BISPECIFIC ANTIBODY, PREPARATION METHOD THEREOF AND APPLICATION THEREOF

Abstract

A bispecific antibody, a preparation method therefor and an application thereof. The bispecific antibody includes a monoclonal antibody unit and a single-chain antibody unit. The single-chain antibody unit includes two complete light chain-heavy chain pairs, and is specifically bound to a surface antigen of a tumor cell. The single-chain antibody unit includes two single-chain antibodies. The single-chain antibody includes a heavy chain variable region and a light chain variable region, and is specifically bound to a surface antigen of an immunocyte. The bispecific antibody can be simultaneously bound to the immunocyte and the tumor cell, can mediate a directed immune response, and can effectively kill the tumor cell.

Claims

1. A bispecific antibody that binds to CD19 and CD3, wherein the bispecific antibody comprises (a) a monoclonal antibody unit and (b) a single-chain antibody unit; the monoclonal antibody unit consists of two complete light chain-heavy chain pairs, and can specifically bind to CD19; the single-chain antibody unit comprises two single-chain antibodies; the single-chain antibody comprises a heavy chain variable region and a light chain variable region, and can specifically bind to CD3; the bispecific antibody has a symmetric structure formed by linkage in any one of the following modes: (1) the C-ends of the two single-chain antibodies are respectively linked to the N-ends of two heavy chains of the monoclonal antibody through a linker peptide; and (2) N-ends of the two single-chain antibodies are respectively linked to C-ends of the two heavy chains of the monoclonal antibody through a linker peptide.

2. The bispecific antibody according to claim 1, wherein the amino acid sequence of the linker peptide is (GGGGX)n, wherein X is Gly or Ser, and n is a natural number selected from 1 to 4; preferably, the amino acid sequence of the linker peptide is represented by SEQ ID NO. 13.

3. The bispecific antibody according to claim 1, wherein, the light chain sequence of the single-chain antibody is represented by SEQ ID NO. 5 or represented by SEQ ID NO. 9; the heavy chain sequence of the single-chain antibody is represented by SEQ ID NO. 6 or represented by SEQ ID NO. 10; preferably, the light chain and the heavy chain of the single-chain antibody constitute a fusion peptide, and the sequence of the fusion peptide is any one of the follows: (1) when C-ends of the two single-chain antibodies are respectively linked to N-ends of two heavy chains of the monoclonal antibody through a linker peptide, the sequence of the fusion peptide is represented by SEQ ID NO. 16; and (2) when N-ends of the two single-chain antibodies are respectively linked to to C-ends of the two heavy chains of the monoclonal antibody through a linker peptide, the sequence of the fusion peptide is represented by SEQ ID NO. 17.

4. The bispecific antibody according to claim 3, wherein the bispecific antibody is a murine antibody, a humanized antibody, a chimeric antibody or a recombinant antibody.

5. The bispecific antibody according to claim 3, wherein the light chain and the heavy chain of the monoclonal antibody are connected by a disulfide bond; Fc fragment of the monoclonal antibody is a Fc fragment of a human or humanized antibody, and the human or humanized antibody is one of IgG1, IgG2, IgG3 or IgG4; preferably, the Fc fragment of the monoclonal antibody is a Fc fragment of a human or humanized IgG4 antibody; more preferably, a full-length sequence of the light chain of the monoclonal antibody is represented by SEQ ID NO. 3; and a full-length sequence of the heavy chain of the monoclonal antibody is represented by SEQ ID NO. 1 or SEQ ID NO. 20.

6. A gene encoding the bispecific antibody claim 1, preferably, a gene sequence coding a full-length light chain of the monoclonal antibody is represented by SEQ ID NO. 4; and/or, a gene sequence coding a full-length heavy chain of the monoclonal antibody is represented by SEQ ID NO. 2 or represented by SEQ ID NO. 21; and/or, when C-ends of the two single-chain antibodies are respectively linked to N-ends of two heavy chains of the monoclonal antibody through a linker peptide, a gene sequence coding the single-chain antibody is represented by SEQ ID NO. 14; and when N-ends of the two single-chain antibodies are respectively linked to C-ends of the two heavy chains of the monoclonal antibody through a linker peptide, a gene sequence coding the single-chain antibody is represented by SEQ ID NO. 15.

7. A biological material comprising the gene of claim 6, wherein the biological material comprises a recombinant DNA, an expression cassette, a vector, a host cell, an engineered bacterium or a cell line.

8. A preparation method of the bispecific antibody according to claim 1, wherein the method comprises: constructing an expression vector containing a coding gene of the single-chain antibody and the monoclonal antibody; introducing the expression vector into a host cell to obtain a host cell stably expressing the bispecific antibody; culturing the host cell, and obtaining the bispecific antibody by separation and purification.

9. A pharmaceutical composition, wherein the pharmaceutical composition comprises the bispecific antibody of claim 1.

10. (canceled)

11. The bispecific antibody according to claim 2, wherein, the light chain sequence of the single-chain antibody is represented by SEQ ID NO. 5 or represented by SEQ ID NO. 9; the heavy chain sequence of the single-chain antibody is represented by SEQ ID NO. 6 or represented by SEQ ID NO. 10; preferably, the light chain and the heavy chain of the single-chain antibody constitute a fusion peptide, and the sequence of the fusion peptide is any one of the follows: (1) when C-ends of the two single-chain antibodies are respectively linked to N-ends of two heavy chains of the monoclonal antibody through a linker peptide, the sequence of the fusion peptide is represented by SEQ ID NO. 16; and (2) when N-ends of the two single-chain antibodies are respectively linked to C-ends of the two heavy chains of the monoclonal antibody through a linker peptide, the sequence of the fusion peptide is represented by SEQ ID NO. 17.

12. The bispecific antibody according to claim 4, wherein the light chain and the heavy chain of the monoclonal antibody are connected by a disulfide bond; Fc fragment of the monoclonal antibody is a Fc fragment of a human or humanized antibody, and the human or humanized antibody is one of IgG1, IgG2, IgG3 or IgG4; preferably, the Fc fragment of the monoclonal antibody is a Fc fragment of a human or humanized IgG4 antibody; more preferably, a full-length sequence of the light chain of the monoclonal antibody is represented by SEQ ID NO. 3; and a full-length sequence of the heavy chain of the monoclonal antibody is represented by SEQ ID NO. 1 or SEQ ID NO. 20.

13. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is used for prevention or treatment of a CD19-expressing B cell-related disease; preferably, the CD19-expressing B cell-related disease include B cell-related tumors and autoimmune diseases caused by B cells.

14. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is used for prevention or treatment of a disease with CD19 as a target.

15. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is used for killing CD19-expressing cells.

16. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is used as a detection reagent for CD19 and/or CD3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1 is a schematic diagram of the structure of the cell surface antigen CD3 molecule in the background of the present invention.

[0065] FIG. 2 is a schematic diagram of the molecular structures of two bispecific antibodies YK001 and YK002 obtained through screening in Example 1 of the present invention, wherein A represents the bispecific antibody YK001; and B represents the bispecific antibody YK002.

[0066] FIG. 3 is the SDS-PAGE electrophoresis diagram of the bispecific antibodies YK001 and YK002 in Example 2 of the present invention, wherein A and C represent reduced SDS-PAGE electrophoresis detection; B and D represent non-reduced SDS-PAGE electrophoresis detection; A and B represent SDS-PAGE electrophoresis results of YK001 bispecific antibody; C and D represent SDS-PAGE electrophoresis results of YK002 bispecific antibody; M represents protein molecular weight marker, and lane 1 represents the target protein.

[0067] FIG. 4 shows HPLC-SEC purity peak graphs of bispecific antibodies YK001 and YK002 in Example 2 of the present invention, wherein A represents the bispecific antibody YK001; and B represents the bispecific antibody YK002.

[0068] FIG. 5 shows the binding efficiency of bispecific antibodies YK001 and YK002 with Raji cells determined based on flow cytometry in Example 3 of the present invention, wherein A represents the negative control NC; B represents the bispecific antibody YK001; C represents the positive control antibody (PC) Anti-CD19; D represents the negative control NC; E represents the bispecific antibody YK002; and F represents the positive control antibody (PC) Anti-CD19.

[0069] FIG. 6 shows the binding efficiency of bispecific antibodies YK001 and YK002 with T cells determined based on flow cytometry in Example 3 of the present invention, wherein A represents the negative control NC; B represents the bispecific antibody YK001; C represents the bispecific antibody YK002, and D represents the positive control (PC) Anti-CD3.

[0070] FIG. 7 is a diagram showing the results in Example 4 of the present invention that the bispecific antibodies YK001 and YK002 effectively mediated PBMC cells to kill Raji tumor cells, wherein (.Math.) represents the bispecific antibody YK001, (∇) represents the bispecific antibody YK002, (.square-solid.) represents Anti-CD19 monoclonal antibody, (⋄) represents irrelevant control 0527×CD3 bispecific antibody (Her2×CD3 bispecific antibody), and (.circle-solid.) represents Anti-CD3 monoclonal antibody.

SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS

[0071] The preferred embodiments of the present invention will be described in detail below in conjunction with Examples. It should be understood that the following Examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. A person skilled in the art can make various modifications and alternatives to the present invention without departing from the aim and spirit of the present invention.

[0072] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0073] The materials and reagents used in the following Examples can be obtained from commercial sources unless otherwise specified.

Example 1: Design of the Structure and Sequence of CD19×CD3 Bispecific Antibody

[0074] In the present Example, the tumor cell surface antigen CD19 and the immune cell surface antigen CD3 were used as targets to design a bispecific antibody.

[0075] Combined with protein structure design software and a lot of artificial experimental screening, a variety of CD19 and CD3 binding bispecific antibody structures were screened in the present invention for bispecific antibody structures with symmetrical structures comprising a single-chain antibody unit and a monoclonal antibody unit, wherein the anti-CD19 monoclonal antibody unit is an IgG antibody, and comprises two complete light chain-heavy chain pairs (i.e., containing complete Fab and Fc domains, and the heavy chain and the light chain are connected by a disulfide bond), the anti-CD3 single-chain antibody unit comprises two single-chain antibodies (ScFv), each single-chain antibody contains a heavy chain variable region domain and a light chain variable region domain, and the heavy chain variable region and the light chain variable region are constructed as a fusion peptide through a linker peptide. The single-chain antibody and the monoclonal antibody are linked by a linker peptide. For the linkage modes between the single-chain antibody and the monoclonal antibody, two different linkage methods were designed to obtain two bispecific antibodies with different symmetric structures:

[0076] (1) The C-ends of the anti-CD3 single-chain antibody were linked to the N-ends of the heavy chain of the anti-CD19 monoclonal antibody through a linker peptide of GGGGSGGGGSGGGGS (represented by SEQ ID NO. 13) to obtain a bispecific antibody YK001 (with the structure schematic diagram as shown in FIG. 2 A); and

[0077] (2) The N-ends of the anti-CD3 single-chain antibody were linked to the C-ends of the heavy chain of the anti-CD19 monoclonal antibody through a linker peptide of GGGGSGGGGSGGGGS (represented by SEQ ID NO. 13) to obtain a bispecific antibody YK002 (with the structure schematic diagram as shown in FIG. 2 B).

[0078] The amino acid sequence of each domain of the above-mentioned bispecific antibody is as follows:

[0079] The amino acid sequence of the heavy chain variable region of the anti-CD19 monoclonal antibody of YK001 is represented by SEQ ID NO. 19, and the amino acid sequence of the full-length heavy chain is represented by SEQ ID NO. 1.

[0080] The amino acid sequence of the heavy chain variable region of the anti-CD19 monoclonal antibody of YK002 is represented by SEQ ID NO. 19, and the amino acid sequence of the full-length heavy chain is represented by SEQ ID NO. 20.

[0081] The amino acid sequence of the light chain variable region of the anti-CD19 monoclonal antibody is represented by SEQ ID NO. 18, and the amino acid sequence of the full-length light chain is represented by SEQ ID NO. 3 (same for YK001 and YK002).

[0082] The amino acid sequence of the anti-CD3 single-chain antibody in YK001 is represented by SEQ ID NO. 16.

[0083] The amino acid sequence of the anti-CD3 single-chain antibody in YK002 is represented by SEQ ID NO. 17.

Example 2: Preparation of a CD19×CD3 Bispecific Antibody

[0084] 1. Design and synthesis of coding genes of the bispecific antibody

[0085] According to the amino acid sequences of the two bispecific antibodies YK001 and YK002 obtained by the design and screening in Example 1, and the codon preference of the host cell, the coding genes of the bispecific antibodies were designed, with the specific sequences as follows:

[0086] the nucleotide sequence coding the heavy chain of the anti-CD19 monoclonal antibody of YK001 was represented by SEQ ID NO. 2;

[0087] the nucleotide sequence coding the heavy chain of the anti-CD19 monoclonal antibody of YK002 was represented by SEQ ID NO. 21;

[0088] the nucleotide sequence coding the light chain of the anti-CD19 monoclonal antibody was represented by SEQ ID NO. 4 (same for YK001 and YK002);

[0089] the nucleotide sequence coding the anti-CD3 single-chain antibody in YK001 was represented by SEQ ID NO. 14; and

[0090] the nucleotide sequence coding the anti-CD3 single-chain antibody in YK002 was represented by SEQ ID NO. 15.

[0091] In order to facilitate the construction of expression vectors, the gene fragment coding the light chain of the anti-CD19 monoclonal antibody (same for YK001 and YK002) and the fusion fragment of the coding gene of the anti-CD3 single-chain antibody and the coding gene of the heavy chain of the anti-CD19 monoclonal antibody (YK001, i.e., the C-end of the anti-CD3 single-chain antibody is linked to the N-end of the heavy chain of the anti-CD19 monoclonal antibody) and the fusion fragment of the coding gene of the heavy chain of the anti-CD19 monoclonal antibody and the coding gene of the anti-CD3 single-chain antibody (YK002, i.e., the N-end of the anti-CD3 single-chain antibody is connected to the C-end of the heavy chain of the anti-CD19 monoclonal antibody) were synthesized.

[0092] 2. Construction of Bispecific Antibody Expression Vectors

[0093] (1) The coding gene of the light chain of the anti-CD19 monoclonal antibody was linked to the expression vector pG4HK by double enzyme digestion with SalI and BsiWI to obtain the expression vector of the light chain of the anti-CD19 monoclonal antibody named as pG4HK19VL.

[0094] (2) The fusion fragment of the coding gene of the anti-CD3 single-chain antibody and the coding gene of the heavy chain of the anti-CD19 monoclonal antibody was linked to the vector pG4HK19VL by double enzyme digestion with Hind III and BstE II to obtain the YK001 bispecific antibody expression vector name as pG4HK-YK001.

[0095] (3) The fusion fragment of the coding gene of the heavy chain of the anti-CD19 monoclonal antibody and the coding gene of the anti-CD3 single-chain antibody coding gene was linked to the vector pG4HK19VL by double enzyme digestion with Hind III and BstE II to obtain the YK002 bispecific antibody expression vector named as pG4HK-YK002.

[0096] 3. Expression of Bispecific Antibodies

[0097] (1) Plasmid was subject to large-scale extraction with an endotoxin-free large-scale extraction kit (Qiagen, 4991083), and the specific operation was carried out according to the instructions of the kit.

[0098] (2) Preparation of Cells for Transfection

[0099] (i) CHO-K1 cells were resuscitated, 6×10.sup.6 cells were inoculated into 12 ml CD-CHO medium (containing 6 mM GlutaMAX) at a density of 0.5×10.sup.6/ml, and the resultant was subjected to shake cultivation in 5% CO.sub.2, at 37° C. and 135 rpm.

[0100] (ii) On the day before transfection, the cell density was adjusted to 0.5×10.sup.6/ml, and the resultant was subjected to shake cultivation in 5% CO.sub.2, at 37° C. and 135 rpm.

[0101] (3) Electroporation transfection

[0102] (i) The cell concentration was measured by cell counting to ensure a cell viability of 95% or more.

[0103] (ii) 1×10.sup.7 cells were taken, centrifuged at 1,000 rpm for 5 min, the supernatant was discarded, the cells were suspended with fresh CD-CHO medium, the resultant was centrifuged at 1,000 rpm for 5 min, and the supernatant was discarded. Washing was repeated once again.

[0104] (iii) Cells were suspended with 0.7 ml CD-CHO medium, 40 μg of expression vector was added to be mix welled and the resultant was transferred to a 0.4 cm electroporation cuvette for electroporation.

[0105] (iv) The cells were quickly transferred to CD-CHO medium (without GlutaMAX) after electroporation, and plated in a 96-well plate, and cultured in 5% CO.sub.2 at 37° C.

[0106] (v) 24 hours after transfection, MSX was added to each well to a final concentration of 50 μM, and the resultant was subjected to cultivation in 5% CO.sub.2 at 37° C.

[0107] (vi) Monoclonal cell strains that highly express bispecific antibodies were picked out to perform fed-batch fermentation and the supernatant was collected after 14 days of culturing.

[0108] 4. Purification of Bispecific Antibodies

[0109] (1) Pretreatment of Feed

[0110] The supernatant of the fermentation culture was centrifuged at 2,000 rpm for 10 min, and then filtered with a 0.22 μM filter membrane.

[0111] (2) Affinity Chromatography

[0112] A Mabselect SuRe affinity chromatography column (purchased from GE, Catalog No. 18-5438-02) was used to capture the antibodies in the pretreated fermentation broth, an equilibration buffer (10 mM PB, 0.1 M NaCl, pH 7.0) was used to fully equilibrate the chromatography column, and the pretreated fermentation broth was allowed to pass through the affinity chromatography column, and elution was performed with an elution buffer (0.1 M citric acid, pH 3.0).

[0113] (3) Cation Exchange Chromatography

[0114] The sample prepared by affinity chromatography was further subjected to purification by SP cation exchange chromatography. The cation exchange column was purchased from GE (17-1014-01, 17-1014-03). After equilibration of the chromatography column with an equilibration buffer (50 mM PBS, pH 5.5), the sample was allowed to pass through the SP column for binding, and then linear elution was performed with 20 column volumes of an elution buffer (50 mM PBS, 1.0 M NaCl, pH 5.5).

[0115] (4) Anion Exchange Chromatography

[0116] After purification by SP cation exchange chromatography, the resultant was further allowed to pass through an ion exchange Q-Sepharose column (purchased from GE, Catalog Nos: 17-1153-01, 17-1154-01), and the buffer used was 50 mM PBS at pH 5.5.

[0117] The purified bispecific antibodies YK001 and YK002 were tested by SDS-PAGE and HPLC-SEC. The result of SDS-PAGE is shown in FIG. 3, the test result of reduced SDS-PAGE electrophoresis of YK001 is shown in A of FIG. 3, and the test result of non-reduced SDS-PAGE electrophoresis of YK001 is shown in B of FIG. 3. The test result of reduced SDS-PAGE electrophoresis of YK002 is shown in C of FIG. 3, and the test result of non-reduced SDS-PAGE electrophoresis of YK002 is shown in D of FIG. 3. The test result of HPLC-SEC is shown in FIG. 4, wherein the SEC test result of YK001 is shown in A of FIG. 4, and the SEC test result of YK002 is shown in B of FIG. 4. The test results show that the bispecific antibodies YK001 and YK002 are successfully prepared after expression and purification, and the purity of the purified bispecific antibodies is 95% or more.

Example 3: Determination of the Binding Activity of Bispecific Antibodies to Tumor Cells and Immune Cells

[0118] Raji cells (purchased from ATCC, CCL-86) were used as CD19-positive cells, T cells were used as CD3-positive cells, and the binding activity of the bispecific antibody of the present invention to target antigens of CD19-expressing tumor cells and CD3-expressing immune cells was detected by flow cytometry.

[0119] 1. Detection of the binding activity of bispecific antibodies to Raji cells by flow cytometry

[0120] (1) Collecting Raji cells: cells were collected at 1×10.sup.6 cells/tube.

[0121] (2) Rinsing the cells: the cells were rinsed once with 1 ml staining buffer (PBS containing 0.5% w/v BSA+2 mM EDTA), the resultant was centrifuged at 350×g at 4° C. for 5 min, and then cells were resuspended with 200 μl staining buffer.

[0122] (3) Bs-antibody binding: bispecific antibodies YK001 and YK002 were added to a concentration of 5 μg/ml, respectively, and the resultant was subjected to incubation on ice for 45 min.

[0123] (4) Rinsing the cells: 1 ml staining buffer was added to the cell suspension to mix well, and centrifuged at 350×g at 4° C. for 5 min, the supernatant was removed, and the resultant was rinsed once again. After centrifugation, cells were resuspended with 100 μl staining buffer.

[0124] (5) 5 μl of Biolegend antibody (PE anti-human IgG Fc Antibody, Biolegend, 409304) was added to a sample tube, isotype control (PE Mouse IgG2a, κ Isotype Ctrl (FC) Antibody, Biolegend, 400213) was added to an isotype control tube, and the resultants were subjected to incubation on ice in dark for 15 min.

[0125] (6) Rinsing the cells: 1 ml staining buffer was added to the cell suspension to mix well, the resultant was centrifuged at 350×g at 4° C. for 5 min, the supernatant was removed, and the resultant was rinsed once again.

[0126] (7) Detection with a flow cytometer: After resuspending the cells with 200 μl PBS, the resultant was subjected to detection with a flow cytometer.

[0127] The results of flow cytometry were shown in FIG. 5, wherein the detection results of binding of YK001 to Raji cells are shown in A, B and C of FIG. 5, and the detection results of binding of YK002 to Raji cells are shown in D, E and F of FIG. 5. The results show that both bispecific antibodies YK001 and YK002 can specifically bind to Raji cells, that is, the bispecific antibody fusion protein retains the binding function of the monoclonal antibody Anti-CD19.

[0128] 2. Detection of the binding activity of bispecific antibodies to T cells by means of flow cytometry

[0129] (1) Collecting T cells: cells were collected at 1×10.sup.6 cells/tube.

[0130] (2) Rinsing the cells: the cells were rinsed once with 1 ml staining buffer (PBS containing 0.5% w/v BSA+2 mM EDTA), the resultant was centrifuged at 350×g at 4° C. for 5 min, and then the cells were resuspended with 200 μl staining buffer.

[0131] (3) Bs-antibody binding: bispecific antibodies YK001 and YK002 were added to a concentration of 5 μg/ml, respectively, and the resultant was subjected to incubation on ice for 45 min.

[0132] (4) Rinsing the cells: 1 ml staining buffer was added to the cell suspension to mix well, the resultant was centrifuged at 350×g at 4° C. for 5 min, the supernatant was removed, and the resultant was rinsed once again. After centrifugation, the cells were resuspended with 100 μl staining buffer.

[0133] (5) 5 μl of Biolegend antibody (PE anti-human IgG Fc Antibody, Biolegend, 409304) was added to a sample tube, isotype control (PE Mouse IgG2a, κ Isotype Ctrl (FC) Antibody, Biolegend, 400213) was added to an isotype control tube, and the resultants were subjected to incubation on ice in dark for 15 min.

[0134] (6) Rinsing the cells: 1 ml staining buffer was added to the cell suspension to mix well, the resultant was centrifuged at 350×g at 4° C. for 5 min, the supernatant was removed, and the resultant was rinsed once again.

[0135] (7) Detection with a flow cytometer: After resuspending the cells with 200 μl PBS, the resultant was subjected to detection with a flow cytometer.

[0136] The results of flow cytometry were shown in FIG. 6, wherein the detection results of binding of YK001 to T cells are shown in A and B of FIG. 6, and the detection results of binding of YK002 to T cells are shown in C and D of FIG. 6. The results show that both bispecific antibodies YK001 and YK002 can specifically bind to T cells, that is, the bispecific antibody fusion protein retains the binding function of the single-chain antibody Anti-CD3.

Example 4: Detection of In-Vitro Cell Killing Efficiency Mediated by Bispecific Antibodies

[0137] In the present Example, Raji-Luc cells were used as target cells, PBMCs were used as immune effector cells, and the effect of killing the target cells mediated by bispecific antibodies YK001 and YK002 was detected, with anti-CD3 monoclonal antibody and anti-CD19 monoclonal antibody and 0527×CD3 bispecific antibody as control.

[0138] 1. Preparation of Target Cells

[0139] As target cells, Raji-Luc cells (luciferase-labeled Raji cells) were counted after mixing well by pipetting up and down, centrifuged at 1,000 rpm for 5 min, and washed once with PBS. After centrifugation and washing of the target cells, the density was adjusted to 0.2×10.sup.6/ml with GT-T551 culture medium, 50 μl of the resultant was added to each well with 10,000 cells in each well.

[0140] 2. Preparation of PBMCs

[0141] PBMCs were used as effector cells. PBMCs frozen in a liquid nitrogen tank were taken out (referring to cell cryopreservation and resuscitation), thawed and added to a 15 ml centrifuge tube containing PBS or GT-T551 culture medium, and centrifuged at 1,000 rpm for 5 min. The cells were washed twice with PBS or GT-T551 culture medium and counted, the activity and density of cells were detected, and the density of living cells was adjusted to 2×10.sup.6/ml. 50 μl of the resultant was added to each well with 100,000 cells in each well.

[0142] 3. Dilution of Antibodies

[0143] The bispecific antibodies YK001 and YK002 were diluted with GT-T551 culture medium, respectively, and the initial concentration of the antibodies YK001 and YK002 was adjusted to 10 nM. The resultant was diluted sequentially at a ratio of 1:5. 100 μl of the diluted antibody was added to the cells prepared above in a 96-well plate to mix well, the 96-well plate was put back into the incubator, and the killing effect was detected after 18 hours.

[0144] 4. Detection

[0145] Since the Luciferase gene was carried by Raji target cells, the efficiency of killing the target cells was detected by the LUMINEX method.

[0146] Steady-GLO (Promega) was used as a substrate. After thawed, the buffer in the kit was added to the substrate powder to mix well, and the resultant was sub-packed with 5 ml or 10 ml for each package to complete reconstruction of the steady-GLO substrate.

[0147] After the co-cultured cells were mixed well by pipetting up and down, 100 μl was taken and transferred to an opaque white plate, then 100 μl of the reconstructed steady-GLO substrate was added, the resultant was tapped to mix well, and detected by a plate reader after standing for 5 minutes. The detection instrument was synergy HT.

[0148] 5. Data Processing

[0149] The calculation formula for the killing ratio of the target cell is as follows:

Killing ratio of target cells=100×(Only target−test well)/Only target.

[0150] The antibody concentration corresponding to the killing ratio of target cells in all detection wells was converted to log 10, which was used as the abscissa, and the killing ratio was used as the ordinate to make a graph. The results were shown in FIG. 7. The results were analyzed by the software Graphpad Prism 7.0, the IC50 of the bispecific antibody was calculated, and the results were shown in Table 2. The results show that compared with control antibodies (anti-CD3 monoclonal antibody, anti-CD19 monoclonal antibody and 0527×CD3 bispecific antibody), both bispecific antibodies YK001 and YK002 can effectively mediate PBMC to kill the tumor cell line Raji-Luc, as a single molecular, both YK001 and YK002 have the biological functions of Anti-CD19 and Anti-CD3 monoclonal antibodies at the same time, and the efficacy of killing target cells mediated by YK001 is higher than that of YK002.

TABLE-US-00002 TABLE 2 IC50 of target cell killing mediated by bispecific antibodies YK001 and YK002 Anti- Anti- 0527 × CD3 CD19 YK001 YK002 CD3 IC50 (nM) 0.02866 N/A 0.0004193 0.002445 ~0.4051

[0151] Although the general description and specific embodiments have been used to describe the present invention in detail above, it is obvious to a person skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

INDUSTRIAL APPLICABILITY

[0152] The present invention provides a bispecific antibody, a preparation method thereof and a use thereof. The bispecific antibody of the present invention comprises a monoclonal antibody unit and a single-chain antibody unit, wherein, the monoclonal antibody unit comprises two complete light chain-heavy chain pairs, and can specifically bind to a surface antigen of a tumor cell; the single-chain antibody unit comprises two single-chain antibodies, and the single-chain antibody comprises a heavy chain variable region and a light chain variable region, and can specifically bind to a surface antigen of an immune cell. The bispecific antibody provided in the present invention is of a symmetric structure formed by linkage in any one of the following modes: (1) C-ends of the two single-chain antibodies are respectively linked to N-ends of two heavy chains of a monoclonal antibody through a linker peptide; (2) N-ends of the two single-chain antibodies are respectively linked to C-ends of the two heavy chains of the monoclonal antibody through a linker peptide. The bispecific antibody of the present invention can simultaneously bind to the immune cell and the tumor cell, mediate a directed immune response, and effectively kill the tumor cell, with good economic value and application prospects.