CN117024594B

CN117024594B - A kind of anti-CD20 nano antibody and its application

Info

Publication number: CN117024594B
Application number: CN202311225671.3A
Authority: CN
Inventors: 张同存
Original assignee: Wuhan Bio Raid Biotechnology Co ltd
Current assignee: Wuhan Bio Raid Biotechnology Co ltd
Priority date: 2023-09-21
Filing date: 2023-09-21
Publication date: 2025-02-28
Anticipated expiration: 2043-09-21
Also published as: CN117024594A

Abstract

The invention discloses a CD 20-targeted nano antibody, which has an amino acid sequence shown in SEQ ID NO. 1. The invention also relates to a CD20 chimeric antigen receptor protein, which comprises a signal peptide, a nano antibody (VHH) of the invention, a strep II tag, a hinge-transmembrane region, at least one co-stimulatory domain and an activating domain from the N end to the C end. Such chimeric antigen receptors can be used to construct the CAR-T cells of the invention that specifically recognize CD20 antigen positive target cells and have specific killing effects.

Description

Anti-CD 20 nano antibody and application thereof

Technical Field

The invention relates to the field of tumor treatment, in particular to a targeting CD20 nanobody, a chimeric antigen receptor containing a nanobody sequence and a T cell (CAR-T cell) expressing the chimeric antigen receptor, which can be used for tumor cell immunotherapy.

Background

CD20 belongs to the non-glycosylated phosphoprotein and is capable of modulating proliferation and differentiation of B lymphocytes. CD20 is restricted in expression in most normal tissues, only in pre-B lymphocytes, immature B lymphocytes, mature B lymphocytes and activated B lymphocytes. However, high CD20 expression is an important molecular feature of B-cell lymphomas, which is highly expressed in more than 95% of B-cell lymphomas and is easily recognized by antibodies for binding, and thus CD20 is an ideal therapeutic target for B-cell lymphomas.

Nanobodies, also known as single domain antibodies, consist of a variable region (VHH) of a natural heavy chain antibody in which the light chain is deleted in camelids (including camels, alpacas and closely related species), which, in comparison to conventional antibodies, lacks a CH1 region between the heavy chain variable region and the hinge region, and comprises only one heavy chain variable region and two conventional CH2 and CH3 regions, in addition to the light chain. The nano antibody has the characteristics of small molecular weight, high affinity and stability, low immunogenicity, high solubility, strong penetrability and the like, and is widely used for antibody immunity and cell immunity treatment at present.

Chimeric antigen receptor-modified T cells (CAR-T) are an artificially modified T lymphocyte. The CAR structure comprises, in order from the N-terminus to the C-terminus, an antibody binding region, a hinge-transmembrane region, a co-stimulatory domain, and an activation domain. Thus 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 a portion of hematological tumors, and a number of CAR-T cell products are currently marketed in bulk. However, the current research on chimeric antigen receptor has some defects, and the problems of high recurrence rate, low safety and the like in the treatment of CAR-T cells. Therefore, there is an urgent need in the art to develop chimeric antigen receptors with good specificity, stable therapeutic effect and small side effects.

Disclosure of Invention

The invention aims to provide a CD 20-targeted nanobody, a chimeric antigen receptor, a CAR-T cell and application thereof, wherein the CD 20-targeted nanobody and the chimeric antigen receptor can specifically bind to a CD20 antigen, and the CD 20-targeted CAR-T cell can target and kill a tumor cell with high expression of CD 20.

In a first aspect of the invention, an anti-human CD20 nanobody is provided, said antibody having the amino acid sequence shown in SEQ ID NO. 1. The framework regions are 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, and the 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.

In a second aspect of the invention, a nucleic acid is provided, and the nucleotide sequence of the SEQ ID NO. 1 nanobody is shown as SEQ ID NO. 9. According to an embodiment of the invention, the nucleic acid is a nucleic acid encoding the nanobody as described above or a complement thereof. Nucleic acids according to embodiments of the invention specifically encode the nanobodies described previously, which are capable of specifically targeting binding to CD20.

It should be noted that, for the nucleic acids mentioned in the claims and the specification of the present application, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, in the claims and specification, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the gene sequences in the present application include DNA forms or RNA forms, one of which is disclosed, meaning the other is also disclosed.

In a third aspect of the invention, the invention provides a chimeric antigen receptor targeting CD20, which is characterized by comprising a CD8a signal peptide, a nanobody, a strep II tag, a CD8a hinge region, a CD28 transmembrane region, a CD28 functional region, a 4-1BB functional region and a CD3 zeta functional region which are sequentially connected from the N end to the C end, wherein the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 24.

In a fourth aspect of the invention, there is provided a gene encoding said chimeric antigen receptor, said gene having the nucleotide sequence shown in SEQ ID NO. 25.

In a fifth aspect of the invention, the invention provides a CD 20-targeting chimeric antigen receptor T cell.

The invention provides the application of the nano antibody, the gene for encoding the nano antibody, the chimeric antigen receptor, the gene for encoding the chimeric antigen receptor and the chimeric antigen receptor T cell in preparing medicaments for treating tumors.

The invention has the beneficial effects that the nano antibody sequence targeting CD20 has stronger binding capacity with CD20 antigen. The chimeric antigen receptor T cells targeting CD20 prepared by the invention have stronger killing effect on CD20 positive tumor cells

Drawings

Table 1 shows the results of ELISA assay titers of CD20 nanobodies in serum of alpaca after immunization of the examples.

Table 2 shows a graph of the results of the CD20 nanobody phage ELISA.

FIG. 1 is a map of a CAR plasmid;

FIG. 2 is a schematic representation of chimeric antigen receptor structure;

FIG. 3 is a graph showing CAR expression rate flow assay results;

FIG. 4 shows the detection result of the killing effect of the CAR-T cells on CD20 positive cells.

Detailed Description

The invention provides a CD 20-targeted nano antibody, the amino acid sequence of which is shown as SEQ ID NO.1, which is specifically as follows:

RRQLVESGGGLVQPGGSLTLSCVASGSFFSIYGSSWYRQVPGKQRELVA SINRSGVTDYTDSVKGRFTISRDNAGNTWWLQMNSLKPEDTAVYYCNARRL WRDYWGQGTQVTVSS.

In the invention, the nano antibody 2A11 comprises 4 framework regions and 3 complementarity determining regions, wherein the framework regions are an FR1 region shown as SEQ ID NO.2, an FR2 region shown as SEQ ID NO. 3, an FR3 region shown as SEQ ID NO. 4 and an FR4 region shown as SEQ ID NO. 5, and the complementarity determining regions are a CDR1 shown as SEQ ID NO. 6, a CDR2 shown as SEQ ID NO. 7 and a CDR3 shown as SEQ ID NO. 8.

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

RRQLVESGGGLVQPGGSLTLSCVASG。

the framework region FR2 amino acid sequence is shown as SEQ ID NO.3, and is specifically as follows:

WYRQVPGKQRE。

the framework region FR3 amino acid sequence is shown as SEQ ID NO.4, and is specifically as follows:

DSVKGRFTISRDNAGNTWWLQMNSLKPEDTAVYYC。

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

WGQGTQVTVSS。

the CDR1 amino acid sequence of the complementarity determining region is shown in SEQ ID NO.6, and is specifically as follows:

SFFSIYGSS。

the CDR2 amino acid sequence of the complementarity determining region is shown as SEQ ID N0.7, and is specifically as follows:

LVASINRSGVTDYT。

the CDR3 amino acid sequence of the complementarity determining region is shown in SEQ ID NO.8, and is specifically as follows:

NARRLWRDY。

in the invention, the nucleotide sequence SEQ ID NO.9 of the single-chain antibody 2A11 gene is shown as follows:

CGTAGGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGACACTCTCCTGTGTAGCCTCTGGAAGCTTCTTCAGTATCTACGGGTCGAGCTGGTACCGCCAGGTTCCAGGGAAGCAGCGCGAGTTAGTCGCGAGTATTAATAGAAGTGGTGTCACGGATTACACTGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCGGGAACACATGGTGGCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTACTACTGTAATGCCCGGAGGCTATGGAGGGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

The invention also provides a chimeric antigen receptor targeting CD20, which comprises a CD8a signal peptide, the nanobody, a strep II label, a CD8a hinge region, a CD28 transmembrane region, a CD28 functional region, a 4-1BB functional region and a CD3 zeta functional region which are connected in sequence.

In the invention, the amino acid sequences of the CD8a signal peptide, the strep II tag, the CD8a hinge region, the CD28 transmembrane region, the CD28 functional region, the 4-1BB functional region and the CD3 zeta functional region are respectively shown as SEQ ID NO. 10-SEQ ID NO.16, and the nucleotide sequences of the genes for correspondingly encoding the CD8a signal peptide, the strep II tag, the CD8a hinge region, the CD28 transmembrane region, the CD28 functional region, the 4-1BB functional region and the CD3 zeta functional region are respectively shown as SEQ ID NO. 17-SEQ ID NO. 23.

In the present invention, when the nanobody is nanobody 2a11, the amino acid sequence of chimeric antigen receptor 2a11 is shown in SEQ ID No.24, which is specifically as follows:

MALPVTALLLPLALLLHAARPRRQLVESGGGLVQPGGSLTLSCVASGSFFSIYGSSWYRQVPGKQRELVASINRSGVTDYTDSVKGRFTISRDNAGNTWWLQMNSLKPEDTAVYYCNARRLWRDYWGQGTQVTVSSNWSHPQFEKGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In the invention, the nucleotide sequence for encoding the chimeric antigen receptor 2A11 is shown as SEQ ID NO.25, and the nucleotide sequence is specifically as follows:

atggccctgcctgtgacagctctgctcctccctctggccctgctgctccatgccgccagaccccgtaggcagctcgtggagtctgggggaggcttggtgcagcctggggggtctctgacactctcctgtgtagcctctggaagcttcttcagtatctacgggtcgagctggtaccgccaggttccagggaagcagcgcgagttagtcgcgagtattaatagaagtggtgtcacggattacactgactccgtgaagggccggttcaccatctccagagacaacgccgggaacacatggtggctgcaaatgaacagcctgaaacctgaggacacggccgtctactactgtaatgcccggaggctatggagggactactggggccaggggacccaggtcaccgtctcctcaaactggagccacccccagttcgagaagggcggtggcggaagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa

The invention provides a recombinant vector targeting CD20, which comprises 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 EcoRI and BamHI sites of the lentiviral vector. The preparation method of the CD 20-targeted recombinant vector is not particularly limited, and the preparation method of a conventional recombinant lentiviral vector in the field is adopted. In the present invention, it is preferable that the method of the present invention is not particularly limited after the CD8a signal peptide, nanobody sequence, strep II tag, CD8a hinge region, CD28 transmembrane region, 4-1BB functional region and CD3 functional region are ligated to the original vector, and the ligation method is conventional in the art.

The invention provides a chimeric antigen receptor T cell targeting CD20, which is obtained by transfecting the T cell with the recombinant vector targeting CD 20. In the present invention, the preparation method of the CD 20-targeted chimeric antigen receptor T cells preferably comprises the steps of 1) isolating and culturing T cells in peripheral blood of a healthy human body, 2) packaging preparation of lentiviruses, 3) obtaining the CD 20-targeted chimeric antigen receptor T cells by infecting the cultured T cells with the lentiviruses, and 4) amplification culture of the CAR-T cells. The method for separating T cells is not particularly limited, and T cell separation methods conventional in the art can be adopted. According to the invention, after T cells are separated, the T cells are cultured by using a complete culture solution of the T cells and IL-2, and meanwhile, CD3/CD28 magnetic beads are added, wherein the culture is preferably performed in an incubator, the temperature of the culture is preferably 37 ℃, the volume concentration of carbon dioxide of the culture is preferably 5%, and the culture time is preferably 48 hours. In the present invention, the ratio of the lentivirus to the T cells after culture is calculated based on the MOI of the virus, which is preferably 5. In the present invention, the infection time is preferably 24 hours. After lentiviral infection, the cells are replaced with fresh T cell complete broth, and the incubation period is preferably 10-20 days.

The invention provides the application of the nano antibody, the gene for encoding the single-chain antibody, the chimeric antigen receptor, the gene for encoding the chimeric antigen receptor, the recombinant vector and the chimeric antigen receptor T cell in preparing medicaments for treating tumors. In the invention, the single-chain antibody has a very good specific targeting function on the CD20 antigen, and the chimeric antigen receptor T cells targeting the CD20 have a relatively strong killing effect on c-Met positive cells, and can be applied to preparing medicines for treating tumors.

The invention is further described below by means of specific examples, but the invention is not limited to only these examples.

Example 1 phage display screening for CD20 nanobodies.

The alpaca is immunized by using the extracellular protein of human CD20, serum titers are detected after immunization for 5 times (table 1), peripheral blood of the alpaca is extracted, PBMC cells are extracted, total RNA is extracted by trizol method, nanobody VHH fragments are amplified by using primers, nanobody gene fragments are cloned into phagemid vector pMED1, and a nanobody phage display library is constructed by electrotransformation of TG1 escherichia coli. Subsequently, phage libraries were screened with immune tube-coated CD20 protein antigen, monoclonal antibodies were obtained by panning-amplification-enrichment, and positive monoclonal antibodies capable of specifically binding to CD20 were screened by phage ELISA experiments, and the ELISA results are shown in table 2. And (3) sequencing the screened positive clones, constructing a VHH eukaryotic expression vector according to a sequencing result, transfecting 293F cells, performing small-scale protein expression, detecting the binding condition of VHH and RAJI cells (high-expression CD 22) by using a flow, selecting a proper VHH sequence, and finally determining a 2A11 phage monoclonal strain, wherein the detection result shows that the 2A11 clone strain (the VHH sequence of which is shown as SEQ ID NO. 1) has extremely strong binding capacity with CD20 antigen, and the very good specific targeting function on the CD20 antigen is demonstrated.

TABLE 1 CD20 nanobody alpaca immunotitres

Dilution factor	1000	5000	1*10⁴	2*10⁴	4*10⁴	8*10⁴
							OD450	3.012	2.642	2.817	1.568	1.033	0.493
Dilution factor	1.6*10⁵	3.2*10⁵	6.4*10⁵	1.28*10⁶	2.56*10⁶	Blank control
							OD450	0.327	0.18	0.09	0.074	0.06	0.043

TABLE 2 ELISA results for CD20 nanobody phage

Wherein A11 is a target sequence obtained by screening.

EXAMPLE 2 CAR plasmid construction (plasmid Structure is shown in FIG. 1)

Primers were designed to amplify 2a11 nanobody gene fragments by PCR. The PTK-CAR vector was digested with EcoRI and BamHI, and subjected to agarose gel electrophoresis, followed by gel cutting to recover the backbone plasmid. Then, connecting the plasmid and VHH fragment in a homologous recombination method and transforming the plasmid and VHH fragment into Stbl3 competent bacteria, wherein the specific steps comprise standing for 20min on ice, carrying out 42 ℃ heat shock for 90s, rapidly putting the mixture into ice for 5min, adding 1ml of LB liquid medium, incubating for 60min at 37 ℃, centrifuging at 3000rpm for 3min, collecting thalli, uniformly coating the thalli on a plate containing antibiotics, and incubating for overnight. The monoclonal strain was picked and sent to sequencing company for sequencing.

EXAMPLE 3 CAR-T cell construction (Structure shown in FIG. 2)

1. Lentivirus package

The 2A11 plasmid which is sequenced correctly is greatly extracted. Premixed plasmids (destination plasmid: BZ1 plasmid: BZ2 plasmid: bz3=12:10:6:5) were transfected into 293T cells with PEI and the cell supernatants were centrifuged at 4000rpm for 10min 72h after transfection. The collected supernatant was filtered with a 0.45 μm filter membrane, and then with an ultracentrifuge. The virus solution is concentrated by centrifugation for 90min at 4 ℃ and 100000 Xg.

T cell isolation

Fresh blood of 20ml of healthy person was taken, and lymphocyte separation liquid (Mediatech) was added to the blood to separate peripheral blood mononuclear cells. T cells were cultured in IL-2 in complete T cell culture broth (OpTmizer^TMCTS^TMT-Cell Expansion Basal Medium,Gibco,A3021201;OpTmizer^TMCTS T-Cell Expansion Supplement,Gibco,A3021601),500IU/mL, while Dynabeads Human T-Activator CD3/CD28 (Gibco, 11132D) was added at 25. Mu.L/106 cells to obtain T cells. Cells were cultured in a 5% CO237℃incubator, 48h later for lentiviral infection.

3. Virus infection of T cells

Adding corresponding slow viruses according to MOI=5, uniformly mixing, placing in a CO2 incubator for incubation, supplementing a complete T cell culture medium after 4 hours, transferring the slow viruses into a fresh complete T cell culture solution after 24 hours, adjusting the density of living cells to be 1.0X10 ⁶/mL, continuing to culture and expand for 10-20 days, observing and counting every day, and carrying out fluid replacement and expansion culture according to the calculated cell number, wherein the cell culture density is always kept to be 1.0X10 ⁶/mL. After 3 days, CAR-T cells were transferred into sterile flow tubes, and the flow antibody detected the CAR expression rate, as shown in fig. 3, with a 2a11CAR expression rate of 65.4%

Example 4 in vitro cell killing

5×10 ⁴ Raji target cells and control human fibroblasts BJ10 cells were inoculated into 96-well plates, respectively, and 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, respectively. After 6h incubation, the specific killing activity of CART cells against hodgkin's lymphoma cells was detected by operating according to the instructions of LDH cytotoxicity assay kit (CAYMANCHEMICAL). The killing results are shown in fig. 4, and the prepared CD20 CAR-T cells all have a stronger killing effect (63.4%) on CD20 positive Raji cells, but have no killing effect on control cells which do not express CD 20. Thus the CART cells have specific killing activity to tumor cells highly expressing CD 20.

Claims

1. An anti-CD 20 antibody is characterized in that the amino acid sequence of the antibody is shown as SEQ ID NO. 1, and the antibody is a nano antibody.

2. A nucleic acid encoding the antibody of claim 1 or a complement thereof.

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

4. A chimeric antigen receptor targeting CD20, comprising, in order from N-terminus to C-terminus, a signal peptide, the antibody of claim 1, a strep ii tag, a hinge-transmembrane region, at least one co-stimulatory domain, and an activation domain.

5. The chimeric antigen receptor according to claim 4, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 24.

6. A gene encoding the chimeric antigen receptor according to claim 4, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 25.

7. CD 20-targeted CAR-T cell, characterized in that it expresses the chimeric antigen receptor according to claim 4.

8. Use of the antibody of claim 1 for the preparation of a medicament for the treatment of a tumor which is a CD20 antigen positive B cell lymphoma.

9. Use of the CAR-T cell of claim 7 in the manufacture of a medicament for the treatment of a tumor that is a CD20 antigen positive B cell lymphoma.