WO2020259151A1 - 一种ctl细胞的制备方法及应用 - Google Patents

一种ctl细胞的制备方法及应用 Download PDF

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WO2020259151A1
WO2020259151A1 PCT/CN2020/091781 CN2020091781W WO2020259151A1 WO 2020259151 A1 WO2020259151 A1 WO 2020259151A1 CN 2020091781 W CN2020091781 W CN 2020091781W WO 2020259151 A1 WO2020259151 A1 WO 2020259151A1
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cells
csf
pap
preparation
tumor antigen
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WO2020259151A8 (zh
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周超
安鸿
卢有德
周玲
杜永彪
涂嘉琦
尹海滨
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广州安捷生物医学技术有限公司
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Priority to US17/621,687 priority Critical patent/US20240123071A1/en
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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a preparation method and application of CTL cells.
  • Prostate cancer is one of the most common malignant tumors of the male genitourinary system. It is a local organ cancerous disease. Its course can be divided into two stages, namely, hormone-dependent and non-hormone-dependent. In the hormone-dependent period, it can be treated with prostatectomy, radiotherapy, and medical castration, which will cause serious side effects and affect the patient's quality of life. After the hormone-dependent phase, the disease will progress to the hormone-independent phase. Almost all patients develop castration-resistant prostate cancer (CRPC), while metastatic castration-resistant prostate cancer (metastatic Castration Resistant) Prostate Cancer (mCRPC) is the main lethal factor of prostate cancer, and the average median survival time of mCRPC patients is less than 2 years. Therefore, it is very urgent to develop a new and effective treatment plan for prostate cancer.
  • CRPC castration-resistant prostate cancer
  • mCRPC metastatic Castration Resistant Prostate Cancer
  • the purpose of the present invention is to perform gene editing on tumor antigen-specific CTLs, knock out the immune checkpoint gene PD-1, and obtain more powerful CTL cells that do not respond to the immunosuppressive microenvironment of solid tumors.
  • a method for preparing CTL cells includes the following steps: using DC cells sensitized with the tumor antigen PAP-GM-CSF to induce the production of CTL cells; knocking out the PD-1 gene of the CTL cells to obtain PD-1 knockout CTL cells.
  • the tumor antigen PAP-GM-CSF is composed of PAP and GM-CSF connected by two amino acids Gly-Ser.
  • the upstream of PAP of the tumor antigen PAP-GM-CSF contains a signal peptide.
  • nucleotide sequence list of the tumor antigen PAP-GM-CSF is shown in SEQ ID NO.:1.
  • amino acid sequence list of the tumor antigen PAP-GM-CSF is shown in SEQ ID NO.: 2.
  • tumor antigen PAP-GM-CSF is expressed by genetic engineering method.
  • the genetic engineering method is selected from one of insect cell baculovirus expression system, HEK293 cell expression system, yeast expression system, and E. coli expression system; preferably, the genetic engineering method is insect cell baculovirus Expression system.
  • tumor antigen PAP-GM-CSF is expressed by genetic engineering method and purified.
  • the purity of the tumor antigen PAP-GM-CSF is not less than 98%.
  • the purification includes ultrafiltration and continuous column chromatography.
  • the continuous column chromatography includes at least one of ion exchange, hydrophobic chromatography, hydroxyapatite chromatography, and affinity chromatography.
  • the continuous column chromatography is one or more of cation column EMD SO 3 -(M) flow-through-anion column EMD TMAE (M)-hydrophobic column Capto Butyl.
  • the preparation method of the tumor antigen PAP-GM-CSF includes the following steps:
  • the preparation method of the tumor antigen PAP-GM-CSF includes the following steps:
  • the DC cells are selected from human peripheral blood mononuclear cells (PBMC), human peripheral blood CD14 + cells or bone marrow.
  • using the tumor antigen PAP-GM-CSF to sensitize DC cells includes the following steps: adding the DC cells to a lymphocyte serum-free medium containing rhGM-CSF and rhIL-4; after culturing, adding the tumor antigen PAP -GM-CSF and TNF- ⁇ are induced to obtain DC cells sensitized by the tumor antigen PAP-GM-CSF.
  • using the tumor antigen PAP-GM-CSF to sensitize DC cells includes the following steps: a blood cell separator or Ficoll separates peripheral blood mononuclear cells (PBMC), and separates CD14 + monocytes by magnetic bead sorting , Add X-VIVO TM 15 (LONZA) lymphocyte serum-free medium (containing 500-1000U/ml rhGM-CSF and 500U/ml rhIL-4), and place in a CO 2 incubator at 37°C and 5% CO 2 Cultured in medium, on the 5th day, PAP-GM-CSF fusion protein was added to stimulate the activation of DC, and 20ng/ml TNF- ⁇ was added to induce DC maturation, and the culture was continued for 48 hours.
  • a blood cell separator or Ficoll separates peripheral blood mononuclear cells (PBMC), and separates CD14 + monocytes by magnetic bead sorting , Add X-VIVO TM 15 (LONZA) lymphocyte serum-free medium (containing
  • using the DC cells sensitized with the tumor antigen PAP-GM-CSF to induce the production of CTL cells includes the following steps: obtaining human peripheral blood mononuclear cells from the same source as the DC cells, and adding them to the tumor antigen PAP -Co-culture in the DC cells sensitized with GM-CSF to induce the CTL cells.
  • one or more of the CRISPR/Cas9 system, the TALEN system or the zinc finger nuclease system is used to knock out the PD-1 gene of the CTL cell; preferably, the CRISPR/Cas9 system is used to knock out The PD-1 gene of the CTL cell.
  • the CRISPR/Cas9 system to knock out the PD-1 gene of the CTL includes the following steps: co-transfecting a Cas9 nuclease element and a gRNA targeting the PD-1 gene into the CTL cell; preferably Specifically, the co-transfection method is electroporation transfection.
  • the Cas9 nuclease element is a plasmid, mRNA or protein.
  • the gRNA targeting the PD-1 gene is composed of a targeting RNA and a guide RNA scaffold.
  • target RNA nucleotide sequence is shown in SEQ ID NO.: 3 to 110
  • guide RNA scaffold nucleotide sequence is shown in SEQ ID NO.: 111.
  • the present invention also provides a kit for obtaining CTL cells, which includes the above-mentioned tumor antigen PAP-GM-CSF; preferably, the above-mentioned Cas9 nuclease element, the above-mentioned PD-1 gene-targeting gRNA, and the kit instructions.
  • the kit instructions describe the above preparation method.
  • the present invention also provides an application of the above-mentioned preparation method of CTL cells in the preparation of drugs for treating prostate cancer.
  • the invention also provides an application of the above kit in the preparation of drugs for treating prostate cancer.
  • the present invention also provides an application of the above-mentioned preparation method of CTL cells in the preparation of drugs for treating PAP-positive prostate cancer.
  • the invention also provides an application of the above kit in the preparation of a medicine for treating PAP-positive prostate cancer.
  • the present invention provides a method for preparing PD-1 knock-out CTL cells.
  • DC cells are sensitized with the tumor antigen PAP-GM-CSF; about 95% of prostate cancers express prostatic acid phosphatase (PAP), and PAP
  • PAP prostatic acid phosphatase
  • the expression is mainly limited to prostate tissue.
  • PAP is widely expressed in prostate cancer patients and has good specificity. Therefore, PAP can be used as a target for therapeutic prostate cancer vaccines.
  • PAP can only mediate CD4 + cells to produce humoral immunity and induce plasma cells to produce antibodies through MHC II exogenous pathways, and cannot mediate CD8 + cells (CTL cells) to produce cytotoxic effects through MHC I endogenous pathways.
  • CTL cells CD8 + cells
  • the complex of PAP and GM-CSF as a tumor antigen can mediate CD8 + cells (CTL cells) to produce cytotoxic effects through the MHC I endogenous pathway.
  • the present invention provides a method for preparing CTL cells that knock out PD-1.
  • the purity of the tumor antigen PAP-GM-CSF is not less than 98%.
  • the immunized mice have immunogenicity, clear and single components, and can be amplified by the purification process. Large-scale production, high stability between batches.
  • the present invention provides a method for preparing PD-1 knockout CTL cells.
  • the obtained PD-1 knockout CTL cells will not cause CTL failure and disability due to PD-L1 expressed by the tumor after being returned to the body , So as to produce high-efficiency and specific cytotoxicity on tumor cells and improve its curative effect.
  • Figure 1 shows the identification of recombinant bacmid PAP-GM-CSF-Bacmid
  • Figure 2 is Western blotting to detect the expression of P2 generation baculovirus D on PAP-GM-CSF;
  • Figure 3 shows the detection of purified PAP-GM-CSF protein by SDS-PAGE
  • FIG. 4 shows HPLC analysis of purified PAP-GM-CSF protein
  • Figure 5 shows the phenotype of sensitized DC cells detected by flow cytometry
  • Figure 6 shows the ratio of DC-induced CTL cells detected by flow cytometry
  • Figure 7 shows the detection of PD-1 expression in CTL cells by flow cytometry
  • Figure 8 shows the detection of PD-1 knockout by sanger sequencing.
  • PD-1 Programmed death receptor 1
  • PD-1 has two ligands, PD-L1 and PD-L2, and Ligand interactions transmit inhibitory signals and play a negative regulatory role in the immune response.
  • Cytotoxic T lymphocytes a subdivision of white blood cells, are specific T cells that secrete various cytokines to participate in the immune function, and have a killing effect on certain viruses, tumor cells and other antigenic substances.
  • Prostatic Acid Phosphatas is a glycoprotein and an isoenzyme of acid phosphatase. It is produced by prostate epithelial cell lysosomes. Under normal conditions, serum levels are low. When prostate disease is destroyed After the blood-prostate barrier, serum PAP increased to varying degrees.
  • Granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF) is a white blood cell growth factor that can stimulate the colony formation of neutrophils and macrophages in vitro, and has the ability to promote early red The function of megakaryocyte and eosinophilic progenitor cell proliferation and development.
  • Dendritic cells are the body's most powerful full-time antigen presenting cells (Antigen presenting cells, APC), which can efficiently ingest, process and present antigens, and immature DCs have strong migration Ability, mature DC can effectively activate initial T cells, which are at the center of initiating, regulating, and maintaining immune response.
  • APC Antigen presenting cells
  • Glycine is a non-essential amino acid for the human body. It has both acidic and basic functional groups in the molecule. It can be ionized in water and has strong hydrophilicity. It is a polar amino acid and is soluble in polar solvents, but insoluble It is a non-polar solvent and has a higher boiling point and melting point.
  • Serine is a non-essential amino acid for the human body. It plays a role in the metabolism of fat and fatty acids and the growth of muscle.
  • Interleukin-4 is a type of cytokine produced by a variety of cells and acting on a variety of cells. It can stimulate the proliferation of activated B cells and T cells, and play a role in regulating humoral immunity and adaptive immunity. Key role.
  • Tumor Necrosis Factor- ⁇ Tumor Necrosis Factor- ⁇ (Tumor Necrosis Factor- ⁇ , TNF- ⁇ ) is a cytokine that can directly kill tumor cells without obvious toxicity to normal cells. It is the biological activity with the strongest direct tumor killing effect found so far One of the factors.
  • PBMC Peripheral blood mononuclear cells
  • Single-stranded guide RNA is a single-stranded RNA with the function of a crRNA-tracrRNA complex that can bind to Cas9 endonuclease and guide the latter to target sites on the genome for binding and cutting.
  • the prostatic acid phosphatase (Prostatic Acid Phosphatas, PAP) and granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF) gene sequences were retrieved from Genbank.
  • PAP and GM-CSF have two
  • the amino acid Gly-Ser connection structure contains a signal peptide upstream of the PAP.
  • the software is used for codon optimization to synthesize the entire DNA sequence.
  • the DNA sequence corresponding to the PAP-GM-CSF fusion protein is shown in SEQ ID NO.: 1, and the corresponding amino acid sequence is shown in SEQ ID NO.: 2.
  • the gene of the vector pFast-Bac1 and the synthetic tumor antigen PAP-GM-CSF is cut, connected, and transformed to construct a recombinant plasmid.
  • the successfully constructed pFast-Bac1-PAP-GM-CSF shuttle plasmid was extracted, transformed into E. coli competent cell DH10bac, and screened for blue and white spots on the LB plate of the third antibody (gentamicin, tetracycline, kanamycin).
  • the leukoplakia that grows is subjected to multiple plate streaking (three antibody LB plate + blue-white spot screening) for purification.
  • FIG. 1 shows the identification result of the recombinant bacmid PAP-GM-CSF-Bacmid, where M represents the nucleic acid marker, and lanes 1, 2, and 3 respectively represent the picked 3 recombinant rods
  • M represents the nucleic acid marker
  • lanes 1, 2, and 3 respectively represent the picked 3 recombinant rods
  • the size of the PAP-GMCSF fragment is about 1500bp, amplified with M13F/R primers increased the size of 2300bp, the theoretical amplified band size should be close to 4000bp, consistent with the figure above, indicating that the recombinant bacmid PAP-GM-CSF-Bacmid was constructed success.
  • the transfection process is as follows (take the transfection of 1 well of a 6-well plate as an example): add 5 ⁇ L of recombinant bacmid PAP-GM-CSF-Bacmid to 100 ⁇ l of Grace's medium and mix well; add 6 ⁇ L of liposome Escort TM IV Transfection (SIGMA) was added to 100 ⁇ l Grace's medium and mixed. Add the latter to the former to mix, and incubate at room temperature for 30 min.
  • SIGMA liposome Escort TM IV Transfection
  • the collected P1 generation virus was used to infect the domesticated suspension insect cells Sf-9, and the P2 generation baculovirus was prepared by expanding the culture. Under the microscope, it can be seen that the P1 generation virus infects Sf9 cells on the 3rd day after the cell pathological changes. Collect the cell supernatant containing the P2 generation virus, and use the collected P2 generation virus to infect the domesticated suspension insect cell Sf-9 for expansion culture to prepare the P3 generation rod At the same time, the expression of P2 generation baculovirus culture superalbumin was detected by western blotting. As shown in Figure 2, the expression of PAP-GM-CSF in the culture supernatant of P2 recombinant baculovirus was detected by Western blotting.
  • Lane 1 PageRuler TM Prestained Protein Ladder
  • Lane 2 Sf-9 cell culture supernatant.
  • Lane 3 Culture supernatant of Sf-9 cells infected with PAP-GM/CSF baculovirus (P2 generation)
  • Lane 4 Culture supernatant concentrate of Sf-9 cells infected with PAP-GM/CSF baculovirus (P2 Generation)
  • lane 5 PAP-GM/CSF protein expressed by HEK293T cells. It can be seen that the size of the tumor antigen PAP-GM-CSF fusion protein of the insect cell baculovirus expression system is 64kD, indicating that it is the target protein.
  • the domesticated suspension insect cells Sf-9 were infected with 5 MOI P3 recombinant baculoviruses, and Sf-900 TM II SFM serum-free insect cell culture medium was used to culture in a 27°C incubator at a rotation speed of 120 r/min. After 96-120h after infection, the supernatant was collected when the cytopathic changes were obvious, detected by western blotting, and purified.
  • the expressed PAP-GM-CSF fusion protein was concentrated 5 times through the Spectral hollow fiber tangential flow filtration system (pore size 30kD), and the buffer (20mM PB, pH7.2) was replaced.
  • Anion column EMD TMAE (M): Equilibrate the column bed with 5 ⁇ CV solution A [20mM PB, pH7.2] (flow rate 4ml/min). Load the flow-through liquid collected in the first step at a flow rate of 4 ml/min. Stepwise wash with Elution buffer 1 [20mM PB, 100mM NaCl, pH7.2], Elution buffer 2 [20mM PB, 200mM NaCl, pH7.2], Elution buffer 3 [20mM PB, 2M NaCl, pH7.2] The flow rate is 4ml/min. Among them, the eluted component of Elution buffer 2 is the target protein.
  • Hydrophobic column Capto Butyl Equilibrate the column bed with 5 ⁇ CV solution B [20mM PB, 2M NaCl, pH7.2] (flow rate 2ml/min). Before equilibrating the column, carefully add NaCl to the elution fraction collected in the second step of Elution buffer 2 to make the concentration reach 2M, and then load the sample at a flow rate of 2ml/min.
  • Respectively Elution buffer 4 [20mM PB, 1M NaCl, pH 7.2], Elution buffer 5 [20 mM PB, 500 mM NaCl, pH 7.2], Elution buffer 6 [20 mM PB, 200 mM NaCl, pH 7.2], Elution buffer 7 [ 20mM PB, pH7.2] for stepwise elution with a flow rate of 2ml/min.
  • the eluted component of Elution buffer 5 is the target protein.
  • Lane 1 PAP-GM-CSF sample
  • lane 2 SO 3 - (M) flow through
  • Lane 3 PageRuler TM Prestained Protein Ladder
  • Lane 4 TMAE(M) flows through
  • lane 5 Elution1, lane 6: Elution2,
  • lane 7 Elution3,
  • lane 8 Capto Butyl (GE) precolumn sample
  • lane 9 PageRuler TM Prestained Protein Ladder
  • lane 10 Capto Butyl (GE) )
  • the purified PAP-GM-CSF fusion protein (Elution5) was analyzed by HPLC. The results are shown in Figure 4. The corresponding parameters of the two components are shown in Table 1. It can be seen that the PAP-GM-CSF after analysis and purification The purity of the fusion protein reaches 98%;
  • the fractions eluted by Elute buffer 5 in the third step are concentrated through the Spectral hollow fiber tangential flow filtration system (pore size 30kD), and the buffer (physiological saline) is replaced, filtered and sterilized, and the protein concentration is measured. Save.
  • the purified protein was divided into different doses to immunize mice and the antiserum titer was measured by ELISA.
  • the measured reading results are shown in Table 2. It can be seen that the antiserum titer is >10000; among them, the experimental components are three groups, and the immune dose They are 150 ⁇ g/mouse, 100 ⁇ g/mouse and 50 ⁇ g/mouse, 5 in each group.
  • the antibody used in the positive antibody group is Anti-GM-CSF antibody (ab54429).
  • Example 2 DC cells sensitized by tumor antigen PAP-GM-CSF
  • PBMC peripheral blood mononuclear cells
  • a blood cell separator or Ficoll Separate peripheral blood mononuclear cells (PBMC) with a blood cell separator or Ficoll, separate CD14 + monocytes by magnetic bead sorting, add X-VIVO TM 15 (LONZA) lymphocyte serum-free medium (containing 500-1000 U/ml) rhGM-CSF and 500U/ml rhIL-4), placed in a CO 2 incubator at 37°C and 5% CO 2 and added tumor antigen PAP-GM-CSF to stimulate the activation of DC on the 5th day, while adding TNF- ⁇ (Final concentration 20ng/ml) induce DC maturation and continue to culture for 48h to obtain DC cells sensitized by tumor antigen PAP-GM-CSF.
  • TNF- ⁇ Federal concentration 20ng/ml
  • Figure 6(B) shows that the ratio of CD3 + CD8 + in the cells induced by flow cytometry is 79.9%, that is, CTL cells are as high as 79.9%.
  • Example 4 Crispr/Cas9 technology knocks out the PD-1 gene of tumor antigen-specific CTL cells
  • the tumor antigen PAP-GM-CSF specific CTL cells prepared in Example 3 were washed 3 times with OPTI-MEM (Thermo) and resuspended in OPTI-MEM, the cell density was 5 ⁇ 10 7 cells/ml.
  • Example 5 PD-1 knock-out prostate antigen-specific CTL cell preparation for the treatment of prostate cancer
  • Example 4 The cells after electroporation in Example 4 were cultured in a 6-well plate overnight and then transferred to a cell culture bag for further culture for 7-10 days. Add X-VIVO TM 15 (LONZA) medium (containing 200-500U/ml rhIL according to the cell growth) -2). Collect the cell suspension and wash it by centrifugation 3 times, resuspend it in 100ml of normal saline, and add 2% human serum albumin to obtain a PD-1 knockout prostate antigen-specific CTL cell preparation for prostate cancer treatment, in which PD-1 The knock-out prostate antigen-specific CTL cells are >1 ⁇ 10 10 .
  • Tumor antigen PAP-GM-CSF gene synthesis same as in Example 1.
  • PAP-GM-CSF expressed by HEK293T cell expression system. The results are shown in Figure 2. Lane 5: PAP-GM/CSF protein expressed by HEK293T cells. PAP-GM-CSF expressed by HEK293 cells can be seen The size of the fusion protein is 75kD, indicating that it is the target protein. PAP-GM-CSF after the expression of the fusion protein concentrate to flow filtration system (pore size of a 30 kD) through the hollow fiber cut, through the cation column EMD SO 3 - (M) flow through anion column EMD TMAE (M) and the hydrophobic column Capto Butyl ( GE) Purification.
  • flow filtration system pore size of a 30 kD
  • PD-1 gene knockout of tumor antigen PAP-GM-CSF-specific CTL cells knockout using TALEN system.
  • Tumor antigen PAP-GM-CSF gene synthesis same as in Example 1.
  • Preparation of tumor antigen PAP-GM-CSF expressed by yeast expression system, concentrated by hollow fiber tangential flow filtration system (pore size 30kD), purified by hydroxyapatite chromatography, hydrophobic chromatography and affinity chromatography.
  • Knockout of PD-1 gene of tumor antigen PAP-GM-CSF-specific CTL cells Knockout using zinc finger nuclease system.
  • a kit for obtaining PD-1 knockout CTL cells including the tumor antigen PAP-GM-CSF prepared in Example 1, human peripheral blood CD14 + cells, Cas9 nuclease elements, and gRNA targeting PD-1 gene And kit instructions;
  • the kit instructions include:
  • CD14 + monocytes add X-VIVO TM 15 (LONZA) lymphocyte serum-free medium (containing 500-1000U/ml rhGM-CSF and 500U/ml rhIL-4), place at 37°C, 5% CO 2 Cultured in a CO 2 incubator, on the 5th day, the tumor antigen PAP-GM-CSF was added to stimulate the activation of the DC, and TNF- ⁇ (final concentration 20ng/ml) was added to induce the maturation of the DC. Continue to culture for 48 hours to obtain the tumor antigen PAP-GM- CSF sensitized DC cells.
  • LONZA lymphocyte serum-free medium
  • TNF- ⁇ final concentration 20ng/ml
  • the prepared tumor antigen PAP-GM-CSF specific CTL cells were washed 3 times with OPTI-MEM (Thermo) and resuspended in OPTI-MEM, the cell density was 5 ⁇ 10 7 cells/ml.
  • the cells are quickly moved to add 2ml of X-VIVO TM 15 (LONZA) preheated at 37°C.
  • Culture medium (containing 200-500U/ml rhIL-2) in a 6-well plate at 37°C and 5% CO 2 in an incubator. After electroporation, the cells were cultured in a 6-well plate overnight and then transferred to a cell culture bag for further culture for 7-10 days, and X-VIVO TM 15 (LONZA) medium (containing 200-500U/ml rhIL-2) was added according to the cell growth.
  • X-VIVO TM 15 (LONZA) medium (containing 200-500U/ml rhIL-2) was added according to the cell growth.

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Abstract

提供一种CTL细胞的制备方法及其应用。该制备方法包括以下步骤：采用肿瘤抗原PAP-GM-CSF致敏的DC细胞诱导产生CTL细胞；敲除该CTL细胞的PD-1基因，得到PD-1敲除的CTL细胞。该制备方法得到的CTL细胞可用于制备治疗前列腺癌的药物，尤其是治疗PAP阳性的前列腺癌。该CTL细胞在回输体内后不会因肿瘤表达的PD-L1而引起CTL细胞衰竭和失能，从而对肿瘤细胞产生高效特异性细胞毒作用，提高其疗效并降低副作用。

Description

一种CTL细胞的制备方法及应用 技术领域
本发明属于生物技术领域，具体涉及一种CTL细胞的制备方法及应用。
背景技术
前列腺癌(prostate cancer，PC)是男性泌尿生殖系统最常见的恶性肿瘤之一，为局部器官性癌变疾病，其病程可以分为两个阶段，即激素依赖期和非激素依赖期。在激素依赖期可以采用前列腺切除手术、放射性疗法、药物去势的方案来治疗，伴随产生严重的毒副作用，影响病人的生活质量。在经过激素依赖期以后病情会进展到激素非依赖期，几乎所有的患者均发展为去势抵抗性前列腺癌(Castration Resistant Prostate Cancer，CRPC)，而转移性去势抵抗性前列腺癌(metastatic Castration Resistant Prostate Cancer，mCRPC)更是前列腺癌的主要致死因素，进展至mCRPC患者平均中位生存时间少于2年。因此开发出新的、有效的前列腺癌治疗方案十分迫切。
细胞治疗作为肿瘤治疗的新手段成为了研究热点，尤其是基于树突状细胞(DC)疫苗的前列腺癌治疗取得了较好的临床效果。通过前列腺癌相关抗原肽致敏DC，能诱导强效的抗肿瘤免疫效应，但是存在输注的DC细胞以及DC诱导的肿瘤抗原特异性CTL被肿瘤免疫抑制性微环境抑制，导致其功能衰竭和丧失的问题。肿瘤微环境是实体瘤治疗中面临的难题，由免疫检查点介导的免疫抑制信号，如PD-1、CTLA-4、LAG-3和TIM-3组成的肿瘤微环境，在促进肿瘤免疫逃逸中发挥重要作用，因此肿瘤疫苗和免疫检查点单克隆抗体联合治疗前列腺癌成为一种有吸引力的策略。在www.clinicaltrials.gov上注册的多个临床试验通过Sipuleucel-T联合免疫检查点抑制剂治疗前列腺癌(如Sipuleucel-T and Ipilimumab for Advanced Prostate Cancer(NCT01832870)、A Randomized Phase 2Trial of Combining Sipuleucel-T With Immediate vs.Delayed CTLA-4 Blockade for Prostate Cancer(NCT01804465))。此外还有多个临床试验通过DNA疫苗联合免疫检查点抑制剂治疗前列腺癌(NCT03600350、NCT02499835、NCT03815942等)。然而免疫检查点抑制剂的长期使用可能破坏免疫耐受，导致严重的副作用。
发明内容
本发明的目的是对肿瘤抗原特异性CTL进行基因编辑，敲除免疫检查点基因PD-1，得到对实体瘤的免疫抑制微环境无反应的更强大的CTL细胞。
本发明所采用的技术方案是：
一种CTL细胞的制备方法，包括以下步骤：采用经肿瘤抗原PAP-GM-CSF致敏的DC细胞诱导产生CTL细胞；敲除所述CTL细胞的PD-1基因，得到PD-1敲除的CTL细胞。
进一步地，所述肿瘤抗原PAP-GM-CSF由PAP和GM-CSF经两个氨基酸Gly-Ser连接构成。
进一步地，所述肿瘤抗原PAP-GM-CSF的PAP上游含有一个信号肽。
进一步地，所述肿瘤抗原PAP-GM-CSF的核苷酸序列表如SEQ ID NO.：1所示。
进一步地，所述肿瘤抗原PAP-GM-CSF的氨基酸序列表如SEQ ID NO.：2所示。
进一步地，所述肿瘤抗原PAP-GM-CSF由基因工程方法表达得到。
进一步地，所述基因工程方法选自昆虫细胞杆状病毒表达系统、HEK293细胞表达系统、酵母表达系统、大肠杆菌表达系统中的一种；优选地，所述基因工程方法为昆虫细胞杆状病毒表达系统。
进一步地，所述肿瘤抗原PAP-GM-CSF由基因工程方法表达后纯化得到。
进一步地，所述肿瘤抗原PAP-GM-CSF的纯度不小于98％。
进一步地，所述纯化包括超滤和连续柱层析。
进一步地，所述连续柱层析包括离子交换、疏水层析、羟基磷灰石层析、亲和层析中的至少一种。
进一步地，所述连续柱层析为阳离子柱EMD SO 3-(M)流穿——阴离子柱EMD TMAE(M)——疏水柱Capto Butyl中的一种或多种。
进一步地，所述肿瘤抗原PAP-GM-CSF的制备方法包括如下步骤：
(1)以pFast-Bac1为骨架载体，构建穿梭质粒pFast-Bac1-PAP-GM-CSF；
(2)将所述穿梭质粒转化进入大肠杆菌感受态细胞，筛选得到重组杆粒PAP-GM-CSF-Bacmid；
(3)将所述重组杆粒转染昆虫细胞，待细胞有明显病变后，收获上清，即为第一代杆状病毒；
(4)将所述第一代杆状病毒感染昆虫细胞，收集第二代或第三代杆状病毒；
(5)用所述第二代或第三代杆状病毒感染驯化的悬浮昆虫细胞，表达PAP-GM-CSF。
进一步地，所述肿瘤抗原PAP-GM-CSF的制备方法包括如下步骤：
(1)以pFast-Bac1为骨架载体，构建穿梭质粒pFast-Bac1-PAP-GM-CSF；
(2)将所述穿梭质粒转化大肠杆菌感受态细胞DH10bac，筛选得到重组杆粒PAP-GM-CSF-Bacmid；
(3)将所述重组杆粒转染昆虫细胞Sf9，待细胞有明显病变后，收获上清，即为第一代杆状病毒；
(4)将所述第一代杆状病毒感染昆虫细胞Sf9，收集第二代或第三代杆状病毒；
(5)用所述第二代或第三代重组杆状病毒感染驯化的悬浮昆虫细胞Sf9，表达PAP-GM-CSF融合蛋白。
进一步地，所述DC细胞选自人外周血单核细胞(PBMC)、人外周血CD14 +细胞或骨髓。
进一步地，采用所述肿瘤抗原PAP-GM-CSF来致敏DC细胞，包括以下步骤：将DC细胞加入含有rhGM-CSF和rhIL-4的淋巴细胞无血清培养基；培养后，加入肿瘤抗原PAP-GM-CSF和TNF-α进行诱导，来得到所述经肿瘤抗原PAP-GM-CSF致敏的DC细胞。
进一步地，采用所述肿瘤抗原PAP-GM-CSF来致敏DC细胞，包括以下步骤：血细胞分离机或Ficoll分离外周血单核细胞(PBMC)，通过磁珠分选法分离CD14 +单核细胞，加入X-VIVO TM15(LONZA)淋巴细胞无血清培养基(含500-1000U/ml rhGM-CSF和500U/ml rhIL-4),37℃、5％CO 2条件下置于CO 2培养箱中培养，第5天加入PAP-GM-CSF融合蛋白刺激活化DC，同时加入20ng/ml TNF-α诱导DC成熟，继续培养48h。
进一步地，采用所述经肿瘤抗原PAP-GM-CSF致敏的DC细胞诱导产生CTL细胞，包括以下步骤：获取与所述DC细胞来源相同的人外周血单核细胞，加入到经肿瘤抗原PAP-GM-CSF致敏的所述DC细胞中共培养，来诱导产生所述CTL细胞。
进一步地，采用所述经肿瘤抗原PAP-GM-CSF致敏的DC细胞诱导产生CTL细胞，包括以下步骤：血细胞分离机或Ficoll分离与DC来源相同的的PBMC，加入到经PAP-GM-CSF刺激且成熟的DC中(DC：PBMC＝1:10)，37℃、5％CO 2的培养箱内共孵育3-5天。
进一步地，采用CRISPR/Cas9系统、TALEN系统或锌指核酸酶系统中的一 种或多种，来敲除所述CTL细胞的PD-1基因；优选地，采用CRISPR/Cas9系统，来敲除所述CTL细胞的PD-1基因。
进一步地，所述CRISPR/Cas9系统敲除所述CTL的PD-1基因，包括以下步骤：将一个Cas9核酸酶元件和一个靶向PD-1基因的gRNA共转染进所述CTL细胞；优选地，所述共转染的方法为电穿孔转染。
进一步地，所述Cas9核酸酶元件为质粒、mRNA或蛋白。
进一步地，所述靶向PD-1基因的gRNA由靶向RNA和向导RNA支架(guide RNA scaffold)构成。
进一步地，所述靶向RNA核苷酸序列如SEQ ID NO.：3～110所示，向导RNA支架核苷酸序列如SEQ ID NO.：111所示。
本发明还提供一种获得CTL细胞的试剂盒，包括上述肿瘤抗原PAP-GM-CSF；优选地，还包括上述Cas9核酸酶元件、上述靶向PD-1基因的gRNA以及试剂盒说明书，所述试剂盒说明书记载有上述制备方法。
本发明还提供一种上述CTL细胞的制备方法在制备治疗前列腺癌药物中的应用。
本发明还提供一种上述试剂盒在制备治疗前列腺癌药物中的应用。
本发明还提供一种上述CTL细胞的制备方法在制备治疗PAP阳性前列腺癌药物中的应用。
本发明还提供一种上述试剂盒在制备治疗PAP阳性前列腺癌药物中的应用。
本发明的有益效果：
1、本发明提供一种敲除PD-1的CTL细胞的制备方法，以肿瘤抗原PAP-GM-CSF致敏DC细胞；约95％的前列腺癌均表达前列腺酸性磷酸酶(PAP)，且PAP表达主要限于前列腺组织，PAP在前列腺癌患者表达广泛，并且特异性好，因此PAP可作为治疗性前列腺癌疫苗的靶标。但是PAP仅能通过MHC II外源性途径介导CD4 +细胞产生体液免疫，诱导浆细胞产生抗体，不能通过MHC I内源性途径介导CD8 +细胞(CTL细胞)产生细胞毒效应。而将PAP与GM-CSF构成复合物作为肿瘤抗原则能通过MHC I内源性途径介导CD8 +细胞(CTL细胞)产生细胞毒效应。
2、本发明提供一种敲除PD-1的CTL细胞的制备方法，肿瘤抗原PAP-GM-CSF纯度不小于98％，经免疫小鼠具有免疫原性，成分明确单一，可进行纯化工艺放大进行大规模生产，批次间稳定性高。
3、本发明提供一种敲除PD-1的CTL细胞的制备方法，得到的敲除PD-1的CTL细胞在回输体内后不会因肿瘤表达的PD-L1而引起CTL衰竭和失能，从而对肿瘤细胞产生高效特异性细胞毒作用，提高其疗效。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1为重组杆粒PAP-GM-CSF-Bacmid的鉴定；
图2为Western blotting检测P2代杆状病毒D对PAP-GM-CSF的表达情况；
图3为SDS-PAGE检测纯化的PAP-GM-CSF蛋白；
图4为HPLC分析纯化的PAP-GM-CSF蛋白；
图5为流式细胞术检测致敏DC细胞的表型；
图6为流式细胞术检测DC诱导CTL细胞的比例；
图7为流式细胞术检测CTL细胞PD-1表达；
图8为sanger测序检测PD-1敲除。
具体实施方式
以下结合附图和具体的实施例对本发明的技术方案做进一步说明，但本发明并不限于这些具体实施方式。实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
程序性死亡受体1(Programmed Death 1，PD-1)，属于免疫球蛋白超家族成员，是一种重要的免疫抑制分子；PD-1有两个配体PD-L1和PD-L2，与配体相互作用传递抑制性信号，在免疫应答中发挥负向调控作用。
细胞毒性T淋巴细胞(cytotoxic lymphocyte，CTL)，是白细胞的亚部，为一种特异T细胞，专门分泌各种细胞因子参与免疫作用，对某些病毒、肿瘤细胞等抗原物质具有杀伤作用。
前列腺酸性磷酸酶(Prostatic Acid Phosphatas，PAP)是一种糖蛋白，是酸性磷酸酶的同工酶，由前列腺上皮细胞溶酶体产生，在正常情况下血清含量均较低，当前列腺病变破坏了血-前列腺屏障以后血清PAP即有不同程度的升高。
粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony  stimulating factor，GM-CSF)，是一种白细胞生长因子，在体外可刺激中性粒细胞和巨噬细胞的集落形成，并具有促进早期红巨核细胞、嗜酸性祖细胞增殖和发育的功能。
树突状细胞(Dendritic cells,DC)是机体功能最强的专职抗原递呈细胞(Antigen presenting cells,APC)，它能高效地摄取、加工处理和递呈抗原，未成熟DC具有较强的迁移能力，成熟DC能有效激活初始T细胞，处于启动、调控、并维持免疫应答的中心环节。
甘氨酸(Glycine，Gly)是人体非必需氨基酸，在分子中同时具有酸性和碱性官能团，在水中可电离，具有很强的亲水性，属于极性氨基酸，溶于极性溶剂，而难溶于非极性溶剂，而且具有较高的沸点和熔点。
丝氨酸(Serine，Ser)是人体非必需氨基酸，它在脂肪和脂肪酸的新陈代谢及肌肉的生长中发挥着作用。
白细胞介素(Interleukin-4，IL-4)是由多种细胞产生并作用于多种细胞的一类细胞因子，能够刺激活化B细胞和T细胞增殖，在调节体液免疫和适应性免疫中起关键作用。
肿瘤坏死因子-α(Tumor Necrosis Factor-α，TNF-α)是一种能够直接杀伤肿瘤细胞而对正常细胞无明显毒性的细胞因子，是迄今为止所发现的直接杀伤肿瘤作用最强的生物活性因子之一。
外周血单个核细胞(peripheral blood mononuclear cell，PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞、树突状细胞和其他少量细胞。
单链引导RNA(single guide RNA，sgRNA)具备crRNA-tracrRNA复合物的功能的单链RNA，能够与Cas9核酸内切酶结合并将后者引导至基因组上的靶位点处进行结合与切割。
实施例1肿瘤抗原PAP-GM-CSF的制备
1、昆虫杆状病毒载体构建
(1)肿瘤抗原PAP-GM-CSF的基因合成
从Genbank里面调取前列腺酸性磷酸酶(Prostatic Acid Phosphatas，PAP)和粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor，GM-CSF)的基因序列，PAP和GM-CSF经两个氨基酸Gly-Ser连接构成，在PAP上游含有一个信号肽，利用软件进行密码子优化，进行DNA 全序列合成。PAP-GM-CSF融合蛋白所对应的DNA序列如SEQ ID NO.：1所示，对应的氨基酸序列如SEQ ID NO.：2所示。
(2)穿梭质粒pFast-Bac1-PAP-GM-CSF的构建
载体pFast-Bac1和合成的肿瘤抗原PAP-GM-CSF的基因通过酶切、连接、转化构建重组质粒。
(3)重组杆粒PAP-GM-CSF-Bacmid的构建及鉴定
抽提构建成功的pFast-Bac1-PAP-GM-CSF穿梭质粒，转化大肠杆菌感受态细胞DH10bac，在三抗LB平板(庆大霉素、四环素、卡那霉素)上进行蓝白斑筛选，对长出的白斑进行多次平板划线(三抗LB平板+蓝白斑筛选)进行纯化。抽提重组杆粒PAP-GM-CSF-Bacmid并以此为模板，M13F(如SEQ ID NO.：112所示)和M13R(如SEQ ID NO.：113所示)为引物对进行PCR扩增，验证重组杆粒的正确性；如图1所示为重组杆粒PAP-GM-CSF-Bacmid的鉴定结果，其中M表示核酸marker，泳道1、2、3分别表示挑取的3个重组杆粒菌抽提杆粒后进行PCR扩增的结果。PAP-GMCSF片段大小约为1500bp，用M13F/R引物扩增增加了2300bp的大小，理论扩增条带大小应接近4000bp，与上图相一致，说明重组杆粒PAP-GM-CSF-Bacmid构建成功。
2、重组杆状病毒的制备和鉴定
(1)第一(P1)代重组杆状病毒的制备
转染前一天将处于对数生长期的昆虫细胞Sf9铺在6孔板，1×10 6cells/孔，贴壁过夜。转染过程如下(以转染6孔板1个孔为例)：将5μL重组杆粒PAP-GM-CSF-Bacmid加到100μl Grace’s培养基并混匀；将6μL的脂质体Escort TMIV Transfection(SIGMA)加到100μl Grace’s培养基并混匀。将后者加入前者混合，室温孵育30min。每孔中加入800μl无血清Grace’s培养基，再滴加脂质体—Bac混合物，27℃孵育5h。去掉转染混合物，加入4ml的Grace’s培养基(含5％FBS)，置于27℃培养箱培养。在显微镜下可以观察到在转染的第4天细胞发生病变，收集含有病毒的培养液，1000g，离心15min，将上清移至新离心管中，避光4℃保存，即为P1代重组杆状病毒。
(2)第二(P2)及第三(P3)代重组杆状病毒的制备
用收集的P1代病毒感染驯化的悬浮昆虫细胞Sf-9，进行扩大培养制备P2代杆状病毒。显微镜下可见P1代病毒感染Sf9细胞后第3天细胞发生病变，收集含有P2代病毒的细胞上清，用收集的P2代病毒感染驯化的悬浮昆虫细胞Sf-9进行扩大培养以制备P3代杆状病毒，同时通过western blotting检测P2 代杆状病毒培养上清蛋白的表达。如图2所示，通过Western blotting检测P2代重组杆状病毒培养上清中PAP-GM-CSF的表达情况，其中泳道1:PageRuler  TMPrestained Protein Ladder，泳道2:Sf-9细胞培养上清，泳道3:感染PAP-GM/CSF杆状病毒的Sf-9细胞的培养上清(P2代)，泳道4:感染PAP-GM/CSF杆状病毒的Sf-9的培养上清浓缩液(P2代)，泳道5:HEK293T细胞表达的PAP-GM/CSF蛋白。可以看出昆虫细胞杆状病毒表达系统的肿瘤抗原PAP-GM-CSF融合蛋白的大小是64kD，说明是目的蛋白。
3、肿瘤抗原PAP-GM-CSF的表达纯化
(1)肿瘤抗原PAP-GM-CSF融合蛋白的表达
以5个MOI的P3代重组杆状病毒感染驯化的悬浮昆虫细胞Sf-9，使用Sf-900 TMII SFM无血清昆虫细胞培养基，在27℃培养箱以120r/min的转速震荡培养。感染后96-120h待细胞病变明显时收集上清，通过western blotting检测并进行纯化。
(2)肿瘤抗原PAP-GM-CSF融合蛋白纯化
通过仕必纯中空纤维切向流过滤系统(孔径30kD)将表达的PAP-GM-CSF融合蛋白浓缩5倍，并置换缓冲液(20mM PB，pH7.2)。
阳离子柱EMD SO 3-(M)流穿：用5×CV溶液A[20mM PB，pH7.2]平衡柱床(流速4ml/min)。将浓缩置换缓冲液的样品按4ml/min的流速上样，收集流穿液，然后以4×CV溶液B[20mM PB，2M NaCl，pH7.2]去除杂质。
阴离子柱EMD TMAE(M)：用5×CV溶液A[20mM PB，pH7.2]平衡柱床(流速4ml/min)。将第一步收集的流穿液按4ml/min的流速上样。分别以Elution buffer 1[20mM PB，100mM NaCl，pH7.2]、Elution buffer 2[20mM PB，200mM NaCl，pH7.2]、Elution buffer 3[20mM PB，2M NaCl，pH7.2]进行分步洗脱，流速4ml/min。其中Elution buffer 2洗脱的组分为目的蛋白。
疏水柱Capto Butyl(GE)：用5×CV溶液B[20mM PB，2M NaCl，pH7.2]平衡柱床(流速2ml/min)。在平衡柱子之前，将第二步收集的Elution buffer2洗脱的组分小心添加NaCl使其浓度达到2M，然后按照2ml/min的流速上样。分别以Elution buffer4[20mM PB，1M NaCl，pH7.2]、Elution buffer 5[20mM PB，500mM NaCl，pH7.2]、Elution buffer 6[20mM PB，200mM NaCl，pH7.2]、Elution buffer 7[20mM PB，pH7.2]进行分步洗脱，流速2ml/min。其中Elution buffer 5洗脱的组分为目的蛋白。
将各组分进行SDS-PAGE检测，如图3所示，泳道1:PAP-GM-CSF样品，泳 道2:SO 3 -(M)流穿，泳道3:PageRuler TMPrestained Protein Ladder，泳道4:TMAE(M)流穿，泳道5:Elution1，泳道6:Elution2，泳道7:Elution3，泳道8:Capto Butyl(GE)柱前样品，泳道9:PageRuler TMPrestained Protein Ladder，泳道10:Capto Butyl(GE)流穿，泳道11:Elution4，泳道12:Elution5，泳道13:Elution6，泳道14:Elution7，泳道15:Elution5超滤浓缩。
分析纯化的PAP-GM-CSF融合蛋白(Elution5)经HPLC分析，结果如图4所示，其中两个组分对应的相关参数如表1所示，可以看出分析纯化后PAP-GM-CSF融合蛋白的纯度达到98％；
表1分析纯化的PAP-GM-CSF融合蛋白的液相色谱数据
Figure PCTCN2020091781-appb-000001
将第三步Elute buffer 5洗脱的组分通过仕必纯中空纤维切向流过滤系统(孔径30kD)进行浓缩，并置换缓冲液(生理盐水)，过滤除菌，测蛋白浓度，分装冻存。
(3)PAP-GM-CSF融合蛋白的免疫原性
用纯化的蛋白分不同剂量免疫小鼠通过ELISA测得抗血清效价，测得的读数结果如表2所示，可知抗血清效价>10000；其中，其中，实验组分三组，免疫剂量分别为150μg/只、100μg/只和50μg/只，每组5只。阳性抗体组所用抗体是Anti-GM-CSF抗体(ab54429)。
表2不同剂量PAP-GM-CSF免疫小鼠的抗血清效价测定结果
Figure PCTCN2020091781-appb-000002
Figure PCTCN2020091781-appb-000003
实施例2肿瘤抗原PAP-GM-CSF致敏的DC细胞
血细胞分离机或Ficoll分离外周血单核细胞(PBMC)，通过磁珠分选法分离CD14 +单核细胞，加入X-VIVO TM15(LONZA)淋巴细胞无血清培养基(含500-1000U/ml rhGM-CSF和500U/ml rhIL-4)，37℃、5％CO 2条件下置于CO 2培养箱中培养，第5天加入肿瘤抗原PAP-GM-CSF刺激活化DC，同时加入TNF-α(终浓度20ng/ml)诱导DC成熟，继续培养48h，得到肿瘤抗原PAP-GM-CSF致敏的DC细胞，如图5所示，通过流式细胞术检测到DC细胞的CD86的比例为54.6％。
实施例3致敏的DC细胞诱导产生CTL细胞
血细胞分离机或Ficoll分离与DC来源相同的的PBMC，加入到实施例2经PAP-GM-CSF融合蛋白刺激且成熟的DC中(DC：PBMC＝1:10)，37℃、5％CO 2的培养箱内共孵育3-5天，得到肿瘤抗原PAP-GM-CSF特异性CTL细胞，如图6所示，其中图6(A)为流式细胞术检测所诱导细胞的CD3 +CD4 +的比例为19.0％，图6(B)为流式细胞术检测所诱导细胞的CD3 +CD8 +的比例为79.9％，即CTL细胞高达79.9％。
实施例4 Crispr/Cas9技术敲除肿瘤抗原特异性CTL细胞的PD-1基因
将实施例3制备的肿瘤抗原PAP-GM-CSF特异性CTL细胞用OPTI-MEM (Thermo)洗涤3次并用OPTI-MEM重悬，细胞密度为5×10 7cells/ml。预先将20μg Cas9蛋白和10μg体外转录的靶向PD-1基因的gRNA混匀，室温孵育15min。将Cas9RNP和100μL细胞悬液混匀后加入2mm电击杯中，并通过BTX ECM830(Harvard)进行电穿孔转染，将细胞迅速移至添加2ml 37℃预热的X-VIVO TM15(LONZA)培养基(含200-500U/ml rhIL-2)的6孔板中，37℃、5％CO 2的培养箱内培养。电转后第3天通过流式细胞术检测CTL细胞的PD-1的表达，结果如图7所示，其中对照组(A)：电转Cas9蛋白的CTL细胞，实验组(B)：电转Cas9RNP的CTL细胞，可以看出CTL细胞的PD-1表达水平从50％降到13.8％，敲除效率达到72.4％。通过sanger测序验证CTL细胞PD-1结果如图8所示，从sgRNA靶序列开始出现明显套峰，可以看出肿瘤抗原PAP-GM-CSF特异性CTL细胞的PD-1被敲除。
实施例5前列腺癌治疗用PD-1敲除的前列腺抗原特异性CTL细胞制剂
将实施例4电转后的细胞在6孔板培养过夜后转移至细胞培养袋继续培养7-10天，根据细胞生长情况添加X-VIVO TM15(LONZA)培养基(含200-500U/ml rhIL-2)。收取细胞悬液并离心洗涤3次，以100ml生理盐水重悬，加入2％人血清白蛋白，即可获得前列腺癌治疗用PD-1敲除的前列腺抗原特异性CTL细胞制剂，其中PD-1敲除的前列腺抗原特异性CTL细胞>1×10 10。
实施例6
肿瘤抗原PAP-GM-CSF基因合成：同实施例1。
肿瘤抗原PAP-GM-CSF制备：由HEK293T细胞表达系统进行表达，结果如图2所示，泳道5:HEK293T细胞表达的PAP-GM/CSF蛋白，可以看出HEK293细胞表达的PAP-GM-CSF融合蛋白的大小是75kD，说明是目的蛋白。表达后的PAP-GM-CSF融合蛋白经中空纤维切向流过滤系统(孔径30kD)浓缩，经阳离子柱EMD SO 3 -(M)流穿，阴离子柱EMD TMAE(M)和疏水柱Capto Butyl(GE)纯化。
肿瘤抗原PAP-GM-CSF致敏的DC细胞的制备：同实施例2。
肿瘤抗原PAP-GM-CSF致敏的DC细胞诱导产生CTL细胞的制备：同实施例3。
肿瘤抗原PAP-GM-CSF特异性CTL细胞的PD-1基因敲除：采用TALEN系统进行敲除。
实施例7
肿瘤抗原PAP-GM-CSF基因合成：同实施例1。
肿瘤抗原PAP-GM-CSF制备：由酵母表达系统进行表达，经中空纤维切向流过滤系统(孔径30kD)浓缩，经羟基磷灰石层析，疏水层析和亲和层析纯化。
肿瘤抗原PAP-GM-CSF致敏的DC细胞的制备：同实施例2。
肿瘤抗原PAP-GM-CSF致敏的DC细胞诱导产生CTL细胞的制备：同实施例3。
肿瘤抗原PAP-GM-CSF特异性CTL细胞的PD-1基因敲除：采用锌指核酸酶系统进行敲除。
实施例8
一种获得敲除PD-1的CTL细胞的试剂盒，包括实施例1制备得到的肿瘤抗原PAP-GM-CSF、人外周血CD14 +细胞、Cas9核酸酶元件、靶向PD-1基因的gRNA和试剂盒说明书；
试剂盒说明书包括：
肿瘤抗原PAP-GM-CS致敏的DC细胞的制备
CD14 +单核细胞，加入X-VIVO TM15(LONZA)淋巴细胞无血清培养基(含500-1000U/ml rhGM-CSF和500U/ml rhIL-4)，37℃、5％CO 2条件下置于CO 2培养箱中培养，第5天加入肿瘤抗原PAP-GM-CSF刺激活化DC，同时加入TNF-α(终浓度20ng/ml)诱导DC成熟，继续培养48h，得到肿瘤抗原PAP-GM-CSF致敏的DC细胞。
致敏的DC细胞诱导产生CTL细胞的制备
血细胞分离机或Ficoll分离与DC来源相同的的PBMC，加入经PAP-GM-CSF融合蛋白刺激且成熟的DC中(DC：PBMC＝1:10)，37℃、5％CO 2的培养箱内共孵育3-5天，得到肿瘤抗原PAP-GM-CSF特异性CTL细胞。
Crispr/Cas9技术敲除肿瘤抗原特异性CTL细胞的PD-1基因
将制备的肿瘤抗原PAP-GM-CSF特异性CTL细胞用OPTI-MEM(Thermo)洗涤3次并用OPTI-MEM重悬，细胞密度为5×10 7cells/ml。预先将20μg Cas9蛋白和10μg体外转录的靶向PD-1基因的gRNA混匀，室温孵育15min。将Cas9RNP和100μL细胞悬液混匀后加入2mm电击杯中，并通过BTX ECM 830(Harvard)进行电穿孔转染，将细胞迅速移至添加2ml 37℃预热的X-VIVO TM15(LONZA)培养基(含200-500U/ml rhIL-2)的6孔板中，37℃、5％CO 2的培养 箱内培养。电转后的细胞在6孔板培养过夜后转移至细胞培养袋继续培养7-10天，根据细胞生长情况添加X-VIVO TM15(LONZA)培养基(含200-500U/ml rhIL-2)。收取细胞悬液并离心洗涤3次，以100ml生理盐水重悬，加入2％人血清白蛋白，即可获得前列腺癌治疗用PD-1敲除的前列腺抗原特异性CTL细胞制剂，其中PD-1敲除的前列腺抗原特异性CTL细胞>1×10 10。
本领域技术人员应该理解的是，本发明的使用不受限于上述特定应用。就本文描述或描绘的特定元素和/或特征而言，本发明也不局限于其优选实施方案。应当理解的是，本发明不限于所公开的实施方案例或各个实施方案，且在不脱离由以下权利要求所阐述和限定的本发明的范围的情况下能够进行许多重新布置、修改和替换。

Claims (14)

  1. 一种CTL细胞的制备方法，其特征在于，包括以下步骤：
    采用经肿瘤抗原PAP-GM-CSF致敏的DC细胞诱导产生CTL细胞；
    敲除所述CTL细胞的PD-1基因，得到PD-1敲除的CTL细胞。
  2. 根据权利要求1所述的制备方法，其特征在于，所述肿瘤抗原PAP-GM-CSF由PAP和GM-CSF经两个氨基酸Gly-Ser连接构成；优选地，所述肿瘤抗原PAP-GM-CSF的PAP上游含有信号肽。
  3. 根据权利要求1或2所述的制备方法，其特征在于，所述肿瘤抗原PAP-GM-CSF的核苷酸如SEQ ID NO.：1所示；所述肿瘤抗原PAP-GM-CSF的氨基酸如SEQ ID NO.：2所示。
  4. 根据权利要求1所述的制备方法，其特征在于，所述肿瘤抗原PAP-GM-CSF由基因工程方法表达得到，所述基因工程方法选自昆虫细胞杆状病毒表达系统、HEK293细胞表达系统、酵母表达系统、大肠杆菌表达系统中的一种；优选地，所述肿瘤抗原PAP-GM-CSF由基因工程方法表达后纯化得到；所述基因工程方法为昆虫细胞杆状病毒表达系统。
  5. 根据权利要求4所述的制备方法，其特征在于，所述纯化的方法为超滤和连续柱层析。
  6. 根据权利要求5所述的制备方法，其特征在于，所述连续柱层析选自离子交换、疏水层析、羟基磷灰石层析、亲和层析中的至少一种；优选地，所述连续柱层析为阳离子柱EMD SO 3 -(M)流穿、阴离子柱EMD TMAE(M)和疏水柱Capto Butyl中的一种或多种。
  7. 根据权利要求1或2或4～6任一项所述的制备方法，其特征在于，所述肿瘤抗原PAP-GM-CSF纯度不小于98％。
  8. 根据权利要求1所述的制备方法，其特征在于，所述肿瘤抗原PAP-GM-CSF的制备方法，包括如下步骤：
    (1)以pFast-Bac1为骨架载体，构建穿梭质粒pFast-Bac1-PAP-GM-CSF；
    (2)将所述穿梭质粒转化进入大肠杆菌，筛选得到重组杆粒PAP-GM-CSF-Bacmid；
    (3)将所述重组杆粒转染昆虫细胞，待细胞有明显病变后，收获上清，即为第一代杆状病毒；
    (4)将所述第一代杆状病毒感染昆虫细胞，收集第二代或第三代杆状病毒；
    (5)用所述第二代或第三代杆状病毒感染驯化的悬浮昆虫细胞，表达 PAP-GM-CSF。
  9. 根据权利要求1所述的制备方法，其特征在于，所述DC细胞选自人外周血单核细胞、人外周血CD14 +细胞或骨髓。
  10. 根据权利要求1所述的制备方法，其特征在于，采用所述肿瘤抗原PAP-GM-CSF来致敏DC细胞，包括以下步骤：将DC细胞加入含有rhGM-CSF和rhIL-4的淋巴细胞无血清培养基；培养后，加入肿瘤抗原PAP-GM-CSF和TNF-α进行诱导，来得到所述经肿瘤抗原PAP-GM-CSF致敏的DC细胞。
  11. 根据权利要求1所述的制备方法，其特征在于，采用所述经肿瘤抗原PAP-GM-CSF致敏的DC细胞诱导产生CTL细胞，包括以下步骤：获取与所述DC细胞来源相同的人外周血单核细胞，加入到经肿瘤抗原PAP-GM-CSF致敏的所述DC细胞中共培养，来诱导产生所述CTL细胞。
  12. 根据权利要求1所述的制备方法，其特征在于，采用CRISPR/Cas9系统、TALEN系统或锌指核酸酶系统中的一种或多种，来敲除所述CTL细胞的PD-1基因；优选地，采用CRISPR/Cas9系统，来敲除所述CTL细胞的PD-1基因。
  13. 一种获得CTL细胞的试剂盒，其特征在于，所述试剂盒包括如权利要求1～9任一项所述的肿瘤抗原PAP-GM-CSF；优选地，还包括Cas9核酸酶元件和靶向PD-1基因的gRNA以及试剂盒说明书，所述试剂盒说明书记载有如权利要求10、11和12所述的制备方法。
  14. 根据权利要求1～12任一项所述的CTL细胞的制备方法或根据权利要求13所述的获得CTL细胞的试剂盒在制备治疗PAP阳性前列腺癌药物中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725281A (zh) * 2021-01-28 2021-04-30 广州润生细胞医药科技有限责任公司 一种个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法及应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110358735B (zh) * 2019-06-24 2021-09-21 广州安捷生物医学技术有限公司 一种ctl细胞的制备方法及应用
CN113430202A (zh) * 2021-08-26 2021-09-24 山东兴瑞生物科技有限公司 一种具有高敲除率的人PD1基因sgRNA及含有该sgRNA的质粒、T细胞

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105586320A (zh) * 2016-02-01 2016-05-18 厚朴生物科技（苏州）有限公司 一种重组腺相关病毒及其构建方法和应用
CN105647970A (zh) * 2016-02-22 2016-06-08 河北利同康生物科技有限公司 前列腺癌特异性pap-gm-csf-il-6基因重组融合蛋白及其制备方法
CN105745538A (zh) * 2013-09-05 2016-07-06 丹特安制药公司 用癌症抗原特异的主动免疫疗法治疗之后，针对肿瘤抗原的体液免疫应答及其与改善的临床结果的联系
CN108753817A (zh) * 2018-04-13 2018-11-06 北京华伟康信生物科技有限公司 增强细胞的抗癌能力的方法及采用该方法获得的增强型细胞
CN110358735A (zh) * 2019-06-24 2019-10-22 广州安捷生物医学技术有限公司 一种ctl细胞的制备方法及应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6080409A (en) * 1995-12-28 2000-06-27 Dendreon Corporation Immunostimulatory method
CN106480097A (zh) * 2016-10-13 2017-03-08 南京凯地生物科技有限公司 利用CRISPR/Cas9技术敲除人PD‑1基因构建可靶向MSLN新型CAR‑T细胞的方法及其应用
CN106399375A (zh) * 2016-08-31 2017-02-15 南京凯地生物科技有限公司 利用CRISPR/Cas9敲除人PD‑1基因构建靶向CD19CAR‑T细胞的方法
CN106480027A (zh) * 2016-09-30 2017-03-08 重庆高圣生物医药有限责任公司 CRISPR/Cas9 靶向敲除人PD‑1基因及其特异性gRNA

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105745538A (zh) * 2013-09-05 2016-07-06 丹特安制药公司 用癌症抗原特异的主动免疫疗法治疗之后，针对肿瘤抗原的体液免疫应答及其与改善的临床结果的联系
CN105586320A (zh) * 2016-02-01 2016-05-18 厚朴生物科技（苏州）有限公司 一种重组腺相关病毒及其构建方法和应用
CN105647970A (zh) * 2016-02-22 2016-06-08 河北利同康生物科技有限公司 前列腺癌特异性pap-gm-csf-il-6基因重组融合蛋白及其制备方法
CN108753817A (zh) * 2018-04-13 2018-11-06 北京华伟康信生物科技有限公司 增强细胞的抗癌能力的方法及采用该方法获得的增强型细胞
CN110358735A (zh) * 2019-06-24 2019-10-22 广州安捷生物医学技术有限公司 一种ctl细胞的制备方法及应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHI ZHANG ET AL.: "Genetic abrogation of immune checkpoints in antigen-specific cytotoxic T-lymphocyte as a potential alternative to blockade immunotherapy", SCIENTIFIC REPORTS, vol. 8, no. 1, 3 April 2018 (2018-04-03), XP055770826, ISSN: 2045-2322, DOI: 20200728154250 *
PATRICK A. BURCH ET AL.: "Priming Tissue-specific Cellular Immunity in a Phase I Trial of Autologous Dendritic Cells for Prostate Cancer", CLINICAL CANCER RESEARCH, vol. 6, no. 6, 30 June 2000 (2000-06-30), XP055770829, ISSN: 1078-0432, DOI: 20200729121309Y *
PATRICK A. BURCH ET AL.: "Priming Tissue-specific Cellular Immunity in a Phase I Trial of Autologous Dendritic Cells for Prostate Cancer", CLINICAL CANCER RESEARCH, vol. 6, no. 6, 30 June 2000 (2000-06-30), XP055770829, ISSN: 1078-0432, DOI: 20200729121321Y *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725281A (zh) * 2021-01-28 2021-04-30 广州润生细胞医药科技有限责任公司 一种个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法及应用

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