WO2020164167A1 - Vecteur viral adéno-associé recombiné destiné à être utilisé dans la préparation de cellules car-t universelles, son procédé de construction et son utilisation - Google Patents

Vecteur viral adéno-associé recombiné destiné à être utilisé dans la préparation de cellules car-t universelles, son procédé de construction et son utilisation Download PDF

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WO2020164167A1
WO2020164167A1 PCT/CN2019/075717 CN2019075717W WO2020164167A1 WO 2020164167 A1 WO2020164167 A1 WO 2020164167A1 CN 2019075717 W CN2019075717 W CN 2019075717W WO 2020164167 A1 WO2020164167 A1 WO 2020164167A1
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sequence
associated virus
car
recombinant adeno
virus vector
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芦志华
朱滨
董明洁
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北京门罗生物科技有限公司
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    • 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
    • A61K39/461—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
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/30—Mixture of cells

Definitions

  • the present invention relates to the field of biomedicine, in particular to a recombinant adeno-associated virus vector used for CAR-T preparation and its construction method and application.
  • CAR-T uses antibody fragments that can bind to specific antigens to recognize antigens on the surface of tumor cells.
  • CD19 antigen-specific CAR-T cells have been used in clinical trials for the treatment of B-cell leukemia and lymphoma, and have shown sustained disease relief effects.
  • Chimeric antigen receptors (CAR) endow T cells with the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-modified T cells to recognize a wider range of targets than the natural T cell surface receptor TCR.
  • CAR-T technology has had significant effects in the treatment of acute leukemia and non-Hodgkin’s lymphoma, and is considered to be one of the most promising tumor treatment methods.
  • CAR-T treatment is as follows: through genetic engineering modification, the T cells of cancer patients isolated and collected in vitro express chimeric antigen receptors (CAR) that recognize a single tumor antigen, and after a large number of CAR-T cells are expanded in vitro It is returned to cancer patients for cellular immunotherapy.
  • CAR as a chimeric protein expressed by genes, contains the antigen-binding domain of an antibody (eg, single-chain antibody scFv) connected to the T cell signaling domain.
  • an antibody eg, single-chain antibody scFv
  • the CAR-T cell adoptive immunotherapy system uses genetic modification of the patient's own T cells, and uses the principle of antigen-antibody binding to circumvent the MHC-restricted antigen presentation, thereby achieving precise targeting. At the same time, it overcomes the possible immune escape of tumor cells by down-regulating the expression of MHC molecules to reduce antigen presentation.
  • CAR-T cancer-associated antigen
  • CAR-T can achieve very good curative effects in the treatment of blood system diseases, it can only be autologously infused, that is, T cells are extracted from the patient, genetically modified and amplified, and then returned to the patient. Therefore, this treatment cannot be used as widely as drugs.
  • Some patients cannot undergo CAR-T reinfusion because they cannot obtain a sufficient number of T cells, thus losing the possibility of treatment.
  • Plasmid vectors express genes or DNA sequences at multiple cloning sites into proteins in cells. Plasmid vector DNA is artificially modified and contains other DNA components such as gene sequences for expressing antibiotics, promoter sequences, multiple cloning sites (MCS), etc., and it can be genetically engineered with others
  • the helper plasmid is automatically assembled in engineered cells (such as 293T cells) into a lentivirus capable of infection.
  • Such a lentivirus has the function of expressing CAR protein in cells. Adding such a lentivirus to the culture medium for culturing T cells can infect T cells, that is, enter the T cells, and then use the elements in the T cells to express CAR proteins. After these CAR proteins are expressed, they will be anchored in the T cells. surface.
  • virus-mediated gene expression technologies are usually used, such as lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV), etc.
  • lentiviruses and retroviruses are inserted into the genome randomly after entering cells, and adenoviruses and adeno-associated viruses also have a certain probability. Inserting into the genome may destroy the genes in the cell, resulting in cell abnormalities, and may even transform the cell into a tumor cell, which in turn causes tumors. Therefore, using these viral technologies to prepare CART for cellular immunotherapy has the risk of causing tumors.
  • the present invention relates to a recombinant adeno-associated virus vector
  • the adeno-associated virus vector contains the following operatively linked sequence elements: 5'-end inverted repeat sequence, 3'-end inverted repeat sequence and sequence encoding CAR gene .
  • the sequence encoding the CAR gene is CD19CAR (4-1BB).
  • the adeno-associated virus vector may further include the following operatively linked sequence elements: SA sequence, 2A sequence, polyA sequence, 5'genome homologous sequence (5'HA), 3'genome homologous sequence (3' HA).
  • the 5'HA sequence includes the sequence shown in SEQ ID NO:1;
  • the SA sequence includes the sequence shown in SEQ ID NO: 2;
  • the 2A sequence includes the sequence shown in SEQ ID NO: 3;
  • the CD19CAR (4-1BB) sequence includes the sequence shown in SEQ ID NO: 4;
  • the polyA sequence includes the sequence shown in SEQ ID NO: 5;
  • the 3’HA sequence includes the sequence shown in SEQ ID NO: 6
  • the 5'end inverted repeat sequence includes the sequence shown in SEQ ID NO: 7
  • the 3'end inverted repeat sequence includes the sequence shown in SEQ ID NO: 8.
  • the recombinant adeno-associated virus vector comprises nucleotides having at least about 70%, at least about 80%, at least about 90% sequence identity or more sequence identity with the sequence shown in SEQ ID NO: 9 sequence.
  • the present invention also relates to a method for preparing the recombinant adeno-associated virus vector.
  • the method includes the following steps: providing a packaging cell line of the aforementioned viral vector; and recovering the recombinant AAV virus from the supernatant of the packaging cell line.
  • the present invention also relates to a recombinant adeno-associated virus, which is obtained by packaging any of the aforementioned recombinant adeno-associated virus vectors.
  • the present invention also relates to a method for expressing CAR genes, the method comprising providing a nucleotide sequence comprising any of the foregoing recombinant adeno-associated virus (AAV); combining with CRISPR/cas9 gene editing technology, The AAV homologously recombines into the genome of the T cell, and the AAV expresses the CAR gene in the T cell.
  • AAV adeno-associated virus
  • the present invention also relates to the application of any of the aforementioned recombinant adeno-associated virus vectors and recombinant adeno-associated viruses in the preparation of CAR-T cells or anti-tumor drugs.
  • the present invention also relates to a method for preparing CAR-T cells of the aforementioned recombinant adeno-associated virus vector, which is characterized in that the method includes the following steps:
  • the first step is to construct any of the aforementioned recombinant adeno-associated virus vectors
  • the second step is virus packaging
  • the third step is T cell isolation, activation and amplification, CRISPR/cas9 gene editing, AAV virus-mediated gene recombination;
  • Any of the aforementioned recombinant adeno-associated virus vectors are expressed on T cells isolated and collected from the peripheral blood of cancer patients or healthy people through gene editing methods to obtain CAR-T cells.
  • the present invention also relates to CAR-T cells prepared by the above method.
  • the present invention also relates to a kit, which contains any of the foregoing recombinant adeno-associated virus vector, recombinant adeno-associated virus, or the CAR-T cell.
  • the invention also relates to the application of the CAR-T cell or the kit in the preparation of anti-tumor drugs.
  • this application uses gene editing technology, combined with gene recombination technology, from the expression
  • the structure of the AAV vector has been improved, so as to achieve the targeted and precise integration of CAR gene fragments, ensuring the continuous and stable expression of the CAR gene, and avoiding the risk of causing tumors.
  • the expression of CAR is under the control of the endogenous promoter, so that the expression of CAR is regulated by normal physiology, significantly reducing the side effects of treatment, and obtaining a physiological universal CAR-T.
  • the AAV vector with improved structure obtained in the present invention is non-pathogenic
  • the general-purpose T cells and general-purpose CAR-T cells of the invention can be applied to the treatment of malignant tumors or infectious diseases by allogeneic reinfusion, which greatly reduces the treatment cost;
  • the prepared CAR is integrated before the exon of the TCR constant region and is regulated by an endogenous promoter, ensuring that the expression of CAR is physiologically regulated and the expression is uniform. Due to this physiological and uniformity, CAR will not be overexpressed, and the expression level of CAR is consistent with the expression level of the original TCR, and the individual differences between CAR-T cells are small;
  • CAR is accurately integrated into the designated TCR constant region gene through homologous recombination, rather than randomly integrated into the genome of T cells, so there will be no triggering The risk of tumors.
  • operably linked refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • the “element” refers to a series of functional nucleic acid sequences useful for protein expression.
  • the “element” is systematically constructed to form an expression construct.
  • the sequence of the “element” may be those provided in the present invention, and also include their variants, as long as these variants basically retain the function of the "element” by inserting or deleting some bases (such as 1-50bp; preferably 1-30bp, more preferably 1-20bp, more preferably 1-10bp), or by random or site-directed mutagenesis.
  • Adeno-associated virus (adeno-associated virus, AAV) vector is a vector that can be artificially modified by genetic engineering using certain characteristics of naturally occurring adeno-associated virus.
  • Adeno-associated virus (AAV) is a virus that cannot replicate itself and has low immunogenicity.
  • serotypes of AAV There are currently about 10 serotypes of AAV, and different serotypes of AAV can selectively target different tissues.
  • the loading capacity of AAV virus vectors is limited, not exceeding 5.0 kb.
  • variants of the element described above that have been appropriately changed and still retain their original functions are also included in the present invention.
  • the full-length nucleotide sequence of the gene pointed to by each element of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences.
  • the upstream and downstream positions of the aforementioned elements in the vector may also include restriction enzyme cleavage sites, which facilitates the organic connection of the elements.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.
  • the vector containing the above-mentioned appropriate polynucleotide sequence and appropriate promoter or control sequence can be used for virus packaging.
  • the present invention also provides a kit containing the recombinant adeno-associated virus vector expressing CAR or a virus packaged by the vector.
  • Other reagents commonly used for virus packaging, transfection, injection, etc. can also be included in the kit for the convenience of those skilled in the art.
  • the kit may also include instructions for instructing those skilled in the art to operate.
  • Figure 1- Figure 3 are schematic diagrams of the three plasmid structures, in order: AAV6-TCR-CD19CAR (4-1BB) (experimental vector), AAV6-TCR-GFP (negative control), LV-EF-1a-CD19CAT (4- 1BB)-mCherry (positive control);
  • Figure 4 A diagram of the gene-edited AAV6-TCR-CD19CAR(4-1BB) plasmid structure
  • FIG. 5 Flow cytometry results of TCR knockout of T cells from two different individuals after gene editing
  • Figure 6 Flow cytometry results of CD19CAR (4-1BB) homologous recombination mediated by AAV6;
  • Figure 7 Flow cytometric test results of tumor killing ability of AAV6-TCR-CD19CAR-T;
  • Figure 8 Detection and comparison results of cytokine concentrations at different time points, where the values of the bars from left to right in the bar chart represent Kmix, LV, AAV, LV+Kmix, AAV+Kmix, respectively;
  • Figure 9 Flow cytometric detection and comparison results of the failure markers of CAR-T cells, where the upper broken line in the broken line chart is LV CD19 CAR-T+Kmix, and the lower broken line is AAV CD19 CAR-T+Kmix;
  • Figure 10a, b CAR-T anti-tumor in vivo experimental results, where Figure 10b is the survival curve, lentivirus-transduced LV CAR-T and AAV-transduced AAV-TCR-CAR-T both significantly prolong the survival time of mice , There is no significant difference between the two groups.
  • the plasmid constructed in this example includes
  • AAV6-TCR-CD19CAR(4-1BB) (experimental carrier, structure diagram is shown in Figure 1, 4)
  • AAV6-TCR-GFP negative control, structure diagram is shown in Figure 2
  • the functional region of the plasmid LTR-EF-1a-CD19CAT(4-1BB)-mCherry-LTR for gene synthesis.
  • This sequence was cloned into the lentiviral vector plasmid.
  • the structure of the plasmid functional region is: ITR-5’HA-SA-2A-GFP-polyA-3’HA-ITR
  • GFP primers were designed, and GFP fragments were obtained by PCR.
  • Poisonous plasmids include: pSLQ5367, pCMV-dR8.91, pMD2-G, Reagent, Opti MEM.
  • the specific ratio is in accordance with the 10-cm plate in Table 1.
  • AAV virus collection virus particles exist in both packaging cells and culture supernatant.
  • PBMC peripheral blood mononuclear cells
  • step 2) Place the blood sample in step 2) on the upper layer of the solution in step 1), minimize the mixing of blood and Ficoll-PaqueTM PLUS, and centrifuge at 400g for 30 minutes at room temperature, and then slow down naturally;
  • step 3 Discard the upper plasma after centrifugation in step 3, and take the boundary layer between the plasma and Ficoll-PaqueTM PLUS solution is the peripheral blood mononuclear cells (the tube is divided into four layers after centrifugation, from top to bottom in turn are plasma, Peripheral blood mononuclear cells, Ficoll-PaqueTM PLUS fluid, red blood cell and granulocyte layer).
  • the peripheral blood mononuclear cells the tube is divided into four layers after centrifugation, from top to bottom in turn are plasma, Peripheral blood mononuclear cells, Ficoll-PaqueTM PLUS fluid, red blood cell and granulocyte layer.
  • T memory stem cells Tmsc
  • This kit is a negative selection kit.
  • T cell culture medium IL-2: OpTmizer TM CTS TM T-cell Expansion SFM+5% CTS TM Immune Cell SR+1% Penicillin-Streptomycin 100X Solution+1% L-glutamine+IL-2 200IU/mL .
  • T cell culture medium (IL-7/15): OpTmizer TM CTS TM T-cell Expansion SFM+5% CTS TM Immune Cell SR+1% Penicillin-Streptomycin 100X Solution+1% L-glutamine+IL-710ng/ ml+IL-15 10ng/mL.
  • the initial cell concentration is 1M/mL, and the height of the medium liquid level in Flask is not higher than 1cm.
  • T cell activation use Dynabeads Human T-Activator CD3/CD28 magnetic beads
  • T cell culture medium (IL-7/15) was changed to activate cell culture at a starting cell density of 1M/mL for activated T cells.
  • the medium was supplemented every 2 days and appropriate cytokines were added.
  • AAV6 virus-mediated CAR gene homologous recombination 48-72 hours after T cell activation in Example 3, AAV6 virus-mediated CAR gene homologous recombination, in situ insertion of the cut site for genetic modification, immediate transduction, AAV6 virus MOI: 2.5 ⁇ 10e5-10e6.
  • the sgRNA sequence is as follows:
  • the CAR gene homologous recombination mediated by AAV6 virus inserts the splice site in situ. Transduction immediately after electroporation, AAV6MOI: 2.5 ⁇ 10e5-10e6.
  • T cells were from two different individuals.
  • Flow cytometry detects the expression of TCR and CD3.
  • the TCR knockout rates of T cells in two different individuals were 87.2% and 68.6%, respectively.
  • FIG. 6 shows: T cells are from two different individuals. The expression of CAR by flow cytometry was 69.1% and 67.5% respectively. Compared with CAR-T transduced by lentivirus, the expression of CAR in this CAR-T cell is more uniform.
  • K562-CD19+/K562-CD19- cells are co-cultured, the initial cell number is 5 ⁇ 10e4 each, of which CD19+ cells express mCherry fluorescent protein
  • NegativeCtrl 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1
  • PositiveCtrl 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1
  • Test 1 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1
  • Test 2 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1
  • Cytotoxicity test take 20ul culture medium supernatant at different time points to measure cytokines: IL-2, IFN-r, TNF-a
  • the markers of T cell failure were detected at different time points: PD-1, TIM-3, LAG-3
  • T cell failure markers PD-1, LAG-3, and TIM-3 were detected.
  • the expression of failure markers of AAV-TCR-CD19CAR-T cells was significantly lower than that of the control group.

Abstract

L'invention concerne un vecteur viral adéno-associé recombiné destiné à être utilisé dans la préparation de cellules CAR-T universelles physiologiques, et son procédé de construction et son utilisation en thérapie antitumorale. Le vecteur viral adéno-associé recombiné peut être utilisé pour l'expression d'un gène CAR et la préparation d'une cellule CAR-T.
PCT/CN2019/075717 2019-02-15 2019-02-21 Vecteur viral adéno-associé recombiné destiné à être utilisé dans la préparation de cellules car-t universelles, son procédé de construction et son utilisation WO2020164167A1 (fr)

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