CN108264559B

CN108264559B - Tri-functional molecule combined with CD19, CD3 and T cell positive co-stimulatory molecule and application thereof

Info

Publication number: CN108264559B
Application number: CN201611258691.0A
Authority: CN
Inventors: 陈帅; 廖远平; 朱化星
Original assignee: Huihe Biotechnology Shanghai Co ltd
Current assignee: Huihe Biotechnology Shanghai Co ltd
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2021-08-10
Anticipated expiration: 2036-12-30
Also published as: CN108264559A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a tri-functional molecule combined with CD19, CD3 and a T cell positive co-stimulatory molecule and application thereof. The trifunctional molecule comprises a first domain capable of binding to CD19, a second domain capable of binding to and activating CD3, and a third domain capable of binding to and activating a T cell positive costimulatory molecule in its structure. The molecule has obvious advantages in the aspects of preparation process and practical application: the efficacy of activating T cells is further improved while the T cells are endowed with targeting to CD19 positive cells, the mediated killing effect of the T cells to CD19 positive target cells is better than that of an anti-CD 19/anti-CD 3 BiTE bispecific antibody when the T cells are added independently, and the application convenience is better than that of a CD19 targeted CAR-T technology.

Description

Tri-functional molecule combined with CD19, CD3 and T cell positive co-stimulatory molecule and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a tri-functional molecule combined with CD19, CD3 and a T cell positive co-stimulatory molecule and application thereof.

Background

The human CD19 antigen is a transmembrane glycoprotein of 95kDa in size, belonging to the immunoglobulin superfamily, and CD19 is highly expressed in B cell malignancies in addition to being expressed on the surface of normal B lymphocytes, so an anti-CD 19 monoclonal full-length antibody has been developed for use in the treatment of acute/chronic lymphocytic leukemia and B cell lymphoma (Wang K et al, Experimental Hematology & Oncology, 1:36-42, 2012). Given that anti-CD 19 monoclonal antibodies are unable to effectively recruit Cytotoxic T lymphocytes (CTLs, such CD3/CD8 double positive T cells specifically recognize antigen peptide/MHC class I molecule complexes on the surface of target cells, release perforin (Peforin) upon self-activation, cause lytic death of target cells, and also secrete DNA damage to target cell nuclei due to cytotoxins and granzymes (Granzyme), etc., causing apoptosis of target cells), bispecific antibodies (Bi-specific antibodies, BsAb) that can engage T cells and lymphoma B cells, as well as genetically engineered Chimeric antigen receptor T-cell immunotherapy (CAR-T) (Zhukovsky et al, Current Opinion in Immunology, 40: 24-35, 2016) were further designed and developed.

One current relatively mature type of bispecific antibody targeting CD19 is the Bi-specific T cell engager (BiTE) against CD 19/CD 3, whose structure is two Single-chain variable fragments (scFv) domains covalently linked in series by a linking peptide fragment (Linker) with flexibility (Goebeler ME et al, leukamia & Lymphoma, 57: 1021-. In the cellular immune process of an organism, specific recognition is carried out on a TCR/CD3 complex on the surface of a CD8 positive T cell and an endogenous Antigen peptide/MHC I molecule complex on the surface of an Antigen Presenting Cell (APC), so that CD3 interacts with a cytoplasmic segment of a co-receptor CD8, protein tyrosine kinase connected with a cytoplasmic segment tail is activated, tyrosine phosphorylation in an Immunoreceptor tyrosine kinase activation motif (ITAM) of a CD3 cytoplasmic region is enabled, a signal transduction molecular cascade reaction is started, and a transcription factor is activated, so that the T cell is initially activated. The anti-CD 19/anti-CD 3 BiTE bispecific antibody has the binding activity of two antigens of human CD3 and CD19, can form cell engagement between T cells and tumor B cells, and simultaneously gives a primary activation signal to the T cells, so that the killing targeting of the bispecific antibody to the tumor cells is improved. However, the BiTE bispecific antibody does not have an Fc fragment of a full-length antibody, has a small protein molecular weight (54 kDa), can cross the urinary and cerebral blood barriers during tumor therapy, has low bioavailability, needs to be administered by intravenous injection, and has certain neurotoxicity.

Furthermore, dual signaling pathways are required for T cell activation in humans (Baxter AG et al, Nature Reviews Immunology, 2: 439-446, 2002). First, the interaction of the antigenic peptide-MHC molecule complex on the surface of APC with the TCR/CD3 complex on the surface of T cell generates a first signal, which leads to the primary activation of T cell, and then the interaction of Co-stimulatory molecule ligands (e.g., CD80, CD86, 4-1BBL, B7RP-1, OX40L, GITRL, CD40, CD70, PD-L1, PD-L2, etc.) on the surface of APC with the corresponding Co-stimulatory molecules (Co-stimulatory molecules, e.g., CD28, 4-1BB, ICOS, OX40, GITR, CD40L, CD27, CTLA-4, PD-1, LAG-3, TIM-3, tig, and BTLA, etc.) on the surface of T cell generate a second signal (Co-stimulatory signal): wherein CD28, 4-1BB, ICOS, OX40, GITR, CD40L, CD27 and the like belong to the positive costimulatory molecules, which produce a second signal (positive costimulatory signal) that leads to complete activation of T cells; CTLA-4, PD-1, LAG-3, TIM-3, TIGIT and BTLA belong to negative costimulatory molecules, and their actions are mainly to down-regulate and terminate activation of T cells (negative costimulatory signal). It has been shown that the first signaling pathway alone does not sufficiently activate T cells, but rather leads to their incapacitation and even Activation-induced T cell death (AICD). To address this problem, bispecific antibodies against tumor antigen/anti-T cell positive (negative) costimulatory molecules can be used in combination with bispecific antibodies against tumor antigen/anti-CD 3 to increase the T cell activation and tumor cell killing efficiency (Jung G et al, Int J Cancer, 91: 225-. However, this method has many inconveniences in practical operation, such as increased workload for expression and purification of the recombinant bispecific antibody and production cost, and optimization of the relative ratio of the two bispecific antibodies during activation and expansion of T cells. In contrast, CAR-T technology better addresses the problem of T cell activation. The construction of a CAR typically includes: a tumor-associated antigen binding region (e.g., a CD19 antigen binding region, typically derived from a scFv fragment of a monoclonal full-length antibody against CD 19), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Wherein the intracellular signaling region is responsible for mediating the activation of T cells, on one hand, the first stimulation signal is completed through a tyrosine activation motif on a zeta chain of CD3, on the other hand, the expansion of the first stimulation signal is realized through a positive costimulatory signal of CD28, the proliferation and the activation of the T cells are promoted, and the secretion of cytokines is increased, the secretion of anti-apoptotic proteins is increased, the death of the cells is delayed, and the like. However, the CAR-T technology itself has some disadvantages: firstly, the technology relies on virus transfection to carry out gene modification on T cells, the steps are complex, and the requirements on experimental conditions are high; secondly, when in specific use, the CAR-T cells after in-vitro amplification and activation need to be infused back into a patient body, and the control of the dosage is more difficult than that of antibody drugs; in addition, a dramatic increase in the number of CAR-T cells after entry into a patient can lead to Cytokine storms (cytokines storms) that produce excessive amounts of cytokines within a short period of time, causing side effects such as high fever, low pressure, shock, and even death.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a trifunctional molecule capable of simultaneously binding CD19, CD3 and a T cell positive costimulatory 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 trifunctional molecule comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T-cell surface CD3 molecule, and a third domain capable of binding to and activating a T-cell positive costimulatory molecule.

Preferably, the trifunctional molecule is capable of binding to and activating the T cell surface CD3 molecule and the T cell positive costimulatory molecule while recognizing CD19, thereby generating the first and second signals required for T cell activation.

Preferably, the first domain is an antibody against CD19, the second domain is an antibody against CD3, and the third domain is an antibody against a T cell positive co-stimulatory molecule.

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 linker1, and the second domain and the third domain are linked by a linker 2.

Preferably, the connecting segment 1 and the connecting segment 2 are selected from the group consisting of connecting segments with the unit of G4S or hinge region segments of 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 by a linker1 in G4S, and the second domain and the third domain are linked by a linker2 in G4S. The connecting fragment 1 contains a G4S unit, and the amino acid sequence of the connecting fragment is shown as SEQ ID NO. 1. The connecting fragment 2 contains three G4S units, and the amino acid sequence of the connecting fragment is shown as SEQ ID NO. 3.

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 illustrated, wherein the first domain is linked to the second domain by a linker1 in G4S, and the second domain is linked to the third domain by a hinge region fragment of an immunoglobulin IgD, which is the hinge Ala90-Val170 of the immunoglobulin IgD. The connecting fragment 1 contains a G4S unit, and the amino acid sequence of the connecting fragment is shown as SEQ ID NO. 5. The amino acid sequence of the connecting segment 2 is shown as SEQ ID NO. 7. The connecting segments 2 may be linked to each other by disulfide bonds to form a dimer.

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

Preferably, the first domain is a single chain antibody against CD19, the second domain is a single chain antibody against CD3, and the third domain is a single chain antibody against a T cell positive co-stimulatory molecule, the 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-CD 19 single-chain antibody is shown in SEQ ID NO. 41. The amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 42. The amino acid sequence of the heavy chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 44. The amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 45.

Preferably, the single chain antibody against the T cell positive co-stimulatory molecule may be any one of a single chain antibody against 4-1BB, a single chain antibody against ICOS, a single chain antibody against OX40, a single chain antibody against GITR, a single chain antibody against CD40L, or a single chain antibody against CD 27.

Preferably, the amino acid sequence of the heavy chain variable region of the anti-4-1 BB single-chain antibody is shown in SEQ ID NO. 47. The amino acid sequence of the light chain variable region of the anti-4-1 BB single-chain antibody is shown in SEQ ID NO. 48.

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

The amino acid sequence of the heavy chain variable region of the anti-OX 40 single-chain antibody is shown in SEQ ID No. 53. The amino acid sequence of the variable region of the light chain of the anti-OX 40 single-chain antibody is shown in SEQ ID No. 54.

The amino acid sequence of the heavy chain variable region of the anti-GITR single-chain antibody is shown as SEQ ID NO. 56. The amino acid sequence of the light chain variable region of the anti-GITR single-chain antibody is shown as SEQ ID NO. 57.

The amino acid sequence of the heavy chain variable region of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 59. The amino acid sequence of the light chain variable region of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 60.

The amino acid sequence of the heavy chain variable region of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 62. The amino acid sequence of the light chain variable region of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 63.

In some embodiments of the invention, the amino acid sequence of the anti-CD 19 single chain antibody is shown in SEQ ID NO. 40. The amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 43. The amino acid sequence of the anti-4-1 BB single-chain antibody is shown in SEQ ID NO. 46. The amino acid sequence of the single-chain antibody for resisting ICOS is shown in SEQ ID NO. 49. The amino acid sequence of the anti-OX 40 single-chain antibody is shown in SEQ ID NO. 52. The amino acid sequence of the anti-GITR single-chain antibody is shown as SEQ ID NO. 55. The amino acid sequence of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 58. The amino acid sequence of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 61.

In a preferred embodiment, the amino acid sequence of the trifunctional molecule in monomeric form is as shown in any one of SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28, SEQ ID NO.32 or SEQ ID NO. 36.

In a preferred embodiment, the amino acid sequence of the trifunctional molecule in the form of a dimer is as shown in any one of SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.26, SEQ ID NO.30, SEQ ID NO.34 or SEQ ID NO. 38.

In a second aspect of the invention, there is provided a polynucleotide encoding the aforementioned trifunctional 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 aforementioned trifunctional molecule, comprising: constructing an expression vector containing a gene sequence of the trifunctional molecules, then transforming the expression vector containing the gene sequence of the trifunctional molecules into host cells for induction expression, and separating the expression product to obtain the trifunctional molecules.

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, the use of the aforementioned trifunctional molecules for preparing a medicament for treating tumors is provided.

In the seventh aspect of the present invention, a pharmaceutical composition for treating tumor is provided, which contains the above three functional molecules and at least one pharmaceutically acceptable carrier or excipient. The tumor is a tumor with a cell surface positive for CD 19.

In the eighth aspect of the invention, a method for treating tumors in vitro is disclosed, which comprises the step of administering the three-functional molecule or the tumor treatment pharmaceutical composition to a tumor patient. The method may be for non-therapeutic purposes. The tumor is a tumor with a cell surface positive for CD 19.

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

(1) the three-functional molecule fuses a first functional domain capable of being combined with CD19, a second functional domain capable of being combined with and activating a CD3 molecule on the surface of a T cell and a third functional domain capable of being combined with and activating a positive co-stimulatory molecule of the T cell into the same protein peptide chain, is produced by adopting a eukaryotic cell expression system, has a single structure of an expression product, is simple and convenient in purification process, has high protein yield, and is stable in preparation process and product and convenient to use; and if the anti-CD 19/anti-CD 3 bispecific antibody and the anti-CD 19/anti-T cell positive co-stimulatory molecule bispecific antibody are used in combination, the two bispecific antibodies need to be expressed and purified respectively, the preparation process is more complicated, the workload and the production cost are obviously increased, and the relative proportion of the two antibodies needs to be optimized when the antibodies are used.

(2) The tri-functional molecule can generate a second (positive) stimulation signal for activating the T cell, further improves the activation effect on the T cell while endowing the T cell with targeting property, increases the secretion of cytokines and anti-apoptosis protein, effectively avoids the phenomenon of incapability and death of the T cell, and can achieve the effect even better than that of an anti-CD 19/anti-CD 3 BiTE bispecific antibody on the mediated killing of the T cell on a CD19 positive target cell, and the using amount of the protein is less.

(3) Compared with CAR-T technology of targeting CD19, the three-functional molecule of the invention does not relate to operation steps such as virus-mediated transgene, in-vitro T cell culture and reinfusion, is more convenient to use, has controllable dosage, has small risk of causing excessive release of cytokines after entering a patient organism, and avoids toxic and side effects when CAR-T is used.

Drawings

FIG. 1: A. a structural diagram of a monomeric anti-CD 19/anti-CD 3/anti-T cell positive co-stimulatory molecule trispecific antibody; B. a structural diagram of a dimeric form of anti-CD 19/anti-CD 3/anti-T cell positive costimulatory molecule trispecific antibody.

FIG. 2: A. purified CD19-CD3-4-1BB TsAb _ M SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-4-1BB TsAb _ M; lane 3: non-reducing CD19-CD3-4-1BB TsAb _ M; B. purified CD19-CD3-4-1BB TsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-4-1BB TsAb _ D; lane 3: non-reducing CD19-CD3-4-1BB TsAb _ D.

FIG. 3A: the ELISA identification results of CD19-CD3-4-1BB TsAb _ M are shown in the graph, and the curves represent 4 detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

coating 1 mu g/ml of recombinant antigen CD 3-hFc; coating 1 microgram/ml recombinant antigen 4-1 BB-hFc;

assay results without any antigen coating.

FIG. 3B: the ELISA identification results of CD19-CD3-4-1BB TsAb _ D are shown in the graph as representing the results of 4 detections: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

assay results without any antigen coating.

FIG. 4: CD19-CD3-4-1BB trispecific antibody mediated cell killing experiment, Raji lymphoma cells are used as CD19 positive target cells, CIK (cytokine induced killers) cells are used as CD3 positive killing effector cells, and the killing efficiency of the CIK cells on the Raji cells mediated by CD19-CD3-4-1BB TsAb _ M, CD19-CD3-4-1BB TsAb _ D and CD19-CD3BsAb at different concentrations is respectively detected; effector cells: target cells (E: T ratio) 1: 1, killing time: and 3 h.

FIG. 5: A. SDS-PAGE analysis of purified CD19-CD3-ICOS TsAb _ M, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-ICOS TsAb _ M; lane 3: non-reducing CD19-CD3-ICOS TsAb _ M; B. purified CD19-CD3-ICOS TsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-ICOS TsAb _ D; lane 3: non-reducing CD19-CD3-ICOS TsAb _ D.

FIG. 6A: the ELISA identification results of CD19-CD3-ICOS TsAb _ M are shown in the graph as representing the results of 4 detections: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

coating 1 mu g/ml of recombinant antigen CD 3-hFc; coating the tangle-solidup with 1 mu g/ml recombinant antigen ICOS-hFc;

assay results without any antigen coating.

FIG. 6B: the ELISA identification results of CD19-CD3-ICOS TsAb _ D are shown in the graph as representing the results of 4 detections: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

assay results without any antigen coating.

FIG. 7: CD19-CD3-ICOS trispecific antibody mediated cell killing experiment, Raji lymphoma cells are used as CD19 positive target cells, CIK cells are used as CD3 positive killing effector cells, and the killing efficiency of the CIK cells mediated by CD19-CD3-ICOS TsAb _ M, CD19-CD3-ICOS TsAb _ D and CD19-CD3BsAb on the Raji cells at different concentrations is respectively detected; effector cells: target cells (E: T ratio) 1: 1, killing time: and 3 h.

FIG. 8: A. purified CD19-CD3-OX40TsAb _ M SDS-PAGE analysis map, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-OX40TsAb _ M; lane 3: non-reducing CD19-CD3-OX40TsAb _ M; B. purified CD19-CD3-OX40TsAb _ D SDS-PAGE analysis map, lane 1: a molecular weight protein Marker; lane 2: non-reducing CD19-CD3-OX40TsAb _ D; lane 3: reducing CD19-CD3-OX40TsAb _ D.

FIG. 9A: the ELISA identification results of CD19-CD3-OX40TsAb _ M are shown in the graph, and the curves in the graph represent 4 detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

coating 1 mu g/ml of recombinant antigen CD 3-hFc; coating 1 mug/ml recombinant antigen OX40-hFc with tangle solidup;

assay results without any antigen coating.

FIG. 9B: the ELISA identification results of CD19-CD3-OX40TsAb _ D are shown in the graph, and the curves in the graph represent 4 detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

assay results without any antigen coating.

FIG. 10: CD19-CD3-OX40 trispecific antibody mediated cell killing experiments. Raji lymphoma cells are used as CD19 positive target cells, CIK cells are used as CD3 positive killing effector cells, and the killing efficiency of the CIK cells on the Raji cells mediated by CD19-CD3-OX40TsAb _ M, CD19-CD3-OX40TsAb _ D and CD19-CD3BsAb at different concentrations is detected; effector cells: target cells (E: T ratio) 1: 1, killing time: and 3 h.

FIG. 11: A. purified CD19-CD3-GITR TsAb _ M SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-GITR TsAb _ M; lane 3: non-reducing CD19-CD3-GITR TsAb _ M; B. purified CD19-CD3-GITR TsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-GITR TsAb _ D; lane 3: non-reducing CD19-CD3-GITR TsAb _ D.

FIG. 12A: ELISA identification result of CD19-CD3-GITR TsAb _ M, figureThe curves in (a) represent the results of 4 assays, respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

coating 1 mu g/ml of recombinant antigen CD 3-hFc; coating the tangle-solidup with 1 mu g/ml recombinant antigen GITR-hFc;

assay results without any antigen coating.

FIG. 12B: the ELISA identification results of CD19-CD3-GITR TsAb _ D are shown in the graph as representing the results of 4 detections: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

assay results without any antigen coating.

FIG. 13: in the CD19-CD3-GITR trispecific antibody mediated cell killing experiment, Raji lymphoma cells are used as CD19 positive target cells, CIK cells are used as CD3 positive killing effector cells, and the killing efficiency of the CIK cells on the Raji cells mediated by CD19-CD3-GITR TsAb _ M, CD19-CD3-GITR TsAb _ D and CD19-CD3BsAb at different concentrations is respectively detected; effector cells: target cells (E: T ratio) 1: 1, killing time: and 3 h.

FIG. 14: A. purified CD19-CD3-CD40L TsAb _ M SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-CD40L TsAb _ M; lane 3: non-reducing CD19-CD3-CD40L TsAb _ M; B. purified CD19-CD3-CD40L TsAb _ D SDS-PAGE analysis, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-CD40L TsAb _ D; lane 3: non-reducing CD19-CD3-CD40L TsAb _ D.

FIG. 15A: the ELISA identification results of CD19-CD3-CD40L TsAb _ M are shown in the graph as follows, wherein the curves represent 4 detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

coating 1 mu g/ml of recombinant antigen CD 3-hFc; coating 1 mug/ml recombinant antigen CD 40L-hFc;

assay results without any antigen coating.

FIG. 15B: the ELISA identification results of CD19-CD3-CD40L TsAb _ D are shown in the graph, and the curves in the graph represent 4 detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

assay results without any antigen coating.

FIG. 16: CD19-CD3-CD40L trispecific antibody mediated cell killing experiment, Raji lymphoma cells are used as CD19 positive target cells, CIK cells are used as CD3 positive killing effector cells, and the killing efficiency of CIK cells on Raji cells mediated by CD19-CD3-CD40L TsAb _ M, CD19-CD3-CD40L TsAb _ D and CD19-CD3BsAb at different concentrations is respectively detected; effector cells: target cells (E: T ratio) 1: 1, killing time: and 3 h.

FIG. 17: A. purified CD19-CD3-CD27TsAb _ M SDS-PAGE analysis map, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-CD27TsAb _ M; lane 3: non-reducing CD19-CD3-CD27TsAb _ M; B. purified CD19-CD3-CD27TsAb _ D SDS-PAGE analysis map, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-CD27TsAb _ D; lane 3: non-reducing CD19-CD3-CD27TsAb _ D.

FIG. 18A: the ELISA identification results of CD19-CD3-CD27TsAb _ M are shown in the graph, and the curves in the graph represent 4 detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

coating 1 mu g/ml of recombinant antigen CD 3-hFc; coating the tangle-solidup with 1 microgram/ml recombinant antigen CD 27-hFc;

assay results without any antigen coating.

FIG. 18B: the ELISA identification results of CD19-CD3-CD27TsAb _ D are shown in the graph, and the curves in the graph represent 4 detection results respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc;

assay results without any antigen coating.

FIG. 19: CD19-CD3-CD27 trispecific antibody mediated cell killing experiment, Raji lymphoma cells are used as CD19 positive target cells, CIK cells are used as CD3 positive killing effector cells, and the killing efficiency of CIK cells mediated by different concentrations of CD19-CD3-CD27TsAb _ M, CD19-CD3-CD27TsAb _ D and CD19-CD3BsAb to the Raji cells is detected respectively; effector cells: target cells (E: T ratio) 1: 1, killing time: and 3 h.

Detailed Description

First, terms and abbreviations:

CTL: cytotoxic T lymphocytes (cytoxic T lymphocytes)

BsAb: bispecific Antibody (Bi-specific Antibody)

TsAb: trispecific Antibody (Tri-specific Antibody)

BiTE: bi-specific T cell engager (Bi-specific T cell engage)

And (3) TiTE: trispecific T cell adaptor (Tri-specific T cell engage)

Fab: antigen binding Fragment (Fragment of antigen binding)

Fv: variable region fragments (Variable fragment)

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

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

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

Linker 1: connecting fragment 1

Linker 2: connecting fragment 2

Excellar domain: extracellular region

CD19-CD3-4-1BB TsAb _ M: monomeric anti-CD 19/anti-CD 3/anti-4-1 BB trispecific antibody

CD19-CD3-4-1BB TsAb _ D: dimeric form of anti-CD 19/anti-CD 3/anti-4-1 BB trispecific antibody

CD19-CD3-ICOS TsAb _ M: monomeric anti-CD 19/anti-CD 3/anti-ICOS trispecific antibody

CD19-CD3-ICOS TsAb _ D: dimeric form of anti-CD 19/anti-CD 3/anti-ICOS trispecific antibody

CD19-CD3-OX40TsAb _ M: monomeric anti-CD 19/anti-CD 3/anti-OX 40 trispecific antibody

CD19-CD3-OX40TsAb _ D: dimeric form of anti-CD 19/anti-CD 3/anti-OX 40 trispecific antibody

CD19-CD3-GITR TsAb _ M: monomeric anti-CD 19/anti-CD 3/anti-GITR trispecific antibody

CD19-CD3-GITR TsAb _ D: dimeric form of anti-CD 19/anti-CD 3/anti-GITR trispecific antibody

CD19-CD3-CD40L TsAb _ M: monomeric anti-CD 19/anti-CD 3/anti-CD 40L trispecific antibody

CD19-CD3-CD40L TsAb _ D: dimeric form of anti-CD 19/anti-CD 3/anti-CD 40L trispecific antibody

CD19-CD3-CD27TsAb _ M: monomeric anti-CD 19/anti-CD 3/anti-CD 27 trispecific antibody

CD19-CD3-CD27TsAb _ D: dimeric form of anti-CD 19/anti-CD 3/anti-CD 27 trispecific antibody

Di-and tri-functional molecules

The trifunctional molecules of the invention comprise a first domain capable of binding to CD19, a second domain capable of binding to and activating a T cell surface CD3 molecule, and a third domain capable of binding to and activating a T cell positive costimulatory molecule.

Further, the trifunctional molecules are capable of binding and activating the T cell surface CD3 molecule and the T cell positive costimulatory molecule while recognizing CD19, thereby generating a first signal and a second signal required for T cell activation.

The T cell positive co-stimulatory molecule includes, but is not limited to, human CD28, 4-1BB, ICOS, OX40, GITR, CD40L, or CD 27.

The first domain, the second domain and the third domain of the present invention are not particularly limited as long as they can bind to and activate the T cell surface CD3 molecule and the T cell costimulatory molecule while recognizing CD19, thereby generating the first signal and the second signal required for T cell activation. For example, the first domain may be an antibody against CD19, the second domain may be an antibody against CD3, and the third domain may be an antibody against a T cell positive co-stimulatory molecule. 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 antibodies consist of an intact 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 functional domain and the second functional domain are connected through a linker1, and the second functional domain and the third functional domain are connected through a linker 2. 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 C-terminus of the second domain is linked to the N-terminus of the third domain. The present invention is not particularly limited to the linker1 and the linker2, either, as long as the object of the present invention is not limited.

Further, the connecting segment 1 and the connecting segment 2 are selected from the group consisting of a connecting segment with a G4S unit or a hinge region segment of immunoglobulin IgD.

In a preferred embodiment of the present invention, the structure of the trifunctional molecule is schematically shown in FIG. 1. The trifunctional molecules may be in monomeric or dimeric form. The structure of the trifunctional molecule of the invention in a monomeric form is schematically shown in fig. 1A, and the trifunctional molecule has a structure comprising a first domain capable of binding to CD19 antigen, a second domain capable of binding to CD3 antigen, and a third domain capable of binding to T cell costimulatory molecule antigen, wherein the first domain is a single-chain antibody (scFv) capable of binding to CD19 antigen, the second domain is a single-chain antibody (scFv) capable of binding to CD3 antigen, and the third domain is a single-chain antibody (scFv) capable of binding to T cell costimulatory molecule antigen. The structure of the trifunctional molecule in a dimeric form according to the present invention is schematically shown in fig. 1B, and the trifunctional molecule has a structure comprising two first domains binding to CD19 antigen, two second domains binding to CD3 antigen, and two third domains binding to T cell costimulatory molecule antigen, wherein the first domains are single-chain antibodies (scFv) binding to CD19 antigen, the second domains are single-chain antibodies (scFv) binding to CD3 antigen, and the third domains are single-chain antibodies (scFv) binding to T cell costimulatory molecule antigen. The antigen binding potency of the dimeric form of the trifunctional molecules of the invention is twice that of the monomeric form. The first signal (CD3) and the second signal (T cell positive co-stimulatory molecule) of T cell activation are doubled, so that the T cell activation is more complete, and the killing effect on target cells is stronger; the doubling of the CD19 single-chain antibody domain makes the recognition of target cells more accurate, so that the dimer has better use effect than the monomer.

The T cell positive co-stimulatory molecule may be CD28, 4-1BB, ICOS, OX40, GITR, CD40L, CD27 or the like.

The amino acid sequence of the extracellular region of the T cell positive co-stimulatory molecule human CD28 is shown as SEQ ID NO.9, and specifically comprises the following steps:

NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP。

the amino acid sequence of the T cell positive co-stimulatory molecule human 4-1BB extracellular region is shown in SEQ ID NO.10, and specifically comprises the following steps:

LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ。

the amino acid sequence of the T cell positive co-stimulatory molecule human ICOS extracellular region is shown as SEQ ID NO.11, and specifically comprises the following steps:

EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLK。

the amino acid sequence of the T cell positive co-stimulatory molecule human OX40 extracellular region is shown as SEQ ID NO.12, and specifically comprises the following steps:

LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRA。

the amino acid sequence of the T cell positive co-stimulatory molecule human GITR extracellular region is shown as SEQ ID NO.13, and specifically comprises the following steps:

QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEP。

the amino acid sequence of the extracellular region of the T cell costimulatory molecule human CD40L is shown in SEQ ID NO.14, and specifically comprises the following steps:

HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL。

the amino acid sequence of the extracellular region of the T cell positive co-stimulatory molecule human CD27 is shown in SEQ ID NO.15, and specifically comprises the following steps:

ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR。

in particular, the first domain is a single chain antibody against CD 19. The anti-CD 19 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-CD 19 single-chain antibody is shown in SEQ ID NO. 41. The amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 42. Further, the amino acid sequence of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 40.

The second functional 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. 44. The amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 45. Further, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 43.

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

The single chain antibody against the T cell positive co-stimulatory molecule may be any one of a single chain antibody against 4-1BB, a single chain antibody against ICOS, a single chain antibody against OX40, a single chain antibody against GITR, a single chain antibody against CD40L, or a single chain antibody against CD 27.

The amino acid sequence of the heavy chain variable region of the anti-4-1 BB single-chain antibody is shown in SEQ ID NO. 47. The amino acid sequence of the light chain variable region of the anti-4-1 BB single-chain antibody is shown in SEQ ID NO. 48. The amino acid sequence of the anti-4-1 BB single-chain antibody is shown in SEQ ID NO. 46.

The amino acid sequence of the heavy chain variable region of the single-chain antibody for resisting ICOS is shown as SEQ ID NO. 50. The amino acid sequence of the light chain variable region of the anti-ICOS single-chain antibody is shown in SEQ ID NO. 51. The amino acid sequence of the single-chain antibody for resisting ICOS is shown in SEQ ID NO. 49.

The amino acid sequence of the heavy chain variable region of the anti-OX 40 single-chain antibody is shown in SEQ ID No. 53. The amino acid sequence of the variable region of the light chain of the anti-OX 40 single-chain antibody is shown in SEQ ID No. 54. The amino acid sequence of the anti-OX 40 single-chain antibody is shown in SEQ ID NO. 52.

The amino acid sequence of the heavy chain variable region of the anti-GITR single-chain antibody is shown as SEQ ID NO. 56. The amino acid sequence of the light chain variable region of the anti-GITR single-chain antibody is shown as SEQ ID NO. 57. The amino acid sequence of the anti-GITR single-chain antibody is shown as SEQ ID NO. 55.

The amino acid sequence of the heavy chain variable region of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 59. The amino acid sequence of the light chain variable region of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 60. The amino acid sequence of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 58.

The amino acid sequence of the heavy chain variable region of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 62. The amino acid sequence of the light chain variable region of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 63. The amino acid sequence of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 61.

In a preferred embodiment, the amino acid sequence of the trifunctional molecule in monomeric form is as shown in any one of SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28, SEQ ID NO.32 or SEQ ID NO. 36. The amino acid sequence of the three-functional molecule in a dimer form is shown in any one of SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.26, SEQ ID NO.30, SEQ ID NO.34 or SEQ ID NO. 38. But are not limited to, the specific forms set forth in the preferred embodiment of the invention.

Polynucleotides encoding trifunctional molecules

The polynucleotide of the present invention encoding the trifunctional 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 trifunctional molecules of the invention may be prepared by any suitable technique 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-CD 19 is shown in SEQ ID NO. 65. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-CD 19 is shown in SEQ ID NO. 66. The nucleotide sequence of the single-chain antibody for encoding the anti-CD 19 is shown in SEQ ID NO. 64.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-CD3 is shown in SEQ ID NO. 68. 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. 69. The nucleotide sequence of the single-chain antibody for encoding the anti-CD3 is shown as SEQ ID NO. 67.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-4-1 BB is shown as SEQ ID NO. 71. The nucleotide sequence of the light chain variable region of the single-chain antibody for encoding the anti-4-1 BB is shown as SEQ ID NO. 72. The nucleotide sequence of the single-chain antibody for coding the anti-4-1 BB is shown as SEQ ID NO. 70.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-ICOS is shown as SEQ ID NO. 74. The nucleotide sequence of the light chain variable region of the single-chain antibody for encoding the anti-ICOS is shown as SEQ ID NO. 75. The nucleotide sequence of the single-chain antibody for coding the ICOS is shown as SEQ ID NO. 73.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-OX 40 is shown as SEQ ID NO. 77. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-OX 40 is shown as SEQ ID NO. 78. The nucleotide sequence for coding the anti-OX 40 single-chain antibody is shown as SEQ ID NO. 76.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-GITR is shown as SEQ ID NO. 80. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-GITR is shown as SEQ ID NO. 81. The nucleotide sequence for coding the anti-GITR single-chain antibody is shown as SEQ ID NO. 79.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-CD 40L is shown as SEQ ID NO. 83. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-CD 40L is shown as SEQ ID NO. 84. The nucleotide sequence of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 82.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-CD 27 is shown in SEQ ID NO. 86. The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-CD 27 is shown as SEQ ID NO. 87. The nucleotide sequence for coding the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 85.

The nucleotide sequence of the connecting segment 1 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 2 with the coding amino acid sequence shown as SEQ ID NO.3 is shown as SEQ ID NO. 4.

The nucleotide sequence of the connecting segment 1 with the coding amino acid sequence shown as SEQ ID NO.5 is shown as SEQ ID NO. 6.

The nucleotide sequence of the connecting segment 2 with the coding amino acid sequence shown as SEQ ID NO.7 is shown as SEQ ID NO. 8.

Further, the nucleotide sequence encoding the trifunctional molecule in monomeric form is shown in any one of SEQ ID NO.17, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.29, SEQ ID NO.33 or SEQ ID NO. 37. The nucleotide sequence of the three-functional molecule in a form of encoding dimer is shown in any one of SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27, SEQ ID NO.31, SEQ ID NO.35 or SEQ ID NO. 39.

Fourth, expression vector

The expression vectors of the invention contain polynucleotides encoding the trifunctional molecules. 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 tri-functional molecule

The method for preparing the three-functional molecule comprises the following steps: constructing an expression vector containing a gene sequence of the trifunctional molecules, then transforming the expression vector containing the gene sequence of the trifunctional molecules into host cells for induction expression, and separating the expression product to obtain the trifunctional molecules. 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-and trifunctional molecules

The tri-functional molecule of the invention can be used for tumor treatment drugs. The tumor is a tumor with a cell surface positive for CD 19.

In the preferred embodiment of the invention, experiments show that the tri-functional molecules of the invention have the in vitro binding activity with the recombinant antigens of CD19, CD3 and corresponding T cell positive co-stimulatory molecules, can promote the targeted killing of the T cells on CD19 positive target cells, and the dimer has better effect than the monomer.

Seven, tumor treating medicine composition

The tumor treatment medicine composition contains the three functional molecules and at least one pharmaceutically acceptable carrier or excipient. The tumor is a tumor with a cell surface positive for CD 19.

The pharmaceutical composition provided by the invention can exist in various dosage forms, such as injections for intravenous injection and the like, percutaneous absorbents for subcutaneous injection, external application of epidermis and the like, sprays for nose, throat, oral cavity, epidermis, mucous membrane and the like, drops for nose, eye, ear and the like, suppositories, tablets, powders, granules, capsules, oral liquid, ointment, cream and the like for anorectal and the like, pulmonary administration preparations and other compositions for parenteral administration. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field. Flavoring agent, sweetener, etc. can also be added into the medicinal composition.

The pharmaceutical preparation can be clinically used for mammals including human and animals, and can be administered by intravenous injection or oral, nasal, skin, lung inhalation and the like. The preferable weekly dosage of the above drugs is 0.1-5mg/kg body weight, and the preferable course of treatment is 10-30 days. The administration is carried out once or in several times. Regardless of the method of administration, the optimal dosage for an individual human will depend on the particular treatment.

Method for treating tumor in vitro

The method for treating tumors in vitro comprises the step of administering the trifunctional molecules or the tumor treatment pharmaceutical composition to a tumor patient. The tumor is a tumor with a cell surface positive for CD 19. The method may be for non-therapeutic purposes. In the preferred embodiment of the invention, experiments show that the tri-functional molecules of the invention have the in vitro binding activity with the recombinant antigens of CD19, CD3 and corresponding T cell positive co-stimulatory molecules, can promote the targeted killing of the T cells on CD19 positive target cells, and the dimer has better effect than the monomer.

Aiming at the defects of an anti-CD 19/anti-CD 3 BiTE bispecific antibody and a CAR-T technology of targeting CD19, the invention constructs a tri-functional molecule capable of simultaneously recognizing CD19, CD3 and any T cell costimulatory molecule by genetic engineering and antibody engineering methods. The molecule has obvious advantages in the aspects of preparation process and practical application: the efficacy of activating T cells is further improved while the T cells are endowed with targeting to CD19 positive cells, the mediated killing effect of the T cells to CD19 positive target cells is better than that of an anti-CD 19/anti-CD 3 BiTE bispecific antibody when the T cells are added independently, and the application convenience is better than that of a CD19 targeted CAR-T technology.

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 eukaryotic expression vectors for CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ D

In the present invention, the TITE trispecific antibody targeting the human CD19 protein on the surface of lymphoma B cells, the human CD3 on the surface of T cells and the 4-1BB protein, which is a T cell costimulatory molecule, was named CD19-CD3-4-1BB TsAb.

First, CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD19-CD3-4-1BB TsAb _ M is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-4-1 BB scFv are linked by a Linker (Linker), specifically, the sequences of the anti-CD 19 scFv and the anti-CD3 scFv are linked by a Linker 1(Linker 1), and the sequences of the anti-CD3 scFv and the anti-4-1 BB scFv are linked by a Linker 2(Linker 2).

The specific construction scheme of the dimer form of CD19-CD3-4-1BB TsAb _ D is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-4-1 BB scFv are connected by a Linker (Linker), specifically, the sequences of the anti-CD 19 scFv and the anti-CD3 scFv are connected by a Linker 1(Linker 1), and the sequences of the anti-CD3 scFv and the anti-4-1 BB scFv are connected by an IgD hinge region (Ala 90-Val 170) as a Linker 2(Linker 2).

For expression of the trispecific antibody in mammalian cells, the sequence of anti-CD 19 scFv, anti-CD3 scFv, and anti-4-1 BB scFv were codon optimized for mammalian system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of the anti-CD 19 scFv is shown in SEQ ID No.65, specifically:

CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

the nucleotide sequence of the variable region of the light chain of the anti-CD 19 scFv is shown in SEQ ID NO.66, and specifically comprises the following steps:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG。

the nucleotide sequence of the anti-CD 19 scFv is shown as SEQ ID NO.64, and specifically comprises the following steps:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

the nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID NO.68, and specifically comprises the following steps:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

the nucleotide sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID NO.69, and specifically comprises the following steps:

GACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

the nucleotide sequence of the anti-CD3 scFv is shown as SEQ ID NO.67, and specifically comprises the following steps:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

the nucleotide sequence of the heavy chain variable region of the anti-4-1 BB scFv is shown in SEQ ID NO.71, and specifically comprises the following steps:

CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGC。

the nucleotide sequence of the light chain variable region of the anti-4-1 BB scFv is shown in SEQ ID NO.72, and specifically comprises the following steps:

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the anti-4-1 BB scFv is shown as SEQ ID NO.70, and specifically comprises the following steps:

CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric CD19-CD3-4-1BB TsAb _ M connecting segment 1(Linker 1) is shown as SEQ ID NO.2, and specifically comprises the following steps: GGTGGCGGAGGGTCC are provided.

The nucleotide sequence of the monomeric CD19-CD3-4-1BB TsAb _ M connecting fragment 2(Linker 2) is shown as SEQ ID NO.4, and specifically comprises the following steps:

GGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGT。

the nucleotide sequence of the dimer-form CD19-CD3-4-1BB TsAb _ D connecting fragment 1(Linker 1) is shown as SEQ ID NO.6, and specifically comprises the following steps: GGTGGCGGAGGGTCC are provided.

The nucleotide sequence of the dimer form of CD19-CD3-4-1BB TsAb _ D junction fragment 2(Linker 2) is shown as SEQ ID NO.8, and specifically comprises the following steps:

GCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTG。

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

The amino acid sequence of the secretory expression signal peptide is shown as SEQ ID NO.88, and specifically comprises the following steps:

MTRLTVLALLAGLLASSRA。

the nucleotide sequence of the secretory expression signal peptide is shown as SEQ ID NO.89, and specifically comprises the following components:

ATGACCCGGCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCTCCTCCAGGGCC。

secondly, construction of eukaryotic expression vectors of CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ D

The construction and expression of the tri-specific antibody of the invention select a transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.) of mammalian cell protein. To construct monospecific and dimeric forms of trispecific antibodies, primers as shown in table 1 were designed, all of which were synthesized by sumizia jingzhi biotechnology limited and gene templates for amplification were synthesized by sumizia hong kong technology limited, respectively.

Cloning construction for CD19-CD3-4-1BB TsAb _ M, signal peptide fragments were first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3- (GGGGS) were used respectively₃Amplifying anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) from-4-1 BB-F and pcDNA3.1-4-1BB-R₃The gene sequence of Linker 2+ anti-4-1 BB scFv; cloning of CD19-CD3-4-1BB TsAb _ D was carried out by first amplifying signal peptide fragments using primers pcDNA3.1-Sig-F and Sig-R, and then amplifying gene sequences of anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, IgD hinge region Linker2 and anti-4-1 BB scFv using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-4-1BB-F and pcDNA3.1-4-1BB-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 monomer and dimer three-specificity antibodies, seamlessly clones the full-length gene sequences to a pcDNA3.1 expression vector which is subjected to EcoRI and HindIII linearization treatment, transforms Escherichia coli DH5 alpha, performs positive cloning identification by colony PCR, and performs sequencing identification on recombinants (recombinant plasmids) which are identified to be 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 the monomeric form of CD19-CD3-4-1BB TsAb _ M and the dimeric form of CD19-CD3-4-1BB TsAb _ D were correct and consistent with the expectations.

Specifically, the nucleotide sequence of the monomeric CD19-CD3-4-1BB TsAb _ M is shown as SEQ ID NO.17, and specifically comprises:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the dimeric form of CD19-CD3-4-1BB TsAb _ D is shown in SEQ ID NO.19, and specifically comprises the following components:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。

TABLE 1 primers used in the cloning of CD19-CD3-4-1BB trispecific antibody genes

Example 2: expression and purification of CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ D

Expression of CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ 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 project (CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ 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;

tu Ji plusInto 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 CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ 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 CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ 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, the CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ D recombinant proteins were all > 95% pure following Protein L affinity column purification: wherein the theoretical molecular weight of the recombinant CD19-CD3-4-1BB TsAb _ M protein is 80.6kDa, 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 trispecific antibody is in a monomer form (FIG. 2A); the theoretical molecular weight of the recombinant CD19-CD3-4-1BB TsAb _ D protein is 88.4kDa, 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 (about 180kDa) under non-reducing conditions (FIG. 2B), indicating that the two protein molecules can form a disulfide bond via the IgD hinge region to each other, so that the trispecific 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 no reading frame and are consistent with the theoretical N/C terminal amino acid sequence, and mass spectrometry further confirms that the CD19-CD3-4-1BB TsAb _ M is in a monomer form, and the CD19-CD3-4-1BB TsAb _ D is in a dimer form.

Therefore, it can be known that the amino acid sequence of the monomeric form of CD19-CD3-4-1BB TsAb _ M is shown in SEQ ID NO.16, and specifically:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。

the amino acid sequence of the dimeric form of CD19-CD3-4-1BB TsAb _ D is shown in SEQ ID NO.18, and specifically comprises the following components:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。

the amino acid sequence of the anti-CD 19 scFv is shown as SEQ ID NO.40, and specifically comprises the following steps:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS。

the amino acid sequence of the heavy chain variable region of the anti-CD 19 scFv is shown in SEQ ID NO.41, and specifically comprises the following steps:

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS。

the amino acid sequence of the light chain variable region of the anti-CD 19 scFv is shown in SEQ ID NO.42, and specifically comprises the following steps:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK。

the amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO.43, 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.44, 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.45, and specifically comprises the following steps:

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。

the amino acid sequence of the anti-4-1 BB scFv is shown as SEQ ID NO.46, and specifically comprises the following steps:

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。

the amino acid sequence of the heavy chain variable region of the anti-4-1 BB scFv is shown in SEQ ID NO.47, and specifically comprises the following steps:

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS。

the amino acid sequence of the light chain variable region of the anti-4-1 BB scFv is shown in SEQ ID NO.48, and specifically comprises the following steps:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。

the amino acid sequence of the monomeric CD19-CD3-4-1BB TsAb _ M connecting segment 1(Linker 1) is shown as SEQ ID NO.1, and specifically comprises the following steps: GGGGS.

The amino acid sequence of the monomeric CD19-CD3-4-1BB TsAb _ M connecting segment 2(Linker 2) is shown as SEQ ID NO.3, and specifically comprises the following steps: GGGGSGGGGSGGGGS.

The amino acid sequence of the dimer-form CD19-CD3-4-1BB TsAb _ D connecting fragment 1(Linker 1) is shown as SEQ ID NO.5, and specifically comprises the following components: GGGGS.

The amino acid sequence of the dimer-form CD19-CD3-4-1BB TsAb _ D connecting fragment 2(Linker 2) is shown as SEQ ID NO.7, and specifically comprises the following components:

ASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGV。

example 3: ELISA for detection of antigen binding Activity of CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD19-hFc, human CD3-hFc and human 4-1BB-hFc fusion protein (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 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 times of PBS plate washing, purified trispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation method: taking 10 μ g/ml purified CD19-CD3-4-1BB TsAb _ M or CD19-CD3-4-1BB TsAb _ D as the initial concentration, carrying out a double dilution of 6 gradients, each gradient being provided with 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: add stop solution (1M HCl), 100. mu.l/well, read the absorbance (OD) at 450nm wavelength on a microplate reader₄₅₀)。

The ELISA results are shown in fig. 3A and 3B: FIG. 3A illustrates that CD19-CD3-4-1BB TsAb _ M has in vitro binding activity to the recombinant antigens CD19-hFc, CD3-hFc, and 4-1BB-hFc, wherein 4-1BB binding activity is highest, CD19 binding activity is second lowest, and CD3 binding activity is weaker; FIG. 3B illustrates that CD19-CD3-4-1BB TsAb _ D also has in vitro binding activity with recombinant antigens CD19-hFc, CD3-hFc, and 4-1BB-hFc, with the highest 4-1BB binding activity and the second lowest CD19 binding activity, and the weaker CD3 binding activity.

Example 4: CD19-CD3-4-1BB trispecific antibody mediated cell killing experiment

Using human Peripheral Blood Mononuclear Cells (PBMC) as an experimental material, CIK cells (CD3) prepared by respectively acting on human PBMC of the same donor origin with the monomeric form of the TiTE trispecific antibody (CD19-CD3-4-1BB TsAb _ M), the dimeric form of the TiTE trispecific antibody (CD19-CD3-4-1BB TsAb _ D) and the anti-CD 19/anti-CD 3 BiTE bispecific antibody (CD19-CD 3BsAb, available from WUJIANG Yonghai protein technology Co., Ltd.) prepared according to the present invention⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺Purchased from ATCC), cell death was detected, and the difference in the killing efficiency of CCL-86Raji target cells by three antibody-mediated CIK effector cells was compared.

Cell killing experiment step:

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

CIK cell culture and expansion: PBMC were resuspended in CIK basal medium (90% X-vivo15+ 10% FBS) (available from Gbico Co.) to a cell density of 1X 10⁶Perml, added to full-length antibody Anti-CD3(5ug/ml), full-length antibody Anti-CD28(5ug/ml) and NovoNectin (25ug/ml) coated T25 flasks (both full-length antibody and NovoNectin are available from Youjiang Korea protein technology Co., Ltd.) and added simultaneouslyThe cytokines IFN-. gamma.200 ng/ml from Wujiang Yuan-Shi protein technology Co., Ltd and IL-1. beta.2 ng/ml from Wujiang Yuan-Shi protein technology Co., Ltd were placed in an incubator at a saturated humidity of 37 ℃ and 5.0% CO₂Culturing under the conditions of (1). After overnight culture, adding 500U/ml IL-2 (purchased from Wujiang near-shore protein technology Co., Ltd.) to continue culture, counting every 2-3 days and adding 500U/ml IL-2 into CIK basal medium according to the ratio of 1 × 10⁶Cell passage is carried out at a density of/ml;

killing efficiency of CIK cells against Raji cells: performing cell killing experiment in 96-well plate with reaction volume of 100uL, collecting the above cultured CIK cells at 1 × 10⁵Adding Raji cells at 1X 10⁵Adding CD19-CD3BsAb, CD19-CD3-4-1BB TsAb _ M and CD19-CD3-4-1BB TsAb _ D antibody samples with different final concentrations (25, 12.5, 6.25 and 3.125ng/ml) respectively (CIK effector cells: Raji target cells (E: T ratio) are 1: 1), uniformly mixing at room temperature for 3-5 min, co-culturing at 37 ℃ for 3h, adding 10 mu l of CCK-8 into each hole, continuously reacting at 37 ℃ for 2-3 h, and then measuring OD (optical density) by using an enzyme reader₄₅₀The cell killing efficiency was calculated according to the following formula, and each set of experiments was tested 3 times repeatedly; the cell killing efficiency without any added antibody was also used as a blank.

The results are shown in FIG. 4: when CIK effector cells: raji target cells (E: T ratio) 1: 1, under the condition of not adding any antibody, the killing efficiency of the CIK cells to Raji cells for 3h is about 23 percent; under the condition of adding higher concentration of antibody (25, 12.5 and 6.25ng/ml), the killing efficiency of the CIK cells on Raji cells is remarkably improved, wherein the cell killing effect mediated by CD19-CD3-4-1BB TsAb _ D is the best, the killing efficiency is about 96 percent, 96 percent and 92 percent respectively, the killing efficiency is the second best of the CD19-CD3-4-1BB TsAb _ M, the killing efficiency is about 92 percent, 90 percent and 86 percent, the killing efficiency of CD19-CD3BsAb is the weakest, and the killing efficiency is about 80 percent, 54 percent and 54 percent respectively; under the condition of adding lower concentration antibody (3.125ng/ml), the killing efficiency of CIK cells on Raji cells mediated by CD19-CD3-4-1BB TsAb _ D and CD19-CD3-4-1BB TsAb _ M is still obviously improved, the killing efficiency is respectively about 87% and 80%, and CD19-CD3BsAb has no effect basically compared with a blank control. The results show that the target killing activity of T cells on CD19 positive tumor cells mediated by two forms of CD19-CD3-4-1BB TiTE trispecific antibodies is better than that of CD19-CD3 BiTE bispecific antibodies, wherein the dimeric form has better effect than the monomeric form.

Example 5: construction of CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D eukaryotic expression vectors

In the present invention, the TiTE trispecific antibody targeting the human CD19 protein on the surface of lymphoma B cells, the human CD3 on the surface of T cells and the ICOS protein, the T cell costimulatory molecule, was named CD19-CD3-ICOS TsAb.

First, CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD19-CD3-ICOS TsAb _ M is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-ICOS scFv are connected by a Linker (Linker), specifically, the sequences of the anti-CD 19 scFv and the anti-CD3 scFv are connected by a Linker 1(Linker 1), and the sequences of the anti-CD3 scFv and the anti-ICOS scFv are connected by a Linker 2(Linker 2).

The specific construction scheme of the dimer form of CD19-CD3-ICOS TsAb _ D is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-ICOS scFv were linked by a Linker (Linker), specifically, the anti-CD 19 scFv and the anti-CD3 scFv were linked by a Linker 1(Linker 1), and the anti-CD3 scFv and the anti-ICOS scFv were linked by an IgD hinge region (Ala 90-Val 170) as a Linker 2(Linker 2).

For expression of the trispecific antibody in mammalian cells, the mammalian system expression was codon optimized for each of the anti-CD 19 scFv, anti-CD3 scFv, and anti-ICOS scFv sequences.

Specifically, the nucleotide sequence of the heavy chain variable region of the anti-CD 19 scFv is shown in SEQ ID NO. 65.

The nucleotide sequence of the light chain variable region of the anti-CD 19 scFv is shown in SEQ ID NO. 66.

The nucleotide sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 64.

The nucleotide sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 68.

The nucleotide sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 69.

The nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 67.

The nucleotide sequence of the heavy chain variable region of the anti-ICOS scFv is shown as SEQ ID NO.74, and specifically comprises the following steps:

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC。

the nucleotide sequence of the variable region of the light chain of the anti-ICOS scFv is shown as SEQ ID NO.75, and specifically comprises the following steps:

GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

the nucleotide sequence of the ICOS scFv is shown as SEQ ID NO.73, and specifically comprises the following steps:

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

the nucleotide sequence of the monomeric CD19-CD3-ICOS TsAb _ M junction fragment 1(Linker 1) is shown as SEQ ID NO. 2.

The nucleotide sequence of the monomeric form of CD19-CD3-ICOS TsAb _ M junction fragment 2(Linker 2) is shown as SEQ ID NO. 4.

The nucleotide sequence of the dimeric form of CD19-CD3-ICOS TsAb _ D junction fragment 1(Linker 1) is shown in SEQ ID NO. 6.

The nucleotide sequence of the dimeric form of CD19-CD3-ICOS TsAb _ D junction fragment 2(Linker 2) is shown in SEQ ID NO. 8.

The amino acid sequence of the secretory expression signal peptide is shown as SEQ ID NO. 88.

The nucleotide sequence of the secretory expression signal peptide is shown as SEQ ID NO. 89.

II, construction of eukaryotic expression vectors of CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D

The construction and expression of the tri-specific antibody of the invention select a transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.) of mammalian cell protein. To construct monospecific and dimeric forms of trispecific antibodies, primers as shown in table 2 were designed, all of which were synthesized by sumizia jingzhi biotechnology limited and gene templates for amplification were synthesized by sumizia hong kong technology limited, respectively.

Cloning construction for CD19-CD3-ICOS TsAb _ M Signal peptide fragments were first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3- (GGGGS)₃ICOS-F and pcDNA3.1-ICOS-R amplified anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS)₃ Linker 2+ gene sequence against ICOS scFv; clonotypes for CD19-CD3-ICOS TsAb _ DSimilarly, signal peptide fragments were amplified first using primers pcDNA3.1-Sig-F and Sig-R, and then gene sequences for anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, IgD hinge region Linker2, anti-ICOS scFv were amplified using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-ICOS-F and pcDNA3.1-ICOS-R, respectively. After amplification, the amplified DNA is used

Sequencing revealed that the full-length gene sequences of the monomeric form of CD19-CD3-ICOS TsAb _ M and the dimeric form of CD19-CD3-ICOS TsAb _ D were correct and consistent with expectations.

Specifically, the nucleotide sequence of the monomeric form of CD19-CD3-ICOS TsAb _ M is shown as SEQ ID NO.21, and specifically comprises:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

the nucleotide sequence of the dimeric form of CD19-CD3-ICOS TsAb _ D is shown in SEQ ID NO.23, and specifically comprises:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

TABLE 2 primers used in the cloning of CD19-CD3-ICOS trispecific antibody genes

Example 6: expression and purification of CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D

Expression of CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D

1.3. preparation of transfection complex: for each project (CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D), two centrifuge tubes/culture flasks were prepared, each containing 20ml of the recombinant plasmid prepared in example 5:

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

Purification of CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ 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 CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D were analyzed by SDS-PAGE and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 5. As can be seen from the figure, the recombinant proteins, CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D, were all > 95% pure following Protein L affinity column purification: wherein the theoretical molecular weight of the recombinant CD19-CD3-ICOS TsAb _ M protein is 80.7kDa, 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 trispecific antibody is in a monomer form (FIG. 5A); the theoretical molecular weight of the recombinant CD19-CD3-ICOS TsAb _ D protein was 88.6kDa, the electrophoretic band of the protein exhibited a molecular weight consistent with that of the monomer under reducing conditions, and the electrophoretic band exhibited a molecular weight consistent with that of the dimer (about 180kDa) under non-reducing conditions (FIG. 5B), indicating that the two protein molecules can form disulfide bonds through the IgD hinge region to each other, and thus the trispecific antibody was in a dimeric form.

In addition, the purified recombinant protein samples were subjected to N/C terminal sequence analysis, and the results showed that the expressed recombinant protein samples were all in frame and consistent with the theoretical N/C terminal amino acid sequence, and mass spectrometry further confirmed that CD19-CD3-ICOS TsAb _ M was in a monomeric form and CD19-CD3-ICOS TsAb _ D was in a dimeric form.

Therefore, it can be seen that the amino acid sequence of the monomeric form of CD19-CD3-ICOS TsAb _ M is shown in SEQ ID NO.20, specifically:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。

the amino acid sequence of the dimeric form of CD19-CD3-ICOS TsAb _ D is shown in SEQ ID NO.22, and specifically comprises the following components:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。

the amino acid sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 40.

The amino acid sequence of the heavy chain variable region of the anti-CD 19 scFv is shown in SEQ ID NO. 41.

The amino acid sequence of the light chain variable region of the anti-CD 19 scFv is shown in SEQ ID NO. 42.

The amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 43.

The amino acid sequence of the heavy chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 44.

The amino acid sequence of the light chain variable region of the anti-CD3 scFv is shown in SEQ ID NO. 45.

The amino acid sequence of the ICOS scFv is shown as SEQ ID NO.49, and specifically comprises the following steps:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。

the amino acid sequence of the heavy chain variable region of the anti-ICOS scFv is shown as SEQ ID NO.50, and specifically comprises the following steps:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSS。

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

DIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。

the amino acid sequence of the monomeric CD19-CD3-ICOS TsAb _ M connecting fragment 1(Linker 1) is shown as SEQ ID NO. 1.

The amino acid sequence of the monomeric CD19-CD3-ICOS TsAb _ M junction fragment 2(Linker 2) is shown as SEQ ID NO. 3.

The amino acid sequence of the dimeric form of CD19-CD3-ICOS TsAb _ D junction fragment 1(Linker 1) is shown in SEQ ID NO. 5.

The amino acid sequence of the dimeric form of CD19-CD3-ICOS TsAb _ D junction fragment 2(Linker 2) is shown in SEQ ID NO. 7.

Example 7: ELISA detection of antigen binding Activity of CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD19-hFc, human CD3-hFc and human ICOS-hFc fusion protein (purchased from Wujiang near-shore protein technologies Co., 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 times of PBS plate washing, purified trispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation method: using 10 ug/ml purified CD19-CD3-ICOS TsAb _ M or CD19-CD3-ICOS TsAb _ D as initial concentration, performing double dilution of 6 gradients, each gradient having 2 duplicate wells;

The ELISA results are shown in fig. 6A and 6B: FIG. 6A illustrates that CD19-CD3-ICOS TsAb _ M has in vitro binding activity to the recombinant antigens CD19-hFc, CD3-hFc, and ICOS-hFc, with the highest ICOS binding activity and the second lowest CD19 binding activity, and the weaker CD3 binding activity; FIG. 6B illustrates that CD19-CD3-ICOS TsAb _ D also has in vitro binding activity with the recombinant antigens CD19-hFc, CD3-hFc and ICOS-hFc, with the highest ICOS binding activity and the second lowest CD19 binding activity, and the weaker CD3 binding activity.

Example 8: CD19-CD3-ICOS trispecific antibody mediated cell killing experiments

Using human Peripheral Blood Mononuclear Cells (PBMC) as experimental material, the monomeric form of the TiTE trispecific antibody (CD19-CD3-ICOS TsAb _ M), the dimeric form of the TiTE trispecific antibody (CD19-CD3-ICOS TsAb _ D) and the anti-CD 19/anti-CD 3 BiTE bispecific antibody (CD19-CD 3BsAb, available from WUJIANG Yonghai protein technology Co., Ltd.) prepared by the present invention were allowed to act on human PBMC (CD3) prepared from the same donor source⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺Purchased from ATCC), cell death was detected, and the difference in the killing efficiency of CCL-86Raji target cells by three antibody-mediated CIK effector cells was compared.

Cell killing experiment step:

CIK cell culture and expansion: PBMC were resuspended in CIK basal medium (90% X-vivo15+ 10% FBS) (available from Gbico Co.) to a cell density of 1X 10⁶Adding to full-length antibody Anti-CD3(5ug/ml), full-length antibody Anti-CD28(5ug/ml) and NovoNectin (25ug/ml) coated T25 flasks (both full-length antibody and NovoNectin are from Youjiang Yoshiki protein technology Co., Ltd.), adding cytokine IFN-gamma (200 ng/ml) and IL-1 beta (2 ng/ml) to the flasks, placing in an incubator, and culturing at saturation humidity, 37 deg.C and 5.0% CO₂Culturing under the conditions of (1). After overnight incubation, addCulturing 500U/ml IL-2 (purchased from Wujiang near-shore protein science and technology Co., Ltd.), counting every 2-3 days and adding 500U/ml IL-2 into CIK basal medium according to the ratio of 1 × 10⁶Cell passage is carried out at a density of/ml;

killing efficiency of CIK cells against Raji cells: performing cell killing experiment in 96-well plate with reaction volume of 100uL, collecting the above cultured CIK cells at 1 × 10⁵Adding Raji cells at 1X 10⁵Respectively adding CD19-CD3BsAb, CD19-CD3-ICOS TsAb _ M and CD19-CD3-ICOS TsAb _ D antibody samples with different final concentrations (25, 12.5, 6.25 and 3.125ng/ml) (the ratio of CIK effector cells to Raji target cells (E: T is 1: 1), uniformly mixing at room temperature for 3-5 min, co-culturing at 37 ℃ for 3h, adding 10 mu l of CCK-8 into each well, continuously reacting at 37 ℃ for 2-3 h, and measuring OD (optical density) by using an enzyme reader₄₅₀The cell killing efficiency was calculated according to the following formula, and each set of experiments was tested 3 times repeatedly; the cell killing efficiency without any added antibody was also used as a blank.

The results are shown in FIG. 7: when CIK effector cells: raji target cells (E: T ratio) 1: 1, under the condition of not adding any antibody, the killing efficiency of the CIK cells to Raji cells for 3h is about 23 percent; under the condition of adding higher concentration of antibody (25, 12.5 and 6.25ng/ml), the killing efficiency of the CIK cells on Raji cells is remarkably improved, wherein the cell killing effect mediated by CD19-CD3-ICOS TsAb _ D is the best, the killing efficiency is about 94 percent, 94 percent and 82 percent respectively, the killing efficiency is the second best of the killing efficiency of CD19-CD3-ICOS TsAb _ M, the killing efficiency is about 92 percent, 86 percent and 84 percent, the killing efficiency of CD19-CD3BsAb is the weakest, and the killing efficiency is about 80 percent, 54 percent and 54 percent respectively; with the addition of lower concentrations of antibody (3.125ng/ml), the killing efficiency of the CIK cells on Raji cells mediated by CD19-CD3-ICOS TsAb _ D and CD19-CD3-ICOS TsAb _ M is still significantly improved, and the killing efficiency is about 76% and 71%, respectively, while the killing efficiency of CD19-CD3BsAb is basically not effective compared with the blank control. The results show that the target killing activity of T cells on CD19 positive tumor cells mediated by two forms of CD19-CD3-ICOS TiTE trispecific antibodies is better than that of CD19-CD3 BiTE bispecific antibodies, wherein the dimeric form has better effect than the monomeric form.

Example 9: construction of eukaryotic expression vectors for CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D

In the present invention, the TiTE trispecific antibody targeting the human CD19 protein on the surface of lymphoma B cells, the human CD3 on the surface of T cells and the positive co-stimulatory molecule of T cells OX40 protein was named CD19-CD3-OX40 TsAb.

First, CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD19-CD3-OX40TsAb _ M is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-OX 40scFv are linked by a Linker (Linker), specifically, the sequences of the anti-CD 19 scFv and the anti-CD3 scFv are linked by a Linker 1(Linker 1), and the sequences of the anti-CD3 scFv and the anti-OX 40scFv are linked by a Linker 2(Linker 2).

The specific construction scheme of the dimer form of CD19-CD3-OX40TsAb _ D is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-OX 40scFv were linked by a Linker (Linker), specifically, the anti-CD 19 scFv and the anti-CD3 scFv were linked by a Linker 1(Linker 1), and the anti-CD3 scFv and the anti-OX 40scFv sequences were linked by an IgD hinge region (Ala 90-Val 170) as a Linker 2(Linker 2).

For expression of the trispecific antibody in mammalian cells, the mammalian system expression was codon optimized for each of the anti-CD 19 scFv, anti-CD3 scFv, and anti-OX 40scFv sequences.

The nucleotide sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 64.

The nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 67.

The nucleotide sequence of the heavy chain variable region of the anti-OX 40scFv is shown as SEQ ID NO.77, and specifically comprises the following steps:

CAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGC。

the nucleotide sequence of the light chain variable region of the anti-OX 40scFv is shown as SEQ ID NO.78, and specifically comprises the following steps:

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

the nucleotide sequence of the anti-OX 40scFv is shown as SEQ ID NO.76, and specifically comprises the following steps:

CAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric CD19-CD3-OX40TsAb _ M connecting fragment 1(Linker 1) is shown as SEQ ID NO. 2.

The nucleotide sequence of the monomeric CD19-CD3-OX40TsAb _ M connecting fragment 2(Linker 2) is shown as SEQ ID NO. 4.

The nucleotide sequence of the dimeric form of CD19-CD3-OX40TsAb _ D connecting fragment 1(Linker 1) is shown in SEQ ID NO. 6.

The nucleotide sequence of the dimeric form of CD19-CD3-OX40TsAb _ D connecting fragment 2(Linker 2) is shown in SEQ ID NO. 8.

II, construction of CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D eukaryotic expression vectors

The construction and expression of the tri-specific antibody of the invention select a transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.) of mammalian cell protein. To construct monospecific and dimeric forms of trispecific antibodies, primers as shown in Table 3 were designed, all of which were synthesized by Suzhou Jinzhi Biotech, Inc., and gene templates for amplification were synthesized by Suzhou Hongxin Tech, Inc., respectively.

Cloning construction for CD19-CD3-OX40TsAb _ M Signal peptide fragments were first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3- (GGGGS) were used respectively₃OX40-F and pcDNA3.1-OX40-R amplified anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS)₃ Linker 2+ anti-OX 40 scFv; cloning construction of the cDNA for CD19-CD3-OX40TsAb _ D was carried out by first amplifying signal peptide fragments using primers pcDNA3.1-Sig-F and Sig-R, and then amplifying anti-CD 19 s cFv, GGGGS Linker 1+ anti-CD3 scFv, IgD hinge region Linker2 and anti-OX 40scFv using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-OX40-F and pcDNA3.1-OX40-R, respectivelyAnd (4) columns. After amplification, the amplified DNA is used

Sequencing revealed that the full-length gene sequences of the monomeric form of CD19-CD3-OX40TsAb _ M and the dimeric form of CD19-CD3-OX40TsAb _ D were correct and consistent with the expectations.

Specifically, the nucleotide sequence of the monomeric form of CD19-CD3-OX40TsAb _ M is shown in SEQ ID NO.25, and specifically comprises:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

the nucleotide sequence of the dimeric form of CD19-CD3-OX40TsAb _ D is shown in SEQ ID NO.27, and specifically comprises the following components:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

TABLE 3 primers used in the cloning of CD19-CD3-OX40 trispecific antibody genes

Example 10: expression and purification of CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D

Expression of CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D

1.3. preparation of transfection complex: for each project (CD19-CD3-OX40 TsAb _ M and CD19-CD3-OX40TsAb _ D), two centrifuge tubes/culture flasks were prepared, each placed at 20ml, and the recombinant plasmids prepared in example 9 were:

Purification of CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ 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 CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D were analyzed by SDS-PAGE, and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 8. As can be seen from the figure, the purity of both CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D recombinant proteins was > 95% after purification on Protein L affinity chromatography columns: wherein the theoretical molecular weight of the recombinant CD19-CD3-OX40TsAb _ M protein is 80.1kDa, 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 trispecific antibody is in a monomeric form (FIG. 8A); the theoretical molecular weight of the recombinant CD19-CD3-OX40TsAb _ D protein was 88.0kDa, the electrophoretic band of the protein exhibited a molecular weight consistent with that of the monomer under reducing conditions, and the electrophoretic band exhibited a molecular weight consistent with that of the dimer (about 180kDa) under non-reducing conditions (FIG. 8B), indicating that the two protein molecules can form disulfide bonds through the IgD hinge region to each other, and thus the trispecific antibody was in a dimeric 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 no reading frame and are consistent with the theoretical N/C terminal amino acid sequence, and mass spectrometry further confirms that CD19-CD3-OX40TsAb _ M is in a monomer form, and CD19-CD3-OX40TsAb _ D is in a dimer form.

Therefore, it can be known that the amino acid sequence of the monomeric form of CD19-CD3-OX40TsAb _ M is shown in SEQ ID NO.24, and specifically:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。

the amino acid sequence of the dimeric form of CD19-CD3-OX40TsAb _ D is shown in SEQ ID NO.26, and specifically comprises the following components: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR are provided.

The amino acid sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 40.

The amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 43.

The amino acid sequence of the anti-OX 40scFv is shown as SEQ ID NO.52, and specifically comprises the following steps:

QLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。

the amino acid sequence of the heavy chain variable region of the anti-OX 40scFv is shown in SEQ ID NO.53, and specifically comprises the following steps:

QLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSS。

the amino acid sequence of the light chain variable region of the anti-OX 40scFv is shown as SEQ ID NO.54, and specifically comprises the following steps:

DIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。

the amino acid sequence of the monomeric CD19-CD3-OX40TsAb _ M connecting fragment 1(Linker 1) is shown as SEQ ID NO. 1.

The amino acid sequence of the monomeric CD19-CD3-OX40TsAb _ M connecting fragment 2(Linker 2) is shown as SEQ ID NO. 3.

The amino acid sequence of the dimeric form of CD19-CD3-OX40TsAb _ D connecting fragment 1(Linker 1) is shown in SEQ ID NO. 5.

The amino acid sequence of the dimeric form of CD19-CD3-OX40TsAb _ D Linker 2(Linker 2) is shown in SEQ ID NO. 7.

Example 11: ELISA for detecting the antigen binding activity of CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD19-hFc, human CD3-hFc and human OX40-hFc fusion proteins (purchased from Wujiang near-shore protein technologies, 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 conditions of 1 hour at 37 ℃ or overnight at 4 ℃, with 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 times of PBS plate washing, purified trispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation method: taking 10 μ g/ml purified CD19-CD3-OX40TsAb _ M or CD19-CD3-OX40TsAb _ D as initial concentration, performing double dilution on 6 gradients, and setting 2 duplicate wells in each gradient;

The ELISA results are shown in fig. 9A and 9B: FIG. 9A illustrates that CD19-CD3-OX40TsAb _ M has in vitro binding activity to recombinant antigens CD19-hFc, CD3-hFc, and OX40-hFc, with the highest CD19 binding activity, the next lowest OX40 binding activity, and the weaker CD3 binding activity; FIG. 9B illustrates that CD19-CD3-OX40TsAb _ D also has in vitro binding activity with recombinant antigens CD19-hFc, CD3-hFc and OX40-hFc, with the highest CD19 binding activity and the next lowest OX40 binding activity and the weaker CD3 binding activity.

Example 12: CD19-CD3-OX40 trispecific antibody mediated cell killing experiments

Using human Peripheral Blood Mononuclear Cells (PBMC) as experimental material, the monomeric form of the TiTE trispecific antibody (CD19-CD3-OX40 TsAb _ M), the dimeric form of the TiTE trispecific antibody (CD19-CD3-OX40 TsAb _ D) and the anti-CD 19/anti-CD 3 BiTE bispecific antibody (CD19-CD 3BsAb, available from Wujiang Korea protein technology Co., Ltd.) prepared according to the present invention were allowed to act on human PBMC (CD3 PBMC) of the same donor source⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺Purchased from ATCC), cell death was detected, and the difference in the killing efficiency of CCL-86Raji target cells by three antibody-mediated CIK effector cells was compared.

Cell killing experiment step:

CIK cell culture and expansion: PBMC were resuspended in CIK basal medium (90% X-vivo15+ 10% FBS) (available from Gbico Co.) to a cell density of 1X 10⁶Adding to full-length antibody Anti-CD3(5ug/ml), full-length antibody Anti-CD28(5ug/ml) and NovoNectin (25ug/ml) coated T25 flasks (both full-length antibody and NovoNectin are from Youjiang Yoshiki protein technology Co., Ltd.), adding cytokine IFN-gamma (200 ng/ml) and IL-1 beta (2 ng/ml) to the flasks, placing in an incubator, and culturing at saturation humidity, 37 deg.C and 5.0% CO₂Culturing under the conditions of (1). After overnight culture, adding 500U/ml IL-2 (purchased from Wujiang near-shore protein technology Co., Ltd.) to continue culture, counting every 2-3 days and adding 500U/ml IL-2 into CIK basal medium according to the ratio of 1 × 10⁶Cell passage is carried out at a density of/ml;

killing efficiency of CIK cells against Raji cells: performing cell killing experiment in 96-well plate with reaction volume of 100uL, collecting the above cultured CIK cells at 1 × 10⁵Adding Raji cells at 1X 10⁵Separately (CIK effector cells: Raji target cells (E: T ratio) 1: 1), CD19-CD3BsAb, CD19-CD3-OX40TsAb _ M and CD19-CD3-OX40TsAb _ D antibody samples with different final concentrations (25, 12.5, 6.25 and 3.125ng/ml) are added, the mixture is mixed for 3-5 min at room temperature, 10 mu l of CCK-8 is added to each well after 3h of co-culture at 37 ℃, the reaction is continued for 2-3 h at 37 ℃, and then OD (cell density) is measured by an ELISA reader₄₅₀The cell killing efficiency was calculated according to the following formula, and each set of experiments was tested 3 times repeatedly; the cell killing efficiency without any added antibody was also used as a blank.

The results are shown in FIG. 10: when CIK effector cells: raji target cells (E: T ratio) 1: 1, under the condition of not adding any antibody, the killing efficiency of the CIK cells to Raji cells for 3h is about 23 percent; under the condition of adding higher concentration of antibody (25, 12.5 and 6.25ng/ml), the killing efficiency of the CIK cells on Raji cells is remarkably improved, wherein the cell killing effect mediated by CD19-CD3-OX40TsAb _ D is the best, the killing efficiency is about 96 percent, 92 percent and 86 percent respectively, the killing efficiency is the second best of the killing efficiency of CD19-CD3-OX40TsAb _ M, the killing efficiency is about 89 percent, 82 percent and 80 percent, the killing efficiency of CD19-CD3BsAb is the weakest, and the killing efficiency is about 80 percent, 54 percent and 54 percent respectively; under the condition of adding lower concentration antibody (3.125ng/ml), the killing efficiency of CIK cells on Raji cells mediated by CD19-CD3-OX40TsAb _ D and CD19-CD3-OX40TsAb _ M is still obviously improved, the killing efficiency is about 72 percent and 68 percent respectively, and CD19-CD3BsAb has no effect basically compared with a blank control. The results show that the target killing activity of T cells on CD19 positive tumor cells mediated by two forms of CD19-CD3-OX40 TiTE trispecific antibodies is better than that of CD19-CD3 BiTE bispecific antibodies, wherein the dimeric form has better effect than the monomeric form.

Example 13: construction of eukaryotic expression vectors for CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D

In the present invention, the TiTE trispecific antibody targeting the human CD19 protein on the surface of lymphoma B cells, the human CD3 on the surface of T cells and the GITR protein, the T cell positive costimulatory molecule, was named CD19-CD3-GITR TsAb.

First, CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD19-CD3-GITR TsAb _ M is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-GITR scFv were linked by a Linker (Linker), specifically, the anti-CD 19 scFv and the anti-CD3 scFv were linked by a Linker 1(Linker 1), and the anti-CD3 scFv and the anti-GITR scFv were linked by a Linker 2(Linker 2).

The specific construction scheme of the dimer form of CD19-CD3-GITR TsAb _ D is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-GITR scFv were linked by a Linker (Linker), specifically, the anti-CD 19 scFv and the anti-CD3 scFv were linked by a Linker 1(Linker 1), and the anti-CD3 scFv and the anti-GITR scFv were linked by an IgD hinge region (Ala 90-Val 170) as a Linker 2(Linker 2).

For expression of the trispecific antibody in mammalian cells, the mammalian system expression was codon optimized for each of the anti-CD 19 scFv, anti-CD3 scFv, and anti-GITR scFv sequences.

The nucleotide sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 64.

The nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 67.

The nucleotide sequence of the heavy chain variable region of the anti-GITR scFv is shown as SEQ ID NO.80, and specifically comprises the following steps:

CAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

the nucleotide sequence of the variable region of the light chain of the anti-GITR scFv is shown as SEQ ID NO.81, and specifically comprises the following steps:

GACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。

the nucleotide sequence of the anti-GITR scFv is shown as SEQ ID NO.79, and specifically comprises the following steps:

CAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。

the nucleotide sequence of the monomeric CD19-CD3-GITR TsAb _ M junction fragment 1(Linker 1) is shown as SEQ ID NO. 2.

The nucleotide sequence of the monomeric CD19-CD3-GITR TsAb _ M junction fragment 2(Linker 2) is shown as SEQ ID NO. 4.

The nucleotide sequence of the dimeric form of CD19-CD3-GITR TsAb _ D junction fragment 1(Linker 1) is shown in SEQ ID NO. 6.

The nucleotide sequence of the dimeric form of CD19-CD3-GITR TsAb _ D junction fragment 2(Linker 2) is shown in SEQ ID NO. 8.

II, construction of CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D eukaryotic expression vectors

The construction and expression of the tri-specific antibody of the invention select a transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.) of mammalian cell protein. To construct monospecific and dimeric forms of trispecific antibodies, primers as shown in Table 4 were designed, all of which were synthesized by Suzhou Jinzhi Biotech, Inc., and gene templates for amplification were synthesized by Suzhou Hongxin Tech, Inc., respectively.

Cloning construction for CD19-CD3-GITR TsAb _ M, signal peptide fragments were first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3- (GGGGS) were used respectively₃Amplification of anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) by-GITR-F and pcDNA3.1-GITR-R₃Linker 2+ gene sequence of anti-GITR scFv; cloning construction for CD19-CD3-GITR TsAb _ D, signal peptide fragments were also first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then anti-CD 19 scFv, GGGGGGS Linker 1+ anti-CD3 scFv, IgD hinge region Linker2, anti-GITR scFv were amplified using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-GITR-F and pcDNA3.1-GITR-R, respectively. After amplification, the amplified DNA is used

PCR one-step directional cloning kit(purchased from Wujiang near-shore protein science and technology Co., Ltd.) were spliced to full-length gene sequences of the monospecific antibody and the dimeric form respectively and cloned seamlessly into the expression vector pcDNA3.1 which was linearized with EcoRI and HindIII, and transformed into Escherichia coli DH5 alpha, and colony PCR was used to perform positive cloning identification, and the recombinants (recombinant plasmids) identified as positive were subjected to sequencing identification. 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 CD19-CD3-GITR TsAb _ M and the dimeric form of CD19-CD3-GITR TsAb _ D were correct and consistent with expectations.

Specifically, the nucleotide sequence of the monomeric form of CD19-CD3-GITR TsAb _ M is shown as SEQ ID NO.29, and specifically comprises:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。

the nucleotide sequence of the dimeric form of CD19-CD3-GITR TsAb _ D is shown as SEQ ID NO.31, and specifically comprises:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。

TABLE 4 primers used in the cloning of CD19-CD3-GITR trispecific antibody genes

Example 14: expression and purification of CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D

Expression of CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D

1.3. preparation of transfection complex: for each project (CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D), two centrifuge tubes/culture flasks were prepared, each placed in 20ml, and the recombinant plasmids prepared in example 13 were taken:

1.6. at 37 ℃ CO₂Culturing transfected cells at 8% concentration and 130rpm of shaking table, and collecting after 5 days

Culture supernatant for target protein expression detection

Purification of CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ 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 CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D recombinant proteins were analyzed by SDS-PAGE and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 11. As can be seen from the figure, the recombinant proteins, CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D, were all > 95% pure following Protein L affinity column purification: wherein the theoretical molecular weight of the recombinant protein CD19-CD3-GITR TsAb _ M is 80.1kDa, and the protein presents a single electrophoretic band under both reducing and non-reducing conditions, and the molecular weight is consistent with that of a monomer, so that the trispecific antibody is in a monomeric form (FIG. 11A); the theoretical molecular weight of the recombinant CD19-CD3-GITR TsAb _ D protein was 88.0kDa, the electrophoretic band of the protein exhibited a molecular weight consistent with that of the monomer under reducing conditions, and the electrophoretic band exhibited a molecular weight consistent with that of the dimer (about 180kDa) under non-reducing conditions (FIG. 11B), indicating that the two protein molecules can form disulfide bonds through the IgD hinge region to each other, and thus the trispecific antibody was in a dimeric 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 the mass spectrum analysis further confirms that the CD19-CD3-GITR TsAb _ M is in a monomer form, and the CD19-CD3-GITR TsAb _ D is in a dimer form.

Therefore, it can be seen that the amino acid sequence of the monomeric form of CD19-CD3-GITR TsAb _ M is shown in SEQ ID NO.28, specifically:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。

the amino acid sequence of the dimeric form of CD19-CD3-GITR TsAb _ D is shown in SEQ ID NO.30, and specifically comprises: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK are provided.

The amino acid sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 40.

The amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 43.

The amino acid sequence of the anti-GITR scFv is shown as SEQ ID NO.55, and specifically comprises the following steps:

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。

the amino acid sequence of the heavy chain variable region of the anti-GITR scFv is shown as SEQ ID NO.56, and specifically comprises the following steps:

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSS。

the amino acid sequence of the variable region of the light chain of the anti-GITR scFv is shown as SEQ ID NO.57, and specifically comprises the following steps:

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。

the amino acid sequence of the monomeric CD19-CD3-GITR TsAb _ M connecting segment 1(Linker 1) is shown as SEQ ID NO. 1.

The amino acid sequence of the monomeric CD19-CD3-GITR TsAb _ M junction fragment 2(Linker 2) is shown as SEQ ID NO. 3.

The amino acid sequence of the dimeric form of CD19-CD3-GITR TsAb _ D junction fragment 1(Linker 1) is shown in SEQ ID NO. 5.

The amino acid sequence of the dimeric form of CD19-CD3-GITR TsAb _ D junction fragment 2(Linker 2) is shown in SEQ ID NO. 7.

Example 15: ELISA detection of antigen binding Activity of CD19-CD3-GITR TsAb _ M and CD19-CD3-GITR TsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD19-hFc, human CD3-hFc and human GITR-hFc fusion protein (purchased from Wujiang near-shore protein technologies Co., 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 times of PBS plate washing, purified trispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation method: taking 10 μ g/ml purified CD19-CD3-GITR TsAb _ M or CD19-CD3-GITR TsAb _ D as the initial concentration, carrying out a dilution of 6 gradients in a multiple ratio, each gradient being provided with 2 duplicate wells;

The ELISA results are shown in fig. 12A and 12B: FIG. 12A illustrates that CD19-CD3-GITR TsAb _ M has in vitro binding activity to the recombinant antigens CD19-hFc, CD3-hFc, and GITR-hFc, wherein both GITR and CD19 binding activity is higher and CD3 binding activity is weaker; FIG. 12B illustrates that CD19-CD3-GITR TsAb _ D also has in vitro binding activity with the recombinant antigens CD19-hFc, CD3-hFc and GITR-hFc, where both GITR and CD19 binding activity were higher and CD3 binding activity was weaker.

Example 16: CD19-CD3-GITR trispecific antibody mediated cell killing assay

Using human Peripheral Blood Mononuclear Cells (PBMC) as experimental material, the monomeric form of the TiTE trispecific antibody (CD19-CD3-GITR TsAb _ M), the dimeric form of the TiTE trispecific antibody (CD19-CD3-GITR TsAb _ D) and the anti-CD 19/anti-CD 3 BiTE bispecific antibody (CD19-CD 3BsAb, available from WUJIANG Yonghai protein technology Co., Ltd.) were used to act on the same donorCIK cells prepared from human PBMC of origin (CD3)⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺Purchased from ATCC), cell death was detected, and the difference in the killing efficiency of CCL-86Raji target cells by three antibody-mediated CIK effector cells was compared.

Cell killing experiment step:

killing efficiency of CIK cells against Raji cells: performing cell killing experiment in 96-well plate with reaction volume of 100uL, collecting the above cultured CIK cells at 1 × 10⁵Adding Raji cells at 1X 10⁵Each of CD19-CD3BsAb, CD19-CD3-GITR cells was added to each of the cells (CIK effector cells: Raji target cells (E: T ratio) 1: 1) at different final concentrations (25, 12.5, 6.25, 3.125ng/ml)The TsAb _ M and CD19-CD3-GITR TsAb _ D antibody samples are uniformly mixed at room temperature for 3-5 min, after co-culture is carried out for 3h at 37 ℃, 10 mu l of CCK-8 is added into each hole, reaction is continuously carried out for 2-3 h at 37 ℃, and then OD (optical density) is measured by using an enzyme labeling instrument₄₅₀The cell killing efficiency was calculated according to the following formula, and each set of experiments was tested 3 times repeatedly; the cell killing efficiency without any added antibody was also used as a blank.

The results are shown in FIG. 13: when CIK effector cells: raji target cells (E: T ratio) 1: 1, under the condition of not adding any antibody, the killing efficiency of the CIK cells to Raji cells for 3h is about 23 percent; under the condition of adding higher concentration of antibody (25, 12.5 and 6.25ng/ml), the killing efficiency of the CIK cells on Raji cells is remarkably improved, wherein the cell killing effect mediated by CD19-CD3-GITR TsAb _ D is the best, the killing efficiency is about 93 percent, 77 percent and 73 percent respectively, the killing efficiency is the second best of the effects of CD19-CD3-GITR TsAb _ M, the killing efficiency is about 88 percent, 83 percent and 66 percent, the effect of CD19-CD3BsAb is the weakest, and the killing efficiency is about 80 percent, 54 percent and 54 percent respectively; with the addition of lower concentrations of antibody (3.125ng/ml), the killing efficiency of the CIK cells on Raji cells mediated by CD19-CD3-GITR TsAb _ D and CD19-CD3-GITR TsAb _ M is still improved to some extent, and the killing efficiency is about 57% and 49%, respectively, while the CD19-CD3BsAb has no effect basically compared with the blank control. The results show that the target killing activity of T cells on CD19 positive tumor cells mediated by two forms of CD19-CD3-GITR TiTE trispecific antibodies is better than that of CD19-CD3 BiTE bispecific antibodies, wherein the dimeric form has better effect than the monomeric form.

Example 17: construction of eukaryotic expression vectors for CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D

In the present invention, the TiTE trispecific antibody targeting the human CD19 protein on the surface of lymphoma B cells, the human CD3 on the surface of T cells and the T cell positive co-stimulatory molecule CD40L protein was named CD19-CD3-CD40L TsAb.

First, CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD19-CD3-CD40L TsAb _ M is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-CD 40L scFv are connected by a Linker (Linker), specifically, the sequences of the anti-CD 19 scFv and the anti-CD3 scFv are connected by a Linker 1(Linker 1), and the sequences of the anti-CD3 scFv and the anti-CD 40L scFv are connected by a Linker 2(Linker 2).

The specific construction scheme of the dimer form of CD19-CD3-CD40L TsAb _ D is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-CD 40L scFv are connected by a Linker (Linker), specifically, the anti-CD 19 scFv and the anti-CD3 scFv are connected by a Linker 1(Linker 1), and the anti-CD3 scFv and the anti-CD 40L scFv sequences are connected by an IgD hinge region (Ala 90-Val 170) as a Linker 2(Linker 2).

For expression of the trispecific antibody in mammalian cells, the mammalian system expression was codon optimized for the anti-CD 19 scFv, anti-CD3 scFv, and anti-CD 40L scFv sequences.

The nucleotide sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 64.

The nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 67.

The nucleotide sequence of the heavy chain variable region of the anti-CD 40L scFv is shown in SEQ ID NO.83, and specifically comprises the following steps:

GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

the nucleotide sequence of the variable region of the light chain of the anti-CD 40L scFv is shown in SEQ ID NO.84, and specifically comprises the following steps:

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the anti-CD 40L scFv is shown in SEQ ID NO.82, and specifically comprises the following steps:

GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric CD19-CD3-CD40L TsAb _ M connecting fragment 1(Linker 1) is shown as SEQ ID NO. 2.

The nucleotide sequence of the monomeric CD19-CD3-CD40L TsAb _ M connecting fragment 2(Linker 2) is shown as SEQ ID NO. 4.

The nucleotide sequence of the dimeric form of CD19-CD3-CD40L TsAb _ D connecting fragment 1(Linker 1) is shown in SEQ ID NO. 6.

The nucleotide sequence of the dimeric form of CD19-CD3-CD40L TsAb _ D connecting fragment 2(Linker 2) is shown in SEQ ID NO. 8.

Secondly, construction of eukaryotic expression vectors of CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D

The construction and expression of the tri-specific antibody of the invention select a transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.) of mammalian cell protein. To construct monospecific and dimeric forms of trispecific antibodies, primers as shown in Table 5 were designed, all of which were synthesized by Suzhou Jinzhi Biotech, Inc., and gene templates for amplification were synthesized by Suzhou Hongxin Tech, Inc., respectively.

Cloning construction for CD19-CD3-CD40L TsAb _ M Signal peptide fragment was first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3- (GGGGS)₃Amplification of anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) from-CD 40L-F and pcDNA3.1-CD40L-R₃Linker 2+ anti-CD 40L scFv; cloning construction for CD19-CD3-CD40L TsAb _ 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-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, IgD hinge region Linker2 and anti-CD 40L scFv using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CD40L-F and pcDNA3.1-CD40L-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 monomer and dimer three-specificity antibodies, seamlessly clones the full-length gene sequences to a pcDNA3.1 expression vector which is subjected to EcoRI and HindIII linearization treatment, transforms Escherichia coli DH5 alpha, performs positive cloning identification by colony PCR, and performs sequencing identification on recombinants (recombinant plasmids) which are identified to be positive. Then sequencing is positiveThe exact recombinants (recombinant plasmids) were arranged in plasmids and used for transfection of CHO-S cells.

Sequencing revealed that the full-length gene sequences of the monomeric form of CD19-CD3-CD40L TsAb _ M and the dimeric form of CD19-CD3-CD40L TsAb _ D were correct and consistent with the expectations.

Specifically, the nucleotide sequence of the monomeric form of CD19-CD3-CD40L TsAb _ M is shown in SEQ ID NO.33, and specifically comprises:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the dimeric form of CD19-CD3-CD40L TsAb _ D is shown in SEQ ID NO.35, and specifically comprises the following components:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

TABLE 5 primers used in the cloning of the CD19-CD3-CD40L trispecific antibody Gene

Example 18: expression and purification of CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D

Expression of CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D

1.3. preparation of transfection complex: for each project (CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D), two centrifuge tubes/culture flasks were prepared and placed in 20ml containers, respectively, to obtain the recombinant plasmid prepared in example 17:

1.6. at 37 ℃ CO₂Culturing the transfected cells at 8% concentration and 130rpm of shaker, collecting the culture supernatant after 5 days, and detecting the expression of the target protein

Purification of CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ 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 CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ 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, the purity of both the CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D recombinant proteins was > 95% after purification on Protein L affinity chromatography column: wherein the theoretical molecular weight of the recombinant protein CD19-CD3-CD40L TsAb _ M is 79.2kDa, 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 trispecific antibody is in a monomer form (FIG. 14A); the theoretical molecular weight of the recombinant CD19-CD3-CD40L TsAb _ D protein is 87.1kDa, 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 (about 180kDa) under non-reducing conditions (FIG. 14B), indicating that the two protein molecules can form disulfide bonds through the IgD hinge region to each other, and thus the trispecific 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 no correct reading frame and are consistent with the theoretical N/C terminal amino acid sequence, and mass spectrometry further confirms that CD19-CD3-CD40L TsAb _ M is in a monomer form, and CD19-CD3-CD40L TsAb _ D is in a dimer form.

Therefore, it can be known that the amino acid sequence of the monomeric form of CD19-CD3-CD40L TsAb _ M is shown in SEQ ID No.32, specifically:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。

the amino acid sequence of the dimeric form of CD19-CD3-CD40L TsAb _ D is shown in SEQ ID NO.34, and specifically comprises the following components:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。

the amino acid sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 40.

The amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 43.

The amino acid sequence of the anti-CD 40L scFv is shown in SEQ ID NO.58, and specifically comprises the following steps:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。

the amino acid sequence of the heavy chain variable region of the anti-CD 40L scFv is shown in SEQ ID NO.59, and specifically comprises the following steps:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSS。

the amino acid sequence of the light chain variable region of the anti-CD 40L scFv is shown in SEQ ID NO.60, and specifically comprises the following steps:

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。

the amino acid sequence of the monomeric CD19-CD3-CD40L TsAb _ M connecting fragment 1(Linker 1) is shown as SEQ ID NO. 1.

The amino acid sequence of the monomeric CD19-CD3-CD40L TsAb _ M connecting fragment 2(Linker 2) is shown as SEQ ID NO. 3.

The amino acid sequence of the dimeric form of CD19-CD3-CD40L TsAb _ D connecting fragment 1(Linker 1) is shown in SEQ ID NO. 5.

The amino acid sequence of the dimeric form of CD19-CD3-CD40L TsAb _ D connecting fragment 2(Linker 2) is shown in SEQ ID NO. 7.

Example 19: ELISA for detecting the antigen binding activity of CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD19-hFc, human CD3-hFc and human CD40L-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 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 to 1mol/L HCl or 1mol/L NaOH7.4, supplementing water to 1L;

3. sample adding: after 4 times of PBS plate washing, purified trispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation method: taking 10 mu g/ml purified CD19-CD3-CD40L TsAb _ M or CD19-CD3-CD40L TsAb _ D as a starting concentration, carrying out a double-ratio dilution of 6 gradients, wherein each gradient is provided with 2 multiple wells;

The ELISA results are shown in fig. 15A and 15B: FIG. 15A illustrates that CD19-CD3-CD40L TsAb _ M has in vitro binding activity to recombinant antigens CD19-hFc, CD3-hFc, and CD40L-hFc, with the highest CD40L binding activity and the next lowest CD19 binding activity and the weaker CD3 binding activity; FIG. 15B illustrates that CD19-CD3-CD40L TsAb _ D has in vitro binding activity as well as recombinant antigens CD19-hFc, CD3-hFc and CD40L-hFc, with the highest CD40L binding activity and the second lowest CD19 binding activity and the weaker CD3 binding activity.

Example 20: CD19-CD3-CD40L trispecific antibody mediated cell killing experiment

Using human Peripheral Blood Mononuclear Cells (PBMC) as experimental material, CIK cells (CD 3-CD 3-CD40L TsAb _ M) prepared by the present invention and prepared by using the above monomer form of TiTE trispecific antibody (CD19-CD3-CD40L TsAb _ M), dimer form of TiTE trispecific antibody (CD19-CD3-CD40L TsAb _ D) and anti-CD 19/anti-CD 3 BiTE bispecific antibody (CD19-CD 3BsAb, available from Wujiang Korea protein technology Co., Ltd.) were respectively acted on human PBMC of the same donor origin⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺Purchased from ATCC), cell death was detected, and the difference in the killing efficiency of CCL-86Raji target cells by three antibody-mediated CIK effector cells was compared.

Cell killing experiment step:

killing efficiency of CIK cells against Raji cells: performing cell killing experiment in 96-well plate with reaction volume of 100uL, collecting the above cultured CIK cells at 1 × 10⁵Adding Raji cells at 1X 10⁵Each of the cells (CIK effector cells: Raji target cells (E: T ratio) 1: 1) was inoculated with CD19-CD3BsAb, CD19-CD3-CD40L TsAb _ M and CD19-CD3-CD40L TsAb _ D antibody samples at different final concentrations (25, 12.5, 6.25, 3.125ng/ml), mixed at room temperature for 3 to 5min, co-cultured at 37 ℃ for 3 hours, and then 1 was added to each well0 mul of CCK-8, continuing the reaction for 2-3 h at 37 ℃, and then measuring OD by using an enzyme-labeling instrument₄₅₀The cell killing efficiency was calculated according to the following formula, and each set of experiments was tested 3 times repeatedly; the cell killing efficiency without any added antibody was also used as a blank.

The results are shown in FIG. 16: when CIK effector cells: raji target cells (E: T ratio) 1: 1, under the condition of not adding any antibody, the killing efficiency of the CIK cells to Raji cells for 3h is about 23 percent; under the condition of adding higher concentration of antibody (25, 12.5 and 6.25ng/ml), the killing efficiency of the CIK cells on Raji cells is remarkably improved, wherein the cell killing effect mediated by CD19-CD3-CD40L TsAb _ D is the best, the killing efficiency is about 94%, 90% and 82%, the killing efficiency is the second best of the CD19-CD3-CD40L TsAb _ M, the killing efficiency is about 91%, 88% and 73%, the killing efficiency of CD19-CD3BsAb is the weakest, and the killing efficiency is about 80%, 54% and 54% respectively; the killing efficiency of the CIK cells on Raji cells mediated by CD19-CD3-CD40L TsAb _ D and CD19-CD3-CD40L TsAb _ M is improved to some extent under the condition of adding lower concentration of antibody (3.125ng/ml), the killing efficiency is about 68% and 61%, respectively, and the killing efficiency of CD19-CD3BsAb is basically ineffective compared with a blank control. The results show that the target killing activity of T cells on CD19 positive tumor cells mediated by two forms of CD19-CD3-CD40L TiTE trispecific antibodies is better than that of CD19-CD3 BiTE bispecific antibodies, wherein the dimeric form has better effect than the monomeric form.

Example 21: construction of eukaryotic expression vectors for CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D

In the present invention, the TiTE trispecific antibody targeting the human CD19 protein on the surface of lymphoma B cells, the human CD3 on the surface of T cells and the T cell positive co-stimulatory molecule CD27 protein was named CD19-CD3-CD27 TsAb.

First, CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD19-CD3-CD27TsAb _ M is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-CD 27 scFv are connected by a Linker (Linker), specifically, the sequences of the anti-CD 19 scFv and the anti-CD3 scFv are connected by a Linker 1(Linker 1), and the sequences of the anti-CD3 scFv and the anti-CD 27 scFv are connected by a Linker 2(Linker 2).

The specific construction scheme of the dimer form of CD19-CD3-CD27TsAb _ D is as follows: the sequences of the anti-CD 19 scFv, the anti-CD3 scFv and the anti-CD 27 scFv are connected by a Linker (Linker), specifically, the anti-CD 19 scFv and the anti-CD3 scFv are connected by a Linker 1(Linker 1), and the anti-CD3 scFv and the anti-CD 27 scFv are connected by an IgD hinge region (Ala 90-Val 170) as a Linker 2(Linker 2).

For expression of the trispecific antibody in mammalian cells, the mammalian system expression was codon optimized for the anti-CD 19 scFv, anti-CD3 scFv, and anti-CD 27 scFv sequences.

The nucleotide sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 64.

The nucleotide sequence of the anti-CD3 scFv is shown in SEQ ID NO. 67.

The nucleotide sequence of the heavy chain variable region of the anti-CD 27 scFv is shown in SEQ ID NO.86, and specifically comprises the following steps:

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

the nucleotide sequence of the light chain variable region of the anti-CD 27 scFv is shown in SEQ ID NO.87, and specifically comprises the following steps:

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

the nucleotide sequence of the anti-CD 27 scFv is shown in SEQ ID NO.85, and specifically comprises the following steps:

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

the nucleotide sequence of the monomeric CD19-CD3-CD27TsAb _ M connecting fragment 1(Linker 1) is shown as SEQ ID NO. 2.

The nucleotide sequence of the monomeric CD19-CD3-CD27TsAb _ M connecting fragment 2(Linker 2) is shown as SEQ ID NO. 4.

The nucleotide sequence of the dimeric form of CD19-CD3-CD27TsAb _ D connecting fragment 1(Linker 1) is shown in SEQ ID NO. 6.

The nucleotide sequence of the dimeric form of CD19-CD3-CD27TsAb _ D connecting fragment 2(Linker 2) is shown in SEQ ID NO. 8.

Secondly, construction of eukaryotic expression vectors of CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D

The construction and expression of the tri-specific antibody of the invention select a transient expression vector pcDNA3.1 (purchased from Shanghai Ying Jun Biotech Co., Ltd.) of mammalian cell protein. To construct monospecific and dimeric forms of trispecific antibodies, primers as shown in Table 6 were designed, all of which were synthesized by Suzhou Jinzhi Biotech, Inc., and gene templates for amplification were synthesized by Suzhou Hongxin Tech, Inc., respectively.

Cloning construction for CD19-CD3-CD27TsAb _ M Signal peptide fragment was first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3- (GGGGS)₃Amplification of anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, (GGGGS) from-CD 27-F and pcDNA3.1-CD27-R₃Linker 2+ anti-CD 27 scFv gene sequence; cloning for CD19-CD3-CD27TsAb _ D was carried out by first amplifying signal peptide fragments using primers pcDNA3.1-Sig-F and Sig-R, and then amplifying gene sequences of anti-CD 19 scFv, GGGGS Linker 1+ anti-CD3 scFv, IgD hinge region Linker2 and anti-CD 27 scFv using primers Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CD27-F and pcDNA3.1-CD27-R, respectively. After amplification, the amplified DNA is used

Sequencing revealed that the full-length gene sequences of the monomeric form of CD19-CD3-CD27TsAb _ M and the dimeric form of CD19-CD3-CD27TsAb _ D were correct and consistent with the expectations.

Specifically, the nucleotide sequence of the monomeric form of CD19-CD3-CD27TsAb _ M is shown in SEQ ID NO.37, and specifically comprises:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

the nucleotide sequence of the dimeric form of CD19-CD3-CD27TsAb _ D is shown in SEQ ID NO.39, and specifically comprises the following components:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

TABLE 6 primers used in the cloning of the CD19-CD3-CD27 trispecific antibody Gene

Example 22: expression and purification of CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D

Expression of CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D

1.3. preparation of transfection complex: for each project (CD19-CD3-CD27 TsAb _ M and CD19-CD3-CD27TsAb _ D), two centrifuge tubes/culture flasks were prepared, each placed at 20ml, and the recombinant plasmids prepared in example 21 were taken:

Purification of CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ 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 CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D were analyzed by SDS-PAGE, and the electrophoretograms under reducing and non-reducing conditions are shown in FIG. 17. As can be seen from the figure, the purity of both the CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D recombinant proteins was > 95% after purification on a Protein L affinity column: wherein the theoretical molecular weight of the recombinant protein CD19-CD3-CD27TsAb _ M is 80.1kDa, 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 trispecific antibody is in a monomer form (FIG. 17A); the theoretical molecular weight of the recombinant CD19-CD3-CD27TsAb _ D protein is 88.0kDa, 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 (about 180kDa) under non-reducing conditions (FIG. 17B), indicating that the two protein molecules can form disulfide bonds through the IgD hinge region to each other, and thus the trispecific 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 no reading frame and are consistent with the theoretical N/C terminal amino acid sequence, and mass spectrometry further confirms that CD19-CD3-CD27TsAb _ M is in a monomer form, and CD19-CD3-CD27TsAb _ D is in a dimer form.

Therefore, it can be known that the amino acid sequence of the monomeric form of CD19-CD3-CD27TsAb _ M is shown in SEQ ID No.36, specifically:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。

the amino acid sequence of the dimeric form of CD19-CD3-CD27TsAb _ D is shown in SEQ ID NO.38, and specifically comprises the following components: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK are provided.

The amino acid sequence of the anti-CD 19 scFv is shown in SEQ ID NO. 40.

The amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO. 43.

The amino acid sequence of the anti-CD 27 scFv is shown as SEQ ID NO.61, and specifically comprises the following steps:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。

the amino acid sequence of the heavy chain variable region of the anti-CD 27 scFv is shown in SEQ ID NO.62, and specifically comprises the following steps:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSS。

the amino acid sequence of the light chain variable region of the anti-CD 27 scFv is shown in SEQ ID NO.63, and specifically comprises the following steps:

DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。

the amino acid sequence of the monomeric CD19-CD3-CD27TsAb _ M connecting fragment 1(Linker 1) is shown as SEQ ID NO. 1.

The amino acid sequence of the monomeric CD19-CD3-CD27TsAb _ M connecting fragment 2(Linker 2) is shown as SEQ ID NO. 3.

The amino acid sequence of the dimeric form of CD19-CD3-CD27TsAb _ D connecting fragment 1(Linker 1) is shown in SEQ ID NO. 5.

The amino acid sequence of the dimeric form of CD19-CD3-CD27TsAb _ D connecting fragment 2(Linker 2) is shown in SEQ ID NO. 7.

Example 23: ELISA for detecting the antigen binding activity of CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D

ELISA operation steps:

1. recombinant antigen coating: human CD19-hFc, human CD3-hFc and human CD27-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;

3. sample adding: after 4 times of PBS plate washing, purified trispecific antibody samples were added, 100. mu.l/well, incubated at 37 ℃ for 1 hour, sample gradient preparation method: taking 10 mu g/ml purified CD19-CD3-CD27TsAb _ M or CD19-CD3-CD27TsAb _ D as a starting concentration, carrying out a dilution by multiple of 6 gradients, wherein each gradient is provided with 2 duplicate wells;

The ELISA results are shown in fig. 18A and 18B: FIG. 18A illustrates that CD19-CD3-CD27TsAb _ M has in vitro binding activity to recombinant antigens CD19-hFc, CD3-hFc, and CD27-hFc, with the highest CD27 binding activity and the second lowest CD19 binding activity, and the weaker CD3 binding activity; FIG. 18B illustrates that CD19-CD3-CD27TsAb _ D also has in vitro binding activity with recombinant antigens CD19-hFc, CD3-hFc, and CD27-hFc, with the highest CD27 binding activity and the second lowest CD19 binding activity, and the weaker CD3 binding activity.

Example 24: CD19-CD3-CD27 trispecific antibody mediated cell killing experiment

Using human Peripheral Blood Mononuclear Cells (PBMC) as experimental material, the monomeric form of the TiTE trispecific antibody (CD19-CD3-CD27 TsAb _ M), the dimeric form of the TiTE trispecific antibody (CD19-CD3-CD27 TsAb _ D) and the anti-CD 19/anti-CD 3 BiTE bispecific antibody (CD19-CD 3BsAb, available from Wujiang Korea protein technology Co., Ltd.) prepared by the present invention were allowed to act on human PBMC (CD3 PBMC) of the same donor source⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺Purchased from ATCC), cell death was detected, and the difference in the killing efficiency of CCL-86Raji target cells by three antibody-mediated CIK effector cells was compared.

Cell killing experiment step:

killing efficiency of CIK cells against Raji cells: performing cell killing experiment in 96-well plate with reaction volume of 100uL, collecting the above cultured CIK cells at 1 × 10⁵Adding Raji cells at 1X 10⁵Separately (CIK effector cells: Raji target cells (E: T ratio) 1: 1), respectively adding CD19-CD3BsAb, CD19-CD3-CD27TsAb _ M and CD19-CD3-CD27TsAb _ D antibody samples with different final concentrations (25, 12.5, 6.25 and 3.125ng/ml), uniformly mixing at room temperature for 3-5 min, co-culturing at 37 ℃ for 3h, adding 10 mul of CCK-8 into each well, continuously reacting at 37 ℃ for 2-3 h, and then measuring OD by using an enzyme reader₄₅₀The cell killing efficiency was calculated according to the following formula, and each set of experiments was tested 3 times repeatedly; the cell killing efficiency without any added antibody was also used as a blank.

The results are shown in FIG. 19: when CIK effector cells: raji target cells (E: T ratio) 1: 1, under the condition of not adding any antibody, the killing efficiency of the CIK cells to Raji cells for 3h is about 23 percent; under the condition of adding higher concentration of antibody (25, 12.5 and 6.25ng/ml), the killing efficiency of the CIK cells on Raji cells is remarkably improved, wherein the cell killing effect mediated by CD19-CD3-CD27TsAb _ D is the best, the killing efficiency is respectively about 89%, 84% and 74%, the killing efficiency is the second best of the killing efficiency of CD19-CD3-CD27TsAb _ M, the killing efficiency is respectively about 89%, 84% and 67%, the killing efficiency of CD19-CD3BsAb is the weakest, and the killing efficiency is respectively about 80%, 54% and 54%; with the addition of lower concentrations of antibody (3.125ng/ml), the killing efficiency of the CIK cells on Raji cells mediated by CD19-CD3-CD27TsAb _ D and CD19-CD3-CD27TsAb _ M is still improved to some extent, the killing efficiency is about 55% and 49%, respectively, and the killing efficiency of CD19-CD3BsAb is basically ineffective compared with the blank control. The results show that the target killing activity of T cells on CD19 positive tumor cells mediated by two forms of CD19-CD3-CD27 TiTE trispecific antibodies is better than that of CD19-CD3 BiTE bispecific antibodies, wherein the dimeric form has better effect than the monomeric form.

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 offshore Biotechnology Ltd

<120> a trifunctional molecule combining CD19, CD3 and T cell positive co-stimulatory molecule and application thereof

<130> 164332

<160> 115

<170> PatentIn version 3.3

<210> 1

<211> 5

<212> PRT

<213> Artificial

<220>

<223> of linker fragment 1 in anti-CD 19/anti-CD 3/anti-T cell positive costimulatory molecule trispecific antibody in monomeric form

Amino acid sequence

<400> 1

Gly Gly Gly Gly Ser

1 5

<210> 2

<211> 15

<212> DNA

<213> Artificial

<220>

Nucleotide sequence

<400> 2

ggtggcggag ggtcc 15

<210> 3

<211> 15

<212> PRT

<213> Artificial

<220>

<223> of linker fragment 2 in monomeric form of anti-CD 19/anti-CD 3/anti-T cell positive costimulatory molecule trispecific antibody

Amino acid sequence

<400> 3

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

1 5 10 15

<210> 4

<211> 45

<212> DNA

<213> Artificial

<220>

Nucleotide sequence

<400> 4

ggaggcggag gttccggcgg tgggggatcg gggggtggag ggagt 45

<210> 5

<211> 5

<212> PRT

<213> Artificial

<220>

<223> linker fragment in dimeric form of anti-CD 19/anti-CD 3/anti-T cell positive costimulatory molecule trispecific antibody

1 amino acid sequence

<400> 5

Gly Gly Gly Gly Ser

1 5

<210> 6

<211> 15

<212> DNA

<213> Artificial

<220>

1 nucleotide sequence

<400> 6

ggtggcggag ggtcc 15

<210> 7

<211> 81

<212> PRT

<213> Artificial

<220>

2, or a pharmaceutically acceptable salt thereof

<400> 7

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> 8

<211> 243

<212> DNA

<213> Artificial

<220>

2, or a pharmaceutically acceptable salt thereof

<400> 8

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> 9

<211> 134

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of extracellular region of T cell positive co-stimulatory molecule human CD28

<400> 9

Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn

1 5 10 15

Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu

20 25 30

Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys

35 40 45

Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr

50 55 60

Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr

65 70 75 80

Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile

85 90 95

Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly

100 105 110

Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe

115 120 125

Pro Gly Pro Ser Lys Pro

130

<210> 10

<211> 163

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of human 4-1BB extracellular domain of T cell positive co-stimulatory molecule

<400> 10

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

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

145 150 155 160

Ser Pro Gln

<210> 11

<211> 120

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of human ICOS extracellular domain of T cell positive co-stimulatory molecule

<400> 11

Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile Phe His Asn Gly

1 5 10 15

Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val Gln Gln Phe Lys

20 25 30

Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp Leu Thr Lys Thr

35 40 45

Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu Lys Phe Cys His

50 55 60

Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu Tyr Asn Leu Asp

65 70 75 80

His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser Ile Phe Asp Pro

85 90 95

Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu His Ile Tyr Glu

100 105 110

Ser Gln Leu Cys Cys Gln Leu Lys

115 120

<210> 12

<211> 186

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of human OX40 extracellular region of T cell positive co-stimulatory molecule

<400> 12

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His

1 5 10 15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln

20 25 30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

35 40 45

Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly

50 55 60

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

65 70 75 80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

85 90 95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

100 105 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

115 120 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

130 135 140

Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr

145 150 155 160

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

165 170 175

Arg Pro Val Glu Val Pro Gly Gly Arg Ala

180 185

<210> 13

<211> 137

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of human GITR extracellular domain of T cell positive co-stimulatory molecule

<400> 13

Gln Arg Pro Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu

1 5 10 15

Gly Thr Gly Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys

20 25 30

Cys Arg Asp Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met

35 40 45

Cys Val Gln Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys

50 55 60

Arg His His Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys

65 70 75 80

Phe Ser Phe Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser

85 90 95

Gly Gly His Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe

100 105 110

Gly Phe Leu Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys

115 120 125

Val Pro Gly Ser Pro Pro Ala Glu Pro

130 135

<210> 14

<211> 215

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of extracellular region of T cell positive co-stimulatory molecule human CD40L

<400> 14

His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp

1 5 10 15

Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser

20 25 30

Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe

35 40 45

Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser

50 55 60

Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val

65 70 75 80

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

85 90 95

Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly

100 105 110

Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln

115 120 125

Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile

130 135 140

Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu

145 150 155 160

Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser

165 170 175

Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe

180 185 190

Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr

195 200 205

Ser Phe Gly Leu Leu Lys Leu

210 215

<210> 15

<211> 172

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of extracellular region of T cell positive co-stimulatory molecule human CD27

<400> 15

Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala Gln

1 5 10 15

Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val Lys

20 25 30

Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile Pro

35 40 45

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

50 55 60

Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile Thr

65 70 75 80

Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp Lys

85 90 95

Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala Arg

100 105 110

Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro Tyr

115 120 125

Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr Leu

130 135 140

Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp Pro

145 150 155 160

Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

165 170

<210> 16

<211> 759

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-4-1BB TsAb _ M in monomer form

<400> 16

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp

545 550 555 560

Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu

565 570 575

Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

580 585 590

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

595 600 605

Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Thr Lys Val Glu Ile Lys Arg

755

<210> 17

<211> 2277

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-4-1BB TsAb _ M in monomer form

<400> 17

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaagggaggc 1500

ggaggttccg gcggtggggg atcggggggt ggagggagtc aggtgcagct gcagcagtgg 1560

ggcgccggcc tgctgaagcc cagcgagacc ctgagcctga cctgcgccgt gtacggcggc 1620

agcttcagcg gctactactg gagctggatc cgccagagcc ccgagaaggg cctggagtgg 1680

atcggcgaga tcaaccacgg cggctacgtg acctacaacc ccagcctgga gagccgcgtg 1740

accatcagcg tggacaccag caagaaccag ttcagcctga agctgagcag cgtgaccgcc 1800

gccgacaccg ccgtgtacta ctgcgcccgc gactacggcc ccggcaacta cgactggtac 1860

ttcgacctgt ggggccgcgg caccctggtg accgtgagca gcggcggcgg cggcagcggc 1920

ggcggcggca gcggcggcgg cggcagcgag atcgtgctga cccagagccc cgccaccctg 1980

agcctgagcc ccggcgagcg cgccaccctg agctgccgcg ccagccagag cgtgagcagc 2040

tacctggcct ggtaccagca gaagcccggc caggcccccc gcctgctgat ctacgacgcc 2100

agcaaccgcg ccaccggcat ccccgcccgc ttcagcggca gcggcagcgg caccgacttc 2160

accctgacca tcagcagcct ggagcccgag gacttcgccg tgtactactg ccagcagcgc 2220

agcaactggc cccccgccct gaccttctgc ggcggcacca aggtggagat caagcgc 2277

<210> 18

<211> 825

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-4-1BB TsAb _ D in dimer form

<400> 18

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Leu Gly Val Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys

580 585 590

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

595 600 605

Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu

610 615 620

Glu Trp Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro

625 630 635 640

Ser Leu Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln

645 650 655

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

660 665 670

Tyr Cys Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp

675 680 685

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

Gly Gly Thr Lys Val Glu Ile Lys Arg

820 825

<210> 19

<211> 2475

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-4-1BB TsAb _ D in dimeric form

<400> 19

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaaggccagc 1500

aagagcaaga aggagatctt ccgctggccc gagagcccca aggcccaggc cagcagcgtg 1560

cccaccgccc agccccaggc cgagggcagc ctggccaagg ccaccaccgc ccccgccacc 1620

acccgcaaca ccggccgcgg cggcgaggag aagaagaagg agaaggagaa ggaggagcag 1680

gaggagcgcg agaccaagac ccccgagtgc cccagccaca cccagcccct gggcgtgcag 1740

gtgcagctgc agcagtgggg cgccggcctg ctgaagccca gcgagaccct gagcctgacc 1800

tgcgccgtgt acggcggcag cttcagcggc tactactgga gctggatccg ccagagcccc 1860

gagaagggcc tggagtggat cggcgagatc aaccacggcg gctacgtgac ctacaacccc 1920

agcctggaga gccgcgtgac catcagcgtg gacaccagca agaaccagtt cagcctgaag 1980

ctgagcagcg tgaccgccgc cgacaccgcc gtgtactact gcgcccgcga ctacggcccc 2040

ggcaactacg actggtactt cgacctgtgg ggccgcggca ccctggtgac cgtgagcagc 2100

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgagat cgtgctgacc 2160

cagagccccg ccaccctgag cctgagcccc ggcgagcgcg ccaccctgag ctgccgcgcc 2220

agccagagcg tgagcagcta cctggcctgg taccagcaga agcccggcca ggccccccgc 2280

ctgctgatct acgacgccag caaccgcgcc accggcatcc ccgcccgctt cagcggcagc 2340

ggcagcggca ccgacttcac cctgaccatc agcagcctgg agcccgagga cttcgccgtg 2400

tactactgcc agcagcgcag caactggccc cccgccctga ccttctgcgg cggcaccaag 2460

gtggagatca agcgc 2475

<210> 20

<211> 760

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-ICOS TsAb _ M in monomeric form

<400> 20

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Met Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys

565 570 575

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala

580 585 590

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

595 600 605

Cys Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp Ala

610 615 620

Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

645 650 655

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

660 665 670

Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu Leu Ala Trp

675 680 685

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Val Ala

690 695 700

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

705 710 715 720

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

725 730 735

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

740 745 750

Gln Gly Thr Lys Val Glu Ile Lys

755 760

<210> 21

<211> 2280

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-ICOS TsAb _ M in monomeric form

<400> 21

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaagggaggc 1500

ggaggttccg gcggtggggg atcggggggt ggagggagtc aggtgcagct ggtgcagagc 1560

ggcgccgagg tgaagaagcc cggcgccagc gtgaaggtga gctgcaaggc cagcggctac 1620

accttcaccg gctactacat gcactgggtg cgccaggccc ccggccaggg cctggagtgg 1680

atgggctgga tcaaccccca cagcggcggc accaactacg cccagaagtt ccagggccgc 1740

gtgaccatga cccgcgacac cagcatcagc accgcctaca tggagctgag ccgcctgcgc 1800

agcgacgaca ccgccgtgta ctactgcgcc cgcacctact actacgacag cagcggctac 1860

taccacgacg ccttcgacat ctggggccag ggcaccatgg tgaccgtgag cagcggcggc 1920

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatccagat gacccagagc 1980

cccagcagcg tgagcgccag cgtgggcgac cgcgtgacca tcacctgccg cgccagccag 2040

ggcatcagcc gcctgctggc ctggtaccag cagaagcccg gcaaggcccc caagctgctg 2100

atctacgtgg ccagcagcct gcagagcggc gtgcccagcc gcttcagcgg cagcggcagc 2160

ggcaccgact tcaccctgac catcagcagc ctgcagcccg aggacttcgc cacctactac 2220

tgccagcagg ccaacagctt cccctggacc ttcggccagg gcaccaaggt ggagatcaag 2280

<210> 22

<211> 826

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-ICOS TsAb _ D in dimeric form

<400> 22

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Glu Trp Met Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala

625 630 635 640

Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser

645 650 655

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

660 665 670

Tyr Tyr Cys Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His

675 680 685

Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

690 695 700

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

705 710 715 720

Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp

725 730 735

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu Leu

740 745 750

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

755 760 765

Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

770 775 780

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

785 790 795 800

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp Thr

805 810 815

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

820 825

<210> 23

<211> 2478

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-ICOS TsAb _ D in dimeric form

<400> 23

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaaggccagc 1500

aagagcaaga aggagatctt ccgctggccc gagagcccca aggcccaggc cagcagcgtg 1560

cccaccgccc agccccaggc cgagggcagc ctggccaagg ccaccaccgc ccccgccacc 1620

acccgcaaca ccggccgcgg cggcgaggag aagaagaagg agaaggagaa ggaggagcag 1680

gaggagcgcg agaccaagac ccccgagtgc cccagccaca cccagcccct gggcgtgcag 1740

gtgcagctgg tgcagagcgg cgccgaggtg aagaagcccg gcgccagcgt gaaggtgagc 1800

tgcaaggcca gcggctacac cttcaccggc tactacatgc actgggtgcg ccaggccccc 1860

ggccagggcc tggagtggat gggctggatc aacccccaca gcggcggcac caactacgcc 1920

cagaagttcc agggccgcgt gaccatgacc cgcgacacca gcatcagcac cgcctacatg 1980

gagctgagcc gcctgcgcag cgacgacacc gccgtgtact actgcgcccg cacctactac 2040

tacgacagca gcggctacta ccacgacgcc ttcgacatct ggggccaggg caccatggtg 2100

accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgac 2160

atccagatga cccagagccc cagcagcgtg agcgccagcg tgggcgaccg cgtgaccatc 2220

acctgccgcg ccagccaggg catcagccgc ctgctggcct ggtaccagca gaagcccggc 2280

aaggccccca agctgctgat ctacgtggcc agcagcctgc agagcggcgt gcccagccgc 2340

ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcagcct gcagcccgag 2400

gacttcgcca cctactactg ccagcaggcc aacagcttcc cctggacctt cggccagggc 2460

accaaggtgg agatcaag 2478

<210> 24

<211> 755

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-OX40TsAb _ M in monomer form

<400> 24

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys

565 570 575

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

580 585 590

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

595 600 605

Arg Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp Gly Gln

610 615 620

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

625 630 635 640

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

645 650 655

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

660 665 670

Ser Gln Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu

675 680 685

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

690 695 700

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

705 710 715 720

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

725 730 735

Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu

740 745 750

Ile Lys Arg

755

<210> 25

<211> 2265

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-OX40TsAb _ M in monomer form

<400> 25

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaagggaggc 1500

ggaggttccg gcggtggggg atcggggggt ggagggagtc agctggtgga gagcggcggc 1560

ggcctggtgc agcccggcgg cagcctgcgc ctgagctgcg ccgccagcgg cttcaccttc 1620

agcagctaca gcatgaactg ggtgcgccag gcccccggca agggcctgga gtgggtgagc 1680

tacatcagca gcagcagcag caccatctac tacgccgaca gcgtgaaggg ccgcttcacc 1740

atcagccgcg acaacgccaa gaacagcctg tacctgcaga tgaacagcct gcgcgacgag 1800

gacaccgccg tgtactactg cgcccgcggc gtgtaccaca acggctggag cttcttcgac 1860

tactggggcc agggcaccct gctgaccgtg agcagcggcg gcggcggcag cggcggcggc 1920

ggcagcggcg gcggcggcag cgacatccag atgacccaga gccccagcag cctgagcgcc 1980

agcgtgggca accgcgtgac catcacctgc cgcgccagcc aggacatcag cagctggctg 2040

gcctggtacc agcagaagcc cgagaaggcc cccaagagcc tgatctacgc cgccagcagc 2100

ctgcagagcg gcgtgcccag ccgcttcagc ggcagcggca gcggcaccga cttcaccctg 2160

accatcagca gcctgcagcc cgaggacttc gccacctact actgccagca gtacaacagc 2220

taccccctga ccttcggcca gggcacccgc ctggagatca agcgc 2265

<210> 26

<211> 821

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-OX40TsAb _ D in dimeric form

<400> 26

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser

625 630 635 640

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

645 650 655

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

660 665 670

Cys Ala Arg Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp

675 680 685

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

690 695 700

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

Leu Glu Ile Lys Arg

820

<210> 27

<211> 2463

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-OX40TsAb _ D in dimeric form

<400> 27

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaaggccagc 1500

aagagcaaga aggagatctt ccgctggccc gagagcccca aggcccaggc cagcagcgtg 1560

cccaccgccc agccccaggc cgagggcagc ctggccaagg ccaccaccgc ccccgccacc 1620

acccgcaaca ccggccgcgg cggcgaggag aagaagaagg agaaggagaa ggaggagcag 1680

gaggagcgcg agaccaagac ccccgagtgc cccagccaca cccagcccct gggcgtgcag 1740

ctggtggaga gcggcggcgg cctggtgcag cccggcggca gcctgcgcct gagctgcgcc 1800

gccagcggct tcaccttcag cagctacagc atgaactggg tgcgccaggc ccccggcaag 1860

ggcctggagt gggtgagcta catcagcagc agcagcagca ccatctacta cgccgacagc 1920

gtgaagggcc gcttcaccat cagccgcgac aacgccaaga acagcctgta cctgcagatg 1980

aacagcctgc gcgacgagga caccgccgtg tactactgcg cccgcggcgt gtaccacaac 2040

ggctggagct tcttcgacta ctggggccag ggcaccctgc tgaccgtgag cagcggcggc 2100

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatccagat gacccagagc 2160

cccagcagcc tgagcgccag cgtgggcaac cgcgtgacca tcacctgccg cgccagccag 2220

gacatcagca gctggctggc ctggtaccag cagaagcccg agaaggcccc caagagcctg 2280

atctacgccg ccagcagcct gcagagcggc gtgcccagcc gcttcagcgg cagcggcagc 2340

ggcaccgact tcaccctgac catcagcagc ctgcagcccg aggacttcgc cacctactac 2400

tgccagcagt acaacagcta ccccctgacc ttcggccagg gcacccgcct ggagatcaag 2460

cgc 2463

<210> 28

<211> 754

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-GITR TsAb _ M in monomeric form

<400> 28

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr

530 535 540

Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu

545 550 555 560

Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro

565 570 575

Ser Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln

580 585 590

Val Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ala Ala Thr Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys

645 650 655

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

660 665 670

Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly

675 680 685

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

690 695 700

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

705 710 715 720

Thr Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln

725 730 735

Gln Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

740 745 750

Ile Lys

<210> 29

<211> 2262

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-GITR TsAb _ M in monomeric form

<400> 29

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaagggaggc 1500

ggaggttccg gcggtggggg atcggggggt ggagggagtc aggtgaccct gaaggagagc 1560

ggccccggca tcctgaagcc cagccagacc ctgagcctga cctgcagctt cagcggcttc 1620

agcctgagca ccagcggcat gggcgtgggc tggatccgcc agcccagcgg caagggcctg 1680

gagtggctgg cccacatctg gtgggacgac gacaagtact acaaccccag cctgaagagc 1740

cagctgacca tcagcaagga caccagccgc aaccaggtgt tcctgaagat caccagcgtg 1800

gacaccgccg acgccgccac ctactactgc gcccgcaccc gccgctactt ccccttcgcc 1860

tactggggcc agggcaccct ggtgaccgtg agcagcggcg gcggcggcag cggcggcggc 1920

ggcagcggcg gcggcggcag cgacatcgtg atgacccaga gccagaagtt catgagcacc 1980

agcgtgggcg accgcgtgag cgtgacctgc aaggccagcc agaacgtggg caccaacgtg 2040

gcctggtacc agcagaagcc cggccagagc cccaaggccc tgatctacag cgccagctac 2100

cgctacagcg gcgtgcccga ccgcttcacc ggcagcggca gcggcaccga cttcaccctg 2160

accatcaaca acgtgcacag cgaggacctg gccgagtact tctgccagca gtacaacacc 2220

gaccccctga ccttcggcgc cggcaccaag ctggagatca ag 2262

<210> 30

<211> 820

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-GITR TsAb _ D in dimeric form

<400> 30

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys

610 615 620

Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr

625 630 635 640

Asn Pro Ser Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg

645 650 655

Asn Gln Val Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ala Ala

660 665 670

Thr Tyr Tyr Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp

675 680 685

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

690 695 700

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser

705 710 715 720

Gln Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys

725 730 735

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys

740 745 750

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

755 760 765

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

770 775 780

Thr Leu Thr Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr Phe

785 790 795 800

Cys Gln Gln Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys

805 810 815

Leu Glu Ile Lys

820

<210> 31

<211> 2460

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-GITR TsAb _ D in dimeric form

<400> 31

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaaggccagc 1500

aagagcaaga aggagatctt ccgctggccc gagagcccca aggcccaggc cagcagcgtg 1560

cccaccgccc agccccaggc cgagggcagc ctggccaagg ccaccaccgc ccccgccacc 1620

acccgcaaca ccggccgcgg cggcgaggag aagaagaagg agaaggagaa ggaggagcag 1680

gaggagcgcg agaccaagac ccccgagtgc cccagccaca cccagcccct gggcgtgcag 1740

gtgaccctga aggagagcgg ccccggcatc ctgaagccca gccagaccct gagcctgacc 1800

tgcagcttca gcggcttcag cctgagcacc agcggcatgg gcgtgggctg gatccgccag 1860

cccagcggca agggcctgga gtggctggcc cacatctggt gggacgacga caagtactac 1920

aaccccagcc tgaagagcca gctgaccatc agcaaggaca ccagccgcaa ccaggtgttc 1980

ctgaagatca ccagcgtgga caccgccgac gccgccacct actactgcgc ccgcacccgc 2040

cgctacttcc ccttcgccta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 2100

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatcgtgat gacccagagc 2160

cagaagttca tgagcaccag cgtgggcgac cgcgtgagcg tgacctgcaa ggccagccag 2220

aacgtgggca ccaacgtggc ctggtaccag cagaagcccg gccagagccc caaggccctg 2280

atctacagcg ccagctaccg ctacagcggc gtgcccgacc gcttcaccgg cagcggcagc 2340

ggcaccgact tcaccctgac catcaacaac gtgcacagcg aggacctggc cgagtacttc 2400

tgccagcagt acaacaccga ccccctgacc ttcggcgccg gcaccaagct ggagatcaag 2460

<210> 32

<211> 752

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-CD40L TsAb _ M in monomer form

<400> 32

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

565 570 575

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

580 585 590

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

595 600 605

Cys Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

610 615 620

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

625 630 635 640

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

645 650 655

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

<210> 33

<211> 2256

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-CD40L TsAb _ M in monomer form

<400> 33

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaagggaggc 1500

ggaggttccg gcggtggggg atcggggggt ggagggagtg aggtgcagct gctggagagc 1560

ggcggcggcc tggtgcagcc cggcggcagc ctgcgcctga gctgcgccgc cagcggcttc 1620

accttcagca gctacgccat gagctgggtg cgccaggccc ccggcaaggg cctggagtgg 1680

gtgagcgcca tcagcggcag cggcggcagc acctactacg ccgacagcgt gaagggccgc 1740

ttcaccatca gccgcgacaa cagcaagaac accctgtacc tgcagatgaa cagcctgcgc 1800

gccgaggaca ccgccgtgta ctactgcgcc aagagctacg gcgccttcga ctactggggc 1860

cagggcaccc tggtgaccgt gagcagcggc ggcggcggca gcggcggcgg cggcagcggc 1920

ggcggcggca gcgacatcca gatgacccag agccccagca gcctgagcgc cagcgtgggc 1980

gaccgcgtga ccatcacctg ccgcgccagc cagagcatca gcagctacct gaactggtac 2040

cagcagaagc ccggcaaggc ccccaagctg ctgatctacg ccgccagcag cctgcagagc 2100

ggcgtgccca gccgcttcag cggcagcggc agcggcaccg acttcaccct gaccatcagc 2160

agcctgcagc ccgaggactt cgccacctac tactgccagc agagctacag cacccccaac 2220

accttcggcc agggcaccaa ggtggagatc aagcgc 2256

<210> 34

<211> 818

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-CD40L TsAb _ D in dimer form

<400> 34

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

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

645 650 655

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

660 665 670

Tyr Tyr Cys Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly

675 680 685

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

690 695 700

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

705 710 715 720

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

785 790 795 800

Ser Tyr Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

805 810 815

Lys Arg

<210> 35

<211> 2454

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-CD40L TsAb _ D in dimer form

<400> 35

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaaggccagc 1500

aagagcaaga aggagatctt ccgctggccc gagagcccca aggcccaggc cagcagcgtg 1560

cccaccgccc agccccaggc cgagggcagc ctggccaagg ccaccaccgc ccccgccacc 1620

acccgcaaca ccggccgcgg cggcgaggag aagaagaagg agaaggagaa ggaggagcag 1680

gaggagcgcg agaccaagac ccccgagtgc cccagccaca cccagcccct gggcgtggag 1740

gtgcagctgc tggagagcgg cggcggcctg gtgcagcccg gcggcagcct gcgcctgagc 1800

tgcgccgcca gcggcttcac cttcagcagc tacgccatga gctgggtgcg ccaggccccc 1860

ggcaagggcc tggagtgggt gagcgccatc agcggcagcg gcggcagcac ctactacgcc 1920

gacagcgtga agggccgctt caccatcagc cgcgacaaca gcaagaacac cctgtacctg 1980

cagatgaaca gcctgcgcgc cgaggacacc gccgtgtact actgcgccaa gagctacggc 2040

gccttcgact actggggcca gggcaccctg gtgaccgtga gcagcggcgg cggcggcagc 2100

ggcggcggcg gcagcggcgg cggcggcagc gacatccaga tgacccagag ccccagcagc 2160

ctgagcgcca gcgtgggcga ccgcgtgacc atcacctgcc gcgccagcca gagcatcagc 2220

agctacctga actggtacca gcagaagccc ggcaaggccc ccaagctgct gatctacgcc 2280

gccagcagcc tgcagagcgg cgtgcccagc cgcttcagcg gcagcggcag cggcaccgac 2340

ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagcag 2400

agctacagca cccccaacac cttcggccag ggcaccaagg tggagatcaa gcgc 2454

<210> 36

<211> 754

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-CD27TsAb _ M in monomer form

<400> 36

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Cys Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln

610 615 620

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

625 630 635 640

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

645 650 655

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

725 730 735

Gln Tyr Asn Thr Tyr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu

740 745 750

Ile Lys

<210> 37

<211> 2262

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-CD27TsAb _ M in monomer form

<400> 37

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaagggaggc 1500

ggaggttccg gcggtggggg atcggggggt ggagggagtc aggtgcagct ggtggagagc 1560

ggcggcggcg tggtgcagcc cggccgcagc ctgcgcctga gctgcgccgc cagcggcttc 1620

accttcagca gctacgacat gcactgggtg cgccaggccc ccggcaaggg cctggagtgg 1680

gtggccgtga tctggtacga cggcagcaac aagtactacg ccgacagcgt gaagggccgc 1740

ttcaccatca gccgcgacaa cagcaagaac accctgtacc tgcagatgaa cagcctgcgc 1800

gccgaggaca ccgccgtgta ctactgcgcc cgcggcagcg gcaactgggg cttcttcgac 1860

tactggggcc agggcaccct ggtgaccgtg agcagcggcg gcggcggcag cggcggcggc 1920

ggcagcggcg gcggcggcag cgacatccag atgacccaga gccccagcag cctgagcgcc 1980

agcgtgggcg accgcgtgac catcacctgc cgcgccagcc agggcatcag ccgctggctg 2040

gcctggtacc agcagaagcc cgagaaggcc cccaagagcc tgatctacgc cgccagcagc 2100

ctgcagagcg gcgtgcccag ccgcttcagc ggcagcggca gcggcaccga cttcaccctg 2160

accatcagca gcctgcagcc cgaggacttc gccacctact actgccagca gtacaacacc 2220

tacccccgca ccttcggcca gggcaccaag gtggagatca ag 2262

<210> 38

<211> 820

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-CD27TsAb _ D in dimer form

<400> 38

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

705 710 715 720

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

725 730 735

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

740 745 750

Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln

755 760 765

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

770 775 780

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

785 790 795 800

Cys Gln Gln Tyr Asn Thr Tyr Pro Arg Thr Phe Gly Gln Gly Thr Lys

805 810 815

Val Glu Ile Lys

820

<210> 39

<211> 2460

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-CD27TsAb _ D in dimeric form

<400> 39

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaaggccagc 1500

aagagcaaga aggagatctt ccgctggccc gagagcccca aggcccaggc cagcagcgtg 1560

cccaccgccc agccccaggc cgagggcagc ctggccaagg ccaccaccgc ccccgccacc 1620

acccgcaaca ccggccgcgg cggcgaggag aagaagaagg agaaggagaa ggaggagcag 1680

gaggagcgcg agaccaagac ccccgagtgc cccagccaca cccagcccct gggcgtgcag 1740

gtgcagctgg tggagagcgg cggcggcgtg gtgcagcccg gccgcagcct gcgcctgagc 1800

tgcgccgcca gcggcttcac cttcagcagc tacgacatgc actgggtgcg ccaggccccc 1860

ggcaagggcc tggagtgggt ggccgtgatc tggtacgacg gcagcaacaa gtactacgcc 1920

gacagcgtga agggccgctt caccatcagc cgcgacaaca gcaagaacac cctgtacctg 1980

cagatgaaca gcctgcgcgc cgaggacacc gccgtgtact actgcgcccg cggcagcggc 2040

aactggggct tcttcgacta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 2100

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatccagat gacccagagc 2160

cccagcagcc tgagcgccag cgtgggcgac cgcgtgacca tcacctgccg cgccagccag 2220

ggcatcagcc gctggctggc ctggtaccag cagaagcccg agaaggcccc caagagcctg 2280

atctacgccg ccagcagcct gcagagcggc gtgcccagcc gcttcagcgg cagcggcagc 2340

ggcaccgact tcaccctgac catcagcagc ctgcagcccg aggacttcgc cacctactac 2400

tgccagcagt acaacaccta cccccgcacc ttcggccagg gcaccaaggt ggagatcaag 2460

<210> 40

<211> 250

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-CD 19 scFv

<400> 40

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

245 250

<210> 41

<211> 124

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-CD 19 scFv

<400> 41

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

1 5 10 15

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

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 42

<211> 111

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-CD 19 scFv

<400> 42

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

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

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 43

<211> 243

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-CD3 scFv

<400> 43

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> 44

<211> 119

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-CD3 scFv

<400> 44

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> 45

<211> 106

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-CD3 scFv

<400> 45

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> 46

<211> 246

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-4-1 BB scFv

<400> 46

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 Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

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

225 230 235 240

Lys Val Glu Ile Lys Arg

245

<210> 47

<211> 121

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-4-1 BB scFv

<400> 47

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 Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 110

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-4-1 BB scFv

<400> 48

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

Ala Leu Thr Phe Cys Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

<210> 49

<211> 247

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-ICOS scFv

<400> 49

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 Gly Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp Ala Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

130 135 140

Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

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

165 170 175

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

180 185 190

Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Gly Thr Lys Val Glu Ile Lys

245

<210> 50

<211> 125

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of ICOS scFv

<400> 50

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 Gly Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp Ala Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 51

<211> 107

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of ICOS-resistant scFv

<400> 51

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 52

<211> 242

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-OX 40scFv

<400> 52

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

65 70 75 80

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

85 90 95

Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

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

145 150 155 160

Gln Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys

165 170 175

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

180 185 190

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

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile

225 230 235 240

Lys Arg

<210> 53

<211> 119

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-OX 40scFv

<400> 53

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

65 70 75 80

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

85 90 95

Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Leu Thr Val Ser Ser

115

<210> 54

<211> 108

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-OX 40scFv

<400> 54

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

1 5 10 15

Asn Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

100 105

<210> 55

<211> 241

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-GITR scFv

<400> 55

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr

85 90 95

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

100 105 110

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys Phe

130 135 140

Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser

145 150 155 160

Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val

180 185 190

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

195 200 205

Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln

210 215 220

Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 56

<211> 119

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-GITR SCFV

<400> 56

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 57

<211> 107

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-GITR scFv

<400> 57

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Val His Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Thr Asp Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 58

<211> 239

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-CD 40L scFv

<400> 58

Glu Val Gln Leu Leu 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 Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 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 Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile

145 150 155 160

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

165 170 175

Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235

<210> 59

<211> 116

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-CD 40L scFv

<400> 59

Glu Val Gln Leu Leu 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 Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 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 Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 60

<211> 108

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-CD 40L scFv

<400> 60

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn

85 90 95

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

100 105

<210> 61

<211> 241

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-CD 27 scFv

<400> 61

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

Asp 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 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 Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

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

225 230 235 240

Lys

<210> 62

<211> 119

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-CD 27 scFv

<400> 62

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

Asp 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 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 Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 63

<211> 107

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-CD 27 scFv

<400> 63

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 64

<211> 750

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-CD 19 scFv

<400> 64

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc 750

<210> 65

<211> 372

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-CD 19 scFv

<400> 65

caggtgcagc tgcagcagag cggcgccgag ctggtgcgcc ccggcagcag cgtgaagatc 60

agctgcaagg ccagcggcta cgccttcagc agctactgga tgaactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggccag atctggcccg gcgacggcga caccaactac 180

aacggcaagt tcaagggcaa ggccaccctg accgccgacg agagcagcag caccgcctac 240

atgcagctga gcagcctggc cagcgaggac agcgccgtgt acttctgcgc ccgccgcgag 300

accaccaccg tgggccgcta ctactacgcc atggactact ggggccaggg caccaccgtg 360

accgtgagca gc 372

<210> 66

<211> 333

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-CD 19 scFv

<400> 66

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aag 333

<210> 67

<211> 729

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-CD3 scFv

<400> 67

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> 68

<211> 357

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-CD3 scFv

<400> 68

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> 69

<211> 318

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-CD3 scFv

<400> 69

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> 70

<211> 738

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-4-1 BB scFv

<400> 70

caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60

acctgcgccg tgtacggcgg cagcttcagc ggctactact ggagctggat ccgccagagc 120

cccgagaagg gcctggagtg gatcggcgag atcaaccacg gcggctacgt gacctacaac 180

cccagcctgg agagccgcgt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgaccgc cgccgacacc gccgtgtact actgcgcccg cgactacggc 300

cccggcaact acgactggta cttcgacctg tggggccgcg gcaccctggt 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 ccccccgccc tgaccttctg cggcggcacc 720

aaggtggaga tcaagcgc 738

<210> 71

<211> 363

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-4-1 BB scFv

<400> 71

caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60

acctgcgccg tgtacggcgg cagcttcagc ggctactact ggagctggat ccgccagagc 120

cccgagaagg gcctggagtg gatcggcgag atcaaccacg gcggctacgt gacctacaac 180

cccagcctgg agagccgcgt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgaccgc cgccgacacc gccgtgtact actgcgcccg cgactacggc 300

cccggcaact acgactggta cttcgacctg tggggccgcg gcaccctggt gaccgtgagc 360

agc 363

<210> 72

<211> 330

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-4-1 BB scFv

<400> 72

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 ggccccccgc cctgaccttc 300

tgcggcggca ccaaggtgga gatcaagcgc 330

<210> 73

<211> 741

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-ICOS scFv

<400> 73

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc ggctactaca tgcactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatgggctgg atcaaccccc acagcggcgg caccaactac 180

gcccagaagt tccagggccg cgtgaccatg acccgcgaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt actactgcgc ccgcacctac 300

tactacgaca gcagcggcta ctaccacgac gccttcgaca tctggggcca gggcaccatg 360

gtgaccgtga gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 420

gacatccaga tgacccagag ccccagcagc gtgagcgcca gcgtgggcga ccgcgtgacc 480

atcacctgcc gcgccagcca gggcatcagc cgcctgctgg cctggtacca gcagaagccc 540

ggcaaggccc ccaagctgct gatctacgtg gccagcagcc tgcagagcgg cgtgcccagc 600

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 660

gaggacttcg ccacctacta ctgccagcag gccaacagct tcccctggac cttcggccag 720

ggcaccaagg tggagatcaa g 741

<210> 74

<211> 375

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of ICOS scFv

<400> 74

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc ggctactaca tgcactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatgggctgg atcaaccccc acagcggcgg caccaactac 180

gcccagaagt tccagggccg cgtgaccatg acccgcgaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt actactgcgc ccgcacctac 300

tactacgaca gcagcggcta ctaccacgac gccttcgaca tctggggcca gggcaccatg 360

gtgaccgtga gcagc 375

<210> 75

<211> 321

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of ICOS scFv

<400> 75

gacatccaga tgacccagag ccccagcagc gtgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gggcatcagc cgcctgctgg cctggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgtg gccagcagcc tgcagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag gccaacagct tcccctggac cttcggccag 300

ggcaccaagg tggagatcaa g 321

<210> 76

<211> 726

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-OX 40scFv

<400> 76

cagctggtgg agagcggcgg cggcctggtg cagcccggcg gcagcctgcg cctgagctgc 60

gccgccagcg gcttcacctt cagcagctac agcatgaact gggtgcgcca ggcccccggc 120

aagggcctgg agtgggtgag ctacatcagc agcagcagca gcaccatcta ctacgccgac 180

agcgtgaagg gccgcttcac catcagccgc gacaacgcca agaacagcct gtacctgcag 240

atgaacagcc tgcgcgacga ggacaccgcc gtgtactact gcgcccgcgg cgtgtaccac 300

aacggctgga gcttcttcga ctactggggc cagggcaccc tgctgaccgt gagcagcggc 360

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcca gatgacccag 420

agccccagca gcctgagcgc cagcgtgggc aaccgcgtga ccatcacctg ccgcgccagc 480

caggacatca gcagctggct ggcctggtac cagcagaagc ccgagaaggc ccccaagagc 540

ctgatctacg ccgccagcag cctgcagagc ggcgtgccca gccgcttcag cggcagcggc 600

agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 660

tactgccagc agtacaacag ctaccccctg accttcggcc agggcacccg cctggagatc 720

aagcgc 726

<210> 77

<211> 357

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-OX 40scFv

<400> 77

cagctggtgg agagcggcgg cggcctggtg cagcccggcg gcagcctgcg cctgagctgc 60

gccgccagcg gcttcacctt cagcagctac agcatgaact gggtgcgcca ggcccccggc 120

aagggcctgg agtgggtgag ctacatcagc agcagcagca gcaccatcta ctacgccgac 180

agcgtgaagg gccgcttcac catcagccgc gacaacgcca agaacagcct gtacctgcag 240

atgaacagcc tgcgcgacga ggacaccgcc gtgtactact gcgcccgcgg cgtgtaccac 300

aacggctgga gcttcttcga ctactggggc cagggcaccc tgctgaccgt gagcagc 357

<210> 78

<211> 324

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-OX 40scFv

<400> 78

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcaa ccgcgtgacc 60

atcacctgcc gcgccagcca ggacatcagc agctggctgg cctggtacca gcagaagccc 120

gagaaggccc ccaagagcct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacagct accccctgac cttcggccag 300

ggcacccgcc tggagatcaa gcgc 324

<210> 79

<211> 723

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-GITR scFv

<400> 79

caggtgaccc tgaaggagag cggccccggc atcctgaagc ccagccagac cctgagcctg 60

acctgcagct tcagcggctt cagcctgagc accagcggca tgggcgtggg ctggatccgc 120

cagcccagcg gcaagggcct ggagtggctg gcccacatct ggtgggacga cgacaagtac 180

tacaacccca gcctgaagag ccagctgacc atcagcaagg acaccagccg caaccaggtg 240

ttcctgaaga tcaccagcgt ggacaccgcc gacgccgcca cctactactg cgcccgcacc 300

cgccgctact tccccttcgc ctactggggc cagggcaccc tggtgaccgt gagcagcggc 360

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcgt gatgacccag 420

agccagaagt tcatgagcac cagcgtgggc gaccgcgtga gcgtgacctg caaggccagc 480

cagaacgtgg gcaccaacgt ggcctggtac cagcagaagc ccggccagag ccccaaggcc 540

ctgatctaca gcgccagcta ccgctacagc ggcgtgcccg accgcttcac cggcagcggc 600

agcggcaccg acttcaccct gaccatcaac aacgtgcaca gcgaggacct ggccgagtac 660

ttctgccagc agtacaacac cgaccccctg accttcggcg ccggcaccaa gctggagatc 720

aag 723

<210> 80

<211> 357

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-GITR scFv

<400> 80

caggtgaccc tgaaggagag cggccccggc atcctgaagc ccagccagac cctgagcctg 60

acctgcagct tcagcggctt cagcctgagc accagcggca tgggcgtggg ctggatccgc 120

cagcccagcg gcaagggcct ggagtggctg gcccacatct ggtgggacga cgacaagtac 180

tacaacccca gcctgaagag ccagctgacc atcagcaagg acaccagccg caaccaggtg 240

ttcctgaaga tcaccagcgt ggacaccgcc gacgccgcca cctactactg cgcccgcacc 300

cgccgctact tccccttcgc ctactggggc cagggcaccc tggtgaccgt gagcagc 357

<210> 81

<211> 321

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-GITR scFv

<400> 81

gacatcgtga tgacccagag ccagaagttc atgagcacca gcgtgggcga ccgcgtgagc 60

gtgacctgca aggccagcca gaacgtgggc accaacgtgg cctggtacca gcagaagccc 120

ggccagagcc ccaaggccct gatctacagc gccagctacc gctacagcgg cgtgcccgac 180

cgcttcaccg gcagcggcag cggcaccgac ttcaccctga ccatcaacaa cgtgcacagc 240

gaggacctgg ccgagtactt ctgccagcag tacaacaccg accccctgac cttcggcgcc 300

ggcaccaagc tggagatcaa g 321

<210> 82

<211> 717

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-CD 40L scFv

<400> 82

gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtgagcgcc atcagcggca gcggcggcag cacctactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caagagctac 300

ggcgccttcg actactgggg ccagggcacc ctggtgaccg tgagcagcgg cggcggcggc 360

agcggcggcg gcggcagcgg cggcggcggc agcgacatcc agatgaccca gagccccagc 420

agcctgagcg ccagcgtggg cgaccgcgtg accatcacct gccgcgccag ccagagcatc 480

agcagctacc tgaactggta ccagcagaag cccggcaagg cccccaagct gctgatctac 540

gccgccagca gcctgcagag cggcgtgccc agccgcttca gcggcagcgg cagcggcacc 600

gacttcaccc tgaccatcag cagcctgcag cccgaggact tcgccaccta ctactgccag 660

cagagctaca gcacccccaa caccttcggc cagggcacca aggtggagat caagcgc 717

<210> 83

<211> 348

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-CD 40L scFv

<400> 83

gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtgagcgcc atcagcggca gcggcggcag cacctactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caagagctac 300

ggcgccttcg actactgggg ccagggcacc ctggtgaccg tgagcagc 348

<210> 84

<211> 324

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-CD 40L scFv

<400> 84

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gagcatcagc agctacctga actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag agctacagca cccccaacac cttcggccag 300

ggcaccaagg tggagatcaa gcgc 324

<210> 85

<211> 723

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-CD 27 scFv

<400> 85

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacgaca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc ccgcggcagc 300

ggcaactggg gcttcttcga ctactggggc cagggcaccc tggtgaccgt gagcagcggc 360

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcca gatgacccag 420

agccccagca gcctgagcgc cagcgtgggc gaccgcgtga ccatcacctg ccgcgccagc 480

cagggcatca gccgctggct ggcctggtac cagcagaagc ccgagaaggc ccccaagagc 540

ctgatctacg ccgccagcag cctgcagagc ggcgtgccca gccgcttcag cggcagcggc 600

agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 660

tactgccagc agtacaacac ctacccccgc accttcggcc agggcaccaa ggtggagatc 720

aag 723

<210> 86

<211> 357

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-CD 27 scFv

<400> 86

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacgaca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc ccgcggcagc 300

ggcaactggg gcttcttcga ctactggggc cagggcaccc tggtgaccgt gagcagc 357

<210> 87

<211> 321

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-CD 27 scFv

<400> 87

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gggcatcagc cgctggctgg cctggtacca gcagaagccc 120

gagaaggccc ccaagagcct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacacct acccccgcac cttcggccag 300

ggcaccaagg tggagatcaa g 321

<210> 88

<211> 19

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of secretory expression signal peptide

<400> 88

Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser

1 5 10 15

Ser Arg Ala

<210> 89

<211> 57

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence for secretory expression of signal peptide

<400> 89

atgacccggc tgaccgtgct ggccctgctg gccggcctgc tggcctcctc cagggcc 57

<210> 90

<211> 59

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-Sig-F

<400> 90

gtgctggata tctgcagaat tcgccgccac catgacccgg ctgaccgtgc tggccctgc 59

<210> 91

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Sig-R

<400> 91

ggccctggag gaggccagca ggccggccag cagggccagc acggtcagc 49

<210> 92

<211> 42

<212> DNA

<213> Artificial

<220>

<223> Sig-CD19-F

<400> 92

ctgctggcct cctccagggc cgacatccag ctgacccaga gc 42

<210> 93

<211> 23

<212> DNA

<213> Artificial

<220>

<223> CD19-R

<400> 93

gctgctcacg gtcacggtgg tgc 23

<210> 94

<211> 56

<212> DNA

<213> Artificial

<220>

<223> CD19-G4S-CD3-F

<400> 94

ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag cagagc 56

<210> 95

<211> 20

<212> DNA

<213> Artificial

<220>

<223> CD3-R

<400> 95

cttcagctcc agcttggtgc 20

<210> 96

<211> 72

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-4-1BB-F

<400> 96

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccaggt gc 72

<210> 97

<211> 51

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-4-1BB-R

<400> 97

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51

<210> 98

<211> 41

<212> DNA

<213> Artificial

<220>

<223> CD3-IgD-F

<400> 98

gcaccaagct ggagctgaag gccagcaaga gcaagaagga g 41

<210> 99

<211> 21

<212> DNA

<213> Artificial

<220>

<223> IgD-R

<400> 99

cacgcccagg ggctgggtgt g 21

<210> 100

<211> 42

<212> DNA

<213> Artificial

<220>

<223> IgD-4-1BB-F

<400> 100

cacacccagc ccctgggcgt gcaggtgcag ctgcagcagt gg 42

<210> 101

<211> 86

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-ICOS-F

<400> 101

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccaggt gcagctggtg cagagc 86

<210> 102

<211> 50

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-ICOS-R

<400> 102

ctgatcagcg gtttaaactt aagctttcac ttgatctcca ccttggtgcc 50

<210> 103

<211> 42

<212> DNA

<213> Artificial

<220>

<223> IgD-ICOS-F

<400> 103

cacacccagc ccctgggcgt gcaggtgcag ctggtgcaga gc 42

<210> 104

<211> 85

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-OX40-F

<400> 104

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccagct ggtggagagc ggcgg 85

<210> 105

<211> 52

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-OX40-R

<400> 105

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaggcgggt gc 52

<210> 106

<211> 45

<212> DNA

<213> Artificial

<220>

<223> IgD-OX40-F

<400> 106

gccacaccca gcccctgggc gtgcagctgg tggagagcgg cggcg 45

<210> 107

<211> 85

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-GITR-F

<400> 107

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccaggt gaccctgaag gagag 85

<210> 108

<211> 52

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-GITR-R

<400> 108

ctgatcagcg gtttaaactt aagctttcac ttgatctcca gcttggtgcc gg 52

<210> 109

<211> 43

<212> DNA

<213> Artificial

<220>

<223> IgD-GITR-F

<400> 109

gccacaccca gcccctgggc gtgcaggtga ccctgaagga gag 43

<210> 110

<211> 87

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-CD40L-F

<400> 110

ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60

ggcagcgagg tgcagctgct ggagagc 87

<210> 111

<211> 51

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-CD40L-R

<400> 111

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51

<210> 112

<211> 43

<212> DNA

<213> Artificial

<220>

<223> IgD-CD40L-F

<400> 112

gccacaccca gcccctgggc gtggaggtgc agctgctgga gag 43

<210> 113

<211> 86

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-CD27-F

<400> 113

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccaggt gcagctggtg gagagc 86

<210> 114

<211> 51

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-CD27-R

<400> 114

ctgatcagcg gtttaaactt aagctttcac ttgatctcca ccttggtgcc c 51

<210> 115

<211> 43

<212> DNA

<213> Artificial

<220>

<223> IgD-CD27-F

<400> 115

gccacaccca gcccctgggc gtgcaggtgc agctggtgga gag 43

Claims

1. A trifunctional molecule comprising a first domain capable of binding to CD19, a second domain capable of binding to and activating a T-cell surface CD3 molecule, and a third domain capable of binding to and activating a T-cell positive costimulatory molecule,

wherein the first domain is a single chain antibody against CD19, the second domain is a single chain antibody against CD3, the third domain is a single chain antibody against a T cell costimulatory molecule selected from the group consisting of a single chain antibody against 4-1BB, a single chain antibody against ICOS, a single chain antibody against OX40, a single chain antibody against GITR, a single chain antibody against CD40L, and a single chain antibody against CD27, the single chain antibody comprising a heavy chain variable region and a light chain variable region,

the first domain and the second domain are connected by a linker1, the second domain and the third domain are connected by a linker2,

the connecting segment 1 and the connecting segment 2 are respectively a connecting segment taking G4S as a unit and a hinge region segment of immunoglobulin IgD shown as SEQ ID NO. 7.

2. The trifunctional molecule of claim 1, wherein the trifunctional molecule is capable of binding to and activating a T-cell surface CD3 molecule and a T-cell positive costimulatory molecule, while recognizing CD19, thereby generating a first signal and a second signal required for T-cell activation.

3. Trifunctional molecule according to claim 1, characterized in that the amino acid sequence of the linker fragment in G4S units is as shown in any of SEQ ID No.1, SEQ ID No.3, SEQ ID No. 5.

4. The trifunctional molecule of claim 1, wherein the anti-CD 19 single-chain antibody has the heavy chain variable region amino acid sequence as set forth in SEQ ID No. 41; the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 42; the amino acid sequence of the heavy chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 44; the amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 45; the amino acid sequence of the heavy chain variable region of the anti-4-1 BB single-chain antibody is shown in SEQ ID NO. 47; the amino acid sequence of the light chain variable region of the anti-4-1 BB single-chain antibody is shown in SEQ ID NO. 48; the amino acid sequence of the heavy chain variable region of the single-chain antibody for resisting ICOS is shown as SEQ ID NO. 50; the amino acid sequence of the light chain variable region of the anti-ICOS single-chain antibody is shown in SEQ ID NO. 51; the amino acid sequence of the heavy chain variable region of the anti-OX 40 single-chain antibody is shown as SEQ ID No. 53; the amino acid sequence of the light chain variable region of the anti-OX 40 single-chain antibody is shown as SEQ ID No. 54; the amino acid sequence of the heavy chain variable region of the anti-GITR single-chain antibody is shown as SEQ ID NO. 56; the amino acid sequence of the light chain variable region of the anti-GITR single-chain antibody is shown as SEQ ID NO. 57; the amino acid sequence of the heavy chain variable region of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 59; the amino acid sequence of the light chain variable region of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 60; the amino acid sequence of the heavy chain variable region of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 62; the amino acid sequence of the light chain variable region of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 63.

5. The trifunctional molecule according to claim 4, wherein the anti-CD 19 single-chain antibody has an amino acid sequence as shown in SEQ ID No. 40; the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 43; the amino acid sequence of the single-chain antibody for resisting 4-1BB is shown as SEQ ID NO. 46; the amino acid sequence of the single-chain antibody for resisting ICOS is shown as SEQ ID NO. 49; the amino acid sequence of the anti-OX 40 single-chain antibody is shown as SEQ ID NO. 52; the amino acid sequence of the anti-GITR single-chain antibody is shown as SEQ ID NO. 55; the amino acid sequence of the anti-CD 40L single-chain antibody is shown in SEQ ID NO. 58; the amino acid sequence of the anti-CD 27 single-chain antibody is shown in SEQ ID NO. 61.

6. Trifunctional molecule according to claim 1, characterized in that the amino acid sequence of the trifunctional molecule is as shown in any of SEQ ID No.18, SEQ ID No.22, SEQ ID No.26, SEQ ID No.30, SEQ ID No.34 or SEQ ID No. 38.

7. A polynucleotide encoding a trifunctional molecule according to any one of claims 1-6.

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

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

10. A method of preparing a trifunctional molecule according to any one of claims 1-6, comprising: constructing an expression vector containing a gene sequence of the trifunctional molecules, then transforming the expression vector containing the gene sequence of the trifunctional molecules into host cells for induction expression, and separating the expression product to obtain the trifunctional molecules.

11. Use of the trifunctional molecules of any one of claims 1-6 for the preparation of a medicament for the treatment of tumors.