A kind of anti-CD20 nano antibody and its application

Abstract

The present invention discloses a nano antibody targeting CD20, which has an amino acid sequence shown in SEQ ID NO: 1. The present invention also relates to a CD20 chimeric antigen receptor protein, which comprises, from N-terminus to C-terminus: a signal peptide, a nano antibody (VHH) of the present invention, a StrepII tag, a hinge-transmembrane region, at least one co-stimulatory domain, and an activation domain. This chimeric antigen receptor can be used to construct the CAR-T cell of the present invention, which can specifically recognize CD20 antigen-positive target cells and has a specific killing effect.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, and specifically, the present invention relates to a CD20-targeting nanoantibody, a chimeric antigen receptor containing the nanoantibody sequence, and a T cell (CAR-T cell) expressing the chimeric antigen receptor, which can be used for tumor cell immunotherapy. Background Art

[0002] CD20 is a non-glycosylated phosphoprotein that can regulate the proliferation and differentiation of B lymphocytes. CD20 expression is limited in most normal tissues and is only expressed in pre-B lymphocytes, immature B lymphocytes, mature B lymphocytes, and activated B lymphocytes. However, high expression of CD20 is an important molecular feature of B cell lymphoma. It is highly expressed in more than 95% of B cell lymphomas and is easily recognized and bound by antibodies. Therefore, CD20 is an ideal therapeutic target for B cell lymphoma.

[0003] Nanobodies, also known as single-domain antibodies, are composed of the variable region (VHH) of natural heavy chain antibodies lacking light chains from camelids (including camels, alpacas and their close relatives). Compared with ordinary antibodies, nanobodies lack light chains and have no CH1 region between the heavy chain variable region and the hinge region. They only contain one heavy chain variable region and two conventional CH2 and CH3 regions. Nanobodies have the characteristics of small molecular weight, high affinity and stability, low immunogenicity, high solubility and strong penetration, and are currently widely used in antibody immunity and cell immunotherapy.

[0004] Chimeric antigen receptor modified T cells (CAR-T) are artificially modified T lymphocytes. The CAR structure includes an antibody binding region, a hinge-transmembrane region, a co-stimulatory domain, and an activation domain from the N-terminus to the C-terminus. Therefore, CAR-T cells have both the targeting effect of antibodies and the effector killing ability of T lymphocytes. CAR-T cell therapy has made breakthrough progress in the treatment of some blood tumors, and many CAR-T cell products have been approved for marketing. However, there are still some shortcomings in the current research on chimeric antigen receptors, and there are problems such as high recurrence rate and low safety in CAR-T cell therapy. Therefore, there is an urgent need to develop chimeric antigen receptors with good specificity, stable efficacy, and few side effects in this field. Summary of the invention

[0005] The purpose of the present invention is to provide a nano antibody targeting CD20, a chimeric antigen receptor, a CAR-T cell and an application thereof; the nano antibody targeting CD20 and the chimeric antigen receptor can specifically bind to the CD20 antigen, and the CAR-T cell targeting CD20 can target and kill tumor cells with high expression of CD20.

[0006] In the first aspect of the present invention, an anti-human CD20 nanobody is provided, the antibody having the amino acid sequence shown in SEQ ID NO: 1. Its framework region is FR1 shown in SEQ ID NO: 2, FR2 shown in SEQ ID NO: 3, FR3 shown in SEQ ID NO: 4, and FR4 shown in SEQ ID NO: 5; its complementary determining region is CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8.

[0007] In the second aspect of the present invention, a nucleic acid is provided, and the nucleotide sequence encoding the nanobody of SEQ ID NO: 1 is shown in SEQ ID NO: 9. According to an embodiment of the present invention, the nucleic acid is a nucleic acid encoding the nanobody described above or its complementary sequence. The nucleic acid according to an embodiment of the present invention specifically encodes the nanobody described above, and the nanobody can specifically target and bind to CD20.

[0008] It should be noted that, for the nucleic acids mentioned in the claims and the specification of the present invention, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, in the claims and the specification, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the gene sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0009] In the third aspect of the present invention, the present invention provides a chimeric antigen receptor targeting CD20, characterized in that it includes a CD8a signal peptide, a nanobody, a StrepII tag, a CD8a hinge region, a CD28 transmembrane region, a CD28 functional region, a 4-1BB functional region and a CD3ζ functional region connected sequentially from the N-terminus to the C-terminus, and the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.24.

[0010] In the fourth aspect of the present invention, the present invention provides a gene encoding the chimeric antigen receptor, and the nucleotide sequence of the gene is shown in SEQ ID NO.25.

[0011] In the fifth aspect of the present invention, the present invention provides a chimeric antigen receptor T cell targeting CD20.

[0012] The nanobody, the gene encoding the nanobody, the chimeric antigen receptor, the gene encoding the chimeric antigen receptor, and the chimeric antigen receptor T cell provided by the present invention are used in the preparation of drugs for treating tumors.

[0013] Beneficial effects of the present invention: The CD20-targeting nanoantibody sequence provided by the present invention has a strong binding ability with the CD20 antigen. The CD20-targeting chimeric antigen receptor T cells prepared by the present invention have a strong killing effect on CD20-positive tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Table 1 shows the results of the ELISA test for the titer of CD20 nanoantibodies in the immunized alpaca serum of the embodiment.

[0015] Table 2 shows the results of CD20 nanobody phage ELISA.

[0016] Figure 1 is the CAR plasmid map;

[0017] Figure 2 Schematic diagram of the chimeric antigen receptor structure;

[0018] Figure 3 The results of flow cytometry detection of CAR expression rate;

[0019] Figure 4 This is the test result of the killing effect of CAR-T cells on CD20 positive cells. DETAILED DESCRIPTION

[0020] The present invention provides a nanobody targeting CD20, the amino acid sequence of the nanobody is shown in SEQ ID NO.1, and is as follows:

[0021] RRQLVESGGGLVQPGGSLTLSCVASGSFFSIYGSSWYRQVPGKQRELVA SINRSGVTDYTDSVKGRFTISRDNAGNTWWLQMNSLKPEDTAVYYCNARRL WRDYWGQGTQVTVSS.

[0022] In the present invention, the Nanobody 2A11 comprises 4 framework regions and 3 complementarity determining regions; its framework region is the FR1 region shown in SEQ ID NO: 2, the FR2 region shown in SEQ ID NO: 3, the FR3 region shown in SEQ ID NO: 4, and the FR4 shown in SEQ ID NO: 5; its complementarity determining regions are CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8.

[0023] The amino acid sequence of the framework region FR1 is shown in SEQ ID NO.2, and is as follows:

[0024] RRQLVESGGGLVQPGGSLTLSCVASG.

[0025] The amino acid sequence of the framework region FR2 is shown in SEQ ID NO.3, and is as follows:

[0026] WYRQVPGKQRE.

[0027] The amino acid sequence of the framework region FR3 is shown in SEQ ID NO.4, and is as follows:

[0028] DSVKGRFTISRDNAGNTWWLQMNSLKPEDTAVYYC.

[0029] The amino acid sequence of the framework region FR4 is shown in SEQ ID NO.5, and is specifically as follows:

[0030] WGQGTQVTVSS.

[0031] The complementary determining region CDR1 amino acid sequence is shown in SEQ ID NO.6, and is specifically as follows:

[0032] SFFSIYGSS.

[0033] The complementary determining region CDR2 amino acid sequence is shown in SEQ ID NO.7, and is specifically as follows:

[0034] LVASINRSGVTDYT.

[0035] The complementary determining region CDR3 amino acid sequence is shown in SEQ ID NO.8, and is specifically as follows:

[0036] NARRLWRDY.

[0037] In the present invention, the nucleotide sequence encoding the single-chain antibody 2A11 gene is shown in SEQ ID NO.9, which is as follows:

[0038] CGTAGGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGACACTCTCCTGTGTAGCCTCTGGAAGCTTCTTCAGTATCTACGGGTCGAGCTGGTACCGCCAGGTTCCAGGGAAGCAGCGCGAGTTAGTCGCGAGTATTAATAGAAGTGGTGTCACGGA TTACACTGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCGGGAACACATGGTGGCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTACTACTGTAATGCCCGGAGGCTATGGAGGGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

[0039] The present invention also provides a chimeric antigen receptor targeting CD20, comprising a CD8a signal peptide, the nanobody of the present invention, a StrepII tag, a CD8a hinge region, a CD28 transmembrane region, a CD28 functional region, a 4-1BB functional region and a CD3ζ functional region connected in sequence.

[0040] In the present invention, the amino acid sequences of the CD8a signal peptide, StrepII tag, CD8a hinge region, CD28 transmembrane region, CD28 functional region, 4-1BB functional region and CD3ζ functional region are shown as SEQ ID NO.10 to SEQ ID NO.16, respectively, and the corresponding nucleotide sequences encoding the CD8a signal peptide, StrepII tag, CD8a hinge region, CD28 transmembrane region, CD28 functional region, 4-1BB functional region and CD3ζ functional region genes are shown as SEQ ID NO.17 to SEQ ID NO.23.

[0041] In the present invention, when the nanobody is nanobody 2A11, the amino acid sequence of the chimeric antigen receptor 2A11 is shown in SEQ ID NO.24, specifically as follows:

[0042] MALPVTALLLPLALLLHAARPRRQLVESGGGLVQPGGSLTLSCVASGSFFSIYGSSWYRQVPGKQRELVASINRSGVTDYTDSVKGRFTISRDNAGNTWWLQMNSLKPEDTAVYYCNARRLWRDYWGQGTQVTVSSNWSHPQFEKGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0043] In the present invention, the nucleotide sequence encoding the chimeric antigen receptor 2A11 is as shown in SEQ ID NO.25, which is as follows:

[0044]

[0045] The present invention provides a recombinant vector targeting CD20, comprising the chimeric antigen receptor gene and an initial vector. In the present invention, the initial vector is preferably a lentiviral vector, and the chimeric antigen receptor gene is preferably recombined between the EcoR I and BamH I sites of the lentiviral vector. The present invention does not specifically limit the preparation method of the recombinant vector targeting CD20, and the conventional preparation method of the recombinant lentiviral vector in the art can be used. In the present invention, preferably after the CD8a signal peptide, nano antibody sequence, StrepII tag, CD8a hinge region, CD28 transmembrane region, 4-1BB functional region and CD3 functional region are connected to the initial vector, the present invention does not specifically limit the method of the connection, and the conventional connection method in the art can be used.

[0046] The present invention provides a chimeric antigen receptor T cell targeting CD20, which is obtained by transfecting T cells with the recombinant vector targeting CD20. In the present invention, the preparation method of the chimeric antigen receptor T cell targeting CD20 preferably includes the following steps: 1) isolating T cells from healthy human peripheral blood and culturing; 2) packaging and preparing lentivirus; 3) infecting the cultured T cells with the lentivirus to obtain the chimeric antigen receptor T cell targeting CD20; 4) amplification and culture of CAR-T cells. The present invention does not specifically limit the separation method of the T cells, and the conventional T cell separation method in the art can be used. After the T cells are separated, the present invention uses T cell complete culture fluid and IL-2 to culture, and CD3 / CD28 magnetic beads are added at the same time; the culture is preferably carried out in an incubator, the culture temperature is preferably 37°C, the carbon dioxide volume concentration of the culture is preferably 5%, and the culture time is preferably 48h. In the present invention, the ratio of the lentivirus to the cultured T cells is calculated according to the MOI of the virus, and the MOI is preferably 5. In the present invention, the infection time is preferably 24h. After lentiviral infection, the cells are replaced with fresh T cell complete culture medium, and the culture time is preferably continued for 10-20 days.

[0047] The present invention provides the use of the nano antibody, the gene encoding the single-chain antibody, the chimeric antigen receptor, the gene encoding the chimeric antigen receptor, the recombinant vector, and the chimeric antigen receptor T cell in the preparation of a drug for treating tumors. In the present invention, the single-chain antibody has a good specific targeting function for the CD20 antigen; the chimeric antigen receptor T cell targeting CD20 has a relatively strong killing effect on c-Met positive cells, and can be used in the preparation of a drug for treating tumors.

[0048] The present invention is further described below by means of specific examples, but the present invention is not limited to the examples.

[0049] Example 1: Phage display screening of CD20 nanobodies.

[0050] Alpacas were immunized with human CD20 extracellular segment protein, and serum titers were detected after 5 immunizations (Table 1). Peripheral blood was drawn from alpacas, PBMC cells were extracted, and total RNA was extracted using the trizol method. Nanobody VHH fragments were amplified using primers, and the nanobody gene fragments were cloned into the phagemid vector pMED1. The nanobody phage display library was constructed by electrotransferring TG1 Escherichia coli. Subsequently, the phage library was screened by coating the CD20 protein antigen with an immunotube, and monoclonal antibodies were obtained by panning-amplification-enrichment. Positive monoclonal antibodies that can specifically bind to CD20 were screened by phage ELISA experiments, and the ELISA results are shown in Table 2. The positive clones obtained by screening were sent for sequencing, and the VHH eukaryotic expression vector was constructed according to the sequencing results. After transfection into 293F cells, small-scale protein expression was carried out. The binding of VHH to RAJI cells (highly expressing CD22) was detected by flow cytometry, and the appropriate VHH sequence was selected. Finally, the 2A11 phage monoclonal strain was determined. The test results showed that the 2A11 clone (whose VHH sequence is shown in SEQ ID NO.1) had a very strong binding ability with the CD20 antigen, indicating that it had a good specific targeting function for the CD20 antigen.

[0051] Table 1 CD20 nanoantibody immune titer in alpacas

[0052] Dilution multiple 1000 5000 <![CDATA[1*10 4 ]]> <![CDATA[2*10 4 ]]> <![CDATA[4*10 4 ]]> <![CDATA[8*10 4 ]]> OD450 3.012 2.642 2.817 1.568 1.033 0.493 Dilution multiple <![CDATA[1.6*10 5 ]]> <![CDATA[3.2*10 5 ]]> <![CDATA[6.4*10 5 ]]> <![CDATA[1.28*10 6 ]]> <![CDATA[2.56*10 6 ]]> Blank control OD450 0.327 0.18 0.09 0.074 0.06 0.043

[0053] Table 2 CD20 nanobody phage ELISA results

[0054]

[0055] Among them, A11 is the target sequence obtained by screening.

[0056] Example 2: CAR plasmid construction (plasmid structure as shown in Figure 1 (shown)

[0057] The designed primers were used to amplify the 2A11 nanobody gene fragment by PCR. The PTK-CAR vector was treated with EcoRI and BamHI double enzymes, and the backbone plasmid was recovered by cutting the gel after agarose gel electrophoresis. After that, the plasmid and VHH fragment were connected by homologous recombination method and transformed into Stbl3 competent bacteria. The specific steps included: standing on ice for 20 minutes; heat shock at 42℃ for 90 seconds; quickly put in ice for 5 minutes; add 1ml LB liquid culture medium, incubate at 37℃ for 60 minutes; centrifuge at 3000rpm for 3 minutes to collect the bacteria, and evenly spread the bacteria on the antibiotic-containing plate and incubate overnight. Pick the monoclonal strain and send it to the sequencing company for sequencing.

[0058] Example 3: CAR-T cell construction (structure as Figure 2 (shown)

[0059] 1. Lentiviral packaging

[0060] The 2A11 plasmid that was sequenced correctly was extracted. The premixed plasmid (target plasmid: BZ1 plasmid: BZ2 plasmid: BZ3 = 12:10:6:5) was transfected into 293T cells using PEI. 72 hours after transfection, the cell supernatant was centrifuged at 4000 rpm for 10 minutes. The collected supernatant was filtered with a 0.45 μm filter membrane, and then concentrated using an ultracentrifuge. 4°C, 100,000 × g, centrifuged for 90 minutes.

[0061] 2. T Cell Isolation

[0062] Take 20 ml of fresh blood from a healthy person and add lymphocyte separation medium (Mediatech) to separate peripheral blood mononuclear cells. Use T cell complete culture medium (OpTmizer TM CTS TM T-Cell Expansion Basal Medium, Gibco, A3021201; OpTmizer TM CTS T-Cell Expansion Supplement, Gibco, A3021601), 500 IU / mL IL-2, and Dynabeads Human T-ActivatorCD3 / CD28 (Gibco, 11132D) were added at 25 μL / 106 cells to obtain T cells. The cells were cultured in a 5% CO2 37°C incubator and used for lentiviral infection after 48 hours.

[0063] 3. Virus-infected T cells

[0064] The corresponding lentivirus was added at an MOI of 5, mixed and placed in a CO2 incubator for incubation. After 4 hours, T cell complete medium was added. After 24 hours of lentiviral transduction, the cells were replaced with fresh T cell complete medium and the live cell density was adjusted to 1.0×10 6 / mL, continue to culture and expand for 10-20 days, observe and count every day, and replenish fluid and expand the culture according to the counted cell number, always maintaining the cell culture density of 1.0×10 6 After 3 days, the CAR-T cells were transferred to a sterile flow cytometry tube and the CAR expression rate was detected by flow cytometry antibody. The test results were as follows: Figure 3 As shown, the expression rate of 2A11CAR was 65.4%

[0065] Example 4: Cell killing in vitro

[0066] Take 5×10 4 Raji target cells and control human fibroblast BJ10 cells were inoculated in a 96-well plate. After overnight culture, CAR-T cells, control virus-modified T cells (GFP-T) and unmodified T cells (T) were added to the cell culture wells at a ratio of effector cells (E): target cells (T) = 10:1. After incubation for 6 hours, the LDH cytotoxicity assay kit (Cayman Chemical) was used to detect the specific killing activity of CART cells against Hodgkin lymphoma cells. The killing results are shown in Figure 4 As shown, the prepared CD20 CAR-T cells have a strong killing effect on CD20-positive Raji cells (63.4%), but no killing effect on control cells that do not express CD20. Therefore, CART cells have specific killing activity against tumor cells that highly express CD20.

Claims

1. An anti-CD20 antibody, characterized in that The amino acid sequence of the antibody is shown in SEQ ID NO: 1, and the antibody is a nanobody.

2. A nucleic acid, characterized in that The nucleic acid is: a nucleic acid encoding the antibody according to claim 1 or its complementary sequence.

3. A nucleic acid, characterized in that: The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:9 or the complementary sequence of the sequence shown in SEQ ID NO:

9.

4. A chimeric antigen receptor targeting CD20, characterized in that: From the N-terminus to the C-terminus, there are a signal peptide, the antibody according to claim 1, a StrepII tag, a hinge-transmembrane region, at least one co-stimulatory domain and an activation domain.

5. The chimeric antigen receptor according to claim 4, characterized in that The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.

24.

6. The gene encoding the chimeric antigen receptor according to claim 4, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

25.

7. A CAR-T cell targeting CD20, characterized in that: It expresses the chimeric antigen receptor according to claim 4.

8. Use of the antibody according to claim 1 in the preparation of a drug for treating a tumor, wherein the tumor is a CD20 antigen-positive B-cell lymphoma.

9. Use of the CAR-T cells of claim 7 in the preparation of a drug for treating a tumor, wherein the tumor is a CD20 antigen-positive B-cell lymphoma.

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