Anti-CD19 Antibody and Its Applications

Abstract

The present invention discloses anti-CD19 antibodies and their applications, belonging to the technical field of monoclonal antibodies. There are three kinds of anti-CD19 antibodies, namely anti-CD19 antibody 1011, anti-CD19 antibody 1012, and anti-CD19 antibody 1013. The amino acid sequences of their VH and VL and the nucleotide sequences of the encoding genes are shown in SEQ ID NO.1-12. The application of the anti-CD19 antibody in the preparation of anti-tumor drugs. The present invention screens out fully human single-chain antibodies specifically binding to CD19 from a fully human phage library, which have good affinity with human CD19 protein, can all bind to NALM6 cells, and do not bind to HEK293T cells, and can be used for the treatment of tumors. The present invention enriches fully human single-chain antibodies specifically binding to CD19, provides potential new antibody drugs for the clinical treatment of tumors, and has 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 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 the VH of anti-CD19 antibody 1012 is shown in SEQ ID NO.6, and the amino acid sequence of the VL is shown in SEQ ID NO.8;

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

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

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

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

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

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

[0016] A transformant comprising the above recombinant expression vector.

[0017] 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 lymphoma.

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

[0020] The various terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Schematic diagram of the phage titer after three rounds of screening.

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

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

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

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

[0026] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can 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 are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels without special instructions. The experimental methods, detection methods, etc. involved in the following embodiments are all conventional experimental methods, detection methods, etc. existing in the prior art without special instructions.

[0028] Experiment 1 Screening of anti-CD19 antibodies

[0029] 1. Experimental method

[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 respectively, perform electrophoresis, and purify and recover the VH and VL fragments.

[0032] 1.1.2 Connect the recovered VH and VL fragments to the phage vector through genetic engineering techniques. Transfer the constructed vector into Escherichia coli by electroporation. The phagemid vector can self-replicate in the successfully transduced host bacteria. When the host bacteria grow to the logarithmic phase, infect with helper phage at an MOI of 10, culture overnight to amplify the phage, and obtain the phage library by centrifugal precipitation.

[0033] 1.2 Screening of the phage antibody library

[0034] The steps are as follows:

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

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

[0037] (3) Remove the antigen solution on the ELISA plate, wash it 3 times with PBS, then add 2% skim milk powder to block 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; ensure that the volume of the added phage library is between 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 elution buffer with pH 2.0, place it at room temperature for 10 minutes, and immediately add 60 μl of Tris-HCl buffer with pH 8.0 for neutralization. Repeat the elution once.

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

[0042] (8) Take 10 μl of the bacterial solution, dilute it 100-fold, 1000-fold, and 10000-fold respectively, and inoculate it on a 2YT plate with resistance for calculating the phage titer.

[0043] (9) Inoculate all the remaining bacterial solution on a 2YT plate (containing 100 μg / ml ampicillin antibiotic and 1% glucose); incubate it in a 37°C incubator for 16 hours.

[0044] (10) Scrape all the clones on the overnight plate with 2YT medium (100 μg / ml ampicillin antibiotic and 1% glucose), and place it in a 37°C incubator until the OD600 reaches 0.4.

[0045] (11) Add helper phage, place it in a 37°C incubator and let it stand for 30 minutes, then at 220 rpm for 60 minutes.

[0046] (12) Centrifuge to collect the bacterial cell precipitate, and culture overnight at 30 °C and 220 rpm in 2YT culture medium (100 μg / ml ampicillin antibiotic, 50 μg / ml kanamycin, and 0.1% glucose).

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

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

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

[0050] The steps are as follows:

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

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

[0053] (3) After washing the ELISA plate 3 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 the 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 of TMB substrate solution, develop color for 10 - 15 minutes, and then terminate the color development reaction with 2M sulfuric acid.

[0058] (8) Read the plate at a wavelength of 450 nm on an enzyme-linked immunosorbent assay reader, and then analyze the results.

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

[0060] (1) Suspend the cells at a concentration of 1×10 6 cells / ml in 5% BSA solution, aliquot 1 ml per tube;

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

[0062] (3) Centrifuge at 400 g for 5 min to collect the cells, and wash once with cold PBS;

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

[0064] (5) Centrifuge at 400g for 5 min to collect the cells, and wash them three times with cold PBS;

[0065] (6) Centrifuge at 400g for 5 min to collect the cells, resuspend the cells in 1 ml of PBS solution, and perform flow cytometry 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 to screen for the target of human CD19. In this experiment, CD19 protein at a concentration of 10 μg / ml was used as an antigen and coated on an ELISA plate, as Figure 1 shown. After three rounds of screening, with the same input amount, the titer of the obtained phages was significantly enriched.

[0069] After three rounds of screening, monoclonal 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, coated overnight at 4°C. After washing once with PBST, skim milk powder was added for blocking, and then the blocked Phage supernatant was added and incubated at room temperature for 1 h. After washing 4 times with PBST, the secondary antibody was added for incubation, washed and developed color, and the OD450 was detected with an ELISA reader. The results were as Figure 2 shown.

[0070] The positive clones were selected and sent for sequencing, showing that 3 independent clones (1011, 1012, 1013) had a relatively high enrichment. The gene sequences and amino acid sequences of the VH and VL of these 3 antibodies are as follows.

[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 the VH of 1011 is shown below, as shown in SEQ ID NO.2:

[0074] QVQLVESGGGLVQSGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGRSRNKANSYTTEYAASVKGRFTISRDD SKNSLYLQMNSLKTEDTAVYYCRVGKVWGQGTMVTVSS。

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

[0076] gaaattgtgctgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgcaggtctagtcaaagcctcgtatacagtaatggaaacacctacttgaattggtttcagcagaggccaggccaatctccaaggcgcctaatttatagggtttctaaccgggactctggggtcccagacagattcagcgccagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggaatttattactgcatgcaaggtacacactggcctccgacgttcggccaagggaccaagctggagatcaaa。

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

[0078] EIVLTQSPLSLPVTLGQPASISCRSSQSLVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSASGSGTDF TLKISRVEAEDVGIYYCMQGTHWPPTFGQGTKLEIK。

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

[0080] gaggtgcagctgttggagtctggggggggcttggtccagcctggggggtccctgagactctcctgtgcggcctccggactcaccataagtggcaactacatgacctgggtccgccaggctccagggaaggggctggagtgggtctcagttatttataccggtggtgacacaaactacgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacatggtgtatcttcaaatgaacagcctgagacctgacgacacggctgtctattattgtgcgagaaggttgccggattactactacatggacgtctggggcaaagggaccacggtcaccgtctcgagt。

[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 shown below (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 the VH of 1013 is shown below, as shown in SEQ ID NO.10:

[0090] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCAKHRDDGIDYWGQGTLVTVSS。

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

[0092] gatatccagatgacccagagccctagctctctgagcgcaagcgtgggtgatcgcgtgaccattacctgccgcgccagccagggcattcgcaatgatctgggctggtatcagcagaaaccagggaaagcacctaaacgcctgatttatgcagcatcttctctgcagagcggtgtgccaagccgcttcagcggcagcggctctggtaccgaattcaccctgaccattagcagcctgcagcctgaagatttcgcaacctattactgcctgcagggccgtgcgattccgccgaccttcggtcagggtaccaaactggaaatcaaa。

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

[0094] DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQGRAIPPTFGQGTKLEIK。

[0095] The 3 independent clones obtained by screening were induced for expression, and periplasmic proteins (scFv) were extracted. The binding activity of the periplasmic proteins to CD19 was detected by ELISA. The specific steps were as follows: Coat CD19 protein at a concentration of 1 μg / ml and an irrelevant protein (NC) at a concentration of 1 μg / ml, 100 μl per well, overnight at 4°C. After washing once with PBST, add skim milk powder for blocking, then add the periplasmic supernatant for incubation at room temperature for 1 h. After washing 4 times with PBST, add the secondary antibody for incubation, washing and color development, and detect OD450 with an ELISA reader. The results are as Figure 3 shown, indicating that 1011, 1012, and 1013 can all bind to the target protein CD19 but not to the irrelevant protein NC.

[0096] Experiment 2: Flow cytometry detection

[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 heavy-chain expression vectors and light-chain expression vectors by genetic engineering methods. The heavy-chain expression plasmid and the light-chain expression plasmid were co-transfected with PEI for antibody expression (IgG), and the expression supernatant was obtained for detection by flow cytometry. The specific steps were as follows: Add the IgG expression supernatants of 1011, 1012, and 1013 and cell culture medium (NC group) to 1×10 6 cells / ml cell suspensions (experimental group: NALM6 cells, control group: HEK293T cells) respectively. After incubation on ice for 1 h, wash and add a fluorescently labeled secondary antibody for incubation and washing, then resuspend in PBS for flow cytometry analysis. The analysis results are as Figure 4 and Figure 5 shown. The results show that 1011, 1012, and 1013 can all bind to NALM6 cells and do not bind to HEK293T cells.

[0098] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An anti-CD19 antibody, characterized in that: It is 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. Anti-CD19 antibody, characterized in that: It is 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. Anti-CD19 antibody, characterized in that: It is 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 described in any one of claims 1 to 3.

5. The isolated nucleic acid according to claim 4, wherein: 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: It comprises the isolated nucleic acid described in claim 4 or 5.

7. A transformant, characterized in that: It comprises the recombinant expression vector described in claim 6.

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