Anti-CD19 antibody and application thereof

Abstract

The invention discloses an anti-CD19 antibody and application thereof, and belongs to the technical field of monoclonal antibodies. The number of the anti-CD19 antibodies is three, the anti-CD19 antibodies are respectively an anti-CD19 antibody 1011, an anti-CD19 antibody 1012 and an anti-CD19 antibody 1013, and amino acid sequences of VH and VL of the anti-CD19 antibodies and nucleotide sequences of coding genes of the anti-CD19 antibodies are shown as SEQ ID NO.1-12. The invention also discloses an application of the anti-CD19 antibody in preparation of anti-tumor drugs. According to the present invention, the completely human single-chain antibody specifically bound with CD19 is screened from the completely human bacteriophage library, has good affinity with the human CD19 protein, can be bound with NALM6 cells, is not bound with HEK293T cells, and can be used for tumor treatment. According to the invention, the fully humanized single-chain antibody specifically bound with CD19 is enriched, a potential new antibody drug is provided for clinical treatment of tumors, and the antibody has relatively high potential application value and significance.

Description

Technical Field

[0001] The present invention relates to anti-CD19 antibodies and applications thereof, and belongs to the technical field of monoclonal antibodies. Background Art

[0002] Tumor immunotherapy based on monoclonal antibodies is one of the current research hotspots in biological therapy. Currently available monoclonal antibody targeted therapy drugs have good therapeutic effects on patients.

[0003] The CD19 molecule is a type I transmembrane protein on the surface of B cells. The CD19 molecule begins to be expressed in the B-lineage progenitor cells in the bone marrow and continues to be expressed until the maturity of the B cells. Studies have shown that CD19 is highly expressed in a variety of B-cell-related malignancies such as B-lymphocytic leukemia and non-Hodgkin's lymphoma, so CD19 is an ideal target for the diagnosis and treatment of B-lymphocytic leukemia. Currently, the drug forms developed for the CD19 target include monoclonal antibodies, bispecific antibodies, ADC CAR-T, etc., and these drug forms are developed based on monoclonal antibodies targeting CD19.

[0004] At present, there are two main ways to obtain relatively mature monoclonal antibodies: hybridoma technology and phage antibody display technology. Hybridoma technology is to fuse mouse spleen cells immunized with antigens with mouse myeloma cells in vitro. The fused hybrid cells have both the ability of parent spleen cells to secrete antibodies and the ability of tumor cells to proliferate indefinitely. The mouse antibodies obtained by hybridoma technology generally have high affinity, but mouse antibodies are immunogenic and will produce HAMA effects when used in clinical practice. Phage display technology uses phage as a carrier, and connects the protein encoded by the antibody gene to the capsid protein PⅢ through genetic engineering, and expresses it on the surface of the phage in the form of a fusion protein. This antibody displayed by the phage can recognize specific antigens, and antibodies that specifically bind to specific antigens can be enriched through 3 to 5 rounds of screening. Phage display technology does not require immunization and can quickly obtain antibodies in large quantities in an in vitro environment. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides several anti-CD19 antibodies and applications thereof, belonging to the technical field of monoclonal antibodies.

[0006] The present invention is achieved through the following technical solutions:

[0007] There are three types of anti-CD19 antibodies, namely anti-CD19 antibody 1011, anti-CD19 antibody 1012, and anti-CD19 antibody 1013.

[0008] The amino acid sequence of the heavy chain variable region (VH) of the anti-CD19 antibody 1011 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO.4;

[0009] The amino acid sequence of VH of anti-CD19 antibody 1012 is shown in SEQ ID NO.6, and the amino acid sequence of VL is shown in SEQ ID NO.8;

[0010] The amino acid sequence of VH of the anti-CD19 antibody 1013 is shown in SEQ ID NO.10, and the amino acid sequence of VL is shown in SEQ ID NO.12.

[0011] An isolated nucleic acid encoding the above-mentioned anti-CD19 antibody.

[0012] Further, the nucleotide sequence encoding VH of the anti-CD19 antibody 1011 is shown in SEQ ID NO.1, and the nucleotide sequence encoding VL is shown in SEQ ID NO.3;

[0013] The nucleotide sequence encoding VH of the anti-CD19 antibody 1012 is shown in SEQ ID NO.5, and the nucleotide sequence encoding VL is shown in SEQ ID NO.7;

[0014] The nucleotide sequence encoding VH of the anti-CD19 antibody 1013 is shown in SEQ ID NO.9, and the nucleotide sequence encoding VL is shown in SEQ ID NO.11.

[0015] A recombinant expression vector comprises the isolated nucleic acid.

[0016] A transformant comprises the above-mentioned recombinant expression vector.

[0017] The use of the above anti-CD19 antibody, isolated nucleic acid, recombinant expression vector and transformant in the preparation of anti-tumor drugs.

[0018] Furthermore, the tumor is selected from B lymphocytic leukemia and / or non-Hodgkin's lymphoma.

[0019] The present invention screens out fully human single-chain antibodies that specifically bind to CD19 from a fully human phage library, has good affinity with human CD19 protein, can bind to NALM6 cells, and does not bind to HEK293T cells, and can be used for tumor treatment. The present invention enriches fully human single-chain antibodies that specifically bind to CD19, provides potential new antibody drugs for the clinical treatment of tumors, and has high potential application value and significance.

[0020] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Schematic diagram of phage titers in three rounds of screening.

[0022] Figure 2 : Schematic diagram of phage ELISA results.

[0023] Figure 3 : Schematic diagram of periplasmic ELISA identification results.

[0024] Figure 4 : Schematic diagram of flow cytometry analysis results (NALM6 cells).

[0025] Figure 5 : Schematic diagram of flow cytometry analysis results (HEK293T cells). DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0027] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0028] Experiment 1 Screening of anti-CD19 antibodies

[0029] 1. Experimental Methods

[0030] 1.1 Construction of a fully human phage antibody library

[0031] 1.1.1 Collect B cells from healthy individuals and extract total RNA. Amplify the VH and VL genes of the antibody by RT-PCR, perform electrophoresis, and purify and recover the VH and VL fragments.

[0032] 1.1.2 The recovered VH and VL fragments are connected to the phage vector through genetic engineering technology, and the constructed vector is transferred to Escherichia coli by electroporation. The phagemid vector can replicate itself in the successfully transduced host bacteria. When the host bacteria grow to the logarithmic phase, helper phage is added at an MOI of 10 for infection, and the phage is amplified by culturing overnight, and the phage library is obtained by centrifugal precipitation.

[0033] 1.2 Phage antibody library screening

[0034] Here are the steps:

[0035] (1) Coat 10 μg / ml CD19 protein on an ELISA plate and incubate at 4°C overnight; inoculate the stored E. coli TG1 and culture at 37°C and 220 rpm overnight.

[0036] (2) Transfer overnight grown E. coli TG1 at a ratio of 1:100 until the OD600 reaches 0.4.

[0037] (3) Remove the antigen solution from the ELISA plate, wash it with PBS three times, add 2% skim milk powder and block it at room temperature for 1 hour. At the same time, block the phage antibody library with 2% skim milk powder solution at room temperature for 1 hour.

[0038] (4) Add the blocked phage library to the wells containing the antigen and incubate at room temperature for 1 hour; the volume of the phage library added should be 10 11 ~10 12 .

[0039] (5) Remove the phage solution and wash the ELISA plate 15 times with PBST.

[0040] (6) Add 200 μl of pH 2.0 elution buffer and leave at room temperature for 10 minutes. Immediately add 60 μl of pH 8.0 Tris-HCl buffer for neutralization and repeat the elution once.

[0041] (7) Collect the eluted phages and take 250 μl to infect 10 ml of TG1 bacterial solution grown to the logarithmic phase. Let it stand at 37°C for half an hour, and then put it in a shaker at 37°C, 220 rpm, for 30 minutes.

[0042] (8) Take 10 μl of bacterial solution and dilute it 100 times, 1000 times, and 10,000 times respectively, and then inoculate it on a 2YT plate with resistance to calculate the phage titer.

[0043] (9) The remaining bacterial suspension was inoculated onto a 2YT plate (containing 100 μg / ml ampicillin and 1% glucose) and cultured in a 37°C incubator for 16 hours.

[0044] (10) Scrape all the colonies from the overnight plate using 2YT medium (100 μg / ml ampicillin and 1% glucose) and place in a 37°C incubator until the OD600 reaches 0.4.

[0045] (11) Add helper phage and place in an incubator at 37°C for 30 min and 220 rpm for 60 min.

[0046] (12) The bacterial precipitate was collected by centrifugation and cultured overnight in 2YT culture medium (100 μg / ml ampicillin, 50 μg / ml kanamycin and 0.1% glucose) at 30°C and 220 rpm.

[0047] (13) Centrifuge the overnight culture and collect the supernatant, which will serve as the phage for the second round of screening.

[0048] (14) Repeat the above screening process for at least 3 rounds until the phage titer is significantly improved.

[0049] 1.3 Enzyme-linked immunosorbent assay (ELISA)

[0050] Here are the steps:

[0051] (1) Dilute the antigen with PBS and coat the plate with 100 μl per well at 4°C overnight.

[0052] (2) After washing the ELISA plate once with PBST, 200 μl of 2% skim milk powder was added and blocked at room temperature for 1 hour.

[0053] (3) Wash the ELISA plate three times with PBST, add the diluted primary antibody, and incubate at room temperature for 1 hour.

[0054] (4) Wash the ELISA plate 4 times with PBST.

[0055] (5) Add diluted HRP-labeled secondary antibody and incubate at room temperature for 1 hour.

[0056] (6) Wash the ELISA plate 4 times with PBST.

[0057] (7) Add 100 μl / well TMB substrate solution, allow color to develop for 10 to 15 minutes, and then terminate the color development reaction with 2 M sulfuric acid.

[0058] (8) Read the plate at 450 nm on a microplate reader and analyze the results.

[0059] 1.4 Flow cytometric analysis of antibody binding to cell membrane surface antigens

[0060] (1) Cells were cultured at 1×10 6 The concentration of 100 / ml was suspended in 5% BSA solution and 1 ml was dispensed into each tube;

[0061] (2) After adding the diluted primary antibody, incubate on ice for 1 hour;

[0062] (3) Collect cells by centrifugation at 400 g for 5 min and wash once with cold PBS;

[0063] (4) Centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cells in 1 ml of 5% BSA solution, add fluorescently labeled secondary antibody, and incubate on ice for 30 min;

[0064] (5) Collect cells by centrifugation at 400 g for 5 min and wash three times with cold PBS;

[0065] (6) Collect the cells by centrifugation at 400 g for 5 min, resuspend the cells in 1 ml of PBS solution, and perform flow cytometric analysis.

[0066] 2. Experimental results

[0067] 2.1 Screening of fully human antibodies binding to CD19 protein

[0068] In this study, a fully human antibody (scFv) library constructed in our laboratory was used for screening against the human CD19 target. In this experiment, a CD19 protein with a concentration of 10 μg / ml was used as an antigen and coated on an ELISA plate. Figure 1 As shown, after three rounds of screening, with the same input amount, the titer of the obtained phage was significantly enriched.

[0069] After three rounds of screening, single clones were randomly selected for phage ELISA. The specific steps included: the coating concentration of CD19 protein was 1 μg / ml, 100 μl per well, coating at 4°C overnight, washing once with PBST, adding skim milk powder to block, then adding the blocked Phage supernatant, incubating at room temperature for 1 hour, washing 4 times with PBST, adding secondary antibody for incubation, washing and color development, and detecting OD450 with an enzyme marker. The results are as follows Figure 2 shown.

[0070] Positive clones were selected and sent for sequencing, which showed that three independent clones (1011, 1012, and 1013) were highly enriched. The gene sequences and amino acid sequences of the VH and VL of these three antibodies are shown below.

[0071] The gene sequence of VH of 1011 is as follows (direction 5'-3'), as shown in SEQ ID NO.1:

[0072] caggtgcagctggtggagtctgggggaggcttggtccagtctggagggtccctgagactctcctgtgcagcctctggattcaccttcagtgaccactacatggactgggtccgccaggctccaggaaaggggctggagtgggttggccgtagtagaaacaaagctaacagttacaccacagaatacgccgcgtctgtgaaaggcagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtcgggtcggaaaagtctggggccaagggacaatggtcaccgtctcgagt。

[0073] The amino acid sequence of VH of 1011 is shown below, as shown in SEQ ID NO.2:

[0074] QVQLVESGGGLVQSGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGRSRNKANSYTTEYAASVKGRFTISRDD SKNSLYLQMNSLKTEDTAVYYCRVGKVWGQGTMVTVSS。

[0075] The gene sequence of VL of 1011 is shown below (direction 5'-3'), as shown in SEQ ID NO.3:

[0076] gaaattgtgctgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgcaggtctagtcaaagcctcgtatacagtaatggaaacacctacttgaattggtttcagcagaggccaggccaatctccaaggcgcctaatttatagggtttctaaccgggactctggggtcccagacagattcagcgccagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggaatttattactgcatgcaaggtacacactggcctccgacgttcggccaagggaccaagctggagatcaaa。

[0077] The amino acid sequence of VL of 1011 is as follows, as shown in SEQ ID NO.4:

[0078] EIVLTQSPLSLPVTLGQPASISCRSSQSLVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSASGSGTDF TLKISRVEAEDVGIYYCMQGTHWPPTFGQGTKLEIK.

[0079] The gene sequence of VH of 1012 is as follows (direction 5'-3'), as shown in SEQ ID NO.5:

[0080] gaggtgcagctgttggagtctggggggggcttggtccagcctggggggtccctgagactctcctgtgcggcctccggactcaccataagtggcaactacatgacctgggtccgccaggctccagggaaggggctggagtgggtctcagttatttataccggtggtgacacaaactac gcagactccgtgaagggccgattcaccatctccagagacaattccaagaacatggtgtatcttcaaatgaacagcctgagacctgacgacacggctgtctattattgtgcgagaaggttgccggattactactacatggacgtctggggcaaagggaccacggtcaccgtctcgagt.

[0081] The amino acid sequence of VH of 1012 is shown below, as shown in SEQ ID NO.6:

[0082] EVQLLESGGGLVQPGGSLRLSCAASGLTISGNYMTWVRQAPGKGLEWVSVIYTGGDTNYADSVKGRFTISRDNSKN MVYLQMNSLRPDDTAVYYCARRLPDYYYMDVWGKGTTVTVSS.

[0083] The gene sequence of VL of 1012 is as follows (direction 5'-3'), as shown in SEQ ID NO.7:

[0084] cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgatttatgatgtcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaagcagcagcactttcaatgtggtattcggcggagggaccgagctgaccgtccta。

[0085] The amino acid sequence of the VL of 1012 is shown below, as shown in SEQ ID NO.8:

[0086] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLT ISGLQAEDEADYYCSSYTSSSTFNVVFGGGTELTVL。

[0087] The gene sequence of the VH of 1013 is shown below (direction 5'-3'), as shown in SEQ ID NO.9:

[0088] gaggtgcagctgctggaaagcggtggcggcctggtgcagcctggtggcagcctgcgcctgagctgcgcagccagcggtttcacctttagcagctatgccatgagctgggttcgccaggcaccaggtaaaggtctggaatgggtgagcgcaattagcggtagcggtggcagcacctactacgcagatagcgtgaaaggccgcttcaccatcagccgcgataattccaagaacacgctgtatctgcagatgaacagcctgcgcgccgaagataccgccgtgtattactgcgccaaacatcgcgatgatggcattgattattggggccagggcaccctggtcaccgtctcgagt。

[0089] The amino acid sequence of VH of 1013 is shown below, as shown in SEQ ID NO.10:

[0090] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCAKHRDDGIDYWGQGTLVTVSS.

[0091] The gene sequence of VL of 1013 is as follows (direction 5'-3'), as shown in SEQ ID NO.11:

[0092] gatatccagatgacccagagccctagctctctgagcgcaagcgtgggtgatcgcgtgaccattacctgccgcgccagggcattcgcaatgatctgggctggtatcagcagaaaccagggaaagcacctaaacgcctgatttatgcagcatcttctct gcagagcggtgtgccaagccgcttcagcggcagcggctctggtaccgaattcaccctgaccattagcagcctgcagcctgaagatttcgcaacctattactgcctgcagggccgtgcgattccgccgaccttcggtcagggtaccaaactggaaatcaaa.

[0093] The amino acid sequence of VL of 1013 is as follows, as shown in SEQ ID NO.12:

[0094] DIQMTQSPSSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQGRAIPPTFGQGTKLEIK.

[0095] The three independent clones obtained by screening were induced to express, and the periplasmic protein (scFv) was extracted. The binding activity of the periplasmic protein with CD19 was detected by ELISA. The specific steps are: coating with a concentration of 1μg / ml CD19 protein and 1μg / ml irrelevant protein (NC), 100μl per well, 4℃ overnight, washing once with PBST, adding skim milk powder to block, and then adding periplasmic supernatant to incubate at room temperature for 1h, washing 4 times with PBST, adding secondary antibody to incubate, washing and coloring, and detecting OD450 with an enzyme marker. The results are as follows Figure 3 As shown, it was shown that 1011, 1012, and 1013 could bind to the target protein CD19 but not to the irrelevant protein NC.

[0096] Experiment 2 Flow cytometry

[0097] To further determine whether the three clones 1011, 1012, and 1013 obtained by screening bind to the CD19 high-expressing cell line NALM6 (human B-cell leukemia cell line), the heavy and light chains of the three antibodies were loaded onto the heavy chain expression vector and the light chain expression vector by genetic engineering, and the heavy chain expression plasmid and the light chain expression plasmid were co-transfected with PEI to express antibodies (IgG), and the expression supernatant was obtained and detected by flow cytometry. The specific steps were as follows: 1×10 6 1011, 1012, 1013 IgG expression supernatants and cell culture medium (NC group) were added to the cell suspension (NALM6 cells in the experimental group and HEK293T cells in the control group) at 100 cells / ml, respectively. After incubation on ice for 1 hour, the cells were washed, incubated with fluorescently labeled secondary antibodies, washed, resuspended in PBS, and analyzed by flow cytometry. The analysis results are shown in Figure 4 , Figure 5 As shown, the results showed that 1011, 1012, and 1013 could bind to NALM6 cells, but not to HEK293T cells.

[0098] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. An anti-CD19 antibody, characterized in that: It is the anti-CD19 antibody 1011, the amino acid sequence of its VH is shown in SEQ ID NO.2, and the amino acid sequence of its VL is shown in SEQ ID NO.

4.

2. An anti-PD-L1 antibody, characterized in that: It is the anti-CD19 antibody 1012, the amino acid sequence of its VH is shown in SEQ ID NO.6, and the amino acid sequence of its VL is shown in SEQ ID NO.

8.

3. An anti-PD-L1 antibody, characterized in that: It is the anti-CD19 antibody 1013, the amino acid sequence of its VH is shown in SEQ ID NO.10, and the amino acid sequence of its VL is shown in SEQ ID NO.

12.

4. An isolated nucleic acid, characterized in that: It encodes the anti-CD19 antibody according to any one of claims 1 to 3.

5. The isolated nucleic acid according to claim 4, characterized in that: The nucleotide sequence encoding VH of the anti-CD19 antibody 1011 is shown in SEQ ID NO.1, and the nucleotide sequence encoding VL is shown in SEQ ID NO.3; The nucleotide sequence encoding VH of the anti-CD19 antibody 1012 is shown in SEQ ID NO.5, and the nucleotide sequence encoding VL is shown in SEQ ID NO.7; The nucleotide sequence encoding VH of the anti-CD19 antibody 1013 is shown in SEQ ID NO.9, and the nucleotide sequence encoding VL is shown in SEQ ID NO.

11.

6. A recombinant expression vector, characterized in that: Comprising the isolated nucleic acid of claim 4 or 5.

7. A transformant, characterized in that: Comprising the recombinant expression vector according to claim 6.

8. Use of the anti-CD19 antibody according to any one of claims 1 to 3, the isolated nucleic acid according to claim 4 or 5, the recombinant expression vector according to claim 6, or the transformant according to claim 7 in the preparation of an anti-tumor drug.

9. The use according to claim 8, characterized in that: The tumor is selected from B-lymphocytic leukemia and / or non-Hodgkin's lymphoma.

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