WO2020042648A1 - 改进的慢病毒载体 - Google Patents

改进的慢病毒载体 Download PDF

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WO2020042648A1
WO2020042648A1 PCT/CN2019/084828 CN2019084828W WO2020042648A1 WO 2020042648 A1 WO2020042648 A1 WO 2020042648A1 CN 2019084828 W CN2019084828 W CN 2019084828W WO 2020042648 A1 WO2020042648 A1 WO 2020042648A1
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lentiviral vector
nucleotide sequence
cells
seq
receptor
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PCT/CN2019/084828
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English (en)
French (fr)
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李贤秀
郑南哲
王歈
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法罗斯疫苗株式会社
北京永泰瑞科生物科技有限公司
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Application filed by 法罗斯疫苗株式会社, 北京永泰瑞科生物科技有限公司 filed Critical 法罗斯疫苗株式会社
Priority to SG11202102012SA priority Critical patent/SG11202102012SA/en
Priority to US17/272,197 priority patent/US20220042039A1/en
Priority to JP2021510858A priority patent/JP7386848B2/ja
Priority to KR1020217008649A priority patent/KR20210087017A/ko
Priority to CA3110926A priority patent/CA3110926A1/en
Priority to EP19855599.7A priority patent/EP3845656A4/en
Publication of WO2020042648A1 publication Critical patent/WO2020042648A1/zh

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Definitions

  • the invention belongs to the field of biomedicine.
  • the present invention relates to an improved lentiviral vector, a method for its preparation and use.
  • the invention relates to a lentiviral vector that is particularly suitable for the production of therapeutic T cells.
  • T cells are the key immune cells that perform tumor cell killing and virus infection cell killing in vivo.
  • T cells have been used, including antigen-specific T cells derived from in vitro induced or tumor infiltrating lymphocytes, genetically modified chimeric antigen receptor T cells (CAR-T cells), and genetically modified T cell receptor T cells (TCR-T cells) for the treatment of malignant tumors, has shown significant tumor clearance and control in some clinical patients.
  • CAR-T cells genetically modified chimeric antigen receptor T cells
  • TCR-T cells genetically modified T cell receptor T cells
  • TGF- ⁇ is an important T cell inhibitory factor, which causes the killing effect of T cells on target cells to weaken or disappear.
  • TGF- ⁇ is widely expressed in a variety of tumor tissues, and significantly inhibits the killing activity of tumor-specific T cells on tumor cells, which is an important reason for the failure of immunotherapy.
  • the dominant negative TGF- ⁇ type II receptor (dominant TGF- ⁇ receptor type II (DNRII)) is a negative regulator of TGF- ⁇ , which can inhibit the inhibitory effect of TGF- ⁇ on T cells.
  • tumor cells also express TGF- ⁇ , which leads to the problem that CAR-T cells and TCR-T cell functions are inhibited. It is also desirable in the art to introduce DNRII into CAR-T cells or TCR-T cells.
  • CAR / TCR and DNRII are co-expressed on the same T cell for the treatment of tumors. This may be due to the low co-expression efficiency of the two proteins by the existing expression system, which is difficult to meet the clinical needs.
  • Figure 1 shows the lentiviral vector structure and integrity identification strategy for CAR-19 expression.
  • A Old vector pPVLV1 containing P EF1 ⁇ - L (long promoter, 531 bp);
  • B New vector pPVLV2 including P EF1 ⁇ -S (short promoter, 212 bp).
  • the expected PCR products (F1-F5: PCR fragments) were generated from cDNA reverse transcribed using random hexamer primers, and the integrity of the viral vector was identified.
  • Figure 2 shows the difference between pPVLV1 and pPVLV2.
  • A The expected DNA fragment was amplified from the reverse transcription cDNA of the viral genome. Defective loci were observed in the viral gene fragment containing PEF1 ⁇ - L. DNA fragments with unexpected sizes are indicated by arrows (left panel).
  • B Comparison of the percentage of CAR-19-expressing cells and
  • C Titration of each vector 48 hours after transduction of 293T cells.
  • Figure 3 shows the structure and luciferase activity of CAR-19-Fluc.
  • A and (B) used a bicistronic construct encoding CAR-19 cloned upstream of the P2A-Fluc box.
  • C Schematic representation of the expressed CAR-19 and Fluc molecules.
  • D Luciferase activity measured 48 hours after lentiviral vector transduction of 293T cells.
  • Figure 4 shows the structure of CAR-19-DNRII and the viral vector.
  • A) and (B) show vector maps of CAR-19 co-expressing truncated TGFBRII (DNRII).
  • C Schematic diagram of the co-expressed CAR-19 and DNRII molecules.
  • Figure 5 shows the transduction efficiency of CAR-19 and DNRII expression in transduced 293T cells.
  • the numbers in the figure indicate the percentage of positive cells of CAR-19 (top) or DNRII (bottom) relative to the negative control of untransduced 293T cells. Results from representative experiments from ten independent experiments are given.
  • Figure 6 shows the expression of CAR-19 and DNRII in transduced T cells.
  • Activated T cells were transduced with a lentiviral vector to express CAR-19 or CAR-19-DNRII and evaluated by flow cytometry.
  • the numbers in the figure indicate the percentage of positive cells of CAR-19 (top) or DNRII (bottom) relative to the negative control of untransduced T cells. The results represent three independent experiments.
  • Figure 7 shows cell viability and counts after transduction of CAR-19 or CAR-19-DNRII vectors. Data are expressed as mean ⁇ SD.
  • Figure 8 shows that DNRII reduces TGF- ⁇ 1-induced SMAD2 phosphorylation.
  • Figure 9 shows mRNA levels of IFN- ⁇ and TNF- ⁇ in CAR-T-19 and CAR-T-19-DNRII cells. Data are expressed as mean ⁇ SEM.
  • FIG. 10 shows the antigen-specific killing of CD19 + tumor cells by CAR-T-19 and CAR-T-19-DNRII cells in the presence of TGF- ⁇ 1.
  • EuTDA cytotoxicity assay measures cell lysis activity. T cells were taken 3 days before the measurement and cultured with rhTGF- ⁇ 1 (10 ng / ml) for 72 hours. Target cells were labeled with BATDA reagent for 15 minutes, and then transduced T cells were added as effector cells at the specified E: T ratio. After 4 hours of incubation, lysis was determined.
  • the inventors have surprisingly found that transduction of cells, such as T cells, using a lentiviral vector containing a long EF1 ⁇ promoter (such as SEQ ID NO 7), an abnormality in this promoter region can occur, resulting in the introduction of foreign genes into the cell (In particular, a gene encoding a CAR or a fusion protein thereof) has a low expression rate.
  • a truncated EF1 ⁇ promoter can avoid this phenomenon and significantly increase the expression rate of the introduced foreign gene.
  • the invention provides a lentiviral vector comprising a truncated EF1 ⁇ promoter for directing expression of a nucleotide sequence encoding a polypeptide of interest in a host cell.
  • the truncated EF1 ⁇ promoter is an EF1 ⁇ core promoter comprising the nucleotide sequence shown in SEQ ID NO: 13.
  • a "lentiviral vector” refers to a non-replicating vector that is used to transduce a transgene comprising a cis-acting lentiviral RNA or DNA sequence to a host cell, and requires the lentiviral protein to be provided in trans form ( (Eg Gag, Pol and / or Env).
  • Lentiviral vectors lack coding sequences for functional Gag, Pol, and Env proteins. Lentiviral vectors can exist in the form of RNA or DNA molecules, depending on the stage of production or development of the retroviral vector.
  • Lentiviral vectors can be in the form of a recombinant DNA molecule, such as a plasmid (eg, a transfer plasmid vector).
  • Lentiviral vectors can be in the form of lentiviral particle vectors, such as RNA molecules in a complex of lentivirus and other proteins.
  • a lentiviral vector corresponding to a modified or recombinant lentiviral particle comprises a genome consisting of two copies of single-stranded RNA.
  • RNA sequences can be obtained by transcription from a double-stranded DNA sequence (proviral vector DNA) inserted into the genome of a host cell, or can be obtained by transient expression of plasmid DNA (plasmid vector DNA) in a transformed host cell.
  • a lentiviral vector may also refer to a DNA sequence integrated into a host cell.
  • Lentiviral vectors can be derived from lentiviruses, especially human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), goat arthritis encephalitis virus ( CAEV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV), which are modified to remove genetic determinants involved in pathogenicity and introduced into foreign expression cassettes.
  • HIV-1 or HIV-2 human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • EIAV equine infectious encephalitis virus
  • CAEV goat arthritis encephalitis virus
  • BIV bovine immunodeficiency virus
  • FV feline immunodeficiency virus
  • the lentiviral vector further comprises at least one element selected from the group consisting of 5'LTR, ⁇ element, RRE element, cPPT / CTS element, and a nucleotide sequence encoding a polypeptide of interest. Multiple cloning site, WPRE element and 3'LTR.
  • the lentiviral vector comprises an operably linked 5'LTR, ⁇ element, RRE element, cPPT / CTS element, the truncated EF1 ⁇ promoter, WPRE element, and 3'LTR, and optionally Ground cloning sites for inserting a nucleotide sequence encoding a polypeptide of interest.
  • the 5'LTR comprises the nucleotide sequence shown in SEQ ID NO: 3 or 11; the ⁇ element comprises the nucleotide sequence shown in SEQ ID NO: 4 or 12; and the RRE element comprises The nucleotide sequence shown in SEQ ID NO: 5; the cPPT / CTS element contains the nucleotide sequence shown in SEQ ID NO: 6; the WPRE element contains the nucleotide sequence shown in SEQ ID NO: 9 or 14; Wherein, the 3'LTR comprises the nucleotide sequence shown in SEQ ID NO: 10 or 15.
  • the lentiviral vector comprises an operably linked 5'LTR comprising the nucleotide sequence shown in SEQ ID NO: 11, a ⁇ element comprising the nucleotide sequence shown in SEQ ID NO: 12, comprising
  • the RRE element of the nucleotide sequence shown in SEQ ID NO: 5 includes the cPPT / CTS element of the nucleotide sequence shown in SEQ ID NO: 6 and the truncated EF1 ⁇ of the nucleotide sequence shown in SEQ ID NO: 13
  • a promoter comprising the WPRE element of the nucleotide sequence shown in SEQ ID NO: 14, the 3'LTR of the nucleotide sequence shown in SEQ ID NO: 15, and optionally for inserting a gene encoding a polypeptide of interest Multiple cloning sites for nucleotide sequences.
  • the lentiviral vector is derived from SEQ ID NO: 2.
  • the nucleotide sequence encoding CAR-19 at position 2,042-3,499 of SEQ ID NO: 2 may be replaced by the coding sequence of other polypeptides of interest.
  • the lentiviral vector further comprises a nucleotide sequence encoding a polypeptide of interest.
  • the polypeptide of interest is a fusion polypeptide comprising a plurality of proteins, the plurality of proteins in the fusion polypeptide being separated by a self-cleaving peptide.
  • the polypeptide of interest is a fusion polypeptide comprising a first protein and a second protein, the fusion polypeptide comprising a self-cleaving peptide between the first protein and the second protein.
  • the lentiviral vector further comprises a nucleotide sequence encoding a self-cleaving peptide.
  • the coding nucleotide sequence of the self-cleaving peptide is used for co-expression of two or more different proteins by the lentiviral vector.
  • Self-cleaving peptide as used herein means a peptide that can achieve self-cleaving within a cell.
  • the self-cleaving peptide may contain a protease recognition site so that it is recognized and specifically cleaved by a protease in the cell.
  • the self-cleaving peptide may be a 2A polypeptide.
  • 2A polypeptides are a class of short peptides derived from viruses that undergo self-cleaving during translation. When 2A polypeptide is used to link two different proteins of interest in the same reading frame, the two proteins of interest are generated at a ratio of almost 1: 1.
  • Commonly used 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thoseaasigna virus, and E2A from equine rhinitis A virus And F2A from foot-and-mouth disease virus. Among them, P2A has the highest cutting efficiency and is therefore preferred.
  • a variety of functional variants of these 2A polypeptides are also known in the art, and these variants can also be used in the present invention.
  • the first protein and the second protein are separated by a 2A polypeptide, placed in the same open reading frame, and driven by the same promoter, which can ensure the obtained transduced cells to express both proteins to the greatest extent. Because if the two proteins are transduced to cells in different vectors, some cells may only express the first protein and some cells only express the second protein, and the proportion of cells co-expressing the two proteins will be very low. In addition, if the expression of two proteins is driven by different promoters in the same vector, due to the difference in promoter efficiency, the proportion of cells that simultaneously express the two proteins will also be reduced.
  • the first protein is a cancer-associated antigen-specific receptor protein.
  • the second protein is a dominant negative TGF- ⁇ type II receptor.
  • the dominant negative TGF- ⁇ type II receptor means capable of competing with TGF- ⁇ RII for binding to a TGF- ⁇ ligand (such as TGF- ⁇ 1), but cannot perform TGF- ⁇ RII signaling A variant of a functional TGF- ⁇ type II receptor.
  • the intracellular signaling domain of the dominant negative TGF- ⁇ type II receptor is mutated, thereby losing intracellular signaling capabilities.
  • the dominant negative TGF- ⁇ type II receptor lacks the intracellular signaling domain of the TGF- ⁇ type II receptor.
  • the dominant negative TGF- ⁇ type II receptor comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the “cancer-associated antigen-specific receptor protein” described in the present invention may be a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the cancer-related antigens include, but are not limited to, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion Epidermal molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72 , Glycosphingolipid, glioma-associated antigen, ⁇ -human chorionic gonadotropin, alpha fetal globulin (AFP), exogenous lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX , Human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal
  • T cell receptor is also called T cell antigen receptor. It is a molecular structure that T cells specifically recognize and bind to the antigen peptide-MHC molecule. It usually exists on the surface of T cells in the form of a complex with CD3 molecules.
  • the TCR of most T cells consists of ⁇ and ⁇ peptide chains, and the TCR of a few T cells consists of ⁇ and ⁇ peptide chains.
  • the TCR is a TCR that specifically binds a cancer-associated antigen (usually comprising a chain that typically contains alpha and beta).
  • CAR Chimeric antigen receptor
  • the CAR may comprise an extracellular antigen-binding domain against a cancer-associated antigen.
  • the extracellular antigen-binding domain may be, for example, a monoclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single-domain antibody, an antibody single-chain variable region (scFV), and an antigen-binding fragment thereof.
  • the extracellular antigen-binding domain may be derived from one or more known antibodies including any commercially available antibody, such as FMC63, rituximab, alemtuzumab, elixir Epratuzumab, trastuzumab, bivatuzumab, cetuximab, labetuzumab, parizumab ( palivizumab), sevirumab, tuvirumab, basiliximab, daclizumab, infliximab, omalimumab (omalizumab), efalizumab, keliximab, siplizumab, natalizumab, clenoliximab, pemalimumab ( pemtumomab), Edrecolomab, Cantuzumab, and the like.
  • FMC63 FMC63
  • rituximab alemtuzumab
  • the CAR further comprises a transmembrane domain and an intracellular signal transduction domain.
  • the intracellular signal transduction domain of the CAR according to the present invention is responsible for intracellular signal transduction after the extracellular ligand binding domain binds to the target, leading to activation of immune cells and immune responses.
  • the intracellular signal transduction domain has the ability to activate at least one normal effector function of a CAR-expressing immune cell.
  • the effector function of a T cell may be cytolytic activity or auxiliary activity, including secretion of cytokines.
  • the intracellular signal transduction domain for CAR can be a cytoplasmic sequence, such as, but not limited to, a cytoplasmic sequence of a T cell receptor and a co-receptor (they work in concert to initiate signal transduction after antigen receptor junction), As well as derivatives or variants of any of these sequences and any synthetic sequences having the same functional capacity.
  • the intracellular signal transduction domain includes two different types of cytoplasmic signal transduction sequences: those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or costimulatory signals.
  • the primary cytoplasmic signal transduction sequence may include a signal transduction motif called an immune receptor tyrosine activation motif called ITAM.
  • Non-limiting examples of ITAM used in the present invention may include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signal transduction domain of the CAR may include a CD3 ⁇ signal transduction domain.
  • the intracellular signal transduction domain of the CAR of the invention further comprises a co-stimulatory domain.
  • the costimulatory domain is selected from a 41BB costimulatory domain or a CD28 costimulatory domain.
  • a CAR is expressed on the surface of cells. Therefore, a CAR can include a transmembrane domain.
  • Suitable transmembrane domains of the CARs of the present invention have the ability to: (a) be expressed on the cell surface, preferably immune cells, such as but not limited to lymphocytes or natural killer (NK) cells, and (b) a ligand binding domain It interacts with intracellular signal transduction domains to direct the immune cells to respond to predetermined target cells.
  • Transmembrane domains can be derived from natural or synthetic sources. The transmembrane domain can be derived from any membrane-bound protein or transmembrane protein.
  • a transmembrane domain can be derived from a subunit of a T cell receptor, such as an ⁇ subunit, a ⁇ subunit, a ⁇ , or a ⁇ subunit, a polypeptide constituting the CD3 complex, and p55 of the IL-2 receptor ( ⁇ chain), p75 ( ⁇ chain) or ⁇ , a subunit chain of the Fc receptor, especially the Fc ⁇ receptor III or CD protein.
  • the transmembrane domain may be synthetic and may include mainly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from a human CD8 ⁇ chain.
  • the transmembrane domain may further include a hinge region between an extracellular ligand-binding domain and the transmembrane domain.
  • the hinge region is, for example, from the extracellular region of CD8, CD4 or CD28. In some embodiments, the hinge region is part of a human CD8 ⁇ chain.
  • the CAR used in the present invention may include extracellular antigen-binding domains such as scFv, CD8 hinge and transmembrane domain, CD3 ⁇ signal transduction domain, and 4-1BB that specifically bind to cancer-associated antigens. Costimulatory domain.
  • the CAR comprises an extracellular antigen-binding domain directed against CD19.
  • the CAR comprises the amino acid sequence shown in SEQ ID NO: 16.
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • TGF- ⁇ T cell receptor
  • the lentiviral vector of the present invention is particularly suitable for co-expressing T cell receptor (TCR) or chimeric antigen receptor (CAR) and dominant negative TGF- ⁇ type II receptor on T cells.
  • the invention provides a method for preparing a lentiviral vector particle, the method comprising:
  • lentiviral vector of the present invention co-transfecting the lentiviral vector of the present invention, one or more packaging vectors expressing Gag and / or Pol, and an envelope vector expressing an envelope protein such as VSV-G;
  • the vector is a plasmid.
  • Suitable host cells for preparing lentiviral vector particles include, but are not limited to, 293T cells.
  • the present invention provides a lentiviral vector particle comprising the lentiviral vector of the present invention or prepared by the above method of the present invention.
  • the present invention provides the use of a lentiviral vector particle of the present invention in the preparation of a therapeutic T cell, wherein the therapeutic T cell expresses a cancer-associated antigen-specific receptor protein, such as a T cell receptor (TCR) Or chimeric antigen receptor (CAR), and optionally a dominant negative TGF- ⁇ type II receptor.
  • a cancer-associated antigen-specific receptor protein such as a T cell receptor (TCR) Or chimeric antigen receptor (CAR), and optionally a dominant negative TGF- ⁇ type II receptor.
  • the invention provides a method for preparing a therapeutic T cell, comprising transducing a T cell with a lentiviral vector particle of the invention. Transduction of the lentiviral vector particles will cause the therapeutic T cells to express cancer-associated antigen-specific receptor proteins, such as T-cell receptor (TCR) or chimeric antigen receptor (CAR), and optionally a Negative TGF- ⁇ type II receptor.
  • TCR T-cell receptor
  • CAR chimeric antigen receptor
  • the T cells of the invention can be obtained from a number of non-limiting sources by a variety of non-limiting methods, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors.
  • the cells can be derived from a healthy donor or from a patient diagnosed with cancer.
  • the cells can be part of a mixed population of cells that exhibit different phenotypic characteristics.
  • T cells can be obtained by isolating peripheral blood mononuclear cells (PBMC), and then activating and expanding with specific antibodies.
  • PBMC peripheral blood mononuclear cells
  • the T cell is derived from an autologous cell of a subject.
  • autologous means that the cell, cell line, or cell population used to treat a subject is derived from the subject.
  • the T cells are derived from an allogeneic cell, for example, derived from a donor compatible with the subject's human leukocyte antigen (HLA). Donor-derived cells can be transformed into non-allogene-reactive cells using standard protocols and replicated as needed to produce cells that can be administered to one or more patients.
  • HLA human leukocyte antigen
  • the present invention provides a kit for producing lentiviral vector particles, comprising the lentiviral vector of the present invention, a suitable packaging vector, a suitable envelope vector, and / or a suitable host cell such as 293T cells .
  • the kit may further include a cell transfection reagent.
  • the invention provides a kit for expressing a polypeptide of interest in a cell, comprising a lentiviral vector particle of the invention.
  • Lentiviral vectors used to transduce CAR should contain the desired CAR transgene and be capable of being expressed in cells.
  • Two third-generation lentiviral vectors for CAR expression were designed, the old vector pPVLV1 ( Figure 1A) and the new vector pPVLV2 ( Figure 1B).
  • pPVLV1 contains a 531 bp long human elongation factor 1 ⁇ (EF1 ⁇ ) promoter and pPVLV1 contains a 212 bp truncated human EF1 ⁇ promoter.
  • Table 1 The various elements contained in the two vectors and their descriptions are shown in Table 1 below.
  • the CAR to be expressed in the examples of the present application includes a scFv targeting CD19, a hinge and transmembrane domain of human CD8, an intracellular domain 4-1BB, and CD3 ⁇ .
  • the amino acid of the CD19-targeting CAR is shown in SEQ ID NO: 16, and the nucleotide sequence is shown in SEQ ID NO: 8.
  • pPVLV1 vector including EF1 ⁇ long promoter (5,320bp, SEQ ID NO: 1)
  • pPVLV2 vector including EF1 ⁇ short promoter (4,417bp, SEQ ID NO: 2)
  • Lentiviral supernatants were generated by transfecting 293T cells with a gag / pol packaging plasmid, a VSV-G envelope plasmid, and a transfer construct containing a lentiviral vector sequence as described above. Briefly, the DNA mixture was mixed in Opti-MEM (Life Technologies, Gaithersburg, MD, USA) and mixed with an equal volume of Opti-MEM containing Lipofectamine 3000 (Life Technologies). After 15 minutes of incubation at room temperature, the resulting mixture was applied to 293T cells. Lentivirus-containing medium was collected 24 hours after transfection. After each collection, the supernatant was filtered through a PVDF membrane (0.45 ⁇ m). The lentiviral harvests were combined and stored at 4 ° C, followed by ultracentrifugation at 20,000 xg for 1 hour and 30 minutes. The lentiviral particles were resuspended in PBS.
  • Figure 1 shows a schematic diagram of the structure of two lentiviral vectors and a strategy for verifying the integrity of the lentivirus by overlapping PCR products.
  • Appropriate primers were designed to amplify overlapping fragments F1-F5 from cDNA reverse transcribed using random primers.
  • a PCR product of the expected size can demonstrate the integrity of the lentivirus.
  • FIG. 2A shows each DNA fragment amplified from the cDNA reversely transcribed from the viral genome.
  • defective gene loci were observed in viral gene fragments containing P EF1 ⁇ -L (long promoter). Arrows indicate unexpected DNA fragments (left). This phenomenon was not observed in viral gene fragments containing P EF1 ⁇ -S (short promoter). Such defective viral genomes may affect titer and transduction efficiency.
  • lentiviral titration 2 ⁇ 10 6 293T cells were seeded into each well of a 6-well plate and transduced with a series of volumes of concentrated lentivirus. 48 hours after transduction, 293T cells were isolated from the plate. The presence of CAR was detected by flow cytometry using Alexa Fluor 488 labeled goat anti-human IgG F (ab) 2 . Viral genomic RNA from 5'LTR to 3'LTR was examined using conventional PCR.
  • the results are shown in Figures 2B and C.
  • the transduction efficiency (proportion of CAR-expressing cells) of the virus with P EF1 ⁇ - L (based on pPVLV1 vector) was only 9.95%, which was far lower than 70.4% of the virus with P EF1 ⁇ - S (based on pPVLV2 vector).
  • the titer of the virus with P EF1 ⁇ - L (based on the pPVLV1 vector) was also significantly lower than that of the virus with P EF1 ⁇ - S (based on the pPVLV2 vector). It is shown that pPVLV2 vector is superior to pPVLV1 vector, and this may be caused by EF1 ⁇ promoters of different lengths.
  • FIG. 3D shows the luciferase activity measured 48 hours after transduction of two lentiviral vectors into 293T cells. The results showed that cells transduced with the lentiviral vector with P EF1 ⁇ - S had significantly stronger fluorescence than cells transduced with the lentiviral vector with P EF1 ⁇ - L. It was further proved that P EF1 ⁇ -S significantly improved the expression of the transgene in cells.
  • TGF- ⁇ is an important T cell inhibitory factor, which may cause the killing effect of therapeutic T cells on target cells to weaken or disappear.
  • TGF- ⁇ is widely expressed in a variety of tumor tissues, and significantly inhibits the killing activity of tumor-specific T cells on tumor cells, which is an important reason for the failure of immunotherapy.
  • the dominant negative TGF- ⁇ receptor type II (dominant, TGF- ⁇ receptor type II, DNRII) is a negative regulator of TGF- ⁇ , which can inhibit the inhibitory effect of TGF- ⁇ on T cells.
  • the following examples investigate the effect of co-expression of CAR and DNRII in T cells.
  • the amino acid sequence of DNRII is shown in SEQ ID NO: 17, and its nucleotide sequence is shown in SEQ ID NO: 18.
  • FIG. 4 shows a schematic diagram of the structure of the CAR-19 and DNRII molecules.
  • DNRII lacks the intracellular serine / threonine kinase domain of TGFBRII and cannot transmit signals downstream.
  • CAR-19 and DNRII coding sequences are separated by 2A coding sequences, placed in the same open reading frame, and driven by the same promoter, which can ensure that the obtained transduced cells express CAR-19 and DNRII at the same time. Because if CAR-19 and DNRII are transduced to cells in different vectors, some cells may only express CAR-19, some cells only express DNRII, and the proportion of cells co-expressing the two proteins will be very low. In addition, if the expression of two proteins is driven by different promoters in the same vector, due to the difference in promoter efficiency, the proportion of cells that simultaneously express the two proteins will also be reduced.
  • the two CAR-19-DNRII lentiviral vectors were transduced to 293T cells with equal MOI (multiple infections).
  • CAR or DNRII expression was detected by flow cytometry using a labeled goat anti-human IgG F (ab) 2 or anti-DNRII antibody using a MACSQuant analyzer 10 and the data was analyzed with FlowJo software.
  • PBMCs Peripheral blood mononuclear cells
  • Cells were suspended at 3 ⁇ 10 5 cells / ml in a medium containing rhIL-2 (200IU / mL), fresh medium was replaced every 2 to 3 days, and cultured for 12 days to obtain a CAR expressing a CAR-19 molecule.
  • -T-19 cells and CAR-T-19-DNRII cells co-expressing CAR-19 molecules and DNRII molecules.
  • PBMC cultured under the same culture conditions but without gene transduction was used as a control (NC).
  • Flow cytometry was used to detect the expression of each protein molecule of CAR-T cells obtained after transduction.
  • Cells were stained with propidium iodide (PI) every 2-3 days, and cell viability was measured by flow cytometry. During the cell culture, trypan blue staining was performed every 2-3 days (three replicates per sample), and the number of cells was calculated (mean ⁇ SD).
  • PI propidium iodide
  • this example determines that the backbone of the pPVLV2 vector (including P EF1 ⁇ -S) is particularly suitable for CAR expression in cells, such as T cells, and is particularly suitable for co-expression of CAR and other proteins such as DNRII.
  • placing the CAR and DNRII coding sequences in the same open reading frame can achieve a high co-expression rate of the two molecules.
  • TFG- ⁇ The inhibitory effect of TFG- ⁇ on T cells is achieved through the phosphorylation of SMAD2 molecules after TFG- ⁇ binds to its receptor.
  • CAR-T-19 cells and CAR-T-19-DNRII cells 9 days after transduction were incubated with recombinant human TFG- ⁇ 1 (10ng / ml) for 24 hours to perform expression of phosphorylated SMAD2 (pSMAD2). analysis.
  • pSMAD2 phosphorylated SMAD2
  • the Bradford assay kit (Sigma-Aldrich) was used to measure the protein concentration of whole cell lysates. An equal amount of protein was loaded into the wells of an SDS-PAGE gel, and the separated protein was transferred to a PVDF membrane (Thermo Scientific). The membrane was blocked with 10% (w / v) skim milk in TBST and then incubated with primary antibodies (anti-pSMAD2 and anti-SMAD2; from Cellsignaling Technologies, Danvers, MA, USA; all diluted 1: 1000) overnight at 4 ° C. .
  • the membrane was then washed with TBST and incubated with HRP-conjugated goat anti-rabbit IgG (1: 2000 dilution; Cellular Signaling Technologies) at room temperature for 2 hours.
  • the membrane was then exposed to ECL reagent (Thermo Scientific) and the resulting signal was detected using a luminescence image analyzer (LAS-4000, Fuji Film, Tokyo, Japan).
  • Fig. 8 The level of pSAMD2 in CAR-T-19-DNRII cells was significantly lower than that of CAR-T-19 cells. This indicates that the expression of DNRII inhibits the phosphorylation of SMAD2, a key signaling molecule in the TGF- ⁇ signaling pathway.
  • Example 5 Expression of IFN- ⁇ and TNF- ⁇ in CAR-T-19-DNRII cells and CAR-T-19 cells treated with recombinant human TGF- ⁇ 1
  • IFN- ⁇ and TNF- ⁇ are the hallmark cytokines that T cells kill target cells.
  • the high expression levels of these two cytokines indicate that T cells have high killing potential for target cells, and conversely, low killing potential.
  • RNA samples were analyzed using specific primers and One-step SensiFAST SYBR Low-ROX kit (Bioline, Maryland, USA)
  • Real-time quantitative RT-PCR analysis was performed using a QuantStudio3 real-time PCR detection system (Applied Biosystems, Foster City, CA, USA). 18s rRNA was amplified as an internal control. The expression level was calculated by the ⁇ Ct method, and the expression multiple was obtained using the formula 2- ⁇ Ct. All experiments were performed in triplicate.
  • Example 6 CAR-T-19-DNRII cells and CAR-T-19 cells treated with recombinant human TGF- ⁇ 1 specifically kill tumor target cells
  • Target cell killing experiments were performed using CAR-T-19 cells and CAR-T-19-DNRII cells transduced for 12 days.
  • a TDA release assay was performed to determine the cytotoxic activity of CAR-T-19 cells and CAR-T-19-DNRII cells to K562 or CD19 + -K562 in the presence of TGF- ⁇ 1.
  • CAR-T-19 cells and CAR-T-19-DNRII cells were incubated with recombinant human TGF- ⁇ 1 (10ng / ml) for 72 hours, respectively.
  • Target cells were labeled with BA-TDA (Perkin Elmer, Norwalk, Connecticut, USA) for 15 minutes, and the effector cells (T cells) were 20: 1, 10: 1, 5: 1, and 2.5: 1: target cells (tumor Cells were mixed with effector cells, and TDA release (target cell lysis) was detected after 4 hours of incubation.

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Abstract

本发明公开了改进的慢病毒载体及其制备方法和用途。具体地，本发明公开了适合于制备治疗性T细胞的慢病毒载体。

Description

改进的慢病毒载体 技术领域
本发明属于生物医药领域。具体而言，本发明涉及改进的慢病毒载体及其制备方法和用途。具体而言，本发明涉及特别适合于制备治疗性T细胞的慢病毒载体。
发明背景
T细胞是体内执行肿瘤细胞杀伤和病毒感染细胞杀伤的关键免疫细胞。近年来，使用T细胞，包括来源于体外诱导或肿瘤浸润淋巴细胞的抗原特异性T细胞、基因改造的嵌合抗原受体T细胞(CAR-T细胞)、基因改造的T细胞受体T细胞(TCR-T细胞)进行恶性肿瘤的治疗，在部分临床患者中显示了显著的肿瘤清除和控制作用。然而，由于患者体内肿瘤的免疫逃逸作用，部分肿瘤患者对回输的T细胞产生抵抗作用，导致T细胞不能发挥应有的抗肿瘤作用。
体内体外的研究均表明，TGF-β是重要的T细胞抑制因子，导致T细胞对靶细胞的杀伤作用减弱或消失。在临床上，TGF-β广泛表达于多种肿瘤组织中，显著抑制肿瘤特异性T细胞对肿瘤细胞的杀伤活性，是免疫治疗失败的重要原因。而显性负性TGF-βII型受体(dominant negative TGF-βreceptor type II,DNRII)是TGF-β的负性调节受体，可以抑制TGF-β对T细胞的抑制作用。在动物体内，通过给予或者表达T细胞特异性的DNRII，或者给予可溶性TGF-β RII，干扰TGF-β信号通路，可以显著提高T细胞对肿瘤的杀伤作用。美国贝勒医学院Catherin M Bollard领导的研究团队发现，给予患者EB病毒特异性的T细胞(EBV-CTL)治疗，对EBV感染导致的霍杰金和非霍杰金淋巴瘤均有一定的效果。然而，在这些疾病中，由于肿瘤组织表达TGF-β，EBV-CTL的疗效受到干扰。该研究团队通过基因转导的方式，将DNRII表达于EBV-CTL细胞表面，用于治疗复发的霍杰金淋巴瘤。在7名患者可以评价的患者中，4名患者获得了完全缓解，其中2例患者完全缓解持续4年，其中1例是在未经DNRII基因修饰的EBV-CTL治疗后未能获得完全缓解的患者。然而，这些EBV-CTL仅仅表达DNRII一种外源蛋白。
在目前应用与临床的CAR-T细胞和TCR-T细胞治疗中，同样面临肿瘤细胞表达TGF-β，导致CAR-T细胞和TCR-T细胞功能受到抑制的问题。本领域也期望将DNRII导入CAR-T细胞或TCR-T细胞中。然而，迄今为止，尚未有将CAR/TCR与DNRII共表达于同一T细胞用于治疗肿瘤的报道。这可能由于现有的表达系统对两种蛋白的共表达效率低，难以满足临床需要。
为了解决该问题，需要新的改进的表达载体，如改进的慢病毒载体，其特别适合于两种或更多种蛋白的高效共表达。
附图简述
图1示出用于表达CAR-19的慢病毒载体结构及完整性鉴定策略。(A)包含P EF1α-L(长启动子，531bp)的老载体pPVLV1；(B)包括P EF1α-S(短启动子，212bp)的新载体pPVLV2。从使用随机六聚体引物逆转录的cDNA产生预期的PCR产物(F1-F5：PCR片段)，鉴定病毒载体的完整性。
图2示出pPVLV1和pPVLV2的差别。(A)从病毒基因组的逆转录cDNA扩增出预期的DNA片段。在含有P EF1α-L的病毒基因片段中观察到缺陷的基因位点。用箭头指示具有意外大小的DNA片段(左图)。(B)比较表达CAR-19的细胞的百分比和(C)转导293T细胞48小时后各载体的滴度。
图3示出CAR-19-Fluc的结构和荧光素酶活性。(A)和(B)使用的编码克隆在P2A-Fluc盒上游的CAR-19的双顺反子构建体。(C)表达的CAR-19和Fluc分子的示意图。(D)在慢病毒载体转导293T细胞后48小时测定的荧光素酶活性。
图4示出CAR-19-DNRII的结构和病毒载体。(A)和(B)显示共表达截短的TGFBRII(DNRII)的CAR-19的载体图谱。(C)共表达的CAR-19和DNRII分子的结构示意图。
图5示出转导的293T细胞中CAR-19和DNRII表达的转导效率。图中的数字表示CAR-19(上)或DNRII(下)的阳性细胞相对于未转导的293T细胞的阴性对照的百分比。给出了来自十个独立实验的代表性实验的结果。
图6示出转导的T细胞中CAR-19和DNRII的表达。用慢病毒载体转导活化的T细胞以表达CAR-19或CAR-19-DNRII，并通过流式细胞术评估。图中的数字表示CAR-19(上)或DNRII(下)的阳性细胞相对于未转导的T细胞的阴性对照的百分比。结果代表三个独立实验。
图7示出CAR-19或CAR-19-DNRII载体转导后的细胞活力和计数。数据表示为平均值±SD。
图8示出DNRII减少TGF-β1诱导的SMAD2磷酸化。
图9示出CAR-T-19和CAR-T-19-DNRII细胞中的IFN-γ和TNF-α的mRNA水平。数据表示为平均值±SEM。
图10示出CAR-T-19和CAR-T-19-DNRII细胞在TGF-β1存在下对CD19 +肿瘤细胞的抗原特异性杀死。在CAR T初始活化细胞12天后，通过
Figure PCTCN2019084828-appb-000001
EuTDA细胞毒性测定法测定细胞裂解活性。在测定前3天取T细胞，并用rhTGF-β1(10ng/ml)培养72小时。用BATDA试剂标记靶细胞15分钟，随后以指定的E:T比加入作为效应细胞的经转导的T细胞。孵育4小时后测定裂解。
发明内容
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook et al.,“Molecular Cloning:A Laboratory Manual”；Lewin,“Genes VIII”；及Roitt et al.,“Immunology”(第8版)，以 及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
本发明人令人意外地发现，用包含长EF1α启动子(如SEQ ID NO 7)的慢病毒载体转导细胞例如T细胞，会出现该启动子区域的异常，导致导入细胞的外源基因(特别是编码CAR或其融合蛋白的基因)的低表达率。而更意想不到的是，使用截短的EF1α启动子则可以避免该现象，显著提高所导入外源基因的表达率。
因此，在第一方面，本发明提供一种慢病毒载体，其包含用于指导编码感兴趣的多肽的核苷酸序列在宿主细胞中表达的截短的EF1α启动子。在一些实施方案中，所述截短的EF1α启动子是包含SEQ ID NO:13所示核苷酸序列的EF1α核心启动子。
在本发明范围内，“慢病毒载体”指非复制型载体，其用于将包含顺式作用慢病毒RNA或DNA序列的转基因转导至宿主细胞，且需要以反式形式提供慢病毒蛋白(例如Gag、Pol和/或Env)。慢病毒载体缺少功能性Gag、Pol和Env蛋白的编码序列。慢病毒载体可以RNA或DNA分子的形式存在，这取决于所述逆转录病毒载体的产生或发展阶段。
慢病毒载体可以是重组DNA分子的形式，如质粒(例如转移质粒载体)。慢病毒载体可以是慢病毒颗粒载体的形式，如慢病毒和其他蛋白质的复合物中的RNA分子。通常，对应于修饰或重组的慢病毒颗粒的慢病毒载体包含由两个单链RNA拷贝组成的基因组。这些RNA序列可通过从插入宿主细胞基因组的双链DNA序列(原病毒载体DNA)转录获得，或者可由转化的宿主细胞中质粒DNA(质粒载体DNA)的瞬时表达获得。慢病毒载体也可以指整合进宿主细胞的DNA序列。
慢病毒载体可以来源于慢病毒，尤其是人免疫缺陷病毒(HIV-1或HIV-2)、猿免疫缺陷病毒(SIV)、马传染性脑炎病毒(EIAV)、山羊关节炎脑炎病毒(CAEV)、牛免疫缺陷病毒(BIV)和猫免疫缺陷病毒(FIV)，其经修饰以除去涉及致病性的遗传决定簇并导入外源表达盒。
在一些实施方案中，所述慢病毒载体还包含选自以下的至少一种元件：5’LTR、ψ元件、RRE元件、cPPT/CTS元件、用于插入编码感兴趣的多肽的核苷酸序列的多克隆位点、WPRE元件和3’LTR。
在一些实施方案中，所述慢病毒载体包含可操作连接的5’LTR、ψ元件、RRE元件、cPPT/CTS元件、所述截短的EF1α启动子、WPRE元件和3’LTR，以及任选地，用于插入编码感兴趣的多肽的核苷酸序列的多克隆位点。
在一些实施方案中，所述5’LTR包含SEQ ID NO:3或11所示核苷酸序列；所述ψ元件包含SEQ ID NO:4或12所示核苷酸序列；所述RRE元件包含SEQ ID NO:5所示核苷酸序列；所述cPPT/CTS元件包含SEQ ID NO:6所示核苷酸序列；所述WPRE元件包含SEQ ID NO:9或14所示核苷酸序列；其中所述3’LTR包含SEQ ID NO:10或15所示核苷酸序列。
在一些实施方案中，所述慢病毒载体包含可操作连接的包含SEQ ID NO:11所示核苷酸序列的5’LTR，包含SEQ ID NO:12所示核苷酸序列的ψ元件，包含SEQ ID NO:5所示核苷酸序列的RRE元件，包含SEQ ID NO:6所示核苷酸序列的cPPT/CTS元件，包含SEQ ID NO:13所示核苷酸序列的截短的EF1α启动子，包含SEQ ID NO:14所示核苷酸序列的WPRE元件，包含SEQ ID NO:15所示核苷酸序列的3’LTR，以及任选地，用于插入编码感兴趣的多肽的核苷酸序列的多克隆位点。
在一些实施方案中，所述慢病毒载体衍生自SEQ ID NO:2。其中SEQ ID NO:2的2,042-3,499位的编码CAR-19的核苷酸序列可以被其他感兴趣的多肽的编码序列取代。
在一些实施方案中，所述慢病毒载体还包含编码感兴趣的多肽的核苷酸序列。
在一些实施方案中，所述感兴趣的多肽是包括多种蛋白的融合多肽，所述融合多肽中的所述多种蛋白由自裂解肽分隔开。
在一些实施方案中，所述感兴趣的多肽是包括第一蛋白和第二蛋白的融合多肽，所述融合多肽在第一蛋白和第二蛋白之间包含自裂解肽。
因此，在一些实施方案中，所述慢病毒载体还包含自裂解肽的编码核苷酸序列。所述自裂解肽的编码核苷酸序列用于通过所述慢病毒载体共表达两种或更多种不同蛋白。
如本文所用“自裂解肽”意指可以在细胞内实现自剪切的肽。例如，所述自裂解肽可以包含蛋白酶识别位点，从而被细胞内的蛋白酶识别并特异性切割。
或者，所述自裂解肽可以是2A多肽。2A多肽是一类来自病毒的短肽，其自切割发生在翻译期间。当用2A多肽连接两种不同目的蛋白在同一读码框表达时，几乎以1:1的比例生成两种目的蛋白。常用的2A多肽可以是来自猪捷申病毒(porcine techovirus-1)的P2A、来自明脉扁刺蛾β四体病毒(Thosea asigna virus)的T2A、马甲型鼻病毒(equine rhinitis A virus)的E2A和来自口蹄疫病毒(foot-and-mouth disease virus)的F2A。其中P2A的切割效率最高，因此是优选的。本领域也已知多种这些2A多肽的功能性变体，这些变体也可以用于本发明。
将例如第一蛋白和第二蛋白由2A多肽隔开，置于同一开放读码框，由同一启动子驱动表达，可以最大程度确保所获得的经转导的细胞同时表达两种蛋白。因为如果两种蛋白在不同载体中分别转导至细胞，将可能造成部分细胞仅表达第一蛋白，部分细胞仅表达第二蛋白，而共表达两种蛋白的细胞的比例将很低。另外，如果两种蛋白的表达由同一载体中不同启动子驱动表达，由于启动子效率的差异，同样会降低同时表达两种蛋白的细胞的比例。
在一些实施方案中，所述第一蛋白是癌症相关抗原特异性受体蛋白。在一些实施方案中，所述第二蛋白是显性负性TGF-β II型受体。
如本发明所用，“显性负性TGF-β II型受体”是指能够与TGF-β RII竞争结合TGF-β配体(如TGF-β1)，但不能执行TGF-β RII的信号传导功能的TGF-β II型受体的变体。在一些实施方案中，所述显性负性TGF-β II型受体的胞内信号传导结构域被突变，从而丧失胞内信号传导能力。在一些实施方案中，所述显性负性TGF-β II型受体缺失TGF-β II 型受体的胞内信号传导结构域。在一些具体实施方案中，所述显性负性TGF-β II型受体包含SEQ ID NO:18所示的氨基酸序列。
本发明所述“癌症相关抗原特异性受体蛋白”可以是T细胞受体(TCR)或者是嵌合抗原受体(CAR)。
所述癌症相关抗原包括但不限于CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
“T细胞受体(TCR)”又称T细胞抗原受体，是T细胞特异性识别和结合抗原肽-MHC分子的分子结构，通常与CD3分子呈复合物形式存在于T细胞表面。大多数T细胞的TCR由α和β肽链组成，少数T细胞的TCR由γ和δ肽链组成。例如，所述TCR是特异性结合癌症相关抗原的TCR(通常包含通常包含α和β链)。
“嵌合型抗原受体(CAR)”又称人工T细胞受体、嵌合型T细胞受体、嵌合型免疫受体，是一种人工设计的受体，可以赋予免疫效应细胞某一种特异性。普遍来讲，这一技术被用于赋予T细胞特异性识别肿瘤表面抗原的特性。通过这种方法，可以产生大量的靶向肿瘤杀伤细胞。
所述CAR可以包含针对癌症相关抗原的细胞外抗原结合结构域。所述细胞外抗原结合结构域例如可以是单克隆抗体、合成的抗体、人抗体、人源化抗体、单域抗体、抗体单链可变区(scFV)，以及其抗原结合片段。例如，所述细胞外抗原结合结构域可以衍生自一或多种已知抗体包括任何商业可获得的抗体，如FMC63、利妥昔单抗(rituximab)、阿仑珠单抗(alemtuzumab)、依帕珠单抗(epratuzumab)、曲妥珠单抗(trastuzumab)、比伐珠单抗(bivatuzumab)、西妥昔单抗(cetuximab)、拉贝珠单抗(labetuzumab)、帕利珠单抗(palivizumab)、司韦单抗(sevirumab)、妥韦单抗(tuvirumab)、巴利昔单抗(basiliximab)、 达克珠单抗(daclizumab)、英利昔单抗(infliximab)、奥马珠单抗(omalizumab)、依法珠单抗(efalizumab)、凯利昔单抗(Keliximab)、西利珠单抗(siplizumab)、那他珠单抗(natalizumab)、克立昔单抗(clenoliximab)、培马单抗(pemtumomab)、依屈洛单抗(Edrecolomab)、坎妥珠单抗(Cantuzumab)等。
在本发明各方面的一些实施方案中，所述CAR还包括跨膜结构域和细胞内信号转导结构域。根据本发明的CAR的细胞内信号转导结构域负责细胞外配体结合结构域与靶结合之后的细胞内信号转导，导致激活免疫细胞和免疫应答。细胞内信号转导结构域具有激活表达CAR的免疫细胞的至少一种正常效应子功能的能力。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
用于CAR的细胞内信号转导结构域可以是细胞质序列，例如但不限于T细胞受体和共同受体(它们一致地起作用从而在抗原受体接合之后启动信号转导)的细胞质序列，以及任何这些序列的衍生物或变体以及具有相同功能能力的任何合成序列。细胞内信号转导结构域包括两种不同类型的细胞质信号转导序列：那些启动抗原依赖性初级激活的序列，以及那些以抗原非依赖性方式作用以提供次级或共刺激信号的序列。初级细胞质信号转导序列可以包括称为ITAM的免疫受体酪氨酸激活基序的信号转导基序。本发明中使用的ITAM的非限制性实例可以包括衍生自TCRζ、FcRγ、FcRβ、FcRε、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。在一些实施方案中，CAR的细胞内信号转导结构域可以包括CD3ζ信号转导结构域。在一些实施方案中，本发明的CAR的细胞内信号转导结构域还包括共刺激结构域。在一些实施方案中，所述共刺激结构域选自41BB共刺激结构域或CD28共刺激结构域。
CAR在细胞的表面上表达。因此，CAR可以包括跨膜结构域。本发明的CAR的合适跨膜结构域具有以下能力：(a)在细胞表面表达，优选免疫细胞，例如但不限于淋巴细胞或自然杀伤(NK)细胞，和(b)与配体结合结构域和细胞内信号转导结构域相互作用，用于指导免疫细胞对预定靶细胞的细胞应答。跨膜结构域可以衍生自天然来源或合成来源。跨膜结构域可以衍生自任何膜结合蛋白或跨膜蛋白。作为非限制性实例，跨膜结构域可以衍生自T细胞受体的亚基如α亚基、β亚基、γ或δ亚基，组成CD3复合物的多肽，IL-2受体的p55(α链)、p75(β链)或γ，Fc受体的亚基链，特别是Fcγ受体III或CD蛋白。或者，跨膜结构域可以是合成的，并且可以主要包括疏水性残基，如亮氨酸和缬氨酸。在一些实施方案中，所述跨膜结构域衍生自人CD8α链。跨膜结构域可以进一步包括位于细胞外配体结合结构域和所述跨膜结构域之间的铰链区域。所述铰链区域例如来自CD8、CD4或CD28的胞外区。在一些实施方案中，所述铰链区域是人CD8α链的一部分。
在一些具体实施方案中，本发明中使用的CAR可以包括特异性结合癌症相关抗原的细胞外抗原结合结构域如scFv、CD8铰链和跨膜结构域、CD3ζ信号转导结构域、和4-1BB共刺激结构域。
在一些具体实施方案中，所述CAR包含针对CD19的细胞外抗原结合结构域。在 一些具体实施方案中，所述CAR包含SEQ ID NO:16所示氨基酸序列。
将T细胞受体(TCR)或嵌合抗原受体(CAR)和显性负性TGF-β II型受体在T细胞中共表达，可解除TGF-β对T细胞的抑制，显著提高TCR-T细胞或CAR-T细胞的活性如肿瘤杀伤活性。如实施例所示，本发明的慢病毒载体特别适合于在T细胞共表达T细胞受体(TCR)或嵌合抗原受体(CAR)和显性负性TGF-β II型受体。
在另一方面，本发明提供一种制备慢病毒载体颗粒的方法，所述方法包括：
a)使本发明的慢病毒载体、表达Gag和/或Pol的一或多种包装载体、表达包膜蛋白如VSV-G的包膜载体共转染合适的宿主细胞；
b)培养转染的所述宿主细胞以使所述慢病毒载体包装成慢病毒载体颗粒；以及
c)收获步骤b)中产生的慢病毒载体颗粒。
本领域已知并且本领域技术人员可以容易获得合适的表达Gag和/或Pol的一或多种包装载体、表达包膜蛋白如VSV-G的包膜载体。在一些实施方式中，所述载体是质粒。
用于制备慢病毒载体颗粒的合适的宿主细胞包括但不限于293T细胞。
在另一方面，本发明提供一种慢病毒载体颗粒，其包含本发明的慢病毒载体或通过本发明上述的方法制备。
在另一方面，本发明提供本发明的慢病毒载体颗粒在制备治疗性T细胞中的用途，其中所述治疗性T细胞表达癌症相关抗原特异性受体蛋白，例如T细胞受体(TCR)或嵌合抗原受体(CAR)，以及任选的显性负性TGF-β II型受体。
在另一方面，本发明提供一种制备治疗性T细胞的方法，包括用本发明的慢病毒载体颗粒转导T细胞。所述慢病毒载体颗粒的转导将导致所述治疗性T细胞表达癌症相关抗原特异性受体蛋白，例如T细胞受体(TCR)或嵌合抗原受体(CAR)，以及任选的显性负性TGF-β II型受体。
本发明的T细胞可以通过各种非限制性方法从许多非限制性来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、腹水、胸腔积液、脾组织和肿瘤。在一些实施方案中，细胞可以衍生自健康供体或来自诊断为癌症的患者。在一些实施方案中，细胞可以是呈现不同表型特征的细胞的混合群体的一部分。例如，T细胞可以通过分离外周血单个核细胞(PBMC)，然后用特异性抗体活化、扩增获得。
在本发明各方面的一些实施方案中，所述T细胞衍生自对象的自体细胞。如本文所用，“自体”是指用于治疗对象的细胞、细胞系或细胞群源自所述对象。在一些实施方案中，所述T细胞衍生自异体细胞，例如源自与所述对象人类白细胞抗原(HLA)相容的供体。可以使用标准方案将来自供体的细胞转化为非同种异体反应性细胞，并根据需要进行复制，从而产生可以施用至一个或多个患者的细胞。
在另一方面，本发明提供一种用于产生慢病毒载体颗粒的试剂盒，其包含本发明的慢病毒载体、合适的包装载体、合适的包膜载体和/或合适的宿主细胞如293T细胞。所述试剂盒还可以包括细胞转染试剂。在另一方面，本发明提供一种用于在细胞中表达感兴趣的多肽的试剂盒，其包含本发明的慢病毒载体颗粒。
实施例
实施例中的统计学分析使用GraphPad软件(GraphPad Prism v5.0；GraphPad Software，San Diego，CA，USA)进行。通过配对t检验然后进行Newman-Keuls检验分析数据。结果表示为平均值±SEM。p值<0.05被认为是显著的。
实施例1、用于表达CAR的慢病毒载体的优化
用于转导CAR的慢病毒载体应含有所需的CAR转基因并能够在细胞中表达。设计了两种用于表达CAR的第三代慢病毒载体，分别为旧载体pPVLV1(图1A)和新载体pPVLV2(图1B)。pPVLV1含有531bp的长的人延伸因子1α(EF1α)启动子，pPVLV1含有212bp的截短的人EF1α启动子。两种载体中包含的各种元件及其描述见于下表1。
本申请实施例中待表达的CAR包含靶向CD19的scFv、人CD8的铰链和跨膜结构域、细胞内结构域4-1BB和CD3ζ。该靶向CD19的CAR的氨基酸如SEQ ID NO:16所示，核苷酸序列如SEQ ID NO:8所示。
表1.pPVLV1和pPVLV2慢病毒载体上的相关元件
Figure PCTCN2019084828-appb-000002
1)包括EF1α长启动子的pPVLV1载体(5,320bp，SEQ ID NO:1)
2)包括EF1α短启动子的pPVLV2载体(4,417bp，SEQ ID NO:2)
通过用gag/pol包装质粒、VSV-G包膜质粒和包含如上所述慢病毒载体序列的转移构建体转染293T细胞产生慢病毒上清液。简言之，将DNA混合物在Opti-MEM(Life  Technologies，Gaithersburg，MD，USA)混合，并与等体积的含有Lipofectamine 3000(Life Technologies)的Opti-MEM混合。在室温下孵育15分钟后，将所得混合物施加到293T细胞。在转染后24小时收集含有慢病毒的培养基。每次收集后，将上清液通过PVDF膜(0.45μm)过滤。将慢病毒收获物合并，并在4℃下储存，然后在20,000xg下超速离心1小时30分钟。将慢病毒颗粒重悬于PBS中。
图1示出了两种慢病毒载体的结构示意图，以及通过重叠的PCR产物检验慢病毒完整性的策略。设计了合适的引物，从使用随机引物逆转录的cDNA扩增重叠的片段F1-F5。具有预期大小的PCR产物可以证明慢病毒的完整性。
图2A示出了从病毒基因组逆转录出的cDNA扩增出的各DNA片段。令人意想不到的是，在含有P EF1α-L(长启动子)的病毒基因片段中观察到缺陷的基因位点。箭头指示具有非预期的DNA片段(左)。在含有P EF1α-S(短启动子)的病毒基因片段中没有观察到此现象。此种缺陷的病毒基因组可能影响滴度和转导效率。
为此，测试了两种慢病毒的滴度和转导效率。对于慢病毒滴定，将2×10 6个293T细胞接种到6孔板的每个孔中，并用一系列体积的浓缩慢病毒转导。转导后48小时后，将293T细胞从平板上分离。使用Alexa Fluor 488标记的山羊抗人IgG F(ab) 2通过流式细胞术检测CAR的存在。使用常规PCR检查5’LTR至3’LTR的病毒基因组RNA。
结果如图2B和C所示。具有P EF1α-L的病毒(基于pPVLV1载体)的转导效率(表达CAR的细胞比例)仅为9.95％，远远低于具有P EF1α-S的病毒(基于pPVLV2载体)的70.4％。此外，具有P EF1α-L的病毒(基于pPVLV1载体)的滴度也显著低于具有P EF1α-S的病毒(基于pPVLV2载体)的慢病毒。表明pPVLV2载体优于pPVLV1载体，而这可能是由于不同长度的EF1α启动子造成的。
实施例2、启动子影响CAR基因的转导和表达
为了进一步证明不同启动子的影响，基于pPVLV2构建了图3两种CAR-荧光素酶报告载体，其区别仅仅在与驱动转基因表达的启动子，其中CAR-19被克隆至P2A-Fluc(萤火虫荧光素酶)盒的上游，形成双顺反子(图3A和B)。
在所述载体转导进细胞中后，由于存在P2A自剪切肽的编码序列，在同一细胞将以大约1:1的比例表达出CAR-19和荧光素酶两种分子，其中荧光强度可反映出CAR-19转导效率(见图3C原理示意图)。图3D示出将两种慢病毒载体转导进293T细胞48小时后测定的荧光素酶活性。结果显示，用具有P EF1α-S的慢病毒载体转导的细胞荧光显著强于用具有P EF1α-L的慢病毒载体转导的细胞。进一步证明P EF1α-S显著改善了转基因在细胞的表达。
本实施例证明，常规的强启动子，531bp的EF1α启动子，在慢病毒载体中用于蛋白表达时，将令人意想不到地导致低转导效率。通过使用截短的EF1α启动子(212bp)可以显著提高转导效率，改善外源蛋白例如CAR在细胞中的表达。
实施例3、在细胞中共表达CAR和DNRII
TGF-β是重要的T细胞抑制因子，可能导致治疗性T细胞对靶细胞的杀伤作用减弱或消失。在临床上，TGF-β广泛表达于多种肿瘤组织中，显著抑制肿瘤特异性T细胞对肿瘤细胞的杀伤活性，是免疫治疗失败的重要原因。而显性负性TGF-β II型受体(dominant negative TGF-β receptor type II,DNRII)是TGF-β的负性调节受体，可以抑制TGF-β对T细胞的抑制作用。以下实施例研究在T细胞中共表达CAR和DNRII的效果。DNRII的氨基酸序列示于SEQ ID NO:17，其核苷酸序列示于SEQ ID NO:18。
首先，类似于实施例2，基于pPVLV2构建了图4(A和B)中两种CAR-19-DNRII载体，其区别仅仅在驱动转基因表达的启动子。其中CAR-19和DNRII位于同一开放读码框，中间包含2A多肽编码序列。图4C示出CAR-19和DNRII分子的结构示意图。其中DNRII缺失TGFBRII的胞内丝氨酸/苏氨酸激酶结构域，无法向下游传导信号。
将CAR-19和DNRII编码序列由2A编码序列隔开，置于同一开放读码框，由同一启动子驱动表达，可以最大程度确保所获得的经转导的细胞同时表达CAR-19和DNRII。因为如果CAR-19和DNRII在不同载体中分别转导至细胞，将可能造成部分细胞仅表达CAR-19，部分细胞仅表达DNRII，而共表达两种蛋白的细胞的比例将很低。另外，如果两种蛋白的表达由同一载体中不同启动子驱动表达，由于启动子效率的差异，同样会降低同时表达两种蛋白的细胞的比例。
将两种CAR-19-DNRII慢病毒载体以相等的MOI(感染复数)转导至293T细胞。使用标记的山羊抗人IgG F(ab) 2或抗DNRII抗体通过流式细胞术使用MACSQuant分析仪10检测CAR或DNRII的表达，并用FlowJo软件分析数据。
结果如图5所示，用具有P EF1α-S的慢病毒载体转导的293T细胞中CAR-19和DNRII表达均显著高于用具有P EF1α-L的慢病毒载体转导的293T细胞。
此外，还测试了两种CAR-19慢病毒载体以及两种CAR-19-DNRII慢病毒载体转导T细胞后的CAR-19和DNRII的表达。
健康供者来源的人外周血单个核细胞(PBMC)经过抗CD3/CD28 Dynabeads磁珠活化2天(珠：细胞＝3：1)，以1×10 6个细胞/ml重悬于补充有rhIL-2(200IU/mL)的IMSF100无血清培养基(LONZA，比利时)中。分别加入CAR-19和CAR-19-DNRII慢病毒上清液进行转导。之后在1,200×g下在32℃下离心2小时。24小时后，除去含有病毒载体的上清液。将细胞以3×10 5个细胞/ml悬浮于含rhIL-2(200IU/mL)的培养基，每2至3天更换新鲜培养基，扩增培养12天，获得表达CAR-19分子的CAR-T-19细胞和共表达CAR-19分子和DNRII分子的CAR-T-19-DNRII细胞。以在同样培养条件下进行培养，但未进行基因转导的PBMC为对照(NC)。使用流式细胞仪检测转导后获得的CAR-T细胞的各蛋白分子的表达。细胞每2-3天进行一次碘化丙啶(PI)染色，通过流式细胞仪检测细胞活性。细胞培养过程中，每2-3天使用台盼蓝染色法进行细胞计数(每个样本三次重复)，并计算细胞数量(均值±SD)。
结果如图6所示，未经转导的细胞(NC)不表达CAR-19或DNRII。使用包含P EF1α-S 的pPVLV2载体的CAR-T-19表达CAR-19(表达率为67.4％)；CAR-T-19-DNRII细胞既表达CAR-19(表达率为62.9％)，也表达DNRII(表达率为62.3％)。说明CAR-T-19-DNRII细胞中CAR-19和DNRII为共表达，且转导效率与单独转导CAR-19相当。使用P EF1α-L载体时，CAR-T-19-DNRII细胞中CAR-19和DNRII也为共表达，但是表达率显著降低；而在CAR-T-19细胞中，CAR-19的表达率也显著降低。
此外，如图7所示，CAR-T-19-DNRII细胞的活性与数量与CAR-T-19没有差异。
因此，本实施例确定pPVLV2载体的骨架(包含P EF1α-S)特别适合用于CAR在细胞中例如T细胞中的表达，尤其适合于CAR和其他蛋白如DNRII的共表达。此外，将CAR和DNRII编码序列至于同一开放读码框，可以实现两种分子的高共表达率。
实施例4、DNRII的表达降低TFG-β1诱导的SMAD2分子的磷酸化
TFG-β对T细胞的抑制作用是通过TFG-β与其受体结合后，进行SMAD2分子磷酸化实现的。
将转导9天后的CAR-T-19细胞和CAR-T-19-DNRII细胞与重组人TFG-β1(10ng/ml)共孵育24小时，进行被磷酸化的SMAD2(pSMAD2)的表达水平的分析。以GAPDH和未磷酸化的SMAD2分子作为对照，通过Western blot进行pSMAD2分子的相对定量。
具体而言，使用Bradford测定试剂盒(Sigma-Aldrich)测量全细胞裂解物的蛋白质浓度。将等量的蛋白质加载到SDS-PAGE凝胶的孔中，并将分离的蛋白质转移到PVDF膜(Thermo Scientific)。用TBST中的10％(w/v)脱脂乳封闭膜，然后与一抗(抗pSMAD2和抗SMAD2；来自Cell signaling Technologies，Danvers，MA，USA；全部以1:1000稀释)一起4℃孵育过夜。然后用TBST洗涤膜，并与HRP缀合的山羊抗兔IgG(1:2000稀释；Cell Signaling Technologies)在室温下孵育2小时。然后将膜暴露于ECL试剂(Thermo Scientific)，并使用发光图像分析仪(LAS-4000，Fuji Film，Tokyo，Japan)检测所得信号。
结果如图8所示，CAR-T-19-DNRII细胞中pSAMD2的水平较CAR-T-19细胞显著下降。说明DNRII的表达抑制了TGF-β信号通路关键信号传导分子SMAD2的磷酸化。
实施例5、经重组人TGF-β1处理的CAR-T-19-DNRII细胞和CAR-T-19细胞中IFN-γ和TNF-α表达
IFN-γ和TNF-α是T细胞对靶细胞进行杀伤的标志性细胞因子。这两种细胞因子表达水平高，说明T细胞对靶细胞的杀伤潜能高，反之，则杀伤潜能低。
将转导9天后的CAR-T-19细胞和CAR-T-19-DNRII细胞与或不与重组人TGF-β1(10ng/ml)共孵育24小时后，两种细胞分别与表达CD19分子的CD19 +-K562靶细胞共孵育24小时。为了定量IFN-γ和TNF-α的mRNA水平，收获每种混合细胞并使用PureLink RNA Mini试剂盒(Thermo Scientific，Waltham，MA，USA)提取总RNA。在DNase消化和使用Agilent 2100生物分析仪(Agilent Technologies，Palo Alto，USA)进 行浓度测定后，使用特异性引物和One-step SensiFAST SYBR Low-ROX试剂盒(Bioline，Maryland，USA)对总RNA样品进行实时定量RT-PCR分析，使用QuantStudio3实时PCR检测系统(Applied Biosystems，Foster City，CA，USA)。扩增18s rRNA为内部对照。通过ΔΔCt法计算表达水平，并使用公式2-ΔΔCt获得表达倍数。所有实验一式三份进行。
结果显示，经重组人TGF-β1处理后，CAR-T-19-DNRII细胞中IFN-γ和TNF-α表达显著高于CAR-T-19细胞(图9)。
实施例6、经重组人TGF-β1处理的CAR-T-19-DNRII细胞和CAR-T-19细胞对肿瘤靶细胞特异性杀伤
靶细胞杀伤实验使用转导12天的CAR-T-19细胞和CAR-T-19-DNRII细胞进行。
进行TDA释放测定以确定TGF-β1存在下CAR-T-19细胞和CAR-T-19-DNRII细胞对K562或CD19 +-K562的细胞毒活性。CAR-T-19细胞和CAR-T-19-DNRII细胞分别与重组人TGF-β1(10ng/ml)共孵育72小时。靶细胞用BA-TDA(Perkin Elmer，Norwalk，Connecticut，USA)标记15分钟后，按分别20:1、10:1、5:1、2.5:1的效应细胞(T细胞):靶细胞(肿瘤细胞)比例与效应细胞混合，共孵育4小时后检测TDA的释放(靶细胞裂解)。使用时间分辨荧光(TRF)读数器(Thermo Scientific)检测测定上清液的TDA释放。如下计算特异性裂解：％裂解＝(实验裂解-自发裂解)/(最大裂解-自发裂解)×100。
结果如图10所示，经过重组人TGF-β1处理后，CAR-T-19细胞对表达CD19的K562靶细胞杀伤作用降低至未加CAR-T细胞的背景水平(图10A)；而CAR-T-19-DNRII细胞对表达CD19的K562靶细胞杀伤作用几乎没有下降，与未加CAR-T细胞的杀伤作用有显著性差异(图10B)。说明DNRII有效逆转了TGF-β对T细胞杀伤的抑制作用。
Figure PCTCN2019084828-appb-000003
Figure PCTCN2019084828-appb-000004
Figure PCTCN2019084828-appb-000005
Figure PCTCN2019084828-appb-000006

Claims (18)

  1. 一种慢病毒载体，其包含用于指导编码感兴趣的多肽的核苷酸序列在宿主细胞中表达的截短的EF1α启动子，例如，所述截短的EF1α启动子是包含SEQ ID NO:13所示核苷酸序列的EF1α核心启动子。
  2. 权利要求1的慢病毒载体，其是非复制型慢病毒载体。
  3. 权利要求1或2的慢病毒载体，其还包含选自以下的至少一种元件：5’LTR、ψ元件、RRE元件、cPPT/CTS元件、用于插入编码感兴趣的多肽的核苷酸序列的多克隆位点、WPRE元件和3’LTR。
  4. 权利要求1-3中任一项的慢病毒载体，其包含可操作连接的5’LTR、ψ元件、RRE元件、cPPT/CTS元件、所述截短的EF1α启动子、WPRE元件和3’LTR，以及任选地，用于插入编码感兴趣的多肽的核苷酸序列的多克隆位点。
  5. 权利要求3或4的慢病毒载体，其中所述5’LTR包含SEQ ID NO:3或11所示核苷酸序列；所述ψ元件包含SEQ ID NO:4或12所示核苷酸序列；所述RRE元件包含SEQ ID NO:5所示核苷酸序列；所述cPPT/CTS元件包含SEQ ID NO:6所示核苷酸序列；所述WPRE元件包含SEQ ID NO:9或14所示核苷酸序列；其中所述3’LTR包含SEQ ID NO:10或15所示核苷酸序列。
  6. 权利要求1-5中任一项的慢病毒载体，其包含可操作连接的包含SEQ ID NO:11所示核苷酸序列的5’LTR，包含SEQ ID NO:12所示核苷酸序列的ψ元件，包含SEQ ID NO:5所示核苷酸序列的RRE元件，包含SEQ ID NO:6所示核苷酸序列的cPPT/CTS元件，包含SEQ ID NO:13所示核苷酸序列的截短的EF1α启动子，包含SEQ ID NO:14所示核苷酸序列的WPRE元件，包含SEQ ID NO:15所示核苷酸序列的3’LTR，以及任选地，用于插入编码感兴趣的多肽的核苷酸序列的多克隆位点。
  7. 权利要求1-6中任一项的慢病毒载体，其还包含编码感兴趣的多肽的核苷酸序列。
  8. 权利要求1-7中任一项的慢病毒载体，其中所述感兴趣的多肽是包括多种蛋白的融合多肽，所述融合多肽中的所述多种蛋白由自裂解肽分隔开。
  9. 权利要求1-8中任一项的慢病毒载体，其中所述感兴趣的多肽是包括第一蛋白和第二蛋白的融合多肽，所述融合多肽在第一蛋白和第二蛋白之间包含自裂解肽。
  10. 权利要求8或9的慢病毒载体，其中所述自裂解肽是2A多肽，例如，所述自裂解肽选自P2A、F2A、E2A或T2A多肽，或其功能性变体。
  11. 权利要求9或10的慢病毒载体，其中所述第一蛋白是癌症相关抗原特异性受体蛋白，例如T细胞受体(TCR)或嵌合抗原受体(CAR)。
  12. 权利要求9-11中任一项的慢病毒载体，其中所述第二蛋白是显性负性TGF-β II型受体。
  13. 权利要求12的慢病毒载体，其中所述显性负性TGF-β II型受体缺失TGF-β II 型受体的胞内信号传导结构域，例如，所述显性负性TGF-β II型受体包含SEQ ID NO:18所示的氨基酸序列。
  14. 一种制备慢病毒载体颗粒的方法，所述方法包括：
    a)使权利要求1-13中任一项所述的慢病毒载体、表达Gag和/或Pol的一或多种包装载体、表达包膜蛋白如VSV-G的包膜载体共转染合适的宿主细胞；
    b)培养转染的所述宿主细胞以使所述慢病毒载体包装成慢病毒载体颗粒；以及
    c)收获步骤b)中产生的慢病毒载体颗粒。
  15. 一种慢病毒载体颗粒，其包含权利要求1-13中任一项的慢病毒载体或通过权利要求14的方法制备。
  16. 权利要求15的慢病毒载体颗粒在制备治疗性T细胞中的用途，其中所述治疗性T细胞表达癌症相关抗原特异性受体蛋白，例如T细胞受体(TCR)或嵌合抗原受体(CAR)，以及任选的显性负性TGF-β II型受体。
  17. 一种制备治疗性T细胞的方法，包括用权利要求15的慢病毒载体颗粒转导T细胞。
  18. 权利要求17的方法，其中所述慢病毒载体颗粒的转导导致所述治疗性T细胞表达癌症相关抗原特异性受体蛋白，例如T细胞受体(TCR)或嵌合抗原受体(CAR)，以及任选的显性负性TGF-β II型受体。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021183643A1 (en) * 2020-03-11 2021-09-16 Immatics US, Inc. Wpre mutant constructs, compositions and methods thereof
US12123013B2 (en) 2023-11-03 2024-10-22 Immatics US, Inc. WPRE mutant constructs, compositions, and methods thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113832191B (zh) * 2021-11-26 2022-04-19 和元生物技术（上海）股份有限公司 慢病毒载体、系统及其应用
CN114107396B (zh) * 2021-11-26 2024-02-02 和元智造(上海)基因技术有限公司 慢病毒载体、系统及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104583231A (zh) * 2012-03-30 2015-04-29 免疫设计公司 具有对表达dc-sign的细胞改善的转导效率的慢病毒载体粒子
CN107099546A (zh) * 2017-06-07 2017-08-29 胜武（北京）生物科技有限公司 一种调控嵌合抗原受体表达的方法
WO2017207979A1 (en) * 2016-06-01 2017-12-07 University Of Sheffield Inhibitors of srsf1 to treat neurodegenerative disorders
CN108174604A (zh) * 2015-08-07 2018-06-15 西雅图儿童医院（Dba西雅图儿童研究所） 用于实体瘤靶向的双特异性car t细胞
CN108203720A (zh) * 2016-12-20 2018-06-26 上海生博生物医药科技有限公司 能靶向Her2并封闭PD-L1降低肿瘤免疫逃逸的CAR-T载体及其构建方法和应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1438075A4 (en) * 2001-10-02 2006-04-19 Inst Clayton De La Rech METHOD AND COMPOSITIONS ASSOCIATED WITH LENTIVIRAL VECTORS WITH LIMITED EXPRESSION AND ITS APPLICATIONS
WO2017103613A1 (en) * 2015-12-18 2017-06-22 Ucl Business Plc Treatment
AU2017230011A1 (en) * 2016-03-11 2018-09-27 2Seventy Bio, Inc. Genome edited immune effector cells
CN105602992B (zh) * 2016-03-17 2019-06-21 上海优卡迪生物医药科技有限公司 一种基于复制缺陷性重组慢病毒的car-t转基因载体及其构建方法和应用
US20190153064A1 (en) * 2016-04-15 2019-05-23 The Trustees Of The University Of Pennsylvania Compositions and methods of chimeric alloantigen receptor t cells
EP3487991B1 (en) * 2016-07-25 2022-09-07 The United States of America, as represented by The Secretary, Department of Health and Human Services Methods of producing modified natural killer cells and methods of use
WO2018071576A1 (en) * 2016-10-14 2018-04-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Treatment of tumors by inhibition of cd300f
AU2019261361A1 (en) * 2018-04-23 2020-11-19 Duke University Downregulation of SNCA expression by targeted editing of DNA-methylation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104583231A (zh) * 2012-03-30 2015-04-29 免疫设计公司 具有对表达dc-sign的细胞改善的转导效率的慢病毒载体粒子
CN108174604A (zh) * 2015-08-07 2018-06-15 西雅图儿童医院（Dba西雅图儿童研究所） 用于实体瘤靶向的双特异性car t细胞
WO2017207979A1 (en) * 2016-06-01 2017-12-07 University Of Sheffield Inhibitors of srsf1 to treat neurodegenerative disorders
CN108203720A (zh) * 2016-12-20 2018-06-26 上海生博生物医药科技有限公司 能靶向Her2并封闭PD-L1降低肿瘤免疫逃逸的CAR-T载体及其构建方法和应用
CN107099546A (zh) * 2017-06-07 2017-08-29 胜武（北京）生物科技有限公司 一种调控嵌合抗原受体表达的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KLOSS, C. C. ET AL.: "Dominant-Negative TGF-beta Receptor Enhances PSMA-Targeted Human CAR T Cell Proliferation And Augments Prostate Cancer Eradication", MOLECULAR THERAPY, vol. 26, no. 7, 5 July 2018 (2018-07-05), pages 1855 - 1866, XP055649123, DOI: 10.1016/j.ymthe.2018.05.003 *
ROITT ET AL., IMMUNOLOGY
SAMBROOKLEWIN ET AL.: "Molecular Cloning: A Laboratory Manual", article "Genes VIII"
See also references of EP3845656A4

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021183643A1 (en) * 2020-03-11 2021-09-16 Immatics US, Inc. Wpre mutant constructs, compositions and methods thereof
US11851673B2 (en) 2020-03-11 2023-12-26 Immatics US, Inc. WPRE mutant constructs, compositions, and methods thereof
US12123013B2 (en) 2023-11-03 2024-10-22 Immatics US, Inc. WPRE mutant constructs, compositions, and methods thereof

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