CN113088540B - A GM-CSF knockdown T cell and its use - Google Patents

Abstract

The present invention relates to a chimeric antigen receptor expression vector and a chimeric antigen receptor T cell transfected with the expression vector, wherein the chimeric antigen receptor expression vector has a sequence encoding a chimeric antigen receptor and a sequence encoding a shRNA capable of knocking down human GM-CSF. The chimeric antigen receptor T cell has high efficiency in chimeric antigen receptor expression and tumor killing activity while knocking down the expression level of GM-CSF, and is expected to reduce side effects such as CRS generated during chimeric antigen receptor T cell therapy, and has broad application.

Description

GM-CSF knockdown T cell and application thereof

Technical Field

The invention relates to a genetically modified T cell, in particular to a chimeric antigen receptor T cell knocked down by GM-CSF and application thereof, belonging to the field of biological pharmacy.

Background

Chimeric antigen Receptor-T cell (CAR-T) therapy refers to an immunotherapeutic approach that is genetically engineered to specifically target T cells to kill tumor cells, and can be used to treat relapsed refractory malignancies in the blood system and other systems. The CAR-T cell therapy most widely studied and used in clinical practice is to treat a relapsed refractory CD19 ⁺ B cell malignancy with anti-CD 19-CART cells, the total remission rate can reach 50-90%, and some patients can achieve sustained disease remission. Kymriah from North is the first CAR-T treatment product available worldwide for the treatment of Acute Lymphoblastic Leukemia (ALL) and adult relapsed or refractory diffuse large B-cell lymphoma (DLBCL) in pediatric and adolescent patients.

However, some characteristic toxic and side effects which are distinct from those in traditional chemotherapy and small molecule targeted drug therapy appear in the CAR-T cell therapy, which greatly influences the popularization and application of CAR-T cell therapy. Two of the most common toxic side effects following CAR-T cell infusion include cytokine release syndrome (CRS, major symptoms are hyperthermia, hypotension, hypoxia and/or multi-organ toxicity) and CART cell-related encephalopathy syndrome (CRES, which is mainly manifested by confusion, delirium, occasional epileptic attacks and cerebral oedema). The incidence of these toxic side effects is between 37% and 93%, although controllable in most patients, few patients need intensive care therapy and few die. Despite extensive research on the mechanism of toxic and side effects of cell therapy, the complete mechanism and prevention of CRS/CRES remains a scientific challenge due to the complex network of actions involved between many immune cells and inflammatory factors. It is therefore necessary to trace back to the source of cytokine secretion to prevent CRS/CRES from occurring. The IL-6 receptor blocking drug tolizumab (Tocilizumab) is now routinely used clinically to control CRS and if ineffective or neurotoxic reactions occur, the use of glucocorticoids is recommended. However, the presence of toxic side effects inevitably increases the medication burden and hospitalization time of patients, so that some patients must receive intensive care and treatment, and even a few patients cause uncontrollable toxic reactions. In addition to IL-6, many clinical studies have found that inflammatory factors that are elevated in patient serum following infusion of CAR-T cells include sIL-6R, sIL R alpha, IFN-gamma, GM-CSF, MCP-1, IL-1 beta, and the like. It has been shown that CAR-T cells, after contact with tumor cells and activation, secrete pro-inflammatory cytokines such as GM-CSF and IFN- γ, which are then contacted with immune cells such as monocytes, thereby promoting the secretion of a large number of pathogenic inflammatory factors such as IL-6 and IL-1 by monocytes. It has been reported that CAR-T-induced myeloid cells secrete IL-6 and IL-1 as the main causes of CRS. Whereas GM-CSF secreted by CAR-T cells is the primary factor in myeloid cell activation.

There have been research teams that use TALEN or CRISPR/Cas9 techniques to knock out GM-CSF genes in CAR-T cells, thereby reducing CAR-T cell GM-CSF secretion (2018, sachdeva and Sterner). Their findings indicate that knocking out GM-CSF in CAR-T cells can prevent CRS from occurring while not compromising CAR-T cell function. However, the technical means they employ required either prior electrotransfection of mRNA encoding the GM-CSF TALEN arm in T cells, or additional transfection of LENTICRISPRV plasmid-packaged lentiviruses while preparing CAR-T cells. These additional procedures not only increase the difficulty and expense of preparation, but also impair the viability and proliferation of CAR-T cells. In addition, uncertainty in transfection efficiency also makes the efficiency of GM-CSF knockdown difficult to predict. Therefore, there is a need for a more convenient and efficient technique for preparing GM-CSF under-expressed CAR-T cells.

Disclosure of Invention

In combination with the shortcomings in the prior art, a novel GM-CSF knock-down type CAR expression vector is designed and constructed, and a short hairpin RNA (shRNA) expression frame for knocking down the GM-CSF is combined on the CAR expression vector to realize the co-expression of the CAR structure and the shRNA. Thus, the CAR is synchronously expressed and GM-CSF expression after the activation of the CAR-T cells is reduced without adding any drug load and additional preparation flow.

In particular, one aspect of the present invention provides a chimeric antigen receptor expression vector comprising

1) At least one protein coding sequence encoding a chimeric antigen receptor, and

2) At least one RNA coding sequence encoding an interfering RNA targeting human GM-CSF;

The interfering RNA is shRNA targeting the sequence shown as SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4. Preferably, the RNA coding sequence comprises the sequence shown as SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9. More preferably, the RNA coding sequence comprises the sequence set forth in SEQ ID NO. 9.

In a preferred embodiment, the chimeric antigen receptor targets CD19, and more preferably, the protein coding sequence comprises the sequence set forth in SEQ ID NO. 23.

In another preferred embodiment, the promoter of the interfering RNA is a U6 promoter, the promoter of the chimeric antigen receptor is an EF-1 alpha promoter, preferably, the expression vectors are pL-CAR+shRNA G1, pL-CAR+shRNA G2, pL-CAR+shRNA G3, pL-CAR+shRNA G4, and more preferably, the expression vectors are pL-CAR+shRNA G4.

In a preferred embodiment, the expression vector is a lentiviral vector or an adenoviral vector, preferably a lentiviral vector, more preferably an expression vector constructed based on a pWPXL plasmid.

In another aspect, the invention provides the use of an expression vector as described above for the preparation of a GM-CSF knockdown T cell into which the expression vector is transfected. Preferably, the transfected T cells express the chimeric antigen receptor at a high efficiency and the level of GM-CSF is significantly reduced compared to untransfected cells.

In a further aspect the invention provides a GM-CSF knockdown T cell transfected with an expression vector as described in any of the above. Preferably, the T cells are human peripheral blood mononuclear cells.

In a preferred embodiment, the expression efficiency of CD107a in the T cells is greater than 40%.

In a further aspect the invention provides the use of a T cell as described in any of the above for the preparation of a medicament for the treatment of cancer, preferably the cancer is leukemia, more preferably the cancer is chronic myelogenous leukemia or chronic lymphocytic leukemia.

Cell experiments prove that the expression vector provided by the invention can co-express the shRNA of the CAR structure and the countersunk GM-CSF through one vector, the tumor killing function of the GM-CSF knockdown type CAR-T cell obtained by modifying the expression vector is not damaged, and the secreted GM-CSF level is obviously reduced. The GM-CSF knockdown type CAR-T cell provided by the invention can prevent/reduce toxic and side effects related to cell therapy on the premise of not increasing clinical medication burden and additional preparation flow, so that the GM-CSF knockdown type CAR-T cell has high clinical application value.

Drawings

FIG. 1. Schematic representation of shRNA design of the present invention, showing the location of shRNA target sites on GM-CSF mRNA and the mechanism by which shRNA silences the GM-CSF gene.

FIG. 2 is a schematic representation of a CAR expression vector according to the present invention showing the construction positions of shRNAs and CARs in a pL-CAR+shRNA plasmid.

Figure 3. Knocking down GM-CSF expression did not decrease CAR-T cell CD107a expression.

Figure 4. Knocking down GM-CSF expression did not reduce CAR-T cell tumor cell killing.

FIG. 5. Efficient knockdown of GM-CSF expression following transfection of a CAR expression vector according to the invention.

Fig. 6. Different shrnas differ in efficiency of GM-CSF knockdown of CAR T cells.

FIG. 7 GM-CSF secretion levels from each group of cells after activation.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.

Chimeric antigen receptor (CHIMERIC ANTIGEN receptor, CAR)

Chimeric antigen receptors generally consist of an extracellular antigen recognition domain, a hinge region, a transmembrane region, a co-stimulatory domain and an activation domain. The extracellular antigen recognition domain specifically binds to a target molecule expressed on the surface of a tumor cell and then provides an activation signal to a T cell, and mainly comprises a single-chain variable region fragment (SINGLE CHAIN variable fragment, scFv), a single-domain antibody (Single domain antibody) or the like. The intracellular domain typically includes a CD3 zeta chain and one or more costimulatory domains, such as CD28 and/or 4-1BB (CD 137), to promote T cell activation. In classical CARs, scFv fragments of monoclonal antibodies that specifically recognize tumor antigens are implanted on T cells. Nucleic acids encoding the CAR can be introduced into T cells using, for example, lentiviral or retroviral vectors. In this way, a large number of T cells can be generated that specifically recognize a certain protein target.

Granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF)

GM-CSF is a cytokine derived from a variety of immune cells. Macrophages, T cells, mast cells, endothelial cells and fibroblasts all secrete GM-CSF. Functionally, GM-CSF can promote differentiation of myeloid stem cells into common progenitors of the granulocytes (including eosinophils, neutrophils), erythrocytes, megakaryocytes, and monocyte lineage, among others. Has stimulating effect on proliferation and differentiation of stem cells of marrow system to mature granulocytes of various types. The GM-CSF targeted by shRNA of the invention is human GM-CSF, geneBank number is M11220.1.

Cytokine Release Syndrome (CRS)

Cytokine release syndrome is a comprehensive clinical condition in which immune cells release inflammatory cytokines in large amounts in a short period of time, resulting in the production of cytokines by the body. The occurrence of this syndrome is often manifested by fever, hypotension, hypoxia, and the appearance of neurological symptoms. The occurrence of cytokine release syndromes is often associated with the action of certain cytokines.

T cell

T cells, also known as T lymphocytes (T lymphocytes), are composed of a population of heterogeneous lymphocytes, which are called T cells because of their differentiated maturation within the thymus. In the embryonic and primary stages of the human body, a portion of the pluripotent stem cells or pre-T cells in the bone marrow migrate to the thymus where they mature, forming immunocompetent T cells. T cells mature and circulate with blood to surrounding lymphoid organs, and colonize and proliferate in their respective predetermined areas. T cells differentiate and proliferate after being activated by antigen, generating effector cells, and exerting their immune function. Immune effector cells generally refer to immune cells that are involved in the immune response in clearing foreign antigens and performing effector functions. Lymphocytes are a variety of cell types that are produced and mature in the lymphatic system, with immune response functions, including T cells, B cells, natural killer cells (NK), macrophages, etc. The lymphocytes are further preferably T cells.

Sequence(s)

The term "sequence" as used herein is understood to include sequences that are substantially identical to the sequences of the present invention, and the term "substantially identical sequence" refers to a protein/nucleotide sequence that has at least 80%,83%,85%,86%,87%,88%,89%,90%,91%,92%, 93%,94%,95%,96%,97%,98%,99% or 100% identity, and the same or similar function, or that has the same or similar function as the sequence when optimally aligned, e.g., by using the GAP or BESTFIT program, using default GAP values.

Gene knockdown

Gene knockdown is a technique that uses small RNAs to efficiently and specifically bind and degrade mRNA contained in a cell that has a sequence homologous to the small RNA, thereby blocking expression of a target gene in the cell. Cells develop a target gene deletion phenotype after mRNA is degraded.

Interfering RNA

Interfering RNAs can generally include shRNA and siRNA, both of which can reduce protein expression at the RNA level. shRNA and siRNA lead to a decrease in mRNA of the target gene by a similar mechanism, the difference being that siRNA can be directly targeted to the target gene without processing, whereas shRNA comprises a neck ring structure, which is normally processed by intracellular proteins and transported into the cytoplasm after nuclear expression. In the cytoplasm, shRNA is combined with Dicer to remove a circular sequence, then is combined with RISC and removes one RNA chain, and finally target gene mRNA is identified, so that mRNA degradation is caused.

Human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMC) refer to cells with a single nucleus present in peripheral blood, including both lymphocyte and monocyte (monocyte) major classes.

Expression vector

The expression vector is a kind of vector which is added with expression elements such as a promoter, a terminator and the like on the basis of the basic skeleton of the DNA vector so that the target gene on the vector can be expressed, and one of the expression vectors is called plasmid. The chimeric antigen receptor expression vector of the invention can be a lentiviral expression vector or an adenovirus expression vector.

Lentiviral vector

Lentiviral vectors are a class of DNA vectors comprising lentiviral elements. Lentiviral vectors and lentiviral packaging plasmids, which are generally engineered from HIV-1, can be used together to make lentiviruses. The lentivirus carrying the target gene inserts the target gene into the target cell genome after infecting the target cell, thereby allowing the target cell to stably express the target gene. The expression vector of the present invention is preferably a lentiviral vector, more preferably an expression vector constructed based on a pWPXL plasmid.

Cancer of the human body

The term "cancer" refers to a broad class of diseases characterized by uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth results in the formation of malignant tumors or cells that invade adjacent tissues and can also metastasize to distal parts of the body through the lymphatic system or blood flow. Another equivalent description of "treating cancer" in the present invention is "treating a tumor" or "anti-cancer" or "anti-tumor".

Cancer is an uncontrolled condition of cell growth that impedes normal function of body organs and systems. A subject with cancer is a subject with objectively measurable cancer cells present in the body of the subject. Subjects at risk for developing cancer are subjects prone to develop cancer (e.g., based on family history, genetic predisposition), subjects exposed to radiation or other agents that cause cancer.

The T cells of the invention are useful for preventing or treating a variety of cancers. Examples of such cancers may include breast cancer, prostate cancer, lung cancer, ovarian cancer, cervical cancer, skin cancer, melanoma, colon cancer, stomach cancer, liver cancer, esophageal cancer, kidney cancer, throat cancer, thyroid cancer, pancreatic cancer, testicular cancer, brain cancer, bone cancer, and blood cancer (e.g., leukemia, chronic lymphocytic leukemia), and the like. Preferably, the GM-CSF knockdown T cells of the invention are useful for the treatment of chronic myeloid leukemia or chronic lymphocytic leukemia in humans.

The invention is further illustrated by the following examples. These examples are given for illustration only and are not intended to limit the scope of the invention in any way. The abbreviations are as follows, "h" means hours, "min" means minutes, "s" means seconds, "ms" means milliseconds, "d" means days, "μL" means microliters, "mL" means milliliters, "L" means liters, "bp" means base pairs, "mM" means millimoles, "μM" means micromolar.

EXAMPLE 1 construction of GM-CSF knock-down CAR expression vector

GM-CSF shRNA design

Useful shRNA sequence information targeting GM-CSF is looked up using shRNA design tools (www.sigmaaldrich.com). Sequence alignment was performed using BLAST tools (https:// BLAST. Ncbi. Nlm. Nih. Gov/BLAST. Cgi). The target site with the lowest off-target effect was selected, and 4 target sites (shRNA T1, shRNA T2, shRNA T3 and shRNA T4) were selected together, and 1 control target site (shRNA T0) was selected, and the shRNA target site sequences are shown in table 1. The stem-loop structure and the cleavage site (EcoR 1 and BamH 1) are respectively designed aiming at specific target sites to obtain oligonucleotide sequences of shRNAs, four pairs of shRNAs (shRNA G1, shRNA G2, shRNA G3 and shRNA G4) are designed in total, and a non-specific control shRNA (shRNA G0) is designed, wherein the specific shRNA sequences are shown in Table 2. The primers required for the synthesis of shRNA are shown in Table 3 (shRNA primers were synthesized by platinum Biotechnology (Shanghai) Inc.). The location of each shRNA target site on GM-CSF mRNA and the mechanism by which shRNA silences the GM-CSF gene are shown in FIG. 1.

TABLE 1 GM-CSF shRNA target site DNA sequence

Sequence numbering	Target site name	Target site DNA sequence
			SEQ ID NO:1	shRNA T1	GAGATGAATGAAACAGTAGAA
SEQ ID NO:2	shRNA T2	GAAGTCATCTCAGAAATGTTT
			SEQ ID NO:3	shRNA T3	GAAGGACTTTCTGCTTGTCAT
SEQ ID NO:4	shRNA T4	GGAGCTGCTCTCTCATGAAAC
			SEQ ID NO:5	shRNA T0	AGCGTGTAGCTAGCAGAGG

TABLE 2 DNA sequence listing of GM-CSF shRNA

TABLE 3 primer list

Construction of GM-CSF-shRNA expression cassette plasmid

1) The shRNA primer to be annealed was diluted to 50. Mu.M with triple distilled water. The following annealing reaction system is adopted, and various reagents are sequentially added and uniformly mixed.

Annealing reaction system:

the annealing reaction was performed by setting a PCR instrument under the conditions of 95℃for 2 minutes, 0.1℃drop every 8 seconds, 25℃drop, and 4℃hold for a long period of time. The annealed product was diluted 100-fold for use.

2) Plvx-shRNA1 (Clontech) plasmid was digested with EcoRI (Thermo Scientific, # 00565402) and BamHI (Thermo Scientific, # 00566125), loaded onto a 1% agarose gel, electrophoresed at 130V for 30 min, and digested product bands were recovered by digestion to obtain purified digested product. The purified cleavage product was ligated to the annealed product in the following manner:

add enzyme-free water to 20. Mu.l and place in a 24℃water bath for 1 hour.

After ligation was completed, the ligation product was transformed into DH5a competent cells (Tiangen Biochemical technology, # CB 101), 200. Mu.l of the transformation product was plated on LB agar plates with ampicillin resistance, after 37℃overnight, the monoclonal was picked up and shaken and sent to platinum Biotechnology (Shanghai) Inc. for sequencing, and plasmids containing GM-CSF-shRNA expression cassettes were successfully constructed and used for subsequent experiments, with the plasmids numbered Plvx-shRNA1-G1, plvx-shRNA1-G2, plvx-shRNA1-G3, plvx-shRNA1-G4 and Plvx-shRNA1-G0, respectively.

Construction of GM-CSF-shRNA and CAR Co-expression lentiviral vector

The shRNA expression frame is obtained by PCR amplification of U6 promoter-shRNA region of Plvx-shRNA1-G0 (target sequence: SEQ ID NO: 5) and Plvx-shRNA1-G4 plasmid (target sequence: SEQ ID NO: 4), and the amplification primers are SalI-F (SEQ ID NO: 21) and SalI-R (SEQ ID NO: 22) and brought into SalI cleavage site.

AntiCD the antiCD-CAR gene was biosynthesized from gold and cloned into pUC57 vector (gold and silver biotechnology), antiCD-CAR DNA sequence and protein sequence are shown in Table 4 (SEQ ID NO:23 and SEQ ID NO: 24). When the gene is synthesized, the enzyme cutting sites BamH1 and Nde1 are added at two ends of the gene. antiCD19 the 19-CAR gene fragment was digested with a restriction enzyme and ligated into an engineered lentiviral vector (pWPXL, addgene: 12257), and a CAR lentiviral vector was prepared and designated pL-CAR. The constructed plasmid was then digested with SalI (Thermo Scientific, # ER 1021) and the digested product was purified, and the PCR amplified shRNA expression cassette was ligated into pL-CAR. The linking and transformation operations were performed as described above in the experimental procedure to obtain lentiviral vector plasmids with both CAR expression cassettes and GM-CSF shRNA or control shRNA expression cassettes, designated pL-car+shrna G1, pL-car+shrna G2, pL-car+shrna G3, pL-car+shrna G4 and pL-car+shrna G0, respectively. The large plasmids were prepared using QIAGEN HISPEED PLASMID Maxi Kit (# 12662) following the protocol provided by the Kit for subsequent CAR-T cell preparation.

Table 4, antiCD-CAR sequence Listing

Example 2 different shRNAs were shown to be less efficient at GM-CSF knockdown in K562 cells

2.1 Preparation of lentiviruses comprising shRNA

293T cells were seeded one day in advance by seeding cells into 6-well plates at about 1X 10 ⁶/well (4 ml DMEM complete medium). Plasmid transfection was performed until the cell density reached 90-95%. The original culture solution was removed, and replaced with the serum-reduced OPTI-MEM culture solution.

The plasmid complexes were first prepared by adding the following plasmids to 375ul Opti-MEM (Thermo FISHER SCIENTIFIC; 31985-070) and mixing after addition. psPAX2 plasmid (Addgene; cat# 12260) 2.8125ug, pMD2.G plasmid (Addgene; cat# 12259) 0.9375ug, lentiviral vector plasmids (Plvx-shRNA 1-G1, plvx-shRNA 1-G2, plvx-shRNA1-G3, plvx-shRNA1-G4 and Plvx-shRNA 1-G0) 4ug, P3000 15ul.

The transfection reagent complex was prepared by adding 22.5ul Lipofectamine 3000 (Invitrogen; 11668-019) to 375ul Opti-MEM, mixing well after addition, and standing at room temperature for 5min.

Adding the plasmid complex into the transfection reagent complex, mixing, standing for 25min, and adding the transfection complex into cell culture medium, and shaking gently. Transfer to a 37 ℃ cell incubator with 5% co ₂. After 48 hours from the completion of transfection, the supernatant was collected, centrifuged at 3000rpm for 10min to remove cell debris, and filtered through a 0.45um filter. The filtrate was transferred to a special centrifuge tube, trimmed, and ultracentrifuged using an ultracentrifuge 20000rpm for 2-3 hours. The supernatant was decanted, and the lentivirus was resuspended using serum-free medium and stored at-80 ℃ for later use after sub-packaging. Lentiviruses containing shRNA G0, shRNA G1, shRNA G2, shRNA G3 and shRNA G1 were prepared according to this procedure, respectively.

2.2 Knock-out of GM-CSF in K562 cells

K562 cells (human chronic myelogenous leukemia cells) with good growth state are inoculated into 12-well plates according to 5X 10 ⁵/well and divided into six groups of untransfected cells, shRNA1-G1, shRNA1-G2, shRNA1-G3, shRNA1-G4 and shRNA 1-G0, and 600ul of corresponding lentiviral supernatant, 400ul of RPMI 1640 complete medium and polybrene (see section, #40804ES 76) are added to each group, and the mixture is centrifuged for 1.5 hours at 32 ℃. After 6 hours, the complete culture medium is added by centrifugation, after 48 hours, the transfection efficiency is judged by detecting GFP expression of the cells by flow cytometry, and after 72 hours, the cell extract protein is collected.

Extracted protein and Western blotting experiment

1. Protein extraction, namely selecting 1X protein lysis and loading buffer solution (New Saimei, # WB 2010), adding protease inhibitor mixed solution (New Saimei, # P001) before using, centrifuging and collecting cells, and washing with PBS for 1 time. Cells collected in 12-well plates were added to the lysate at a ratio of 100 ul/well and shaken on a vortex shaker until no significant cell pellet was present to lyse the cells. Heating in boiling water bath at 100deg.C for 5-10 min, centrifuging at 12000rpm at 4deg.C for 15 min, collecting supernatant, packaging, and preserving at-80deg.C.

2. Electrophoresis Each pack of Tris-MOPS-SDS running buffer powder (gold Style, # M00138) was dissolved in 1L of deionized water to prepare 1 Xrunning buffer. Taking out a proper volume of protein sample, heating in 100 ℃ water bath for 3-5 minutes to enable the protein to be fully denatured, and loading 10-25ul of protein sample into ExpressPlus ^TM prefabricated glue (gold Style, # M81615C) holes after cooling to room temperature. Electrophoresis was performed at 140V for 50 minutes until the bromophenol blue band had run to the bottom of the gel.

3. Transfer membrane 1000mL of fast transfer buffer (1X) 100mL of fast transfer buffer (10X) +750 mL deionized water+150 mL of methanol was prepared. Taking out the gel after electrophoresis, cutting the gel according to the positions of the protein marker and the strip, and placing the gel on filter paper soaked in the transfer membrane liquid for standby. Cutting PVDF film according to the size of the glue, pouring about 5ml PVDF film infiltration activating solution (Biyun Tian, # P0021S) into a plate, clamping the PVDF film by using flat-head forceps, enabling the film to be infiltrated into uniform and semitransparent state, after 10 seconds of wetting in film transfer solution, assembling a film transfer sandwich, pouring the film transfer solution to a marking line, and placing a film transfer groove into ice water bath. The film was transferred at 100V for 50min.

4. After completion of transfer, blotting membranes were washed in 1 XTBE for 1-2 min, and about 10ml QuickBlock ^TM Western-sealing solution (Biyun, #P0252) was poured into the dishes and blocked for about 20 min on a horizontal shaker. Anti-GM-CSF antibody (Abcam, ab 9741) was diluted 1:1000 with QuickBlock ^TM Western primary Anti-dilution (Biyun, # P0256) and shaken overnight at 4 ℃.

5. Washing the membrane 3 times in 1 XTBE for 10min each time, applying QuickBlock ^TM Western secondary antibody dilution (Biyun day, # P0258) to dilute the secondary antibody, washing 3 times in 1 XTBE for 10min each time, dripping onto PVDF membrane using a hypersensitive ECL chemiluminescence kit (Biyun day, # P0018S), and developing with LAS-4000 type chemiluminescent instrument.

The development results show (figure 3), after 72 hours of virus transfection (transfection efficiency greater than 90%), the protein expression level of GM-CSF in K562 cell line was detected by Western blotting experiment, and shRNA G0 group was used as a control to exclude the interference of transfection, off-target effect, etc. on GM-CSF expression. The results show that the GM-CSF expression of shRNA G0 and shRNA G1 groups is not obviously reduced, and the GM-CSF expression of shRNA G2, shRNA G3 and shRNA G4 groups are obviously reduced, wherein the knocking-down effect of shRNA G4 is most obvious. According to the research requirement, shRNA G4 is selected to construct a GM-CSF knock-down type CAR expression vector.

Example 3 preparation of GM-CSF knock-down CAR lentivirus

293T cells were seeded one day in advance by seeding cells into 6-well plates at about 1X 10 ⁶/well (4 ml DMEM complete medium). Plasmid transfection was performed until the cell density reached 90-95%. The original culture solution was removed, and replaced with the serum-reduced OPTI-MEM culture solution.

The plasmid complexes were first prepared by adding the following plasmids to 375ul Opti-MEM (Thermo FISHER SCIENTIFIC; 31985-070) and mixing after addition. psPAX2 plasmid (Addgene; cat# 12260) 2.8125ug, pMD2.G plasmid (Addgene; cat# 12259) 0.9375ug, 4ug of lentiviral vector plasmid (pL-CAR+shRNA G0/1/2/3/4), P3000 ul.

The transfection reagent complex was prepared by adding 22.5ul Lipofectamine 3000 (Invitrogen; 11668-019) to 375ul Opti-MEM, mixing well after addition, and standing at room temperature for 5min.

Adding the plasmid complex into the transfection reagent complex, mixing, standing for 25min, and adding the transfection complex into cell culture medium, and shaking gently. Transfer to a 37 ℃ cell incubator with 5% co ₂. After 48 hours from the completion of transfection, the supernatant was collected, centrifuged at 3000rpm for 10min to remove cell debris, and filtered through a 0.45um filter. The filtrate was transferred to a special centrifuge tube, trimmed, and ultracentrifuged using an ultracentrifuge 20000rpm for 2-3 hours. The supernatant was decanted, and the lentivirus was resuspended using serum-free medium and stored at-80 ℃ for later use after sub-packaging. Lentiviruses containing CAR, car+shrna G0, car+shrna G1, car+shrna G2, car+shrna G3 and car+shrna G1 were prepared according to this procedure, respectively.

Example 4 preparation of GM-CSF knockdown CAR-T cells

Taking human peripheral blood mononuclear cells frozen in liquid nitrogen (Miaotong biotechnology), recovering and culturing in PRIME-In cell CDM (Irvine, # 91154) medium, the medium was supplemented with an additional 100IU/ml IL-2 (Stemcell technologies). Cells were stimulated by addition of equal proportions of anti-CD 3/CD28 magnetic beads (Gibco, # 1132D) and lentiviral transfection was performed 24 hours later. Three groups of CAR, car+ shRNAG0 and car+ shRNAG4 were assigned and untransfected T cells were set as controls. After 72 hours of lentivirus addition, a volume of cells was taken and incubated for 30 minutes at 4℃with fluorescent labelled CD19 protein (Acrobiosystems). After incubation was completed, the cells were washed once with PBS. Cells were then resuspended using PBS and CAR expression of the cells was detected by flow cytometry. Obtaining a CAR T cell, a car+ shRNAG T cell, and a car+ shRNA G4T cell

The results show that the CAR expression efficiencies of the CAR T cells, the CAR+shRNA G0T cells and the CAR+shRNA G4T cells are respectively 42.5%, 44% and 43.6%, and the three groups of cells reach about 40%. Thus, expression of shRNA did not affect the expression efficiency of CAR (see fig. 4).

Example 5 GM-CSF knockdown CAR-T cell killing ability assay

CD107a expression detection

T cells and tumor cells were labeled with anti-CD 3 (BD Biosciences; 555335) and CD19 antibodies (ebiosciences, 11-0199-42) after incubation for 4 hours at 37℃with 2X 10 ⁵ CAR-T cells and 4X 10 ⁵ Nalm-6 cells (acute human B lymphowhite blood cells, china academy of sciences cell bank), 15. Mu.l CD107a-PE antibody (BD Biosciences; 555801) and BD GolgiSF (BD Biosciences; 554724) diluted in a 1:30,000 ratio, respectively, per well in 24 well plates. The amount of CD107a expression in T cells was detected using a flow cytometer. The results showed that the CD107a expression amount of car+shrnh4t cells was 57.2%, whereas the CD107a expression efficiency of CAR T cells and car+ shRNAG T cells was about 40%. Thus, knocking down GM-CSF expression did not decrease CAR-T cell CD107a expression (see figure 5). Since CD107a is an important indicator of T cell and NK cell activation and killing, it is believed that knockdown of GM-CSF expression does not impair the killing activity of CAR-T cells.

2. Killing experiment

After co-culturing CAR-T cells and Nalm-6 cells carrying luciferase-GFP for 12 hours according to the effective target ratio of 1:1, 0.5:1 and 0.25:1, D-luciferin (Thermo Scientific, 8829) is added as a luminescent substrate to detect bioluminescence. The conversion formula for calculating the killing efficiency is that the killing efficiency= (experimental hole fluorescence value-blank control fluorescence value)/target cell control hole fluorescence value multiplied by 100%. The results show that the killing efficiency of the CAR T cells, the CAR+ shRNAG T cells and the CAR+shRNA G4T cells reaches about 80% when the effective target ratio is 1:1 (see figure 6). Thus, knockdown of GM-CSF expression had no significant effect on CAR T cell killing tumor cell efficacy.

Example 6 detection of GM-CSF knockdown of CAR T cells GM-CSF secretion

The experiments were divided into three groups of CAR T cells, car+ shRNAG 0T cells and car+shrna G4T cells. The supernatants were collected after adding 4X 10 ⁵ CAR-T cells and 4X 10 ⁵ Nalm-6 cells (CD19+), per well in 24 well plates, incubating for 16 hours in a 37℃incubator containing 5% CO 2. The expression level of GM-CSF in the supernatant was measured using a Human GM-CSF ELISA kit (Shenzhen Daidae, # 1117302) according to the experimental procedure provided by the kit. The results show that the GM-CSF levels secreted by car+shrnh4t cells after activation were significantly lower than that of CAR T cells and car+ shRNAG T cells (see fig. 7). The above results demonstrate that we prepared CAR-T cells with knockdown GM-CSF, which did not affect the tumor killing activity of CAR-T.

Although specific embodiments of the invention have been described above for illustrative purposes, those skilled in the art will appreciate that many changes in detail can be made without departing from the invention as described in the claims.

Sequence listing

<110> Shanghai transportation university medical college affiliated Ruijin Hospital

<120> A GM-CSF knockdown T cell and uses thereof

<130> P2020-0026

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 1

gagatgaatg aaacagtaga a 21

<210> 2

<211> 21

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 2

gaagtcatct cagaaatgtt t 21

<210> 3

<211> 21

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 3

gaaggacttt ctgcttgtca t 21

<210> 4

<211> 21

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 4

ggagctgctc tctcatgaaa c 21

<210> 5

<211> 19

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 5

agcgtgtagc tagcagagg 19

<210> 6

<211> 57

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

gagatgaatg aaacagtaga attcaagaga ttctactgtt tcattcatct ctttttt 57

<210> 7

<211> 57

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

gaagtcatct cagaaatgtt tttcaagaga aaacatttct gagatgactt ctttttt 57

<210> 8

<211> 57

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

gaaggacttt ctgcttgtca tttcaagaga atgacaagca gaaagtcctt ctttttt 57

<210> 9

<211> 57

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

ggagctgctc tctcatgaaa cttcaagaga gtttcatgag agagcagctc ctttttt 57

<210> 10

<211> 59

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

agcgtgtagc tagcagaggt tcaagagacc tctgctagct acacgctttt tttacgcgt 59

<210> 11

<211> 63

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

gatccgagat gaatgaaaca gtagaattca agagattcta ctgtttcatt catctctttt 60

ttg 63

<210> 12

<211> 63

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

aattcaaaaa agagatgaat gaaacagtag aatctcttga attctactgt ttcattcatc 60

tcg 63

<210> 13

<211> 63

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

gatccgaagt catctcagaa atgtttttca agagaaaaca tttctgagat gacttctttt 60

ttg 63

<210> 14

<211> 63

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 14

aattcaaaaa agaagtcatc tcagaaatgt tttctcttga aaaacatttc tgagatgact 60

tcg 63

<210> 15

<211> 63

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 15

gatccgaagg actttctgct tgtcatttca agagaatgac aagcagaaag tccttctttt 60

ttg 63

<210> 16

<211> 63

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

aattcaaaaa agaaggactt tctgcttgtc attctcttga aatgacaagc agaaagtcct 60

tcg 63

<210> 17

<211> 63

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

gatccggagc tgctctctca tgaaacttca agagagtttc atgagagagc agctcctttt 60

ttg 63

<210> 18

<211> 63

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

aattcaaaaa aggagctgct ctctcatgaa actctcttga agtttcatga gagagcagct 60

ccg 63

<210> 19

<211> 65

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 19

gatccagcgt gtagctagca gaggttcaag agacctctgc tagctacacg ctttttttac 60

gcgtg 65

<210> 20

<211> 65

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 20

aattcacgcg taaaaaaagc gtgtagctag cagaggtctc ttgaacctct gctagctaca 60

cgctg 65

<210> 21

<211> 34

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 21

atgtcgactc gggtttatta cagggacagc agag 34

<210> 22

<211> 33

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 22

atcagctgga actccatata tgggctatga act 33

<210> 23

<211> 1458

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 23

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgc 1458

<210> 24

<211> 486

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 24

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 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

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

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

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

85 90 95

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

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

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

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

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

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

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

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

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

340 345 350

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

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

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

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

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

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

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

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

Claims (7)

1. A chimeric antigen receptor expression vector comprising

1) A protein coding sequence encoding a chimeric antigen receptor that targets CD19 and which is shown in SEQ ID NO. 23, and

2) An RNA coding sequence encoding an interfering RNA targeting human GM-CSF;

The interfering RNA is shRNA of a sequence shown in a targeted SEQ ID NO. 4, and the RNA coding sequence is shown in a SEQ ID NO. 9.

2. The expression vector of claim 1, wherein the promoter of the interfering RNA is a U6 promoter and the promoter of the chimeric antigen receptor is an EF-1 a promoter.

3. The expression vector of claim 1, wherein the vector is a lentiviral vector.

4. Use of an expression vector according to any one of claims 1 to 3 for the preparation of GM-CSF knockdown T cells, characterized in that the expression vector is transfected into the T cells.

5. A GM-CSF knockdown T cell transfected with the expression vector of any one of claims 1-3.

6. The T cell of claim 5, wherein the T cell is a human peripheral blood mononuclear cell.

7. Use of T cells according to claim 5 or 6 for the preparation of a medicament for the treatment of leukemia.