Method for producing NK cells with PD-1 knockout gene and trail or FAS-ligand overexpression
12359171 ยท 2025-07-15
Assignee
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
A61K40/15
HUMAN NECESSITIES
C12N9/22
CHEMISTRY; METALLURGY
International classification
A61K40/15
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The invention relates to the field of medicine and genetic engineering, specifically to guide RNAs and DNA fragments and to cell selection methods, which can be used in CRISPR-Cas9 systems for producing lines of natural killer cells with a PD-1 knockout gene and increased production of TRAIL or Fas-ligand proteins. Methods are disclosed for producing modified lines of NK cells: with a PD-1 knockout gene and constitutive Fas-ligand overexpression, with a PD-1 knockout gene and constitutive TRAIL overexpression, and also a method is described for selecting modified NK cells with a PD-1 knockout gene, which is carried out using a zeocin selection marker. The invention makes it possible to produce a high yield of modified lines of NK cells that are highly active in inhibiting the growth of tumour cells.
Claims
1. A method of obtaining a modified line of NK-cells with a knockout of the PD-1 gene in order to inactivate the expression of the PD-1 gene, comprising: A) selecting at least one guide RNA sequence specific to the PD-1 gene, the guide RNA sequence being selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4; B) constructing separate expression plasmid vectors encoding each of the selected guide RNA sequences and an endonuclease Cas9; C) synthesizing a fragment of donor DNA encoding one of: i) an expression cassette for Fas-ligand having the sequence according to SEQ ID NO: 16, or ii) an expression cassette for TRAIL having the sequence according to SEQ ID NO: 20, or iii) an expression cassette for resistance of cells to zeocin having the sequence according to SEQ ID NO: 9; D) transfecting a culture of NK-cells with at least one of the expression plasmid vectors of B) and with the donor DNA of C); E) staining the NK-cells with anti-PD-1 antibodies and anti-Fas-ligand antibodies, or staining the NK-cells with anti-PD-1 antibodies and anti-TRAIL antibodies, or culturing the NK-cells in a selective medium with zeocin and staining the surviving cells with anti-PD-1 antibodies; F) carrying out a selection and cloning of cells with a minimum signal for PD-1 and a maximum signal for Fas-ligand or for TRAIL using a flow sorter, or carrying out a selection and cloning of zeocin-cultured cells with a minimum signal for PD-1 using a flow sorter; and G) carrying out a secondary cultivation of cell clones and selection of cells using immunoblotting with anti-PD-1 antibodies and anti-Fas-ligand antibodies, or with anti-PD-1 antibodies and anti-TRAIL antibodies, or with anti-PD-1 antibodies.
2. The method according to claim 1, wherein at least one of the expression plasmid vectors includes: 295-572 bpCBh promoter; 819-5090 bpCas9 endonuclease; 5121-5328 bpPoly A Signal; 5552-6007 bpfl ori; 6289-6393 bpbla promoter; 6394-7254 bpbla; 7425-8013 bpColEl ori; 8075-8315 bpU6 promoter; and 8322-8419 bpsequence encoding a guide RNA.
3. The method of according to claim 1, wherein at least one of the expression plasmid vectors includes: 295-572 bpCBh promoter; 819-5090 bpCas9 endonuclease; 5121-5328 bpPoly A Signal; 5552-6007 bpfl ori; 6289-6393 bpbla promoter; 6394-7254 bpbla; 7425-8013 bpColEl ori; 8075-8315 bpU6 promoter; and 8322-8419 bpsequence encoding a guide RNA.
4. The method according to claim 1, wherein the donor DNA encoding the expression cassette for resistance of cells to zeocin includes: a fragment containing the sequence of the first part of the PD-1 gene with a size of 379 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 10; a fragment containing the sequence of CMV enhancer with a size of 405 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 11; a fragment containing the sequence-hFerL Promoter with a size of 263 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 12; a fragment containing the sequence-Zeo-zeocin resistance gene with a size of 375 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 13; a fragment containing the sequence pGlo poly A Signal with a size of 401 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 14; and a fragment containing the sequence of the second part of the PD-1 gene with a size of 276 base pairs and characterized by the nucleotide sequence shown in SEQ ID NO: 15.
5. The method according to claim 1, wherein at least one of the expression plasmid vectors includes: 295-572 bpCBh promoter; 819-5090 bpCas9 endonuclease; 5121-5328 bpPoly A Signal; 5552-6007 bpP ori; 6289-6393 bpbla promoter; 6394-7254 bpbla; 7425-8013 bpColEl ori; 8075-8315 bpU6 promoter; and 8322-8419 bpsequence encoding a guide RNA.
6. The method according to claim 1, wherein the donor DNA encoding the expression cassette for Fas-ligand includes: a fragment containing the sequence of the first part of the PD-1 gene with a size of 379 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 10; a fragment containing the sequence CMV Promoter with a size of 588 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 17; a fragment containing the sequence Fas-ligand with a size of 846 base pairs characterized by the nucleotide sequence shown in SEQ ID NO: 18; a fragment containing the sequence BGH Poly A Signal with a size of 225 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 19; and a fragment containing the sequence of the second part of the PD-1 gene with a size of 276 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 15.
7. The method according to claim 1, wherein at least one of the expression plasmid vectors includes: 295-572 bpCBh promoter; 819-5090 bpCas9 endonuclease; 5121-5328 bpPoly_A Signal; 5552-6007 bpfl ori; 6289-6393 bpbla promoter; 6394-7254 bpbla; 7425-8013 bpColEl ori; 8075-8315 bpU6 promoter; and 8322-8419 bpsequence encoding a guide RNA.
8. The method according to claim 1, wherein the donor DNA encoding the expression cassette for TRAIL includes: a fragment containing the sequence of the first part of the PD-1 gene with a size of 379 base pairs, characterized by a nucleotide sequence presented in SEQ ID NO: 10; a fragment containing the sequence CMV Promoter with a size of 588 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 17; a fragment containing the sequence TRAIL with a size of 846 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 21; a fragment containing the sequence BGH Poly A Signal with a size of 225 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 19; and a fragment containing the sequence of the second part of the PD-1 gene with a size of 276 base pairs, characterized by the nucleotide sequence shown in SEQ ID NO: 15.
9. A method of inducing apoptosis or lysis of mammalian cancer cells, the method comprising administering an effective amount of a NK-cell obtained from the modified NK-cell line of claim 1 to a mammal.
Description
LIST OF FIGURES
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INVENTION DESCRIPTION
(11) The present invention relates to a cell or NK-cell line of natural killer (NK) cells that have been genetically modified in such a way as to increase their cytotoxicity.
(12) These NK-cells and NK-cell lines can be: a) isolated from peripheral blood, b) isolated from umbilical cord blood, c) obtained from pluripotent and embryonic stem cells (i.e. iPSCs and ESC) using the method of Galat et al [9.10], d) obtained from pluripotent stem cells (iPSCs) using the method of Kaufman et al. [11]. These NK-cells and NK-cell lines can be infiltrated into tissues and tumors. These NK-cells and NK-cell lines can be included in the group consisting of KHYG-1/CVCL_2976, NK-92/CVCL_2142, NK-YS/CVCL_8461, NKL/CVCL_0466, and NK3.3/CVCL_7994, which includes but is not limited to other types of NK-cells and NK-cell lines.
(13) Generation of PD-1 Knockout NK-Cells
(14) The method for obtaining a line of PD-1 knockout NK-cells includes several main steps: 1. Selection of a guide RNA sequence specific to various sites of the PD-1 gene having a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4; 2. Construction of an expression plasmid vector encoding the selected guide RNA and Cas9 endonuclease (pGR301 or pGR302 or pGR303 or pGR304);
(15) The composition of the plasmid vector with a physical map shown in
(16) To select the optimal guide RNA sequences, the analysis of nucleotide sequences was carried out using freely available web resources: CRISPR Design, CHOPCHOP, E-CRISPR. Sequences of guide RNAs were selected having sequences according to SEQ ID NO: 1-4, specific to various sites of the PD-1 gene having a sequence according to SEQ ID NO: 5-8.
(17) TABLE-US-00001 InvectorpGR301ofSEQIDNO:1 GTCTGGGCGGTGCTACAACTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC InvectorpGR302ofSEQIDNO:2 GGGCGGTGCTACAACTGGGCGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC InvectorpGR303ofSEQIDNO:3 GGCGCCCTGGCCAGTCGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC InvectorpGR304ofSEQIDNO:4 GCCCTGGCCAGTCGTCTGGGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC. TargetDNAsequences (PAMsequenceshighlighted): ForvectorpGR301ofSEQIDNO:5 GTCTGGGCGGTGCTACAACTGGG ForvectorpGR302ofSEQIDNO:6 GGGCGGTGCTACAACTGGGCTGG ForthepGR303vectorSEQIDNO:7 GGCGCCCTGGCCAGTCGTCTGGG ForvectorpGR304ofSEQIDNO:8 GCCCTGGCCAGTCGTCTGGGCGG
(18) To obtain plasmid vectors encoding complexes of Cas9 endonuclease and guide RNAs, DNA fragments encoding guide RNAs were synthesized using a polymerase chain reaction with overlapping oligonucleotides. The resulting fragments were cloned into a plasmid vector intended for the expression of the components of the CRISPR-Cas9 system in mammalian cells. The vector was pre-digested at the BbsI restriction endonuclease site. Selected plasmid vectors pGR301, pGR302, pGR303, and pGR304 were sequenced to confirm that the planned genetic constructs were obtained.
(19) NK-cells were transfected with pGR301, pGR302, pGR303, and pGR304 vectors using Lipofectamine 3000 (Thermo Fisher Scientific), cells were incubated in an RPMI medium with 20% fetal bovine serum until a cell density of 4 610.sup.5 cells/ml was reached. A mixture of Lipofectamine 3000 lipagent and DNA was prepared (at a ratio of 3 L lipagent, 4 g pGR301 or pGR302 or pGR303 or pGR304, 8 L P3000 reagent, and 100 L Opti-MEM Medium per 1 ml cell suspension). The mixture was incubated for 5 min and added to the cell suspension. The cells were incubated for 48 h at 37 C. and 5% CO2.
(20) To select PD-1 gene knockout cells 48 h after transfection, the cells were incubated with anti-PD-1 antibodies labeled with fluorescein for 1 h. The cells were washed with a culture medium and separated using a cell sorter. Single cells with minimal or no fluorescent signal were selected in the wells of a 96-well plate with the RPMI medium with 20% fetal bovine serum. The cells were cultured for 2-4 weeks at 37 C. and 5% CO2 with replacement of the culture medium every 3-4 days.
(21) To obtain labeled anti-PD-1 antibodies, we used monoclonal mouse anti-PD-1 antibodies, the clone NAT105 (Manufacturer: Abeam cat. no. ab52587) and Fluorescein-EX Protein Labeling Kit (Manufacturer Invitrogen, cat. no. F10240).
(22) The expression level of PD-1 in the clones was assessed using immunoblotting with anti-PD-1 antibodies. Non-transfected NK-cells were used as a positive control. The cells were lysed using a buffer containing 25 mM Tris-HCl, 150 mM NaCl, mMEDTA, and 1% Triton-X100. Cell lysates were centrifuged at 10,000 g for 10 min. The concentration of total protein in the supernatant was determined and aliquots corresponding to 100 g of protein were separated by electrophoresis under denaturing conditions. Proteins were electrically transferred from the gel to a nitrocellulose membrane. Then the nitrocellulose membrane with immobilized proteins was washed with buffer I (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% twin-20) and incubated for 1 h in a 2% bovine serum albumin solution in buffer I. Then they were incubated for 16 h with primary antibodies to PD1 in 2% BSA in buffer I. After that, the membrane was washed with buffer I and incubated for 1 h with secondary antibodies conjugated with horseradish peroxidase (Manufacturer Bio-Rad) in a 1% milk solution in buffer I.
(23) The membrane was washed with buffer I and stained using a commercial ECL chemiluminescence detection kit. Clones were selected that did not show specific bands corresponding to PD-1.
(24) Generation of PD-1 Knockout NK-Cells and Antibiotic Resistance to Zeocin.
(25) To increase the efficiency of the process of creating cells with a knockout PD-1 gene, a method for selecting NK-cells using a selective marker zeocin is proposed.
(26) The process includes several steps: 1. Selection of a guide RNA sequence specific to the PD-1 gene having a sequence according to SEQ ID NO: 1; 2. Construction of an expression plasmid vector encoding the selected guide RNA and Cas9 endonuclease (pGR301); 3. Synthesis of a donor DNA fragment encoding an expression cassette for cell resistance to zeocin having the sequence according to SEQ ID NO: 9; 4. Transfection of a culture of NK-cells with a mixture of a plasmid vector and a fragment of donor DNA; 5. Cultivation of cells in a selective medium with zeocin. 6. Staining of surviving cells with anti PD-1 antibodies, and selection and cloning of cells with minimal signal using a flow sorter. 7. Culturing clones of cells, secondary selection of cells by immunoblotting with anti PD-1 antibodies.
(27) For transfection of the cells, a mixture of the pGR301 vector and a pd1-Zeo DNA fragment, SEQ ID NO: 9, encoding the zeocin resistance gene was used. To synthesize a DNA fragment and pd1-Zeo polymerase chain reaction was used with overlapping primers. NK-cells were transfected with Lipofectamin 3000 (Thermo Fisher Scientific). For transfection with Lipofectamine 3000, cells were incubated in an RPMI medium with 20% fetal bovine serum until a cell density of 4 610.sup.5 cells/ml was achieved. A mixture of Lipofectamine 3000 lipagent and DNA was prepared (at the ratio of 3 L lipagent, 2 g pGR301 vector, 2 g pd1-Zeo DNA fragment, 8 L P3000 reagent, 100 L Opti-MEM Medium per 1 ml cell suspension). The mixture was incubated for 5 min and added to the cell suspension. The cells were incubated for 48 h at 37 C. and 5% CO2.
(28) To select NK-cells with a knocked-out PD-1 gene using a selective marker zeocin 48 h after transfection, the cells were transferred to a selective medium (RPMI supplemented with zeocin) and cultured for 2-4 weeks. Surviving cells were stained with fluorescein-labeled anti-PD-1 antibodies. Samples of stained cells were separated using a cell sorter. Single cells with minimal or no fluorescent signal were selected in the wells of a 96-well plate with the RPMI medium with 20% fetal bovine serum. The cells were cultured for 2-4 weeks at 37 C. and 5% CO2 with replacement of the culture medium every 3-4 days.
(29) The expression level of PD-1 in clones was assessed by immunoblotting with anti-PD-1 antibodies, similar to the protocol described for obtaining NK-cells with a knockout PD-1 gene.
(30) Generation of NK-Cells with PD-1 Gene Knockout and Constitutive Increased Expression of Fas-Ligand.
(31) To increase the activity of NK-cells, a method is proposed for producing NK-cells with a knockout PD-1 gene and constitutive increased expression of Fas-ligand.
(32) The process includes several steps: 1. Selection of the guide RNA sequence specific to the PD-1 gene having the sequence according to SEQ ID NO: 1; 2. Construction of an expression plasmid vector encoding the selected guide RNA and Cas9 endonuclease (pGR301); 3. Synthesis of a donor DNA fragment encoding an expression cassette for Fas-ligand having the sequence according to SEQ ID NO: 16; 4. Transfection of a culture of NK-cells with a mixture of a plasmid vector and a fragment of donor DNA; 5. Cell staining with anti PD-1 antibodies and anti Fas-ligand antibodies. Selection and cloning of cells with minimal signal for PD-1 and maximal signal for Fas-ligand using a flow sorter. 6. Culturing cell clones, secondary cell selection by immunoblotting with anti PD-1 and anti Fas-ligand antibodies.
(33) For transfection of cells, a mixture of the pGR301 vector and a pd1-FasL DNA fragment having the sequence according to SEQ ID NO: 16, encoding an expression cassette for expressing the Fas-ligand, was used. To synthesize a DNA fragment and pd1-FasL, a polymerase chain reaction with overlapping primers was used.
(34) NK-cells were transfected with Lipofectamin 3000 (Thermo Fisher Scientific). For transfection with Lipofectamine 3000, cells were incubated in an RPMI medium with 20% fetal bovine serum until a cell density of 4-610.sup.5 cells/ml was reached. A mixture of Lipofectamine 3000 lipagent and DNA was prepared (at a ratio of 3 L lipagent, 2 g pGR301 vector, 2 g pd1-FasL DNA fragment, 8 L P3000 reagent, and 100 L Opti-MEM medium per 1 ml cell suspension). The mixture was incubated for 5 min and added to the cell suspension. The cells were incubated for 48 h at 37 C. and 5% CO2.
(35) To select cells with a PD-1 knockout gene and constitutively increased expression of Fas-ligand, 48 h after transfection, cells were stained with a mixture of anti-PD-1 antibodies labeled with fluorescein and anti-Fas-ligand antibodies labeled with Alexa Fluor 610-R-phycoerythrin. Cells with a minimum fluorescent signal of fluorescein and a maximum signal of Alexa Fluor 610-R-phycoerythrin were selected and cloned.
(36) To obtain labeled anti-Fas-ligand antibodies, we used monoclonal mouse anti-Fas-ligand antibodies (Manufacturer: BD Biosciences, clone G-247) and a Zenon Alexa Fluor 610-R-Phycoerythrin Mouse IgG1 Labeling Kit (Manufacturer: Invitrogen, cat. no. Z25020).
(37) The expression level of PD-1 in clones was assessed by immunoblotting with anti-PD-1 antibodies, similar to the protocol described for obtaining NK-cells with the knockout PD-1 gene. In clones with confirmed PD-1 knockout, the expression level of the Fas-ligand was assessed. Immunoblotting with anti-Fas-ligand antibodies (Manufacturer: BD Biosciences, clone G-247) was carried out in a manner similar to PD-1. As a result of the analysis, cells were selected in which the maximum signal of the bands corresponding to the Fas-ligand was detected.
(38) Generation of PD-1 gene knockout NK-cells and constitutive increased TRAIL expression.
(39) To increase the activity of NK-cells, a method is proposed for obtaining NK-cells with knockout PD-1 gene and constitutive increased TRAIL expression.
(40) The process includes several steps: 1. Selection of a guide RNA sequence specific to the PD-1 gene having the sequence according to SEQ ID NO: 1; 2. Construction of an expression plasmid vector encoding the selected guide RNA and Cas9 endonuclease (pGR301); 3. Synthesis of a donor DNA fragment encoding an expression cassette for TRAIL having the sequence according to SEQ ID NO: 20; 4. Transfection of a culture of NK-cells with a mixture of a plasmid vector and a fragment of donor DNA; 5. Cell staining with anti-PD-1 antibodies and anti-TRAIL antibodies. Selection and cloning of cells with a minimum signal for PD-1 and a maximum signal for TRAIL using a flow sorter. 6. Cultivation of cell clones, secondary selection of cells by immunoblotting with anti PD-1 and anti TRAIL antibodies.
(41) A mixture of the pGR301 vector and a pdl-TRAIL DNA fragment, SEQ ID NO: 20, encoding an expression cassette for TRAIL expression, was used to transfect the cells. To synthesize a DNA fragment and pdl-TRAIL, a polymerase chain reaction with overlapping primers was used. NK-cells were transfected using the Lipofectamin 3000 lipagent (Thermo Fisher Scientific). For transfection with Lipofectamine 3000, the cells were incubated in an RPMI medium with 20% fetal bovine serum until a cell density of 4-610.sup.5 cells/ml was reached. A mixture of Lipofectamine 3000 lipagent and DNA was prepared at the ratio of 3 L lipagent, 2 g pGR301 vector, 2 g a pdl-TRAIL DNA fragment, 8 L P3000 reagent, and 100 L Opti-MEM Medium medium per 1 ml cell suspension. The mixture was incubated for 5 min and added to the cell suspension. The cells were incubated for 48 h at 37 C. and 5% CO2.
(42) To select cells with a knockout PD-1 gene and constitutively increased TRAIL expression, 48 h after transfection, the cells were stained with a mixture of anti-PD-1 antibodies labeled with fluorescein and anti-TRAIL antibodies labeled with Alexa Fluor 610-R-phycoerythrin. Cells with a minimal fluorescent signal for fluorescein and a maximal signal for Alexa Fluor 610-R-phycoerythrin were selected and cloned.
(43) To obtain labeled anti-TRAIL antibodies, monoclonal mouse anti-TRAIL antibodies (Manufacturer: Abeam, clone 2E5, cat. ab2219) and a Zenon Alexa Fluor 610-R-Phycoerythrin Mouse IgG1 Labeling Kit (Manufacturer: Invitrogen, cat. no. Z25020) were used.
(44) The expression level of PD-1 in clones was assessed by immunoblotting with anti-PD-1 antibodies, similar to the protocol described for obtaining NK-cells with a knockout PD-1 gene. In clones with confirmed PD-1 knockout, the expression level of TRAIL was assessed. Immunoblotting was performed with anti-TRAIL antibodies (Manufacturer: Abcam, clone 75411.11, cat. no. ab10516) using a method similar to PD-1. As a result of the analysis, cells were selected in which the maximum signal of the bands corresponding to TRAIL was detected.
(45) The cell lines HeLa (human cervical adenocarcinoma), K562 (human myeloid leukemia), A172 (human glioblastoma), and T98G (human glioblastoma) were obtained from the Russian collection of vertebrate cell cultures of the Institute of Cytology, Russian Academy of Sciences (St. Petersburg).
(46) The possibility of using the invention is illustrated by examples confirming the cytotoxic activity of NK92-PD1 and NK92-PD1-TRAIL cells to induce apoptosis or lysis of various types of mammalian cancer cells.
Example 1. Evaluation of the Cytotoxic Activity of NK92 and NK92-PD1 Cells Incubated with Target Cells K562 (Human Myeloid Leukemia), and With Target Cells HeLa (Adenocarcinoma of the Human Cervix)
(47) In the wells of a 96-well plate, 100 L of a suspension of HeLa or K562 cells (2*10.sup.4/well) in DMEM (Gibco) or RPMI 1640 (Gibco) media containing 10% fetal calf serum (Hyclone), respectively, were added and incubated for 16 h at 37 C. and 5% CO2. Then, 50 L of a suspension of NK92 and NK92-PD1 cells (6*10.sup.4/well) in MEM medium supplemented with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 250 U/ml recombinant interleukin 2 (Prospec), and 25% fetal calf serum (Hyclone) were added to the wells with cells. The cells were incubated for 6 hours at 37 C. and 5% CO2 and visualized with a microscope in transmitted light (see
Example 2. Comparative Growth Kinetics of the NK92 and NK92-PD1 Cultures
(48) To assess proliferative activity, 100 L of a suspension of NK92 or NK92-PD1 cells (1*10.sup.4/well) in an MEM medium supplemented with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 400 U/ml recombinant interleukin 2 (Prospec), and 25% fetal bovine serum (Hyclone) were added to the wells of a 96-ell plate and incubated for 72 h at 37 C. and 5% CO2. Every 24 h, aliquots of cells were taken from 5 wells for each type of cells, an equal volume of a 0.4% trypan blue solution was added to them, and the number of living cells was counted using a Goryaev camera. The result of evaluating the growth kinetics of NK92 and NK92-PD1 cultures is shown in
Example 3. Assessment of the Cytotoxic Activity of NK92 and NK92-PD1-TRAIL Cells on A 172 Cells (Human Glioblastoma)
(49) In the wells of a 96-well plate, 100 L of a suspension of A172 cells (2*10.sup.4/well) in an MEM medium supplemented with 10% fetal bovine serum (Hyclone) was added and incubated for 16 h at 37 C. and 5% CO2. Then, 50 L of a suspension of NK92 and NK92-PD1-TRAIL cells (6*10.sup.4/well) was placed in an MEM medium supplemented with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 250 U/ml recombinant interleukin 2 (Prospec), and 25% fetal calf serum (Hyclone). The cells were incubated for 6 h at 37 C. and 5% CO2 and visualized with a microscope in transmitted light. Wells with A172 cells were washed with buffered saline, the remaining cells were stained with a 0.2% solution of neutral red and evaluated for light absorption (wavelength-540 nm) using a plate photometer. The percentage of surviving A172 cells was determined as the ratio of the optical density of the experimental wells (A172 with NK92 or NK92-PD1 PD1-TRAIL) and the optical density of the control wells with A172 cells. The result of evaluating the cytotoxic activity of NK92 and NK92-PD1-TRAIL cells incubated with target A172 cells is shown in
Example 4. Evaluation of the Cytotoxic Activity of NK92 and NK92-PD1-TRAIL Cells on T98G Cells (Human Glioblastoma)
(50) 100 L of a suspension of T98G cells (2*10.sup.4/l) in MEM medium supplemented with 10% fetal bovine serum (Hyclone) was added to the wells of a 96-well plate and incubated for 16 h at 37 C. and 5% CO2. Then, 50 L of a suspension of NK92 and NK92-PD1-TRAIL cells (6*10.sup.4/well) in MEM medium supplemented with 2 mM L-glutamine, sodium bicarbonate (1.5 g/L), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 250 U/ml recombinant interleukin 2 (Prospec), and 25% fetal calf serum (Hyclone). The cells were incubated for 8 h at 37 C. and 5% CO2 and visualized with a microscope in transmitted light. Wells with T98G cells were washed with buffered saline, the remaining cells were stained with 0.2% neutral red solution and evaluated for light absorption (wavelength-540 nm) using a plate photometer. The percentage of surviving cells was determined as the ratio of the optical density of the experimental wells (T98G with NK92 or NK92-PD1 PD1-TRAIL) and the optical density of the control wells (T98G cells). The result of evaluating the cytotoxic activity of NK92 and NK92-PD1-TRAIL cells incubated with T98G target cells is shown in
INDUSTRIAL APPLICABILITY
(51) In accordance with the object of the invention, modified NK-cells, NK-cell lines, or compositions thereof with increased cytotoxicity are intended for use in the treatment of cancer in a patient. In preferred embodiments of the invention, the modified NK-cell, NK-cell line, or a composition thereof is intended for use in the treatment of blood cancer, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). A modified NK-cell and an NK-cell line can be used to treat: Hodgkin's lymphomas and non-Hodgkin's lymphomas, including T-cell lymphomas, B-cell lymphomas, asymptomatic myelomas, smoldering multiple myelomas (SMM), active myelomas, or myeloma light chains.
INFORMATION SOURCES
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