CN116370618A - 一种全肿瘤细胞微载体支架疫苗及其制备方法 - Google Patents
一种全肿瘤细胞微载体支架疫苗及其制备方法 Download PDFInfo
- Publication number
- CN116370618A CN116370618A CN202310152192.7A CN202310152192A CN116370618A CN 116370618 A CN116370618 A CN 116370618A CN 202310152192 A CN202310152192 A CN 202310152192A CN 116370618 A CN116370618 A CN 116370618A
- Authority
- CN
- China
- Prior art keywords
- microcarrier
- tumor cell
- scaffold
- microcarriers
- tumor
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 63
- 210000004881 tumor cell Anatomy 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 210000004027 cell Anatomy 0.000 claims abstract description 40
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 34
- 239000000725 suspension Substances 0.000 claims abstract description 13
- 238000004108 freeze drying Methods 0.000 claims abstract description 10
- 239000002955 immunomodulating agent Substances 0.000 claims abstract description 7
- 229940121354 immunomodulator Drugs 0.000 claims abstract description 7
- 230000002584 immunomodulator Effects 0.000 claims abstract description 6
- 230000002779 inactivation Effects 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000002844 melting Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 206010009944 Colon cancer Diseases 0.000 claims description 6
- KGRVJHAUYBGFFP-UHFFFAOYSA-N 2,2'-Methylenebis(4-methyl-6-tert-butylphenol) Chemical compound CC(C)(C)C1=CC(C)=CC(CC=2C(=C(C=C(C)C=2)C(C)(C)C)O)=C1O KGRVJHAUYBGFFP-UHFFFAOYSA-N 0.000 claims description 5
- 238000004113 cell culture Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000010257 thawing Methods 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 208000014018 liver neoplasm Diseases 0.000 claims description 3
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims description 2
- 229920002101 Chitin Polymers 0.000 claims description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 2
- 108010010803 Gelatin Proteins 0.000 claims description 2
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 claims description 2
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 claims description 2
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 claims description 2
- 239000004793 Polystyrene Substances 0.000 claims description 2
- 229920005830 Polyurethane Foam Polymers 0.000 claims description 2
- 229940072056 alginate Drugs 0.000 claims description 2
- 235000010443 alginic acid Nutrition 0.000 claims description 2
- 229920000615 alginic acid Polymers 0.000 claims description 2
- 239000013043 chemical agent Substances 0.000 claims description 2
- 238000013329 compounding Methods 0.000 claims description 2
- 239000000499 gel Substances 0.000 claims description 2
- 239000008273 gelatin Substances 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 235000011852 gelatine desserts Nutrition 0.000 claims description 2
- 230000002519 immonomodulatory effect Effects 0.000 claims description 2
- 230000005934 immune activation Effects 0.000 claims description 2
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 claims description 2
- 230000001571 immunoadjuvant effect Effects 0.000 claims description 2
- 239000000568 immunological adjuvant Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 239000011496 polyurethane foam Substances 0.000 claims description 2
- 230000003068 static effect Effects 0.000 claims description 2
- 239000000427 antigen Substances 0.000 abstract description 20
- 102000036639 antigens Human genes 0.000 abstract description 20
- 108091007433 antigens Proteins 0.000 abstract description 20
- 210000002865 immune cell Anatomy 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 6
- 238000009169 immunotherapy Methods 0.000 abstract description 6
- 230000005975 antitumor immune response Effects 0.000 abstract description 3
- 238000011068 loading method Methods 0.000 abstract description 3
- 206010027476 Metastases Diseases 0.000 abstract description 2
- 230000009401 metastasis Effects 0.000 abstract description 2
- 238000010254 subcutaneous injection Methods 0.000 abstract description 2
- 239000007929 subcutaneous injection Substances 0.000 abstract description 2
- 230000007365 immunoregulation Effects 0.000 abstract 1
- 230000002980 postoperative effect Effects 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 8
- 239000000463 material Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 238000003501 co-culture Methods 0.000 description 4
- 208000029742 colonic neoplasm Diseases 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 229940124644 immune regulator Drugs 0.000 description 4
- 239000012620 biological material Substances 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000001878 scanning electron micrograph Methods 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940022399 cancer vaccine Drugs 0.000 description 2
- 238000009566 cancer vaccine Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000003194 forelimb Anatomy 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000004626 scanning electron microscopy Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 206010067868 Skin mass Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000746 body region Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000445 field-emission scanning electron microscopy Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000010907 mechanical stirring Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- 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
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- 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
- 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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0631—Mammary 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
- 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
- C12N5/067—Hepatocytes
-
- 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
- C12N5/0679—Cells of the gastro-intestinal tract
-
- 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
- C12N5/0693—Tumour cells; Cancer 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
- C12N2531/00—Microcarriers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Oncology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Dermatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明属于免疫治疗技术领域,具体一种全肿瘤细胞微载体支架疫苗及其制备方法。本发明采用微载体培养肿瘤细胞,收集肿瘤细胞与微载体制备成混合悬液;经过灭活、负载免疫调节和冻干处理,得到全肿瘤细胞微载体支架疫苗。制备过程简单,将全细胞肿瘤抗原的制备、生物支架的制备和抗原的负载集于一体。使用时,以皮下注射的方式给药,其中全肿瘤细胞作为抗原部分,辅以免疫调节剂,而微载体本身所具有的大孔结构可以作为免疫中心招募免疫细胞,从而引发抗肿瘤免疫应答,用于防止肿瘤术后复发和转移。本发明所提供的全肿瘤细胞微载体支架疫苗能发挥很好的疗效,具有广阔的应用前景。
Description
技术领域
本发明属于免疫治疗技术领域,具体涉及一种微载体支架疫苗及其制备方法。
背景技术
传统的肿瘤治疗方法存在着易复发、易耐受和不良反应大等缺陷。近年来随着免疫学研究的进展,肿瘤免疫疗受到越来越多的关注。肿瘤的免疫疗法通过作用于免疫细胞,激活人体免疫系统,减少肿瘤细胞免疫逃逸,从而增强对肿瘤细胞的杀伤作用。肿瘤疫苗作为免疫治疗的分支,具有极大的发展前途及应用潜力。尽管肿瘤疫苗发展迅速,但目前大多数肿瘤疫苗仍然存在治疗效果有限,临床客观响应率低,生产工艺较复杂等问题。肿瘤疫苗效果欠佳的可能原因主要是:①肿瘤抗原的免疫原性不足;②肿瘤内所存在的免疫抑制性微环境,例如肿瘤内高水平的PD-L1表达和缺乏持久的T细胞浸润;③缺乏高效的癌症疫苗递送技术。因此,开发新兴且便捷的肿瘤疫苗递送系统具有很高的临床应用价值。
生物支架材料由于其具有良好的生物相容性已被广泛应用于癌症疫苗递送领域。相比于纳米材料,生物支架材料不仅可以作为抗原和免疫调节剂的递送系统,还可以创造局部生态位,原位募集免疫细胞。
然而,目前基于生物材料支架的肿瘤疫苗制备过程较繁琐,主要包括制备肿瘤抗原,生物材料支架的制备以及抗原的组装和负载。由于肿瘤细胞上存在一系列突变的肿瘤抗原,全肿瘤细胞抗原比通用型肿瘤抗原的免疫原性更强,可以增强机体产生特异性免疫应答。全肿瘤细胞抗原处理时需要经过反复冻融、放疗、化疗等,而在生物支架材料制备完成后,还需要对肿瘤抗原进行额外负载,进一步使过程复杂化,因此,寻找更简单的方法制备基于生物支架的肿瘤疫苗意义重大。
微载体是一种广泛用于干细胞培养的生物支架材料,具有生物可降解性高,生物安全性好等优点。通过利用微载体递送肿瘤抗原和免疫调节剂,能够对抗原和免疫调节剂的释放进行时间和空间的控制,从而实现对招募的树突状细胞进行原位诱导和活化,进而激活T细胞产生强有力的细胞毒性T淋巴细胞(CTL)的免疫反应,提高抗肿瘤效果。
发明内容
本发明针对现有技术所存在的不足,提供一种全肿瘤细胞微载体支架疫苗及其制备方法。
本发明提供的全肿瘤细胞微载体支架疫苗的制备方法,具体步骤为:
(1)利用微载体接种和培养肿瘤细胞:首先在片状微载体上接种肿瘤细胞,每片的接种细胞量为0.2~1×106个细胞;然后培养细胞,培养条件为37℃,pH值为6.8-7.8,微载体上的细胞量可达到初始量的4~6倍;培养完成后,收集肿瘤细胞与微载体,制备成混合悬液;
(2)灭活:将所得的混合悬液于液氮(-196℃)中冷冻;然后水浴复融解冻;其中,冷冻时间为24h~96h;水浴复融温度为25℃~37℃;
(3)负载免疫调节:将复融后的空白微载体疫苗组与一定量的免疫调节剂复配,混匀得到混合溶液;
(4)冻干:将步骤(3)中所得的混合溶液进行冷冻干燥,即得全肿瘤细胞微载体支架疫苗。其中,冻干时间为8h~24h中任意时长。
进一步的,所述微载体选自液体微载体、大孔明胶微载体、聚苯乙烯微载体、甲壳质微载体、聚氨酯泡沫微载体、藻酸盐凝胶微载体以及磁性微载体等。
进一步的,所述的肿瘤细胞培养方法可采取静态培养或者转瓶培养的方式。
进一步的,所述的肿瘤细胞可以为CT26结直肠癌细胞、Hepa1-6肝癌细胞、4T1乳腺癌细胞等肿瘤细胞。
进一步的,所述免疫调节剂包括免疫佐剂、免疫检查点抑制剂、对免疫激活起效的化学药物的一种或多种的组合。
在一个较优的实施例中,所述免疫调节剂为瑞奎莫德(R848)与抗OX40抗体的药物组合。
本发明提供了一种微载体的新应用,传统的微载体是应用在干细胞培养,本发明创新性的利用其大孔的性质,将其作为一种生物支架材料应用于肿瘤免疫治疗领域,具有很好的疗效。
本发明还提供如上所述肿瘤疫苗在制备治疗肿瘤的药物中的应用。
与现有技术相比,本发明的有益效果如下:常规的制备基于生物材料支架的肿瘤疫苗的方法主要包括肿瘤抗原的制备、生物支架材料的制备和抗原的负载,制备过程较繁琐。本发明将这三个步骤融合在一起,一体化制备得到全肿瘤细胞微载体支架疫苗,省去了繁琐的制备过程,提高了制备效率。本发明所选用的全细胞肿瘤抗原,相比通用型肿瘤抗原其所含有的抗原谱更广泛,具有更强的免疫原性且与佐剂相结合使用能协同增强机体产生特异性免疫应答。冻干所获得的微载体支架疫苗因其具有大孔的3D结构可招募免疫细胞并促进其成熟,从而引发抗肿瘤免疫反应。使用时,以皮下注射的方式给药(相比传统皮下植入式支架给药的方式,患者的依从性更好),其中全肿瘤细胞作为抗原部分,辅以免疫调节剂,而微载体本身所具有的大孔结构可以作为免疫中心招募免疫细胞,从而引发抗肿瘤免疫应答,用于防止肿瘤术后复发和转移。本发明所提供的全肿瘤细胞微载体支架疫苗能发挥很好的疗效,具有广阔的应用前景。
附图说明
图1 为微载体支架疫苗的具体制备方法流程图示。
图2 为各组经扫描电镜(SEM)记录的图像。其中,(a)为CT26细胞的SEM图、(b)为微载体的SEM图像,(c)为微载体与CT26细胞共培养后的SEM图像。
图3 为CT26细胞、Hepa1-6细胞、4T1细胞与微载体共培养72小时的激光共聚焦图像。
图4 为CT26细胞在微载体其上培养的CCK-8细胞活性实验的结果示意图。
图5 分别为实施例1中所制备得到的微载体支架疫苗。其中,(a)R848、(b)aOX40(以IgG-FITC代替)在pH=7.4和温度37℃条件下的释放曲线示意图。
图6 为微载体支架疫苗的体内降解行为的示意图。
图7 为微载体支架疫苗诱导招募不同免疫细胞的能力。
图8 为实施例1中提供的微载体支架疫苗治疗性实验的肿瘤抑制效果示意图(n=5)。
具体实施方式
下面通过具体实施例结合附图进一步介绍本发明。
下述实施例中所使用到的微载体为3D TableTrix,购自于北京华龛生物有限公司。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1, CT26全肿瘤细胞微载体支架疫苗的制备
如图1所示,将一定量的CT26结肠癌细胞与微载体(2×107个细胞/片)进行培养,共培养72h后收集微载体与肿瘤细胞悬液,离心,用PBS洗涤一次,重悬在PBS中。经过液氮冷冻12h后,用37℃水浴复融一次,即得空白微载体支架疫苗。随后加入瑞奎莫德(R848,150µg/dose)与抗OX40抗体(a-OX40,15µg/dose),4℃下,机械搅拌12小时后,将混合物溶液进行冷冻干燥即得3D微载体支架疫苗。
实施例2 ,4T1全肿瘤细胞微载体支架疫苗的制备
如图1所示,将一定量的4T1乳腺癌细胞与微载体(2×107个细胞/片)进行培养,共培养72h后收集微载体与肿瘤细胞悬液,离心,用PBS洗涤一次,重悬在PBS中。经过液氮冷冻12h后,用37℃水浴复融一次,即得空白微载体支架疫苗。随后加入瑞奎莫德(R848,150µg/dose)与抗OX40抗体(a-OX40,15µg/dose),4℃下,机械搅拌12小时后,将混合物溶液进行冷冻干燥即得3D微载体支架疫苗。
实施例3 ,Hepa1-6全肿瘤细胞微载体支架疫苗的制备
如图1所示,将一定量的Hepa1-6肝癌细胞与微载体(2×107个细胞/片)()进行培养,共培养72h后收集微载体与肿瘤细胞悬液,离心,用PBS洗涤一次,重悬在PBS中。经过液氮冷冻12h后,用37℃水浴复融一次,即得空白微载体支架疫苗。随后加入瑞奎莫德(R848,150µg/dose)与抗OX40抗体(a-OX40,15µg/dose),4℃下,机械搅拌12小时后,将混合物溶液进行冷冻干燥即得3D微载体支架疫苗。
实施例4 ,微载体可用于培养肿瘤细胞的验证
扫描电子显微镜进行表征:在冷冻场发射扫描电镜(Zeiss Gemini SEM500FESEM)下分别观察CT26结肠癌细胞、微载体、微载体与CT26结肠癌细胞共培养后的形貌,如图2所示,可以看见微载体原本所具有的大孔3D结构变少,一部分被肿瘤细胞所占据,故微载体也可应用于肿瘤细胞培养。
激光共聚焦显微镜进行表征:分别收集与微载体共培养72h的CT26结肠癌细胞、4T1乳腺癌细胞和Hepa1-6肝癌细胞的混合悬液,经5%多聚甲醛固定10min后,PBS洗涤后,用FITC-鬼笔环肽和DAPI进行染色。转移至35mm的共聚焦小皿中并用激光共聚焦显微镜(C2+,Nikon)观察。如图3所示,可以看到,三种肿瘤细胞均能依附于微载体上生存,证明了微载体培养肿瘤细胞的可行性以及普适性。
实施例5, 评价微载体的生物相容性
体外细胞增值活力测定采用标准的Cell Counting Kit-8方法。即收获与微载体共培养后12h、24h、48h、72h以及96h时的混合悬液,待其静置沉降后,取100µL混合悬液转移至96孔板中,每孔加入10µL CCK-8溶液,用酶标仪(ELX800,BioTek)测定450nm处的吸光度。结果如图4所示,可以发现72h之前均呈现增长,且在72h吸光度达到最大,而由于微载体的培养面积有限以及培养体系中的营养物质的小号,故96h吸光度呈现下降的趋势。证明了微载体具有良好的生物相容性。
实施例6, R848和aOX40的体外释放
将实施例1中所制备得到的微载体支架疫苗溶解在2mL含5%(w/v)吐温-80的PBS中,将其转移至透析袋内,用封口夹将透析袋封端。随后将透析袋浸于装有30mL含5%(w/v)吐温-80的PBS的离心管中。置于震荡培养箱中孵育(37℃,120rpm),在预设的时间点,分别从离心管中取出1.5mL溶液到ep管中,并补充等量的释放介质。R848药物释放浓度的检测通过紫外可见分光光度计(Lambda750)检测λ=320nm波长处的吸光度,IgG-FITC药物释放浓度的检测则通过微孔板检测仪(Cytation3)检测。实验测定前先分别测定R848于IgG-FITC药物在溶液中浓度与吸光度的标准曲线。
结果如图5所示,可以发现,通过将微载体与R848与IgG-FITC混进行物理混合吸附后,对R848与IgG-FITC的释放具有一定的缓释作用,这种缓释作用确保了应用到机体内能够对机体部位的免疫细胞进行持续的激活。
实施例7 ,微载体支架疫苗的体内降解分析
将实施例1中所制备得到的微载体支架疫苗溶解在PBS中,在靠近小鼠腋下的部位皮下注射200µL的疫苗制剂,注射部位会形成一个隆起的结节,监测并记录不同时间点所形成的结节的体积。
结果如图6所示,皮下结节的体积起初会呈现短暂的体积增长趋势,推测微载体支架疫苗在注射部位招募了免疫细胞,使得体积呈现短暂的增加趋势。而随着时间的延长,所引起的结节变小,微载体支架疫苗逐渐在体内降解,大约25天,结节消失,微载体支架疫苗具有良好的生物相容性,可以在机体内缓慢降解。
实施例8 ,微载体支架疫苗在注射部位招募免疫细胞
将实施例1中所制备得到的微载体支架疫苗溶解在PBS中,在靠近小鼠腋下的部位皮下注射200µL的疫苗制剂。分别在注射后第一天、第三天和第六天,取下一定大小的注射部位,剪切消化处理制备成单细胞悬液进行进一步分析,将悬浮液中的红细胞完全裂解后,用抗体染色剩余细胞并用流式细胞仪(BD Fortessa)检测分析。APC-Cy7 CD45用于标记募集的白细胞。FITC-CD3、PE-Cy7-CD4、APC-CD8、BV786-NK1.1用于标记微载体支架中的T细胞和NK细胞,PE-CD11b、APC-R700-F4/80、BV650-Ly6C、BV421-Ly6G、BB700-MHC I-A/I-E、BV605-CD11c等分析微载体支架疫苗中的巨噬细胞、树突状细胞、单核细胞和中性粒细胞等。
结果如图7所示,微载体支架疫苗组比空白微载体疫苗组更能招募免疫细胞,且招募的具有细胞毒作用的CD8+ T细胞在免疫反应早期,先激活非特异性免疫应答,而随着时间的延长,再激活机体的特异性免疫应答。
实施例9 ,微载体支架疫苗的体内免疫治疗效果
取对数生长期的CT26细胞,用胰酶消化后收集细胞,调整细胞浓度为1×107/mL。取5~6周龄的雄性Balb/c小鼠,随机分为3组,每组5只,每只小鼠右侧腹部皮下消毒后注射100µL细胞悬液。待肿瘤长至400mm3时,3组小鼠按如下剂量进行给药治疗:(1)对照组(不进行处理);(2)空白微载体支架组:小鼠右侧前肢腋下处皮下注射空白微载体(含10mg微载体粉末,106 cells/dose)。(3)微载体支架疫苗组(实施例1制备的):鼠右侧前肢腋下处皮下注射微载体支架疫苗。(含10mg微载体粉末、106 cells/dose、R848为150µg/dose,a-OX40为15µg/dose)观察并记录小鼠的肿瘤体积大小(肿瘤体积(mm3)= )。
结果如图8所示,可以发现经微载体支架疫苗组免疫后的小鼠的肿瘤生长均较其他组更慢,且瘤体积小,表现出最佳的的抑瘤效果。
Claims (10)
1.一种全肿瘤细胞微载体支架疫苗的制备方法,其特征在于,具体步骤为:
(1)利用微载体接种和培养肿瘤细胞:首先在片状微载体上接种肿瘤细胞,每片的接种细胞量为0.2~1×106个细胞;然后培养细胞,培养条件为37℃,pH值为6.8-7.8,微载体上的细胞量达到初始量的4~6倍;培养完成后,收集肿瘤细胞与微载体,制备成混合悬液;
(2)灭活:将所得的混合悬液于液氮中冷冻;然后水浴复融解冻;
(3)负载免疫调节:将复融后的空白微载体疫苗组与免疫调节剂复配,混匀得到混合溶液;
(4)冻干:将步骤(3)中所得的混合溶液进行冷冻干燥,即得全肿瘤细胞微载体支架疫苗。
2.根据权利要求1所述的全肿瘤细胞微载体支架疫苗的制备方法,其特征在于,所述微载体选自液体微载体、大孔明胶微载体、聚苯乙烯微载体、甲壳质微载体、聚氨酯泡沫微载体、藻酸盐凝胶微载体以及磁性微载体。
3.根据权利要求1所述的全肿瘤细胞微载体支架疫苗的制备方法,其特征在于,所述肿瘤细胞培养采取静态培养或者转瓶培养的方式。
4.根据权利要求1所述的全肿瘤细胞微载体支架疫苗的制备方法,其特征在于,步骤(2)中所述冷冻时间为24h~96h;水浴复融温度为25℃~37℃。
5.根据权利要求1所述的全肿瘤细胞微载体支架疫苗的制备方法,其特征在于,步骤(4)中所述冻干时间为8h~24h。
6.根据权利要求1所述的全肿瘤细胞微载体支架疫苗的制备方法,其特征在于,所述肿瘤细胞为CT26结直肠癌细胞、Hepa1-6肝癌细胞或4T1乳腺癌细胞。
7.根据权利要求1所述的全肿瘤细胞微载体支架疫苗的制备方法,其特征在于,所述免疫调节剂为免疫佐剂、免疫检查点抑制剂、对免疫激活起效的化学药物的一种或多种的组合。
8.根据权利要求1所述的全肿瘤细胞微载体支架疫苗的制备方法,其特征在于,所述免疫调节剂为瑞奎莫德与抗OX40抗体的药物组合。
9.由权利要求1-8之一所述制备方法得到的全肿瘤细胞微载体支架疫苗。
10.如权利要求9所述的全肿瘤细胞微载体支架疫苗在制备治疗肿瘤药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310152192.7A CN116370618A (zh) | 2023-02-23 | 2023-02-23 | 一种全肿瘤细胞微载体支架疫苗及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310152192.7A CN116370618A (zh) | 2023-02-23 | 2023-02-23 | 一种全肿瘤细胞微载体支架疫苗及其制备方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116370618A true CN116370618A (zh) | 2023-07-04 |
Family
ID=86966289
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310152192.7A Pending CN116370618A (zh) | 2023-02-23 | 2023-02-23 | 一种全肿瘤细胞微载体支架疫苗及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116370618A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116763725A (zh) * | 2023-08-16 | 2023-09-19 | 四川大学华西医院 | 一种智能响应型可注射水凝胶及其制备方法和应用 |
-
2023
- 2023-02-23 CN CN202310152192.7A patent/CN116370618A/zh active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116763725A (zh) * | 2023-08-16 | 2023-09-19 | 四川大学华西医院 | 一种智能响应型可注射水凝胶及其制备方法和应用 |
CN116763725B (zh) * | 2023-08-16 | 2023-11-24 | 四川大学华西医院 | 一种智能响应型可注射水凝胶及其制备方法和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10806777B2 (en) | Method for allogeneic cell therapy | |
JP2007500217A5 (zh) | ||
Yang et al. | Preparation and antitumor effects of nanovaccines with MAGE-3 peptides in transplanted gastric cancer in mice | |
CN109998998A (zh) | 一种纳米疫苗及其制备方法 | |
CN112516297B (zh) | 一种基于鱼精蛋白为载体的抗原和佐剂共传递纳米疫苗的制备方法及其应用 | |
CN116370618A (zh) | 一种全肿瘤细胞微载体支架疫苗及其制备方法 | |
CN110787301B (zh) | CTLA-4 Aptamer靶向共负载PD-1 siRNA纳米颗粒及其制备和应用 | |
US20070172490A1 (en) | Leukocyte stimulation matrix | |
CN101626781A (zh) | 制备具有抗肿瘤免疫应答反应的细胞群的方法 | |
CN105031631A (zh) | 一种HLA-A0201限制性抗Sox2特异性CTL的制备方法及其应用 | |
CN111996166B (zh) | 一种负载的dc细胞、dc-cik细胞及在肿瘤细胞治疗方面的应用 | |
CN107254440A (zh) | Cd4阳性th17t细胞培养方法 | |
CN115887640A (zh) | 香菇多糖在制备pH响应型纳米药物载体上的应用 | |
CN116327682A (zh) | 一种负载微囊藻的可注射水凝胶及其制备方法和应用 | |
CN115590887A (zh) | 诱导成纤维细胞呈现免疫抑制表型的方法 | |
CN117860698A (zh) | 靶向肿瘤组织的卡介苗融合囊泡及其制备方法与应用 | |
CN116763725A (zh) | 一种智能响应型可注射水凝胶及其制备方法和应用 | |
CN116889622A (zh) | 一种高效抗癌的肿瘤疫苗及其制备方法 | |
CN116396475A (zh) | 一种基于氟化聚合物的纳米载体及其制备方法和应用 | |
CN117431211A (zh) | 一种DEV@IL-12-aCTLA-4及其制备方法和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |