CN102250245B - Bispecific antibody capable of resisting B cell lymphoma and application thereof - Google Patents

Abstract

The present invention relates to the technical fields of genetic engineering and protein engineering, in particular to DNA encoding a recombinant fusion protein comprising a human CD19 antibody variable region and a human CD3 antibody variable region fragment, the encoded fusion protein, and the production of the fusion protein Methods, pharmaceutical uses of the fusion protein and therapeutic methods of the fusion protein. The present invention provides a bispecific antibody protein comprising human CD19scFv-CD3scFv, which can bind to CD19 and CD3 positive cells, has good biological activity both in vivo and in vitro, and can activate human T lymphocytes and kill B lymphoma cells, It has a good application prospect.

Description

Anti-B-cell lymphoma bispecific antibody and use thereof

technical field

The present invention relates to the technical fields of genetic engineering and protein engineering, in particular, to a DNA encoding a recombinant fusion protein comprising a variable region of a human CD19 antibody and a fragment of a variable region of a human CD3 antibody, the encoded fusion protein, the fusion The production method of the protein, the pharmaceutical use of the fusion protein and the treatment method of the fusion protein.

Background of the invention

B-cell lymphoma is a malignant tumor with a high incidence rate, which seriously endangers human health. At present, its antibody drug therapy has been affirmed, and antibody drugs such as Rituximab have been marketed. Antibody drug targets for B-cell lymphoma are mainly CD molecules with increased specific expression in B-lymphoma cells, such as CD19, CD20, and CD22. Antibody drugs targeting these CD molecules can induce apoptosis of B lymphoma cells, thereby achieving the purpose of treating the disease.

Immunotherapy is also a commonly used anti-tumor treatment method, in which cellular immunity plays a very important role in anti-tumor. T lymphocytes in immune cells are important cells for killing tumors, and there are surface molecules such as CD3 and interleukin-2 receptors on their surfaces. Studies have shown that CD3 antibody and interleukin-2 ligand can effectively activate T lymphocytes, induce their activation and proliferation, thereby increasing the activity of killing tumor cells, which have been applied to tumor immunotherapy. However, due to the lack of the function of targeting tumor cells, it is difficult for activated T lymphocytes to be very effectively enriched in tumor sites, resulting in insufficient tumor killing efficiency. Therefore, molecules that can activate T lymphocytes, such as CD3 antibodies, can be fused with molecules that target B lymphoma cells, such as CD19 antibodies, so that T lymphocytes can be effectively enriched and activated around B lymphoma cells, thereby greatly improving effect on killing tumor cells.

Natural antibodies in the human body are composed of heavy chains and light chains, and the variable region structures of the heavy chains and light chains play a key role in the recognition of antigens. In recent years, the emergence of human antibodies has overcome the occurrence of HAMA (Human antimous antibody) caused by the original mouse antibody therapy, and greatly improved the clinical treatment effect. Compared with humanized antibodies, human antibodies not only have a fully human amino acid sequence, but also have the same spatial structure as human antibodies, and their immunogenicity is very low. It has more advantages and has great clinical application prospects.

In view of this, the present invention provides a bispecific antibody, which can not only target B lymphoma cells, but also activate T lymphocytes, greatly improving the effect of killing B lymphoma cells. Moreover, the bispecific antibody is a fully human protein sequence and has extremely low immunogenicity, which greatly improves the application value of the bispecific antibody of the present invention.

Contents of the invention

One of the technical problems to be solved by the present invention is to provide a human bispecific antibody protein comprising CD19scFv and CD3scFv, which has good biological activity and stability in vivo and in vitro, can bind to CD19 and CD3 positive cells, and It can activate human T lymphocytes and kill B lymphoma cells.

The technical solution of the present invention to solve the technical problem is to provide an anti-B cell lymphoma bispecific antibody. The anti-B-cell lymphoma bispecific antibody is composed of the variable region from the anti-CD19 antibody and the variable region of the anti-CD3 antibody, which are connected by a linker.

Wherein, the variable region of the CD19 antibody in the above-mentioned anti-B-cell lymphoma bispecific antibody is formed by fusion of a light chain variable region (VL) and a heavy chain variable region (VH). The CD19 antibody variable region can be expressed as CD19scFv, and the arrangement of the light chain variable region and the heavy chain variable region of the CD19 antibody variable region can be VL-VH or VH-VL. Further, the light chain variable region (VL) and the heavy chain variable region (VH) are linked by a connecting peptide to form an antibody variable region. The amino acid sequence of the commonly used connecting peptide is GGGGSGGGGSGGGGS, which can be expressed as (G4S)3.

Preferably, the amino acid sequence of the variable region of the CD19 antibody in the above-mentioned anti-B-cell lymphoma bispecific antibody is shown in SEQ ID NO.2.

Wherein, the variable region of the CD3 antibody in the above-mentioned anti-B-cell lymphoma bispecific antibody is formed by fusion of a light chain and a heavy chain. Denoted as CD3scFv.

The CD3 antibody variable region can be expressed as CD3scFv, and the arrangement of the light chain variable region and the heavy chain variable region of the CD3 antibody variable region can be VL-VH or VH-VL. Further, the light chain variable region (VL) and the heavy chain variable region (VH) are connected by a connecting peptide. The amino acid sequence of the commonly used connecting peptide is GGGGSGGGGSGGGGS, which can be expressed as (G4S)3.

Preferably, the amino acid sequence of the variable region of the CD3 antibody in the above-mentioned anti-B-cell lymphoma bispecific antibody is shown in SEQ ID NO.4.

Among them, the purpose of the connecting peptide in the above-mentioned anti-B-cell lymphoma bispecific antibody is to provide better flexibility, so as to avoid the formation of CD19 scFv and CD3 scFv or the light chain variable region and heavy chain light chain variable region in scFv. Due to the steric hindrance in the structure, the amino acid sequence and length of the connecting peptide can be changed to a certain extent. The amino acid sequence of the commonly used connecting peptide is GGGGSGGGGSGGGGS, which can be expressed as (G4S)3.

Further, the above-mentioned bispecific antibody against B-cell lymphoma:

(1) its amino acid sequence is shown in SEQ ID NO.8;

Or: Substitution and/or deletion and/or addition of one or several amino acids in the amino acid sequence of the protein shown in SEQ ID No.8 have the same function as the bispecific antibody shown in SEQ ID No.8 or similar antibodies.

The present invention also provides the nucleotide sequence encoding the above-mentioned anti-B-cell lymphoma bispecific antibody.

Further, the nucleotide sequence encoding the bispecific antibody against B-cell lymphoma:

(1) The nucleotide sequence is shown in SEQ ID NO.7; or the degenerate sequence of SEQ ID NO.7;

Or (2): a nucleotide sequence derived from the nucleotide sequence defined in (1) by substitution, deletion or addition of one or several nucleotides, and has the same coding function as the nucleotide sequence in (1) the same or similar antibodies.

The present invention also provides a gene carrier containing the above-mentioned nucleotide sequence encoding the bispecific antibody against B-cell lymphoma. The vector may be selected from a plasmid or a virus.

The present invention also provides host cells containing the above-mentioned gene carrier. The host cell may be selected from eukaryotic or prokaryotic cells.

The present invention also provides the application of the above-mentioned anti-B-cell lymphoma bispecific antibody in the preparation of anti-B-cell lymphoma drugs.

The present invention also provides an anti-B-cell lymphoma pharmaceutical composition, which uses the above-mentioned anti-B-cell lymphoma bispecific antibody as an active ingredient. Of course, it can add pharmaceutical excipients. The dosage form of the anti-B-cell lymphoma pharmaceutical composition can be common dosage forms such as injection, powder injection, freeze-dried preparation and the like.

Further, the above-mentioned anti-B-cell lymphoma pharmaceutical composition also includes at least one other anti-B-cell lymphoma drug.

The above-mentioned anti-B-cell lymphoma pharmaceutical composition can also be used to treat B-cell lymphoma together with other treatment methods selected from chemotherapy, radiation therapy, and gene therapy.

The present invention designs and constructs a bispecific antibody containing CD19scFv and CD3scFv, so that it has sufficient targeting and biological activity. More importantly, this bispecific antibody has a fully human sequence, which effectively avoids the occurrence of HAMA in clinical treatment applications, thereby more effectively reducing the occurrence of drug resistance and improving drug utilization efficiency and therapeutic effect .

Natural antibodies in the human body are composed of heavy chains and light chains, in which the structures of the variable region of the heavy chain and the variable region of the light chain are particularly important for the binding of antigens. The research of the present invention shows that the fusion protein (that is, the single-chain antibody) composed of the variable region of the heavy chain and the variable region of the light chain still has good antigen-binding activity. The molecular weight of this single-chain antibody is only about 25kD, and the volume is only 1/6 of the natural antibody, so it has better tissue penetration ability. The natural antibody variable region is a heterodimer composed of two peptide chains, the heavy chain variable region and the light chain variable region, while the scFv is a fusion single chain composed of the heavy chain variable region and the light chain variable region, May form steric hindrance, making it difficult to form an effective antigen-binding conformation. Therefore, it is necessary to insert a flexible connecting peptide between the variable region of the heavy chain and the variable region of the light chain, so as to ensure that the single-chain antibody can form a correct spatial configuration and have effective antigen-binding activity. In addition, in order to ensure that the two different scFvs of the bispecific antibody have their own antigen-binding activities, a flexible linking peptide needs to be inserted between the two scFvs to avoid possible steric hindrance between the two scFvs.

In order to effectively purify the bispecific antibody of the present invention, the present invention fuses His6 and Flag tag protein at the N-terminus of the bispecific antibody, because the Flag tag amino acid sequence (DYKDDDDK) contains an enzyme cleavage site for enterokinase (EK enzyme) Click DDDDK, so after the protein is purified using His6 and Flag tags, the His6 and Flag tags can be further cut off with EK enzyme to obtain a bispecific antibody without tag proteins.

The design of the bispecific antibody of the present invention can also be used for the fusion of scFv and CD3 scFv of other anti-tumor antibodies. The formed bispecific antibody can target tumor cells on the one hand, and activate T lymphocytes on the other hand. So as to play its role in killing tumor target cells.

The bispecific antibody described in the present invention can be constructed by conventional gene recombination technology, and the specific experimental steps are as described in the third edition of "Molecular Cloning" (Joseph Sambrook, Science Press) and similar experimental manuals. The CD19 single-chain antibody, CD3 single-chain antibody and connecting peptide used can be respectively:

1. The variable region of the CD19 human antibody is expressed as CD19scFv, the amino acid sequence is shown in SEQ ID NO.2 in the sequence listing, and the coding nucleotide sequence is shown in SEQ ID NO.1.

2. The variable region of the CD3 human antibody is expressed as CD3scFv, the amino acid sequence is shown in SEQ ID NO.4 in the sequence listing, and the coding nucleotide sequence is shown in SEQ ID NO.3.

3. The connecting peptide used between CD19scFv and CD3scFv is represented as (G4S) 3, the amino acid sequence is as described in SEQ ID NO.6 in the sequence listing, and the encoding nucleotide sequence is as shown in SEQ ID NO.5.

The DNA of the above-mentioned bispecific antibody can be obtained by conventional gene recombination technology. The required DNA sequences encoding CD19scFv and CD3scFv are from natural human CD19 and CD3 antibodies, respectively. The DNA sequences encoding the above scFv are obtained by PCR and cloned into vectors respectively, and the vectors used can be plasmids, viruses or DNA fragments commonly used in molecular biology. A protein secretion signal peptide sequence is added to the front of the DNA sequence encoding the above-mentioned bispecific antibody to ensure that the bispecific antibody can be secreted from the cells. The vector sequence includes a promoter for gene expression, protein translation initiation and termination signals, and polyadenylic acid (PolyA) sequence. The vector contains antibiotic resistance genes to facilitate the replication and expression of the vector in host cells such as bacteria and eukaryotic cells. In addition, the vector also includes a eukaryotic cell selection gene for selection of stable transfection host cell lines.

The functional segments of the bispecific antibody of the present invention are two independent scFvs. Therefore, under the condition of ensuring the integrity of the scFv, the amino acid sequence and length at both ends of the scFv can be changed without weakening its biological activity. They all belong to this invention. scope of invention.

The arrangement of the light chain variable region (VL) and the heavy chain variable region (VH) in the scFv of the bispecific antibody of the present invention can be VL-VH or VH-VL, and the arrangement of the two scFv can be It may be CD19scFv-CD3scFv, or it may be CD3scFv-CD19scFv, and they all belong to the scope of the present invention.

The purpose of the internal connecting peptide of the bispecific antibody of the present invention is to provide better flexibility to avoid steric hindrance in the formation of CD19scFv and CD3scFv. Therefore, the amino acid sequence and length of the connecting peptide can have certain changes, and they all belong to this invention. scope of invention.

After the construction of the plasmid containing the DNA sequence encoding the above-mentioned bispecific antibody is completed, the recombinant vector can be used to transfect or transform the host cell to express the corresponding protein. There are various expression systems that can be used to express these bispecific antibodies, which can be eukaryotic cells or prokaryotic cells, including mammalian cells, insect cells, yeast, bacteria, etc. Mammalian cells are the preferred system for expressing bispecific antibodies because prokaryotic cells are prone to formation of inclusion bodies. There are many kinds of mammalian cells that can be used for large-scale expression of bispecific antibodies, such as CHO cells, 293 cells, NSO cells, COS cells, etc., all of which are included in the cells that can be used in the present invention. The recombinant plasmid containing the gene encoding the above-mentioned bispecific antibody can be transfected into the host cell. There are many methods for transfecting the cell, including electroporation, liposome transfection and calcium phosphate transfection.

A preferred method of protein expression is expression by stably transfected host cells. For example, after stably transfecting host cells without neomycin resistance with a recombinant vector containing a neomycin (Neomycin) resistance gene, the concentration of neomycin can be increased in the cell culture medium to select stable cells with high expression For another example, after stably transfecting a host cell lacking DHFR with a recombinant vector containing a dihydrofolate reductase (DHFR) gene, the concentration of methotrexate (MTX) can be increased in the cell culture medium to screen out a stable cell line with high expression .

Other expression systems other than mammalian cells, such as insect cells, yeast, bacteria, etc., can also be used to express the bispecific antibody of the present invention, and they are also included in the list of host cells that can be used in the present invention. These expression systems have higher protein yields than mammalian cells, but are prone to formation of inclusion bodies, thus requiring further protein renaturation.

Since the bispecific antibody of the present invention contains His and Flag tags, after the bispecific antibody is expressed, the protein concentration of the bispecific antibody in the cell culture medium can be measured by enzyme-linked immunosorbent assay (ELISA) or other methods, and can also be used Nickel column or immunoaffinity chromatography to purify the expressed bispecific antibody. In addition, the bispecific antibody of the present invention can be further purified by using it in combination with other protein purification methods such as ion exchange chromatography.

After obtaining the corresponding bispecific antibody from the recombinant culture medium, its binding activity to CD19 and CD3 can be detected by cell ELISA and flow cytometry. The experimental results show that the bispecific antibody of the present invention can bind CD19-positive Cells such as Raji cells can also bind to CD3-positive cells such as Jurkat cells, so the bispecific antibody constructed in the present invention can effectively target B lymphocytes and T lymphocytes.

After the high-purity bispecific antibody is obtained by the purification method, its activation effect on T lymphocytes can be detected in vitro, and its inhibitory effect on the growth of B-cell lymphoma can also be detected by using in vitro cell models and in vivo animal models. In in vitro experiments, methods such as MTT can be used to detect the proliferation effect of the bispecific antibody of the present invention on peripheral blood T lymphocytes with positive expression of CD3, or the growth of lymphoma cells with positive expression of D19 such as Raji cells can be detected by methods such as MTT. inhibition. In vivo experiments, the above-mentioned CD19-positive tumor cells can be used to construct mouse tumor models, and tumor models can be constructed by methods such as ventral and dorsal subcutaneous, abdominal cavity, tail vein, etc., to observe the anti-tumor or anti-metastasis of bispecific antibodies experiment.

The bispecific antibody of the present invention can also be carried and expressed by viral vectors, these viral vectors include but not limited to adenoviral vectors (adenoviral vectors), adeno-associated viral vectors (adeno-associated viral vectors), retroviral vectors (retroviral vectors), herpes simplex virus-based vectors, lentiviral vectors.

The present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared into various forms of pharmaceutical preparations according to conventional pharmacy techniques, more preferably injections, most preferably freeze-dried injections.

The pharmaceutical composition of the present invention also includes any one or several other antitumor drugs with synergistic effect, and the composition can treat tumors together with other treatment methods, and the other treatment methods include chemotherapy and radiotherapy , biological therapy.

The beneficial effect of the present invention is that it provides a human-derived CD19scFv-CD3scFv bispecific antibody, which can bind to CD19 and CD3 positive cells, and can activate human T lymphocytes, so as to achieve outstanding anti-B cell lymphoma effect , has a good application prospect

Description of drawings

Figure 1. Schematic diagram of the structure of the CD19-CD3 bispecific antibody.

Figure 2. Schematic diagram of CD19scFv-CD3scFv/pcDNA3.1(+) recombinant vector.

Figure 3. Identification of recombinant vectors by double enzyme digestion. 1, recombinant vector; 2, vector digested by HindIII/XhoI; M, molecular marker.

Figure 4. Western blot detection of bispecific antibodies secreted and expressed by CHO cells. 1, culture supernatant of untransfected CHO cells; 2: culture supernatant of stably transfected CHO cells.

Figure 5. Highly pure bispecific antibody protein obtained by affinity chromatography. 1, before purification; 2, after purification.

Figure 6. The binding effect of bispecific antibody to Raji detected by flow cytometry. A, PBS; B, CD19scFv-CD3scFv (10ug/ml).

Figure 7. The binding effect of bispecific antibody to Jurkat detected by flow cytometry. A, PBS; B, CD19scFv-CD3scFv (10ug/ml).

Figure 8. The activation effect of the bispecific antibody of the present invention on T lymphocytes (48h).

Figure 9. The in vitro growth inhibitory effect of the bispecific antibody of the present invention on Raji cells. (The number ratio of T cells to Raji cells is 10:1; 96h).

Fig. 10. Experimental results of the bispecific antibody of the present invention inhibiting the growth of xenografted tumors in mice, the ordinate is the tumor volume. 1: PBMC+PBS; 2: PBMC+CD19scFv-CD3scFv (0.1ug); 3: PBMC+CD19scFv-CD3scFv (1ug); 4: PBS; 5: CD19scFv-CD3scFv (1ug).

Figure 11. Experimental results of bispecific antibodies inhibiting the growth of xenografted tumors in mice to prolong the survival time of tumor-bearing mice. The abscissa is time, and the ordinate is the number of mice. 1: PBMC+PBS; 2: PBMC+CD19scFv-CD3scFv (0.1ug); 3: PBMC+CD19scFv-CD3scFv (1ug); 4: PBS; 5: CD19scFv-CD3scFv (1ug).

Fig. 12. The effect of the bispecific antibody of the present invention on the tissues of important organs of mice. The results showed that it had no significant effect on the histological morphology of important organs in mice.

Detailed ways

The following examples describe in detail the construction, testing and application of the bispecific antibody involved in the present invention. However, the content and use of the present invention are not limited to the scope of the examples.

Example 1 Cloning of DNA sequences encoding bispecific antibodies and construction of recombinant vectors

The gene fragment encoding the bispecific antibody in the present invention can be obtained by classical molecular biology techniques, and the gene sequence can be optimized for the mammalian expression system in order to obtain better expression. Recombinant vectors can be obtained by reconnecting bispecific antibody gene fragments with corresponding expression vectors, which are suitable for expression and screening of mammalian cells.

1. Obtain the gene fragment encoding CD19-CD3 bispecific antibody

The variable region gene of the human CD19 antibody and the variable region gene of the human CD3 antibody in the present invention are respectively obtained by screening the whole human antibody library (the construction method of the whole human antibody library can be found in E, et al. Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat Biotechnol. 2000 Aug; 18(8): 852-6. and other known technology implementations). The structure of the bispecific antibody constructed in this preferred embodiment is shown in Figure 1. The bispecific antibody of the present invention is formed by fusion of CD19scFv and CD3scFv, and there is a connecting peptide between CD19scFv and CD3scFv which contains three consecutive (GGGGS) repeating sequences. The N-terminus of the bispecific antibody is added with the secretion signal peptide of interleukin 2 (IL-2) and His6 and Flag tags, which can not only ensure its secretion outside the mammalian cells, but also can be purified by affinity chromatography, and can be further The tag is cleaved using EK enzyme, leaving only the active bispecific antibody protein. The CD19-CD3 bispecific antibody gene fragment in the present invention is obtained by splicing PCR amplification of the above-mentioned several fragments.

2. Construction of recombinant vectors expressing bispecific antibodies

The gene cloning encoding the optimized fusion protein of the present invention is inserted into the HindIII and XhoI restriction sites of the plasmid vector pcDNA3.1 (+) (Invitrogen Company) through the HindIII and XhoI restriction sites by the above-mentioned bispecific antibody fragment including the signal peptide Click the result (see Figure 2). The recombinant plasmid uses the CMV promoter to express the fusion protein, and contains the SV40 polyadenylic acid (PolyA) sequence to ensure its optimal expression. The recombinant plasmid also includes an ampicillin resistance gene to facilitate replication in bacteria, and a neomycin resistance gene to select stably transfected cells. The recombinant plasmid encoding the bispecific antibody was transformed into E.coli (DH5α) and then added to LB medium for overnight culture to obtain a large number of copies of the recombinant plasmid. The plasmid was extracted with a plasmid extraction kit (Qiagen Company) and digested and digested. Sequencing identification (see Figure 3), the obtained DNA sequence encoding the bispecific antibody is shown in SEQ ID NO.7.

Example 2 Expression and purification of the bispecific antibody of the present invention in cells

The bispecific antibody in the present invention is expressed in CHO cells and secreted into the culture medium, and purified by nickel column affinity chromatography.

1. Transient expression of bispecific antibody in CHO cells

After obtaining high-purity recombinant plasmids encoding bispecific antibodies, use Lipofectamine 2000 Plasmid Transfection Kit (Invitrogen) to transfect CHO cells (ATCC) with the recombinant plasmids, and collect CHO cell supernatants after culturing in serum-free medium for three days solution, the expression of the bispecific antibody can be detected by immunoblotting (see Figure 4). This method can be used to quickly obtain a small amount of bispecific antibody protein, and its concentration can be quantitatively detected by ELISA, and the primary antibody used can be anti-His6 or Flag antibody.

2. Stable expression of bispecific antibody in CHO cells

The recombinant plasmid encoding the bispecific antibody was transfected into CHO cells with Lipofectamine 2000 plasmid transfection kit (Invitrogen Company), cultured in serum-free medium for two days, then added neomycin, and the cell clone culture was carried out by limiting dilution method, approximately After 14 days, neomycin-resistant cell clones were picked for cell expansion, and cells in good condition were selected for cryopreservation in liquid nitrogen. Stably transfected CHO cells can be further expanded in rolling cell culture flasks to produce a large amount of bispecific antibody protein. This method can be used to obtain a large amount of bispecific antibody protein, and its concentration can be quantitatively detected by ELISA method. The primary antibody can be anti-His6 or Flag antibody.

3. Purification of bispecific antibodies

The cell culture solution containing the bispecific antibody can be purified by nickel column affinity chromatography (see Figure 5). After equilibrating the nickel chromatography column with buffer solution, inject the supernatant of CHO cell culture fluid concentrated by ultrafilter, monitor with A280 (nm), wash with cleaning solution until all unbound proteins are eluted, The bound protein is then eluted with an eluent. The concentration of the purified bispecific antibody can be detected by ELISA method. The eluate containing the bispecific antibody can be lyophilized after desalting and purification, and can be stored at -20°C for long-term storage after lyophilization.

Example 3 In vitro binding experiment of the bispecific antibody of the present invention and cells

The bispecific antibody of the present invention may be able to bind the corresponding target cells in vitro. In the present invention, Raji cells are used as CD19-positive cells, Jurkat cells are used as CD3-positive cells, and the bispecific antibody of the present invention is used to detect their cell-binding activity.

1. Detection of binding activity of bispecific antibody to Raji cells by flow cytometry

The bispecific antibody was mixed with Raji cells in 1% bovine serum albumin (BSA) and 0.02% sodium azide in PBS (staining buffer), and incubated on ice for 1 hour. Cells were washed twice with PBS buffer, and then incubated with mouse anti-His6 antibody on ice for 1 hour. After the cells were washed with PBS buffer, they were incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse antibody in staining buffer for 30 minutes on ice. Cells were washed twice with flow buffer and analyzed by flow cytometry (ESP Elite, Coulter). The average fluorescence intensity was calculated by multiplying the average logarithmic fluorescence by the percentage of positive population. The results show that the bispecific antibody of the present invention can bind to CD19-positive Raji cells (see Figure 6).

2. Detection of binding activity of bispecific antibody to Jurkat cells by flow cytometry

The process of this example is basically the same as the above example. After the bispecific antibody was incubated with Jurkat cells on ice for 1 hour, they were washed twice with PBS buffer, and then incubated with mouse anti-His6 antibody on ice for 1 hour. After the cells were washed with PBS buffer, they were incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse antibody in staining buffer for 30 minutes on ice. Cells were washed twice with flow buffer and analyzed by flow cytometry (ESP Elite, Coulter). The average fluorescence intensity was calculated by multiplying the average logarithmic fluorescence by the percentage of positive population. The results showed that the bispecific antibody of the present invention can bind to CD3-positive Jurkat cells (see Figure 7).

Example 4: Detection of in vitro tumor cell growth inhibitory activity of the bispecific antibody of the present invention

The invention uses peripheral blood T lymphocytes and Raji cells as cell models to detect the in vitro growth inhibitory effect of bispecific antibodies on Raji cells.

1. Bispecific antibodies effectively activate T lymphocytes

Peripheral blood was drawn from healthy volunteers, and peripheral blood T lymphocytes were separated with T lymphocyte separation medium. T lymphocyte cells (1×10 ⁴ /well, 200ul) were seeded in a 96-well cell culture plate, and bispecific antibodies of different concentrations were added to the cell culture wells. After continuing to culture the cells for 48 hours, add 20 ul of MTT thiazolium blue solution with a concentration of 5 mg/ml to each well. After continuing to incubate for 4 hours, discard the supernatant in the cell culture wells, add 150ul dimethyl sulfoxide (DMSO) to each well, and shake for 10 minutes to fully dissolve the crystals. The absorption value of each well at a wavelength of 490 nm was measured with an enzyme-linked immunosorbent assay, and the cell growth curve was drawn. The results showed that the bispecific antibody of the present invention can effectively activate T lymphocytes (see Figure 8).

2. Bispecific antibody effectively inhibits the growth of Raji cells in vitro

Inoculate Raji cells (1×10 ⁴ /well, 200ul) in a 96-well cell culture plate. After the cells adhere to the wall, add peripheral blood T lymphocytes to the culture plate according to the ratio of T lymphocytes to Raji cells 10:1 Inside, different concentrations of bispecific antibodies were added to the cell culture wells at the same time. After continuing to culture the cells for 96 hours, add 20 ul of MTT thiazolium blue solution with a concentration of 5 mg/ml to each well. After continuing to incubate for 4 hours, discard the supernatant in the cell culture wells, add 150ul dimethyl sulfoxide (DMSO) to each well, and shake for 10 minutes to fully dissolve the crystals. The absorption value of each well at a wavelength of 490 nm was measured with an enzyme-linked immunosorbent assay, and the cell growth curve was drawn. The results show that the bispecific antibody of the present invention can effectively induce T lymphocytes to inhibit the growth of Raji cells (see Figure 9).

Example 5: Detection of growth inhibition of transplanted tumor in mice by bispecific antibody of the present invention

The present invention uses Raji cells and human peripheral blood mononuclear cells (PBMCs) to inoculate SCID mice to detect the in vivo growth inhibitory effect of bispecific antibodies on Raji cells, and to observe the histological morphology of important organs.

1. Bispecific antibodies can inhibit the growth of transplanted tumors in mice

5-6 weeks old female non-pregnant SCID mice (18-20g/body weight) were divided into 2 groups. One group was inoculated with Raji cells (1×10 ⁵ /monkey) on the ventral and dorsal side, and the other group was inoculated with Raji cells (1×10 ⁵ /monkey) and non-activated human peripheral blood mononuclear cells (PBMCs) (1×10 ⁶ / Only). Raji vaccination group received vehicle (PBS) or 1 μg bispecific antibody (CD19/CD3BsAb) treatment; Raji and PBMCs mixed vaccination group received vehicle (PBS), 0.1 μg or 1 μg bispecific antibody treatment; the treatment scheme was continuous tail vein Inject 7 times (1 time/day, 100ul/only). The tumor volume was calculated using the following formula: (width diameter ² ×long diameter)/2. The results showed that the bispecific antibody of the present invention can effectively inhibit the growth of Raji transplanted tumors (see Figure 10), and can prolong the survival of tumor-bearing mice (see Figure 11).

2. The bispecific antibody has no obvious effect on the histological morphology of important organs in mice

The mice treated with the bispecific antibody were sacrificed and the heart, liver, spleen, lung, kidney and other important organs were separated, fixed in 10% neutral formalin for 24 hours, dehydrated with graded alcohol, and embedded in paraffin. After tissue sections were stained with hematoxylin and eosin (HE), the slides were sealed with neutral gum, and the tissue morphology was observed under a microscope. The results showed that the bispecific antibody of the present invention had no significant effect on the histological morphology of important organs of mice (see Figure 12).

The above examples show that the human CD19-CD3 bispecific antibody of the present invention can be expressed in CHO cells and can be further purified by affinity chromatography. The obtained bispecific antibody can bind to CD19-positive Raji cells and CD3-positive Jurkat cells, and can activate human peripheral blood T lymphocytes, thereby achieving the effect of anti-B cell lymphoma. Works great.

Claims (8)

1. The bispecific antibody against B-cell lymphoma is composed of the variable region from the anti-CD19 antibody and the variable region from the anti-CD3 antibody, which are connected by a connecting peptide; the bispecific antibody against B-cell lymphoma The amino acid sequence of the specific antibody is shown in SEQ ID NO.8. 2. A nucleotide sequence encoding the anti-B-cell lymphoma bispecific antibody of claim 1. 3. The nucleotide sequence according to claim 2, characterized in that: the nucleotide sequence is shown in SEQ ID NO.7; or the degenerate sequence of SEQ ID NO.7. 4. A gene vector containing the nucleotide sequence according to any one of claims 2 or 3. 5. A host cell containing the gene vector of claim 4. 6. The application of the anti-B-cell lymphoma bispecific antibody of claim 1 in the preparation of anti-B-cell lymphoma drugs. 7. The anti-B-cell lymphoma pharmaceutical composition, characterized in that the anti-B-cell lymphoma bispecific antibody according to claim 1 is used as an active ingredient. 8. The pharmaceutical composition according to claim 7, further comprising at least one other anti-B-cell lymphoma drug.

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