WO2020192803A1 - 一种嵌合抗原受体重组腺相关病毒颗粒及其应用 - Google Patents

一种嵌合抗原受体重组腺相关病毒颗粒及其应用 Download PDF

Info

Publication number
WO2020192803A1
WO2020192803A1 PCT/CN2020/091185 CN2020091185W WO2020192803A1 WO 2020192803 A1 WO2020192803 A1 WO 2020192803A1 CN 2020091185 W CN2020091185 W CN 2020091185W WO 2020192803 A1 WO2020192803 A1 WO 2020192803A1
Authority
WO
WIPO (PCT)
Prior art keywords
car
cells
aav
chimeric antigen
antigen receptor
Prior art date
Application number
PCT/CN2020/091185
Other languages
English (en)
French (fr)
Inventor
吴喜林
吴稚伟
纳瓦兹瓦卡斯
黄碧莲
Original Assignee
南京安锐生物科技有限公司
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 南京安锐生物科技有限公司 filed Critical 南京安锐生物科技有限公司
Publication of WO2020192803A1 publication Critical patent/WO2020192803A1/zh

Links

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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • 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
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464421—Receptors for chemokines
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464424—CD20
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • 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
    • C12N15/867—Retroviral 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the technical field of chimeric antigen receptors, in particular to a chimeric antigen receptor recombinant adeno-associated virus and applications thereof.
  • CAR-T Chimeric Antigen Receptor (CAR) modified T cell (CAR-T) immunotherapy is a new type of cell therapy that has been improved and used clinically in recent years.
  • CAR-T cell immunotherapy involves separating T cells from peripheral venous blood of patients, expanding them under the induction of various cytokines, and introducing CAR molecules targeting tumor antigens.
  • the modification of CAR molecules can make the T cell's tumor-killing effect not be restricted by the major histocompatibility complex (MHC) when it obtains the targeted killing ability.
  • MHC major histocompatibility complex
  • CAR-T can be injected through intravenous or intradermal injection. The cells are fed back into the patient to kill the tumor.
  • CAR-T cells have continuous expansion ability and tumor-targeted killing activity, which ensures that they have sustained and effective killing power on tumor cells, so that patients can be cured.
  • CAR-T cell therapy has a significant effect, and CAR-T cell immunotherapy is considered to be one of the most promising tumor treatment methods.
  • CAR-T cell therapy can be achieved by directly injecting the drugs into the human body.
  • the chimerization of CAR molecules and killer T cells needs to be transduced by viral vectors, and methods include retroviral systems, lentiviral systems, adenoviruses, DNA plasmids, and RNA transfection systems.
  • retroviruses and lentiviruses can achieve stable integration and high transfection efficiency in CAR-T cell therapy. Retroviruses are not easy to transfect resting T cells; lentiviruses can transfect resting T cells, but the cost is high, and both are genetically toxic and have the risk of inserting cancer. Therefore, there is an urgent need for a safer and effective viral vector Load the CAR molecule.
  • Adeno-associated virus (AAV) vectors are widely used in gene expression and antibody expression in vivo. As AAV virus vectors exist in the cytoplasm in the form of episomes, there are no insertion mutations and can last for a long time. Expression time.
  • the present invention provides a chimeric antigen receptor recombinant adeno-associated virus particle and application thereof to solve the above technical problems.
  • a chimeric antigen receptor recombinant adeno-associated virus particle which is a recombinant adeno-associated virus vector carrying a chimeric antigen receptor gene.
  • the chimeric antigen receptor recombinant adeno-associated virus particles are obtained by packaging the chimeric antigen receptor gene through an adeno-associated virus packaging system.
  • the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain.
  • extracellular domain is an antibody that recognizes the antigen domain.
  • the antibody that recognizes the antigen domain is a scFV antibody or a V H H antibody.
  • the scFV antibody is specific for CD4, CD19, CCR5 or CD20.
  • the invention also provides the application of the above-mentioned chimeric antigen receptor recombinant adeno-associated virus particles in the preparation of drugs for treating tumors.
  • the drug is a targeted drug applied to tumor immunotherapy.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned chimeric antigen receptor recombinant adeno-associated virus particles, and a pharmaceutically acceptable carrier or excipient.
  • the dosage form of the pharmaceutical composition is injection.
  • the present invention utilizes the recombinant adeno-associated virus (AAV virus) to load the chimeric antigen receptor (CAR molecule) to obtain the chimeric antigen receptor recombinant adeno-associated virus particle (AAV-CAR) directly injected into the body.
  • AAV-CAR directly infects T cells or NK cells in the body, and directly forms CAR cells in the body, thus realizing cell therapy of direct target cell killing.
  • AAV viruses (AAV-CD4-CAR, AAV-CCR5-CAR, AAV-CD19-CAR and AAV-CD20-CAR) loaded with different CAR molecules are successfully prepared, and AAV-CAR can directly infect 293TT adherent cells Suspend cells with 293F and display CAR molecules on the cell surface.
  • AAV-CAR can also directly infect PBMC cells and display CAR molecules on the surface of more than 40% of T cells to form CAR-T cells.
  • the CAR-T cells can specifically kill target cells with a killing efficiency of more than 40%.
  • AAV virus loaded with CAR molecules was directly injected into mice.
  • the AAV-CAR of the present invention can be directly injected in vivo, directly skipping the blood collection required for traditional CAR-T cell therapy, PBMC in vitro expansion, virus in vitro infection of T cells and CAR-T autotransfusion and other personalized treatments.
  • the sequence steps only need to directly inject the chimeric antigen receptor recombinant adeno-associated virus particles (AAV-CAR) into the patient's body, realizing the AAV-CAR universal batch cell therapy, which greatly saves the pre-treatment preparation time and treatment cost. Treatment efficiency.
  • Figure 1 is the Western-bloting results of cell lysates after AAV-CD4-CAR infects 293TT adherent cells and 293F suspension cells respectively in Example 3 of the present invention, where M is a marker, and lane 1 is AAV-CD4-CAR infected 293F cells Lysate, lane 2 is the lysate of AAV-CD4-CAR infected 293F cells, lane 3 is the control group;
  • Figure 2 shows the expression of CAR molecules on the cell surface after AAV-CD4-CAR-infected cells were identified by flow cytometry in Example 3 of the present invention.
  • the high-dose AAV-CD4-CAR transfection group is AAV-CD4-CAR(H)
  • the low-dose AAV-CD4-CAR transfection group is AAV-CD4-CAR(L)
  • the AAV-GFP transfection group is Mock;
  • Fig. 3 shows the expression of CAR molecules on CD3+ cell surface after AAV-CD4-CAR infection of PBMC by flow cytometry in Example 3 of the present invention, using Anti-F(ab') 2 -FITC and Anti-CD4-Fc-FITC respectively Detection, the high-dose AAV-CD4-CAR transfection group is AAV-CD4-CAR(H), the low-dose AAV-CD4-CAR transfection group is AAV-CD4-CAR(L), AAV-GFP transfection The group is Mock;
  • Figure 4 shows the specific killing of CD4+ T cells after the flow cytometric detection of AAV-CD4-CAR directly infects PBMC in Example 4 of the present invention, wherein Figure 4A is a schematic diagram of flow analysis, and Figure 4B is AAV-CD4-CAR processing different sources The ratio of CD4+ T cells after the PBMC of the control group; Figure 4C shows the ratio of CD8+ T cells after AAV-CD4-CAR treatment of PBMC from different sources, normalized to the value of the control group AAV-GFP treatment group, set to 100 %;
  • Figure 5 is a flow cytometric detection of AAV-CD4-CAR killing tumor cells in Example 4 of the present invention.
  • Figure 5A is a schematic flow cytometric analysis.
  • Figure 5B shows AAV-CD4-CAR targeting MT-2 and Jurkat tumors after direct infection of PBMC cells The killing ratio of cells is normalized with the ratio of non-specific killing of tumor cells in the Mock group as 0, and the specific cell killing data of the AAV-CD4-CAR treatment group is obtained after normalization of the reference control group;
  • Figure 6 shows the identification of specific targeted killing directly induced by AAV-CD4-CAR virus in humanized mice in Example 5 of the present invention.
  • Figure 6A shows the CD3+ in humanized mice before AV-CD4-CAR virus treatment The actual ratio of CD4+ T cells.
  • Figure 6B shows the expression of CAR molecules that specifically recognize CD4 protein on the surface of human CD3+ T cells after 2 weeks of direct infection of humanized mice by AAV-CD4-CAR.
  • Figure 6C shows Flow cytometry was used to detect the proportion of CD3+CD4+T cells in humanized mice at different time points.
  • Figure 6D shows the weight monitoring of humanized mice in different treatment groups.
  • AAV adeno-associated virus
  • VL-VH Obtained scFv (VL-VH) antibody sequences specific to CD19, CD4, CCR5 and CD20 and the hinge region, transmembrane domain and intracellular signal transduction domain sequence of the third-generation CAR through PubMed and HIV database websites. among them
  • CD19 scFv (VL-VH) antibody is shown in SEQ ID NO:1:
  • amino acids 1-107 of the sequence SEQ ID NO: 1 are the light chain variable region
  • amino acids 108-122 are the connecting peptide
  • amino acids 123-243 are the heavy chain variable region.
  • the amino acid sequence of the CD4 scFv (VL-VH) antibody is shown in SEQ ID NO: 2:
  • amino acids 1-112 of the sequence SEQ ID NO: 2 are the light chain variable region
  • amino acids 113-127 are the connecting peptide
  • amino acids 128-249 are the heavy chain variable region.
  • amino acid sequence of the scFv (VL-VH) antibody of CCR5 is shown in SEQ ID NO: 3:
  • amino acids 1-92 of the sequence SEQ ID NO: 3 are the light chain variable region
  • amino acids 93-107 are the connecting peptide
  • amino acids 108-229 are the heavy chain variable region.
  • the amino acid sequence of the CD20 scFv (VL-VH) antibody is shown in SEQ ID NO: 4
  • amino acids 1-107 of SEQ ID NO: 4 are the light chain variable region
  • amino acids 108-122 are the connecting peptide
  • amino acids 123-243 are the heavy chain variable region.
  • the selection of the hinge region, transmembrane region and intracellular signal transduction domain of CAR is a conventional choice in the art.
  • the structure of the hinge region, transmembrane domain and intracellular signal transduction domain of CAR in this example is: CD28-4 -1BB-CD3zeta, its nucleotide sequence is shown in SEQ ID NO: 5:
  • the scFv (VL-VH) antibody gene and CD28-4-1BB-CD3zeta gene shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 were digested and linked to pAAV -MCS plasmid vector (Cell Biolabs, San Diego, USA), and finally load a scFv(VL-VH)-CD28-4-1BB-CD3zeta single-gene CAR molecule on pAAV-MCS plasmid vector to construct pAAV-CAR (Respectively pAAV-CD19-CAR, pAAV-CD4-CAR and pAAV-CCR5-CAR and pAAV-CD20-CAR).
  • AAV Helper-Free virus packaging system was purchased from Cell Biolabs, San Diego USA.
  • the above-mentioned pAAV-CAR and pHelper and pAAV-DJ plasmids were co-transfected into AAV-293T cells using PEI transfection reagent in a mass ratio of 1:1:1.
  • AAV-CAR chimeric antigen receptor recombinant adeno-associated virus particles
  • AAV-CAR chimeric antigen receptor recombinant adeno-associated virus particles
  • the purified AAV-CAR was determined by Q-PCR for copy number determination.
  • the purified AAV-CAR was pretreated with DNaseI and the primers of CMV enhancer (AAV-Mono-CMV-F and AAV-Mono-CMV-R) were used to determine AAV.
  • -Copy of CAR virus, other reagents are from SYBR Premix Ex Taq II (Takara) kit. The samples were tested in three replicates on the 7300 instrument.
  • the cycle conditions of real-time PCR are as follows: 50°C for 2 min for a cycle, 95°C for 10 min for a cycle, 95°C for 15s and 60°C for 60s for 40 cycles.
  • the copy number of the virus was calculated by the standard curve made by the AAV-GFP plasmid, and finally 1x10 ⁇ 13gc/mL AAV-CAR virus was obtained.
  • sequence of AAV-Mono-CMV-F is shown in SEQ ID NO: 10:
  • AAV-Mono-CMV-R The sequence of AAV-Mono-CMV-R is shown in SEQ ID NO: 11:
  • AAV-CAR virus Different concentrations of AAV-CAR virus were added directly to different cells (293TT cells, 293F cells and PBMC cells). After 24 hours of culture, the expression of CAR molecules was detected by Western-Blot and flow detection methods.
  • the control group was infected with AAV-GFP virus particles to infect 293TT cells. After infection for 24 hours, the cells were collected and lysed, and the mouse antibody was used. Human CD3zeta antibody, the expression of CAR molecules in the cell lysate was detected by Western-Blot. The results are shown in Figure 1.
  • the cell lysate of 393TT cells and 293F cells infected with AAV-CD4-CAR virus particles has a 74kD
  • AAV-CD4-CAR expresses CAR molecules on the cell surface after infection of adherent cells and suspension cells
  • low-dose AAV-CD4-CAR(L) (1x10 ⁇ 4gc/cell) and high-dose AAV- CD4-CAR(H) (1x10 ⁇ 5gc/cell) infects 293TT adherent cells and 293F suspension cells.
  • the control group (Mock) uses AAV-GFP and uses Anti-F(ab') that specifically recognizes the CAR molecule VL-VH 2 -FITC antibody was used for flow cytometry to detect the expression of CAR molecules on the cell surface of 293TT adherent cells and 293F suspension cells after infection. The results are shown in Figure 2.
  • the low-dose AAV-CD4-CAR(L) infected 293TT cells In the high-dose AAV-CD4-CAR(H) treatment group, more than 50% of the 293TT and 293F cells expressed CAR molecules on the surface of 293F and 293F cells, indicating that AAV-CD4- CAR can directly infect adherent cells and suspension cells, and display CAR molecules on the cell surface.
  • AAV-CD4-CAR In order to further verify whether AAV-CD4-CAR can infect PBMC cells and display CAR molecules on the surface of CD3+ T cells to form CAR-T cells, low-dose AAV-CD4-CAR(L)(1x10 ⁇ 4gc/ cell) and high-dose AAV-CD4-CAR(H)(1x10 ⁇ 5gc/cell) to infect PBMC cells.
  • the control group (Mock) uses AAV-GFP, and uses Anti-F(ab ') 2 -FITC antibody was used to detect the expression of CAR molecules on the surface of PBMC cells by flow cytometry. The results are shown in Figure 3. Low-dose AAV-CD4-CAR(L) can make 28% of T cells form CAR-T.
  • AAV-CD4-CAR(H) infecting PBMC can make 49% of T cells become CAR-T.
  • flow cytometry was performed using Anti-CD4-Fc-FITC.
  • the flow cytometry results are shown in Figure 3.
  • CD3+ T cells in PBMCs directly infected with CAR can specifically bind to CD4 protein; in the high- and low-dose AAV-CD4-CAR treatment group, the positive rates of CD3+ T cells that recognize CD4 protein are 75% and 31%, respectively.
  • the positive rate data The antibody test result is roughly similar to that of Anti-F(ab') 2 -FITC. It is suggested that after AAV-CD4-CAR directly infects PBMC, a high proportion of CAR-T cells can be formed, and these CAR-T cells can specifically recognize CD4 protein.
  • the cell killing experiment of CAR-T cells constructed by AAV-CAR is based on the operating procedure of the radioisotope-free luminescence-based CytoTox-Glo kit (Promega) kit.
  • the kit detects dead cells killed by protease activity.
  • the experimental procedure is briefly described as follows: 2-fold serial dilutions of AAV-CAR transduced PBMC in a 96-well plate, the highest cell number is 2.0x10 6 per well, according to the ratio of AAV-CAR PBMC cells: target cells 50:1 Target cells of corresponding volume (target cells can be tumor cells expressing CD4, CD19, CD20 or CCR5, or CD4+ T cells and CD19+ B cells).
  • the cells were incubated for 4 hours at 37°C and 5% CO 2 ; CytoTox-Glo reagent was added to each well, and after 15 minutes at room temperature, the fluorescence value was measured by the instrument; the cell lysate containing target cells but no effect CAR-T cells was used to read The value is taken as 100% lysis kill.
  • AAV-CD4-CAR was infected with PBMC from different sources. After 48 hours, the proportion of CD3+CD4+T cells in the PBMC was detected. The flow cytometry result is shown in Figure 4A.
  • CD3+CD4+CD8-T are CD4+ T cells.
  • the flow cytometry results of CD4+ T cells are shown in Figure 4B.
  • CD4+ T cells in PBMC cells treated with AAV-CD4-CAR were significantly reduced, and the concentration gradient-dependent, high-dose AAV-CD4 -The number of CD4+ T cells treated with CAR is lower than the number of CD4+ T cells in the low-dose AAV-CD4-CAR treatment group; it is suggested that the higher the dose of AAV-CD4-CAR, the stronger the specific killing ability to CD4+T; After the dose of AAV-CD4-CAR directly infects 6 PBMC cells from different sources, the proportion of CD4+ T cells is reduced by about 50%, indicating that direct infection of PBMC by AAV-CD4-CAR can specifically target and kill in different individuals CD4+ T cells are dose-dependent.
  • AAV-CD4-CAR can specifically kill CD4+ tumor cells after direct infection of PBMC.
  • different doses of AAV-CD4-CAR high-dose AAV-CD4-CAR group (H) and low-dose AAV-CD4-CAR Group (L)
  • H high-dose AAV-CD4-CAR group
  • L low-dose AAV-CD4-CAR Group
  • CD4+MT2 or CD4+Jurkat tumor cells were added at a ratio of 1:1; after 48 hours of co-incubation, flow cytometry was performed Specific dead MT-2 cells or Jurkat; the flow cytometry results are shown in Figure 5.
  • PBMC directly infected by AAV-CD4-CAR can significantly and specifically kill CD4+MT2 cells and Jurkat cells. Subtracting non-specific killing, the specific killing ratio is 25%.
  • the dose of AAV-CD4-CAR is increased, the specific killing ratio of MT2 cells and Jurkat cells will also increase, and the highest specific killing ratio of MT2 cells will reach Above 40%, the above results indicate that after AAV-CD4-CAR directly infects PBMC, it can form CAR-T that specifically recognizes CD4 protein, thereby specifically targeting and killing CD4 tumor cells, and is a dose-dependent, efficient and specific targeting Kill.
  • NCG mice Immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice were purchased from Nanjing University Model Animal Institute. Similar to NSG mice, this mouse lacks the IL2 receptor gene on the basis of SCID mice, resulting in no mouse T cells in the body , B cells and a very small amount of NK cells. 1.5x10 7 PBMC was injected intraperitoneally into NCG mice for 4-6 weeks; after three weeks, blood was collected and flow cytometric detection of human T cells, by staining human CD45+, CD3+, CD4+ and CD8+, the proportion of human CD45 positive cells If it reaches 10% or more, it is judged that the mouse humanization is successful.
  • mice contained more than 10% of human CD45+ cells.
  • the actual proportion of human CD4+ T cells in mice is shown in Figure 6A.
  • the proportion of human CD4+ T cells is more than 20%. Inoculate tumor cells and monitor the size of the tumor for four consecutive weeks.
  • AAV-CAR Different concentrations of AAV-CAR were intraperitoneally injected into tumor-inoculated humanized mice, and blood was collected at different time points for testing; flow cytometry was used to detect the trend of changes in human CD4+ T cells; and the weight of the mice was monitored at the same time.
  • the AAV-CD4-CAR virus was injected directly into the humanized mice by intraperitoneal injection, and the expression of CAR molecules on the surface of CD3+ T cells in the humanized mice was detected after 1, 2, and 4 weeks; the flow cytometry results are shown in Figure 6B
  • the humanized mice of the AAV-CD4-CAR treatment group more than 12% of the CD3+ T cells expressed CAR molecules on the surface, while the humanized mice of the control group AAV-CD20-CAR treatment group expressed specific recognition
  • the proportion of CD3+ T cells in the CD4 protein CAR molecule is less than 2%.
  • AAV-CD4-CAR virus can humanize more than 12% of CD3+ T cells in mice into CD4-CAR-T cells that specifically recognize CD4 protein, while the control group AAV-CD20- CAR cannot form CD4-CAR-T cells that specifically recognize CD4 protein in the body.
  • the present invention has successfully constructed a series of AAV-CAR viruses specifically targeting CD4, CCR5, CD19, CD20 and other molecules.
  • the experimental results show that the surface of AAV-CD4-CAR virus-infected cells can express CAR molecules that specifically recognize CD4 protein.
  • AAV-CD4-CAR virus directly infects PBMC, it can form a high proportion of CAR-T cells with specificity Targeted killing of CD4+ T cells and CD4+ tumor cells.
  • AAV-CAR virus can be developed into a universal and efficient new cell therapy.
  • AAV-CAR virus can be widely used in tumor cell therapy by targeting to kill tumor cells. At the same time, it can also achieve cell therapy against virus infection by targeting virus to potentially or latently infected cells.

Landscapes

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

Abstract

提供了一种嵌合抗原受体重组腺相关病毒颗粒，其为携带有嵌合抗原受体基因的重组腺相关病毒颗粒，还提供了该嵌合抗原受体重组腺相关病毒颗粒的应用以及包含有上述嵌合抗原受体重组腺相关病毒颗粒的药物组合物。将装载有嵌合抗原受体的重组腺相关病毒颗粒直接注射体内，在体内该嵌合抗原受体重组腺相关病毒颗粒直接感染T细胞或者NK细胞，在体内直接形成CAR细胞，从而实现直接靶细胞杀伤的细胞治疗。

Description

一种嵌合抗原受体重组腺相关病毒颗粒及其应用 技术领域
本发明涉及嵌合抗原受体技术领域，具体涉及一种嵌合抗原受体重组腺相关病毒及其应用。
背景技术
嵌合抗原受体(Chimeric Antigen Receptor，CAR)修饰的T细胞(CAR-T)免疫疗法是近几年被改良使用到临床上的新型细胞疗法。CAR-T细胞免疫治疗通过采集患者外周静脉血中分离出T细胞，在多种细胞因子诱导下大量扩增，并引入靶向肿瘤抗原的CAR分子。CAR分子的修饰可使T细胞在获得靶向杀伤能力时其肿瘤杀伤作用的发挥不会受到主要组织相容性复合体(MHC)的限制，最后通过静脉或皮内注射等手段将CAR-T细胞回输入患者体内来达到杀伤肿瘤的目的。CAR-T细胞具有持续扩增能力和肿瘤靶向杀伤活性，保证了其对肿瘤细胞造成持续有效的杀伤力，从而使病人得到治愈。目前在急性白血病和非霍奇金淋巴瘤的治疗上，CAR-T细胞治疗有着显著疗效，CAR-T细胞免疫治疗被认为是最有前景的肿瘤治疗方式之一。
然而部分患者由于疾病进展太快，在对T细胞进行基因改造与培养过程中死亡，从而失去CAR-T细胞治疗的机会；另外利用自体T细胞体外扩增，制备自体CAR-T回输进行个性化治疗，大大增加了CAR-T治疗的时间和成本同时也限制了CAR-T治疗的规模。因此，急需开发出可供临床应用的通用型CAR-T细胞或者获得类似的药物，只需要将该药物直接输入人体即可实现CAR-T细胞治疗。
CAR分子与杀伤性T细胞的嵌合需要通过病毒载体转导，方法包括逆 转录病毒系统、慢病毒系统、腺病毒、DNA质粒及RNA转染系统等。目前CAR-T细胞治疗中能达到稳定整合且转染效率高、使用较为广泛的是逆转录病毒及慢病毒。逆转录病毒不易转染静息T细胞；慢病毒可以转染静息T细胞，但成本较高，且两者都存在遗传毒性，有插入致癌的风险，因此急需一种更安全有效的病毒载体装载CAR分子。腺相关病毒(adeno-associated virus，AAV)载体被广泛应用于基因的表达和抗体的体内表达，由于AAV病毒载体以附加体的形式存在于细胞质里，所以不存在插入突变，且能持续很长的表达时间。
发明内容
本发明为解决上述技术问题提供了一种嵌合抗原受体重组腺相关病毒颗粒及其应用。
本发明解决上述技术问题的技术方案如下：一种嵌合抗原受体重组腺相关病毒颗粒，其为携带有嵌合抗原受体基因的重组腺相关病毒载体。
进一步，所述嵌合抗原受体重组腺相关病毒颗粒通过将嵌合抗原受体基因通过腺相关病毒包装系统进行包装得到。
进一步，所述嵌合抗原受体包括细胞外结构域、跨膜结构域和细胞内信号传导结构域。
进一步，所述细胞外结构域为识别抗原结构域的抗体。
进一步，所述识别抗原结构域的抗体为scFV抗体或V HH抗体。
进一步，所述scFV抗体对CD4、CD19、CCR5或CD20是特异性的。
本发明还提供了上述嵌合抗原受体重组腺相关病毒颗粒在制备治疗肿瘤的药物中的应用。
进一步，所述药物为应用于肿瘤免疫疗法的靶向药物。
本发明还提供了一种药物组合物，包括上述嵌合抗原受体重组腺相关病 毒颗粒，以及药学上可接受的载体或赋形剂。
进一步，所述药物组合物的剂型为注射剂。
本发明的有益效果为：本发明利用重组腺相关病毒(AAV病毒)装载嵌合抗原受体(CAR分子)得到的嵌合抗原受体重组腺相关病毒颗粒(AAV-CAR)直接注射体内，在体内AAV-CAR直接感染T细胞或者NK细胞，在体内直接形成CAR细胞，从而实现直接靶细胞杀伤的细胞治疗。本发明里成功的制备了装载不同CAR分子的AAV病毒(AAV-CD4-CAR，AAV-CCR5-CAR，AAV-CD19-CAR和AAV-CD20-CAR)，AAV-CAR能直接感染293TT贴壁细胞和293F悬浮细胞，并在细胞表面展示CAR分子。另外AAV-CAR也能直接感染PBMC细胞，并在40％以上的T细胞表面展示CAR分子，形成CAR-T细胞。该CAR-T细胞可特异性杀伤靶细胞，杀伤效率可达40％以上；另外将制备装载有CAR分子的AAV病毒直接注入小鼠体内，数天后检测发现小鼠体内形成了大量的CAR-T细胞，并且显示特异性杀伤靶细胞。本发明的AAV-CAR可以直接进行体内注射，直接跳过了传统CAR-T细胞治疗所需的血液采集，PBMC体外扩增和病毒体外感染T细胞和CAR-T自体回输等个体化治疗一序列步骤，只需要将嵌合抗原受体重组腺相关病毒颗粒(AAV-CAR)直接注射到患者体内，实现了AAV-CAR通用型批量细胞治疗，大大的节省了治疗前期准备时间和治疗成本以及治疗效率。
附图说明
图1为本发明实施例3中AAV-CD4-CAR分别感染293TT贴壁细胞和293F悬浮细胞后细胞裂解液的Western-bloting结果，其中M为marker，泳道1为AAV-CD4-CAR感染293F细胞裂解液，泳道2为AAV-CD4-CAR感染293F细胞裂解液，泳道3为对照组；
图2为本发明实施例3流式细胞仪鉴定AAV-CD4-CAR感染细胞后细胞表面CAR分子的表达，其中高剂量的AAV-CD4-CAR转染组为AAV-CD4-CAR(H)，低剂量的AAV-CD4-CAR转染组为AAV-CD4-CAR(L)，AAV-GFP转染组为Mock；
图3为本发明实施例3用流式细胞仪鉴定AAV-CD4-CAR感染PBMC后CD3+细胞表面CAR分子的表达，分别用Anti-F(ab’) 2-FITC和Anti-CD4-Fc-FITC检测,其中高剂量的AAV-CD4-CAR转染组为AAV-CD4-CAR(H)，低剂量的AAV-CD4-CAR转染组为AAV-CD4-CAR(L)，AAV-GFP转染组为Mock；
图4为本发明实施例4流式检测AAV-CD4-CAR直接感染PBMC后针对CD4+T细胞的特异性杀伤，其中图4A为流式分析示意图，图4B为AAV-CD4-CAR处理不同来源的PBMC后，CD4+T细胞的比例；图4C为AAV-CD4-CAR处理不同来源的PBMC后，CD8+T细胞的比例，以对照组AAV-GFP处理组的值进行标准化，设定为100％；
图5为本发明实施例4流式检测AAV-CD4-CAR对肿瘤细胞的杀伤，图5A为流式分析示意图，图5B为AAV-CD4-CAR直接感染PBMC细胞后针对MT-2和Jurkat肿瘤细胞的杀伤比例，以Mock组肿瘤细胞的非特异性杀伤的比例标准化为0，AAV-CD4-CAR处理组特异性细胞杀伤数据是经过参照对照组进行标准化处理后所得；
图6为本发明实施例5人源化小鼠体内鉴定AAV-CD4-CAR病毒直接诱导的特异性靶向杀伤，图6A为AV-CD4-CAR病毒处理前人源化小鼠体内的CD3+CD4+T细胞的实际比例，图6B为流式检测AAV-CD4-CAR直接感染人源化小鼠2周后，人源CD3+T细胞表面特异性识别CD4蛋白的CAR分子表达，图6C为流式检测不同时间点人源化小鼠体内CD3+CD4+T细胞比例，图6D为不同处理组人源化小鼠体重的监测。
具体实施方式
以下结合附图及具体实施例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
发明人在前期工作中发现，腺相关病毒(adeno-associated virus，AAV)在体内至少可以持续表达9周以上，另外，clinicaltrials网站显示，目前有178个AAV载体相关的临床试验，至今没有严重副作用的报道，因此选择AAV病毒载体对嵌合抗原受体(CAR)进行装载得到嵌合抗原受体重组腺相关病毒颗粒(AAV-CAR)。
实施例1利用AAV病毒载体装载CAR分子
1、pAAV-CAR质粒构建
通过PubMed以及HIV database网站获得特异性针对CD19、CD4、CCR5和CD20的scFv(VL-VH)抗体序列和第三代CAR的铰链区、跨膜结构域和细胞内信号传导结构域序列。其中
CD19的scFv(VL-VH)抗体的氨基酸序列如SEQ ID NO:1所示：
Figure PCTCN2020091185-appb-000001
其中序列SEQ ID NO:1的第1-107位氨基酸为轻链可变区，108-122位氨基酸为连接肽，123-243位氨基酸为重链可变区。
CD4的scFv(VL-VH)抗体的氨基酸序列如SEQ ID NO:2所示：
Figure PCTCN2020091185-appb-000002
Figure PCTCN2020091185-appb-000003
其中序列SEQ ID NO:2的第1-112位氨基酸为轻链可变区，113-127位氨基酸为连接肽，128-249位氨基酸为重链可变区。
CCR5的scFv(VL-VH)抗体的氨基酸序列如SEQ ID NO:3所示：
Figure PCTCN2020091185-appb-000004
其中序列SEQ ID NO:3的第1-92位氨基酸为轻链可变区，93-107位氨基酸为连接肽，108-229位氨基酸为重链可变区。
CD20的scFv(VL-VH)抗体的氨基酸序列如SEQ ID NO:4所示
Figure PCTCN2020091185-appb-000005
其中序列SEQ ID NO:4的第1-107位氨基酸为轻链可变区，108-122位氨基酸为连接肽，123-243位氨基酸为重链可变区。
CAR的铰链区、跨膜区和细胞内信号传导结构域的选择属于本领域的常规选择，本实施例的CAR铰链区、跨膜结构域和细胞内信号传导结构域的结构为：CD28-4-1BB-CD3zeta，其核苷酸序列如SEQ ID NO:5所示：
Figure PCTCN2020091185-appb-000006
人工合成编码SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的CD19的scFv(VL-VH)抗体、CD4的scFv(VL-VH)抗体、CCR5的scFv(VL-VH)抗体和CD20的scFv(VL-VH)抗体的核苷酸序列，其核苷酸序列分别为SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示。
SEQ ID NO:6
Figure PCTCN2020091185-appb-000007
Figure PCTCN2020091185-appb-000008
SEQ ID NO:7
Figure PCTCN2020091185-appb-000009
Figure PCTCN2020091185-appb-000010
SEQ ID NO:8
Figure PCTCN2020091185-appb-000011
SEQ ID NO:9
Figure PCTCN2020091185-appb-000012
分别将SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9所示的scFv(VL-VH)抗体基因和CD28-4-1BB-CD3zeta基因酶切连接到pAAV-MCS质粒载体(Cell Biolabs,San Diego,USA)上，最终在pAAV-MCS质粒载体上装载一个scFv(VL-VH)-CD28-4-1BB-CD3zeta的单基因CAR分子，构建得到pAAV-CAR(分别为pAAV-CD19-CAR，pAAV-CD4-CAR和pAAV-CCR5-CAR和pAAV-CD20-CAR)。
2、AAV-CAR病毒包装
AAV Helper-Free病毒包装系统购于Cell Biolabs,San Diego USA。将上述pAAV-CAR与pHelper和pAAV-DJ质粒按照质量比1:1:1的方式使用PEI转染试剂共转染AAV-293T细胞。转染后分别于48、72、96和120小时收集上清，并用5x PEG8000(sigma)进行浓缩，最后用1.37g/mL氯化铯密度梯度离心进行纯化，得到装载不同CAR分子的AAV-CAR病毒颗粒，即嵌合抗原受体重组腺相关病毒颗粒(AAV-CAR),包括AAV-CD4-CAR,AAV-CD19-CAR,AAV-CCR5-CAR,和AAV-CD20-CAR，纯化的AAV-CAR病毒颗粒溶解于PBS里，进行鉴定和分装后保存于-80℃。
实施例2 AAV-CAR病毒滴度定量
纯化的AAV-CAR通过Q-PCR进行拷贝数测定，DNaseI先酶切预处理纯化的AAV-CAR，应用CMV enhancer的引物(AAV-Mono-CMV-F和AAV-Mono-CMV-R)测定AAV-CAR病毒的拷贝，其它的试剂来源于SYBR Premix Ex Taq II(Takara)试剂盒。样品在7300仪器上进行三个复孔检测。实时PCR的循环条件如下:50℃2min一个循环，95℃10min一个循环，95℃15s和60℃60s 40个循环。病毒的拷贝数通过AAV-GFP质粒所做的标准曲线来计算得出,最终获得1x10^13gc/mL的AAV-CAR病毒。
其中，AAV-Mono-CMV-F的序列如SEQ ID NO:10所示：
Figure PCTCN2020091185-appb-000013
AAV-Mono-CMV-R的序列如SEQ ID NO:11所示：
Figure PCTCN2020091185-appb-000014
实施例3 AAV-CAR感染细胞
用不同浓度的AAV-CAR病毒液直接添加到不同的细胞(293TT细胞、293F细胞和PBMC细胞)，培养24h后，通过Western-Blot和流式检测等方法检测CAR分子的表达。
以特异性针对CD4蛋白的AAV-CD4-CAR病毒颗粒感染293TT贴壁细 胞和293F悬浮细胞为例，对照组采用AAV-GFP病毒颗粒感染293TT细胞，感染24h后，收集并裂解细胞，利用鼠抗人CD3zeta抗体，通过Western-Blot检测CAR分子在细胞裂解液中的表达，结果如图1所示，用AAV-CD4-CAR病毒颗粒感染的393TT细胞和293F细胞的细胞裂解液中均有一条74kD大小的特异性条带，对照组并没有该条带，表明AAV-CD4-CAR成功的感染了293TT细胞和293F细胞，并在这两个细胞内大量表达了CD4-CAR分子。
为了进一步验证AAV-CD4-CAR感染贴壁细胞和悬浮细胞后是否在细胞表面表达CAR分子，分别用低剂量的AAV-CD4-CAR(L)(1x10^4gc/cell)和高剂量的AAV-CD4-CAR(H)(1x10^5gc/cell)感染293TT贴壁细胞和293F悬浮细胞，对照组(Mock)采用AAV-GFP,利用特异性识别CAR分子VL-VH的Anti-F(ab’) 2-FITC的抗体进行流式细胞检测293TT贴壁细胞和293F悬浮细胞被感染后细胞表面CAR分子的表达，结果如图2所示，在低剂量AAV-CD4-CAR(L)感染的293TT细胞和293F细胞表面，能检测到大于20％的阳性细胞表达CAR分子；在高剂量AAV-CD4-CAR(H)处理组，大于50％的293TT和293F细胞表面表达CAR分子，说明AAV-CD4-CAR能直接感染贴壁细胞和悬浮细胞，并在细胞表面展示CAR分子。
为了进一步验证AAV-CD4-CAR能否感染PBMC细胞，并在CD3+T细胞表面展示CAR分子，最终形成CAR-T细胞，分别用低剂量的AAV-CD4-CAR(L)(1x10^4gc/cell)和高剂量的AAV-CD4-CAR(H)(1x10^5gc/cell)感染PBMC细胞，对照组(Mock)采用AAV-GFP，用特异性识别CAR分子VL-VH的Anti-F(ab’) 2-FITC的抗体进行流式细胞检测PBMC细胞表面CAR分子的表达，结果如图3所示，低剂量AAV-CD4-CAR(L)能使28％的T细胞形成CAR-T，高剂量AAV-CD4-CAR(H)感染PBMC能使49％的T细胞变为CAR-T。为了进一步验证AAV-CD4-CAR感染PBMC形成的CAR-T细胞能特异性识别CD4蛋白，利用Anti-CD4-Fc-FITC进行流 式检测，流式结果如图3所示，被AAV-CD4-CAR直接感染后的PBMC中CD3+T细胞能特异性结合CD4蛋白；高低剂量AAV-CD4-CAR处理组，识别CD4蛋白的CD3+T细胞的阳性率分别为75％和31％，阳性率数据与Anti-F(ab’) 2-FITC的抗体检测结果大致相似。提示AAV-CD4-CAR直接感染PBMC后，能形成高比例的CAR-T细胞，并且这些CAR-T细胞能特异性识别CD4蛋白。
实施例4 AAV-CAR体外细胞功能鉴定
通过AAV-CAR构建成功的CAR-T细胞的细胞杀伤实验依据radioisotope-free luminescence-based CytoTox-Glo kit(Promega)试剂盒的操作流程。该试剂盒通过检测蛋白酶活性来检测杀伤的死细胞。实验流程简述如下：在96孔板里2倍梯度稀释AAV-CAR转导后的PBMC，最高的细胞数为每孔2.0x10 6，根据AAV-CAR PBMC细胞：靶细胞50:1的比例加入相应体积的靶细胞(靶细胞可以是表达CD4，CD19,CD20或者CCR5的肿瘤细胞，也可以是CD4+T细胞和CD19+B细胞)。细胞于37℃5％CO 2共孵育4小时；CytoTox-Glo reagent加到每个孔，室温15分钟后，通过仪器检测荧光值；以含靶细胞而无效应CAR-T细胞的细胞裂解液读值作为100％的裂解杀伤。
将AAV-CD4-CAR感染不同来源的PBMC，48小时后，检测PBMC里CD3+CD4+T细胞的比例，流式结果示意图如图4A所示。CD3+CD4+CD8-T为CD4+T细胞。CD4+T细胞的流式细胞结果如图4B所示，与阴性对照相比，AAV-CD4-CAR处理的PBMC细胞里的CD4+T细胞显著减少，并且成浓度梯度依赖，高剂量AAV-CD4-CAR处理的CD4+T细胞的数量低于低剂量AAV-CD4-CAR处理组的CD4+T细胞数量；提示AAV-CD4-CAR剂量越高，对CD4+T特异性杀伤能力越强；高剂量AAV-CD4-CAR直接感染6个不同来源的PBMC细胞后，CD4+T细胞减少的比例为50％左右，说明通过 AAV-CD4-CAR直接感染PBMC能在不同的个体内特异性靶向杀伤CD4+T细胞，并且呈现剂量依赖，CD8+T细胞的流式细胞结果如图4C所示，6个不同来源的PBMC里CD8+T细胞不仅没出现减少，个别PBMC细胞的CD8+T细胞还出现了上升的趋势，以上结果说明AAV-CD4-CAR直接感染PBMC能特异性杀伤CD4+T细胞，而不会非特异性杀伤CD8+T细胞。
为了进一步验证AAV-CD4-CAR直接感染PBMC后能特异性杀伤CD4+肿瘤细胞，先将不同剂量的AAV-CD4-CAR(高剂量AAV-CD4-CAR组(H)和低剂量AAV-CD4-CAR组(L))直接感染PBMC细胞，对照组(Mock组)采用AAV-GFP，3天后按照1:1的细胞比例添加CD4+MT2或者CD4+Jurkat肿瘤细胞；共同孵育48小时后通过流式检测特异性死亡的MT-2细胞或者Jurkat；流式结果如图5所示，与对照组相比，AAV-CD4-CAR直接感染的PBMC能显著性的特异性杀伤CD4+MT2细胞和Jurkat细胞，减去非特异性杀伤，特异性杀伤的比例为25％，当提高感染AAV-CD4-CAR的剂量时，MT2细胞和Jurkat细胞被特异性杀伤比例也随之提高，最高特异性MT2细胞杀伤比例达到40％以上，以上结果说明AAV-CD4-CAR直接感染PBMC后，能形成特异性识别CD4蛋白的CAR-T，从而特异性靶向杀伤CD4肿瘤细胞，并且呈剂量依赖性的高效特异性靶向杀伤。
实施例5 AAV-CCR体内效果评估
1、构建人源化小鼠的肿瘤模型
Immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NCG)小鼠购自南京大学模式动物所，与NSG小鼠类似，该小鼠在SCID小鼠基础上缺失了IL2受体基因，导致体内没有小鼠T细胞，B细胞以及很少量的NK细胞。1.5x10 7PBMC腹腔注射进4-6周的NCG小鼠体内；三周后，采血流式检测人的T细胞，通过染色人的CD45+,CD3+,CD4+和CD8+，人的CD45阳性细胞的比例到达10％以上，判定为小鼠人源化成功。结果所有的人源化小鼠体内 均含有大于10％的人CD45+细胞，其中小鼠体内人CD4+T细胞的实际比例如图6A所示，，人CD4+T细胞比例多达20％以上，接种肿瘤细胞，连续四周监测肿瘤的大小。
2、人源化小鼠体内验证AAV-CD4-CAR治疗肿瘤的疗效
不同浓度的AAV-CAR腹腔注射到接种肿瘤的人源化小鼠体内，不同时间点采血检测；流式细胞仪检测人CD4+T细胞的变化趋势；同时监测小鼠的体重。
通过腹腔直接注射AAV-CD4-CAR病毒至人源化小鼠体内1，2和4周后分别检测人源化小鼠体内CD3+T细胞表面CAR分子的表达；流式结果如图6B所示，AAV-CD4-CAR处理组的人源化小鼠体内大于12％的CD3+T细胞表面表达CAR分子，而对照组AAV-CD20-CAR处理组的人源化小鼠体内，表达特异性识别CD4蛋白CAR分子的CD3+T细胞的比例小于2％。由此可见直接注射AAV-CD4-CAR病毒可以让人源化小鼠体内的大于12％的CD3+T细胞变成特异性识别CD4蛋白的CD4-CAR-T细胞，而对照组AAV-CD20-CAR则不能在体内形成特异性识别CD4蛋白CD4-CAR-T细胞。
为了进一步验证,AAV-CD4-CAR直接感染小鼠后形成的CAR-T细胞能否特异性杀伤体内人CD4+T细胞，在不同的时间点检测人的CD4+T细胞比例。流式检测结果如图6C所示，与对照组相比，AAV-CD4-CAR处理组的所有小鼠在处理1周后CD4+T均出现了下降，2周后小鼠体内人CD4+T细胞下降比例大于50％，4周后，CD4+T细胞的比例会进一步降低，而对照组AAV-CD20-CAR小鼠体内的CD4+T细胞没有观察到明显的下降。
以上结果提示，通过直接向人源化小鼠体内注射AAV-CD4-CAR病毒，能直接形成高比例的特异性识别CD4蛋白的CAR-T细胞，并且能达到高效靶向杀伤CD4+靶细胞的作用。通过监测AAV-CAR处理后不同时间点的体重变化，结果如图6D所示，AAV-CD4-CAR病毒直接感染后的小鼠体重并 未出现明显的下降，说明AAV-CD4-CAR的病毒感染未对小鼠引起明显的副作用。
综上所述，本发明成功构建了一系列特异性靶向CD4，CCR5，CD19，CD20等分子的AAV-CAR病毒。实验结果显示AAV-CD4-CAR病毒感染的细胞表面能表达特异性识别CD4蛋白的CAR分子，另外AAV-CD4-CAR病毒直接感染PBMC后，能形成高比例的CAR-T细胞，并能特异性靶向杀伤CD4+T细胞和CD4+肿瘤细胞。体内实验结果进一步证实，直接向人源化小鼠体内注射AAV-CD4-CAR病毒，2周后，大于12％的T细胞会成为CAR-T细胞，并且在体内可以高效靶向杀伤CD4+靶细胞，CD4靶细胞的减少比例大于50％以上，并且随着时间延长会进一步降低。以上结果提示，向体内注射的AAV-CAR病毒可以直接感染体内PBMC细胞并在体内形成CAR-T细胞，而CAR-T细胞可以实现特异性杀伤靶细胞。与传统的CAR-T细胞治疗相比，该方法无需进行血液收集，分离PBMC，体外扩增T细胞和体外制备CAR-T以及回输本体等复杂的流程，我们只需要将AAV-CAR病毒直接注射体内即可实现CAR-T细胞治疗。因此AAV-CAR病毒可以发展成为一种通用型的高效新型细胞治疗。AAV-CAR病毒可以通过靶向杀伤肿瘤细胞广泛的应用于肿瘤细胞治疗，同时也可以通过靶向杀伤病毒潜在或潜伏感染细胞达到针对病毒感染的细胞治疗。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种嵌合抗原受体重组腺相关病毒颗粒，其特征在于，为携带有嵌合抗原受体基因的重组腺相关病毒颗粒。
  2. 根据权利要求1所述的一种嵌合抗原受体重组腺相关病毒颗粒，其特征在于，将嵌合抗原受体基因通过腺相关病毒包装系统进行包装得到。
  3. 根据权利要求1所述的一种嵌合抗原受体重组腺相关病毒颗粒，其特征在于，所述嵌合抗原受体基因编码的嵌合抗原受体包括细胞外结构域、跨膜结构域和细胞内信号传导结构域。
  4. 根据权利要求3所述的一种嵌合抗原受体重组腺相关病毒颗粒，其特征在于，所述细胞外结构域为识别抗原结构域抗体。
  5. 根据权利要求4所述的一种嵌合抗原受体重组腺相关病毒颗粒，其特征在于，所述识别抗原结构域的抗体为scFV抗体或V HH抗体。
  6. 根据权利要求5所述的一种嵌合抗原受体重组腺相关病毒颗粒，其特征在于，所述scFV抗体对CD4、CD19、CCR5或CD20是特异性的。
  7. 一种如权利要求1-6任一项所述的嵌合抗原受体重组腺相关病毒颗粒在制备治疗肿瘤的药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述药物为应用于肿瘤免疫疗法的靶向药物。
  9. 一种药物组合物，其特征在于，包括权利要求1-6任一项所述的嵌合抗原受体重组腺相关病毒颗粒，以及药学上可接受的载体或赋形剂。
  10. 根据权利要求9所述的药物组合物，其特征在于，所述药物组合物的剂型为注射剂。
PCT/CN2020/091185 2019-03-22 2020-05-20 一种嵌合抗原受体重组腺相关病毒颗粒及其应用 WO2020192803A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910224056.8 2019-03-22
CN201910224056.8A CN111718957A (zh) 2019-03-22 2019-03-22 一种嵌合抗原受体重组腺相关病毒颗粒及其应用

Publications (1)

Publication Number Publication Date
WO2020192803A1 true WO2020192803A1 (zh) 2020-10-01

Family

ID=72563750

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/091185 WO2020192803A1 (zh) 2019-03-22 2020-05-20 一种嵌合抗原受体重组腺相关病毒颗粒及其应用

Country Status (2)

Country Link
CN (1) CN111718957A (zh)
WO (1) WO2020192803A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114645066B (zh) * 2020-12-21 2023-08-25 康霖生物科技（杭州）有限公司 一种用于艾滋病基因治疗的核酸构建体

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104177499A (zh) * 2013-05-27 2014-12-03 张鸿声 一种嵌合抗原受体、编码基因、表达载体及其应用
US20160130357A1 (en) * 2010-10-08 2016-05-12 The University Of North Carolina At Charlotte Compositions comprising chimeric antigen receptors, t cells comprising the same, and methods of using the same
CN105949317A (zh) * 2016-04-12 2016-09-21 上海优卡迪生物医药科技有限公司 抗cd20嵌合抗原受体、编码基因、重组表达载体及其构建方法和应用
WO2018057904A1 (en) * 2016-09-23 2018-03-29 University Of Southern California Chimeric antigen receptors and compositions and methods of use thereof
CN109111525A (zh) * 2018-05-24 2019-01-01 卢英 一种hla-g嵌合抗原受体、编码序列和表达载体以及应用
CN109825526A (zh) * 2019-02-15 2019-05-31 北京门罗生物科技有限公司 一种用于通用型car-t制备的重组腺相关病毒载体及其构建方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8518405B2 (en) * 2009-10-08 2013-08-27 The University Of North Carolina At Charlotte Tumor specific antibodies and uses therefor

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160130357A1 (en) * 2010-10-08 2016-05-12 The University Of North Carolina At Charlotte Compositions comprising chimeric antigen receptors, t cells comprising the same, and methods of using the same
CN104177499A (zh) * 2013-05-27 2014-12-03 张鸿声 一种嵌合抗原受体、编码基因、表达载体及其应用
CN105949317A (zh) * 2016-04-12 2016-09-21 上海优卡迪生物医药科技有限公司 抗cd20嵌合抗原受体、编码基因、重组表达载体及其构建方法和应用
WO2018057904A1 (en) * 2016-09-23 2018-03-29 University Of Southern California Chimeric antigen receptors and compositions and methods of use thereof
CN109111525A (zh) * 2018-05-24 2019-01-01 卢英 一种hla-g嵌合抗原受体、编码序列和表达载体以及应用
CN109825526A (zh) * 2019-02-15 2019-05-31 北京门罗生物科技有限公司 一种用于通用型car-t制备的重组腺相关病毒载体及其构建方法和应用

Also Published As

Publication number Publication date
CN111718957A (zh) 2020-09-29

Similar Documents

Publication Publication Date Title
Sivanandam et al. Oncolytic viruses and immune checkpoint inhibition: the best of both worlds
WO2021179353A1 (zh) 靶向nkg2d的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用
CN108409840B (zh) 抗cd123单链抗体及其组合的嵌合抗原受体和应用
CN111675765B (zh) 靶向冠状病毒spike的武装嵌合抗原受体细胞及制备方法和应用
WO2019154391A1 (zh) 嵌合抗原受体、表达该嵌合抗原受体的nkg2d car-nk细胞及其制备方法和应用
CN111690050B (zh) 识别ebv-lmp2抗原的tcr及相应的核酸分子、载体、细胞和药物
WO2018006881A1 (zh) 重组免疫检查点受体及其应用
CN109836496A (zh) 一种靶向cd317的单链抗体、嵌合抗原受体t细胞及其制备方法和应用
CN113416260B (zh) 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用
CN107586341A (zh) 重组免疫检查点受体及免疫检查点抑制分子的共表达及应用
WO2015148879A1 (en) Cancer immunotherapy compositions and methods
CN108753773A (zh) 干扰IFN-gama表达的CD19-CAR-T细胞及其应用
CN110526970A (zh) 靶向cd133的单链抗体、嵌合抗原受体t细胞及其制备方法和应用
CN109957018A (zh) 一种靶向cd22的单链抗体、嵌合抗原受体t细胞及其制备方法和应用
WO2020192803A1 (zh) 一种嵌合抗原受体重组腺相关病毒颗粒及其应用
CN108342360A (zh) 共表达抗msln嵌合抗原受体和无功能egfr的转基因淋巴细胞及其用途
CN111549044B (zh) 靶向trbc1 car-t细胞的制备方法与应用
CN115667317A (zh) 全人源的靶向cd19和cd22双特异抗原嵌合受体及其应用
CN109957020A (zh) 一种靶向dr5的单链抗体、嵌合抗原受体t细胞及其制备方法和应用
CN106967684A (zh) 共表达抗EGFRvIII嵌合抗原受体和无功能EGFR受体的转基因淋巴细胞及其用途
CN110526987A (zh) 靶向cd133的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用
CN109957025A (zh) 一种靶向dr5的单链抗体、嵌合抗原受体t细胞及其制备方法和应用
CN109957546A (zh) 一种靶向cd317的嵌合抗原受体t细胞及其制备方法和应用
CN113646426A (zh) 一种包含肿瘤抗原识别受体的免疫细胞及其应用
WO2020025039A1 (zh) 表达嵌合抗原受体的t细胞、该嵌合抗原相关表达载体以及它们的应用

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: 20776560

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: 20776560

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20776560

Country of ref document: EP

Kind code of ref document: A1