RU2712901C1 - Psca-car-yt cell line having surface expression of chimeric antigen receptors and exhibiting cytotoxic activity on psca-positive human cancer cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to immunology. Disclosed is a recombinant cell line PSCA-CAR-YT, having surface expression of chimeric antigenic receptors and exhibiting cytotoxic activity with respect to PSCA-positive human cancer cells.

EFFECT: present invention can find further application in therapy of PSCA-associated cancer.

1 cl, 5 dwg, 5 ex

Description

The invention relates to the field of biotechnology, medicine and genetic engineering, in particular to the production of cell lines used in immunotherapy of prostate adenocarcinoma.

To date, prostate cancer is one of the most common oncological diseases among men. According to global statistics, prostate cancer remains the third leading cause of death from malignant tumors in older men. Due to the use of surgical approaches in combination with hormonal, chemo and / or radiotherapy in the treatment of prostate neoplasms, the survival rate of patients at the 1st-2nd stage of this disease is about 85%. At the same time, due to the absence of pronounced symptoms and reliable diagnosis in the early stages of the disease, approximately 90% of cases of prostate cancer are detected in the late (III-IV) stages of the disease, which leads to death of more than half of patients and continues to be a serious problem all over the world [1].

The incidence of pancreatic cancer is lower than that of prostate cancer, but the first type of cancer can be treated much worse. Five-year survival for patients diagnosed with pancreatic cancer does not exceed 8% [2]. A characteristic feature that combines prostate and pancreatic cancers is the frequent expression of PSCA protein by cancer cells on its surface. In addition to cancer cells, this protein can be expressed at a low level by normal prostate epithelial cells, however, prostate tissue is not critical for life and, according to vital indications, therapy that eliminates PSCA-positive cells can be extremely effective in controlling metastases in advanced cases. pancreatic and prostate cancer.

Thus, the development of new targeted approaches for the treatment of cancer through the elimination of PSCA-positive cells is a highly demanded task.

Adaptive cell immunotherapy using T cells expressing chimeric antigenic receptors (CARs) (hereinafter CAR-T cells) allows redirecting the cytotoxic activity of T lymphocytes against tumor cells carrying the target surface antigen. This approach is based on the delivery of CAR encoding cassettes to the patient's own T-lymphocytes cultivated ex vivo, followed by their introduction back into the patient's body. Propagating in the body, CAR-T cells are able to find and destroy tumor cells that expose a recognized CAR antigen [3].

CAR is an artificially engineered protein molecule. The main functional-structural domains of CAR are: 1) an antigen-recognizing domain, which is often used as variable regions of the light and heavy chains of antibodies (scFv), combined into a polypeptide chain using a short linker. This part of the receptor is responsible for the recognition of the target antigen by the receptor and is often borrowed from therapeutically approved antibodies; 2) the hinge region of CAR is the most mobile part of the receptor, which provides optimal mutual positioning of the target antigen and antigen-recognizing domain in the immunological synapse when the effector cell meets the target cell. Most often, the sequences CD8a, CD28 and IgG1 / IgG4 (hinge-Fc part) are used to create this part of the receptor [3,4]; 3) the transmembrane domain is necessary for fixing the receptor on the membrane; 4) the signal domain activates the CAR host cell. Most often in clinical trials, such signal domain combinations as CD28-CD3ζ or 4-1BB-CD3 are used [3, 5, 6].

In contrast to oncohematological diseases of B-cell nature, for which CAR-T-cell therapy has proved extremely effective, the use of this technology in relation to solid types of cancer, including prostate cancer, faces a number of difficulties, in particular, with immunosuppressive tumor microenvironment, ineffective “homing” and insufficient lifetime of CAR-T cells in the active state in the patient’s body. According to published data, one of the proteins overexpressed by tumors of the prostate [7] and pancreas [8], as well as their metastases [9] in some patients, is a PSCA-GPI-anchored surface protein with a length of 123 amino acid residues, which has an extremely narrow expression pattern in normal tissues [7,10]. PSCA has been successfully used as a target in the context of other immunotherapeutic approaches [11-13].

The use of CAR-T cells specific for PSCA-positive cancer cells, thus, is one of the promising directions of adaptive immunotherapy for prostate and pancreatic cancer, and is consistent with convincing preclinical data on the effectiveness of such therapy.

In addition to autologous CAR-T cells produced individually for each patient, other cellular sources are also considered as CAR carriers, for example, genomically edited “universal” CAR-T cells, peripheral or umbilical cord blood NK cells, iPS-NK cells and immortalized NK cell lines [14-19]. The advantages of using the latter include cheap production, standardization, limited proliferative potential, homogeneous composition, the possibility of combining and using several CAR-NK cell products sequentially or simultaneously.

Several human NK cell lines have been described that can be used as allogeneic universal CAR carriers: NK-92, NKL, YT, YTS, KHYG-1, etc. Thus, in clinical trials of the IL-2-dependent NK-92 line, the safety of their systemic administration after lethal exposure was shown [20-22].

CAR-NK cell systems based on the NK-92, KHYG-1, and YTS cell lines were characterized in detail preclinically and showed their activity against a number of human tumors, xenografted immunodeficient mice [23]. In particular, it was found that the PSCA-specific CAR expressed by YTS cells is able to specifically recognize PSCA-positive target cells and lead to their elimination in mice [24].

Closest to the claimed cell line, the prototype is a YT cell line with surface expression of chimeric antigenic receptors (CARs) that specifically recognize cancer-embryonic antigen (CEA). The known line was obtained on the basis of the human NK cell line, YT, into the cells of which a genetic construct encoding the scBW431-hFc receptor [zeta] was delivered by electroporation. This chimeric receptor consists of a specific single-chain scFv fragment (borrowed from the variable regions of humanized CEA-specific antibodies BW431) connected by a flexible peptide linker. The hinge and transmembrane region of the receptor are the Fc part of the human IgG antibody, and the CD3ζ sequence of the human T-cell receptor is used as the signal domain. The proposed cell line is capable of causing lysis of cells of human colorectal cancer lines [25]. A disadvantage of the known line is the insufficiently stable expression of CAR in the obtained cells, since the chosen method of delivery of the scBW431-hFc [zeta] receptor sequence to the YT cell genome does not ensure stable insertion of this sequence in the genome of receptor carrier cells, respectively, over time, this cell line may lose CAR expression and lose the ability to exert a cytotoxic effect on cancer cells. In addition, the known YT cell line does not have cytotoxic activity against human PSCA-positive cancer cells; it exhibits a cytotoxic effect only against human colorectal cancer cells.

An object of the present invention is to provide a new stable cell line PSCA-CAR-YT having surface expression of chimeric antigenic receptors and exhibiting cytotoxic activity against human PSCA-positive cancer cells.

The technical result of the claimed invention: expanding the collection of recombinant cell lines that can be used for immunotherapy of malignant neoplasms, in particular prostate adenocarcinomas.

The indicated technical result is achieved by transduction of the YT cell line with lentiviral particles previously prepared using calcium phosphate transfection of the HEK293T cell line with the recombinant aPSCA-myc-IgG1v2-CAR plasmid [26], in the structure of which a chimeric antigenic receptor is encoded against the PSCA protein, which ensures stable insertion of the CAR sequence into the genome of the YT cell line, thereby ensuring stable expression of CAR in the obtained cells.

The essence of the claimed invention is as follows.

The recombinant plasmid aPSCA-myc-IgG1v2-CAR, constructed on the basis of the lentiviral vector pCDH (corresponding GenBank number KX757242.1), which has the following characteristics, is constructed:

- has a size of 9869 p .;

- encodes the basic functional and structural elements of CAR specific for the PSCA protein.

- consists of the following elements:

1) a DNA fragment encoding CAR, namely, the sequence of the antigen-recognizing module of the PSCA-specific CAR, which was borrowed from the humanized mouse antibody 2B3 using the scFv DNA coding sequence (VL2B3 and VH2B3) [27]. The hinge region sequence borrowed from the human IgG1 molecule (hinge CH2-CH3), the transmembrane domain from the CD28 molecule (CD28TM) and the signal domain of CD28-CD3z are the cytoplasmic sequence of the signal subunit of the T cell receptor;

2) a hybrid constitutive promoter hEF1a-HTLV, providing constitutive and strong expression of CAR and copGFP;

3) genetic markers:

AMPr is the ampicillin resistance gene (bla), which determines ampicillin resistance during Escherichia coli transformation;

copGFP - the reporter gene of the green fluorescent protein of copepod Pontellina plumata, which determines the luminescence of transformed or transduced mammalian cells when they are irradiated in the blue-green UV range;

myc-epitope - located between the antigen-recognizing and hinge region in the structure of CAR and necessary to detect the release of CAR to the cell surface.

4) a hybrid RSV-5'LTR promoter consisting of Routh sarcoma virus (RSV) and HIV-1 sequences, providing TAT-independent synthesis of viral transcripts in pack cells of lentiviral particles;

5) modified genetic elements of HIV-1 (cpp, gag, env, RRE) necessary for the correct processing of viral RNA and assembly of lentiviral particles;

6) 5'LTR and 3'dLTR sequences of HIV-1, ensuring stable integration of the sequences enclosed between them into the genome of mammalian cells without the possibility of their further re-mobilization;

7) the sequence of WPRE - a regulatory element of the marmot hepatitis virus, providing increased stability and transcription of transcripts;

8) a DNA fragment containing the Kozak sequence;

9) the IRES element - the internal site of the landing of the ribosome of cardiovirus A, which provides cap-independent translation initiation of the copGFP linked reporter.

The advantage of using this design is its low immunogenicity, which is especially important in further work on in vivo models and in the clinic.

A map of the genetic construct of the recombinant plasmid aPSCA-myc-IgG1v2-CAR is shown in FIG. 1, and the circuit of the resulting CAR is shown in FIG. 2.

The PSCA-CAR-YT cell line is obtained on the basis of the YT cell line by its transduction (infection) with lentiviral particles. To obtain lentiviral particles, the DNA of the recombinant plasmid aPSCA-myc-IgG1v2-CAR is mixed with the DNA of auxiliary plasmids pMD.2G and psPAX2 necessary for packaging lentiviral particles [28] in a molar ratio of 4: 1: 3 and transform human embryonic renal epithelium cells HEK293 by calcium phosphate transfection, after which they are grown for 6 hours in IMDM medium with the addition of fetal bovine serum up to 10%, 100 μg / ml streptomycin and 100 units / ml penicillin in an atmosphere of 5% CO ₂ at 37 ° C. 6 hours after transfection, the medium in the plates is replaced with a new one and the cells are incubated for 48 hours. After that, the supernatants of the conditioned medium are filtered through 0.45 μm PES filters and used to infect the YT cell line by spinoculation in the presence of polybrene (8 μg / ml).

Cells obtained in this way express on their surface CARs specific for the PSCA protein (Fig. 3) and possess cytotoxic activity against PSCA-positive cancer cells, in particular human prostate adenocarcinoma cells (Fig. 4, Fig. 5).

The PSCA-CAR-YT cell line is characterized by the following features.

Morphological signs. The cell line consists of round cells of equal size, growing solitary or forming clusters. The suspension cell line.

Cultural signs. The PSCA-CAR-YT cell line is cultured on IMDM growth medium containing 4 mM L-glutamine, 10% fetal calf serum, 100 units / ml penicillin and 100 μg / ml streptomycin in an atmosphere of 5% CO ₂ at 37 ° C. 1 × 10 ⁶ cells are seeded in culture dishes with a diameter of 6 cm in 5 ml of medium. The time of subcultivation is 2-3 days, the sieving ratio is 1: 5-1: 6.

With prolonged observation, bacteria and fungi were not found in the culture. Mycoplasma test is negative.

Cryopreservation. For long-term storage, the cell culture is cryopreserved by freezing in liquid nitrogen. The cells are thawed at a temperature of 37 ° C. Cell viability after thawing is evaluated by the inclusion of trypan blue and is at least 90%.

The invention is illustrated by the following figures:

FIG. 1. Map of the genetic design of the recombinant plasmid aPSCA-myc-IgG1v2-CAR, where: VL2B3, VH2B3, IgG1, CD28 TM, CD28 cyto, CD3 cyto are DNA sequences encoding the basic functional structural elements of CAR, hEF1a-HTLV is a constitutive factor promoter human translation elongation alpha, IRES — internal site of cardiovirus A ribosome landing, copGFP — green copepod fluorescent protein Pontellina plumata, Kozak — Kozak RSV promoter sequence — hybrid promoter, 5′LTR — long terminal repeat HIV-1, gag — shortened gene gag HIV-1, RRE - REV-binding HIV-1 element, WPRE - regulatory element woodchucks hepatitis virus, 3'dLTR -usechenny long terminal repeat of HIV-1, AmpR - the ampicillin resistance gene. The total size of the plasmid 9869 p. About.

FIG. 2. The main structural components of CAR: hEF1a-HTLV —constitutional promoter of the human translation elongation factor alpha, SP — leader sequence, scFv (aPSCA) —specific scFv sequences for PSCA protein, myc — epitope for detection, IgG1-hinge region, CD28- CD3z is the cytoplasmic sequence of the signal subunit of the T cell receptor, IRES is the internal site of the ribosome of the encephalomyocarditis virus, and copGFP is the reporter gene (green fluorescent protein GFP).

FIG. 3. A — Cytometric analysis of surface expression of CAR (against PSCA protein) of the transduced YT line after antibody staining for the c-myc epitope. Negative control - intact line YT;

FIG. 3. B - Cytometric analysis of the surface expression of CAR (against PSCA protein) of the transduced YT line after staining with Protein L. Negative control - intact YT line;

FIG. 4. The cytotoxicity level shown by the PSCA-CAR-YT cell line when incubated with target cells in various ratios (1: 1, 2: 1, 3: 1), analyzed using FACS. Cells expressing CAR specific for another α-PSMA target protein (PSMA-CAR-YT), as well as intact YT cells, were used as a negative control and did not exert cytotoxic effects on target cells (PC3-PSCA cell line). All indicators are presented in the form of "mean ± standard deviation".

FIG. 5. Secretion of IFN-gamma by the PSCA-CAR-YT cell line when it is coincubated with human prostate adenocarcinoma cells ectopically expressing PSCA (PC3-PSCA) (blue color), compared with the PC3 cell line (red color). Intact YT cells not expressing CAR were used as a negative control. All indicators are presented in the form of "mean ± standard deviation".

For a better understanding of the essence of the proposed group of inventions, the following are specific examples of their implementation.

Example 1. Transduction of the cell line YT encoding CAR lentiviral particles

DNA cassettes encoding CAR are delivered to YT cells using lentiviral particles pseudotyped by surface protein G of vesicular stomatitis virus (VSV), for which HEK 293T cells are used. HEK293T cells in a 5 cm diameter dish with a confluency of 60-70% are transfected with plasmid preparations using calcium phosphate transfection. For this, the recombinant plasmid DNA aPSCA-myc-IgG1v2-CAR is mixed with the auxiliary plasmid DNA psPAX2 (packaging plasmid encoding GAG, POL and REV under the control of the strong constitutive promoter CAG) and pMD2.G (packaging plasmid encoding the G envelope protein of the vesicular stomatitis virus ) in a 4: 3: 1 molar ratio (5 μg of aPSCA-myc-IgG1v2-CAR plasmid: 3.7 μg psPAX2: 1.3 μg pMD2.G). The resulting mixture was mixed with 31.2 μl of CaCl ₂ and water was added to 250 μl. After this, the mixture is gradually added to 250 μl of sterile HBS (150 mm NaCl, 20 mm HEPES, pH 7.0), vigorously mixing it. The suspension of calcium phosphate complexes and plasmid DNA obtained in this way is carefully dug up on the surface of a plate with transfected HEK293T cells. 6 hours after transfection, the medium in the plates is replaced with a new one and the cells are incubated for 48 hours. After that, supernatants of conditioned media obtained by centrifugation at 20,000 g for 3 hours are filtered through 0.45 μm PES filters to get rid of cell fragments and aggregates and used for spinduction of YT cell lines. To do this, 10,000 cells per well of YT cell line are introduced into the wells of a 96-well plate, after which lentivirus preparation is added to them in different volumes: 25 and 75 μl (with MOI equal to 1), polybrene is also added to each well (Sigma Aldrich ) to a final concentration of 8 μg / ml. After that, the cells are centrifuged at 500g for 40 minutes at 32 ° C, then incubated at a temperature of 37 ° C and 5% atmosphere of CO ₂ . 24 hours after transduction, the medium in the wells of the plate is replaced with a new one.

Example 2. Analysis of surface expression of CAR in cells of the lines of PSCA-CAR-YT.

The surface expression of CAR of the PSCA-CAR-YT cell line is detected by staining the cells with antibodies against the c-myc epitope contained in the CAR structure or by co-incubation of the obtained cells with biotinylated Protein L capable of binding immunoglobulin kappa chains. For this, cells in the amount of ~ 100,000 cells are transferred to a cytometric tube, and precipitated by centrifugation at 500 g for 4 minutes, after which the cells are washed from the remnants of the culture medium in 2 ml of PBS (pH 7.4: 1.7 mM KH ₂ PO ₄ , 5.2 mM Na ₂ HPO ₄ , 150 mM NaCl) containing 1.5% FCS and 0.06% NaN _3; followed by centrifugation. Cells are then incubated with mouse anti-c-myc monoclonal antibodies (Abeam, clone 9E10) or Protein L-bio (GenScript, M00097) at a concentration of 1 μg / μl for 30 minutes at 4 ° C. At the end of the incubation, the cells are washed twice from the primary antibodies with a PBS solution with 1.5% FCS and 0.06% NaN ₃ and incubated with fluorescently-labeled conjugates of secondary antibodies. Donkey antibodies against mouse IgG labeled with PE (BioLegend) are used as conjugates to anti-c-myc antibodies, and streptavidin-PE (Thermo Fisher Scientific) as conjugates to Protein L. The conjugates used are diluted 1: 800 in PBS with 1.5% FCS and 0.06% NaN ₃ . Incubation was carried out for 30 minutes at 4 ° C, after which the samples were washed twice and 1 μg / μl of 7AAD (Biolegend) was added to exclude dead cells from the analysis. Samples were analyzed using a BD FACSCanto II flow cytometer (Becton Dickinson and Company). Analysis of the data obtained is carried out using the BD FACSDiva Software software. The experimental results are presented in FIG. 3 and indicate that, in comparison with intact YT cells, the obtained PSCA-CAR-YT cell line has a high level of surface expression of CAR.

Example 3. Cultivation of the cell line PSCA-CAR-YT

The PSCA-CAR-YT cell line was cultured in IMDM growth medium (Sigma) containing 4 mM L-glutamine, 10% fetal calf serum (HyClone), 100 units / ml penicillin and 100 μg / ml streptomycin in an atmosphere of 5% CO ₂ at 37 ° C. Cells are subcultured once every two days or one day before transduction.

For long-term storage, the cells are cryopreserved by freezing in liquid nitrogen. For this, the cells are resuspended in a freezing medium — IMDM growth medium supplemented with fetal calf serum 10% and 10% DMSO. The temperature is reduced at a rate of 1 ° C per minute to minus 70 ° C, then a quick freezing of the cells in liquid nitrogen is carried out.

Cell thawing is rapid, at a temperature of 37 ° C, after which the cells are diluted in 5 ml of culture medium and precipitated by centrifugation at 500 g. After that, the cell pellet is taken up in 5 ml of fresh culture medium and transferred to a culture dish with a diameter of 6 cm. Cell viability is assessed by the inclusion of trypan blue. Cell viability after thawing is at least 90%.

Example 4. Evaluation of the cytotoxic activity of the PSCA-CAR-YT cell line in relation to human prostate adenocarcinoma cells.

To analyze the cytotoxic activity, PSCA-CAR-YT cell lines are co-incubated with a human prostate adenocarcinoma cell line with ectopic expression of PSCA - PC3-PSCA in various effector: target ratios, followed by analysis of the number of dead target cells using FACS. The higher the percentage of death of target cells, the higher the cytotoxic activity of PSCA-specific effector cells. For this purpose, PC3-PSCA target cells were pre-stained by adding 4 μg / μl of DID fluorescent dye (ThermoFisher Scientific) to the culture medium and incubated for 7 minutes. Then, the labeled target cells, like the PSCA-CAR-YT effector cells, are washed from the old culture medium. Then, 20,000 labeled target cells per well of the plate are introduced into a 96-well plate, and effector cells are added to them in the ratios effector: target: 1: 1, 2: 1, 3: 1. In one well, target cells without effectors are left as control, and YT-CAR cells with different specificity (CAR specific for PSMA protein) are added to target cells as a negative control. The experiment is carried out in two biological repeats for each effector: target ratio. Cells are incubated for 4 hours in an atmosphere of 5% CO ₂ at 37 ° C. Then add vital dye PI (propidium iodide). The percentage of dead target cells is measured on a BD FACSCantoII cytofluorimeter. Analysis of the data obtained is carried out using the BD FACSDiva Software software. Presented in FIG. 4 results indicate that the obtained PSCA-CAR-YT line has a cytotoxic effect on PSCA-positive target cells and causes the death of human prostate cancer cells.

Example 5. Measurement of the level of IFN-gamma secretion by the PSCA-CAR-YT cell line during co-incubation with human prostate cancer cells.

PSCA-CAR-YT cells were co-incubated with PC3-PSCA cells in a 24-well plate for 4 hours in a 1: 1 ratio. For incubation, IMDM medium (ThermoFisher Scientific) was used with 10% fetal bovine serum (HyClone), 100 units / ml penicillin and 100 μg / ml streptomycin that did not contain human cytokines, incl. IL-2 and IFN-gamma. After incubation, the supernatants of the conditioned media were filtered through a filter with a pore diameter of 0.45 microns to get rid of cell fragments and aggregates. The filtered supernatant was used to perform ELISA analysis using a gamma-Interferon-IFA-BEST kit (Vector-BEST JSC, Russia). For accurate concentration determination, IFN-gamma concentration standards included in the kit were used. The obtained data presented in FIG. 5 show a high level of IFN-gamma secretion in PSCA-CAR-YT cells compared to cells of the original YT line not transduced with lentiviral particles encoding CAR.

Thus, the obtained PSCA-CAR-YT cell line has surface expression of CAR and exhibits cytotoxic activity against PSCA-positive cancer cells. This cell line can be used in the development of cell therapy for patients with PSCA-positive prostate cancer.

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Claims (11)

Recombinant cell line PSCA-CAR-YT with surface expression of chimeric antigenic receptors and exhibiting cytotoxic activity against human PSCA-positive cancer cells obtained by transduction of the YT cell line with lentiviral particles based on the recombinant plasmid aPSCA-myc-IgG1v2-CAR, characterized in accordance with the physical map shown in FIG. 1, by the following features: it has a size of 9869 bp, encodes the basic functional and structural elements of CAR specific for the PSCA protein, consists of the following elements: 1) a DNA fragment encoding CAR; 2) a hybrid constitutive promoter hEF1a-HTLV, providing constitutive and strong expression of CAR and copGFP; 3) genetic markers: AMPr - gene for ampicillin resistance (bla), copGFP - reporter gene for the green fluorescent copepod protein Pontellina plumata; 4) myc-epitope - a service module located between the antigen-recognizing and hinge region in the structure of CAR. 5) a hybrid RSV-5'LTR promoter consisting of Routh sarcoma virus (RSV) and HIV-1 sequences, providing TAT-independent synthesis of viral transcripts in pack cells of lentiviral particles; 6) modified genetic elements of HIV-1 (cpp, gag, env, RRE); 7) 5'LTR and 3'dLTR sequences of HIV-1; 8) WPRE sequences - a regulatory element of the marmot hepatitis virus; 9) a DNA fragment containing the Kozak sequence; 10) the IRES element - the internal site of landing of the ribosome of cardiovirus A.

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