WO2020227885A1 - 搭载抗pd-1单链抗体的基因工程化红细胞及其制备方法 - Google Patents

搭载抗pd-1单链抗体的基因工程化红细胞及其制备方法 Download PDF

Info

Publication number
WO2020227885A1
WO2020227885A1 PCT/CN2019/086602 CN2019086602W WO2020227885A1 WO 2020227885 A1 WO2020227885 A1 WO 2020227885A1 CN 2019086602 W CN2019086602 W CN 2019086602W WO 2020227885 A1 WO2020227885 A1 WO 2020227885A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
red blood
lin
genetically engineered
blood cells
Prior art date
Application number
PCT/CN2019/086602
Other languages
English (en)
French (fr)
Inventor
高晓飞
黄彦杰
李丹丹
Original Assignee
西湖生物医药科技(杭州)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 西湖生物医药科技(杭州)有限公司 filed Critical 西湖生物医药科技(杭州)有限公司
Priority to US17/595,183 priority Critical patent/US20240043802A1/en
Publication of WO2020227885A1 publication Critical patent/WO2020227885A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • 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
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/14—Erythropoietin [EPO]
    • 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]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • 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]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • 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
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • 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
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • 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
    • C12N2501/998—Proteins not provided for elsewhere
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1114—T 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1128—Erythrocytes
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system 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
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • 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
    • 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

Definitions

  • the invention belongs to the field of medical biology, and specifically relates to genetically engineered red blood cells carrying anti-PD-1 single-chain antibodies and a preparation method thereof.
  • Immunotherapy has become one of the most powerful means to treat cancer. More and more drugs are approved for immunotherapy, and more and more projects are in clinical and pre-clinical development. However, immunotherapy is a wide range of treatment strategies for the systemic immune system, so the key is still the regulation of the immune system.
  • the main problems of current immunotherapy are side effects such as autoimmunity and non-specific inflammation. How to improve the therapeutic effect and control adverse reactions by increasing the response rate of immunotherapy has become the key. How to more effectively deliver drugs used in immunotherapy (for example: anti-PD-1, CTLA-4 monoclonal antibody, IL-2, TNF- ⁇ , etc.) in vivo and reduce toxic side effects remains to be explored and urgently to be resolved .
  • red blood cells make it a very attractive research target for the delivery of natural and synthetic ingredients in vivo.
  • the circulation range is wide and the quantity is large, which can be used for drug carrying; 2)
  • the senile or damaged red blood cells can be removed through the reticuloendothelial system, which can achieve complete biodegradation without producing toxic substances; 3) With good Biocompatibility, especially when using autologous red blood cells; 4)
  • Programmed cell death protein 1 also known as PD-1 and CD279, is a protein on the cell surface that binds to its ligand PD-L1 to down-regulate the immune system's response to human cells. By inhibiting the inflammatory activity of T cells, it regulates the immune system and promotes self tolerance. This can prevent autoimmune diseases, but it can also prevent the immune system from killing cancer cells.
  • PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms. First, it promotes the apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Secondly, it reduces the apoptosis of regulatory T cells (anti-inflammatory, inhibitory T cells).
  • inhibitors that block the interaction of PD-L1 with PD-1 receptors can prevent cancer from evading the immune system in this way, thereby achieving tumor treatment.
  • anti-PD-1 monoclonal antibodies clinically significant therapeutic effects can be achieved for cancers including melanoma, head and neck cancer, renal cell carcinoma, non-small cell lung cancer, bladder cancer, and colorectal cancer.
  • the present invention provides genetically engineered red blood cells carrying anti-PD-1 single-chain antibodies, with the purpose of solving the problem that the delivery of anti-PD-1 monoclonal antibodies cannot be targeted to tumor tissues and the utilization efficiency is low. And technical problems that cause high systemic side effects of tumor immune drugs.
  • the invention also provides a method for preparing genetically engineered red blood cells carrying the anti-PD-1 single-chain antibody.
  • the preparation method of genetically engineered red blood cells carrying anti-PD-1 single-chain antibody includes the following steps:
  • S1 Construct the target fragment sequence in a lentiviral expression vector to obtain the target sequence vector, the target fragment is Anti-PD-1 scFv, and the Anti-PD-1 scFv represents an anti-PD-1 single-chain variable region fragment;
  • step S2 Carry out lentivirus packaging on the target sequence vector in step S1, and obtain a high-titer chronic disease concentrate after centrifugal concentration;
  • step S5 Use the lentivirus concentrate in step S2 to infect Lin-CD34- cells in step S4 to obtain anti-PD-1 scFv-Lin-CD34- cells;
  • the Anti-PD-1 scFv nucleotide sequence is as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.5 and/or SEQ ID NO.6;
  • the lentiviral expression vector is pCDH-MCS-T2A-copGFP-MSCV.
  • the lentivirus packaging adopts a three-plasmid packaging system
  • the three-plasmid packaging system includes a target sequence vector, a PSPAX2 plasmid, and a VSVG plasmid, the target sequence vector, the PSPAX2 plasmid
  • the ratio of the pMD2G plasmid is 2:1:1
  • the lentivirus packaging uses HEK 293T cells as the lentivirus packaging cells.
  • the rotation speed of the centrifugal concentration is 70,000 RCF, the time is 2 hours, and the temperature is 4°C.
  • step S3 the cells are resuspended in a cell separation buffer supplemented with bovine serum albumin and EDTA, and biotin-labeled antibodies and streptavidin-labeled magnetic beads are added to remove special Cells with surface markers, Lin-CD34-cells were isolated;
  • the cytokine composition enriched in Lin-CD34- cells includes 50-100ng/ml recombinant human fms-like tyrosine kinase 3 ligand, 50-100ng/ml recombinant human Stem cell factor, 50-100ng/ml recombinant human interleukin three, 200-800pg/ml recombinant human interleukin six.
  • the first stage of differentiation medium includes IMDM, 10-15% fetal bovine serum, 5-10% human plasma, 1-4 mM glutamine, 1-2% bovine Serum albumin, 300-600 ⁇ g/ml human transferrin, 8-13 ⁇ g/ml recombinant human insulin, 2% penicillin, 3-5ng/ml recombinant human interleukin three, 4-7U/ml recombinant human Erythropoietin and 100ng/ml recombinant human stem cell factor.
  • the infection step is as follows: resuspend Lin-CD34-cells with the first stage of differentiation medium, calculate the infection volume and the amount of virus, and according to the infection volume according to 10 ⁇ g/ml Add polybrene to the lentivirus concentrate and incubate for 5 min. Add the incubated lentivirus concentrate to the cells and mix well.
  • the final concentration of the lentivirus concentrate is 5 ⁇ 10 7 TU/ml-5 ⁇ 10 8 TU /ml; use a horizontal rotor centrifuge for centrifugal infection, rotating speed 500 ⁇ g, temperature 32°C, time 90min, after centrifugation, place anti-PD-1 scFv-Lin-CD34- cells at 37°C, 5% CO 2 Under cultivation.
  • the second-stage differentiation medium includes but is not limited to IMDM, 15% fetal bovine serum, 5-10% human plasma, 1-4 mM glutamine, 1-2% Bovine serum albumin, 300-600 ⁇ g/ml human transferrin, 8-13 ⁇ g/ml recombinant human islets, 2% penicillin and 1-5U/ml recombinant human erythropoietin.
  • the present invention also provides genetically engineered red blood cells carrying anti-PD-1 single-chain antibodies, and genetically engineered red blood cells carrying anti-PD-1 single-chain antibodies prepared based on the above method.
  • the present invention has the following beneficial effects: many unique characteristics of red blood cells: 1) a wide range of circulation and a large number, which can be used for drug carrying; 2) senescent or damaged red blood cells can be removed through the reticuloendothelial system, and complete biological Degraded and will not produce toxic substances; 3) Has good biocompatibility, especially when using autologous red blood cells; 4) Compared with other synthetic carriers, it has a longer circulating half-life (about 120 days for humans); 5) No The nucleus, without mitochondria and any DNA, any form of genetic engineering modification to the precursors of red blood cells will be eliminated after enucleation, so the cells will not cause abnormal growth or tumor formation after being imported into the receptor; 6) They It protects the encapsulated substance from premature inactivation and degradation, and protects the organism from the toxic effects of drugs.
  • the method for preparing genetically engineered erythrocytes carrying anti-PD-1 single-chain antibody uses the unique characteristics of erythrocytes as a transport carrier, and uses genetic engineering to make the surface of the erythrocyte membrane carry antibody drugs for tumor immunotherapy (ie, anti-PD -1 single-chain antibody), and then induce the engineered precursor cells to differentiate into mature red blood cells by means of erythroid induction and differentiation, to obtain genetically engineered red blood cells carrying anti-PD-1 single-chain antibody.
  • tumor immunotherapy ie, anti-PD -1 single-chain antibody
  • the genetically engineered erythrocytes carrying anti-PD-1 single-chain antibody provided by the present invention target the delivery of anti-PD-1 single-chain antibody to tumor tissue so that it can more effectively exert the T cell activation function, and can significantly reduce the dosage of drugs and relieve The side effects of systemic tumor immune drugs.
  • Figure 1 is a flow chart of the method for preparing genetically engineered red blood cells carrying anti-PD-1 single chain antibody provided by the present invention
  • Fig. 2 The cell proliferation curve of mature red blood cells prepared in vitro using peripheral blood in Example 1 of the present invention. In the present invention, data from two donors are used as comparison;
  • Figure 3 is a schematic diagram of the expression levels of CD235a and CD117 of two donors at the stage of enriching Lin-CD34- cells;
  • Figure 4 is a schematic diagram showing the identification of red blood cell denucleation levels after co-staining with CD71 and hoechst33342/CD235a from two donors at the stage of enriching Lin - CD34 - cells;
  • Figure 5 is a schematic diagram of the expression levels of CD235a and CD117 of two donors at the stage of Lin - CD34 - cell differentiation;
  • Figure 6 is a schematic diagram showing the identification of erythrocyte denucleation level after co-staining with CD71 and hoechst33342/CD235a from two donors at the stage of Lin - CD34 - cell differentiation;
  • Figure 7 is a schematic diagram of the expression levels of CD235a and CD117 of two donors during the denucleation and maturation stage of red blood cells;
  • Figure 8 is a schematic diagram showing the identification of the level of red blood cell denucleation after two donors CD71 and hoechst33342/CD235a are co-stained during the mature stage of red blood cell denucleation;
  • Figure 9 is the CD235a expression curve of cells in the differentiation culture stage
  • Figure 10 is the CD71 expression change curve of cells in the differentiation culture stage
  • Figure 11 is the CD117 expression curve of cells in the differentiation culture stage
  • Figure 12 shows the denucleation of red blood cells during the preparation process
  • Figure 13 is the smear staining of the cells provided by donor 1 after the differentiation is completed, and the red blood cell morphology is detected under the microscope;
  • Figure 14 is a smear stain on the cells provided by donor 2 after the differentiation is completed, and the red blood cell morphology is detected under the microscope;
  • Figure 15 is a detection diagram of the conversion efficiency of anti-PD-1 scFv by luciferase
  • Figure 16 is a flow cytometry detection diagram of the expression efficiency of anti-PD-1 scFv in red blood cells
  • Figure 17 is a distribution diagram of anti-PD-1 scFv-red blood cells in mice.
  • Figure 18 is the distribution diagram of anti-PD-1 scFv-red blood cells in the organs of mice;
  • Figure 19 is a microscopic examination of anti-PD-1 scFv-red blood cells and T cells co-cultured;
  • Figure 20 is a flow cytometry detection diagram of anti-PD-1 scFv-red blood cells and T cells co-cultured.
  • Peripheral blood is blood other than bone marrow. Some methods are commonly used clinically to release hematopoietic stem cells in the bone marrow into the blood, and then extract and separate hematopoietic stem cells from the blood. We call the stem cells obtained in this way as peripheral blood stem cells .
  • Peripheral blood mononuclear cells are cells with mononuclear cells in peripheral blood, including lymphocytes and monocytes. At present, the main separation method of peripheral blood mononuclear cells is Ficoll-hypaque (Ficoll-hypaque) density gradient centrifugation law.
  • Ficoll-hypaque Ficoll-hypaque density gradient centrifugation law.
  • Lin - CD34 - cells are cells that have not differentiated into various hematopoietic lineages, CD34 - are cells that do not express CD34 surface markers, Lin - CD34 - cells are not differentiated into hematopoietic lineages and do not express CD34 Surface markers of cells.
  • Biotin-labeled antibody Biotin is attached to the antibody.
  • the biotin labeling reaction is simple, mild and rarely inhibits the activity of the antibody.
  • Covalently binding biotin to the antibody is a very simple and direct labeling method.
  • Hematopoietic stem cell expansion medium StemSpan TM SFEM serum-free expansion medium is a hematopoietic stem cell expansion medium that does not contain serum. After adding hematopoietic growth factors and/or other stimulating factors selected by the user, it can promote The expansion or differentiation of hematopoietic stem/progenitor cells (HSPC) to a specific lineage.
  • HSPC hematopoietic stem/progenitor cells
  • Recombinant human fms-like tyrosine kinase 3 ligand (rhFlt3L): FMS-like tyrosine kinase 3 ligand (Flt-3 ligand), also known as FL, Flt3L and FLT3LG, is an ⁇ that promotes the differentiation of multiple hematopoietic cell lineages -Spiral cytokine.
  • FLT3LG is structurally homologous to stem cell factor (SCF) and colony stimulating factor 1 (CSF-1).
  • SCF stem cell factor
  • CSF-1 colony stimulating factor 1
  • Kit ligand also known as stem cell factor (SCF)
  • SCF stem cell factor
  • Kit ligand also known as stem cell factor (SCF)
  • SCF belongs to the type I transmembrane glycoprotein of the SCF family.
  • KITLG is the ligand of the receptor type protein tyrosine kinase KIT.
  • SCF plays an important role in regulating cell survival and proliferation, hematopoiesis, stem cell maintenance, cell development, migration and function.
  • rhIL-3 Recombinant human interleukin-3
  • rhIL-3 is a glycoprotein belonging to the family of hematopoietic growth factors, which exhibits multi-lineage activity in preclinical in vitro and in vivo studies. Hematopoietic progenitor cells proliferate and differentiate into mature red blood cells, mast cells, megakaryocytes and granulocytes with the help of IL-3 protein.
  • Recombinant human interleukin-6 (rhIL-6): is a multifunctional cytokine that regulates immune response, hematopoietic function, acute phase response and inflammatory response. Cooperate with IL-3 to promote cell proliferation of hematopoietic cells.
  • Recombinant human erythropoietin is the main erythropoietic factor, and it interacts with various other growth factors (eg, IL-3, IL-6, glucocorticoid and SCF) that develop the red blood cell lineage from pluripotent progenitors Synergy.
  • Burst-forming unit-erythrocyte (BFU-E) cells begin to express erythropoietin receptors and are sensitive to erythropoietin. It is an important erythroid hematopoietic cytokine.
  • Human transferrin (holo human transferrin): It is the main iron-containing protein in plasma, which can form a complex with iron ions for the production of hemoglobin in red blood cells.
  • Streptavidin It is the secreted product during the culture of Streptomyces avicllrdi bacteria. It is mainly purified by 2-iminobiotin affinity chromatography. 1L of culture medium contains 10-60mg of protein.
  • CD235a Glycophorin A, a single transmembrane glycoprotein, expressed in mature red blood cells and erythroid precursor cells, and is a special marker protein on the surface of red blood cells.
  • the expression of CD235a indicates that the cells differentiate into erythroid cells.
  • the analysis of flow cytometry shows that the cells do not express CD235a in the SFEM stage, indicating that the cells have not entered erythroid differentiation, and the cells are cultured in the differentiation stage 1 After basic, the cytokines in the culture medium induce the cells to differentiate into the erythroid, and the cells begin to express CD235a, and the proportion increases with the differentiation time.
  • the cells After changing to the differentiation stage 2 medium, the cells have completely entered the erythroid, so almost all The cells all expressed CD235a, indicating that almost all cells are erythroid cells.
  • the cell indicates that the marker indicates that our differentiation system is successful in the induction of erythroid cells in vitro.
  • CD117 also known as c-kit, is the SCF stem cell growth factor receptor, expressed on the surface of hematopoietic stem cells and other cells. SCF plays an important role in regulating cell survival and proliferation, hematopoiesis, stem cell maintenance, cell development, migration and function. The change in the expression of its receptor reflects the change in the ability of cells to utilize SCF. CD117 is not expressed under SFEM conditions, indicating that Lin-cells did not use SCF during the culture stage in SFEM. When the cells enter the differentiation stage 1 medium, the cells enter the erythroid differentiation, and the expression of CD117 rises rapidly and reaches the peak.
  • SCF is added to the medium to regulate cell survival and proliferation, hematopoiesis, stem cell maintenance, and cell development.
  • the expression of CD117 gradually decreases.
  • Transferrin receptor 1 is a transmembrane glycoprotein consisting of two disulfide bonds connected by two disulfide bonds. Each monomer binds a holotransferrin molecule to produce an iron-transferrin-transferrin receptor complex, which enters the cell through endocytosis and is used for hemoglobin production during the development of the erythroid. Under SFEM conditions, CD71 did not express, indicating that Lin-cells did not use transferrin during the culture stage in SFEM, and the cells did not enter the erythroid system and began to take up iron to synthesize hemoglobin.
  • the cells After the cells enter the differentiation stage 1 medium, because the cells enter the erythroid differentiation, they need to take up a large amount of transferrin, so the expression of CD71 rises rapidly to meet the uptake of cell transferrin.
  • the cells enter the differentiation stage 2 medium since the erythroid cells have synthesized enough hemoglobin, the expression of CD71 gradually decreases and the erythrocytes mature.
  • Hoechst33342 is a fluorescent dye used to stain DNA. The dye can pass through the cell membrane and bind to DNA. If the cell is not denucleated, the dye binds to the DNA can be detected as positive by flow cytometry. If the cell is denucleated, the signal is detected by drain cytometry. Negative.
  • CD235a is a surface marker of red blood cells
  • the co-expression of the two markers was detected by flow cytometry.
  • CD235a positive Hoechst 33342 positive are erythroid non-denucleated cells
  • CD235a positive Hoechst 33342 negative are mature red blood cells.
  • the cells entered erythroid differentiation from Lin-cells, and the expression of CD235a was increased, but the cells were not denucleated, so the cells were Hoechet 33342 positive.
  • the cells After the cells enter the differentiation stage 2 medium, the cells begin to mature and denucleate, and Hoechst 33342 negative cells appear, indicating that the red blood cells are mature.
  • the present invention can identify the morphology, structure and function of mature red blood cells.
  • GFP Green fluorescent protein. This protein is a fluorescent reporter gene fused with the target gene anti-PD-1 scFv constructed when constructing MSCV-anti-PD-1 scFv. The detection of this signal indicates that anti-PD-1 scFv is also expressed at the same time, so it can be used as a detection Target gene detection signal. If GFP is positive, it means that the cell expresses anti-PD-1 scFv.
  • Anti-PD-1 single-chain variable region fragments are designed, and the nucleotide sequence of Anti-PD-1 scFv is one or more of SEQ ID NO.1-SEQ ID NO.6
  • This combination was synthesized by conventional biosynthetic methods, and vector construction was carried out by molecular cloning.
  • the Anti-PD-1 scFv was constructed on the lentiviral vector pCDH-MCS-T2A-copGFP-MSCV to obtain the target sequence vector (MSCV- anti-PD-1 scFv), used for virus packaging.
  • a 6-well plate was used.
  • the plasmid is transfected into HEK 293T cells for virus packaging. The solution was changed 12 hours after transfection to remove calcium phosphate, the supernatant was collected at 48 hours and 72 hours, and the cell culture supernatant was filtered through a 0.45 ⁇ m filter.
  • the collected and filtered cell culture supernatant was concentrated by ultracentrifugation at a speed of 70,000 RCF for 2 hours and a temperature of 4°C. After centrifugation, the supernatant was removed, resuspended in differentiation stage 1 medium, and the virus titer was quantified by ELISA, and stored at -80°C for use.
  • Lin - CD34 - cells were inoculated into hematopoietic stem cell expansion medium, and cytokine combinations and penicillin were added.
  • the cells The combination of factors and penicillin constitute an enriched cell culture medium.
  • the components of the enriched cell culture medium are as shown in Table 2.
  • the stage is defined as the enriched Lin - CD34 - cell stage after culturing to the 4th day.
  • StemSpan Serum-Free Medium 1 ⁇ 1 ⁇ 1L Recombinant human fms-like tyrosine kinase 3 ligand (Flt3L) 200mM 2mM 10ml Recombinant human stem cell factor (SCF) 100 ⁇ g/ml 100ng/ml 1ml Recombinant Human Interleukin 3 (IL-3) 100 ⁇ g/ml 100ng/ml 1ml Recombinant Human Interleukin 6 (IL-6) 800 ⁇ g/ml 800pg/ml 1ul
  • SCF human stem cell factor
  • the culture system was changed, and the medium was changed to the first-stage differentiation medium (as shown in Table 3), and the expansion culture was carried out at 37°C and 5% CO 2 .
  • the cells were infected with MSCV-anti-PD-1 scFv prepared and frozen at -80°C before use on the 6th day.
  • Infection method Resuspend the cells in a differentiation stage 1 medium at a density of 1 ⁇ 106. Calculate the infection volume and virus dosage. The virus dosage is calculated at a final concentration of 5 ⁇ 107TU/ml-5 ⁇ 108TU/ml.
  • polybrene is added to the concentrated virus solution and incubated for 5 minutes. Add the concentrated virus solution after incubation to the cells and mix. And use a horizontal rotor centrifuge for centrifugal infection, rotating speed 500 ⁇ g, temperature 32 °C, time 90 minutes. After centrifugation, place it at 37°C and 5% CO 2 for cultivation.
  • cytokines related to erythroid development are provided under the medium to ensure the proliferation and differentiation of Lin - CD34 - cells to erythroid.
  • virus infection is carried out at the fastest proliferation and differentiation stage to ensure the target gene ( anti-PD-1 scFv) is inserted and expressed on the cell membrane.
  • the culture system was changed on the 14th day, and the medium was changed to the second-stage differentiation medium (as shown in Table 4), and the culture was incubated at 37°C and 5% CO 2 until the 22nd day to obtain the anti-PD- 1 Single-chain antibody genetically engineered erythrocytes.
  • This stage is defined as the denucleation and maturation stage of erythrocytes.
  • the medium of the corresponding stage is changed every 2-4 days from 0-22 days.
  • the number of cells was measured on days 0-22. Every 3-4 days, the cells in the culture system were fully resuspended, and 10 ⁇ l of cell suspension was mixed with 10 ⁇ l of trypan blue staining solution and counted by a cell counter.
  • the cell proliferation is as follows, and the starting cell number is standardized to 1 ⁇ 10 6 at this time:
  • the cell proliferation curve is drawn with the obtained cell number, as shown in Fig. 1, which shows that under this culture condition, red blood cells can proliferate and differentiate normally, and can achieve 1000-fold proliferation.
  • the cells in the enriched Lin - CD34 - cell stage, Lin - CD34 - cell differentiation stage, and red blood cell denucleation mature stage were respectively taken for analysis and the specific operation process.
  • the cells in the culture system are fully resuspended, and 50-100 ⁇ l of the cell suspension is added to 500 ⁇ l of phosphate buffer.
  • Add 0.5 ⁇ l of mouse anti-human CD235a-APC antibody mouse Anti-human CD235a APC
  • 2 ⁇ l of mouse anti-human CD71-PerCP Cy5.5 antibody mouse Anti-human CD71-PerCP Cy5.5
  • mouse anti-human CD117-PE Cy7 antibody mouse Anti-human CD117-PE Cy7 antibody
  • Hoechst 33342 dye 0.5 ⁇ l, mix well, incubate in the dark for 20 minutes, and test on flow cytometer.
  • Analyze the expression level of specific surface markers on the target cells Draw the change curve through the analysis at different time points, and get the schematic diagrams as shown in Figure 2 to Figure 11.
  • this ratio can reflect the denucleation of cells.
  • Figure 15 shows the conversion efficiency of anti-PD-1 scFv detected by luciferase.
  • the light field in the figure represents the microscope image of anti-PD-1 scFv-red blood cells in bright field (normal light source), and GFP represents the GFP fluorescent protein excitation light source.
  • Anti-PD-1 scFv-red blood cell microscope image, Merge represents the overlapping effect image of bright field and GFP fluorescent protein excitation light source anti-PD-1 scFv-red blood cell microscope image.
  • the peripheral blood Lin-cells are genetically engineered, and GFP is used as the reporter gene.
  • Anti-PD-1 scFv-red blood cells account for 95% of the total cells, indicating that the genetic engineering efficiency is more than 95%.
  • this ratio can reflect the expression efficiency of anti-PD-1 scFv in red blood cells.
  • the red blood cells stained by Dir were injected into the tail vein of the mouse, and the red blood cells were imaged in vivo at different times (1.5h/4h/5h/24h) to detect the distribution of red blood cells in the mouse body.
  • the mice were sacrificed after 5 days for tissue and organ imaging detection The distribution of red blood cells in the organs of mice.
  • Figure 17 is the distribution map of anti-PD-1 scFv-erythrocytes in mice
  • Figure 18 is the distribution map of anti-PD-1 scFv-erythrocytes in the organs of mice
  • mouse No. 1 is injected with Dir staining solution in the tail vein.
  • Mouse No. 2 was injected with normal red blood cells without Dir staining in the tail vein
  • mouse No. 3 was injected with normal red blood cells stained with Dir
  • mouse No. 4 was injected with anti-PD-1 scFv-red blood cells stained with Dir.
  • the distribution of anti-PD-1 scFv red blood cells and normal red blood cells in mice, including internal organs is consistent.
  • Anti-PD-1 scFv-red blood cells are co-cultured with T cells to detect the activation function of anti-PD-1 scFv-red blood cells on T cells.
  • anti-PD-1 scFv-red blood cells activate T cells under in vitro cell co-cultivation conditions, and make T cells proliferate in large numbers.
  • the results of flow cytometry showed that CD25 expression increased.
  • the method for preparing genetically engineered red blood cells carrying anti-PD-1 single-chain antibodies uses red blood cells as a transport carrier, and genetic engineering is used to make the surface of the red blood cell membrane carry antibody drugs for tumor immunotherapy ( That is, anti-PD-1 single-chain antibody), and then the engineered precursor cells are induced to differentiate into mature red blood cells by means of erythroid induction and differentiation, to obtain genetically engineered red blood cells carrying the anti-PD-1 single-chain antibody.
  • the genetically engineered erythrocytes carrying anti-PD-1 single-chain antibody provided by the present invention target the delivery of anti-PD-1 single-chain antibody to tumor tissue so that it can more effectively exert the T cell activation function, and can significantly reduce the dosage of drugs and relieve The side effects of systemic tumor immune drugs.

Landscapes

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

Abstract

提供搭载抗PD-1单链抗体的基因工程化红细胞的制备方法及搭载抗PD-1单链抗体的基因工程化红细胞。制备方法包括如下步骤：将目的片段序列构建于慢病毒表达载体；将目的序列载体进行慢病毒包装，获得高滴度的慢病浓缩液；从外周血单个核细胞中分离和富集Lin-CD34-细胞；诱导Lin-CD34-细胞向红系分化并进行扩增；使用慢病毒浓缩液对Lin-CD34-细胞进行感染；通过红细胞脱核得到成熟的anti-PD-1scFv-红细胞。搭载抗PD-1单链抗体的基因工程化红细胞，可将抗PD-1单链抗体靶向递送至肿瘤组织。

Description

搭载抗PD-1单链抗体的基因工程化红细胞及其制备方法 技术领域
本发明属于医药生物领域，具体涉及搭载抗PD-1单链抗体的基因工程化红细胞及其制备方法。
背景技术
免疫疗法已成为治疗癌症非常有力的手段之一。用于免疫治疗的药物获批越来越多，处于临床和临床前开发的项目也越来越多。然而，免疫疗法是一种广泛的针对全身免系统的治疗策略，所以关键仍然是免疫系统受到的调控，目前的免疫疗法的主要存在的问题是诸如自身免疫和非特异性炎症等副作用。而如何通过提高免疫疗法的反应率来提高治疗效果以及控制不良反应成为了关键所在。如何更有效的将用于免疫治疗的药物(例如：抗PD-1、CTLA-4单克隆抗体、IL-2、TNF-α等)进行体内递送并且减少毒副作用是有待探索和急待解决的。
红细胞的许多独特特征使其成为体内递送天然和合成成分非常有吸引力的研究目标。1)循环范围广，且数量多，可以用于药物携带；2)通过网状内皮系统可以除去衰老或受损的红细胞，可以实现完全的生物降解，不会产生有毒物质；3)具有良好的生物相容性，特别是在使用自体红细胞时；4)与其他合成载体相比具有较长的循环半衰期(人类约120天)；5)没有细胞核，没有线粒体和任何DNA，对红细胞的前体进行任何形式的基因工程修饰，在其去核后都会被消除，故该细胞在输入受体后不会导致异常生长或成瘤。6)它们保护包封的物质免于过早失活和降解，并保护生物体免受药物的毒性作用，同时也可以成为蛋白类药物的生物反应器。
程序性细胞死亡蛋白1，也称为PD-1和CD279，是细胞表面的一种蛋白质，通过与其配体PD-L1结合向下调节免疫系统对人体细胞的反应。通过抑制T细胞炎症活动来调节免疫系统并促进自身耐受。这可以预防自身免疫性疾病，但它也可以防止免疫系统杀死癌细胞。PD-1是一种免疫检查点，通过两种机制防止自身免疫。首先，它促进淋巴结中抗原特异性T细胞的凋亡(程序性细胞死亡)。其次，它减少了调节性T细胞(抗炎，抑制性T细胞)的细胞凋亡。在肿瘤疾病状态中，肿瘤细胞上的PD-L1与T细胞上的PD-1的相互作用降低了T细胞功能信号，从而阻止免疫系统攻击肿瘤细胞。阻断PD-L1与PD-1受体相互作用的抑制剂(单克隆抗体)的使用可以防止癌症以这种方式逃避免疫系统，从而实现肿瘤治疗。通过使用抗PD-1单克隆抗体可以在临床上针对包括黑色素瘤、头颈部癌、肾细胞癌、非小细胞肺癌、膀胱癌、结直肠癌等癌症中取得了明显的治疗效果。然而在这些适应症中，只有约20～30％的晚期癌症患者对抗PD-1单克隆抗体治疗有响应，大多数患者对抗PD-1疗法并不敏 感，主要原因是药物不能靶向递送至肿瘤组织，药物自身免疫原性，利用率低等，同时造成较高的全身性的肿瘤免疫药物副作用。
发明内容
针对上述现有技术中存在的不足，本发明提供了搭载抗PD-1单链抗体的基因工程化红细胞，目的在于解决抗PD-1单克隆抗体递送不能靶向至肿瘤组织，利用效率低下，以及造成较高的全身性的肿瘤免疫药物副作用的技术问题。本发明还提供了搭载抗PD-1单链抗体的基因工程化红细胞的制备方法。
具体技术方案如下：
搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，包括如下步骤：
S1：将目的片段序列构建于慢病毒表达载体，获得目的序列载体，所述目的片段为Anti-PD-1 scFv，所述Anti-PD-1 scFv表示抗PD-1单链可变区片段；
S2：将步骤S1中的目的序列载体进行慢病毒包装，经离心浓缩后，获得高滴度的慢病浓缩液；
S3：从外周血单个核细胞中分离Lin-CD34-细胞和富集Lin-CD34-细胞；
S4：将所述Lin-CD34-细胞培养基更换为分化第一阶段培养基中，诱导所述Lin-CD34-细胞向红系分化并进行扩增；
S5：使用步骤S2中的慢病毒浓缩液对步骤S4中的Lin-CD34-细胞进行感染，获得anti-PD-1 scFv-Lin-CD34-细胞；
S6：将所述anti-PD-1 scFv-Lin-CD34-细胞的培养基更换为分化第二阶段培养基，所述anti-PD-1 scFv-Lin-CD34-细胞通过红细胞脱核得到成熟的anti-PD-1 scFv-红细胞，即为搭载抗PD-1单链抗体的基因工程化红细胞。
在某些实施方式中，在步骤S1中，所述Anti-PD-1 scFv核苷酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5和/或SEQ ID NO.6所示；所述慢病毒表达载体为pCDH-MCS-T2A-copGFP-MSCV。
在某些实施方式中，在步骤S2中，所述慢病毒包装采用三质粒包装系统，所述三质粒包装系统包括目的序列载体、PSPAX2质粒和VSVG质粒，所述目的序列载体、所述PSPAX2质粒、所述pMD2G质粒的比例为2：1：1，所述慢病毒包装采用HEK 293T细胞作为慢病毒的包装细胞。
所述离心浓缩的转速为70000RCF，时间2小时，温度4℃。
在某些实施方式中，在步骤S3中，利用添加牛血清白蛋白和EDTA的细胞分离缓冲液重悬细胞，添加生物素标记的抗体以及链霉亲和素标记的磁珠，去除带有特殊表面标志物的细 胞，分离出Lin-CD34-细胞；
在某些实施方式中，在步骤S3中，富集Lin-CD34-细胞中的细胞因子组合物包括50-100ng/ml重组人类fms样酪氨酸激酶3配体、50-100ng/ml重组人干细胞因子、50-100ng/ml重组人白细胞介素三、200-800pg/ml重组人白细胞介素六。
在某些实施方式中，在步骤S4中，所述分化第一阶段培养基包括IMDM，10-15％胎牛血清，5-10％人血浆，1-4mM谷氨酰胺，1-2％牛血清白蛋白，300-600μg/ml人转铁蛋白，8-13μg/ml重组人胰岛素，2％青链霉素,3-5ng/ml重组人白细胞介素三，4-7U/ml重组人促红细胞生成素以及100ng/ml重组人干细胞因子。
在某些实施方式中，在步骤S5中，所述感染的步骤如下：用分化第一阶段培养基重悬Lin-CD34-细胞，计算感染体积及病毒用量，并根据感染体积按照10μg/ml向慢病毒浓缩液中加入凝聚胺混合孵育5min，将孵育后的慢病毒浓缩液加入细胞中，混匀，所述慢病毒浓缩液的终浓度为5×10 7TU/ml-5×10 8TU/ml；使用水平转子离心机进行离心感染，转速500×g，温度32℃，时间90min，离心完成后将anti-PD-1 scFv-Lin-CD34-细胞置于37℃，5％CO 2条件下进行培养。
在某些实施方式中，在步骤S6中，所述分化第二阶段培养基包括但不限于IMDM，15％胎牛血清，5-10％人血浆，1-4mM谷氨酰胺，1-2％牛血清白蛋白，300-600μg/ml人转铁蛋白，8-13μg/ml重组人胰岛，2％青链霉素以及1-5U/ml重组人促红细胞生成素。
本发明还提供了搭载抗PD-1单链抗体的基因工程化红细胞，基于上述方法制备得到的搭载抗PD-1单链抗体的基因工程化红细胞。
本发明具有以下有益效果：红细胞的许多独特特征：1)循环范围广，且数量多，可以用于药物携带；2)通过网状内皮系统可以除去衰老或受损的红细胞，可以实现完全的生物降解，不会产生有毒物质；3)具有良好的生物相容性，特别是在使用自体红细胞时；4)与其他合成载体相比具有较长的循环半衰期(人类约120天)；5)没有细胞核，没有线粒体和任何DNA，对红细胞的前体进行任何形式的基因工程修饰，在其去核后都会被消除，故该细胞在输入受体后不会导致异常生长或成瘤；6)它们保护包封的物质免于过早失活和降解，并保护生物体免受药物的毒性作用，同时也可以成为蛋白类药物的生物反应器。以上这些特征使红细胞成为体内递送成分天然、合成成分重要生物运输载体。本发明提供的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，利用红细胞独特特征作为运输载体，采用基因工程改造使红细胞膜表面携带有用于肿瘤免疫治疗的抗体药物(即抗PD-1单链抗体)，而后通过红系诱导分化手段诱导该工程化前体细胞向成熟红细胞分化，获得搭载抗PD-1单链抗体的基因工程化红细胞。本发明提供的搭载抗PD-1单链抗体的基因工程化红细胞将抗PD-1单链抗体靶向递送 至肿瘤组织使其更有效的发挥T细胞激活功能，并能明显减少药物用量，缓解全身性的肿瘤免疫药物带来的副作用。
附图说明
图1是本发明提供的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法的流程图；
图2本发明的实施例1的利用外周血体外制备成熟红细胞的细胞增殖曲线，本发明中以两个捐献者的数据作为比对；
图3是在富集Lin-CD34-细胞阶段，两个捐献者的CD235a以及CD117表达水平的示意图；
图4是在富集Lin -CD34 -细胞阶段，两个捐献者的CD71以及hoechst33342/CD235a共染后鉴定红细胞脱核水平示意图；
图5是在Lin -CD34 -细胞分化阶段，两个捐献者的CD235a以及CD117表达水平的示意图；
图6是在Lin -CD34 -细胞分化阶段，两个捐献者CD71以及hoechst33342/CD235a共染后鉴定红细胞脱核水平示意图；
图7是在红细胞脱核成熟阶段，两个捐献者的CD235a以及CD117表达水平的示意图；
图8是在红细胞脱核成熟阶段，两个捐献者CD71以及hoechst33342/CD235a共染后鉴定红细胞脱核水平示意图；
图9是分化培养阶段细胞CD235a表达变化曲线；
图10是分化培养阶段细胞CD71表达变化曲线；
图11是分化培养阶段细胞CD117表达变化曲线；
图12是制备过程中的红细胞脱核情况；
图13是捐献者1提供的细胞分化完成后对细胞进行涂片染色，镜下检测红细胞形态；
图14是捐献者2提供的细胞分化完成后对细胞进行涂片染色，镜下检测红细胞形态；
图15是荧光素酶检测anti-PD-1 scFv的转化效率检测图；
图16是流式细胞术检测anti-PD-1 scFv在红细胞中的表达效率检测图；
图17是anti-PD-1 scFv-红细胞在小鼠体内分布图；
图18是anti-PD-1 scFv-红细胞在小鼠体内器官分布图；
图19是anti-PD-1 scFv-红细胞与T细胞共培养显微镜检测图；
图20是anti-PD-1 scFv-红细胞与T细胞共培养流式细胞术检测图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图 1-20，对本发明进一步详细说明。
本方案中的专业术语介绍如下：
外周血：外周血是除骨髓之外的血液，临床上常用一些方法把骨髓中的造血干细胞释放到血液中，再从血液中提取分离得到造血干细胞，我们把这样得到的干细胞称为外周血干细胞。
外周血单个核细胞：是外周血中具有单个核的细胞，包括淋巴细胞和单核细胞，目前外周血单个核细胞主要的分离方法是Ficoll-hypaque(聚蔗糖－泛影葡胺)密度梯度离心法。
Lin -CD34 -细胞：Lin -细胞是指并未进入各个造血谱系分化的细胞，CD34 -是指细胞表面不表达CD34表面标志物，Lin -CD34 -细胞是并未进入造血谱系分化且不表达CD34表面标志物的细胞。
生物素标记的抗体:是在抗体上连接生物素(biotin)，生物素标记反应简单、温和且很少抑制抗体活性，将生物素与抗体共价结合是一种非常简便、直接的标记方法。
造血干细胞扩增培养基：StemSpan TM SFEM无血清扩增培养基，是一款造血干细胞扩增培养基，不含血清，在加入造血生长因子和/或用户选择的其他刺激因子后，可促进人造血干/祖细胞(HSPC)的扩增或向特定谱系的分化。
重组人类fms样酪氨酸激酶3配体(rhFlt3L)：FMS样酪氨酸激酶3配体(Flt-3配体)也称为FL，Flt3L和FLT3LG，是促进多种造血细胞谱系分化的α-螺旋细胞因子。FLT3LG在结构上与干细胞因子(SCF)和集落刺激因子1(CSF-1)同源。FLT3LG作为生长因子，通过激活造血祖细胞来增加细胞的数量。
重组人干细胞因子(rhSCF)：Kit配体(KITLG)也称为干细胞因子(SCF)，属于SCF家族的I型跨膜糖蛋白。KITLG是受体型蛋白酪氨酸激酶KIT的配体。SCF在调节细胞存活和增殖，造血，干细胞维持，细胞发育，迁移和功能中起重要作用。
重组人白细胞介素三(rhIL-3)：是属于造血生长因子家族的糖蛋白，其在临床前体外和体内研究中表现出多谱系活性。造血祖细胞在IL-3蛋白的帮助下增殖和分化成成熟的红细胞，肥大细胞，巨核细胞和粒细胞。
重组人白细胞介素六(rhIL-6)：是一种调节免疫反应，造血功能，急性期反应和炎症反应的多功能细胞因子。与IL-3协同促进细胞造血细胞增殖。
重组人促红细胞生成素(rhEpo)：是主要的红细胞生成因子，其与从多能祖细胞发育红细胞谱系的各种其他生长因子(例如，IL-3，IL-6，糖皮质激素和SCF)协同作用。爆式形成单位-红细胞(BFU-E)细胞开始促红细胞生成素受体表达并且对促红细胞生成素敏感。是重要的红系造血细胞因子。
人转铁蛋白(holo human transferrin)：是血浆中主要含铁蛋白，可以与铁离子形成复合物，供红细胞中血红蛋白生成。
链霉亲和素：是Streptomyces avicllrdi菌培养过程中的分泌产物，主要通过2一亚氨基生物素亲和层析法提纯，1L培养液中含蛋白10～60mg.
CD235a：血型糖蛋白A，是一种单跨膜糖蛋白，表达于成熟红细胞和红系前体细胞，为红细胞表面特殊的标记蛋白。CD235a表达说明该细胞向红系细胞分化，从流式细胞术结果分析表明，在SFEM阶段，细胞不表达CD235a，说明该细胞并未进入红系分化，而在将细胞换液为分化1阶段培养基后，培养基中的细胞因子诱导细胞向红系分化，细胞开始表达CD235a，且比例随分化时间不断增加，在更换为分化2阶段培养基后，由于细胞已经完全进入红系，故几乎所有细胞均表达CD235a，表明几乎所有细胞均为红系细胞。该细胞表明标志物说明我们的分化体系在体外培养红系细胞的诱导过程中是成功的。
CD117：又称c-kit，是SCF干细胞生长因子受体，表达于造血干细胞及其他细胞表面。SCF在调节细胞存活和增殖，造血，干细胞维持，细胞发育，迁移和功能中起重要作用。其受体的表达量变化反应出细胞利用SCF能力的变化，在SFEM条件下CD117并无表达，表明Lin-细胞在SFEM中培养阶段并未利用SCF。当细胞进入分化1阶段培养基后细胞进入红系分化，CD117表达快速上升，达到顶峰，此时培养基中加入SCF以调节细胞存活和增殖，造血，干细胞维持，细胞发育。而当细胞进入分化2阶段培养基后，由于细胞进入成熟脱核阶段，故CD117表达又逐渐下降。
CD71：转铁蛋白受体1，是一种跨膜糖蛋白，由两个通过两个二硫键连接的二硫键连接的单体组成。每个单体结合一个全转铁蛋白分子，产生铁-转铁蛋白-转铁蛋白受体复合物，其通过胞吞作用进入细胞，供细胞向红系发育过程中血红蛋白生成。在SFEM条件下CD71并无表达，表明Lin-细胞在SFEM中培养阶段并未利用转铁蛋白，细胞并未进入红系开始摄取铁合成血红蛋白。当细胞进入分化1阶段培养基后，由于细胞进入红系分化，需要摄取大量的转铁蛋白，故CD71表达迅速上升，满足细胞转铁蛋白的摄取。当细胞进入分化2阶段培养基后，由于红系细胞已经合成了足够多的血红蛋白，故CD71表达逐渐下降，红细胞走向成熟。
Hoechst33342：是用于染色DNA的荧光染料。该染料可以穿过细胞膜与DNA相结合，如果细胞未脱核，则该染料与DNA结合可以通过流式细胞术检测到信号为阳性，如果该细胞脱核，则通过流失细胞术检测到信号为阴性。
同时由于CD235a是红细胞的表面标志物，通过流式细胞术同时检测检测两种标志物共表达的情况，CD235a阳性Hoechst 33342阳性为红系未脱核细胞，CD235a阳性Hoechst 33342 阴性为成熟红细胞。在SFEM和分化1阶段培养基中，细胞由Lin-细胞进入红系分化，CD235a表达升高，但并未脱核，故该细胞为Hoechet 33342阳性。当细胞进入分化2阶段培养基后，细胞开始成熟脱核，出现Hoechst 33342阴性细胞，表明该红细胞成熟，本发明可以对成熟红细胞进行形态、结构和功能鉴定。
GFP：绿色荧光蛋白。该蛋白为构建MSCV-anti-PD-1 scFv时构建的与目的基因anti-PD-1 scFv融合的荧光报告基因，检测到该信号表明anti-PD-1 scFv也同时表达了，故可以作为检测目的基因检测信号。若GFP为阳性则表明该细胞表达anti-PD-1 scFv。
实施例1
一、含anti-PD-1 scFv慢病毒载体的构建
抗PD-1单链可变区片段(Anti-PD-1 scFv)进行设计，Anti-PD-1 scFv的核苷酸序列为SEQ ID NO.1-SEQ ID NO.6中的一种或几种组合，采用常规生物合成方法进行合成，并通过分子克隆手段进行载体构建，将Anti-PD-1 scFv构建于慢病毒载体pCDH-MCS-T2A-copGFP-MSCV上，得到目的序列载体(MSCV-anti-PD-1 scFv)，用于病毒包装。
二、慢病毒浓缩液的制备
采用MSCV-anti-PD-1 scFv:pSPAX2:VSVG＝2：1：1比例进行慢病毒包装包装，本实施例采用6孔板，使用磷酸钙转染法按目的序列载体(MSCV-anti-PD-1 scFv):pSPAX2:VSVG＝2μg：1μg：1μg将质粒转染至HEK 293T细胞内进行病毒包装。转染后12小时换液去除磷酸钙，48小时及72小时收集上清，并将细胞培养上清经过0.45μm滤膜过滤。将收集并过滤的细胞培养上清进行超速离心浓缩，转速70000RCF，时间2小时，温度4℃。离心后去除上清，使用分化1阶段培养基进行重悬，使用ELISA对病毒滴度进行定量，并保存与-80℃备用。
三、外周血中Lin -CD34 -细胞的获取：
取用全血，使用磷酸盐缓冲液1：1稀释全血后，使用淋巴细胞分离液(LymphoprepTM，STEMCELL Technologies)以及淋巴细胞分离管，在1200×g离心15分钟，用毛细血管吸取单个核细胞。
将获取的外周血单个核细胞在300×g离心10分钟后，使用添加了2％牛血清白蛋白，1mM EDTA的磷酸盐缓冲液的细胞分离缓冲液，按1×10 6个/毫升的比例重悬细胞，得到细胞悬浮液。
使用细胞悬液10倍体积的细胞分离缓冲液对生物素标记(如表1所示)的抗体进行稀释，往细胞悬浮液中以20-40μg/ml添加生物素标记的抗体，加入300μl的链霉亲和素标记的磁珠，在4℃孵育30分钟；以及随后将溶液置于磁力架内6-8分钟，进行谱系细胞分离，收集未被 吸附分离的分离缓冲液，在300×g离心10分钟，得到Lin-CD34-细胞。
表1 1ml生物素标记的抗体的成分
成分 体积
生物素标记的鼠抗人CD3抗体 20ul
生物素标记的鼠抗人CD14抗体 20ul
生物素标记的鼠抗人CD16抗体 20ul
生物素标记的鼠抗人CD19抗体 20ul
生物素标记的鼠抗人CD41a抗体 20ul
生物素标记的鼠抗人CD56抗体 20ul
生物素标记的鼠抗人CD235a抗体 20ul
四、富集Lin-CD34-细胞：
在第0天，将0.1-0.5×10 5的Lin -CD34 -细胞，接种到造血干细胞扩增培养基中，并添加细胞因子组合以及青链霉素，其中造血干细胞扩增培养基中，细胞因子组合以及青链霉素组成富集细胞培养基，所述富集细胞培养基的成分如表2所示，培养到第4天，该阶段被定义为富集Lin -CD34 -细胞阶段。
表2 配置1L富集细胞培养基需要的成分：
成分 储存液浓度 终浓度 体积
StemSpan无血清培养基(STEMCELL Technologies) 1× 1× 1L
重组人类fms样酪氨酸激酶3配体(Flt3L) 200mM 2mM 10ml
重组人干细胞因子(SCF) 100μg/ml 100ng/ml 1ml
重组人白细胞介素3(IL-3) 100μg/ml 100ng/ml 1ml
重组人白细胞介素六(IL-6) 800μg/ml 800pg/ml 1ul
五、诱导所述Lin-CD34-细胞向红系分化并进行扩增
在第5天更换培养体系，将培养基更换为分化第一阶段培养基(如表3所示)，在置于37℃，5％CO 2条件下进行扩增培养。
表3 配置1L分化第一阶段培养基需要的成分
成分 终浓度 体积
IMDM 85％ 850ml
胎牛血清(FBS，Gibco) 10％ 100ml
人血浆(Plasma) 5％ 50ml
谷氨酰胺 2nM 10
牛血清白蛋白 2％ 20ml
人转铁蛋白 500μg/ml 100
重组人胰岛素 10μg/ml 100μl
青链霉素 2％ 20ml
重组人白细胞介素3 100ng/ml 1ml
重组人促红细胞生成素 100ng/ml 1ml
重组人干细胞因子 100ng/ml 1ml
六、慢病毒浓缩液感染Lin -CD34 -细胞
在第6天使用之前制备并冻存于-80℃的MSCV-anti-PD-1 scFv对细胞进行感染。感染方法：将细胞按照1×106密度使用分化1阶段培养基重悬。计算感染体积及病毒用量，病毒使用量按终浓度5×107TU/ml-5×108TU/ml计算，并根据感染体积按照10μg/ml向浓缩病毒液中加入凝聚胺(polybrene)混合孵育5分钟。将孵育后的浓缩病毒液加入细胞中，混匀。并使用水平转子离心机进行离心感染，转速500×g，温度32℃，时间90分钟。离心完成后置于37℃，5％CO 2条件下进行培养。
分化第一阶段培养基下只提供了向红系发育相关的细胞因子，保证了Lin -CD34 -细胞向红系的增殖分化，同时在其增殖分化最快的阶段进行病毒感染，保证目的基因(anti-PD-1 scFv)的插入并表达至细胞膜上。
七、红细胞脱核得到成熟的anti-PD-1 scFv-红细胞
在第14天更换培养体系，将培养基更换为分化第二阶段培养基(如表4所示)，在置于37℃，5％CO 2条件下培养至第22天，获得搭载抗PD-1单链抗体的基因工程化红细胞，该阶段被定义为红细胞脱核成熟阶段，从0-22天全期间每2-4天更换相应阶段培养基。
表4 配置1L分化第二阶段培养基需要的成分：
成分 终浓度 体积
IMDM 85％ 850ml
胎牛血清(FBS，Gibco) 10％ 100ml
人血浆(Plasma) 5％ 50ml
谷氨酰胺 2mM 10ml
牛血清白蛋白 2％ 20ml
人转铁蛋白 500μg/ml 100μl
重组人胰岛素 10μg/ml 100μl
青链霉素 2％ 20ml
重组人促红细胞生成素 2U/ml 20ul
八、红细胞的鉴定与检测
(一)细胞扩增数量分析：
在第0-22天分别对细胞数量进行检测，每3-4天，将培养体系中的细胞充分重悬，取10μl细胞悬液与10μl台盼蓝染液混合，通过细胞计数仪计数。
具体地，细胞增殖情况如下所述，此时将起始细胞数量标准化到1×10 6:
天数 0 4 7 11 13 17 19 22
捐献者1 1 2.69 21.53 89.20 209.17 738.24 996.62 1011.38
捐献者2 1 2.33 13.84 36.91 126.73 406.03 501.92 517.22
将得到的细胞数量绘制细胞增殖曲线，如图1所示，可见，在该培养条件下红细胞能够正常的增殖分化，且能够实现1000倍增殖。
(二)细胞表型分析：
分别取用富集Lin -CD34 -细胞阶段、Lin -CD34 -细胞分化阶段以及红细胞脱核成熟阶段的细胞进行分析，具体的操作过程。
将培养体系中的细胞充分重悬，取50-100μl细胞悬液加入500μl磷酸盐缓冲液中。分别加入鼠抗人CD235a-APC抗体(mouse Anti-human CD235a APC)0.5μl,鼠抗人CD71-PerCP Cy5.5抗体(mouse Anti-human CD71-PerCP Cy5.5)2μl,鼠抗人CD117-PE Cy7抗体(mouse Anti-human CD117-PE Cy7)2μl,Hoechst 33342染料0.5μl，充分混匀后避光孵育20分钟，流式细胞仪上机检测。对目的细胞进行特定的表面标记物表达水平进行分析。通过不同时间点的分析绘制变化曲线，得到如图2到如11所示的示意图。
(三)红细胞脱核情况的分析：
根据流式细胞术分析CD235a阳性/Hoechst 33342阴性细胞的比例，该比例可以反映细胞脱核情况。
得到如图12所示的示意图，由图可知，细胞从第13天开始脱核水平明显提高。
(四)细胞形态分析：
取0.5-1×10 6细胞，使用血细胞离心涂片机进行离心涂片。
使用-20℃预冷的甲醇室温固定2分钟。
将固定后的血涂片水洗3次，每次5分钟，并置于室温风干。
使用10ml磷酸盐缓冲液溶解对二氨基联苯片剂(Benzidine,Sigma)，添加10μl过氧化氢溶液，过滤。使用300-500μl该溶液对血涂片进行染色，室温1小时。
将染色后的血涂片水洗3次，每次5分钟，并置于室温风干。
使用吉姆萨染液对血涂片二次染色，室温35-40分钟。
将染色后的血涂片水洗3次，每次5分钟，并置于室温风干。
使用封片剂封片，镜下观察拍照。
得到如图13、图14所示的示意图，由图可知，在第22天细胞分化完成后，明显出现成熟的红细胞。
九、搭载抗PD-1单链抗体的基因工程化红细胞的检测与鉴定
(一)荧光素酶检测anti-PD-1 scFv的转化效率
将GFP基因构建在目的片段(anti-PD-1 scFv)下游，使目的序列片段具有荧光素酶报告基因，通过GFP荧光显色来指示anti-PD-1 scFv在红细胞中的表达，从而判断anti-PD-1 scFv的转化效率。将anti-PD-1 scFv-红细胞分别置于正常光源和GFP荧光蛋白激发光源下进行显微镜观察。
图15为荧光素酶检测anti-PD-1 scFv的转化效率，图中Light field表示，在明场(正常 光源)下anti-PD-1 scFv-红细胞显微镜图，GFP表示GFP荧光蛋白激发光源下anti-PD-1 scFv-红细胞显微镜图，Merge表示明场和GFP荧光蛋白激发光源anti-PD-1 scFv-红细胞显微镜图重叠的效果图。如图所示，对外周血Lin-细胞进行基因工程改造，同时使用GFP为报告基因，anti-PD-1 scFv-红细胞占总体细胞的95％，说明基因工程改造效率达95％以上。
(二)流式细胞术检测anti-PD-1 scFv在红细胞中的表达效率
根据流式细胞术分析CD235a阳性/GFP阳性细胞的比例，该比例可以反映anti-PD-1 scFv在红细胞中的表达效率。
如图16所示，anti-PD-1 scFv-红细胞在分化末期成为成熟的红细胞后依然有约85％细胞为GFP阳性，表明anti-PD-1 scFv-红细胞在分化至末期依然高水平表达具有免疫激活能力的anti-PD-1 scFv。
(三)小鼠Dir染色实验检测anti-PD-1 scFv-红细胞的分布
向小鼠尾静脉注射经Dir染色后的红细胞，并在不同时间(1.5h/4h/5h/24h)进行活体成像检测红细胞在小鼠体内的分布情况，5天后处死小鼠进行组织器官成像检测红细胞在小鼠体内器官的分布情况。
图17为anti-PD-1 scFv-红细胞在小鼠体内分布图，图18为anti-PD-1 scFv-红细胞在小鼠体内器官分布图，图中1号小鼠尾静脉注射Dir染色液，2号小鼠尾静脉注射未经Dir染色正常的红细胞，3号小鼠尾静脉注射经Dir染色后正常的红细胞，4号小鼠尾静脉注射经Dir染色后anti-PD-1 scFv-红细胞。如图17、18所示，anti-PD-1 scFv-红细胞与正常的红细胞在小鼠体内包括体内器官分布情况是一致的。
(三)体外T细胞激活功能实验
anti-PD-1 scFv-红细胞与T细胞进行共培养，检测anti-PD-1 scFv-红细胞对T细胞激活功能。
如图19、20所示，anti-PD-1 scFv-红细胞在体外细胞共培养条件下，激活T细胞，使T细胞大量增殖，流式细胞术的检测结果显示CD25表达增高。
综上所述，本发明提供的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，利用红细胞作为运输载体，采用基因工程改造使红细胞膜表面携带有用于肿瘤免疫治疗的抗体药物(即抗PD-1单链抗体)，而后通过红系诱导分化手段诱导该工程化前体细胞向成熟红细胞分化，获得搭载抗PD-1单链抗体的基因工程化红细胞。本发明提供的搭载抗PD-1单链抗体的基因工程化红细胞将抗PD-1单链抗体靶向递送至肿瘤组织使其更有效的发挥T细胞激活功能，并能明显减少药物用量，缓解全身性的肿瘤免疫药物带来的副作用。
上述仅为本发明较佳可行实施例，并非是对本发明的限制，本发明也并不限于上述举例。 本技术领域的技术人员，在本发明的实质范围内，所作出的变化、改型、添加或替换，也应属于本发明的保护范围。

Claims (9)

  1. 搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，其特征在于：包括如下步骤：
    S1：将目的片段序列构建于慢病毒表达载体，获得目的序列载体，所述目的片段为Anti-PD-1scFv，所述Anti-PD-1scFv表示抗PD-1单链可变区片段；
    S2：将步骤S1中的目的序列载体进行慢病毒包装，经离心浓缩后，获得高滴度的慢病浓缩液；
    S3：从外周血单个核细胞中分离Lin-CD34-细胞和富集Lin-CD34-细胞；
    S4：将所述Lin-CD34-细胞培养基更换为分化第一阶段培养基中，诱导所述Lin-CD34-细胞向红系分化并进行扩增；
    S5：使用步骤S2中的慢病毒浓缩液对步骤S4中的Lin-CD34-细胞进行感染，获得anti-PD-1scFv-Lin-CD34-细胞；
    S6：将所述anti-PD-1scFv-Lin-CD34-细胞的培养基更换为分化第二阶段培养基，所述anti-PD-1scFv-Lin-CD34-细胞通过红细胞脱核得到成熟的anti-PD-1scFv-红细胞，即为搭载抗PD-1单链抗体的基因工程化红细胞。
  2. 根据权利要求1所述的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，其特征在于：在步骤S1中，所述Anti-PD-1scFv核苷酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5和/或SEQ ID NO.6所示；所述慢病毒表达载体为pCDH-MCS-T2A-copGFP-MSCV。
  3. 根据权利要求1所述的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，其特征在于：在步骤S2中，所述慢病毒包装采用三质粒包装系统，所述三质粒包装系统包括目的序列载体、PSPAX2质粒和VSVG质粒，所述目的序列载体、所述PSPAX2质粒、所述pMD2G质粒的比例为2：1：1，所述慢病毒包装采用HEK 293T细胞作为慢病毒的包装细胞；
    所述离心浓缩的转速为70000RCF，时间2小时，温度4℃。
  4. 根据权利要求1所述的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，其特征在于：在步骤S3中，利用添加牛血清白蛋白和EDTA的细胞分离缓冲液重悬细胞，添加生物素标记的抗体以及链霉亲和素标记的磁珠，去除带有特殊表面标志物的细胞，分离出Lin-CD34-细胞。
  5. 根据权利要求1所述的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，其特征在于：在步骤S3中，富集Lin-CD34-细胞中的细胞因子组合物包括50-100ng/ml重组人类fms样酪氨酸激酶3配体、50-100ng/ml重组人干细胞因子、50-100ng/ml重组人白细胞介素三、200-800pg/ml重组人白细胞介素六。
  6. 根据权利要求1所述的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，其特 征在于：在步骤S4中，所述分化第一阶段培养基包括IMDM，10-15％胎牛血清，5-10％人血浆，1-4mM谷氨酰胺，1-2％牛血清白蛋白，300-600μg/ml人转铁蛋白，8-13μg/ml重组人胰岛素，2％青链霉素,3-5ng/ml重组人白细胞介素三，4-7U/ml重组人促红细胞生成素以及100ng/ml重组人干细胞因子。
  7. 根据权利要求1所述的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，其特征在于：在步骤S5中，所述感染的步骤如下：用分化第一阶段培养基重悬Lin-CD34-细胞，计算感染体积及病毒用量，并根据感染体积按照10μg/ml向慢病毒浓缩液中加入凝聚胺混合孵育5min，将孵育后的慢病毒浓缩液加入细胞中，混匀，所述慢病毒浓缩液的终浓度为5×10 7TU/ml-5×10 8TU/ml；使用水平转子离心机进行离心感染，转速500×g，温度32℃，时间90min，离心完成后将anti-PD-1scFv-Lin-CD34-细胞置于37℃，5％CO 2条件下进行培养。
  8. 根据权利要求1所述的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法，其特征在于：在步骤S5中，所述分化第二阶段培养基包括但不限于IMDM，15％胎牛血清，5-10％人血浆，1-4mM谷氨酰胺，1-2％牛血清白蛋白，300-600μg/ml人转铁蛋白，8-13μg/ml重组人胰岛，2％青链霉素以及1-5U/ml重组人促红细胞生成素。
  9. 搭载抗PD-1单链抗体的基因工程化红细胞，其特征在于：基于权利要求1至8任一项所述的搭载抗PD-1单链抗体的基因工程化红细胞的制备方法制备得到的搭载抗PD-1单链抗体的基因工程化红细胞。
PCT/CN2019/086602 2019-05-10 2019-05-13 搭载抗pd-1单链抗体的基因工程化红细胞及其制备方法 WO2020227885A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/595,183 US20240043802A1 (en) 2019-05-10 2019-05-13 Genetically engineered erythrocytes carrying anti-pd-1 single chain antibody and preparation method therefor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910387639.2A CN110129273B (zh) 2019-05-10 2019-05-10 搭载抗pd-1单链抗体的基因工程化红细胞及其制备方法
CN201910387639.2 2019-05-10

Publications (1)

Publication Number Publication Date
WO2020227885A1 true WO2020227885A1 (zh) 2020-11-19

Family

ID=67576965

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/086602 WO2020227885A1 (zh) 2019-05-10 2019-05-13 搭载抗pd-1单链抗体的基因工程化红细胞及其制备方法

Country Status (3)

Country Link
US (1) US20240043802A1 (zh)
CN (1) CN110129273B (zh)
WO (1) WO2020227885A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023284742A1 (en) * 2021-07-13 2023-01-19 Westlake Therapeutics (Hangzhou) Co. Limited Cells modified by conjugated n-terminal glycine and uses thereof

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114786710A (zh) * 2019-10-17 2022-07-22 西湖生物医药科技(杭州)有限公司 工程改造红细胞用于治疗溶酶体贮积病
WO2021073613A1 (en) * 2019-10-18 2021-04-22 Westlake Therapeutics (Hangzhou) Co. Limited Engineered red blood cells presenting specific cancer neoantigen with artificial mhc
WO2021083278A1 (en) * 2019-10-29 2021-05-06 Westlake Therapeutics (Hangzhou) Co. Limited Engineering red blood cells for treating gout and hyperuricemia diseases
WO2022089605A1 (en) * 2020-10-30 2022-05-05 Westlake Therapeutics (Hangzhou) Co. Limited Modified red blood cells and uses thereof for delivering agents
WO2022166720A1 (zh) * 2021-02-05 2022-08-11 华南理工大学 基于血清白蛋白的融合蛋白、纳米组装体及其制备方法和应用
WO2023169528A1 (zh) * 2022-03-11 2023-09-14 西湖生物医药科技(上海)有限公司 一种靶向pd-1的工程化红细胞
WO2024017192A1 (zh) * 2022-07-18 2024-01-25 西湖生物医药科技(上海)有限公司 包含血细胞的药物递送系统
CN117965442A (zh) * 2022-10-26 2024-05-03 北京睿脉医药科技有限公司 一种将治疗性分子偶联至成熟红细胞表面的方法及应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109609533A (zh) * 2017-12-27 2019-04-12 郑州大学第附属医院 基于人源化cd276抗体的car慢病毒表达载体构建及其应用
CN109652369A (zh) * 2019-02-28 2019-04-19 西湖生物医药科技(杭州)有限公司 利用外周血体外制备成熟红细胞的方法及制剂

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109609533A (zh) * 2017-12-27 2019-04-12 郑州大学第附属医院 基于人源化cd276抗体的car慢病毒表达载体构建及其应用
CN109652369A (zh) * 2019-02-28 2019-04-19 西湖生物医药科技(杭州)有限公司 利用外周血体外制备成熟红细胞的方法及制剂

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
REN J.: "A versatile system for rapid multiplex genome-edited CAR T cell generation", ONCOTARGET, vol. 8, no. 10, 7 March 2017 (2017-03-07), XP055565031, ISSN: 1949-2553, DOI: 10.18632/oncotarget.15218 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023284742A1 (en) * 2021-07-13 2023-01-19 Westlake Therapeutics (Hangzhou) Co. Limited Cells modified by conjugated n-terminal glycine and uses thereof

Also Published As

Publication number Publication date
CN110129273A (zh) 2019-08-16
CN110129273B (zh) 2020-09-08
US20240043802A1 (en) 2024-02-08

Similar Documents

Publication Publication Date Title
WO2020227885A1 (zh) 搭载抗pd-1单链抗体的基因工程化红细胞及其制备方法
US11913021B2 (en) Methods and compositions for promoting survival and proliferation of endothelial cells and stimulating angiogenesis
CA2898180C (en) Reprogramming of human endothelium into hematopoietic multi-lineage progenitors by defined factors
JP6288615B2 (ja) ヒト赤血球前駆細胞株及びヒト脱核赤血球の製造方法
US20210154231A1 (en) Method for producing t cells modified by chimeric antigen receptor
Singh et al. Vaccinia virus infection modulates the hematopoietic cell compartments in the bone marrow
Silvestri et al. Persistence of drug-resistant leukemic stem cells and impaired NK cell immunity in CML patients depend on MIR300 antiproliferative and PP2A-activating functions
KR20160075676A (ko) 방법
Perlingeiro et al. A role for thrombopoietin in hemangioblast development
KR20190142257A (ko) 조건부 성숙이 가능한 불멸화 적혈구전구세포주 확립
Suzuki et al. Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL
US20220233665A1 (en) Medicinal composition
JP2023504075A (ja) Ｃａｒ－ｎｋ細胞を得る方法
RU2469086C1 (ru) Способ экспансии cd34+ гематопоэтических клеток
JPWO2005056778A1 (ja) 造血幹細胞の分化抑制又は増殖方法
WO2024010317A1 (ko) 유전자 과발현 조합을 이용한 적혈구 분화능이 우수한 불멸화 적혈구전구세포주 확립, 이의 제조방법 및 용도
US20210403867A1 (en) Method for Producing Erthyroid Cells
WO2006085482A1 (ja) 造血幹細胞の自己複製因子及び増幅方法
WO2023156774A1 (en) Generating bone marrow organoids
US20090169523A1 (en) Hsc self-renewal
KR20240005600A (ko) 유전자 과발현 조합을 이용한 적혈구 분화능이 우수한불멸화 적혈구전구세포주 확립, 이의 제조방법 및 용도
CN117866894A (zh) 一种成纤维细胞转分化为巨噬细胞的方法
Lee Engineering Macrophages for Cell-Based Therapies
CN114746545A (zh) 应用人工mhc呈递特异性癌症新抗原的工程化红细胞
Kim Regulation of Erythroid Self-Renewal

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: 19928912

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19928912

Country of ref document: EP

Kind code of ref document: A1