CN111763264A

CN111763264A - PSCA (phosphosilicate antigen) -targeted chimeric antigen receptor and application thereof

Info

Publication number: CN111763264A
Application number: CN202010761425.XA
Authority: CN
Inventors: 汤朝阳; 秦乐; 吴迪; 邓殷健; 吴海鹏; 王翠花; 冯世忠; 冯嘉昆; 其他发明人请求不公开姓名
Original assignee: Guangdong Zhaotai In Vivo Biomedical Technology Co ltd
Current assignee: Guangdong Zhaotai In Vivo Biomedical Technology Co ltd
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-10-13

Abstract

The invention provides a PSCA (phosphosilicate antigen) -targeted chimeric antigen receptor and application thereof, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and a signal transduction domain; the antigen binding domain comprises an anti-PSCA single chain antibody; the signaling domains include OX40, CD3 ζ, and TLR 1. The chimeric antigen receptor disclosed by the invention not only has a targeting effect on PSCA positive tumor cells, but also can efficiently activate T cells, eliminate the immunosuppression effect of regulatory T cells and promote the formation of memory T cells, and the constructed T cells expressing the chimeric antigen receptor have the remarkably enhanced tumor cell killing capability.

Description

PSCA (phosphosilicate antigen) -targeted chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and relates to a PSCA (phosphosilicate antigen) targeted chimeric antigen receptor and application thereof.

Background

The chimeric antigen receptor T cell is also called CAR-T cell, and is characterized in that a single chain variable region (scFv) which specifically recognizes an antigen is connected in series with T cell costimulation and activation signals (such as CD28, 4-1BB, OX40, CD3 zeta and the like) by using a genetic engineering technology and is expressed on the T cell, so that the T cell is endowed with the function of specifically recognizing a target protein and has a killing effect on cells expressing the target protein. The prepared CAR-T cell is infused back into a tumor patient body, and the aim of eliminating the tumor cell is fulfilled by exerting the effect of targeting the CAR-T cell to a specific tumor antigen.

In recent years, with the development of tumor immunology theory and clinical technology, Chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapy. At present, CAR-T cell therapy has been widely applied to the treatment of B cell malignant tumors, and CAR-T cells targeting CD19 are precursors for the treatment of B cell malignant tumors by CAR-T therapy, and provide an effective scheme for the treatment of B cell malignant tumors. However, CAR-T therapy is always faced with problems of tumor recurrence, drug resistance.

Therefore, there is a need for further improvement of chimeric antigen receptor, which not only improves the target binding ability of the chimeric antigen receptor to tumor antigen, but also improves the active activation function of the chimeric antigen receptor to T cells.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a PSCA-targeted chimeric antigen receptor and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a signaling domain;

the antigen binding domain comprises an anti-PSCA single chain antibody;

the signaling domains include OX40, CD3 ζ, and TLR 1.

In the invention, the anti-PSCA single-chain antibody is used as an antigen binding domain and is connected with a signal conduction domain to construct a chimeric antigen receptor, so that the chimeric antigen receptor has an obvious targeting effect on tumor cells and has a high-efficiency activation effect on T cells.

Preferably, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO:1：

DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSEPKSCDKTHTCPPC。

preferably, the transmembrane domain comprises CD28 and/or CD8 a.

Preferably, the chimeric antigen receptor further comprises a signal peptide.

Preferably, the signal peptide comprises a CD8 a signal peptide.

Preferably, the chimeric antigen receptor consists of a CD8 a signal peptide, an anti-PSCA single chain antibody, CD8 a, OX40, CD3 ζ and TLR1 in tandem.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 2;

SEQ ID NO:2：

MALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSEPKSCDKTHTCPPCTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRNIPLEELQRNLQFHAFISYSGHDSFWVKNELLPNLEKEGMQICLHERNFVPGKSIVENIITCIEKSYKSIFVLSPNFVQSEWCHYELYFAHHNLFHEGSNSLILILLEPIPQYSIPSSYHKLKSLMARRTYLEWPKEKSKRGLFWANLRAAINIKLTEQAKK。

in a second aspect, the present invention provides a coding gene encoding the chimeric antigen receptor of the first aspect.

Preferably, the coding gene comprises an anti-PSCA single chain antibody coding sequence.

Preferably, the coding gene further comprises a CD8 a signal peptide coding sequence, a CD8 a coding sequence, an OX40 coding sequence, a CD3 ζ coding sequence and a TLR1 coding sequence.

In a third aspect, the present invention provides an expression vector, wherein the expression vector is a lentiviral vector comprising the coding gene of the second aspect.

In a fourth aspect, the present invention provides a recombinant lentivirus which is a mammalian cell transfected with an expression vector and a helper plasmid as described in the third aspect.

In a fifth aspect, the present invention provides a chimeric antigen receptor T cell expressing the chimeric antigen receptor of the first aspect.

Preferably, the chimeric antigen receptor T cell has the coding gene of the second aspect integrated into its genome.

Preferably, the chimeric antigen receptor T cell comprises the expression vector of the third aspect and/or the recombinant lentivirus of the fourth aspect.

In a sixth aspect, the present invention provides a method for producing a chimeric antigen receptor T cell according to the fifth aspect, the method comprising the step of introducing a gene encoding the chimeric antigen receptor according to the first aspect into a T cell.

In a seventh aspect, the present invention provides a use of the chimeric antigen receptor of the first aspect, the coding gene of the second aspect, the expression vector of the third aspect, the recombinant lentivirus of the fourth aspect, or the chimeric antigen receptor T cell of the fifth aspect, in the preparation of a medicament for treating a disease.

Preferably, the disease comprises a tumor.

Preferably, the tumor includes any one or a combination of at least two of liver cancer, lung cancer, breast cancer, stomach cancer, nephroblastoma, glioma, neuroblastoma, melanoma, nasopharyngeal carcinoma, mesothelioma, islet cell tumor, retinoblastoma, pancreatic cancer, uterine fibroids, cervical cancer, or thyroid cancer.

Compared with the prior art, the invention has the following beneficial effects:

(1) the chimeric antigen receptor constructed by the invention adopts anti-PSCA single-chain antibody as an antigen binding domain, is connected with the signal transduction domains OX40, CD3 zeta and TLR1 to construct the chimeric antigen receptor, has obvious targeting effect on tumor cells, has obviously improved activation activity on T cells, eliminates the immunosuppression effect of regulatory T cells and promotes the formation of memory T cells;

(2) the T cell expressing the chimeric antigen receptor constructed by the invention has obvious effect of eliminating and killing PSCA positive tumor cells.

Drawings

FIG. 1 is the killing efficiency of WT, alpha PSCA-CAR-T1-T on K562 and K562-PSCA;

FIG. 2 is IL-2 secretion following co-culture of WT, alpha PSCA-CAR-T1-T with K562-PSCA;

FIG. 3 shows the secretion of IFN-. gamma.after co-culture of WT, α PSCA-CAR-T1-T and K562-PSCA.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 construction of CAR molecular vectors

In this example, a chimeric antigen receptor α PSCA-CAR-T1 with an antigen binding domain of anti-PSCA single chain antibody and a signaling domain of OX40, CD3 ζ and TLR1 was constructed, and the amino acid sequence is shown in SEQ ID No. 2;

firstly, synthesizing a coding gene of alpha PSCA-CAR-T1 by gene, and respectively adding enzyme cutting sites Pme1 and Spe1 and protective basic groups thereof at the 5 'end and the 3' end of the coding gene;

carrying out double enzyme digestion on the coding gene by using restriction enzymes Pme1 and Spe1, incubating for 30min in water bath at 37 ℃, and carrying out agarose gel electrophoresis recovery to obtain an enzyme digestion product containing a sticky end;

the digestion products are connected into a linearized pWPXld-eGFP plasmid (containing sticky ends) which is also subjected to double digestion by Pme1 and Spe1, and the connection system is shown in the table 1, so that a lentiviral vector containing the coding gene of the alpha PSCA-CAR-T1 is obtained.

TABLE 1

Components	Dosage (mu L)
		pWPXld-eGFP plasmid	2(50ng)
CAR gene	10(150ng)
		T4 DNA ligation buffer	2
T4 DNA ligase (NEB)	1
		ddH₂O	5

In this example, alpha PSCA-CAR (antipSCA scFv-CD8 alpha-OX 40-CD3 zeta) was constructed at the same time, and a corresponding lentiviral vector was constructed.

Example 2 Lentiviral packaging

Transferring the lentiviral vector constructed in the embodiment 1 into escherichia coli, selecting positive monoclonal for overnight culture, extracting the lentiviral vector by using a plasmid extraction kit, and packaging viruses;

viral packaging was performed using 293T cells, formulating a packaging system as shown in table 2, with a pwxld-expression plasmid comprising a lentiviral vector containing the gene encoding alpha PSCA-CAR-T1 and a lentiviral vector containing the gene encoding alpha PSCA-CAR, the pwxld-eGFP plasmid being an empty vector containing no CAR encoding gene;

TABLE 2

Adding 36 μ g PEI into another 500 μ L opti-MEM medium, mixing, and standing at room temperature for 5 min;

mixing the plasmid mixed solution shown in the table 2 with PEI, blowing, beating and uniformly mixing, and standing at room temperature for 25-30 min;

dropwise adding the mixed solution to 293T cells cultured in a 100mm culture dish;

after culturing for 6h, changing the culture medium into DMEM containing 1% fetal calf serum, and adding the DMEM into a culture dish with the volume of 7mL/100 mm;

collecting virus supernatant 24h, 48h and 72h after packaging, and simultaneously supplementing a culture medium to 293T cells, wherein the addition amount is 7mL/100mm culture dish;

centrifugation at 1000g for 10min, filtration through a 0.45 μm filter, to obtain recombinant lentivirus expressing CAR or a blank control eGFP lentivirus, and storage at 4 ℃ for future use.

Example 3T cell activation and lentivirus transfection

Isolating mononuclear cells (PBMCs) from adult peripheral blood, sorting out total T cells using a T cell sorting kit, and adding the CAR molecule over-expressing recombinant lentivirus prepared in example 2 after 24 hours of in vitro stimulation with CD3 and CD28 antibodies;

after 12h of transduction, the T cells were centrifuged and the solution was changed; three days after transduction, CAR-T ratios were assessed using flow cytometry to determine GFP positive cell content;

2 × 10 was cultured in per ml of the medium⁶The CAR-T cells were expanded in density culture of individual cells to obtain α PSCA-CAR-T1-T, α PSCA-CAR-T and WT (transfection blank eGFP lentivirus) cells, which were cryopreserved ten days later for use.

Example 4 functional assessment of CAR-T cells

This example used CAR-T cells prepared in example 3 to perform in vitro killing, as follows:

firstly, counting PSCA negative cells K562, PSCA positive cells K562-PSCA, alpha PSCA-CAR-T1-T cells, alpha PSCA-CAR-T cells and WT cells, inoculating the CAR-T cells and the K562 or K562-PSCA in a white flat-bottom 96-well plate according to the proportion of 1:1, setting 3 multiple wells in each group, centrifuging for 5min at 250g, and co-culturing for 24h by using 200 mu L of IMDM culture medium containing 5% fetal bovine serum;

after the co-culture is finished, adding 100 mu L/well of Luciferase substrate (1 x) into a 96-well plate, suspending and mixing the cells evenly, immediately measuring RLU (relative light unit) by a multifunctional microplate reader for 1 second, comparing the killing effect of WT, alpha PSCA-CAR-T and alpha PSCA-CAR-T1-T on K562-PSCA in vitro by using a Luciferase (Luciferase) quantitative killing efficiency evaluation method, wherein the killing proportion calculation formula is as follows:

100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)

As shown in fig. 1, the killing efficiency of the α PSCA-CAR-T1-T cells against PSCA-expressing tumor target cells was significantly higher than that of the α PSCA-CAR-T cells and WT cells, and the α PSCA-CAR-T2-T cells had strong tumor killing activity.

Supernatants of WT, alpha PSCA-CAR-T and alpha PSCA-CAR-T1-T were co-cultured with K562 or K562-PSCA cells for 18h, respectively, and IL-2 levels in the supernatants were assayed by ELISA.

As shown in figure 2, the levels of IL-2 secreted by α PSCA-CAR-T1-T cells were higher than α PSCA-CAR-T and WT, suggesting that TLR1 of the chimeric antigen receptor increased the ability of CAR-T cells to secrete IL-2, i.e., TLR1 promoted T cell proliferation.

Example 5

K562 and K562-PSCA cells were separately prepared according to 5 × 10⁵The cells/well density were inoculated into 24-well plates, followed by addition of WT, α PSCA-CAR-T or α PSCA-CAR-T1-T, co-culture in incubator for 12h, and detection of co-culture supernatants was performed using IFN- γ ELISA assay kit.

As shown in FIG. 3, the level of IFN-gamma secretion of alpha PSCA-CAR-T1-T cells is higher than that of alpha PSCA-CAR-T and WT, indicating that the alpha PSCA-CAR-T1-T can secrete cytokines to play a killing function while killing tumor cells.

In conclusion, the anti-PSCA single-chain antibody is used as an antigen binding domain and is connected with a signal transduction domain containing TLR1 to construct a chimeric antigen receptor, so that the chimeric antigen receptor has an obvious targeting effect on tumor cells and has a high-efficiency activation effect on T cells; the immune cell tumor cell expressing the chimeric antigen receptor has obvious effect of eliminating and killing.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangdong Shoutai biomedical science and technology Co., Ltd

<120> PSCA-targeted chimeric antigen receptor and application thereof

<130>2020

<160>2

<170>PatentIn version 3.3

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Claims

1. A chimeric antigen receptor, comprising an antigen binding domain, a transmembrane domain, and a signaling domain;

the antigen binding domain comprises an anti-PSCA single chain antibody;

the signaling domains include OX40, CD3 ζ, and TLR 1.

2. The chimeric antigen receptor according to claim 1, wherein the antigen binding domain comprises the amino acid sequence shown in SEQ ID No. 1.

3. The chimeric antigen receptor according to claim 1 or 2, wherein the transmembrane domain comprises CD28 and/or CD8 a;

preferably, the chimeric antigen receptor further comprises a signal peptide;

preferably, the signal peptide comprises a CD8 a signal peptide.

4. The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that it consists of a CD8 a signal peptide, an anti-PSCA single chain antibody, CD8 a, OX40, CD3 ζ and TLR1 in tandem;

preferably, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 2.

5. A coding gene encoding the chimeric antigen receptor of any one of claims 1 to 4;

preferably, the coding gene comprises an anti-PSCA single chain antibody coding sequence;

6. An expression vector, wherein the expression vector is a lentiviral vector comprising the coding gene of claim 5.

7. A recombinant lentivirus, wherein the recombinant lentivirus is a mammalian cell transfected with the expression vector of claim 6 and a helper plasmid.

8. A chimeric antigen receptor T cell, wherein said chimeric antigen receptor T cell expresses the chimeric antigen receptor of any one of claims 1-4;

preferably, the chimeric antigen receptor T cell has the coding gene of claim 5 integrated into its genome;

preferably, the chimeric antigen receptor T cell comprises the expression vector of claim 6 and/or the recombinant lentivirus of claim 7.

9. A method for producing a chimeric antigen receptor T cell according to claim 8, which comprises the step of introducing the gene encoding the chimeric antigen receptor according to any one of claims 1 to 4 into a T cell.

10. Use of the chimeric antigen receptor of any one of claims 1 to 4, the coding gene of claim 5, the expression vector of claim 6, the recombinant lentivirus of claim 7 or the chimeric antigen receptor T cell of claim 8 for the preparation of a medicament for the treatment of a disease;

preferably, the disease comprises a tumor;