CN114958765A - CAR-NK cell with function of targeted removal of IL1RAP expression and preparation and application thereof - Google Patents

Abstract

The invention discloses a CAR-NK cell with the effect of eliminating IL1RAP expression in a targeted manner and preparation and application thereof, wherein the gene sequence of the CAR-NK cell is shown in NO.1, the cell has the effect of eliminating CML-LSC of IL1RAP in a targeted manner, can be applied to adoptive cell immunotherapy and can eliminate tiny residual lesion of CML leukemia, so that a patient can be stopped and cured clinically without side effects or worrying about HLA barrier problems, and the CAR-NK cell has the characteristics of obvious treatment effect, safe treatment process and remarkable anti-tumor effect.

Description

CAR-NK cell with function of targeted removal of IL1RAP expression and preparation and application thereof

Technical Field

The invention relates to the technical field of biotechnology, and particularly relates to CAR-NK cells with an effect of targeted removal of IL1RAP expression, and preparation and application thereof.

Background

Chronic Myelogenous Leukemia (CML) is a Chronic disease in which a p210BCR-ABL1 oncoprotein is expressed via a BCR-ABL1 fusion transcript, leading to abnormal expansion of myeloid lineage cells; tyrosine Kinase Inhibitors (TKIs) significantly improve the survival rate of CML patients, and deep responders may consider stopping treatment; however, detection of the BCR-ABL1 breakpoint fusion gene by remote or reverse PCR and cell division studies revealed that after TKI treatment, the quiescent naive CML stem cell compartment persisted by remaining insensitive, presenting a source of recurrence; and more than 50% of patients relapse and re-initiate TKI treatment with subsequent appearance of unknown toxicity; targeted therapies from radiation and chemotherapy to TKIs and IFN- α -related allogeneic stem cell transplantation, if not cured, can lead to persistent, if not permanent, disease remission, although leading to treatment-related mortality, to the extent that most patients have a normal life expectancy; therefore, while allo-SCT is avoided as much as possible, a new method for eliminating the durable TKIs-resistant resting CML precursor cells needs to be explored; in addition to allo-SCT, which is well known for its graft versus leukemia effect, numerous other features also suggest that CML is an immune-sensitive disease; these features include immune surveillance avoidance following down-regulation of MHC-II expression by CML cells, BCR-ABL fusion domain peptides eliciting CML-specific T cell responses, potential for autologous dendritic cell vaccination, Natural Killer (NK) cell effects, anti-BCR-ABL effects by T-helper or cytotoxic T lymphocytes, and restoration of immune control associated with programmed cell death 1 inhibition in deep molecular response CML patients;

besides a plurality of methods such as cytokine therapy, gene therapy, molecular targeted therapy, antibody therapy and the like, the biological immunotherapy has particularly good effect and application prospect of the adoptive immunotherapy of immune cells; the immune cell adoptive therapy mainly comprises lymphokine-activated Killer cells (LAK), tumor-infiltrating lymphocytes (TIL), Cytotoxic T Lymphocytes (CTL), Natural Killer cells (NK) and the like, which play a very important role in killing tumor cells; the Chimeric Antigen Receptor (CAR) is a fusion protein consisting of an extracellular antigen recognition region, a transmembrane region, and an intracellular signaling region; as the name implies, extracellular antigen recognition regions can recognize specific antigens on tumor cells, usually single chain variable fragments (scFv); intracellular signaling domains, such as CD28, 4-1BB (CD137), OX40, and CD3 ζ, are typically designed to enhance T cell activation and killing; CAR-modified T cells can directly recognize tumor-associated antigens (TAAs) and kill tumor cells; CAR-T cell therapy has enjoyed great success in hematological tumors such as Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL) and lymphoma; notably, CD19CAR-T treatment was reported to show a Complete Remission (CR) rate of 90% in children and adults with all diseases; although the rapid development of CAR-modified T cells has achieved tremendous success in the treatment of malignancies, particularly hematologic malignancies, there are still some problems in clinical applications:

(1) CAR-T cell therapy is not very effective in the treatment of solid tumors;

(2) furthermore, most CAR-T cell therapies require autologous adoptive cell transplantation, as allogeneic T cells may cause Graft Versus Host Disease (GVHD) unless the HLA barrier is addressed;

(3) in addition, CAR-T cell therapy may also produce life threatening side effects in patients, such as Cytokine Release Syndrome (CRS);

CAR-modified NK cells have been shown to overcome the above-mentioned disadvantages of CAR-T cells, exerting a significant antitumor effect; at present, preclinical and clinical studies indicate that CAR-NK cell therapy may have significant anti-tumor effects and is safer than CAR-T cell therapy; however, CAR-NK cell therapy still faces challenges of primary NK cell expansion and activation in vitro, difficult storage and transport of NK cell products, low transduction efficiency, etc.; therefore, optimization of CAR-NK cell therapy is still in need of further investigation; the construction of better CAR-NK cells has important significance for improving the treatment effect, and the combined treatment provides a new direction for immunotherapy based on NK cells.

Disclosure of Invention

Aiming at the existing problems, the CAR-NK cell can remove CML-LSC expressing IL1RAP in a targeting manner, can remove minimal residual lesion of CML leukemia, and has the characteristics of obvious treatment effect, safe treatment process and remarkable anti-tumor effect.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the CAR-NK cell has the function of targeted clearing of IL1RAP expression, is the IL1RAP-CAR cell, has a nucleic acid sequence shown as SEQ ID NO.1 and a protein sequence shown as SEQ ID NO. 2, and is used for treating the diseases of the human body and the human body.

A method for preparing CAR-NK cells with effect of targeted clearing of IL1RAP expression comprises the steps

S1, constructing a recombinant plasmid vector for stably expressing IL1RAP-CAR and packaging GMP (good manufacturing practice) lentivirus

S101, designing an scFv nucleic acid chain by using a monoclonal antibody targeting a human IL-1RAP molecule according to the sequence of 'signal peptide-heavy chain variable region- (G4S) 3-light chain variable region-IgG 1';

s102, amplifying an scFv nucleic acid chain, and performing homologous recombination on an scFv target sequence synthesized by amplification to construct a recombinant plasmid vector completely containing the scFv target sequence;

s103, stably transforming the constructed recombinant plasmid vector in a competent cell stal-3, and storing the recombinant plasmid vector in a glycerol strain state;

s104, carrying out shake bacteria amplification on the plasmid competent cells after homologous recombination, and obtaining virus packaging plasmids with large quality particles meeting GMP requirements;

s2, performing NK cell and human Peripheral Blood Mononuclear Cell (PBMC) derived NK cell infection on the prepared lentivirus, preparing CAR-NK cells expressing IL1RAP-CAR and establishing a CAR-NK cell lentivirus infection system.

Preferably, the design process of the scFv nucleic acid strand in step S101 comprises:

(1) connecting the parts of the IL1RAP sequence according to the sequence of 'signal peptide-heavy chain variable region- (G4S) 3-light chain variable region-IgG 1' to obtain the complete sequence of the scFv protein;

(2) after obtaining the complete sequence of the scFv protein, calculating the physicochemical properties of the molecular weight, isoelectric point, hydrophily and lipophilicity, half-life period and stability of the scFv molecule;

(3) and (3) constructing a protein three-dimensional structure of the scFv protein, and checking the feasibility and the integrity of scFv sequence design to obtain an integral and satisfactory scFv molecule.

Preferably, the amplification process of the scFv nucleic acid strand nucleic acid molecule in step S102 comprises:

(1) according to codon preference, mRNA secondary structure, expression influencing fractal action elements, restriction enzyme site optimization and GC content of scFv molecules, codon optimization is carried out on scFv, an AA sequence is converted into a DNA sequence, and DNA synthesis is carried out;

(2) preparing a linearized vector: carrying out enzyme digestion reaction by XbaI and NotI restriction enzymes to obtain a linearized vector;

(3) obtaining of the inserted target fragment: taking a 20bp sequence at the tail end of the linearized vector as a homologous sequence, and respectively adding the homologous sequence to the 5' end of a gene specificity forward/reverse amplification primer sequence to obtain an insert with the homologous sequence by the amplification of the pair of primers;

(4) and (3) recombination reaction: the linearized vector and the insert are mixed in proportion, and the recombination reaction can be completed by reacting for 5min at 50 ℃ under the catalysis of Exnase, so that the in-vitro cyclization of two linearized DNAs is realized.

Preferably, the transformation and preservation process of the IL1RAP-scFv-CAR recombinant plasmid vector in the competent cell stal-3 in the step S103 comprises the following steps:

(1) putting 50 mu L of Stbl-3 competent cells on ice, adding the ligation product into the competent cell suspension, gently dialing the mixture by hands for several times, and standing the mixture on ice for 30 min; rapidly transferring the competent cells to 42 ℃ water bath, thermally shocking for 45s, and then rapidly transferring to ice for cooling for 5 min;

(2) adding 500 μ L of liquid LB culture medium recovered to room temperature, shaking bacteria at 37 deg.C and 220rpm for 1h for amplification; taking 50-100 mu L of bacterial liquid, coating the bacterial liquid on a solid LB plate containing 20 mu g/mL ampicillin according to a scribing method, and culturing overnight at 37 ℃;

(3) observing the growth condition of colonies on the plate, selecting a single colony, inoculating the single colony into 10mL of liquid LB culture medium containing 20 mu g/mL of ampicillin, and shaking the bacteria overnight;

(4) extracting plasmids by using a plasmid small-extraction medium-amount kit, and carrying out DNA sequencing to identify whether the plasmids are constructed successfully;

(5) for the plasmid with correct identification, 20% of sterile glycerol is added into the bacterial liquid, and the mixture is frozen and stored at minus 80 ℃ after being uniformly mixed, and is reserved as a glycerol strain.

Preferably, the process for preparing GMP viral packaging plasmid of IL1RAP-scFv-CAR recombinant vector of step S104 comprises:

(1) selecting a small amount of glycerol bacteria for identifying the correct plasmid, adding the glycerol bacteria into 100mL of liquid LB culture medium containing 20 mu g/mL of ampicillin, shaking the bacteria at 37 ℃, and culturing overnight;

(2) centrifuging at 3000rpm at room temperature for 10min, collecting bacterial precipitate, adding 10mLP1 solution according to kit instructions, fully blowing and mixing the bacterial precipitate uniformly, and then adding 10mL of lysate P2;

(3) standing at room temperature for 5min, adding 10mL of neutralizing solution P4, immediately and gently turning up and down for 6-8 times, and fully and uniformly mixing to obtain white flocculent precipitate;

(4) centrifuging at 8000rpm for 10min to separate white flocculent precipitate to the bottom of the tube, collecting supernatant to clean centrifuge tube; adding 3mL of red endotoxin removing solution ER, and turning over and mixing uniformly to obtain a uniform and transparent yellow solution;

(5) adding isopropanol with the volume of 0.3 time to precipitate out DNA, reversing and mixing uniformly, and transferring the solution to a DNA adsorption silica gel column; centrifuging at 8000rpm for 2min, discarding filtrate, and adsorbing plasmid onto silica gel column; eluting the column with protein eluent and salt eluent twice, and finally eluting with sterile ultrapure water to obtain plasmid water solution;

(6) the concentration and purity of the plasmid solution are detected by using Nanodrop 2000, and the plasmid solution is sent out to package viruses at proper concentration.

Preferably, the CAR-NK cell preparation process of step S2 includes:

day 1: seeding cells, and selecting proper cell density for plating;

Day2：viral transduction, using MOI10 for viral transduction, and Lenti ^BOOST P is added to the total volume at a standard concentration of 1: 100;

day 3: replacing the culture medium, removing the culture medium supernatant containing the virus particles, and replacing the culture medium with a normal culture medium with a proper volume;

day 4: maintaining culture, if the cell confluency is higher, diluting and plate-separating culture on the fourth day, and then changing culture medium and subculturing cells according to conventional process.

Preferably, the optimization process of the MOIs according to the step Day2 includes the steps of

Recovering lentivirus at 4 ℃;

② directly adding calculated Lenti into cells ^BOOST -volume amount of P, gently mix;

thirdly, calculating the required dosage of the slow virus particles according to the following table, adding the slow virus particles into a cell culture medium, and slightly and uniformly mixing;

fourthly, Spinoculate: centrifuging the culture plate at room temperature of 800g for 90 min;

culturing the cells overnight under normal culture conditions.

Preferably, the construction process of the slow virus infection system of the CAR-N cell comprises the following steps:

removing a culture medium in cells;

adding culture medium containing antibiotic in certain concentration;

③ supplementing a fresh culture medium containing IL-2 and 5mmol/L nicotinamide every 3 days, collecting the cell number to monitor the activity of NK cells, and continuing to culture for 14 days.

The application of CAR-NK cells with the function of targeted clearing of IL-1RAP expression, wherein the AR-NK cells can be applied to adoptive cellular immunotherapy.

The beneficial effects of the invention are: the invention discloses CAR-NK cells with the function of targeted removal of IL1RAP expression and preparation and application thereof, and compared with the prior art, the invention has the following improvements:

the CAR-NK cell capable of eliminating the CML-LSC effect expressing IL1RAP in a targeted manner is prepared, experiments prove that the cell has the effect of eliminating the CML-LSC (chronic granulocytic leukemia) effect expressing IL1RAP in a targeted manner, can be applied to adoptive cell immunotherapy and can eliminate tiny residual lesion of the CML leukemia, so that a patient can be stopped and cured clinically, and the CAR-NK cell has no side effect, does not need to worry about the HLA barrier problem, and has the advantages of obvious treatment effect, safe treatment process and obvious anti-tumor effect.

Drawings

FIG. 1 is a process diagram of development and preparation of IL1RAP-CAR-NK cells of the present invention.

FIG. 2 shows the establishment of the NK culture system for peripheral blood derived mononuclear cells and the verification of the killing activity of the NK culture system.

FIG. 3 is a complete 3D structural diagram and a recombinant vector plasmid map of the IL1RAP-scFv protein of the present invention.

FIG. 4 shows the plasmid integrity verification of the IL1RAP-CAR recombinant vector of the present invention.

FIG. 5 shows the verification of the killing effect of IL1RAP-CAR-NK of the present invention on target expression of IL1 RAP.

Wherein: in FIG. 2, a graph (a) is a graph of NK cell growth colonies at each time node during establishment of a peripheral blood-derived monocyte NK cell culture system, a graph (b) is a graph of increase in cell number during establishment of a peripheral blood-derived monocyte NK cell amplification culture system, and a graph (c) is a graph of cell amplification rate during establishment of a peripheral blood-derived monocyte NK cell amplification culture system; graphs (d, e, f, g, h) are histograms of killing activity of NK cells cultured with peripheral blood-derived mononuclear cells at different target-potency ratios for Jurkat, U937, THP1, NB4, K562, respectively;

in FIG. 3, panel (a) is a diagram of the complete 3D structure of 4 different IL1RAP-scFv proteins and panel (e) is a diagram of the recombinant plasmid vector for IL1 RAP-CAR;

in FIG. 4, the diagram (a) is a diagram of a double digestion experiment during the verification process of the recombinant vector, thereby verifying the integrity of the IL1RAP-CAR recombinant plasmid; FIG. (b) is a two-way sequencing map during the validation of the recombinant vector, thereby validating the presence or absence of a mutation site in the IL1RAP-CAR recombinant plasmid during homologous recombination;

in FIG. 5, the graph (a) shows the basic expression of IL1RAP protein detected in KU812, KG-1, Nalm-20, Jurkat, Raji, M phi, K562 cell lines of several common blood diseases; the graphs (b, c and d) respectively detect the killing effects of the three hematological disease cells of Jurkat, Raji and K562 on different effective target ratios of IL1RAP-CAR-NK92 cells 1:1, 2:1 and 3:1, so as to verify the influence of the expression of IL1RAP protein on the killing performance of IL1RAP-CAR-NK92 cells; panel (e) is IL1RAP-CAR-NK92 cell killing on the expression of granulosa CD107 at a potent target ratio of 3:1, thereby feeding back its activation in the presence of IL1RAP target cells; the graphs (f, g) show that NK92 cells and IL1RAP-CAR-NK92 cells secrete killer factors INF-gamma and TNF-alpha, and IL1RAP-CAR-NK92 is obviously superior to NK92 cells; and (i) graphs (i) and (h) show the secretory expression conditions of granzyme GzmB and CD107a after the NK92 cell and the IL1RAP-CAR-NK92 cell are respectively co-cultured with the K562 cell for 3h and 6h, the killing effect is obviously improved along with the prolonging of the action time, and the results have significant differences.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following further describes the technical solution of the present invention with reference to the drawings and the embodiments.

Example 1: referring to figures 1-5, the method for preparing CAR-NK cells with effect of targeted removal of IL-1RAP expression comprises

The method comprises the following steps: preparation of research materials and instruments

1.1 Experimental materials preparation:

(1) Ficoll-Hypaque lymphocyte separation liquid (Dake is-A);

(2) human IFN-gamma, IL-10, IL-4 cytokine ELISA detection kit (Dake is);

(3) DNA synthesis (kasry);

(4) endotoxin-free plasmid miniprep kit (Tiangen);

(5) endotoxin-free plasmid macroextraction kit (Tiangen);

(6) CFSE (5(6) -carboxyfluorescein diacetate succinimidyl ester) (eBioscience);

(7)1L cell culture bag (Tianjin sea);

(8) LDH apoptosis detection kit (bi yun day);

(9) centrifuge tubes, elisa plates (Corning);

(10) pCDH-EF1-MCS-PGK-Puro (vast Ling Bio);

(11) pCDH-EF1-MCS-T2A-Puro (vast Lingzbiol);

(12) LentiBOOST lentiviral transduction enhancers (Sirion Biotech, Germany);

(13) NotI, XbaI restriction enzyme (Takara);

(14) yeast Extract Yeast powder (OXOID);

(15) tryptone (OXOID);

(16) r2AAgarR R2A agar (OXOID);

(17) disposable bacterial culture dishes (biologies);

1.2 laboratory Instrument preparation:

(1) flow cytometry (BD);

(2) optical microscopes (Leica);

(3) biological safety cabinets (Thermo);

(4) ultra-low temperature refrigerators (Thermo);

(5) thermostated cell culture chambers (Thermo);

(6) gel imager (Bio-Rad);

(7) PCR instrument (Thermo);

(8) gel electrophoresis apparatus (Bio-Rad);

(9) a multifunctional microplate reader (Thermo);

(10) high capacity low temperature high speed centrifuges (Thermo);

1.3 preparation of Experimental specimens

1.3.1NK cell preparation:

(1) the culture of NK cells adopts a pure factor culture scheme, and source cells are prepared by in vitro culture of fresh peripheral blood leucoderma cells of healthy blood donors according to a certain initial dose;

(2) NK cell lines such as NK-92 and KHYG-1 were purchased from Shanghai cell Bank, and were all suspension-grown cell lines, and cultured in RPMI-1640 medium containing 10% heat-inactivated FBS.

1.3.2 tumor cells

(1) Human erythroleukemia cells K562, lymphoma cells Raji and the like are all cryopreserved cells in the laboratory, are suspension growth cell lines and are cultured in RPMI-1640 culture medium containing 10% heat-inactivated FBS.

Step two: preparation of CAR-NK cells

S1, constructing a recombinant plasmid vector for stably expressing IL1RAP-CAR and packaging GMP (good manufacturing practice) lentivirus

S101.scFv Molecular Design (scFv Molecular Design)

(1) Searching a monoclonal antibody targeting a human IL-1RAP molecule in an IMGT website mAb-DB database, simultaneously searching an experimental sequence in a clinical test (NCT02842320), and connecting all parts according to the sequence of 'signal peptide-heavy chain variable region- (G4S) 3-light chain variable region-IgG 1' to obtain a complete sequence of the scFv protein;

(2) after obtaining the complete sequence of the scFv protein, the Expasy website ProtParam module is used for calculating the physicochemical properties of the scFv molecule, such as molecular weight, isoelectric point, hydrophilicity and lipophilicity, half-life period, stability and the like, and the detection results are shown in Table 1:

table 1: scFv physical and chemical properties analysis table

(3) Constructing a protein three-dimensional structure through swiss-model, and checking the feasibility and integrity of scFv sequence design to obtain an integral and satisfactory scFv molecule, wherein the integral 3D structure diagram of the IL1RAP-scFv protein and the plasmid map of the recombinant vector are shown in figure 3;

s102, amplifying scFv nucleic acid sequence and constructing homologous recombination vector

(1) After obtaining the complete sequence of the scFv protein, carrying out codon optimization on the scFv according to the codon preference, the mRNA secondary structure, the expression influencing fractional action element, restriction enzyme site optimization, GC content and the like of the scFv molecule, converting the AA sequence into a DNA sequence, and carrying out DNA synthesis;

(2) preparation of linearized vector: obtaining a linearized vector by XbaI and NotI restriction enzyme digestion or reverse PCR;

(3) obtaining an insert: taking a 20bp sequence at the tail end of the linearized vector as a homologous sequence, and respectively adding the homologous sequence to the 5' end of a gene specificity forward/reverse amplification primer sequence to obtain an insert with the homologous sequence by the amplification of the pair of primers;

(4) and (3) recombination reaction: mixing the linearized vector and the insert in proportion, and reacting at 50 ℃ for 5min under the catalysis of Exnase to complete the recombination reaction, thereby realizing the in-vitro cyclization of two linearized DNAs;

s103. transformation preservation of IL1RAP-scFv-CAR recombinant vector

(1) Putting 50 mu L of Stbl-3 competent cells on ice, adding the ligation product into the competent cell suspension, gently dialing the mixture by hands for several times, and standing the mixture on ice for 30 min; rapidly transferring the competent cells to 42 ℃ water bath, thermally shocking for 45s, and then rapidly transferring to ice for cooling for 5 min;

(2) adding 500 μ L of liquid LB culture medium recovered to room temperature, shaking bacteria at 37 deg.C and 220rpm for 1h for amplification; taking 50-100 mu L of bacterial liquid, coating the bacterial liquid on a solid LB plate containing 20 mu g/mL ampicillin according to a scribing method, and culturing overnight at 37 ℃;

(3) observing the growth condition of colonies on the plate, selecting a single colony, inoculating the single colony into 10mL of liquid LB culture medium containing 20 mu g/mL of ampicillin, and shaking the bacteria overnight;

(4) extracting plasmids by using a plasmid small-extraction medium-amount kit, and carrying out DNA sequencing to identify whether the plasmids are constructed successfully;

(5) adding 20% of sterile glycerol into the bacterial liquid of the plasmid with correct identification, uniformly mixing, and freezing and storing at-80 ℃ to be used as a glycerol strain;

s104. preparation of GMP Virus-packaging plasmid for IL1RAP-scFv-CAR recombinant vector

(1) Selecting a small amount of glycerol bacteria for identifying the correct plasmid, adding the glycerol bacteria into 100mL of liquid LB culture medium containing 20 mu g/mL of ampicillin, shaking the bacteria at 37 ℃, and culturing overnight;

(2) centrifuging at 3000rpm at room temperature for 10min, collecting bacterial precipitate, adding 10mLP1 solution according to kit instructions, fully blowing and mixing the bacterial precipitate uniformly, and then adding 10mL of lysate P2;

(3) standing at room temperature for 5min, adding 10mL of neutralizing solution P4, immediately and gently turning over for 6-8 times, and fully and uniformly mixing to obtain white flocculent precipitate;

(4) centrifuging at 8000rpm for 10min to separate white flocculent precipitate to the bottom of the tube, collecting supernatant to clean centrifuge tube; adding 3mL of red endotoxin removing solution ER, and turning over and mixing uniformly to obtain a uniform and transparent yellow solution;

(5) adding isopropanol with the volume of 0.3 time to precipitate out DNA, reversing and mixing uniformly, and transferring the solution to a DNA adsorption silica gel column; centrifuging at 8000rpm for 2min, discarding filtrate, and adsorbing plasmid onto silica gel column; respectively eluting the column with protein eluent and salt eluent twice, and finally eluting with sterile ultrapure water to obtain plasmid aqueous solution;

(6) the concentration and purity of the plasmid solution are detected by using Nanodrop 2000, and the plasmid solution is sent out to package viruses at proper concentration.

S2. preparation of CAR-NK cells and establishment of a Lentiviral infection System of CAR-N cells (as shown in FIG. 1)

Day 1: seeding the cells, plating at an appropriate cell density (note: if the cells are maintained under a certain selective pressure, plating requires removal of the antibiotics for selection until day 3);

day 2: viral transduction with MOI10 and Lenti ^BOOST -P is measured at 1: standard concentration of 100 added to total volume (medium + virus); recovering lentivirus at 4 ℃; ② directly adding calculated Lenti into cells ^BOOST -volume amount of P, gently mix; thirdly, calculating the required dosage of the slow virus particles according to the following table, adding the slow virus particles into a cell culture medium, and slightly and uniformly mixing; (iv) Spinoculate (optional): centrifugation of the plates at 800g for 90min at room temperature (note: the spinolation step is supported by literature findings that increase transduction efficiency for most cells; centrifugation using cell culture plates is necessary to reduce shear); culturing cells overnight under normal culture conditions;

day 3: replacing the culture medium, removing the culture medium supernatant containing the virus particles, and replacing the culture medium with a normal culture medium with a proper volume;

day 4: maintaining culture, if the cell confluence is higher, diluting and plate-dividing culture on the fourth day, and subsequently replacing culture medium and carrying out cell passage according to the conventional process;

day 5: selection of stable cell lines (optional): removing a culture medium in cells; adding a culture medium containing screened antibiotics with certain concentration; supplying a fresh culture medium containing IL-2 and 5mmol/L nicotinamide every 3 days, collecting the number of cells to monitor the activity of NK cells, and continuing to culture for 14 days. Total viable cell counts were counted using a cytometer and the absolute NK cell counts were calculated by multiplying the total viable cell counts by the percentage of CD3-CD56+ cells determined by flow cytometry. Evaluating the proliferation fold by dividing the number of NK cells produced at the end of the culture by the number of NK cells at the beginning of the culture; the recombinant vector for IL1RAP-CAR is shown in FIG. 4.

The CAR-NK cell is the IL1RAP-CAR cell, the nucleic acid sequence of the IL1RAP-CAR cell is shown as SEQ ID NO.1, and the protein sequence is shown as SEQ ID NO. 2.

Example 2: stable killing of CAR-NK cells by CML-LSC targeting IL-1RAP

1. Detection of CAR-NK cell Activity by flow cytometry (GzmB, CD107a FACS detection)

Activated NK cells can release GzmB stored inside cells into the intercellular space, and are one of the important means for killing tumor cells by the NK cells; similarly, NK cells also release cytotoxic granules within the cytoplasm upon stimulation by tumor cells or other target cells; at this time, the lysosome-related membrane protein-1 (CD107a) is transported to the surface of the cell membrane, and the activation level and cytotoxicity level of NK cells can be directly reflected; we further explored the mechanism of effector cell killing of tumors by measuring changes in levels of effector cell secretion GzmB and levels of transport of CD107 a;

for the assay of GzmB and CD107a, two time points of 3h and 6h were selected on the basis of co-incubation of effector cells and tumor cells at an effective target ratio of 1:1, and changes of GzmB and CD107a levels of NK92 cells and IL1RAP-CAR-NK92 cells at two time periods were determined, respectively, and the detection results are shown in fig. 2.

2. In vitro bioactivity test (cytotoxin LDHAssay)

Cytoxicity LDHAssay is an experiment for measuring cell damage by measuring the Lactate Dehydrogenase (LDH) activity released from cells into a culture medium; LDH is an enzyme present in the cytoplasm, and is released into the culture medium when the cell membrane is damaged; since the released LDH is stable, measuring the amount of LDH in the medium can be used as an index for determining the number of dead and damaged cells. Target cells were incubated at 1X 105/mL/well in U-bottom-96-well plates for 4 hours, 3 replicates, at different effector-target ratios (E: T) with the expanded NK cells; setting a natural release group as a negative control group, setting a maximum release group added with NP-40 as a positive control group, and setting 3 times of repetition; and (3) measuring the cell killing toxicity of the CAR-NK cells of the patients at the wavelength of 549nm by using a multifunctional enzyme-labeling instrument.

3. In vitro tumor killing Assay (IFN-. gamma., TNF-. alpha.assay)

Gamma interferon is a very important cytokine involved in anti-tumor reaction, has the functions of resisting virus, regulating immunity, resisting tumor and the like, and plays a central role in identifying and eliminating tumor cells. Tumor necrosis factor alpha (TNF- α) is a pleiotropic cytokine produced by many types of cells, involved in a wide range of pathological processes; TNF-alpha has the functions of killing tumor cells and inhibiting cell proliferation in vivo and in vitro; secretion of TNF- α is one of the ways NK cells exert cytotoxic effects; aiming at the determination of IFN-gamma and TNF-alpha, the method selects to determine the change of the levels of IFN-gamma and TNF-alpha of NK92 cells and IL1RAP-CAR-NK92 cells after incubating effector cells and tumor cells for 3h on the basis of the effective target ratio of 1:1, and the experimental result is shown in figure 5, and the IL1RAP-CAR-NK92 is obviously superior to the NK92 cells through figure 5; and (i) graphs (i) and (h) show the secretory expression conditions of granzyme GzmB and CD107a after the NK92 cell and the IL1RAP-CAR-NK92 cell are respectively co-cultured with the K562 cell for 3h and 6h, the killing effect is obviously improved along with the prolonging of the action time, and the results have significant differences.

It can be seen by verification of the above embodiment that: the CAR-NK cell has the effect of removing CML-LSC (chronic myelocytic leukemia) of IL1RAP (interleukin-1 receptor accessory protein) in a targeted manner, namely the CAR-NK cell can be applied to adoptive cell immunotherapy to remove tiny residual lesion of CML leukemia, so that a patient can achieve the purposes of stopping medicine and even clinically curing.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> Saian International medicine center, Inc

<120> CAR-NK cell with IL1RAP expression targeted removal effect and preparation and application thereof

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gcagggggtt ccggcggcgg cggctctggt ggagggggct ccgttgacat ggagtcacag 540

attcaggtct ttgtattcgt gtttctctgg ttgtctggtg ttgacggaga cattgtgatg 600

acccagtctc acaaattcat gtccacatca gtaggagaca gggtcaccat cacctgcaag 660

gccagtctgg atgtgagtac tgctgtggcc tggtatcaac agaaaccagg acaatctcct 720

aaactactga tttactcggc atcctaccgg tacactggag tccctgatcg cttcactggc 780

agtggatctg ggacggattt cactttcacc atcagcagtg tgcaggctga agacctggca 840

gtttattact gtcagcaaca ttatagtcct ccattcacgt tcggctcggg gacaaacttg 900

gagataaaac gcagagccca aatcccctga taagacacat acatgtcctc cctgccctaa 960

ggatcctaag ttctgggtgc ttgttgtcgt cgggggtgtg ttggcttgct attctctgct 1020

cgtcacagtc gcatttataa ttttctgggt gaaacgagga cgaaagaaac ttttgtatat 1080

tttcaagcag ccctttatga gacccgtcca aactacacag gaagaggacg gctgcagctg 1140

taggttcccg gaagaggagg agggtggctg tgagttgcgc gtgaaattca gtagaagtgc 1200

tgatgccccc gcttaccagc aaggccaaaa tcagctgtat aatgaactga atttgggtcg 1260

cagggaggaa tatgacgtgc tcgacaagag acgagggcgc gatccagaaa tgggtggtaa 1320

gccacgcaga aagaaccctc aggagggatt gttcaatgag ctccagaagg acaagatggc 1380

tgaggcattc tctgagatag aatgaagggc gaacgacggc gaggtaaagg acatgatggc 1440

ctgttccaag gactctctac tgccaccaaa gacacctttg acgcacttca tatgcaagcg 1500

ctcccaccac gc 1512

<210> 2

<211> 346

<212> DNA

<213> protein Sequence (Artificial Sequence)

<400> 2

mtdtwvwvgs tgmgwscvat atgvnsvgam mgasvkvsca sgyttdswmh wvkrggwgad 60

sdsyttynkt gkatsvdssn taymsstsds avyycaryys gsnysywggt vtvsaggsgg 120

ggsggggsvd msvvvwsgvd gdvmtshkms tsvgdrvttc kasdvstava wykgskysas 180

yryrytgvdr tgsgsgtdtt ssvadavyyc hystgsgtnk aksdkthtcc kdkwvvvvgg 240

vacysvtvaw vkrgrkkykm rvttdgcscr ggcrvksrsa daaygnynng rrydvdkrrg 300

rdmggkrrkn gnkdkmaasg mkgrrrgkgh dggstatkdt dahmar 346

Claims (10)

1. CAR-NK cell with targeted clearance of IL1RAP expression characterized by: the CAR-NK cell is an IL1RAP-CAR cell, the nucleic acid sequence of the IL1RAP-CAR cell is shown as SEQ ID NO.1, and the protein sequence is shown as SEQ ID NO. 2.

2. The method for producing CAR-NK cells with targeted clearance of IL1RAP expression according to claim 1, characterized in that: comprising the steps of

S1, constructing a recombinant plasmid vector for stably expressing IL1RAP-CAR and packaging GMP (good manufacturing practice) lentivirus

S101, designing an scFv nucleic acid chain by using a monoclonal antibody targeting a human IL-1RAP molecule according to the sequence of 'signal peptide-heavy chain variable region- (G4S) 3-light chain variable region-IgG 1';

s102, amplifying an scFv nucleic acid chain, and performing homologous recombination on an scFv target sequence synthesized by amplification to construct a recombinant plasmid vector completely containing the scFv target sequence;

s103, stably transforming the constructed recombinant plasmid vector in a competent cell stal-3, and storing the recombinant plasmid vector in a glycerol strain state;

s104, carrying out shake bacteria amplification on the plasmid competent cells after homologous recombination, and obtaining virus packaging plasmids with large quality particles meeting GMP requirements;

s2, performing NK cell and human Peripheral Blood Mononuclear Cell (PBMC) derived NK cell infection on the prepared lentivirus, preparing CAR-NK cells expressing IL1RAP-CAR and establishing a CAR-NK cell lentivirus infection system.

3. The method for producing CAR-NK cells with targeted clearance of IL1RAP expression according to claim 2, characterized in that: the design process of the scFv nucleic acid strand described in step S101 comprises:

(1) connecting the parts of the IL1RAP sequence according to the sequence of 'signal peptide-heavy chain variable region- (G4S) 3-light chain variable region-IgG 1' to obtain the complete sequence of the scFv protein;

(2) after obtaining the complete sequence of the scFv protein, calculating the physicochemical properties of the molecular weight, isoelectric point, hydrophily and lipophilicity, half-life period and stability of the scFv molecule;

(3) and (3) constructing a protein three-dimensional structure of the scFv protein, and checking the feasibility and the integrity of scFv sequence design to obtain an integral and qualified scFv molecule.

4. The method for producing CAR-NK cells with targeted clearance of IL1RAP expression according to claim 2, characterized in that: the amplification process of the scFv nucleic acid strand nucleic acid molecule in step S102 comprises:

(1) according to codon preference, mRNA secondary structure, expression influencing fractal action elements, restriction enzyme site optimization and GC content of scFv molecules, codon optimization is carried out on scFv, an AA sequence is converted into a DNA sequence, and DNA synthesis is carried out;

(2) preparing a linearized vector: carrying out enzyme digestion reaction by XbaI and NotI restriction enzymes to obtain a linearized vector;

(3) obtaining of the inserted target fragment: taking a 20bp sequence at the tail end of the linearized vector as a homologous sequence, and respectively adding the homologous sequence to the 5' end of a gene specificity forward/reverse amplification primer sequence to obtain an insert with the homologous sequence by the amplification of the pair of primers;

(4) and (3) recombination reaction: the linearized vector and the insert are mixed in proportion, and the recombination reaction can be completed by reacting for 5min at 50 ℃ under the catalysis of Exnase, so that the in-vitro cyclization of two linearized DNAs is realized.

5. The method for producing CAR-NK cells with targeted clearance of IL1RAP expression according to claim 2, characterized in that: the transformation and preservation process of the IL1RAP-scFv-CAR recombinant plasmid vector in the competent cell stal-3 in the step S103 comprises the following steps:

(1) putting 50 mu L of Stbl-3 competent cells on ice, adding the ligation product into the competent cell suspension, gently dialing the mixture by hands for several times, and standing the mixture on ice for 30 min; rapidly transferring the competent cells to 42 ℃ water bath, thermally shocking for 45s, and then rapidly transferring to ice for cooling for 5 min;

(2) adding 500 μ L of liquid LB culture medium recovered to room temperature, shaking bacteria at 37 deg.C and 220rpm for 1h for amplification; taking 50-100 mu L of bacterial liquid, coating the bacterial liquid on a solid LB plate containing 20 mu g/mL ampicillin according to a scribing method, and culturing overnight at 37 ℃;

(3) observing the growth condition of colonies on the plate, selecting a single colony, inoculating the single colony into 10mL of liquid LB culture medium containing 20 mug/mL of ampicillin, and shaking the colonies overnight;

(4) extracting plasmids by using a small and medium plasmid extraction kit, and carrying out DNA sequencing to identify whether the plasmids are constructed successfully;

(5) for the plasmid with correct identification, 20% of sterile glycerol is added into the bacterial liquid, and the mixture is frozen and stored at minus 80 ℃ after being uniformly mixed, and is reserved as a glycerol strain.

6. The method for producing CAR-NK cells with targeted clearance of IL1RAP expression according to claim 2, characterized in that: the process for preparing the GMP virus packaging plasmid of the IL1RAP-scFv-CAR recombinant vector in the step S104 comprises the following steps:

(1) selecting a small amount of glycerol bacteria for identifying the correct plasmid, adding the glycerol bacteria into 100mL of liquid LB culture medium containing 20 mu g/mL of ampicillin, shaking the bacteria at 37 ℃, and culturing overnight;

(2) centrifuging at 3000rpm at room temperature for 10min, collecting bacterial precipitate, adding 10mLP1 solution according to kit instructions, fully blowing and mixing the bacterial precipitate uniformly, and then adding 10mL of lysate P2;

(3) standing at room temperature for 5min, adding 10mL of neutralizing solution P4, immediately and gently turning up and down for 6-8 times, and fully and uniformly mixing to obtain white flocculent precipitate;

(4) centrifuging at 8000rpm for 10min to separate white flocculent precipitate to the bottom of the tube, collecting supernatant to clean centrifuge tube; adding 3mL of red endotoxin removing solution ER, and turning over and mixing uniformly to obtain a uniform and transparent yellow solution;

(5) adding isopropanol with the volume of 0.3 time to precipitate out DNA, reversing and mixing uniformly, and transferring the solution to a DNA adsorption silica gel column; centrifuging at 8000rpm for 2min, discarding filtrate, and adsorbing plasmid onto silica gel column; eluting the column with protein eluent and salt eluent twice, and finally eluting with sterile ultrapure water to obtain plasmid water solution;

(6) the concentration and purity of the plasmid solution are detected by using Nanodrop 2000, and the plasmid solution is sent out to package viruses at proper concentration.

7. The method for producing CAR-NK cells with targeted clearance of IL1RAP expression according to claim 2, characterized in that: the CAR-NK cell preparation process of the step S2 comprises the following steps:

day 1: seeding cells, and selecting proper cell density for plating;

day 2: viral transduction, using MOI10 for viral transduction, and Lenti ^BOOST P is added to the total volume at a standard concentration of 1: 100;

day 3: replacing the culture medium, removing the culture medium supernatant containing the virus particles, and replacing the culture medium with a normal culture medium with a proper volume;

day 4: maintaining culture, if the cell confluency is higher, diluting and plate-separating culture on the fourth day, and then changing culture medium and subculturing cells according to conventional process.

8. The method for producing CAR-NK cells with targeted clearance of IL1RAP expression according to claim 7, characterized in that: the process for optimizing MOIs according to step Day2 includes the steps of

Recovering lentivirus at 4 ℃;

② directly adding calculated Lenti into cells ^BOOST -volume amount of P, gently mix;

thirdly, calculating the required dosage of the slow virus particles according to the following table, adding the slow virus particles into a cell culture medium, and slightly and uniformly mixing;

fourthly, Spinoculate: centrifuging the culture plate at room temperature of 800g for 90 min;

culturing the cells overnight under normal culture conditions.

9. The method for producing CAR-NK cells with targeted clearance of IL1RAP expression according to claim 7, characterized in that: the construction process of the slow virus infection system of the CAR-N cell comprises the following steps:

removing a culture medium in cells;

adding culture medium containing antibiotic in certain concentration;

③ supplementing a fresh culture medium containing IL-2 and 5mmol/L nicotinamide every 3 days, collecting the cell number to monitor the activity of NK cells, and continuing to culture for 14 days.

10. Use of a CAR-NK cell with targeted clearance of IL1RAP expression according to claim 1 characterized in that: the AR-NK cell can be applied to adoptive cell immunotherapy.

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