CN106589129B

CN106589129B - Tri-functional molecule combined with CD19, CD3 and CD28 and application thereof

Info

Publication number: CN106589129B
Application number: CN201611258643.1A
Authority: CN
Inventors: 陈帅; 朱化星; 廖远平
Original assignee: Shanghai Novoprotein Biotechnology Co ltd
Current assignee: Huihe Biotechnology (Shanghai) Co.,Ltd.
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2021-03-05
Anticipated expiration: 2036-12-30
Also published as: CN106589129A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a tri-functional molecule combined with CD19, CD3 and CD28 and application thereof. The invention constructs three functional molecules capable of simultaneously recognizing CD19, CD3 and CD28 by using methods of genetic engineering and antibody engineering. The molecule has obvious advantages in the aspects of preparation process and practical application: the targeting effect of the T cells to CD19 positive cells is endowed, the efficacy of activating the T cells is further improved, the mediated combination and killing effect of the T cells to the CD19 positive target cells when the T cells are added independently are better than that of an anti-CD 19/anti-CD 3BiTE bispecific antibody, and the application convenience is better than that of a CD19 targeted CAR-T technology.

Description

Tri-functional molecule combined with CD19, CD3 and CD28 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 CD28 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 CD19/CD3, 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 3BiTE 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, a first signal is generated by the interaction of the antigen peptide-MHC molecule complex on the surface of APC with the TCR/CD3 complex on the surface of T cell, and then a second signal is generated by the interaction of the co-stimulatory molecule ligand (e.g., CD80, CD86) on the surface of antigen presenting cell with the co-stimulatory molecule (e.g., CD28) on the surface of T cell. 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, a bispecific anti-tumor antigen/anti-CD 28 antibody can be used in combination with a bispecific anti-tumor antigen/anti-CD 3 antibody 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 signal 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 CD28 costimulatory signal, 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 CD28 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 surface CD28 molecule.

Preferably, the trifunctional molecule is capable of binding to and activating both a T cell surface CD3 molecule and a CD28 molecule while recognizing CD19, thereby generating a first signal and a second signal 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 CD 28.

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. 23. The connecting fragment 2 contains three G4S units, and the amino acid sequence of the connecting fragment is shown as SEQ ID NO. 25.

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. 27. The amino acid sequence of the connecting segment 2 is shown as SEQ ID NO. 29. 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 CD28, 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. 6. The amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 7. The amino acid sequence of the heavy chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 9. The amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 10. The amino acid sequence of the heavy chain variable region of the anti-CD28 single-chain antibody is shown in SEQ ID NO. 12. The amino acid sequence of the light chain variable region of the anti-CD28 single-chain antibody is shown in SEQ ID NO. 13.

In some embodiments of the invention, the amino acid sequence of the anti-CD 19 single chain antibody is shown in SEQ ID NO. 5. The amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 8. The amino acid sequence of the anti-CD28 single-chain antibody is shown in SEQ ID NO. 11.

In some embodiments of the invention, the amino acid sequence of the trifunctional molecules in monomeric form is shown in SEQ ID NO. 1. The amino acid sequence of the three-functional molecule in the form of a dimer is shown in SEQ ID NO. 3.

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 T cell surface CD3 molecule and a third functional domain capable of being combined with and activating a T cell surface CD28 molecule 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-CD 28 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 stimulation signal for activating the T cell, the activation effect on the T cell is further improved while the T cell is endowed with targeting property, the secretion of cytokines and anti-apoptosis protein is increased, the phenomenon of incapability and death of the T cell is effectively avoided, the mediated combination and killing of the T cell on CD19 positive target cells can achieve the effect even better than that of an anti-CD 19/anti-CD 3BiTE bispecific antibody, and the protein dosage 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. the structure of the monomeric anti-CD 19/CD 3/anti-CD 28 trispecific antibody (CD19-CD3-CD28TsAb _ M); B. a structural diagram of a dimeric form of anti-CD 19/anti-CD 3/anti-CD 28 trispecific antibody (CD19-CD3-CD28TsAb _ D).

FIG. 2: A. purified CD19-CD3-CD28TsAb _ M SDS-PAGE analysis map, lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-CD28TsAb _ M; lane 3: non-reducing CD19-CD3-CD28TsAb _ M; B. SDS-PAGE analysis of purified CD19-CD3-CD28TsAb _ D; lane 1: a molecular weight protein Marker; lane 2: reducing CD19-CD3-CD28TsAb _ D; lane 3: non-reducing CD19-CD3-CD28TsAb _ D.

FIG. 3A: the ELISA identification results of CD19-CD3-CD28TsAb _ 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 ug/ml recombinant antigen CD 3-hFc; coating the tangle-solidup with 1 microgram/ml recombinant antigen CD 28-hFc;

assay results without any antigen coating.

FIG. 3B: ELISA identification results of CD19-CD3-CD28TsAb _ D; the curves in the figure represent the results of 4 tests, respectively: ■ coating 1 ug/ml recombinant antigen CD 19-hFc; ● coating 1 ug/ml recombinant antigen CD 3-hFc; recombinant antibody with coating of 1 microgram/mlpro-CD 28-hFc;

assay results without any antigen coating.

FIG. 4: the cell engagement activity of CD19-CD3-CD28TsAb _ M, CD19-CD3-CD28TsAb _ D and CD19-CD3BsAb at different concentrations is respectively detected by using Raji lymphoma cells as CD19 positive target cells and Jurkat cells as CD3 and CD28 positive effector cells in a cell engagement experiment mediated by a trispecific antibody and a bispecific antibody; a: blank control with no added antibody; b: experimental groups with high concentrations of CD19-CD3BsAb (45 ng/ml); c: experimental groups with high concentrations of CD19-CD3-CD28TsAb _ M (45 ng/ml); d: experimental groups with high concentrations of CD19-CD3-CD28TsAb _ D (45 ng/ml); e: experimental groups with medium concentrations of CD19-CD3BsAb (0.45ng/ml) added; f: experimental groups with medium concentrations of CD19-CD3-CD28TsAb _ M (0.45ng/ml) added; g: experimental groups with medium concentrations of CD19-CD3-CD28TsAb _ D (0.45ng/ml) added; h: experimental groups with low concentrations of CD19-CD3BsAb (0.0045 ng/ml); i: experimental groups with low concentrations of CD19-CD3-CD28TsAb _ M (0.0045 ng/ml); j: experiment groups with low concentrations of CD19-CD3-CD28TsAb _ D (0.0045ng/ml) added.

FIG. 5A: in a cell killing experiment mediated by a tri-specific antibody and a bispecific antibody, Raji lymphoma cells are used as CD19 positive target cells, CIK (cytokine induced killers) cells are used as CD3 and CD28 positive killing effector cells, and the killing efficiency of the CIK cells on the Raji cells mediated by CD19-CD3-CD28TsAb _ M, CD19-CD3-CD28TsAb _ D and CD19-CD3BsAb at different concentrations is respectively detected; effector cells: target cells (E: T ratio) 1: 5, killing time: and 3 h.

FIG. 5B: in a cell killing experiment mediated by a tri-specific antibody and a bispecific antibody, Raji lymphoma cells are used as CD19 positive target cells, CIK cells are used as CD3 and CD28 positive killing effector cells, and the killing efficiency of the CIK cells mediated by CD19-CD3-CD28TsAb _ M, CD19-CD3-CD28TsAb _ D and CD19-CD3BsAb on the Raji cells at different concentrations 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)

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

Linker: connecting segment

Linker 1: connecting fragment 1

Linker2 Linker2

CD19-CD3-CD28TsAb _ M: monomeric anti-CD 19/anti-CD 3/anti-CD 28 trispecific antibody

CD19-CD3-CD28TsAb _ D: dimeric form of anti-CD 19/anti-CD 3/anti-CD 28 trispecific antibody

Di-and tri-functional molecules

The trifunctional molecule comprises a first functional domain capable of binding to CD19, a second functional domain capable of binding to and activating a T cell surface CD3 molecule, and a third functional domain capable of binding to and activating a T cell surface CD28 molecule.

Further, the trifunctional molecules are capable of binding and activating the T cell surface CD3 molecule and CD28 molecule while recognizing CD19, thereby generating a first signal and a second signal required for T cell activation.

The first domain, the second domain and the third domain are not particularly limited as long as the CD19 can be recognizedAt the same time, the first and second signals required for T cell activation are generated by binding and activating the T cell surface CD3 molecule and the CD28 molecule. For example, the first domain may be an anti-CD 19 antibody, the second domain may be an anti-CD3 antibody, and the third domain may be an anti-CD28 antibody. 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 CD28 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 CD28 antigen. The structure of the trifunctional molecule in a dimer form according to the 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 CD28 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 CD28 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 (CD28) are doubled in T cell activation, so that the T cells are more fully activated 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.

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. 6. The amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 7. Further, the amino acid sequence of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 5.

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. 9. The amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 10. Further, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO. 8.

The third functional domain is a single chain antibody against CD 28. The anti-CD28 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-CD28 single-chain antibody is shown in SEQ ID NO. 12. The amino acid sequence of the light chain variable region of the anti-CD28 single-chain antibody is shown in SEQ ID NO. 13. The amino acid sequence of the anti-CD28 single-chain antibody is shown in SEQ ID NO. 11.

In a preferred embodiment, the amino acid sequence of the trifunctional molecule in monomeric form is shown in SEQ ID NO. 1. The amino acid sequence of the three-functional molecule in the form of a dimer is shown in SEQ ID NO. 3. 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 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. 15.

The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-CD 19 is shown as SEQ ID NO. 16.

The nucleotide sequence of the single-chain antibody for encoding the anti-CD 19 is shown in SEQ ID NO. 14.

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. 18.

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. 19.

The nucleotide sequence of the single-chain antibody for encoding the anti-CD3 is shown in SEQ ID NO. 17.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody for encoding the anti-CD28 is shown as SEQ ID NO. 21.

The nucleotide sequence of the variable region of the light chain of the single-chain antibody for encoding the anti-CD28 is shown as SEQ ID NO. 22.

The nucleotide sequence of the single-chain antibody for encoding the anti-CD28 is shown as SEQ ID NO. 20.

The nucleotide sequence of the connecting segment 1 with the coding amino acid sequence shown as SEQ ID NO.23 is shown as SEQ ID NO. 24.

The nucleotide sequence of the connecting segment 2 with the coding amino acid sequence shown as SEQ ID NO.25 is shown as SEQ ID NO. 26.

The nucleotide sequence of the connecting segment 1 with the coding amino acid sequence shown as SEQ ID NO.27 is shown as SEQ ID NO. 28.

The nucleotide sequence of the connecting segment 2 with the coding amino acid sequence shown as SEQ ID NO.29 is shown as SEQ ID NO. 30.

Further, the nucleotide sequence of the trifunctional molecule in the form of a coded monomer is shown in SEQ ID NO. 2. The nucleotide sequence of the trifunctional molecule in the form of a code dimer is shown in SEQ ID NO. 4.

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 a preferred embodiment of the present invention, CIK cells (CD3) prepared by respectively acting on human PBMC of the same donor origin with human Peripheral Blood Mononuclear Cells (PBMC) as the experimental material, using the above-mentioned trifunctional molecules prepared in monomeric form, trifunctional molecules prepared in dimeric form, and the purchased anti-CD 19/anti-CD 3BiTE bispecific antibody (CD19-CD3BsAb) prepared in the present invention⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺). As a result, after the trifunctional molecules disclosed by the invention are added, the killing efficiency of the CIK cells on Raji cells is remarkably improved, and the targeted killing activity on CD19 positive tumor cells is better than that of anti-CD 19/anti-CD 3BiTE bispecific antibodies (CD19-CD3 BsAb).

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 a preferred embodiment of the present invention, CIK cells (CD3) prepared by respectively acting on human PBMC of the same donor origin with human Peripheral Blood Mononuclear Cells (PBMC) as the experimental material, using the above-mentioned trifunctional molecules prepared in monomeric form, trifunctional molecules prepared in dimeric form, and the purchased anti-CD 19/anti-CD 3BiTE bispecific antibody (CD19-CD3BsAb) prepared in the present invention⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺). As a result, after the trifunctional molecules disclosed by the invention are added, the killing efficiency of the CIK cells on Raji cells is remarkably improved, and the targeted killing activity on CD19 positive tumor cells is better than that of anti-CD 19/anti-CD 3BiTE bispecific antibodies (CD19-CD3 BsAb).

Aiming at the defects of an anti-CD 19/anti-CD 3BiTE bispecific antibody and a CAR-T technology of targeting CD19, the invention constructs a tri-functional molecule capable of simultaneously recognizing CD19, CD3 and CD28 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 binding and killing effects of the T cells to CD19 positive target cells when the T cells are added independently are better than that of an anti-CD 19/anti-CD 3BiTE bispecific antibody, and the application convenience is better than that of a CAR-T technology for targeting CD 19.

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-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D

In the present invention, the TiTE trispecific antibody targeting the human CD19 protein on the surface of lymphoma B cells, the human CD3 and CD28 proteins on the surface of T cells was named CD19-CD3-CD28 TsAb.

First, CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D construction scheme design

The specific construction scheme of the monomer form of CD19-CD3-CD28TsAb _ M is as follows: the sequences of the anti-CD 19scFv, the anti-CD 3scFv and the anti-CD28 scFv are connected by a Linker (Linker), specifically, the sequences of the anti-CD 19scFv and the anti-CD 3scFv are connected by a Linker 1(Linker 1), and the sequences of the anti-CD 3scFv and the anti-CD28 scFv are connected by a Linker 2(Linker 2).

The specific construction scheme of the dimer form of CD19-CD3-CD28TsAb _ D is as follows: the sequences of the anti-CD 19scFv, the anti-CD 3scFv and the anti-CD28 scFv are connected by a Linker (Linker), specifically, the sequences of the anti-CD 19scFv and the anti-CD 3scFv are connected by a Linker 1(Linker 1), and the sequences of the anti-CD 3scFv and the anti-CD28 scFv are connected by an IgD hinge region (Ala90-Val170) 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 19scFv, anti-CD 3scFv, and anti-CD28 scFv sequences.

Specifically, the nucleotide sequence of the heavy chain variable region of the anti-CD 19scFv is shown in SEQ ID No.15, specifically:

CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

the nucleotide sequence of the light chain variable region of the anti-CD 19scFv is shown as SEQ ID NO.16, and specifically comprises the following steps:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG。

the nucleotide sequence of the anti-CD 19scFv is shown as SEQ ID NO.14, and specifically comprises the following steps:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

the nucleotide sequence of the heavy chain variable region of the anti-CD 3scFv is shown as SEQ ID NO.18, and specifically comprises the following steps:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

the nucleotide sequence of the light chain variable region of the anti-CD 3scFv is shown as SEQ ID NO.19, and specifically comprises the following steps:

GACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

the nucleotide sequence of the anti-CD 3scFv is shown as SEQ ID NO.17, and specifically comprises the following steps:

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

the nucleotide sequence of the heavy chain variable region of the anti-CD28 scFv is shown as SEQ ID NO.21, and specifically comprises the following steps:

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

the nucleotide sequence of the light chain variable region of the anti-CD28 scFv is shown as SEQ ID NO.22, and specifically comprises the following steps:

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the anti-CD28 scFv is shown as SEQ ID NO.20, and specifically comprises the following steps:

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the monomeric CD19-CD3-CD28TsAb _ M connecting fragment 1(Linker 1) is shown as SEQ ID NO.24, and specifically comprises the following steps:

GGTGGCGGAGGGTCC。

the nucleotide sequence of the monomeric CD19-CD3-CD28TsAb _ M connecting fragment 2(Linker 2) is shown as SEQ ID NO.26, and specifically comprises the following steps:

GGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGT。

the nucleotide sequence of the dimer-form CD19-CD3-CD28TsAb _ D connecting fragment 1(Linker 1) is shown as SEQ ID NO.28, and specifically comprises the following steps:

GGTGGCGGAGGGTCC。

the nucleotide sequence of the dimer form of CD19-CD3-CD28TsAb _ D connecting fragment 2(Linker 2) is shown as SEQ ID NO.30, 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.31, and specifically comprises the following steps:

MTRLTVLALLAGLLASSRA。

the nucleotide sequence of the secretory expression signal peptide is shown as SEQ ID NO.32, and specifically comprises the following components:

ATGACCCGGCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCTCCTCCAGGG

CC。

II, construction of eukaryotic expression vectors of CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ 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-CD28TsAb _ 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)₃Amplification of anti-CD 19scFv, GGGGS Linker 1+ anti-CD 3scFv, (GGGGS) from-CD 28-F and pcDNA3.1-CD28-R₃Linker 2+ anti-CD28 scFv gene sequence; cloning construction for CD19-CD3-CD28TsAb _ D, signal peptide fragments were also first amplified using primers pcDNA3.1-Sig-F and Sig-R, and then anti-CD 19scFv, GGGGGGS Linker 1+ anti-CD 3scFv, IgD hinge region Linker2, anti-CD28 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-CD28-F, and pcDNA3.1-CD28-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-CD28TsAb and the dimeric form of CD19-CD3-CD28TsAb _ D were correct and consistent with expectations.

Specifically, the nucleotide sequence of the monomeric CD19-CD3-CD28TsAb _ M is shown as SEQ ID NO.2, and specifically comprises the following components:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

the nucleotide sequence of the dimeric form of CD19-CD3-CD28TsAb _ D is shown in SEQ ID NO.4, and specifically comprises the following components:

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

TABLE 1 primers used in the cloning of trispecific antibody genes

Example 2 expression and purification of CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D expression of CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ 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-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D), two centrifuge tubes/culture flasks were prepared, each placed in 20ml, and the recombinant plasmids prepared in example 1 were taken:

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

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

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

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

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

Purification of CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ 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;

2.2 Protein 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-CD28TsAb _ M and CD19-CD3-CD28TsAb _ 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 recombinant proteins, CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D, were all > 95% pure following Protein L affinity column purification: wherein the theoretical molecular weight of the recombinant protein CD19-CD3-CD28TsAb _ M is 81.3kDa, and the protein presents a single electrophoresis band under reducing and non-reducing conditions, and the molecular weight is consistent with that of a monomer, so that the trispecific antibody is in a monomer form (FIG. 2A); the theoretical molecular weight of the recombinant CD19-CD3-CD28TsAb _ D protein is 89.1kDa, the electrophoretic band of the protein under reducing conditions has a molecular weight consistent with that of a monomer, and the electrophoretic band under non-reducing conditions has a molecular weight consistent with that of a dimer (-180 kDa) (FIG. 2B), indicating that two protein molecules can form disulfide bonds through the IgD hinge region to form a disulfide bond with 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 the mass spectrometry further confirms that the CD19-CD3-CD28TsAb _ M is in a monomer form, and the CD19-CD3-CD28TsAb _ D is in a dimer form.

Therefore, it can be known that the amino acid sequence of the monomeric form of CD19-CD3-CD28TsAb _ M is shown in SEQ ID NO.1, and specifically:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。

the amino acid sequence of the dimer form of CD19-CD3-CD28TsAb _ D is shown in SEQ ID NO.3, and specifically comprises the following components:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。

the amino acid sequence of the anti-CD 19scFv is shown as SEQ ID NO.5, and specifically comprises the following steps:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS。

the amino acid sequence of the heavy chain variable region of the anti-CD 19scFv is shown as SEQ ID NO.6, and specifically comprises the following steps:

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS。

the amino acid sequence of the heavy chain variable region of the anti-CD 19scFv is shown as SEQ ID NO.7, and specifically comprises the following steps:

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK。

the amino acid sequence of the anti-CD 3scFv is shown as SEQ ID NO.8, and specifically comprises the following steps:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。

the amino acid sequence of the heavy chain variable region of the anti-CD 3scFv is shown as SEQ ID NO.9, and specifically comprises the following steps:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS。

the amino acid sequence of the light chain variable region of the anti-CD 3scFv is shown as SEQ ID NO.10, and specifically comprises the following steps:

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。

the amino acid sequence of the anti-CD28 scFv is shown as SEQ ID NO.11, and specifically comprises the following steps:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。

the amino acid sequence of the heavy chain variable region of the anti-CD28 scFv is shown as SEQ ID NO.12, and specifically comprises the following steps:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS。

the amino acid sequence of the light chain variable region of the anti-CD28 scFv is shown as SEQ ID NO.13, and specifically comprises the following steps:

DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。

the amino acid sequence of the connecting fragment 1(Linker 1) in the monomeric CD19-CD3-CD28TsAb _ M is shown as SEQ ID NO.23, and specifically comprises the following steps: GGGGS.

The amino acid sequence of the connecting fragment 2(Linker 2) in the monomeric CD19-CD3-CD28TsAb _ M is shown as SEQ ID NO.25, and specifically comprises the following steps: GGGGSGGGGSGGGGS.

The amino acid sequence of the connecting fragment 1(Linker 1) in the dimer form of CD19-CD3-CD28TsAb _ D is shown as SEQ ID NO.27, and specifically comprises the following components: GGGGS.

The amino acid sequence of the connecting fragment 2(Linker 2) in the dimer form of CD19-CD3-CD28TsAb _ D is shown as SEQ ID NO.29, and specifically comprises the following steps:

ASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGV。

example 3: ELISA for detecting antigens of CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D

Binding Activity

ELISA operation steps:

1. recombinant antigen coating: human CD19-hFc, human CD3-hFc and human CD28-hFc fusion proteins (purchased from Wujiang near-shore protein technologies, Ltd.) were coated on 96-well plates, respectively, with an antigen concentration of 1. mu.g/ml and a coating volume of 100. mu.l/well, under the conditions of 1 hour at 37 ℃ or overnight at 4 ℃, and the formulation of coating buffer (PBS) was: 3.58g Na₂HPO₄，0.24g NaH₂PO₄，0.2g KCl，8.2g NaCl，950ml H₂O, adjusting the pH value to 7.4 by using 1mol/L HCl or 1mol/L NaOH, and supplementing water to 1L;

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

3. sample adding: after 4 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-CD28TsAb _ M or CD19-CD3-CD28TsAb _ D as the initial concentration, carrying out a double dilution of 6 gradients, each gradient being provided with 2 duplicate wells;

4. color development: after washing the plate 4 times with PBST (PBS + 0.05% Tween-20(V/V)), the HRP-labeled anti-His was diluted 1/5000 with blocking solution PBSA₆Fusion tag antibody (purchased from Abcam) was added at 100. mu.l/well and incubated at 37 ℃ for 1 hour. After washing the plate for 4 times with PBS, adding a color developing solution TMB (purchased from KPL company) with 100 mul/hole, and developing for 5-10 minutes at room temperature in a dark place;

5. termination reaction and result determination: stop solution (1M HCl) was added at 100. mu.l/well, and absorbance (OD450) was read at a wavelength of 450nm on a microplate reader.

The ELISA results are shown in fig. 3A and 3B: FIG. 3A illustrates that CD19-CD3-CD28TsAb _ M has in vitro binding activity to the recombinant antigens CD19-hFc, CD3-hFc, and CD28-hFc, with the highest CD28 binding activity and the second lowest CD19 binding activity, and the weaker CD3 binding activity; FIG. 3B illustrates that CD19-CD3-CD28TsAb _ D also has in vitro binding activity with the recombinant antigens CD19-hFc, CD3-hFc and CD28-hFc, with the highest CD28 binding activity and the second lowest CD19 binding activity and the weaker CD3 binding activity.

Example 4: tri-specific antibody and bispecific antibody-mediated cell engagement experiments

CCL-86Raji lymphoma cells (purchased from ATCC) as CD19 positive target cells and TIB-152Jurkat cells (purchased from ATCC) as CD3 and CD28 positive effector cells were compared with the differences in cell-mediated engagement activities of a monomeric form of a TiTE trispecific antibody (CD19-CD3-CD28TsAb _ M), a dimeric form of a TiTE trispecific antibody (CD19-CD3-CD28TsAb _ D) and an anti-CD 19/anti-CD 3BiTE bispecific antibody (CD19-CD3BsAb, purchased from Wujiang Kongshi protein Ltd.).

Cell linking experiment:

1. taking Raji cell-1X 10⁵ Setting 3 experimental groups with high, medium and low concentrations, respectively adding CD19-CD3BsAb, CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D with final concentrations of 45, 0.45 and 0.0045ng/ml, and standing for 5 min; cells without any added antibody were used as blank control;

2. respectively adding the same number of Jurkat cells into the Raji cell sample, placing in an incubator at 37 ℃ for 1h, taking out the cells, gently shaking for 30s, standing for 2min, observing the cell clustering condition under a microscope and taking a picture;

the results are shown in FIG. 4: raji cells did not aggregate with Jurkat cells without any antibody addition (fig. 4A), indicating that there was no non-specific engagement between the two cells; raji cells and Jurkat cells in 3 experimental groups were significantly clustered with the addition of high concentrations of antibody (45ng/ml) (FIGS. 4B-D), indicating that the cell engagement activity of the two forms of the TiTE trispecific antibody (CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D) was comparable to that of the anti-CD 19/anti-CD 3BiTE bispecific antibody (CD19-CD3BsAb) at higher concentrations; under the condition of adding medium-concentration antibody (0.45ng/ml), CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D can still cause Raji cells to obviously agglomerate with Jurkat cells, and CD19-CD3BsAb can cause two cells to slightly agglomerate (FIG. 4E-G), which indicates that the cell engagement activity of two forms of the TiTE trispecific antibody is better than that of the BiTE bispecific antibody under medium concentration; under the condition of adding low concentration antibody (0.0045ng/ml), CD19-CD3-CD28TsAb _ D can still cause Raji cells to obviously agglomerate with Jurkat cells, CD19-CD3-CD28TsAb _ M can cause two cells to agglomerate slightly, and CD19-CD3BsAb cannot agglomerate the cells (FIG. 4H-J), which shows that the cell engagement activity of the dimeric form of the TiTE trispecific antibody is better than that of the monomeric form of the TiTE trispecific antibody under low concentration, and the BiTE bispecific antibody has no cell engagement activity under low concentration.

Example 5: trispecific antibody and bispecific 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-CD28TsAb _ M, the dimeric form of the TiTE trispecific antibody CD19-CD3-CD28TsAb _ D and the purchased anti-CD 19/anti-CD 3BiTE bispecific antibody (CD19-CD3BsAb) prepared by the invention are respectively acted on CIK cells (CD3) prepared by human PBMC of the same donor source⁺CD56⁺) And CCL-86Raji lymphoma cells (CD 19)⁺) And detecting the cell death condition, and comparing the killing efficiency difference of the CIK effector cells mediated by the three antibodies on CCL-86Raji target cells.

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 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, the cultures were continued with 500U/ml IL-2 (purchased from Wujiang Korea protein technology Ltd.) and counted every 2-3 days and cultured in 1X 10 CIK basal medium supplemented with 500U/ml IL-2⁶Cell 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 5X 10⁵(CIK Effector cells: Raji target cells (E: T ratio) 1: 5) or 1X 10⁵Separately (E: T ratio 1: 1), CD19-CD3BsAb, CD19-CD3-CD28TsAb _ M and CD19-CD3-CD28TsAb _ D antibody samples with different final concentrations (25, 12.5, 6.25, 3.125ng/ml) are added, mixed for 3-5min at room temperature, co-cultured for 3h at 37 ℃, 10uL of CCK8 is added to each well, reaction is continued for 2-3h at 37 ℃, and then OD is measured by a microplate 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. 5: when CIK effector cells: raji target cells (E: T ratio) were 1: 5 and 1: 1, 3h cell killing efficiency was about 17% (fig. 5A) and 21% (fig. 5B) without any antibody addition; the killing efficiency of the CIK cells to Raji cells is remarkably improved under the condition of adding higher concentrations of antibodies (25, 12.5 and 6.25ng/ml), wherein the cell killing effect mediated by CD19-CD3-CD28TsAb _ D is the best, and when E: the ratio T is 1: 5, the killing efficiency was about 36%, 29% and 30%, respectively, when E: the ratio T is 1: 1, killing efficiencies were approximately 85%, 90% and 85%, respectively, with CD19-CD3-CD28TsAb _ M being the second to the effects when E: the ratio T is 1: 5, the killing efficiency was about 30%, 23% and 26%, respectively, when E: the ratio T is 1: 1, killing efficiency was approximately 86%, 82% and 81%, with the least effective of CD19-CD3BsAb when E: the ratio T is 1: 5, the killing efficiency was about 23%, 22% and 22%, respectively, when E: the ratio T is 1: 1, the killing efficiency is respectively about 80%, 55% and 56%; 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-CD28TsAb _ D and CD19-CD3-CD28TsAb _ M is still improved to some extent, when E: t is 1: 5, the killing efficiency was about 23% and 22%, respectively, when E: the ratio T is 1: 1, the killing efficiency was about 82% and 70%, respectively, while CD19-CD3BsAb had substantially no effect compared to the blank control, which indicates that both forms of TiTE trispecific antibody mediated targeted killing activity of T cells against CD19 positive tumor cells is superior to the BiTE bispecific antibody, with the dimeric form having 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> tri-functional molecule binding to CD19, CD3 and CD28 and application thereof

<130> 163905

<160> 43

<170> PatentIn version 3.3

<210> 1

<211> 756

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-CD28TsAb _ M in monomer form

<400> 1

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 Ser

530 535 540

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

545 550 555 560

Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys

565 570 575

Phe Lys Asp Arg Ala Thr Leu Thr Val 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 Phe

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His

660 665 670

Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln Lys Pro

675 680 685

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Glu Ile Lys Arg

755

<210> 2

<211> 2268

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-CD28TsAb _ M in monomer form

<400> 2

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

accttcacca gctactacat ccactgggtg cgccaggccc ccggccaggg cctggagtgg 1680

atcggctgca tctaccccgg caacgtgaac accaactaca acgagaagtt caaggaccgc 1740

gccaccctga ccgtggacac cagcatcagc accgcctaca tggagctgag ccgcctgcgc 1800

agcgacgaca ccgccgtgta cttctgcacc cgcagccact acggcctgga ctggaacttc 1860

gacgtgtggg gccagggcac caccgtgacc gtgagcagcg gcggcggcgg cagcggcggc 1920

ggcggcagcg gcggcggcgg cagcgacatc cagatgaccc agagccccag cagcctgagc 1980

gccagcgtgg gcgaccgcgt gaccatcacc tgccacgcca gccagaacat ctacgtgtgg 2040

ctgaactggt accagcagaa gcccggcaag gcccccaagc tgctgatcta caaggccagc 2100

aacctgcaca ccggcgtgcc cagccgcttc agcggcagcg gcagcggcac cgacttcacc 2160

ctgaccatca gcagcctgca gcccgaggac ttcgccacct actactgcca gcagggccag 2220

acctacccct acaccttcgg cggcggcacc aaggtggaga tcaagcgc 2268

<210> 3

<211> 822

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CD19-CD3-CD28TsAb _ D in dimer form

<400> 3

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 Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

610 615 620

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

625 630 635 640

Glu Lys Phe Lys Asp Arg Ala Thr Leu Thr Val 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 Phe Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val

675 680 685

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

690 695 700

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

705 710 715 720

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

725 730 735

Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr

805 810 815

Lys Val Glu Ile Lys Arg

820

<210> 4

<211> 2466

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of CD19-CD3-CD28TsAb _ D in dimer form

<400> 4

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 cttcaccagc tactacatcc actgggtgcg ccaggccccc 1860

ggccagggcc tggagtggat cggctgcatc taccccggca acgtgaacac caactacaac 1920

gagaagttca aggaccgcgc caccctgacc gtggacacca gcatcagcac cgcctacatg 1980

gagctgagcc gcctgcgcag cgacgacacc gccgtgtact tctgcacccg cagccactac 2040

ggcctggact ggaacttcga cgtgtggggc cagggcacca ccgtgaccgt gagcagcggc 2100

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcca gatgacccag 2160

agccccagca gcctgagcgc cagcgtgggc gaccgcgtga ccatcacctg ccacgccagc 2220

cagaacatct acgtgtggct gaactggtac cagcagaagc ccggcaaggc ccccaagctg 2280

ctgatctaca aggccagcaa cctgcacacc ggcgtgccca gccgcttcag cggcagcggc 2340

agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 2400

tactgccagc agggccagac ctacccctac accttcggcg gcggcaccaa ggtggagatc 2460

aagcgc 2466

<210> 5

<211> 250

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-CD 19scFv

<400> 5

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> 6

<211> 124

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-CD 19scFv

<400> 6

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> 7

<211> 111

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-CD 19scFv

<400> 7

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> 8

<211> 243

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-CD 3scFv

<400> 8

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> 9

<211> 119

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-CD 3scFv

<400> 9

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> 10

<211> 106

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-CD 3scFv

<400> 10

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> 11

<211> 243

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of anti-CD28 scFv

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val 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 Phe Cys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ala

145 150 155 160

Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly

180 185 190

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

195 200 205

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

210 215 220

Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Arg

<210> 12

<211> 120

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain variable region of anti-CD28 scFv

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val 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 Phe Cys

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 13

<211> 108

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain variable region of anti-CD28 scFv

<400> 13

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 His Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr

85 90 95

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

100 105

<210> 14

<211> 750

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-CD 19scFv

<400> 14

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> 15

<211> 372

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-CD 19scFv

<400> 15

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> 16

<211> 333

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-CD 19scFv

<400> 16

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> 17

<211> 729

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-CD 3scFv

<400> 17

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> 18

<211> 357

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-CD 3scFv

<400> 18

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagc 357

<210> 19

<211> 318

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-CD 3scFv

<400> 19

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> 20

<211> 729

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of anti-CD28 scFv

<400> 20

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctactaca tccactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatcggctgc atctaccccg gcaacgtgaa caccaactac 180

aacgagaagt tcaaggaccg cgccaccctg accgtggaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt acttctgcac ccgcagccac 300

tacggcctgg actggaactt cgacgtgtgg ggccagggca ccaccgtgac cgtgagcagc 360

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgacat ccagatgacc 420

cagagcccca gcagcctgag cgccagcgtg ggcgaccgcg tgaccatcac ctgccacgcc 480

agccagaaca tctacgtgtg gctgaactgg taccagcaga agcccggcaa ggcccccaag 540

ctgctgatct acaaggccag caacctgcac accggcgtgc ccagccgctt cagcggcagc 600

ggcagcggca ccgacttcac cctgaccatc agcagcctgc agcccgagga cttcgccacc 660

tactactgcc agcagggcca gacctacccc tacaccttcg gcggcggcac caaggtggag 720

atcaagcgc 729

<210> 21

<211> 360

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of heavy chain variable region of anti-CD28 scFv

<400> 21

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctactaca tccactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatcggctgc atctaccccg gcaacgtgaa caccaactac 180

aacgagaagt tcaaggaccg cgccaccctg accgtggaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt acttctgcac ccgcagccac 300

tacggcctgg actggaactt cgacgtgtgg ggccagggca ccaccgtgac cgtgagcagc 360

<210> 22

<211> 324

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of light chain variable region of anti-CD28 scFv

<400> 22

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc acgccagcca gaacatctac gtgtggctga actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacaag gccagcaacc tgcacaccgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag ggccagacct acccctacac cttcggcggc 300

ggcaccaagg tggagatcaa gcgc 324

<210> 23

<211> 5

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of Linker fragment 1(Linker 1) in CD19-CD3-CD28TsAb _ M in monomeric form

<400> 23

Gly Gly Gly Gly Ser

1 5

<210> 24

<211> 15

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of Linker fragment 1(Linker 1) in CD19-CD3-CD28TsAb _ M in monomeric form

<400> 24

ggtggcggag ggtcc 15

<210> 25

<211> 15

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of Linker fragment 2(Linker 2) in CD19-CD3-CD28TsAb _ M in monomeric form

<400> 25

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

1 5 10 15

<210> 26

<211> 45

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of Linker fragment 2(Linker 2) in CD19-CD3-CD28TsAb _ M in monomeric form

<400> 26

ggaggcggag gttccggcgg tgggggatcg gggggtggag ggagt 45

<210> 27

<211> 5

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of Linker fragment 1(Linker 1) in CD19-CD3-CD28TsAb _ D in dimer form

<400> 27

Gly Gly Gly Gly Ser

1 5

<210> 28

<211> 15

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of Linker fragment 1(Linker 1) in CD19-CD3-CD28TsAb _ D in dimer form

<400> 28

ggtggcggag ggtcc 15

<210> 29

<211> 81

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of Linker fragment 2(Linker 2) in CD19-CD3-CD28TsAb _ D in dimer form

<400> 29

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> 30

<211> 243

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of Linker fragment 2(Linker 2) in CD19-CD3-CD28TsAb _ D in dimer form

<400> 30

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> 31

<211> 19

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of secretory expression signal peptide

<400> 31

Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser

1 5 10 15

Ser Arg Ala

<210> 32

<211> 57

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence for secretory expression of signal peptide

<400> 32

atgacccggc tgaccgtgct ggccctgctg gccggcctgc tggcctcctc cagggcc 57

<210> 33

<211> 59

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-Sig-F

<400> 33

gtgctggata tctgcagaat tcgccgccac catgacccgg ctgaccgtgc tggccctgc 59

<210> 34

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Sig-R

<400> 34

ggccctggag gaggccagca ggccggccag cagggccagc acggtcagc 49

<210> 35

<211> 42

<212> DNA

<213> Artificial

<220>

<223> Sig-CD19-F

<400> 35

ctgctggcct cctccagggc cgacatccag ctgacccaga gc 42

<210> 36

<211> 23

<212> DNA

<213> Artificial

<220>

<223> CD19-R

<400> 36

gctgctcacg gtcacggtgg tgc 23

<210> 37

<211> 56

<212> DNA

<213> Artificial

<220>

<223> CD19-G4S-CD3-F

<400> 37

ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag cagagc 56

<210> 38

<211> 20

<212> DNA

<213> Artificial

<220>

<223> CD3-R

<400> 38

cttcagctcc agcttggtgc 20

<210> 39

<211> 86

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-CD28-F

<400> 39

gcaccaagct ggagctgaag ggaggcggag gttccggcgg tgggggatcg gggggtggag 60

ggagtcaggt gcagctggtg cagagc 86

<210> 40

<211> 51

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-CD28-R

<400> 40

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51

<210> 41

<211> 41

<212> DNA

<213> Artificial

<220>

<223> CD3-IgD-F

<400> 41

gcaccaagct ggagctgaag gccagcaaga gcaagaagga g 41

<210> 42

<211> 21

<212> DNA

<213> Artificial

<220>

<223> IgD-R

<400> 42

cacgcccagg ggctgggtgt g 21

<210> 43

<211> 42

<212> DNA

<213> Artificial

<220>

<223> IgD-CD28-F

<400> 43

cacacccagc ccctgggcgt gcaggtgcag ctggtgcaga gc 42

Claims

1. A trifunctional molecule comprising a structure having 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 surface CD28 molecule, wherein the trifunctional molecule is capable of binding to and activating a T-cell surface CD28 molecule

The first functional domain is a single-chain antibody of anti-CD 19, the second functional domain is a single-chain antibody of anti-CD3, the third functional domain is a single-chain antibody of anti-CD28, and the single-chain antibody comprises 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 fragment 1 is a connecting fragment with G4S as a unit, the amino acid sequence of the connecting fragment is shown in any one of SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO.27, and

the connecting segment 2 is a hinge region segment of immunoglobulin IgD, and the amino acid sequence of the connecting segment is shown as SEQ ID NO. 29.

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 CD28 molecule while recognizing CD19, thereby generating a first signal and a second signal required for T-cell activation.

3. The trifunctional molecule of claim 1, wherein the anti-CD 19 single-chain antibody has the amino acid sequence of the heavy chain variable region as set forth in SEQ ID number 6; the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown as SEQ ID number 7; the amino acid sequence of the heavy chain variable region of the anti-CD3 single-chain antibody is shown as SEQ ID number 9; the amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown as SEQ ID number 10; the amino acid sequence of the heavy chain variable region of the anti-CD28 single-chain antibody is shown as SEQ ID number 12; the amino acid sequence of the light chain variable region of the anti-CD28 single-chain antibody is shown in SEQ ID number 13.

4. The trifunctional molecule according to claim 3, wherein the anti-CD 19scFv has an amino acid sequence as set forth in SEQ ID number 5; the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID number 8; the amino acid sequence of the anti-CD28 single-chain antibody is shown in SEQ ID number 11.

5. The trifunctional molecule of claim 1, wherein the trifunctional molecule has an amino acid sequence as set forth in SEQ ID number 3.

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

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

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

9. A method of preparing a trifunctional molecule according to any one of claims 1-5, 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.

10. Use of the trifunctional molecules of any one of claims 1-5 for the preparation of a medicament for the treatment of tumors.