplification multiple of the cells, and drawing a growth curve;

the detection result is shown in fig. 20, the single use of the CD3-TIGIT bispecific antibody in monomer and dimer forms has better proliferation effect on CIK cells than the combined use of the Anti-CD3/Anti-CD28 monoclonal full-length antibody, after 18 days of culture, a great amount of cell death occurs in the combination of Anti-CD3/Anti-CD28, and the cell expansion fold is obviously reduced; the addition of CD3-TIGIT BsAb _ M in monomer form or CD3-TIGIT BsAb _ D in dimer form did not result in cell death, but the cell expansion rate was relatively slowed. Therefore, both forms of the CD3-TIGIT bispecific antibody prepared by the invention can effectively expand and prolong the survival of CIK cells, and the dimer form has better effect.

Example 25: CIK cell IFN-gamma secretion induced by CD3-TIGIT bispecific antibody

The method comprises the following operation steps:

1. the supernatant of CIK cells obtained after 25 days of culture in example 24 (adjusted to the same cell density, cell number 2X 10)⁵One) 100 μ l, incubated at 37 ℃ for 45min, and tested by the Human IFN- γ ELISA Kit (purchased from Boshide Biotech), three samples were taken for each of the three experiments and repeated;

The results are shown in FIG. 21: wherein the quantity of IFN-gamma secreted by CIK cells cultured by Anti-CD3/Anti-CD28 full-length antibody in a combined mode is defined as 1, the relative secretion quantity of IFN-gamma of CIK cells cultured by adding a monomer form of CD3-TIGIT BsAb _ M in a solution state is 1.66, and the relative secretion quantity of IFN-gamma of CIK cells cultured by adding a dimer form of CD3-TIGIT BsAb _ D in a solution state is 2.30, so that the two forms of CD3-TIGIT bispecific antibodies prepared by the invention are both more favorable for activating the CIK cells and inducing the secretion of the IFN-gamma, and the effect of the dimer form is better.

Example 26: construction of CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D eukaryotic expression vectors

In the present invention, bispecific antibodies targeting a T cell surface human CD3 protein and a T cell negative costimulatory molecule BTLA protein were designated CD3-BTLA BsAb.

First, CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD3-BTLA BsAb _ M is as follows: between anti-CD3 scFv and anti-BTLA scFv sequences through (GGGGS)₃And linking with a Linker.

The specific construction scheme of the dimer form of CD3-BTLA BsAb _ D is as follows: the anti-CD3 scFv and the anti-BTLA scFv are connected by an IgD hinge region as a Linker.

For expression of the bispecific antibody in mammalian cells, the sequences for anti-CD3 scFv, anti-BTLA scFv and Linker fragment (Linker) were codon optimized for mammalian system expression.

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

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCATCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCACCGGCAAGGGCCTGGAGTGGGTGAGCGTGATCGGCCCCGCCGGCGACACCTACTACCCCGGCAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGGCGACACCGCCGTGTACTACTGCGCCCGCGAGGGCATGGCCGCCCACAACTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

specifically, the nucleotide sequence of the light chain variable region of the anti-BTLA scFv is shown in SEQ ID No.76, specifically:

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCATCACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the anti-BTLA scFv is shown in SEQ ID NO.74, and specifically comprises:

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCATCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCACCGGCAAGGGCCTGGAGTGGGTGAGCGTGATCGGCCCCGCCGGCGACACCTACTACCCCGGCAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGGCGACACCGCCGTGTACTACTGCGCCCGCGAGGGCATGGCCGCCCACAACTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCATCACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric form of the CD3-BTLA BsAb _ M junction fragment is shown in SEQ ID NO. 2.

The nucleotide sequence of the dimeric form of the CD3-BTLA BsAb _ D junction fragment is shown in SEQ ID NO. 4.

Second, CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D eukaryotic expression vector construction

The bispecific antibody of the invention is constructed and expressed by selecting a mammalian cell protein transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.). To construct bispecific antibodies in monomeric and dimeric forms, primers as shown in table 6 were designed, respectively, and all primers were synthesized by seuzhou jingzhi biotechnology limited and gene templates for amplification were synthesized by seuzhou hong jun technology limited.

Cloning construction for CD3-BTLA BsAb _ M, signal peptide fragments were first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD3-F and CD3-R, CD3- (GGGGS)₃Amplification of anti-CD3 scFv, (GGGGS) from-BTLA-F and pcDNA3.1-BTLA-R₃Linker, gene sequence of anti-BTLA scFv; cloning construction for CD3-BTLA BsAb _ D, signal peptide fragments were first amplified using the primers pcDNA3.1-Sig-F and Sig-R, and anti-CD3 scF was amplified using the primers Sig-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-BTLA-F and pcDNA3.1-BTLA-R, respectivelyv, IgD hinge region, anti-BTLA scFv. After amplification, the amplified DNA is used

Sequencing revealed that the full-length gene sequences of the monomeric form of CD3-BTLA BsAb _ M and the dimeric form of CD3-BTLA BsAb _ D were correct and consistent with the expectations.

Specifically, the nucleotide sequence of the monomeric form of CD3-BTLA BsAb _ M is shown in SEQ ID NO.32, and specifically comprises:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCATCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCACCGGCAAGGGCCTGGAGTGGGTGAGCGTGATCGGCCCCGCCGGCGACACCTACTACCCCGGCAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGGCGACACCGCCGTGTACTACTGCGCCCGCGAGGGCATGGCCGCCCACAACTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCATCACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

specifically, the nucleotide sequence of the dimeric form of CD3-BTLA BsAb _ D is shown in SEQ ID NO.34, and specifically comprises:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCATCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCACCGGCAAGGGCCTGGAGTGGGTGAGCGTGATCGGCCCCGCCGGCGACACCTACTACCCCGGCAGCGTGAAGGGCCGCTTCACCATCAGCCGCGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGGCGACACCGCCGTGTACTACTGCGCCCGCGAGGGCATGGCCGCCCACAACTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCATCACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

TABLE 6 primers used in the cloning of the CD3-BTLA bispecific antibody Gene

Example 27: expression and purification of CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D

Expression of CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D

1.3. preparation of transfection complex: for each item (CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D), two centrifuge tubes/culture flasks were prepared, each containing 20ml of the recombinant plasmid prepared in example 26:

II, purification of CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D

2.1 sample pretreatment

2.2Protein L affinity column purification

Buffer a (buffer a): PBS, pH7.4

Buffer b (buffer b): 0.1M Glycine, pH3.0

Buffer c (buffer c): 0.1M Glycine, pH2.7

The final purified recombinant proteins CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D were analyzed by SDS-PAGE and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 22. As can be seen from the figure, after purification by Protein L affinity chromatography column, the purity of CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D recombinant proteins is both > 95%; wherein the theoretical molecular weight of the recombinant protein CD3-BTLA BsAb _ M is 53.1kDa, and the protein presents a single electrophoretic band under reducing and non-reducing conditions, and the molecular weight is consistent with that of a monomer, so that the bispecific antibody is in a monomer form (FIG. 22A); the theoretical molecular weight of the CD3-BTLA BsAb _ D recombinant protein is 61.0kDa, the electrophoretic band of the protein under reducing conditions exhibits a molecular weight consistent with that of the monomer, and the electrophoretic band under non-reducing conditions exhibits a molecular weight consistent with that of the dimer (FIG. 22B), indicating that the two protein molecules can be linked to each other by disulfide bonds, and thus the bispecific antibody is in a dimer form.

In addition, the purified recombinant protein samples are subjected to N/C terminal sequence analysis, and the results show that the expressed recombinant protein samples have correct reading frames and are consistent with the theoretical N/C terminal amino acid sequence, and mass spectrometry further confirms that the CD3-BTLA BsAb _ M is in a monomer form, and the CD3-BTLA BsAb _ D is in a dimer form.

Therefore, it can be seen that the amino acid sequence of the monomeric form of CD3-BTLA BsAb _ M is shown in SEQ ID No.31, specifically:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTISSYDMHWVRQATGKGLEWVSVIGPAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAREGMAAHNYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIKR。

the amino acid sequence of the dimer form of CD3-BTLA BsAb _ D is shown in SEQ ID NO.33, and specifically comprises:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVEVQLVESGGGLVQPGGSLRLSCAASGFTISSYDMHWVRQATGKGLEWVSVIGPAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAREGMAAHNYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIKR。

the amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 35.

The amino acid sequence of the BTLA scFv is shown as SEQ ID NO.53, and specifically comprises the following steps:

EVQLVESGGGLVQPGGSLRLSCAASGFTISSYDMHWVRQATGKGLEWVSVIGPAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAREGMAAHNYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIKR。

the amino acid sequence of the heavy chain variable region of the anti-BTLA scFv is shown as SEQ ID NO.54, and specifically comprises the following steps:

EVQLVESGGGLVQPGGSLRLSCAASGFTISSYDMHWVRQATGKGLEWVSVIGPAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAREGMAAHNYYGMDVWGQGTTVTVSS。

the amino acid sequence of the variable region of the light chain of the BTLA scFv is shown as SEQ ID NO.55, and specifically comprises the following steps:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIKR。

the amino acid sequence of the connecting fragment in the monomeric form of CD3-BTLA BsAb _ M is shown in SEQ ID NO. 1.

The amino acid sequence of the connecting fragment in the dimeric form of CD3-BTLA BsAb _ D is shown in SEQ ID NO. 3.

Example 28: ELISA for detecting antigen binding activity of CD3-BTLA BsAb _ M and CD3-BTLA BsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD3-hFc and human BTLA-hFc fusion protein (purchased from Wujiang near-shore protein technologies, Ltd.) were coated on 96-well plates, respectively, with an antigen concentration of 1. mu.g/ml and a coating volume of 100. mu.l/well, under the conditions of 1 hour at 37 ℃ or overnight at 4 ℃, and the formulation of coating buffer (PBS) was: 3.58g Na₂HPO4，0.24g NaH₂PO4，0.2g KCl，8.2g NaCl，950ml H₂O, adjusting the pH value to 7.4 by using 1mol/L HCl or 1mol/L NaOH, and supplementing water to 1L;

3. sample adding: after 4 PBS washes, purified bispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation: taking 10 μ g/ml purified CD3-BTLA BsAb _ M or CD3-BTLA BsAb _ D as the initial concentration, performing multiple dilution for 6 gradients, each gradient being provided with 2 multiple wells;

The ELISA results are shown in fig. 23A and 23B: FIG. 23A illustrates that CD3-BTLA BsAb _ M has in vitro binding activity to both recombinant antigens CD3-hFc and BTLA-hFc, where BTLA binding activity is higher than that of CD 3; FIG. 23B illustrates that CD3-BTLA BsAb _ D has in vitro binding activity as well as recombinant antigens CD3-hFc and BTLA-hFc, with BTLA binding activity being higher.

Example 29: CD3-BTLA bispecific antibody mediated CIK cell proliferation

The bispecific antibody CD3-BTLA BsAb _ M in monomer form, the bispecific antibody CD3-BTLA BsAb _ D in dimer form and the Anti-CD3/Anti-CD28 monoclonal full-length antibody combination (Anti-CD3/Anti-CD28) prepared by the invention respectively act on human PBMC of the same donor source by taking PBMC of human Peripheral Blood Mononuclear Cells (PBMC), and counting and comparing the amplification times after cell culture.

CIK cell culture and expansion: the PBMC were resuspended in CIK basal medium (90% X-vivo15+ 10% FBS) and the cell density was adjusted to 1X 10⁶Ml, the following 3 experimental groups were designed: control groups (Anti-CD 35 ug/ml and Anti-CD285ug/ml coated cell culture plates, full length antibody purchased from Wujiang near-shore protein technology, Inc.); experimental group 1 (bispecific antibody CD3-BTLA BsAb _ M10ng/ml added in solution); experimental group 2 (bispecific antibody CD3-BTLA BsAb _ D10 ng/ml was added in solution). In addition, three groups of experimental cells were simultaneously added with cytokines IFN-. gamma. (200ng/ml, ex WUJIANG NEIGINIAN PROTEIN TECHNOLOGY Co., Ltd.) and IL-1. beta. (2ng/ml, ex WUJIANG NEIGINIAN PROTEIN TECHNOLOGY Co., Ltd.), placed in an incubator at saturated humidity, 37 deg.C, and 5.0% CO₂Culturing under the conditions of (1). After overnight incubation, the cultures were continued with 500U/ml IL-2 (purchased from Wujiang Korea protein technology Ltd.) and counted every 2-3 days and cultured in 1X 10 CIK basal medium supplemented with 500U/ml IL-2⁶Cell passaging was performed at a density of/ml. Culturing for 30 days by the method, finally counting the amplification multiple of the cells, and drawing a growth curve;

the detection result is shown in fig. 24, the single use of the CD3-BTLA bispecific antibody in monomer and dimer forms has better proliferation effect on CIK cells than the combined use of the Anti-CD3/Anti-CD28 monoclonal full-length antibody, after 18 days of culture, a great amount of cell death occurs in the combination of Anti-CD3/Anti-CD28, and the cell expansion fold is obviously reduced; the addition of CD3-BTLA BsAb _ M in monomeric form or CD3-BTLA BsAb _ D in dimeric form did not result in cell death, but the rate of cell expansion was relatively slow. Therefore, the two forms of the CD3-BTLA bispecific antibody prepared by the invention can effectively expand and prolong the survival period of CIK cells, and the dimer form has better effect.

Example 30: CD3-BTLA bispecific antibody induced IFN-gamma secretion from CIK cells

The method comprises the following operation steps:

1. the supernatant of CIK cells cultured for 25 days in example 29 (adjusted to the same cell density and cell number of 2X 10)⁵One) 100 μ l, incubated at 37 ℃ for 45min, and tested by the Human IFN- γ ELISA Kit (purchased from Boshide Biotech), three samples were taken for each of the three experiments and repeated;

The results are shown in FIG. 25: wherein the quantity of IFN-gamma secreted by CIK cells cultured by Anti-CD3/Anti-CD28 full-length antibody in a combined mode is defined as 1, the relative secretion quantity of IFN-gamma of CIK cells cultured by adding a monomer form of CD3-BTLA BsAb _ M in a solution state is 1.54, and the relative secretion quantity of IFN-gamma of CIK cells cultured by adding a dimer form of CD3-BTLA BsAb _ D in a solution state is 2.24, so that the two forms of CD3-BTLA bispecific antibodies prepared by the invention are both more favorable for activating the CIK cells and inducing the secretion of the IFN-gamma, and the effect of the dimer form is better.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> Shanghai Xin Bainuo Biotech Co., Ltd

<120> bifunctional molecule combining CD3 and T cell negative co-stimulatory molecule and application thereof

<130> 164635

<160> 102

<170> PatentIn version 3.3

<210> 1

<211> 15

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of connecting fragment in monomeric anti-CD 3/anti-T cell negative co-stimulatory molecule bispecific antibody

Column(s) of

<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> nucleotide sequence of connecting fragment in monomeric anti-CD 3/anti-T cell negative co-stimulatory molecule bispecific antibody

Column(s) of

<400> 2

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45

<210> 3

<211> 81

<212> PRT

<213> Artificial

<220>

<223> amino group of linker fragment in dimeric form of anti-CD 3/anti-T cell negative co-stimulatory molecule bispecific antibody

Sequences of

<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> nucleosides of linker fragments in anti-CD 3/anti-T cell negative co-stimulatory molecule bispecific antibodies in dimeric form

Sequences of

<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> amino acid sequence of human PD-1 extracellular domain of T cell negative co-stimulatory molecule

<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> amino acid sequence of human CTLA-4 extracellular domain of T cell negative co-stimulatory molecule

<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> amino acid sequence of human LAG-3 extracellular region of T cell negative co-stimulatory molecule

<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> amino acid sequence of human TIM-3 extracellular region of T cell negative co-stimulatory molecule

<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> amino acid sequence of human TIGIT extracellular region of T cell negative co-stimulatory molecule

<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> amino acid sequence of human BTLA extracellular domain of T cell negative co-stimulatory molecule

<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> amino acid sequence of CD3-PD-1 BsAb _ M in monomer 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> nucleotide sequence of CD3-PD-1 BsAb _ M in monomer 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> amino acid sequence of CD3-PD-1 BsAb _ D in dimer form

<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> nucleotide sequence of CD3-PD-1 BsAb _ D in dimeric form

<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> amino acid sequence of CD3-CTLA-4 BsAb _ M in monomer 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> nucleotide sequence of CD3-CTLA-4 BsAb _ M in monomer 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> amino acid sequence of CD3-CTLA-4 BsAb _ D in dimer form

<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> nucleotide sequence of CD3-CTLA-4 BsAb _ D in dimeric form

<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> amino acid sequence of CD3-LAG-3BsAb _ M in monomer 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> nucleotide sequence of CD3-LAG-3BsAb _ M in monomer 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> amino acid sequence of CD3-LAG-3BsAb _ D in dimeric form

<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> nucleotide sequence of CD3-LAG-3BsAb _ D in dimeric form

<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> amino acid sequence of CD3-TIM-3 BsAb _ M in monomer 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> nucleotide sequence of CD3-TIM-3 BsAb _ M in monomer 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> amino acid sequence of CD3-TIM-3 BsAb _ D in dimer form

<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> nucleotide sequence of CD3-TIM-3 BsAb _ D in dimeric form

<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> amino acid sequence of CD3-TIGIT BsAb _ M in monomer 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> nucleotide sequence of CD3-TIGIT BsAb _ M in monomer 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> amino acid sequence of CD3-TIGIT BsAb _ D in dimer form

<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> nucleotide sequence of CD3-TIGIT BsAb _ D in dimeric form

<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> amino acid sequence of CD3-BTLA BsAb _ M in monomer 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> nucleotide sequence of CD3-BTLA BsAb _ M in monomer 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> amino acid sequence of CD3-BTLA BsAb _ D in dimer form

<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> nucleotide sequence of CD3-BTLA BsAb _ D in dimeric form

<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> amino acid sequence of anti-CD3 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> amino acid sequence of heavy chain variable region of anti-CD3 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> amino acid sequence of light chain variable region of anti-CD3 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> 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> amino acid sequence of 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> amino acid sequence of 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> 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> amino acid sequence of 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> amino acid sequence of 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> 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> amino acid sequence of 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> amino acid sequence of 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> 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> amino acid sequence of 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> amino acid sequence of 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> 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> amino acid sequence of 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> amino acid sequence of 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> 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> amino acid sequence of 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> amino acid sequence of 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> anti-CD3 scfv nucleotide sequence

<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> nucleotide sequence of heavy chain variable region of anti-CD3 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> nucleotide sequence of light chain variable region of anti-CD3 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> 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> nucleotide sequence of 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> nucleotide sequence of 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> 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> nucleotide sequence of 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> nucleotide sequence of 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> 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> nucleotide sequence of 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> nucleotide sequence of 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> nucleotide 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> nucleotide sequence of 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> nucleotide sequence of 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> 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> nucleotide sequence of 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> nucleotide sequence of 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> 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> nucleotide sequence of 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> nucleotide sequence of 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> amino acid sequence of secretory expression 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> nucleotide sequence for secretory expression of 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 bifunctional molecule comprising in its structure a first domain capable of binding to and activating the T cell surface CD3 molecule and a second domain capable of binding to and blocking a T cell negative co-stimulatory molecule,

the first functional domain is a single chain antibody against CD3,

the second functional domain is a single-chain antibody of an anti-T cell negative co-stimulatory molecule, the single-chain antibody comprises a heavy chain variable region and a light chain variable region, and the single-chain antibody of the anti-T cell negative co-stimulatory molecule is selected from any one of a single-chain antibody of anti-PD-1, a single-chain antibody of anti-CTLA-4, a single-chain antibody of anti-LAG-3, a single-chain antibody of anti-TIM-3, a single-chain antibody of anti-TIGIT or a single-chain antibody of anti-BTLA;

the first functional domain and the second functional domain are connected through a connecting fragment, and the connecting fragment is a hinge region fragment of the immunoglobulin IgD shown in SEQ ID NO. 3.

2. The bifunctional molecule of claim 1, wherein the bifunctional molecule is capable of binding to and blocking a T cell negative co-stimulatory molecule while binding to and activating a T cell surface CD3 molecule, thereby generating a first signal and a second signal required for T cell activation.

3. The bifunctional molecule of claim 2, wherein the amino acid sequence of the heavy chain variable region of the anti-CD3 single chain antibody is represented by SEQ ID No. 36; the amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 37; the amino acid sequence of the heavy chain variable region of the single-chain antibody for resisting PD-1 is shown as SEQ ID NO. 39; the amino acid sequence of the light chain variable region of the anti-PD-1 single-chain antibody is shown in SEQ ID NO. 40; the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 single-chain antibody is shown in SEQ ID NO. 42; the amino acid sequence of the light chain variable region of the anti-CTLA-4 single-chain antibody is shown in SEQ ID NO. 43; the amino acid sequence of the heavy chain variable region of the anti-LAG-3 single-chain antibody is shown as SEQ ID NO. 45; the amino acid sequence of the variable region of the light chain of the anti-LAG-3 single-chain antibody is shown as SEQ ID NO. 46; the amino acid sequence of the heavy chain variable region of the single-chain antibody for resisting TIM-3 is shown as SEQ ID NO. 48; the amino acid sequence of the variable region of the light chain of the single-chain antibody for resisting TIM-3 is shown as SEQ ID NO. 49; the amino acid sequence of a heavy chain variable region of the anti-TIGIT single-chain antibody is shown as SEQ ID NO. 51; the amino acid sequence of a light chain variable region of the anti-TIGIT single-chain antibody is shown as SEQ ID NO. 52; the amino acid sequence of the heavy chain variable region of the BTLA-resistant single-chain antibody is shown in SEQ ID NO. 54; the amino acid sequence of the variable region of the light chain of the single-chain antibody against BTLA is shown in SEQ ID NO. 55.

4. The bifunctional molecule of claim 3, wherein the amino acid sequence of the anti-CD3 single chain antibody is represented by SEQ ID No. 35; the amino acid sequence of the single-chain antibody for resisting PD-1 is shown in SEQ ID NO. 38; the amino acid sequence of the single-chain antibody for resisting CTLA-4 is shown in SEQ ID NO. 41; the amino acid sequence of the single-chain antibody for resisting the LAG-3 is shown as SEQ ID NO. 44; the amino acid sequence of the single-chain antibody for resisting TIM-3 is shown as SEQ ID NO. 47; the amino acid sequence of the single-chain antibody for resisting TIGIT is shown as SEQ ID NO. 50; the amino acid sequence of the single-chain antibody for resisting BTLA is shown in SEQ ID NO. 53.

5. Bifunctional molecule as claimed in claim 1 having an amino acid sequence as shown in any of 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.

6. A polynucleotide encoding a bifunctional molecule according to any one of claims 1 to 5.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell transformed with the expression vector of claim 7.

9. A method for preparing a bifunctional molecule as claimed in any of claims 1 to 5 comprising: constructing an expression vector containing the gene sequence of the bifunctional molecule, then transforming the expression vector containing the gene sequence of the bifunctional molecule into host cells for inducing expression, and separating the expression product to obtain the bifunctional molecule.

10. Use of a bifunctional molecule according to any one of claims 1 to 5 for the preparation of a T cell ex vivo expansion agent.

11. A method for expanding T cells in vitro, comprising the step of allowing the bifunctional molecule of any one of claims 1 to 5 to act on T cells.