CN114573710A - Immune cell for simultaneously secreting CD47 antibody through target antigen and application thereof - Google Patents

Abstract

The invention discloses an immune cell that targets antigens and simultaneously secretes CD47 antibody and its application in the field of tumor immune cell therapy. One aspect of the present invention provides a chimeric antigen receptor comprising a first signal peptide, an antigen-binding fragment, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, an intracellular domain, a self-cleaving sequence, a second signal The peptide and the anti-CD47 single-chain antibody are formed in series in sequence; on the other hand, it provides an immune cell that targets an antigen and excretes CD47 antibody, especially a chimeric antigen receptor-modified T cell, NK cell or NKT cell. In the present invention, the first nucleic acid encoding the antigen-binding fragment and the second nucleic acid encoding the anti-CD47 single-chain antibody are constructed on the same carrier. It can effectively antagonize the immunosuppressive factors in the tumor immune microenvironment and block the anti-phagocytosis of tumor cells.

Description

An immune cell targeting antigen and exocytosing CD47 antibody and its application

technical field

The invention belongs to the field of tumor immune cell therapy, and in particular relates to an immune cell that targets an antigen and simultaneously secretes CD47 antibody and its application.

Background technique

As a new type of targeted immunotherapy, chimeric antigen receptor-modified T cells (CAR-T) have demonstrated excellent efficacy in hematological malignancies. Chimeric antigen receptors (CARs) are synthetic receptors that relocate T cells to tumor surface antigens, and are mainly composed of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain. The extracellular antigen-binding domain can specifically recognize tumor antigens and connect the intracellular domain through the transmembrane domain, thereby transmitting activation signals and promoting the proliferation and function of T cells. CAR-modified T cells can recognize tumor antigens of tumor cells without the processing and presentation of MHC molecules, and specifically kill tumor cells, which has broad application prospects in clinical. The first generation of CARs usually connect antibody-derived tumor-binding elements to CD3ζ or Fc receptor signaling domains to trigger T cell activation, which can kill tumor cells to a certain extent, but the effect is not very satisfactory, and the survival time of CART cells is short. . Second- and third-generation treatments add costimulatory molecules such as CD28 and 4-1BB, which largely prolong T cell survival and enhance their effector functions.

CAR-T cell therapy is an ideal choice when traditional methods such as chemotherapy, combination of radiotherapy and chemotherapy, and hematopoietic stem cell transplantation are ineffective in the treatment of refractory and relapsed leukemia. At present, CAR-T cells targeting CD19 are the most widely used in the treatment of refractory and relapsed leukemia and lymphoma patients, but patients achieve high remission rates and are accompanied by high risk of relapse. For example, CD19-CAR-T treatment of refractory and relapsed non-Hodgkin lymphoma has achieved significant efficacy, and about 50% of patients can achieve complete remission, but about 55% of them relapse within one year; in addition, due to immunosuppressive tumors Due to limitations such as microenvironment and antigen loss, the efficacy of CD19-CAR-T in solid tumors is poor compared with the efficacy achieved by CD19-CAR-T in acute leukemia. The main mechanisms leading to relapse to CD19-CAR-T therapy include an immunosuppressive tumor microenvironment caused by repeated antigen stimulation and antigen escape caused by tumor alternative splicing. Therefore, in order to reduce the inhibitory effect of immunosuppressive TME on CAR-T cells and reduce the recurrence of treatment, it is necessary to further improve the structure and function of existing CD19-CAR-T cells and treatment strategies in order to obtain better efficacy.

CD47 is a protein widely distributed on the surface of normal cells, and its major ligand, SIRPα, is highly expressed on the membranes of myeloid cells such as macrophages, granulocytes, and monocytes. Normal cells express CD47 and are labeled with self-labeling, which releases the "don't eat me" signal through the CD47/SIRPα axis, inhibits macrophage-mediated phagocytosis, and protects normal cells from destruction. Studies have shown that CD47 is highly expressed in various tumors such as leukemia and lymphoma. Through high expression of CD47, cancer cells can disguise themselves as their own cells to release anti-phagocytic signals, inhibit macrophage-mediated phagocytosis, and cause immune escape, thereby promoting tumor progression and spreading, which is associated with poor prognosis of tumor patients. In addition, the expression of CD47 was positively correlated with the expression of PD-1, Treg marker Foxp3, MDSC markers CD11b and CD33. Anti-CD47 treatment promotes the polarization of macrophages to the M1 subtype in tumor mouse models, and reduces PD-1 expression and increases IFN-γ secretion in effector T cells in tumor mouse models, reducing immunosuppressive cells The number of Tregs and MDSCs can improve the tumor microenvironment and delay tumor growth. However, the inherent characteristics of anti-CD47 single-chain antibodies lead to certain limitations in their application, and there is a risk of targeting normal cells, resulting in cytotoxic reactions.

SUMMARY OF THE INVENTION

In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide an immune cell that targets an antigen and excretes CD47 antibody and its application.

A first aspect of the present invention provides a chimeric antigen receptor comprising a first signal peptide, an antigen-binding fragment, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, an intracellular domain, a self-cleaving sequence, a second signal The peptide and the anti-CD47 single-chain antibody are connected in series.

Further, the antigen recognized and bound by the antigen-binding fragment is a tumor-associated antigen;

Preferably, the antigen-binding fragment is a single-chain antibody;

Preferably, the tumor-associated antigen is selected from CD19, CD20, CD22, CD30, CD123, BCMA or Her2, etc.;

Preferably, the tumor-associated antigen is CD19.

Further, the first signal peptide and the second signal peptide are selected from CD8α signal peptide, IL-2 signal peptide or GM-CSF signal peptide;

Preferably, the first signal peptide is selected from CD8α signal peptide;

Preferably, the second signal peptide is selected from GM-CSF signal peptide;

The hinge region is selected from the CD8α hinge region or the FcRIIIα receptor;

The transmembrane region is selected from T cell receptor subunit, CD8α subunit, CD8β subunit, CD8δ subunit, CD4 transmembrane domain or CD28 transmembrane domain;

The intracellular costimulatory signal domain is selected from one of 4-1BB, CD27, CD28, ICOS, OX40, NKG2D or B7-H3 intracellular costimulatory signal domain; the intracellular costimulatory signal domain is used to conduct extracellular response The stimulatory signal generated by the binding of the antigen-binding domain specific to the antigen-binding fragment to the target antigen causes immune cell activation and immune response;

Preferably, the intracellular costimulatory signal domain is selected from 4-1BB;

The intracellular domain is selected from CD3ζ;

The self-cleaving sequence is selected from T2A or P2A.

The second aspect of the present invention provides a gene encoding the chimeric antigen receptor.

The third aspect of the present invention provides an expression vector for the encoding gene of the chimeric antigen receptor.

Further, the expression vector is selected from lentivirus expression vector, retrovirus expression vector or adenovirus expression vector.

A fourth aspect of the present invention provides a virus comprising the expression vector.

A fifth aspect of the present invention provides an immune cell that targets an antigen and simultaneously excretes a CD47 antibody, comprising the chimeric antigen receptor encoding gene or an expression vector of the chimeric antigen receptor encoding gene;

Preferably, the immune cells are selected from T cells, NK cells or NKT cells;

Preferably, the immune cells are selected from T cells.

The sixth aspect of the present invention provides an application of an immune cell that targets an antigen and simultaneously secretes CD47 antibody in the preparation of a drug for treating lymphoma or leukemia.

The seventh aspect of the present invention provides a pharmaceutical composition, comprising the expression vector of the gene encoding the chimeric antigen receptor or the immune cell that targets the antigen and excretes the CD47 antibody at the same time.

The beneficial effects of the present invention are:

1. The present invention constructs a first nucleic acid encoding an antigen-binding fragment (such as a CD19 single-chain antibody) and a second nucleic acid encoding an anti-CD47 single-chain antibody on the same carrier, and by targeting the antigen and exocytosing the CD47 antibody, the present invention Chimeric antigen receptor-modified immune cells can achieve local delivery of anti-CD47 antibodies, reduce the impact on normal cells, efficiently and specifically target tumor cells expressing the corresponding antigen, and effectively antagonize immunosuppression in the tumor immune microenvironment factor, blocking the anti-phagocytosis of tumor cells, while retaining the anti-tumor activity of immune cells modified by chimeric antigen receptors, reducing tumor immune escape, and improving the persistence and anti-tumor activity of immune cells.

2. The chimeric antigen receptor T cells (CAR-T) targeting CD19 have a high risk of recurrence in the treatment of refractory and recurrent tumors. The novel CAR-T of the present invention can enhance the anti-tumor efficacy of CD19-CAR-T and promote the Phagocytosis of tumor cells by phagocytes, and regulates the proportion of other immunosuppressive cells in the tumor microenvironment, while retaining the anti-tumor activity of CD19-CART, reducing tumor immune escape and improving the persistence and anti-tumor activity of CART cells, Thereby reducing the immune escape of refractory and recurrent tumors to CAR-T cell therapy.

The own characteristics of anti-CD47 single-chain antibody lead to certain limitations in its application, and there is a risk of targeting normal cells, resulting in cytotoxic reactions. The novel CAR-T of the present invention can be used as a medium to achieve local delivery of anti-CD47 single-chain antibody, reducing Risks of targeting normal cells with anti-CD47 single-chain antibodies and circumventing their application limitations.

Description of drawings

Figure 1 shows a schematic diagram of the structure of a chimeric antigen receptor.

Figure 2 shows the vector design scheme of the chimeric antigen receptor CD19-s47-CAR and CD19-CAR.

Figure 3 shows the statistical graph of anti-CD19 and anti-CD47 fragments stably expressed by 293T cells transfected by CD19-CAR and CD19-s47-CAR virus detected by RT-PCR.

Figure 4 shows the transfection efficiency of T cells transfected with CD19-CAR and CD19-s47-CAR virus (MOI=10) and the proportion of CD4, CD8 cells detected by flow cytometry.

Figure 5 shows that the tumor cell lines Raji, Romas and Daudi used in the present invention stably express CD47 by flow cytometry.

Figure 6 shows that the tumor cell lines Raji, Romas and Daudi used in the present invention can stably bind to the exocrine protein s47 by flow cytometry.

Figure 7 shows that T cells transfected with CD19-s47-CAR stably express exocrine protein s47 by western blot.

Figure 8 shows the differentiation and typing of T cells detected by flow cytometry after co-culturing human peripheral blood lymphocytes transfected with vector 19-CAR and CD19-s47-CAR with tumor cell lines for 24 hours.

Figure 9 shows the lysis of tumor cell lines Raji, Romas and Daudi by human peripheral blood lymphocytes transfected with vector 19-CAR and CD19-s47-CAR by cytotoxicity experiments.

Figure 10 shows the expression of IL-2 and TNF-α detected by flow cytometry after co-culture of human peripheral blood lymphocytes transfected with vector 19-CAR and CD19-s47-CAR with tumor cell line Raji for 5 h.

Figure 11 shows the promoting effect of exocrine protein s47 on the phagocytic ability of macrophages.

Figure 12 shows the mechanism by which CD19-s47-CAR blocks anti-phagocytosis of tumor cells.

Detailed ways

For a clearer understanding of the present invention, the present invention will now be further described with reference to the following examples and accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each original reagent material can be obtained commercially, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions suggested by the instrument manufacturer.

Example 1

This embodiment provides a chimeric antigen receptor comprising a signal peptide, an antigen-binding fragment, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, an intracellular domain, a self-cleaving sequence, a signal peptide and an anti-CD47 single chain Antibodies are formed in series.

In a specific embodiment, the antigen recognized and bound by the antigen-binding fragment is a tumor-associated antigen; the tumor-associated antigen can be selected from CD19, CD20, CD22, CD30, CD123, BCMA or Her2; in a preferred embodiment , the tumor-associated antigen is CD19, and the antigen-binding fragment is an anti-CD19 single-chain antibody (ScFv).

In a specific embodiment, the signal peptide is selected from CD8α signal peptide, IL-2 signal peptide or GM-CSF signal peptide; in a preferred embodiment, the first signal peptide is selected from CD8α signal peptide, and the second signal peptide is selected from CD8α signal peptide. Selected from GM-CSF signal peptides.

In a specific embodiment, the hinge region is selected from the CD8α hinge region or the FcRIIIα receptor; in a preferred embodiment, the hinge region is selected from the CD8α hinge region.

In a specific embodiment, the transmembrane region is selected from the group consisting of T cell receptor subunit, CD8α subunit, CD8β subunit, CD8δ subunit, CD4 transmembrane domain or CD28 transmembrane domain; in a preferred embodiment , the transmembrane region is selected from the CD8α subunit.

In a specific embodiment, the intracellular costimulatory signal domain is selected from one of 4-1BB, CD27, CD28, ICOS, OX40, NKG2D or B7-H3 intracellular costimulatory signal domain; in a preferred embodiment , the intracellular costimulatory signaling domain is selected from 4-1BB.

In a specific embodiment, the intracellular domain is selected from CD3ζ.

In a specific embodiment, the self-cleaving sequence is selected from T2A or P2A; in a preferred embodiment, the self-cleaving sequence is selected from T2A.

Figure 1 is a schematic diagram of the structure of one of the preferred embodiments of the chimeric antigen receptor.

Example 2

This example provides the preparation of chimeric antigen receptor T cells. The carrier of the chimeric antigen receptor adopts one of the preferred solutions. In order to facilitate detection, an HA tag is attached to the end of the CD47 single-chain antibody, and the chimeric antigen receptor is denoted as CD19-s47-CAR, other tags such as 6×His, Fc, Myc, GST or Flag can also be used. At the same time, a chimeric antigen receptor excluding self-cleavage sequence, signal peptide and anti-CD47 single-chain antibody was set as a control, and the chimeric antigen receptor was denoted as CD19-CAR. The design scheme of chimeric antigen receptor CD19-s47-CAR and CD19-CAR vector is shown in Figure 2. The sequence used therein is as follows:

CD8αSP (nucleotide sequence):

ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC(SEQID NO.1);

CD8αSP (amino acid sequence):

MALPVTALLLPLALLLHAARP (SEQ ID NO. 2);

CD19 ScFv (nucleotide sequence):

GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.3)；

CD19 ScFv (amino acid sequence):

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYGTYY.SVSSY);

CD8 Hinge (nucleotide sequence):

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAAGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO. 5);

CD8 Hinge (amino acid sequence):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO. 6);

CD8TM (nucleotide sequence):

ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGT (SEQ ID NO. 7);

CD8TM (amino acid sequence):

IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 8);

4-1BB (nucleotide sequence):

AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG (SEQ ID NO. 9)

4-1BB (amino acid sequence):

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO. 10)

CD3ζ (nucleotide sequence):

CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG(SEQ ID NO.11)

CD3ζ (amino acid sequence):

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO. 12)

T2A (nucleotide sequence):

GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCC (SEQ ID NO. 13)

T2A (amino acid sequence):

EGRGSLLTCGDVEENPGP (SEQ ID NO. 14)

GM-CSF SP (nucleotide sequence):

ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCT (SEQ ID NO. 15)

GM-CSF SP (amino acid sequence)

MWLQSLLLLGTVACSIS (SEQ ID NO. 16)

CD47 ScFv (nucleotide sequence):

GACGTGGTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACACCTGGCGAACCTGCCAGCATCAGCTGTAGAAGCAGCCAGAGCATCGTGTACAGCAACGGCAACACCTACCTCGGCTGGTATCTGCAGAAGCCCGGCCAGTCTCCTAAGCTGCTGATCTACAAGGTGTCCAACCGGTTCAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAAGATCTCCAGAGTGGAAGCCGAGGACGTGGGCGTGTACCACTGTTTTCAGGGCAGCCACGTGCCATACACCTTTGGCGGCGGAACAAAGGTGGAAATCAAGGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGCCAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCAACTACAACATGCACTGGGTCCGACAGGCCCCTGGACAAGGACTGGAATGGATCGGCACAATCTACCCCGGCAACGACGACACCAGCTACAACCAGAAGTTCAAGGACAAGGCCACACTGACCGCCGACAAGAGCACAAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCGGCTACAGAGCCATGGACTATTGGGGCCAGGGCACCCTGGTTACCGTTAGCTCT(SEQ ID NO.17)

CD47 ScFv (amino acid sequence):

DVVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHCFQGSHVPYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWIGTIYPGNDDTSYNQKFKDKATLTADKSTSTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS(SEQ ID NO.18)

HA (nucleotide sequence):

TACCCATACGACGTACCAGATTACGCT (SEQ ID NO. 19)

HA (amino acid sequence)

YPYDVPDYA (SEQ ID NO. 20)

The preparation method of immune cells that target antigen and excrete CD47 antibody at the same time is as follows:

1. Construction of Lentiviral Vectors

After the full gene synthesis of the nucleotide sequence of the chimeric antigen receptor, the sequence was amplified by PCR, the amplified fragment was recovered by the Axygen gel recovery kit, and the amplified fragment was combined with the vector pCDH digested with the restriction enzymes BstBI and XbaI. - The EF1-MCS expression vector was connected by homologous recombination, and the CD19-CAR single-target and CD19-s47-CAR dual-target expression vectors were synthesized. Add 5ul of ligation product to 50ul of competent cells DH5α, let stand on ice for 30min, heat shock in 42°C water bath for 90s, cool on ice for 2min, add 500ul of fresh anti-LB liquid medium, shake at 37°C at 150rpm /min shaken for 45min, spread on LB solid plates containing ampicillin, and cultured overnight in a 37°C incubator. After the monoclonal colonies grow, pick 6 monoclonal colonies, add 500ul of LB liquid medium containing ampicillin, and shake them in a shaker at 37°C for 4 hours. For the samples with correct sequence alignment, CD19-CAR single-target and CD19-s47-CAR double-target recombinant expression vectors were obtained, and were cultured in LB liquid medium, followed by endotoxin-free plasmid extraction.

2. Preparation of Lentiviral Vector Virus Liquid

Use Lipofectamine 3000 to mix the recombinant plasmid obtained in step 1 with the packaging plasmids pMD2.G and psPAX2 in a ratio of 4:3:1, and then transfect 293T cells. After 6 hours of transfection, fresh medium was replaced, and cells cultured for 24h and 52h were collected respectively. The supernatant was mixed with virus concentrate at 1:4 and incubated at 4°C overnight, and the virus solution was concentrated by high-speed centrifugation. The supernatant was discarded, and the virus pellet was resuspended in PBS solution to obtain CD19-CAR and CD19-s47-CAR virus solution. .

3. Preparation of Chimeric Antigen Receptor T Cells

Fresh peripheral blood from healthy donors was taken, and fresh peripheral blood mononuclear cells were separated by density gradient centrifugation, and CD3 positive sorting magnetic beads (purchased from Stem cell) were used to separate T cells, and X-vivo serum-free medium ( (purchased from LONZA) resuspended T cells and added CD3/CD28 stimulating antibodies (purchased from Stem cell), IL-2, IL-7 and IL-15, and cultured at 37°C in a 5% CO ₂ incubator for 24h after the addition step The virus solution obtained in 2 was transfected at MOI=10, and CD19-CART and CD19-s47-CART cells were obtained respectively, and the transfection rate of CAR-T cells was detected 3 days after transfection. T cells were always cultured at a density of 1 x 10^6/ml. At the same time, 293T cells were transfected with the virus solution of MOI=1, cultured in a 37°C, 5% CO ₂ incubator, and the cells were collected after 5 days, and the expression of CAR fragments in the virus-transfected 293T was detected by RT-PCR. The results are as follows As shown in Figure 3, CD19-293T cells stably expressed CD19-CAR fragments, while CD19-s47-293T cells stably expressed anti-CD19-CAR and anti-CD47-CAR fragments.

Experimental example

1. Detection of CAR protein expression by flow cytometry

Take an appropriate amount of CD19-CART, CD19-s47-CART cells and control T cells obtained in Example 2, wash 2 times with PBS, resuspend the cells with 50ul PBS solution, and add 0.5ul CAR-GREEN (purchased from Shanghai Ya Science and Technology Co., Ltd.), incubate at 4°C for 30 min in the dark, wash the cells twice, add 200ul PBS to resuspend the cells, and use BD Cytoflex to detect on the computer. The detection results are shown in Figure 4. The expression of CAR molecules was almost undetectable in control T cells, and the anti-CD19 ScFv expression rates of CD19-CART and CD19-s47-CART cells were 50.4% and 60.4%, respectively.

2. Binding detection of exocrine protein s47 to target cells

Take the CD19-CART, CD19-s47-CART cells and control T cells obtained in Example 2, and collect the cell supernatants of CD19-CART, CD19-s47-CART cells and control T cells after 5 days of infection. The lymphoma cell lines Raji, Daudi and Romas expressing CD47 (Fig. 5) were washed twice with PBS and then incubated with the supernatant at 37°C for 30 min respectively. The supernatant was discarded by centrifugation, washed twice with PBS and then washed with 50ul PBS respectively. Resuspend, and add 0.5ul of anti-HA-APC flow antibody (purchased from Biolegend), incubate at room temperature for 25 min in the dark, wash the cells twice, add 200ul PBS to resuspend the cells, and use BDCytoflex to detect on the computer. The detection results are shown in Figure 6. shown. The binding rates of the three lymphoma cell lines Raji, Daudi and Romas to exocrine protein s47 were all close to 100%.

3. Western blot assay to detect the expression of exocrine protein s47

Take the CD19-CART, CD19-s47-CART cells and control T cells obtained in Example 2, and collect the cell supernatants of CD19-CART, CD19-s47-CART cells and control T cells after 5 days of infection. Protein precipitation kit (purchased from Invent) precipitated the protein in the supernatant, quantitatively measured the protein concentration by BCA, added 5x loading buffer in proportion, heated at 100°C for 10min and loaded 30ug of protein into the SDS page well, electrophoresed at 80V for 30min After electrophoresis at 120V for 60min, the protein samples were separated, then the gel was cut and transferred to the membrane, and the flow was constant at 250mA for 2h, and the protein on the gel was transferred to the PVDF membrane. After transfer, the membrane was blocked in 5% nonfat milk powder at room temperature for 2 hours, and then placed in mouse-derived anti-HA monoclonal antibody (1:1000, purchased from Bioworld) and mouse-derived anti-α-Turbulin monoclonal antibody (1:10000, purchased from Proteintech) ) at 4°C overnight, washed three times with PBST, and incubated with HRP-goat anti-mouse Fc (1:10000) for 1 h at room temperature. After washing with PBST for three times, FDbio-Dura ECL luminescent solution was used for color development, and the BioRad Imaging Syatem was used to take pictures. The results are shown in Figure 7.

4. Differentiation function detection of chimeric antigen receptor T cells

The CD19-CART, CD19-s47-CART cells and control T cells obtained in Example 2 were taken, and the T cell differentiation function was detected 5 days after infection. CD19-CART, CD19-s47-CART cells and control T cells and target cells Raji were plated in a 96-well plate at a 1:1 effect-to-target ratio. After culturing in a 37°C, 5% CO ₂ incubator for 24 h, the cells were collected. After washing twice with PBS, resuspend in 50ul PBS, and add 0.5ul CAR-GREEN, anti-CD8-Percp.cy5.5, anti-CD45RO-APC and anti-CCR7-PE flow antibodies (purchased from Biolegend), room temperature After incubating in the dark for 25 min, wash the cells twice, add 200ul PBS to resuspend the cells, and use BD Cytoflex to test on the machine. The test results are shown in Figure 8. Compared with CD19-CART, CD19-s47-CART cells have a higher proportion of central memory T cells (Tcms), a lower proportion of effector memory T cells (Tems).

5. Cytotoxic killing assay of chimeric antigen receptor T cells

Take the CD19-CART, CD19-s47-CART cells and control T cells obtained in Example 2, and after 5 days of infection, take the cells to carry out the target cell killing experiment, and simultaneously take the lymphoma cell lines Raji, Daudi and Romas, and adjust the target cell density to be 1.6×10^5/ml, 50ul per well was inoculated in a round-bottom 96-well plate, and T cells were inoculated according to the effect-target ratio of 20:1, 10:1, 5:1, 2.5:1, using non-radioactive cytotoxicity The detection kit (purchased from Promega) was used to detect the cell killing effect, and the specific operation was referred to the kit instructions, and the experimental results are shown in FIG. 9 . For target cells Raji, Romas, Daudi, CD19-s47-CART cells have stronger tumor killing function than CD19-CART cells.

6. Detection of cytokine secretion by chimeric antigen receptor T cells

The CD19-CART, CD19-s47-CART cells and control T cells obtained in Example 2 were taken. After 5 days of infection, the T cells and target cells were plated according to the ratio of effect to target of 1:1, and Brefeldin A was added at the same time. After culturing in a %CO ₂ incubator for 5 h, the cells were collected for anti-CD8-Percp.cy5.5 flow antibody staining, washed twice with PBS, and then fixed and permeabilized, as well as anti-IL-2-APC and anti-TNF-APC flow-through For antibody staining, incubate the cells at room temperature for 25 minutes in the dark, wash the cells twice, add 200ul PBS to resuspend the cells, and use BD Cytoflex to detect on the computer. The detection results are shown in Figure 10. CD19-s47-CART cells had stronger IL-2 and TNF-α secretion ability than CD19-CART cells.

7. Flow cytometry to detect the effect of exocrine protein s47 on the phagocytic ability of macrophages

Fresh peripheral blood from healthy donors was taken, and fresh peripheral blood mononuclear cells were separated by density gradient centrifugation, and CD14 positive sorting magnetic beads (purchased from Stem cell) were used to separate mononuclear cells. M-CSF (final concentration 25ng/ml) was supplemented in 1640 medium, and mature macrophages were obtained after induction and culture for 5 days in a 37°C, 5% CO ₂ incubator. The target cells Raji were counted after CFSE staining, and the CD19-CART, CD19-s47-CART cells and control T cells obtained in Example 2 were taken at the same time. After 5 days of infection, macrophages, T cells and Raji were divided according to 1:2: The cells were plated at a ratio of 2, cultured for 24 hours, and the cells were collected. After washing twice with PBS, the cells were stained with anti-CD14-Percp cy5.5 by flow-through antibody. BD Cytoflex was tested on the machine, and the test results are shown in Figure 11. CD19-s47-CART cells that can secrete CD47 single-chain antibody can significantly improve the phagocytic ability of macrophages to tumor cells.

Figure 12 shows the mechanism by which CD19-s47-CAR blocks anti-phagocytosis of tumor cells. When CD19-s47-CART reaches tumor tissue, it can bind to CD19 on tumor cells and release perforin, while exocrine anti-CD47 single-chain antibody binds to CD47 on tumor cells, thereby blocking CD47 of tumor cells and macrophages SIRPα binding, that is, blocking the anti-phagocytic pathway of tumor cells, and affecting the polarization direction of macrophages. At the same time, it may also affect the number and function of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs).

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

SEQUENCE LISTING

<110> Pearl River Hospital, Southern Medical University, Biological Island Laboratory

<120> An Immune Cell That Targets Antigen and Excretes CD47 Antibody and Its Application

<130> CP121011105C

<160> 20

<170> PatentIn version 3.3

<210> 1

<211> 63

<212> DNA

<213> Artificial sequences

<400> 1

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccc 63

<210> 2

<211> 21

<212> PRT

<213> Artificial sequences

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 3

<211> 726

<212> DNA

<213> Artificial sequences

<400> 3

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctca 726

<210> 4

<211> 242

<212> PRT

<213> Artificial sequences

<400> 4

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 5

<211> 135

<212> DNA

<213> Artificial sequences

<400> 5

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaagcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 6

<211> 45

<212> PRT

<213> Artificial sequences

<400> 6

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 7

<211> 72

<212> DNA

<213> Artificial sequences

<400> 7

atctacattt gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc 60

actctttact gt 72

<210> 8

<211> 24

<212> PRT

<213> Artificial sequences

<400> 8

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 9

<211> 126

<212> DNA

<213> Artificial sequences

<400> 9

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 60

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 120

gaactg 126

<210> 10

<211> 42

<212> PRT

<213> Artificial sequences

<400> 10

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 11

<211> 336

<212> DNA

<213> Artificial sequences

<400> 11

cgcgtgaaat tcagccgcag cgcagatgct ccagcctacc agcaggggca gaaccagctc 60

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300

tatgacgctc ttcacatgca ggccctgccg cctcgg 336

<210> 12

<211> 112

<212> PRT

<213> Artificial sequences

<400> 12

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 13

<211> 54

<212> DNA

<213> Artificial sequences

<400> 13

gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg cccc 54

<210> 14

<211> 18

<212> PRT

<213> Artificial sequences

<400> 14

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 15

<211> 51

<212> DNA

<213> Artificial sequences

<400> 15

atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc t 51

<210> 16

<211> 17

<212> PRT

<213> Artificial sequences

<400> 16

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser

<210> 17

<211> 732

<212> DNA

<213> Artificial sequences

<400> 17

gacgtggtca tgacacagag ccctctgagc ctgcctgtga cacctggcga acctgccagc 60

atcagctgta gaagcagcca gagcatcgtg tacagcaacg gcaacaccta cctcggctgg 120

tatctgcaga agcccggcca gtctcctaag ctgctgatct acaaggtgtc caaccggttc 180

agcggcgtgc ccgatagatt ttctggcagc ggctctggca ccgacttcac cctgaagatc 240

tccagagtgg aagccgagga cgtgggcgtg taccactgtt ttcagggcag ccacgtgcca 300

tacacctttg gcggcggaac aaaggtggaa atcaagggtg gaggtggcag cggaggaggt 360

gggtccggcg gtggaggaag ccaggttcag ctggttcagt ctggcgccga agtgaagaaa 420

cctggcgcct ctgtgaaggt gtcctgcaag gccagcggct acacctttac caactacaac 480

atgcactggg tccgacaggc ccctggacaa ggactggaat ggatcggcac aatctacccc 540

ggcaacgacg acaccagcta caaccagaag ttcaaggaca aggccacact gaccgccgac 600

aagagcacaa gcaccgccta catggaactg agcagcctga gaagcgagga caccgccgtg 660

tactattgtg ccagaggcgg ctacagagcc atggactatt ggggccaggg caccctggtt 720

accgttagct ct 732

<210> 18

<211> 244

<212> PRT

<213> Artificial sequences

<400> 18

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr His Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

130 135 140

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Asn

145 150 155 160

Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly

165 170 175

Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys Phe Lys

180 185 190

Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met

195 200 205

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Arg Gly Gly Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

225 230 235 240

Thr Val Ser Ser

<210> 19

<211> 27

<212> DNA

<213> Artificial sequences

<400> 19

tacccatacg acgtaccaga ttacgct 27

<210> 20

<211> 9

<212> PRT

<213> Artificial sequences

<400> 20

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

Claims (10)

1. A chimeric antigen receptor is characterized in that the chimeric antigen receptor is formed by connecting a first signal peptide, an antigen binding fragment, a hinge region, a transmembrane region, an intracellular costimulatory signal region, an intracellular region, a self-cleavage sequence, a second signal peptide and an anti-CD 47 single-chain antibody in series in sequence.

2. The chimeric antigen receptor according to claim 1, wherein the antigen recognized and bound by the antigen-binding fragment is a tumor-associated antigen;

preferably, the antigen-binding fragment is a single chain antibody;

preferably, the tumor associated antigen is selected from CD19, CD20, CD22, CD30, CD123, BCMA or Her 2;

preferably, the tumor associated antigen is CD 19.

3. The chimeric antigen receptor according to claim 1, wherein the first and second signal peptides are selected from the group consisting of a CD8 a signal peptide, an IL-2 signal peptide, or a GM-CSF signal peptide;

preferably, the first signal peptide is selected from the group consisting of CD8 a signal peptide;

preferably, the second signal peptide is selected from the group consisting of a GM-CSF signal peptide;

the hinge region is selected from a CD8 a hinge region or FcRIII a receptor;

the transmembrane region is selected from a T cell receptor subunit, a CD8 β subunit, a CD8 α subunit, a CD8 δ subunit, a CD4 transmembrane domain, or a CD28 transmembrane domain;

the intracellular costimulatory signal domain is selected from one of the 4-1BB, CD27, CD28, ICOS, OX40, NKG2D or B7-H3 intracellular costimulatory signal domain;

preferably, the intracellular co-stimulatory signaling domain is selected from the group consisting of 4-1 BB;

the endodomain is selected from CD3 ζ;

the self-splicing sequence is selected from T2A or P2A.

4. A gene encoding the chimeric antigen receptor of any one of claims 1 to 3.

5. An expression vector comprising a gene encoding the chimeric antigen receptor according to claim 4.

6. The expression vector of claim 5, wherein the expression vector is selected from the group consisting of a lentiviral expression vector, a retroviral expression vector, and an adenoviral expression vector.

7. A virus comprising the expression vector of claim 5.

8. An immune cell targeting an antigen while secreting a CD47 antibody, comprising an expression vector of a gene encoding the chimeric antigen receptor of claim 4 or a gene encoding the chimeric antigen receptor of claim 5;

preferably, the immune cell is selected from a T cell, an NK cell, or an NKT cell;

preferably, the immune cells are selected from T cells.

9. An application of an immune cell which targets an antigen and simultaneously secretes a CD47 antibody in preparing a medicament for treating lymphoma or leukemia.

10. A pharmaceutical composition comprising an expression vector of a gene encoding the chimeric antigen receptor of claim 5 or an immune cell of claim 8 targeting an antigen while secreting CD47 antibody.

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