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WO2017031760A1 - Fragment interférent et applications associées - Google Patents

Fragment interférent et applications associées Download PDF

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Publication number
WO2017031760A1
WO2017031760A1 PCT/CN2015/088286 CN2015088286W WO2017031760A1 WO 2017031760 A1 WO2017031760 A1 WO 2017031760A1 CN 2015088286 W CN2015088286 W CN 2015088286W WO 2017031760 A1 WO2017031760 A1 WO 2017031760A1
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cells
expression
immune
cell
lymphocytes
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Chinese (zh)
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杨光华
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention relates to the field of immunobiology technology, in particular to interference fragments and their applications.
  • Malignant tumors are the main type of fatal disease in humans and the mortality rate remains high.
  • the latest statistics from the Ministry of Health indicate that malignant tumors rank first in the top ten causes of death among Chinese residents.
  • the annual medical expenses for patients with malignant tumors account for about 20% of the total health expenditure, which is the largest medical burden for the whole society.
  • the current treatments are still very limited in achieving the goals of curing, prolonging the survival of patients with malignant tumors and improving the quality of life.
  • metastasis whole body Sexual or advanced malignancies are more difficult to cure.
  • systemic treatment includes traditional chemotherapy, molecular targeted drug therapy, biological therapy (including stem cell transplantation therapy and immune cell therapy), and gene therapy.
  • traditional chemotherapy has made some progress in the treatment of certain tumors, it still contributes little to prolonging the survival of cancer patients.
  • the natural resistance of tumor cells to chemotherapeutic drugs and acquired multidrug resistance and chemotherapy toxicity have seriously hindered the development of chemotherapy.
  • Clinical practice over the years has proven that it is not an advantage program for systemic therapy.
  • Immune cell therapy has been developed for nearly two decades. Because of its wide therapeutic range (can be used for solid tumors and leukemia), it is especially effective for small tumor lesions (including metastasis, recurrence, cancer cells in the blood). Side effects, but also applicable to all stages of the tumor (such as late release, chemotherapy can not be used), so it is an advantage of systemic treatment. Immune cell therapy has a significant effect on improving quality of life and prolonging survival. It is highly praised at home and abroad and is one of the best methods for systemic therapy at present. The clinical potential is huge.
  • PD-1 belongs to costimulatory molecule 28 (CD28)/cytotoxic lymphocyte associated antigen (Cytotoxic lymphocyte associated antigen). An inhibitory receptor of the CTLA-4) family. PD-1 is not only a marker of programmed cell death, but also expressed on the surface of B lymphocytes, T lymphocytes, NK cells, NKT cells, and macrophages. In recent years, it has been found that the surface of T lymphocytes activated by antigens transiently expresses PD-1, and the sustained expression of PD-1 may cause T cell dysfunction. The extensive expression and immunosuppressive function of PD-1 has made it the focus of immunotherapy in recent years.
  • PD-1 plays an important role in the treatment of diseases such as cancer, autoimmune diseases, and chronic viral infections.
  • diseases such as cancer, autoimmune diseases, and chronic viral infections.
  • the main application of PD-1 is mainly to develop antibodies that specifically block PD-1 signaling, and then to use such antibody drugs for the treatment of advanced cancer and metastatic cancer.
  • the therapeutic effect is acceptable, it is often Drug-related adverse events occur.
  • Antibody drugs that specifically block PD-1 signaling are used in the treatment of advanced cancer and metastatic cancer. Although the therapeutic effect is acceptable, the total incidence of drug-related adverse events (AEs) is 41%, 3/4. The grade of serious drug AEs is 5%, including: skin (16%), gastrointestinal reactions (12%), and lungs (7%). The incidence of drug-related pneumonia was (6%), and the incidence of grade 3/grade pneumonia was 2% (2/129). Two patients died of pneumonia in the early stage of the trial.
  • Antibodies that block the PD-1 signaling pathway do not act directly on tumor tissue but act by activating anti-tumor immunity. Due to the complexity of the immune response and individual differences, the therapeutic effects of these antibodies vary widely among different types of cancer patients and between individuals. As mentioned earlier, the side effects of PD-1 inhibitors (antibodies) after intravenous reinfusion are present, and patients with systemic immune disease should not use this drug because PD-1 inhibitors can enhance the killing ability of immune cells. Increase the immune response and worsen the condition. There are also some patients who have incomplete response after antibody treatment.
  • an object of the present invention is to provide a means for effectively inhibiting the expression of PD-1 in an immune cell, thereby inhibiting the expression of PD-1 in an immune cell, thereby further suppressing the obtained immune cell of PD-1 expression. It is used for preparing immunotherapeutic drugs and is used for immunotherapy of tumors, thereby effectively solving the above problems and improving the application range and application effect of PD-1 in immunotherapy.
  • the present invention successfully constructs a lentiviral vector targeting shRNA of PD-1 by using genetic engineering technology, and screens a target sequence with an interference efficiency of more than 90%; and then, based on the interference obtained by screening
  • the target sequence is transferred to the immune cells, T lymphocytes (including CTL, TIL, CD8+T, NKT cells, etc.) by the pd-1 shRNA lentiviral vector carrying the corresponding interference fragment.
  • the expression of PD-1 is inhibited for a long period of time, and the immune cells inhibiting the expression of PD-1 are obtained.
  • the expression of the proliferating cell PD1 obtained after the cells enter the human body is also inhibited; further, the inventors return the modified T cells. Human or tumor local, and found that It can effectively improve the effect of immunotherapy and minimize side effects.
  • the invention provides an interference segment.
  • the target of the interference segment is a nucleotide sequence as shown below:
  • the interference fragment of the present invention has good targeting properties against the above-mentioned target, and the interference suppressing effect on PD-1 is outstanding.
  • transferring the above interference fragment into an immune cell T lymphocytes (for example, CTL, TIL, CD8+T, NKT cells, etc.), can cause long-term suppression of PD-1 expression in the immune cell.
  • the immune cells inhibiting the expression of PD-1 are obtained, and the expression of the proliferating cell PD1 obtained after the cells enter the human body is also inhibited, whereby the obtained immune cells inhibiting the expression of PD-1 can be effectively used for the preparation of immunotherapeutic drugs. Furthermore, it is used for immunotherapy of tumors, effectively improving the effect of immunotherapy and minimizing side effects.
  • interference segment according to the above embodiment of the present invention may also have the following additional technical features:
  • the interference segment consists of a sense strand and an antisense strand having the nucleotide sequence:
  • Antisense strand 5'-AATTCAAAAAAGCCTGTGTTCTCTGTGGACTATCTCTTGAATAGTCCAC AGAGAACACAGGCG-3' (SEQ ID NO: 7).
  • the present invention also provides a method of inhibiting PD-1 expression in an immune cell.
  • the method comprises: transferring the interference segment described above into the immune cell.
  • the method can effectively inhibit the expression of PD-1 in immune cells and obtain immune cells with inhibition of PD-1 expression. Furthermore, the obtained immune cells inhibiting the expression of PD-1 can be effectively used for the preparation of immunotherapeutic drugs, thereby obtaining an immunotherapeutic agent having a good therapeutic effect and low side effects; in addition, the immune cells inhibiting the expression of PD-1 can also be directly used. It is used to return to the human body or the tumor, so that it can effectively treat or prevent tumors.
  • the method of transferring the interference fragment into the immune cell is not particularly limited.
  • the interference fragment is transferred into the immune cell using a lentiviral vector system. Thereby, the interference fragment is easily transferred into the immune cell, thereby effectively improving the interference efficiency and realizing the inhibition of PD-1 expression in the immune cell.
  • the immune cell is a lymphocyte, preferably a T cell, a B cell, an NK cell, an NKT cell, more preferably a tumor infiltrating T lymphocyte or a CTL cell.
  • the immune cells inhibiting the expression of PD-1 can be efficiently obtained, and further, the obtained immune cells inhibiting the expression of PD-1 can be used for the preparation of an immunotherapeutic drug, and an immunotherapeutic agent having a good therapeutic effect and a low side effect can be effectively obtained. It can be directly used; the immune cells inhibited by PD-1 expression can also be directly used for reintroduction of human or tumor parts, thereby effectively treating or preventing tumors.
  • immune cells mainly refers to lymphocytes in human blood, such as T lymphocytes, B lymphocytes, NK lymphocytes, and NKT lymphocytes.
  • T lymphocytes may be tumor infiltrating T lymphocytes, and various T cells, such as CTL cells, cultured or induced in vitro.
  • CTL cell refers to a cytotoxic T lymphocyte that is stimulated by autologous lymphocytes by DC cells loaded with tumor antigen and mixed with autologous lymphocytes.
  • the present invention also provides the use of the aforementioned interference fragment for the preparation of an immunotherapeutic drug.
  • the drug comprises an immune cell in which PD-1 expression is inhibited. Therefore, the administration of the drug to a tumor patient can effectively inhibit the expression of PD-1 in the tumor cells of the patient, thereby effectively treating the tumor; in addition, the drug can be effectively used for the prevention of the tumor, and the effect of treating or preventing the tumor is good, and the side effect is good. low.
  • the immune cell in which PD-1 expression is inhibited is obtained by transferring the interference fragment described above into the immune cell to obtain an immune cell in which PD-1 expression is inhibited.
  • immune cells in which PD-1 expression is inhibited can be efficiently obtained.
  • the interference fragment is transferred into the immune cell using a lentiviral vector system.
  • the interference fragment is easily transferred into the immune cell, the interference efficiency is high, and the inhibitory effect on the expression of PD-1 in the immune cell is good.
  • the immune cell is a lymphocyte, preferably a T cell, a B cell, an NK cell, an NKT cell, more preferably a tumor infiltrating T lymphocyte or a CTL cell.
  • a lymphocyte preferably a T cell, a B cell, an NK cell, an NKT cell, more preferably a tumor infiltrating T lymphocyte or a CTL cell.
  • the present invention also provides an immunotherapeutic drug.
  • the immunotherapeutic drug comprises immune cells inhibited by PD-1 expression, and the immune cells inhibited by the PD-1 expression inhibit the immune cells by the method of inhibiting PD-1 expression in the immune cells as described above. Obtained in PD-1 expression. Therefore, the administration of the immunotherapeutic agent of the present invention to a tumor patient can effectively inhibit the expression of PD-1 in the tumor cells of the patient, thereby effectively treating the tumor; in addition, the medicament can be effectively used for the prevention of tumors, and for treating or preventing tumors. Good effect and low side effects.
  • FIG. 1 shows a schematic structural view of an LV3 carrier according to an embodiment of the present invention
  • Figure 2 shows the results of western detection of PD-1 protein in cells of interest transfected with each shRNA lentiviral vector, according to one embodiment of the present invention
  • Figure 3 shows the results of flow cytometry analysis of the number of PD-1 positive cells in the cells of interest transfected with each shRNA lentiviral vector, in accordance with one embodiment of the present invention.
  • a control RNA (negative control) was set up, and three PD-1 shRNA lentiviral vectors were constructed as follows:
  • DNA oligo was designed using Designer3.0 (Genepharma) software, and the synthetic primers were completed by Shanghai Jima Pharmaceutical Technology Co., Ltd.
  • the loop structure in the LV3-shRNA template selected TTCAAGAGA to avoid the formation of a termination signal; the 5' end of the sense strand template added GATCC, which is complementary to the sticky end formed by BamHI digestion; the antisense strand template AATTC was added to the 5' end to complement the sticky ends formed by EcoRI digestion, as follows:
  • shUN the specific information of shUN and three PD-1 shRNA lentiviral vectors are as follows:
  • control shNC The specific information of the control shNC is:
  • Target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 1);
  • Antisense strand sequence
  • Plasmid information of three PD-1 shRNA lentiviral vectors: PDCD1-Homo-694, PDCD1-Homo-791 and PDCD1-Homo-238 are shown in Table 1-3, respectively.
  • the DNA oligo was dissolved with TE (pH 8.0) at a concentration of 100 ⁇ M.
  • TE pH 8.0
  • the corresponding sense chain and antisense strand oligo solution were taken, and the annealing reaction system was configured according to the following ratio:
  • Annealing was carried out on a PCR machine according to the following procedure: 95 ° C for 5 min; 85 ° C for 5 min; 75 ° C for 5 min; 70 ° C for 5 min; 4 ° C for storage. After annealing, a shRNA template was obtained at a concentration of 10 ⁇ M. The resulting template solution was diluted 50-fold to a final concentration of 200 nM for the ligation reaction.
  • the ligation reaction of the vector was carried out according to the following system:
  • Competent cells are prepared by the calcium chloride method, and the specific steps are as follows:
  • the bacteria were transferred to a sterile, single-use, ice-cold 50 ml polypropylene tube, placed on ice for 10 minutes, and the culture was allowed to cool to 0 °C.
  • the cells were recovered by centrifugation at 4000 rpm for 10 minutes at 4 °C.
  • the cells were divided into small portions (100 ⁇ l/branch) and stored frozen at -70 °C.
  • the plate was poured into a 37 ° C incubator and cultured for 16 hours.
  • the positive clones were screened by the following steps and verified by sequencing:
  • the correct sequencing strains were extracted using a high-purity plasmid medium extraction kit, and the resulting plasmids can be used in conventional molecular biology experiments and cytological experiments. If the cytotoxicity is large when used for cell transfection, re-transform into E. coli Top10, and then prepare a higher purity plasmid using the kit or CsCl ultracentrifugation.
  • Lentiviral packaging cell transfection The 293T cells in the logarithmic growth phase were digested with trypsin, plated in 6-well plates, cultured in a 50 mL/L CO 2 incubator at 37 ° C, and the cell density was 60% to 70%. Dyeing. Wash cells twice with PBS before transfection.
  • the cells isolated from 100 ml of peripheral blood collected from 1 AFP-positive liver cancer patient were transferred to a 50 ml centrifuge tube for centrifugation (1800 rpm, 10 min), the supernatant was aspirated, and the supernatant was slowly added to the lymphocyte separation. On a liquid Ficoll solution (GE), the volume ratio is 1:1, centrifuged (2000 rpm, 20 min);
  • the white floc cells of the collection interface were added to the PBS, gently pipetted and mixed, and centrifuged (1500 rpm, 10 min);
  • the collected cells were gently pipetted and mixed, and 20 ml of the basal medium was added and uniformly blown, transferred to a 75 cm 2 culture flask, and cultured at 37 ° C in a 5% CO 2 incubator for 2 hours.
  • the remaining 2 ml of the remaining solution in the original vial (75 cm 2 flask) will be used to induce differentiation of monocytes into DC cells.
  • CTL1 medium preparation methods of CTL1 medium, CTL2 medium and CTL3 medium are:
  • CTL1 medium Take 1 tube of CTL1 factor from -20 degrees, melt at room temperature, add to 20ml of basal medium, And add 10% of the customer's serum, which is filtered using a 0.22um needle filter.
  • CTL2 medium One tube of CTL2 factor was taken out from -20 degrees, melted at room temperature, and added to 20 ml of basal medium, and a 0.22 um needle filter was filtered and used.
  • CTL3 medium One tube of CTL3 factor was taken out from -20 degrees, melted at room temperature, and added to 500 ml of basic medium, wherein a 0.22 um needle filter was used for filtration, and three tubes were used for the entire culture process.
  • CTL1 factor (including gamma interferon, 1000 U/ml): 1 ml, long-term storage at -20 °C, and stored at 4 °C for 2 weeks.
  • CTL2 factor including IL-1, 1000 U/ml; IL-2, 1000 U/ml; CD3 antibody, 100 ng/ml; CD28 antibody, 100 ng/ml: 1 ml, long-term storage at -20 ° C, and stored at 4 ° C for 2 weeks.
  • CTL3 factor (including IL-2, 1000 U/ml; IL-7, 20 ng/ml; IL-15, 20 ng/ml): 1 ml, long-term storage at -20 ° C, and stored at 4 ° C for 2 weeks.
  • the target cells are obtained.
  • the cells of interest were divided into 6-well plates, 10 6 cells per well, and used.
  • the collected virus supernatant was added to the target cells obtained by the above preparation, and an equal volume of fresh medium (CTL3 medium) was added to culture the cells of interest (total 3 ml).
  • the preparation method is as follows:
  • Glycine 2.9g; Tris 5.8g; SDS 0.37g; methanol 200ml; add ddH2O to 1000ml.
  • Blocking solution TBST containing 5% skim milk powder.
  • Electrophoresis An electrophoresis gel was prepared and subjected to SDS-PAGE.
  • the PVDF film was covered with glue, coated with filter paper and sponge, and rolled with a test tube to remove air bubbles.
  • Horseradish peroxidase-conjugated secondary antibody (rat anti-human) (diluted with TBST) was added and shaken smoothly for 2 hr at room temperature.
  • Interference efficiency of PDCD1-Homo-791 (target sequence SEQ ID NO: 10: GCCACCATTGTCTTTCCTAGC): 78%;
  • Interference efficiency of PDCD1-Homo-238 (target sequence SEQ ID NO: 15: GCTAAACTGGTACCGCATGAG): 8%;
  • Interference efficiency of PDCD1-Homo-694 (target sequence SEQ ID NO: 5: GCCTGTGTTCTCTGTGGACTA): 88%.
  • PD791 GCCACCATTGTCTTTCCTAGC (SEQ ID NO: 10),
  • PD694 GCCTGTGTTCTCTGTGGACTA (SEQ ID NO: 5),
  • the PD238 has no interference effect on PD1.
  • Two important PD1 interference targets are obtained in the present invention.
  • the interference fragment carried by the PD694 lentiviral vector was transferred into an immune cell to prepare an immune cell which inhibited the expression of PD-1, and the specific steps were as follows:
  • the cells isolated from 100 ml of peripheral blood collected from 1 AFP-positive liver cancer patient were transferred to a 50 ml centrifuge tube for centrifugation (1800 rpm, 10 min), the supernatant was aspirated, and the supernatant was slowly added to the lymphocyte separation. On a liquid Ficoll solution (GE), the volume ratio is 1:1, centrifuged (2000 rpm, 20 min);
  • the white floc cells of the collection interface were added to the PBS, gently pipetted and mixed, and centrifuged (1500 rpm, 10 min);
  • the collected cells were gently pipetted and mixed, and 20 ml of the basal medium was added and uniformly blown, transferred to a 75 cm 2 culture flask, and cultured at 37 ° C in a 5% CO 2 incubator for 2 hours.
  • the remaining 2 ml of the remaining solution in the original vial (75 cm 2 flask) will be used to induce differentiation of monocytes into DC cells.
  • the cell culture medium was aspirated, washed once with PBS, fresh 15 ml of CTL3 medium was added, and 5*10 8 PD694 lentiviral vector was added to the culture flask for mixing, and the solution was changed after 24 hours.
  • PEI (1 ⁇ g / ⁇ l) Polysciences (CAT#23966-2 formulated into a stock solution) was prepared according to the following steps: First, the endotoxin-free sterile water was heated to about 80 ° C to dissolve the PEI, and cooled to room temperature; Adjust the pH to 7.0, filter and sterilize with a 0.22 ⁇ m filter, store at -20 °C after dispensing, and store the working solution at 4 °C for later use.
  • the centrifuge tube was taken out, and the cell suspension was taken out into a culture flask (75 cm 2 ) with a pipette, and DC medium was added thereto, and the culture was continued for 3 days to obtain mature DC cells.
  • the mature DC cells were mixed with the aforementioned activated cultured lymphocytes at a volume ratio of 1:20.
  • Co-culture with CTL3 medium for seven days induced the production of CTL cells and other killer cells.
  • the immune cell population (PD694-CTL cells) prepared in this example contained not only a large number of CTL cells, but also various immune cells such as NK and CIK cells.
  • the present invention not only prepares AFP-specific CTL cells (PD694-CTL cells) in vitro, but also NKT cells. These lymphocytes have been modified by PD694's lentiviral vector prior to antigen stimulation, and their PD1 has been inhibited by more than 90%.
  • the PD694-CTL cells prepared in Example 1 were returned to the patient, and the reinfusion effect was observed as follows:
  • the interference fragment of the present invention has good targeting property against its target (SEQ ID NO: 5), has outstanding interference inhibition effect on PD-1, and can be effectively used for inhibiting PD-1 expression of immune cells, thereby obtaining PD- 1
  • the expression-inhibited immune cells can be effectively used for the preparation of immunotherapeutic drugs.

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Abstract

La présente invention concerne un fragment interférent et une application associée. La cible d'action de ce fragment interférent est une séquence nucléotidique telle que représentée ci-après : 5'-GCCTGTGTTCTCTGTGGACTA-3' (SEQ ID N° : 5).
PCT/CN2015/088286 2015-08-27 2015-08-27 Fragment interférent et applications associées Ceased WO2017031760A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103820454A (zh) * 2014-03-04 2014-05-28 黄行许 CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA
CN103830725A (zh) * 2005-06-08 2014-06-04 达纳－法伯癌症研究院 通过抑制程序性细胞死亡1（pd-1）途经治疗持续性感染和癌症的方法及组合物
CA2897858A1 (fr) * 2013-02-22 2014-08-28 Curevac Gmbh Combinaison d'une vaccination et de l'inhibition de la voie de pd-1

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103830725A (zh) * 2005-06-08 2014-06-04 达纳－法伯癌症研究院 通过抑制程序性细胞死亡1（pd-1）途经治疗持续性感染和癌症的方法及组合物
CA2897858A1 (fr) * 2013-02-22 2014-08-28 Curevac Gmbh Combinaison d'une vaccination et de l'inhibition de la voie de pd-1
CN103820454A (zh) * 2014-03-04 2014-05-28 黄行许 CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA

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