CN114573710A

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

Info

Publication number: CN114573710A
Application number: CN202210142794.XA
Authority: CN
Inventors: 李玉华; 邱瑛琦; 胡宇行; 胡蓉; 许斌焱; 王浩; 廖佩云
Original assignee: Southern Medical University Zhujiang Hospital; Bioisland Laboratory
Current assignee: Southern Medical University Zhujiang Hospital; Bioisland Laboratory
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-06-03

Abstract

The invention discloses an immune cell for targeting an antigen and simultaneously secreting a CD47 antibody and application thereof, belonging to the field of tumor immune cell treatment. The invention provides a chimeric antigen receptor, which 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 domain, a self-cutting sequence, a second signal peptide and an anti-CD 47 single-chain antibody in series in sequence; another aspect provides an immune cell, in particular a chimeric antigen receptor-modified T cell, NK cell or NKT cell, that targets an antigen while secreting a CD47 antibody. According to the invention, a first nucleic acid for coding an antigen binding fragment and a second nucleic acid for coding an anti-CD 47 single-chain antibody are constructed on the same carrier, and local delivery of the anti-CD 47 antibody can be realized by targeting an antigen and secreting a CD47 antibody, so that the influence on normal cells is relieved, immunosuppressive factors in a tumor immune microenvironment are effectively antagonized, and the anti-phagocytosis effect of tumor cells is blocked.

Description

Immune cell for simultaneously secreting CD47 antibody through target antigen and application thereof

Technical Field

The invention belongs to the field of tumor immune cell therapy, and particularly relates to an immune cell for simultaneously secreting a CD47 antibody by a target antigen and application thereof.

Background

As a novel targeted immunotherapy, chimeric antigen receptor-modified T cells (CAR-T) have shown excellent therapeutic efficacy in hematologic malignancies. Chimeric Antigen Receptors (CARs) are synthetic receptors that relocate T cells to tumor surface antigens and are composed primarily of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain. The extracellular antigen binding domain can specifically recognize tumor antigens and is connected with the intracellular domain through the transmembrane domain, so that an activation signal is transmitted and the proliferation and the function of T cells are promoted. The T cell modified by the CAR can identify the tumor antigen of the tumor cell without the treatment and presentation of MHC molecules, specifically kills the tumor cell, and has wide application prospect in clinic. The first generation of CARs generally linked an antibody-derived tumor binding element to CD3 ζ or Fc receptor signaling domain to trigger T cell activation that could kill tumor cells to some extent, but the effect was not very desirable and the survival time of the car cells was short. Second and third generation therapies increase costimulatory molecules such as CD28, 4-1BB, and the like, greatly extending T cell survival and enhancing effector function.

CAR-T cell therapy is an ideal choice under the condition of poor effect of treating refractory relapsed leukemia by traditional modes such as chemotherapy, radiotherapy and chemotherapy combination, hematopoietic stem cell transplantation and the like. CD 19-targeted CAR-T cells are currently most widely used in the treatment of refractory relapsing leukemia and lymphoma patients, but patients achieve high remission rates with the concomitant risk of high relapse rates. For example, CD19-CAR-T treatment refractory to relapsing non-hodgkin lymphoma has achieved significant efficacy, with about 50% of patients achieving complete remission, but of which about 55% relapse within a year; in addition, due to limitations of the immunosuppressive tumor microenvironment and antigen loss, its therapeutic efficacy in solid tumors is poor compared to that achieved by CD19-CAR-T in acute leukemias. The major mechanisms leading to recurrence of CD19-CAR-T therapy include antigen escape from the immunosuppressive tumor microenvironment resulting from repeated stimulation of the antigen and alternative splicing of the tumor. Therefore, to reduce the inhibitory effect of immunosuppressive TME on CAR-T cells, and to reduce treatment relapse, further improvements in the structure and function of existing CD19-CAR-T cells and treatment strategies are needed in order to achieve better efficacy.

CD47 is a protein widely distributed on the surface of normal cells, and the main ligand SIRP alpha is highly expressed on the membrane of medullary cells such as macrophages, granulocytes, monocytes and the like. Normal cells express CD47 and are marked with self-markers, and release a ' don't eat me ' signal through a CD47/SIRP alpha axis, inhibit macrophage-mediated phagocytosis and protect normal cells from being damaged. The research shows that the CD47 is highly expressed in various tumors such as leukemia and lymphoma. By highly expressing CD47, cancer cells can pretend to be self cells to release anti-phagocytic signals, inhibit macrophage-mediated phagocytosis and generate immune escape, thereby promoting the progression and the diffusion of tumors and being related to poor prognosis of tumor patients. Furthermore, expression of CD47 was positively correlated with expression of PD-1, Treg marker Foxp3, MDSC marker CD11b and CD 33. The anti-CD 47 treatment can promote the macrophage in a tumor mouse model to be polarized to M1 subtype, reduce the expression of PD-1 in effector T cells of the tumor mouse model, increase the secretion of IFN-gamma, and reduce the number of immunosuppressive cells Tregs and MDSCs, thereby improving the tumor microenvironment and delaying the growth of tumor. However, the self-character of the anti-CD 47 single-chain antibody has limited its application and risks targeting normal cells, resulting in a cytotoxic response.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide an immune cell which targets an antigen and simultaneously secretes a CD47 antibody and application thereof.

The first aspect of the invention provides a chimeric antigen receptor, which 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 domain, a self-cleavage sequence, a second signal peptide and an anti-CD 47 single-chain antibody in series in sequence.

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 CD 19.

Further, the first signal peptide and the second signal peptide are selected from 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 a 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 a subunit, a CD8 β subunit, a CD8 δ subunit, a CD4 transmembrane domain, or a 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 co-stimulation signal domain is used for transmitting a stimulation signal generated after the extracellular antigen binding domain specific to the antigen binding fragment is combined with a target antigen to cause immune cell activation and immune response;

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.

In a second aspect, the present invention provides a gene encoding the chimeric antigen receptor.

In a third aspect, the present invention provides an expression vector for the gene encoding the chimeric antigen receptor.

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

In a fourth aspect, the invention provides a virus comprising the expression vector.

The fifth aspect of the present invention provides an immune cell targeting an antigen while secreting a CD47 antibody, comprising the gene encoding the chimeric antigen receptor or an expression vector of the gene encoding the chimeric antigen receptor;

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.

The sixth aspect of the invention provides 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.

The seventh aspect of the present invention provides a pharmaceutical composition comprising an expression vector of the gene encoding the chimeric antigen receptor or an immune cell wherein the targeted antigen simultaneously secretes the CD47 antibody.

The invention has the beneficial effects that:

1. according to the invention, a first nucleic acid for coding an antigen binding fragment (such as a CD19 single-chain antibody) and a second nucleic acid for coding an anti-CD 47 single-chain antibody are constructed on the same carrier, and through targeting an antigen and secreting a CD47 antibody, the immune cell modified by the chimeric antigen receptor can realize local delivery of the anti-CD 47 antibody, reduce the influence on normal cells, efficiently and specifically target tumor cells expressing the corresponding antigen, effectively antagonize immunosuppressive factors in a tumor immune microenvironment, block the anti-phagocytosis effect of the tumor cells, reduce tumor immune escape while retaining the anti-tumor activity of the immune cell modified by the chimeric antigen receptor, and improve the persistence and the anti-tumor activity of the immune cell.

2. The novel CAR-T can enhance the anti-tumor efficacy of CD19-CAR-T, promote the phagocytosis of tumor cells by macrophages, regulate the proportion of other immunosuppressive cells in a tumor microenvironment, retain the anti-tumor activity of CD19-CART, reduce tumor immune escape, improve the persistence and anti-tumor activity of CART cells, and further reduce the immune escape of refractory recurrent tumors to CAR-T cell therapy.

The application of the anti-CD 47 single-chain antibody is limited due to the self-characteristics of the anti-CD 47 single-chain antibody, and the risk of targeting normal cells exists, so that a cytotoxic reaction is caused.

Drawings

FIG. 1 shows a schematic of the structure of a chimeric antigen receptor.

FIG. 2 shows the vector design of the chimeric antigen receptors CD19-s47-CAR and CD 19-CAR.

FIG. 3 shows a statistical plot of the stable expression of anti-CD 19, anti-CD 47 fragments by RT-PCR of 293T cells transfected with CD19-CAR and CD19-s47-CAR virus.

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

FIG. 5 shows the tumor cell lines Raji, Romas, Daudi used in the present invention stably highly express CD47 detected by flow cytometry.

FIG. 6 shows that Raji, Romas, Daudi tumor cell lines used in the present invention stably bind to the secreted protein s47 as detected by flow cytometry.

Figure 7 shows that T cells transfected with CD19-s47-CAR stably expressed the secreted protein s47 by western blot detection.

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

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

FIG. 10 shows the expression of IL-2 and TNF- α in human peripheral blood lymphocytes transfected with vectors 19-CAR and CD19-s47-CAR after 5h of coculture with the tumor cell line Raji, as detected by flow cytometry.

FIG. 11 shows the promotion of phagocytic capacity of macrophages by the secreted protein s 47.

FIG. 12 shows the principle that CD19-s47-CAR blocks anti-phagocytosis of tumor cells.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1

This example provides a chimeric antigen receptor comprising a signal peptide, an antigen-binding fragment, a hinge region, a transmembrane region, an intracellular costimulatory signal region, an intracellular domain, a self-cleaving sequence, a signal peptide, and an anti-CD 47 single-chain antibody, all of which are sequentially linked in series.

In particular embodiments, the antigen recognized and bound by the antigen-binding fragment is a tumor-associated antigen; the tumor associated antigen may be selected from CD19, CD20, CD22, CD30, CD123, BCMA, or Her 2; in a preferred embodiment, the tumor associated antigen is CD19 and the antigen binding fragment is an anti-CD 19 single chain antibody (ScFv).

In specific embodiments, the signal peptide is selected from the group consisting of a CD8 a signal peptide, an IL-2 signal peptide, or a GM-CSF signal peptide; in a preferred embodiment, the first signal peptide is selected from the group consisting of the CD8 α signal peptide and the second signal peptide is selected from the group consisting of the GM-CSF signal peptide.

In particular embodiments, the hinge region is selected from a CD8 a hinge region or FcRIII a receptor; in a preferred embodiment, the hinge region is selected from the group consisting of CD8 a hinge regions.

In particular embodiments, the transmembrane region is selected from a T cell receptor subunit, a CD8 a subunit, a CD8 β subunit, a CD8 δ subunit, a CD4 transmembrane domain, or a CD28 transmembrane domain; in a preferred embodiment, the transmembrane region is selected from the group consisting of the CD8 alpha subunit.

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

In specific embodiments, the endodomain is selected from CD3 ζ.

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

FIG. 1 is a schematic diagram of the structure of a chimeric antigen receptor according to one preferred embodiment.

Example 2

In the embodiment, a preferred scheme is adopted for preparing the chimeric antigen receptor T cells, the HA tag is connected at the tail of a CD47 single-chain antibody for convenient detection, and the chimeric antigen receptor is marked as CD19-s47-CAR, and other tags such as 6 XHis, Fc, Myc, GST or Flag can be selected. Meanwhile, a chimeric antigen receptor not including a self-cleavage sequence, a signal peptide, and an anti-CD 47 single-chain antibody, which was designated as CD19-CAR, was set as a control. Chimeric antigen receptor CD19-s47-CAR and CD19-CAR vector design schemes are shown in FIG. 2. Wherein the sequence adopted is as follows:

CD8 α SP (nucleotide sequence):

ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC(SEQ ID NO.1)；

CD8 α SP (amino acid sequence):

MALPVTALLLPLALLLHAARP(SEQ ID NO.2)；

CD19 ScFv (nucleotide sequence):

GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.3)；

CD19 ScFv (amino acid sequence):

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO.4)；

CD8 Hinge (nucleotide sequence):

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAAGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(SEQ ID NO.5)；

CD8 Hinge (amino acid sequence):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.6)；

CD8 TM (nucleotide sequence):

ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGT(SEQ ID NO.7)；

CD8 TM (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)

CD3 ζ (nucleotide sequence):

CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG(SEQ ID NO.11)

CD3 ζ (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 the immune cell for targeting the antigen and simultaneously secreting the CD47 antibody comprises the following specific steps:

1. construction of Lentiviral vectors

After the chimeric antigen receptor nucleotide sequence is synthesized by a whole gene, the sequence is amplified by PCR, an amplified fragment is recovered by an Axygen gel recovery kit, and is subjected to homologous recombination and connection with a vector pCDH-EF1-MCS expression vector which is subjected to enzyme digestion by restriction enzymes BstBI and XbaI, so that a CD19-CAR single-target and a CD19-s47-CAR double-target expression vector are synthesized. Adding 5ul of the ligation product into 50ul of competent cell DH5 alpha, standing on ice for 30min, heat-shocking in a water bath at 42 ℃ for 90s, cooling on ice for 2min, adding 500ul of fresh non-resistant LB liquid medium, shaking a shaker at 37 ℃ at 150rpm/min for 45min, spreading on an LB solid plate containing ampicillin, and culturing overnight in an incubator at 37 ℃. After the monoclonal colonies grow out, 6 monoclonal colonies are picked, 500ul LB liquid culture medium containing ampicillin is added, after shaking culture is carried out in a shaking table at 37 ℃ for 4h, part of the colonies are sent to a company for sequencing, a sample with correct sequence comparison is selected according to a sequencing result, a CD19-CAR single target point and a CD19-s47-CAR double-target point recombinant expression vector are obtained, mass culture is carried out in the LB liquid culture medium, and then endotoxin-free plasmid extraction is carried out.

2. Preparation of lentivirus vector virus liquid

And (2) mixing the recombinant plasmid obtained in the step 1 with packaging plasmids pMD2.G and psPAX2 according to the ratio of 4:3:1 by using Lipofectamine 3000, transfecting 293T cells, replacing a fresh culture medium after 6h of transfection, collecting cell supernatants after 24h and 52h of culture respectively, mixing the cell supernatants with a virus concentrate according to the ratio of 1:4, incubating overnight at 4 ℃, centrifuging at a high speed to concentrate the virus solution, discarding the supernatant, and re-suspending the virus precipitate by using a PBS solution to obtain CD19-CAR and CD19-s47-CAR virus solutions.

3. Preparation of chimeric antigen receptor T cells

Fresh peripheral blood from healthy donors was taken, fresh peripheral blood mononuclear cells were separated by density gradient centrifugation and T cells were separated using CD3 positive sorting magnetic beads (purchased from Stem cells), resuspended in X-vivo serum-free medium (purchased from LONZA) and added with CD3/CD28 stimulating antibodies (purchased from Stem cells), IL-2, IL-7 and IL-15 at 37 deg.C, 5% CO₂After 24h of culture in an incubator, the virus solution obtained in the step 2 is added to perform transfection with MOI of 10, CD19-CART and CD19-s47-CART cells are obtained respectively, and the CAR-T cell transfection rate is detected after 3 days of transfection. T cells were always cultured at a density of 1X 10^ 6/ml. At the same time, 293T cells were transfected with MOI-1 virus solution,at 37 deg.C, 5% CO₂After 5 days of culture in an incubator, cells were harvested and tested for expression of the CAR fragment in virus-transfected 293T by RT-PCR, with the results shown in figure 3, CD19-293T cells stably expressed the CD19-CAR fragment, while CD19-s47-293T cells stably expressed anti-CD 19-CAR and anti-CD 47-CAR fragments.

Examples of the experiments

1. Detection of expression of CAR protein Using flow cytometry

Appropriate amounts of CD19-CART, CD19-s47-CART cells and control T cells obtained in example 2 were taken, washed with PBS for 2 times, resuspended in 50ul of PBS solution, 0.5ul of CAR-GREEN (purchased from Yake, Shanghai) was added, incubated at 4 ℃ in the dark for 30min, washed for 2 times, 200ul of PBS was added to resuspend the cells, and BD Cytoflex was used for detection, and the detection results are shown in FIG. 4. Control T cells were barely detectable of CAR molecule expression, with anti-CD 19 ScFv expression rates of 50.4% and 60.4% for CD19-CART and CD19-s47-CART cells, respectively.

2. Binding assay for secreted protein s47 to target cells

The cell supernatants of the CD19-CART, CD19-s47-CART cells and the control T cells obtained in example 2 were taken and harvested 5 days after infection, the cell supernatants of the CD19-CART, CD19-s47-CART cells and the control T cells were respectively used, the lymphoma cell strains Raji, Daudi and Romas (FIG. 5) with high expression of CD47 were washed with PBS for 2 times, then incubated with the supernatant at 37 ℃ for 30min, the supernatant was centrifuged and discarded, after washing with PBS for 2 times, the cell supernatants were respectively resuspended with 50ul PBS, 0.5ul of anti-HA-APC flow antibody (purchased from Biolegend) was added, the cell was washed for 2 times after incubation at room temperature for 25min, 200ul of PBS was added to resuspend the cells, and the detection results are shown in FIG. 6 by using a BD Cytoflex machine. The combination rate of three lymphoma cell strains Raji, Daudi and Romas and the exoproteins s47 is close to 100%.

3. Detection of expression quantity of secreted protein s47 by Western immunoblotting experiment

The cell supernatants of the CD19-CART, CD19-s47-CART cells and the control T cells obtained in example 2 are taken and harvested 5 days after infection, respectively, the cell supernatants of the CD19-CART, CD19-s47-CART cells and the control T cells are reserved, the proteins in the supernatant are precipitated by using a high-efficiency protein precipitation kit (purchased from Invent), the protein concentration is quantitatively measured by BCA, 5xloadingbuffer is added in proportion, 30ug of the proteins are loaded into SDS page wells after being heated at 100 ℃ for 10min, the protein samples are separated by electrophoresis at 120V for 60min after electrophoresis at 80V for 30min, then the gel transfer membrane is cut, the flows are balanced at 250mA, and the proteins on the gel are transferred to a PVDF membrane. After membrane transfer, the cells were sealed in 5% skimmed milk powder at room temperature for 2h, incubated overnight at 4 ℃ in a murine anti-HA monoclonal antibody (1:1000, purchased from Bioworld) and a murine anti- α -Turbulin monoclonal antibody (1:10000, purchased from Proteintetech), washed three times with PBST, and incubated with HRP-goat anti-mouse Fc (1:10000) at room temperature for 1 h. PBST was washed three times, developed using FDbio-Dura ECL luminophore, and photographed using a BioRad Imaging Syatem, and the results are shown in FIG. 7.

4. Detection of differentiation function of chimeric antigen receptor T cells

The CD19-CART, CD19-s47-CART cells and control T cells obtained in example 2 were taken and tested for T cell differentiation function 5 days after infection. CD19-CART, CD19-s47-CART cells and control T cells and target cells Raji were plated in 96-well plates at 37 ℃ with 5% CO at an effective target ratio of 1:1₂After 24h incubation in the incubator, the cells were harvested, washed 2 times with PBS, resuspended in 50ul PBS, 0.5ul CAR-GREEN, anti-CD 8-Percp. cy5.5, anti-CD 45RO-APC and anti-CCR 7-PE flow antibody (purchased from Biolegend) were added, the cells were washed 2 times after incubation for 25min in the dark at room temperature, 200ul PBS was added to resuspend the cells, and the cells were detected on a BD Cytoflex machine, as shown in FIG. 8, CD19-s47-CART cells all had a higher proportion of central memory T cells (Tcms) and a lower proportion of effector memory T cells (Tems) than CD 19-CART.

5. Cytotoxic killing experiment of chimeric antigen receptor T cells

CD19-CART, CD19-s47-CART cells and control T cells obtained in example 2 are taken, cells are taken 5 days after infection, target cell killing experiments are carried out, lymphoma cell strains Raji, Daudi and Romas are taken, the target cell density is adjusted to be 1.6 x 10^5/ml, 50ul are inoculated to a round bottom 96-well plate, T cells are inoculated according to the effective target ratio of 20:1,10:1,5:1 and 2.5:1, and a nonradioactive cytotoxicity detection kit (purchased from Promega) is used for cell killing effect detection, and specific operation refers to kit instructions, and the experiment results are shown in figure 9. The target cells Raji, Romas, Daudi and CD19-s47-CART cells have stronger tumor killing function than CD19-CART cells.

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

CD19-CART, CD19-s47-CART cells and control T cells obtained in example 2 were plated 5 days after infection with T cells and target cells at an effective target ratio of 1:1, while Brefeldin A was added at 37 ℃ with 5% CO₂After culturing for 5h in an incubator, collecting cells, performing anti-CD 8-Percp.cy5.5 flow antibody staining, washing for 2 times by PBS, performing fixed membrane rupture, staining by anti-IL-2-APC and anti-TNF-APC flow antibodies, incubating for 25min at room temperature in a dark place, washing for 2 times, adding 200ul PBS to resuspend the cells, and detecting by using a BD Cytoflex machine, wherein the detection result is shown in FIG. 10. CD19-s47-CART cells all have stronger IL-2 and TNF-alpha secretion ability than CD19-CART cells.

7. Flow cytometry for detecting influence of secreted protein s47 on phagocytic capacity of macrophages

Fresh peripheral blood from healthy donors was taken, mononuclear cells from fresh peripheral blood were isolated by density gradient centrifugation and mononuclear cells were isolated using CD14 positive sorting magnetic beads (purchased from Stem cells), supplemented with M-CSF (final concentration 25ng/ml) in 1640 medium containing 10% fetal bovine serum at 37 ℃ with 5% CO₂Mature macrophages can be obtained after 5 days of induction culture in an incubator. Counting target cells Raji after CFSE staining, simultaneously taking CD19-CART, CD19-s47-CART cells and control T cells obtained in example 2, plating macrophages, T cells and Raji according to the ratio of 1:2:2 after infecting for 5 days, collecting cells after culturing for 24h, washing the cells for 2 times by PBS, then carrying out flow antibody anti-CD 14-Percp cy5.5 staining, incubating for 25min at room temperature in a dark place, washing the cells for 2 times, adding 200ul PBS for resuspension of the cells, and detecting by using a BD Cytoflex machine, wherein the detection result is shown in FIG. 11. The CD19-s47-CART cell capable of secreting the CD47 single-chain antibody can obviously improve the phagocytosis capacity of macrophages on tumor cells.

FIG. 12 shows the principle that CD19-s47-CAR blocks anti-phagocytosis of tumor cells. When the CD19-s47-CART reaches tumor tissues, the CD19-s47-CART can bind to CD19 on tumor cells and release perforin, and meanwhile, an secreted anti-CD 47 single-chain antibody binds to CD47 on the tumor cells, so that the CD47 of the tumor cells is blocked from binding with SIRP alpha of macrophages, namely, the anti-phagocytosis pathway of the tumor cells is blocked, and the polarization direction of the macrophages is influenced. Meanwhile, it may have an influence on the number and functions of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and the like.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

SEQUENCE LISTING

<110> Zhujiang Hospital, Biochemical island laboratory of southern medical university

<120> immune cell for targeting antigen and simultaneously secreting CD47 antibody and application thereof

<130> CP121011105C

<160> 20

<170> PatentIn version 3.3

<210> 1

<211> 63

<212> DNA

<213> Artificial sequence

<400> 1

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccc 63

<210> 2

<211> 21

<212> PRT

<213> Artificial sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<400> 7

atctacattt gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc 60

actctttact gt 72

<210> 8

<211> 24

<212> PRT

<213> Artificial sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<400> 13

gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg cccc 54

<210> 14

<211> 18

<212> PRT

<213> Artificial sequence

<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 sequence

<400> 15

atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc t 51

<210> 16

<211> 17

<212> PRT

<213> Artificial sequence

<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 sequence

<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 sequence

<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 sequence

<400> 19

tacccatacg acgtaccaga ttacgct 27

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence

<400> 20

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

Claims

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;

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;

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 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.