WO2019218402A1 - 无血清培养制备嵌合抗原受体t细胞的方法 - Google Patents

无血清培养制备嵌合抗原受体t细胞的方法 Download PDF

Info

Publication number
WO2019218402A1
WO2019218402A1 PCT/CN2018/089801 CN2018089801W WO2019218402A1 WO 2019218402 A1 WO2019218402 A1 WO 2019218402A1 CN 2018089801 W CN2018089801 W CN 2018089801W WO 2019218402 A1 WO2019218402 A1 WO 2019218402A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
antigen receptor
chimeric antigen
serum
culture
Prior art date
Application number
PCT/CN2018/089801
Other languages
English (en)
French (fr)
Inventor
赵荻骏
李超
王飞
吴军峰
刘晓雨
赵佳维
Original Assignee
上海赛比曼生物科技有限公司
无锡赛比曼生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海赛比曼生物科技有限公司, 无锡赛比曼生物科技有限公司 filed Critical 上海赛比曼生物科技有限公司
Priority to JP2020563994A priority Critical patent/JP2021531734A/ja
Priority to AU2018423493A priority patent/AU2018423493A1/en
Priority to US17/054,989 priority patent/US20210214682A1/en
Priority to SG11202011263RA priority patent/SG11202011263RA/en
Priority to EP18919224.8A priority patent/EP3822344A4/en
Priority to KR1020207035736A priority patent/KR20210010509A/ko
Publication of WO2019218402A1 publication Critical patent/WO2019218402A1/zh

Links

Images

Classifications

    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0031—Serum-free culture media
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2301—Interleukin-1 (IL-1)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for preparing chimeric antigen receptor T cells in serum-free culture.
  • Chimeric antigen receptor T cell immunotherapy is a scFv fragment and intracellular signal domain that allows T cells to specifically recognize tumor-associated antigens by gene editing, thereby enhancing T cell targeting, killing and persistence. New medical technology. In recent years, tumor immunotherapy has a good clinical performance, which brings hope to clinical cure of cancer.
  • chimeric antigen receptor T cells The preparation process of chimeric antigen receptor T cells is relatively complicated, involving many operations such as cell activation, sorting, transfection, amplification culture and the like. Insufficient aspects of process flow, equipment and reagent selection will have an important impact on the quality of cell preparation, which in turn affects the viability, yield, safety and clinical effects of chimeric antigen receptor T cells.
  • Serum as an important component of traditional cell culture processes, contains some substances that have toxic side effects on cells. In addition, the quality difference between the batches of serum is large. In addition, risks such as mycoplasma and viruses may be brought into the process of serum sampling, which seriously hinder the development of cell therapy.
  • a method of serum-free preparation of chimeric antigen receptor T cells comprising the steps of:
  • step (b) performing a negative sorting treatment on the PBMC cells in the step (a) to obtain the sorted PBMC cells;
  • step (d) transfecting the activated T cells obtained in step (c) with a viral vector expressing a chimeric antigen receptor to obtain transfected T cells;
  • serum-free medium is used in all steps of the method.
  • step (a) the PBMC cells are revived PBMC cells.
  • the PBMC cells are autologous or allogeneic.
  • the PBMC cells are derived from a human.
  • step (b) the negative sorting process uses CliniMACS.
  • step (c) the activation is carried out by an activation treatment with an antibody against CD3/CD28.
  • step (c) the activation treatment is carried out in an activation medium containing a basal medium and an additive.
  • the base medium selected from the group: LONZA X-VIVO, LIFE CTS AIM V, LIFE OpTmizer SFM, RPMI 1640, ImmunoCult TM -XF,
  • the additive is selected from the group consisting of human serum albumin, recombinant human serum albumin, plant-derived recombinant albumin, or a combination thereof, preferably recombinant albumin.
  • the albumin concentration is from 0.01% to 50% (w/v), preferably from 0.05% to 30% (w/v), more preferably from 0.1% to 20% (W). /V).
  • the activation treatment is selected from the group consisting of antibody coating treatment, free antibody treatment, activated magnetic bead treatment, or a combination thereof.
  • the antibody concentration in the activation treatment is from 10 ng to 100 ng/ml.
  • the ratio of the number of activated magnetic beads to the cell cells is from 0.25 to 5:1, preferably from 0.5 to 3:1.
  • the activation treatment is carried out for a period of from 3 to 8 days.
  • the viral vector is selected from the group consisting of a lentivirus, an adeno-associated virus (AAV), an adenovirus, or a combination thereof.
  • the gene transfection has an infection number MOI of 0.5 to 30, preferably 1 to 20, more preferably 2 to 10.
  • the virus removal treatment comprises: resuspending the transfected cells in a serum-free medium, centrifuging, and aspirating the supernatant to obtain a virus-removed T cell.
  • the predetermined number is 1 ⁇ 10 9 - 1 ⁇ 10 11 , preferably 2 ⁇ 10 9 - 2 ⁇ 10 10 .
  • the amplification medium in step (f) further contains a cytokine.
  • the cytokine is selected from the group consisting of IL-2, IL-15, IL-7, or a combination thereof.
  • each of the cytokine concentrations is independently from 1 to 100 ng/ml, preferably from 2 to 80 ng/ml, more preferably from 5 to 50 ng/ml.
  • step (f) the expansion culture is carried out in a container selected from the group consisting of a culture flask, a culture bag, or a combination thereof.
  • the culture flask is selected from the group consisting of a G-Rex flask, a T75 flask, or a combination thereof.
  • the container is a G-Rex culture flask.
  • the amplification culture is carried out for 8-14 days.
  • step (f) in the expansion culture in the step (f), in the expansion culture, the operation of replacing the container is not performed.
  • the chimeric antigen receptor is directed against a target selected from the group consisting of CD19, CD23, and the like.
  • a chimeric antigen receptor T cell is provided, wherein the chimeric antigen receptor T cell is a method for preparing a chimeric antigen receptor T cell using the serum-free according to claim 1. Prepared.
  • a cell preparation comprising (a) the chimeric antigen receptor T cell of claim 2 and (b) a pharmaceutically acceptable carrier.
  • the cell preparation is a liquid preparation such as an injection.
  • Figure 1 shows the change in growth curve of CAR-T cells in different media.
  • Figure 2 shows the change in growth curve of CAR-T cells in G-Rex flasks, culture bags, and T75 flasks.
  • Figure 3 shows the change in the positive rate curve of CAR-T cells in G-Rex flasks, culture bags, and T75 flasks.
  • Figure 4 shows the effect of different cell culture supplements on CAR-T cell proliferation.
  • Figure 5 shows the proliferation rate of cells in different sorting modes.
  • the serum-free culture method based on the present invention can not only improve the culture success rate and yield of chimeric antigen receptor T cells, but also avoid the toxic side effects of serum on CAR-T cells and significantly reduce or eliminate the risk of introduction due to serum.
  • the present invention has been completed on this basis.
  • a preferred sorting method is a MACS based sorting method.
  • CliniMACS technology can be used for sorting.
  • MACS is a highly specific cell sorting technique.
  • the main components include MACS microspheres, MACS sorting columns and MACS separators.
  • MACS microspheres are superparamagnetic particles coupled to highly specific monoclonal antibodies.
  • the MACS sorting column is placed in a permanent magnetic field MACS separator.
  • Negative sorting is a method of removing magnetic labels from non-target cells from a mixture of cells, ie, non-magnetically labeled cells are target cells.
  • the method of the invention employs CliniMACS or CliniMACS plus sorting techniques (and equipment) for negative sorting to obtain the desired target cells.
  • the sorted cells of the present invention consists essentially of CD3 positive cells.
  • a chimeric antigen receptor includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes an optional signal peptide and a target-specific binding element (also referred to as an antigen binding domain).
  • the intracellular domain includes a costimulatory molecule and a purine chain portion. When CAR is expressed in T cells, the extracellular domain recognizes a specific antigen and then transduces the signal through the intracellular domain, causing activation and proliferation of cells, cytolysis toxicity, and secretion of cytokines such as IL-2 and IFN- ⁇ .
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a sputum chain.
  • the present invention provides a cell preparation, as described in detail in the third aspect of the invention.
  • the cell preparation comprises (a) a chimeric antigen receptor T cell of the second aspect of the invention and (b) a pharmaceutically acceptable carrier.
  • the cell preparation is a liquid preparation such as an injection.
  • the culture method of the present invention uses a serum-free medium and a culture system such as G-Rex to avoid substances which have toxic side effects with cells, and greatly enhance the culture of chimeric antigen receptor T cells. Success rate and security.
  • the culture method of the present invention employs a particularly optimized process flow, thereby significantly improving the culture efficiency of chimeric antigen receptor T cells, particularly significantly increasing the positive rate and yield of harvested CAR-T cells.
  • Serum-free medium basal medium + albumin
  • PBMCs were labeled for 30 minutes using CD19 and CD14 labeled magnetic beads.
  • the required tubes for sorting were installed as required. After the cells were incubated, negative sorting was performed to remove the CD19 and CD14-labeled impurity cells, and the sorted cells with CD3 positive cells as the main components were obtained.
  • the cells obtained by sorting were activated with CD3/CD28, and then serum-free medium (addition of IL-2) containing 1.0% of albumin at a final concentration was used at a cell density of 3 ⁇ 10 6 -6 ⁇ 10 6 /ml.
  • the inoculation culture was carried out and cultured for 2 days.
  • the cells were centrifuged, the supernatant was added to the required volume of lentivirus according to MOI 2-10, resuspended in serum-free medium (containing 1.0% final albumin) and transferred to the flask at 37 ° C, 5 ° Incubate with % CO 2 (12-48 hours).
  • the lentivirus is a viral vector expressing a CAR gene of interest.
  • the cell suspension was centrifuged at 200-300 g for 6-8 minutes. The supernatant was aspirated and resuspended, transferred to serum-free medium (containing a final concentration of 1.0% albumin), and IL-2 concentration was added to 50-500 IU/ml to obtain virus-removed T cells.
  • the virus-depleted T cells were transferred to a G-Rex flask and expanded and cultured at 37 ° C, 5% CO 2 .
  • the G-Rex bottle was taken out from the incubator, and the cells were mixed and sampled. After adding IL-2 concentration to 50-500 IU/ml, the culture was continued until the cell yield reached 1 ⁇ 10 9 . When -1 x 10 10 CAR-T cells were harvested.
  • the CAR-T positive rate was determined to be >20% in the harvested cells.
  • cryopreservation, sorting, cell activation, and gene transduction of cryopreserved PBMC were performed in the same manner as in Example 1.
  • the above cell suspension was centrifuged at 200-300 g for 6-8 minutes. Aspirate the supernatant and gently resuspend the cells to obtain virus-depleted T cells.
  • the virus-depleted T cells were transferred to a medium (OpTmizer SFM + 1.0% albumin), and IL-2 (final concentration of 25 IU/ml) was added, and then transferred to a G-Rex flask, a culture bag, and a T75 culture. The flask was then incubated at 37 ° C, 5% CO 2 .
  • CAR-T cells showed a faster cell proliferation rate from Day3 to Day5 in the G-Rex culture system than in culture bags and culture flasks; as shown in Figure 3: CAR-T cells were in G- Rex can maintain a high level of CAR positive rate, and the decline is smaller than cell culture bags and culture flasks; in summary, for a large number of CAR-T positive cells in a short time, G-Rex has a cell culture bag. And the advantages of the culture bottle with higher proliferation rate and positive rate.
  • Experimental group 1 Experimental group 2
  • Experimental group 3 Experimental group 4
  • Experimental group 5 IL-2 + - + + + + IL-7 - + + + + - IL-15 - + + + - + + - +
  • the combination of the three factors of cytokines IL-2, IL-7 and IL-15 in the culture system has the best effect on cell proliferation and contributes to the increase in the number of CAR-T cells.
  • the resuscitated PBMC were resuspended in sorting buffer and aliquoted for negative sorting CD19+CD14+ and positive sorting CD3+.
  • the sorted cells were inoculated separately with a medium, and added to the activated magnetic beads to be mixed and cultured.
  • the virus was removed by centrifugation on the third day and the activated magnetic beads were removed with a magnetic stand, and the medium was further cultured.
  • the culture was carried out until the 8th day for testing.
  • the ratio of the target cells collected from the PBMC by the negative sorting method is similar to that of the positive sorting, but for the cell proliferation process, the negative sorting method has a significant increase in the positive sorting. In summary, the total amount of CD3 positive cells obtained by the final sorting method was more.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Developmental Biology & Embryology (AREA)

Abstract

提供了一种无血清培养制备嵌合抗原受体T细胞的方法。该方法包括步骤：(a)提供PBMC细胞；(b)对PBMC细胞进行负分选处理获得经分选的PBMC细胞；(c)对经分选的PBMC细胞进行活化处理得到经活化的T细胞；(d)对经活化的T细胞，用表达嵌合抗原受体的病毒载体进行基因转染，从而获得经转染的T细胞；(e)对所述经转染的T细胞，进行去除病毒处理，从而获得去除病毒的T细胞；和(f)将去除病毒的T细胞进行扩增培养，从而获得嵌合抗原受体T细胞。

Description

无血清培养制备嵌合抗原受体T细胞的方法 技术领域
本发明涉及生物技术领域，具体地涉及无血清培养制备嵌合抗原受体T细胞的方法。
背景技术
嵌合抗原受体T细胞免疫疗法是一种利用基因编辑手段使T细胞拥有能特异性识别肿瘤相关抗原的scFv片段和胞内信号域，进而增强T细胞靶向性、杀伤性以及持久性的新型医疗技术。在近年来的肿瘤免疫治疗临床上有着很好的表现，给临床治愈肿瘤带来了希望。
嵌合抗原受体T细胞的制备过程相对复杂，涉及细胞活化、分选、转染、扩增培养等等众多操作。工艺流程、设备设施和试剂选择等任何一个环节的不足都会对于细胞制备的质量产生重要的影响，进而影响嵌合抗原受体T细胞的活力、收率、安全性以及后期的临床效果。
血清，作为传统细胞培养过程中的一个重要成分，由于其本身含有一些对细胞有毒副作用的物质。此外，各批次血清之间质量差异较大。另外，在血清取材过程中可能带入支原体、病毒等风险，这些都严重阻碍了细胞疗法的发展。
目前市场上没有一种令人满意的高效的无血清制备嵌合抗原受体T细胞的工艺，这严重影响了嵌合抗原受体T细胞免疫疗法的进一步开发、推广和应用。
因此，本领域迫切需要开发安全的、基于无血清细胞培养的、且高效制备嵌合抗原受体T细胞的方法。
发明内容
本发明的目的就是提供一种安全的、基于无血清细胞培养的、且高效制备嵌合抗原受体T细胞的方法。
在本发明的第一方面，提供了一种无血清制备嵌合抗原受体T细胞的方法,包括步骤：
(a)提供PBMC细胞，所述PBMC细胞重悬于无血清培养基中；
(b)对步骤(a)中的PBMC细胞进行负分选处理，从而获得经分选的PBMC细胞；
(c)对步骤(b)获得的经分选的PBMC细胞进行活化处理，从而得到经活化的T细胞；
(d)对步骤(c)获得的经活化的T细胞，用表达嵌合抗原受体的病毒载体进行基因转染，从而获得经转染的T细胞；
(e)对所述经转染的T细胞，进行去除病毒处理，从而获得去除病毒的T细胞；和
(f)将去除病毒的T细胞重悬于扩增培养基，并进行扩增培养，从而获得嵌合抗原受体T细胞，并且当所述嵌合抗原受体T细胞的数量达到预定数量时，收获所述嵌合抗原受体T细胞，其中所述扩增培养基是含细胞因子的无血清培养基。
在另一优选例中，在所述方法的所有步骤中使用无血清培养基。
在另一优选例中，在步骤(a)中，所述的PBMC细胞为已复苏的PBMC细胞。
在另一优选例中，所述PBMC细胞是自体的或同种异体的。
在另一优选例中，所述的PBMC细胞来源于人。
在另一优选例中，在步骤(b)中，所述的负分选处理使用CliniMACS。
在另一优选例中，在步骤(c)中，所述活化用抗CD3/CD28的抗体进行活化处理。
在另一优选例中，在步骤(c)中，所述活化处理在活化培养基中进行，所述活化培养基含有基础培养基和添加剂。
在另一优选例中，所述基础培养基选自下组：LONZA X-VIVO、LIFE CTS AIM V、LIFE OpTmizer SFM、RPMI 1640、ImmunoCult TM-XF、
Figure PCTCN2018089801-appb-000001
在另一优选例中，所述添加剂选自下组：人源血清白蛋白、重组人血清白蛋白、植物源重组白蛋白或其组合，优选重组白蛋白。
在另一优选例中，所述白蛋白浓度为0.01％-50％(W/V)，较佳地，0.05％-30％(W/V)，更佳地，0.1％-20％(W/V)。
在另一优选例中，所述活化处理选自下组：抗体包被处理、游离抗体处理、活化磁珠处理、或其组合。
在另一优选例中，在所述活化处理中，抗体浓度在10ng～100ng/ml。
在另一优选例中，在所述活化处理中，活化磁珠数量与细胞细胞的比例在0.25～5:1，较佳地；0.5-3：1。
在另一优选例中，所述活化处理的时间为3～8天。
在另一优选例中，在步骤(d)中，所述的病毒载体选自下组：慢病毒、腺相关 病毒(AAV)、腺病毒、或其组合。
在另一优选例中，所述基因转染的感染倍数MOI为0.5-30，较佳地1-20，更佳地2-10。
在另一优选例中，在步骤(e)中，所述的去除病毒的处理包括：将经转染的细胞重悬于无血清培养基，离心，并吸取上清，从而获得去除病毒的T细胞。
在另一优选例中，在步骤(f)中，所述的预定数量为1×10 9-1×10 11，较佳地2×10 9-2×10 10。
在另一优选例中，在步骤(f)中的扩增培养基还含有细胞因子。
在另一优选例中，所述细胞因子选自下组：IL-2、IL-15、IL-7或其组合。
在另一优选例中，所述各细胞因子浓度各自独立地为1-100ng/ml，较佳地，2-80ng/ml，更佳地，5-50ng/ml。
在另一优选例中，在步骤(f)中，所述扩增培养在一容器中进行，所述容器选自下组：培养瓶、培养袋、或其组合。
在另一优选例中，所述的培养瓶选自下组：G-Rex培养瓶、T75培养瓶、或其组合。
在另一优选例中，所述容器为G-Rex培养瓶。
在另一优选例中，所述扩增培养的时间为8-14天。
在另一优选例中，在步骤(f)中，在所述扩增培养中，不进行更换容器的操作。
在另一优选例中，所述的嵌合抗原受体针对选自下组的靶点：CD19、CD23等。
在本发明的第二方面，提供了一种嵌合抗原受体T细胞，所述的嵌合抗原受体T细胞是用权利要求1所述的无血清制备嵌合抗原受体T细胞的方法制备的。
在本发明的第三方面，提供了一种细胞制剂，所述的细胞制剂含有(a)权利要求2所述的嵌合抗原受体T细胞和(b)药学上可接受的载体。
在另一优选例中，所述的细胞制剂为液体制剂(如注射剂)。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。 限于篇幅，在此不再一一累述。
附图说明
图1显示了CAR-T细胞在不同培养基的生长曲线变化。
图2显示了CAR-T细胞在G-Rex培养瓶、培养袋以及T75培养瓶中的生长曲线变化。
图3显示了CAR-T细胞在G-Rex培养瓶、培养袋以及T75培养瓶中的阳性率曲线变化。
图4显示了不同细胞培养添加物对CAR-T细胞增殖的影响。
图5显示了不同分选方式细胞的增殖速率。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种独特的用于制备嵌合抗原受体T细胞的无血清培养创新工艺。基于本发明的无血清培养方法，不仅可以提高嵌合抗原受体T细胞的培养成功率和产率，而且可以避免血清对CAR-T细胞的毒副作用并显著降低或消除因血清而引入的风险，从而为嵌合抗原受体T细胞免疫疗法的进一步开发和推广提供帮助。在此基础上完成了本发明。
术语
方法
在本发明中，可以采用正分选或负分选。一种优选的分选方法是基于MACS的分选方法。例如可采用CliniMACS技术进行分选。
MACS是一种高度特异的细胞分选技术。主要部件包括MACS微球，MACS分选柱和MACS分离器。MACS微球是与高度特异性单克隆抗体偶联的超顺磁颗粒。MACS分选柱置于永磁场MACS分离器中。负分选是从细胞混合物中去除非靶标细胞中的磁性标记的方法，即非磁性标记的细胞是靶细胞。
优选地，本发明方法采用CliniMACS或CliniMACS plus分选技术(和设备)进行负分选，从而获得所需的靶细胞。
通常，在本发明经分选后的细胞中，基本上由CD3阳性细胞构成。
嵌合抗原受体(CAR)
如本文所用，嵌合免疫抗原受体(Chimeric antigen receptor，CAR)包括细胞外结构域、任选的铰链区、跨膜结构域、和细胞内结构域。胞外结构域包括任选的信号肽和靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激分子和ζ链部分。CAR在T细胞中表达时，胞外段可识别一个特异的抗原，随后通过胞内结构域转导该信号，引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子如IL-2和IFN-γ等，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。
制剂
本发明提供了一种细胞制剂，具体如本发明第三方面所述。在一个实施方式中，所述细胞制剂含有(a)本发明第二方面所述的嵌合抗原受体T细胞和(b)药学上可接受的载体。在一个实施方式中，所述的细胞制剂为液体制剂(如注射剂)。
本发明的主要优点包括：
(a)本发明的培养方法与传统细胞培养相比，采用无血清培养基和G-Rex等培养体系，避免带入与细胞有毒副作用的物质，大大提升了嵌合抗原受体T细胞的培养成功率和安全性。
(b)本发明的培养方法采用特别优化的工艺流程，从而显著提高嵌合抗原受体T细胞的培养有效性，尤其是显著提高了收获的CAR-T细胞的阳性率和产率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
试剂
无血清培养基：基础培养基+白蛋白
实施例1
培养基筛选
1.1 冻存或新鲜PBMC
取冻存或新鲜的300×10 6-1000×10 6个细胞量的PBMC重悬于无血清培养基内。
1.2 分选
使用CD19和CD14标记磁珠对PBMC进行标记孵育30分钟。在CliniMACS上将分选所需管路按照要求进行安装，待细胞孵育结束后进行负分选操作，去除CD19和CD14标记的杂质细胞，获得以CD3阳性细胞为主要成分的分选细胞。
1.3 细胞活化
用CD3/CD28将分选所得细胞进行活化，然后按照3×10 6-6×10 6/ml的细胞密度使用加入含终浓度为1.0％白蛋白的无血清培养基(补加IL-2)进行接种培养，培养2天。
1.4 基因转导
将细胞离心弃上清后按照MOI 2-10加入所需体积的慢病毒，重悬于无血清培养基(含有终浓度为1.0％的白蛋白)后重新转移至培养瓶中，37℃、5％CO 2培养(12-48小时)。
其中，所述的慢病毒为表达目的CAR基因的病毒载体。
1.5 病毒去除
将细胞悬液离心200-300g离心6-8分钟。吸弃上清并重悬细胞后转移至无血清培养基(含有终浓度1.0％白蛋白)中，添加IL-2浓度至50-500IU/ml后，从而获得去除病毒的T细胞。
1.6 扩增培养
将去除病毒的T细胞转移至G-Rex培养瓶中，于37℃、5％CO 2进行扩增培养。
扩增培养3-10天后，将G-Rex瓶从培养箱中取出，混匀细胞后取样计数，补加IL-2浓度至50-500IU/ml后，继续培养至细胞产量达到1×10 9-1×10 10CAR-T细胞时，进行收获。
1.7 结果
经测定，在收获的细胞中，CAR-T阳性率＞20％。
实施例2.
实验耗材筛选
2.1 PBMC复苏、分选、细胞活化、基因转导
使用与实施例1中相同的方法进行冻存PBMC复苏、分选、细胞活化、基因转导。
2.2 病毒去除
将上述细胞悬液200-300g离心6-8分钟。吸弃上清，轻弹重悬细胞，从而获得去除病毒的的T细胞。
2.3 扩增培养
将去除病毒的T细胞转移至培养基(OpTmizer SFM+1.0％白蛋白)中，并添加IL-2(终浓度为25IU/ml)后，分别转移至G-Rex培养瓶、培养袋以及T75培养瓶中，然后置于37℃、5％CO 2继续培养。
继续培养3天后，分别从G-Rex培养瓶、培养袋以及T75培养瓶中取样计数，记录细胞密度和细胞活率。留样检测。
补加IL-2后(终浓度为25IU/ml)，继续放回培养箱培养。
继续培养1天后，分别从G-Rex培养瓶、培养袋以及T75培养瓶中取样计数，记录细胞密度和细胞活率。留样检测。
继续培养2天后，分别从G-Rex培养瓶、培养袋以及T75培养瓶中取样计数，记录细胞密度和细胞活率。留样检测。
2.4 结果
如图2所示：CAR-T细胞在G-Rex培养体系内从Day3至Day5表现出了较 培养袋和培养瓶更快的细胞增殖速率；如图3所示：CAR-T细胞在G-Rex内能保持高水平的CAR阳性率，并且下降幅度较细胞培养袋和培养瓶都要小；综上所述，对于短时间内需要大量CAR-T阳性细胞，G-Rex拥有比细胞培养袋和培养瓶更高增殖率和阳性率的优势。
实施例3.
细胞培养添加物实验
3.1 方法
在本实施例中，在细胞培养基中添加不同的添加物IL-2,IL-7,IL-15或其组合，共设有5个实验组。
表1
实验组1 实验组2 实验组3 实验组4 实验组5
IL-2 + - + + +
IL-7 - + + + -
IL-15 - + + - +
使用与实施例1中相同的方法进行冻存PBMC复苏、分选、磁珠标记活化、基因转导、磁珠去除、病毒去除和扩增培养。
1)使用的培养基为OpTmizer SFM+白蛋白(终浓度为1.0％)。
2)所有添加IL-2的步骤分别替代为表1中实验组2-5对应的细胞因子，进行培养。
3.2 结果
如图4所示：培养体系中细胞因子IL-2、IL-7、IL-15三种因子联用对于细胞增殖的提升效果最好，有助于CAR-T细胞数量的提升。
实施例4.
正分选和负分选的对比实验
4.1 考察对象：
相同PBMC通过正分选或负分选的不同方式对细胞培养的影响对比。
4.2 细胞分选：
将复苏的PBMC重悬于分选缓冲液内并等分两份，分别进行负分选CD19+CD14+和正分选CD3+。
4.3 细胞活化：
将分选后的细胞分别用培养基进行接种，并加入活化磁珠混匀培养。
4.4 病毒转染：
培养至第2天时,计算活化细胞数量，根据MOI(2～10)计算所需慢病毒载体数量，取相应慢病毒载体用培养液重悬均匀，离心去除原细胞上清，加入慢病毒载体悬液重悬并继续培养。
4.5 去除病毒和活化磁珠：
培养至第3天时离心去除病毒和用磁力架去除活化磁珠，加培养基继续培养。
培养至第8天进行检测。
4.6 结果
结果如表2和图5所示：
表2
Figure PCTCN2018089801-appb-000002
采用负分选方式从PBMC中收集到的目的细胞的比例和正分选的比例相近，但是对于细胞增殖过程，负分选方法有较正分选明显的增长。综上所述，采用负分选方式最终收获得到的目的CD3阳性细胞总量更多。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种无血清制备嵌合抗原受体T细胞的方法,其特征在于，包括步骤：
    (a)提供PBMC细胞，所述PBMC细胞重悬于无血清培养基中；
    (b)对步骤(a)中的PBMC细胞进行负分选处理，从而获得经分选的PBMC细胞；
    (c)对步骤(b)获得的经分选的PBMC细胞进行活化处理，从而得到经活化的T细胞；
    (d)对步骤(c)获得的经活化的T细胞，用表达嵌合抗原受体的病毒载体进行基因转染，从而获得经转染的T细胞；
    (e)对所述经转染的T细胞，进行去除病毒处理，从而获得去除病毒的T细胞；和
    (f)将去除病毒的T细胞重悬于扩增培养基，并进行扩增培养，从而获得嵌合抗原受体T细胞，并且当所述嵌合抗原受体T细胞的数量达到预定数量时，收获所述嵌合抗原受体T细胞，其中所述扩增培养基是含细胞因子的无血清培养基。
  2. 如权利要求1所述的方法，所有步骤中使用无血清培养基。
  3. 如权利要求1所述的方法，在步骤(c)中，所述活化处理在活化培养基中进行，所述活化培养基含有基础培养基和添加剂。
  4. 如权利要求1所述的方法，所述基础培养基选自下组：LONZA X-VIVO、LIFE CTS AIM V、LIFE OpTmizer SFM、RPMI 1640、ImmunoCult TM-XF、
    Figure PCTCN2018089801-appb-100001
  5. 如权利要求1所述的方法，所述添加剂选自下组：人源血清白蛋白、重组人血清白蛋白、植物源重组白蛋白或其组合，优选重组白蛋白。
  6. 如权利要求1所述的方法，在步骤(f)中的扩增培养基还含有细胞因子。
  7. 如权利要求1所述的方法，所述细胞因子选自下组：IL-2、IL-15、IL-7或其组合。
  8. 如权利要求1所述的方法，在步骤(f)中，所述扩增培养在一容器中进行，所述容器选自下组：培养瓶、培养袋、或其组合。
  9. 一种嵌合抗原受体T细胞，其特征在于，所述的嵌合抗原受体T细胞是用权利要求1所述的无血清制备嵌合抗原受体T细胞的方法制备的。
  10. 一种细胞制剂，其特征在于，所述的细胞制剂含有(a)权利要求2所述的嵌合抗原受体T细胞和(b)药学上可接受的载体。
PCT/CN2018/089801 2018-05-16 2018-06-04 无血清培养制备嵌合抗原受体t细胞的方法 WO2019218402A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP2020563994A JP2021531734A (ja) 2018-05-16 2018-06-04 無血清培地によりキメラ抗原レセプターｔ細胞を調製する方法
AU2018423493A AU2018423493A1 (en) 2018-05-16 2018-06-04 Method for preparing chimeric antigen receptor T cells by serum-free culture
US17/054,989 US20210214682A1 (en) 2018-05-16 2018-06-04 Method for preparing chimeric antigen receptor t cells by serum-free culture
SG11202011263RA SG11202011263RA (en) 2018-05-16 2018-06-04 Method for preparing chimeric antigen receptor t cells by serum-free culture
EP18919224.8A EP3822344A4 (en) 2018-05-16 2018-06-04 PROCEDURE FOR THE PRODUCTION OF CHIMERA ANTIGEN RECEPTOR T-CELLS BY SERUM-FREE CULTURE
KR1020207035736A KR20210010509A (ko) 2018-05-16 2018-06-04 무혈청 배양에 의해 키메라 항원 수용체 t 세포를 제조하는 방법

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810467017.6 2018-05-16
CN201810467017.6A CN110499291B (zh) 2018-05-16 2018-05-16 无血清培养制备嵌合抗原受体t细胞的方法

Publications (1)

Publication Number Publication Date
WO2019218402A1 true WO2019218402A1 (zh) 2019-11-21

Family

ID=68539350

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/089801 WO2019218402A1 (zh) 2018-05-16 2018-06-04 无血清培养制备嵌合抗原受体t细胞的方法

Country Status (8)

Country Link
US (1) US20210214682A1 (zh)
EP (1) EP3822344A4 (zh)
JP (1) JP2021531734A (zh)
KR (1) KR20210010509A (zh)
CN (1) CN110499291B (zh)
AU (1) AU2018423493A1 (zh)
SG (1) SG11202011263RA (zh)
WO (1) WO2019218402A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112662631A (zh) * 2021-03-16 2021-04-16 合源生物科技(天津)有限公司 一种car-t细胞灌流培养方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438034A (zh) * 2020-11-06 2022-05-06 上海赛比曼生物科技有限公司 一种基因修饰的细胞制备方法
WO2023240483A1 (zh) * 2022-06-15 2023-12-21 上海恒润达生生物科技股份有限公司 一种快速制备t细胞的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399242A (zh) * 2016-09-13 2017-02-15 北京多赢时代转化医学研究院 联合制备CAR‑Vγ9Vδ2T细胞和CAR‑NKT细胞的方法

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10260038B2 (en) * 2013-05-10 2019-04-16 Whitehead Institute For Biomedical Research Protein modification of living cells using sortase
EP3145516A4 (en) * 2014-05-20 2018-06-13 Kiromic, LLC Methods and compositions for treating malignancies with dendritic cells
US10596492B2 (en) * 2014-07-09 2020-03-24 Sudhin Biopharma Particle settling devices
JP6919118B2 (ja) * 2014-08-14 2021-08-18 ノバルティス アーゲー ＧＦＲα−４キメラ抗原受容体を用いる癌の治療
TW201631152A (zh) * 2014-12-29 2016-09-01 諾華公司 製造嵌合抗原受體－表現細胞之方法
CN104829733B (zh) * 2015-05-25 2018-06-05 广州百暨基因科技有限公司 抗原结合单元稳定的嵌合抗原受体及制备方法与应用
CN105087495B (zh) * 2015-08-21 2016-04-27 深圳市茵冠生物科技有限公司 双嵌合抗原受体修饰的t淋巴细胞及其制备方法
AU2016343682A1 (en) * 2015-10-30 2018-06-14 The Regents Of The University Of California Methods of generating T-cells from stem cells and immunotherapeutic methods using the T-cells
US10596274B2 (en) * 2016-03-19 2020-03-24 Exuma Biotech Corp. Methods and compositions for transducing lymphocytes and regulated expansion thereof
CN105950561A (zh) * 2016-05-26 2016-09-21 江苏杰晟生物科技有限公司 一种靶向乳腺癌干细胞的双特异性嵌合抗原受体基因修饰的t淋巴细胞的制备方法及其产品
CN105950554A (zh) * 2016-06-27 2016-09-21 武汉思安医疗技术有限公司 一种高效刺激活化t细胞的方法
CN106047817A (zh) * 2016-07-28 2016-10-26 深圳爱生再生医学科技有限公司 Car‑t细胞及其制备方法与应用
CN106350487B (zh) * 2016-09-13 2019-01-25 北京多赢时代转化医学研究院 联合制备car-nk细胞和car-nkt细胞的方法
TW202340473A (zh) * 2016-10-07 2023-10-16 瑞士商諾華公司 利用嵌合抗原受體之癌症治療
CN107151654B (zh) * 2016-10-11 2020-05-05 深圳宾德生物技术有限公司 一种人源t淋巴细胞的培养基及其制备方法和应用
CN107827991B (zh) * 2017-11-20 2020-10-09 英普乐孚生物技术(上海)有限公司 靶向cd19的嵌合抗原受体t细胞及其应用
AU2018379091A1 (en) * 2017-12-08 2020-06-25 Juno Therapeutics, Inc. Serum-free media formulation for culturing cells and methods of use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399242A (zh) * 2016-09-13 2017-02-15 北京多赢时代转化医学研究院 联合制备CAR‑Vγ9Vδ2T细胞和CAR‑NKT细胞的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GAO, PENG ET AL.: "The Specific Killing Effect of Anti- CD 19 CAR-T Cells on K562 CD 19+ Tumor Cells", CHINESE MEDICAL BIOTECHNOLOGY, vol. 13, no. 2, 30 April 2018 (2018-04-30), pages 145 - 150, XP055767377, ISSN: 1673-713X *
SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3822344A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112662631A (zh) * 2021-03-16 2021-04-16 合源生物科技(天津)有限公司 一种car-t细胞灌流培养方法
CN112662631B (zh) * 2021-03-16 2021-06-29 合源生物科技(天津)有限公司 一种car-t细胞灌流培养方法

Also Published As

Publication number Publication date
JP2021531734A (ja) 2021-11-25
CN110499291A (zh) 2019-11-26
CN110499291B (zh) 2023-11-24
KR20210010509A (ko) 2021-01-27
US20210214682A1 (en) 2021-07-15
SG11202011263RA (en) 2020-12-30
AU2018423493A1 (en) 2020-12-03
EP3822344A1 (en) 2021-05-19
EP3822344A4 (en) 2022-06-08

Similar Documents

Publication Publication Date Title
WO2019218402A1 (zh) 无血清培养制备嵌合抗原受体t细胞的方法
CN112251406B (zh) 一种nk细胞活化阶段的外泌体分选方法
WO2012137538A1 (ja) 細胞傷害性ｔ細胞誘導用組成物
JP4982645B2 (ja) ＴｃＲガンマデルタＴ細胞の産生
CN108165530B (zh) 一种分离提纯小鼠肿瘤组织浸润cd4+cd25+调节性t细胞的方法
CN116426475A (zh) 一种nk细胞的体外激活扩增方法
CN114231488A (zh) 一种体外培养th1细胞的培养液及其应用和th1细胞的体外培养方法
CN115772496A (zh) 一种诱导性多能干细胞制备通用血小板的方法
US20230257704A1 (en) Methods for preparation of immune cells
US20090298045A1 (en) Method For Selectively Expanding, Selecting And Enriching Stem/Progenitor Cell Populations
CN116814731A (zh) 纯化t细胞的方法及其用途
CN113943704A (zh) 一种肿瘤新生抗原特异性t细胞的制备方法
CN112725273A (zh) 一种nk细胞及其制备方法和应用
CN112424342A (zh) 用于培养和扩增细胞的组合物和方法
US20030129166A1 (en) Human circulating dendritic cell compositions and methods
WO2011021503A1 (ja) 一過性生着ｃｔｌを含む医薬品組成物
CN115125203B (zh) 一种Th2细胞体外培养方法
CN109385437B (zh) Dna分子、含有该dna分子的载体以及获得的永生化细胞
CN117384858B (zh) 一种嵌合抗原受体t细胞的制备方法及其应用
CN117965445B (zh) 一种用于细胞培养的组合物
US20230407335A1 (en) Methods for Preparing Genetically Modified Cells
CN117210404A (zh) 一种高纯度的外周血自体nk细胞培养基以及体外培养方法
CN117247900A (zh) 一种γδT混合细胞群的制备方法
CN111647563A (zh) 靶向Survivin全抗原的DC细胞、CTL细胞及其制备方法和应用
CN114561353A (zh) 一种高效分离纯化nkt细胞的免疫学方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18919224

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020563994

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018423493

Country of ref document: AU

Date of ref document: 20180604

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20207035736

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2018919224

Country of ref document: EP

Effective date: 20201216

ENP Entry into the national phase

Ref document number: 2018919224

Country of ref document: EP

Effective date: 20201216

ENP Entry into the national phase

Ref document number: 2018919224

Country of ref document: EP

Effective date: 20201216