WO2019233281A1 - Récepteur antigénique chimérique ayant une structure du signal de récepteur cytotoxique naturel tronqué ou non tronqué, et utilisation associée - Google Patents

Récepteur antigénique chimérique ayant une structure du signal de récepteur cytotoxique naturel tronqué ou non tronqué, et utilisation associée Download PDF

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WO2019233281A1
WO2019233281A1 PCT/CN2019/088051 CN2019088051W WO2019233281A1 WO 2019233281 A1 WO2019233281 A1 WO 2019233281A1 CN 2019088051 W CN2019088051 W CN 2019088051W WO 2019233281 A1 WO2019233281 A1 WO 2019233281A1
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amino acid
acid sequence
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nkp44
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王恩秀
汪晨
张海
武国英
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南京卡提医学科技有限公司
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Definitions

  • the invention relates to the technical field of tumor immunotherapy, in particular to a chimeric antigen receptor carrying a truncated or untruncated natural cytotoxicity receptor signal structure and its application.
  • Chimeric antigen receptor is the core component of CAR-T. Using the characteristics of the ligand binding domain, CAR can redirect the specificity and reactivity of selected immune cells, thus conferring T cells an HLA-independent manner The ability to recognize tumor antigens allows CAR-engineered T cells to recognize a wider range of targets than the native T-cell surface receptor TCR.
  • the basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFV segment of the monoclonal antibody antigen-binding region), an extracellular hinge region, a transmembrane region, and an intracellular signal. Area.
  • TAA tumor-associated antigen
  • CRS cytokine release syndrome
  • CRS cancer-related fibroblasts
  • the traditional CAR-T is directly affected by the antigen-stimulated secretion of cytokines to the severity of CRS.
  • the tumor matrix composed of solid tumor-associated fibroblasts (CAFs) provides a physical barrier for CAR-T cell infiltration.
  • CAFs also secrete extracellular matrix proteins to transport T cells from cancer cells.
  • the metabolic environment of the solid tumor tumor microenvironment is not conducive to the persistence of traditional CAR-T cells, because once tumor formation is activated, tumor cells stop producing ATP through oxidative phosphorylation and convert to aerobic glycolysis Solution, causing the tumor environment to become acidic, which is the so-called "Wattock effect".
  • -Plutonium molecule which weakens the anti-tumor function of traditional CAR-T cells by attracting regulatory T cells (Tregs) to the tumor site. Tregs suppress the immune response, thus limiting the therapeutic effect of traditional CAR-T on solid tumors.
  • An object of the present invention is to provide a chimeric antigen receptor (CAR) carrying a signal structure of a natural cytotoxic receptor NKp44, which has better safety and more remarkable curative effect.
  • CAR chimeric antigen receptor
  • Another object of the present invention is to provide the application of the chimeric antigen receptor.
  • a third object of the present invention is to provide a signaling domain of a chimeric antigen receptor.
  • a fourth object of the present invention is to provide an application of the signal transduction domain.
  • a chimeric antigen receptor comprising: an antigen binding domain (scfv) and a signaling domain, wherein the signaling domain includes a first conductive domain and a second conductive domain, and the first conductive domain And a second conducting domain in tandem with an antigen binding domain.
  • the first conductive domain, antigen binding domain and second conductive domain in tandem in the chimeric antigen receptor according to the present invention become a multi-chain form capable of transmitting an activation signal after the antigen binding domain specifically binds the antigen. , The activation signal is transmitted to the immune cells expressing it, to achieve the role of immunotherapy.
  • the chimeric antigen receptor is a multi-chain structure chimeric antigen receptor. It uses a first conductive domain and a second conductive domain to form a signal transduction domain of the CAR.
  • the antigen binding domain can specifically bind to the target. And induces the activation of immune cells to produce an immune response.
  • the second conductive domain of the present invention is DAP12, and the second conductive domain is connected in series with the antigen-binding domain through T2A.
  • the DAP12 in the present invention is a transmembrane domain, which is widely present on the surface of natural killer cells, granulocytes, monocytes / macrophages, and is used to transmit activation signals. Its nucleotide sequence is as shown in SEQ ID NO.1. The amino acid sequence is shown in SEQ ID NO.2.
  • the T2A according to the present invention is used to tandem a second conductive domain and an antigen-binding domain. Its nucleotide sequence is shown in SEQ ID NO. 3 and its amino acid sequence is shown in SEQ ID NO. 4.
  • the first conductive domain according to the present invention may be truncated or untruncated NKp44, NKp46, NKp30 or NKG2D, wherein the full-length nucleotide sequence of the NKp44 gene can be found in NCBI, GenBank: AJ225109.1, amino acid sequence See NCBI, GenBank: CAB39168.1; For the full-length nucleotide sequence of the NKp46 gene, see NCBI, Accession: NM_004829.6, for the amino acid sequence, see NCBI, Accession: NP_004820; For the full-length nucleotide sequence of the NKp30 gene, see NCBI, Accession: NM_147130.2, amino acid sequence see NCBI, Accession: NP_667341.1; NKG2D gene full-length nucleotide sequence see NCBI, Accession: AF461811, amino acid sequence see NCBI, GenBank: AAL65233.1.
  • the present invention also provides a preferred first conductive domain, which is selected from the truncated NKp44 amino acid sequence and named as NKp44 cut .
  • the NKp44 cut is a polypeptide of 100-160 amino acids at the C-terminus of the full-length NKp44 amino acid sequence, preferably a polypeptide of 120-160 amino acids at the C-terminus of the NKp44 full-length amino acid sequence, and more preferably 140-160 at the C-terminus of the NKp44 full-length amino acid sequence.
  • a polypeptide having an amino acid is either an amino acid sequence having 80% or more identity with the aforementioned polypeptide sequence, or an amino acid sequence having 85% or more identity with the sequence, or an amino acid sequence having 90% or more identity with the sequence, or Or an amino acid sequence having 95% or more identity with the sequence.
  • the present invention also provides a preferred first conductive domain, which is a 160-terminal polypeptide of the full-length amino acid sequence of NKp44.
  • the amino acid sequence is shown in SEQ ID NO.8, and its nucleotide sequence is shown in SEQ ID NO.7. As shown.
  • the antigen-binding domain according to the present invention can be routinely selected in the art according to different tumor targets.
  • the chimeric antigen receptor according to the present invention is DAP12, T2A, an antigen-binding domain, and a first transduction domain, which are tandemly connected in sequence through 2-10 arbitrary amino acids.
  • the sequence and number of 2-10 arbitrary amino acids of the present invention have no significant effect on the efficacy of the chimeric antigen receptor described in the present application, and may be any 2-10 amino acid sequence.
  • the chimeric antigen receptor according to the present invention may use, for example, a retroviral vector to transfer a nucleic acid encoding the chimeric antigen receptor to an immune cell such as a T cell.
  • an immune cell such as a T cell.
  • the chimeric antigen receptor binds a target antigen
  • the first conductive structure The domain and antigen-binding domain are separated to form an activation signal that is transmitted to the immune cells that express it.
  • the invention also provides an immune cell carrying the chimeric antigen receptor.
  • the invention also provides the application of the chimeric antigen receptor or immune cells with the chimeric antigen receptor in tumor immunotherapy.
  • the present invention provides a signal transduction domain including a first transduction domain and a second transduction domain.
  • the first conductive domain and the second conductive domain of the present invention after the antigen-binding domain specifically binds to the antigen, becomes a multi-chain form capable of transmitting an activation signal, and transmits the activation signal to the immune cells expressing it, thereby realizing The role of immunotherapy.
  • the second conductive domain of the present invention is DAP12.
  • the DAP12 in the present invention is a transmembrane domain, which is widely present on the surface of natural killer cells, granulocytes, monocytes / macrophages, and is used to transmit activation signals. Its nucleotide sequence is as shown in SEQ ID NO.1. The amino acid sequence is shown in SEQ ID NO.2.
  • the first conductive domain according to the present invention may be truncated or untruncated NKp44, NKp46, NKp30 or NKG2D, wherein the full-length nucleotide sequence of the NKp44 gene is described in NCBI, GenBank: AJ225109.1, amino acid sequence See NCBI, GenBank: CAB39168.1; For the full-length nucleotide sequence of the NKp46 gene, see NCBI, Accession: NM_004829.6, for the amino acid sequence, see NCBI, Accession: NP_004820; For the full-length nucleotide sequence of the NKp30 gene, see NCBI, Accession: NM_147130.2, amino acid sequence see NCBI, Accession: NP_667341.1; NKG2D gene full-length nucleotide sequence see NCBI, Accession: AF461811, amino acid sequence see NCBI, GenBank: AAL65233.1.
  • the present invention also provides a preferred first transduction domain, which is selected from the truncated NKp44 amino acid sequence and named as NKp44 cut
  • the NKp44 cut according to the present invention is a polypeptide of 100-160 amino acids at the C-terminus of the full-length NKp44 amino acid sequence, preferably a polypeptide of 120-160 amino acids at the C-terminus of the NKp44 full-length amino acid sequence, and more preferably the NKp44 full-length amino acid sequence C.
  • a 140-160 amino acid terminal polypeptide or an amino acid sequence having 80% or more identity with the aforementioned polypeptide sequence, or an amino acid sequence having 85% or more identity with the sequence, or 90% or more identity with the sequence Or an amino acid sequence with 95% or more identity to the sequence.
  • the present invention also provides a preferred first conduction domain, when the NKp44 full-length amino acid sequence of the 160-terminal polypeptide of 160 amino acids, the amino acid sequence is shown in SEQ ID NO. 8, and its nucleotide sequence is shown in SEQ ID ID NO. 7 is shown.
  • the transmembrane receptor DAP12 is combined with the first conductive domain to form the signaling domain of the CAR.
  • the CAR specifically binds to a ligand in the target, it can induce the activation of immune cells To produce an immune response.
  • the invention also provides the application of the signaling domain in a chimeric antigen receptor or tumor immunotherapy.
  • the CAR structure involved in the present invention has confirmed its significant effect on solid tumors through in vitro functional experiments. Therefore, the present invention can not only be applied to the treatment of blood tumors, but also expand the application of CAR-T in the treatment of solid tumors;
  • the CAR structure involved in the present invention has stronger killing ability in vitro and positive antitumor activity.
  • Figure 1 is a lentiviral vector containing different signal domain structures
  • Figure 2 shows the positive expression rate of CAR structure that recognizes MSLN antigen on the surface of T cells by flow cytometry after 7 days of MSLN4CAR-T cell infection with lentivirus;
  • Figure 3 shows the proliferation of CAR-T cells after lentivirus infection
  • Figure 4 is the IL-2 secretion of MSLN4CAR-T cells under antigen stimulation
  • FIG. 5 shows the secretion of IFN- ⁇ by MSLN4CAR-T cells under antigen stimulation
  • Figure 6 is the secretion of IL-2 and IFN- ⁇ by CAR-T in different groups under antigen stimulation
  • Figure 7 shows the killing effect of CAR-T in different groups on antigen-positive tumor cell lines.
  • test materials used in the following examples were purchased from conventional biochemical reagent stores.
  • NKp44 cut truncated natural cytotoxicity The body (referred to as NKp44 cut ), its structure is shown in Figure 1.
  • the nucleotide sequence of DAP12 is shown in SEQ ID NO.1, the amino acid sequence is shown in SEQ ID NO.2, the nucleotide sequence of T2A is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4
  • the nucleotide sequence of the anti-human mesothelin single chain antibody (MSLN) scfv is shown in SEQ ID NO.5
  • the amino acid sequence is shown in SEQ ID NO.6
  • the full-length nucleotide sequence of the NKp44 gene is shown in NCBI, GenBank: AJ225109.1, amino acid sequence see NCBI, GenBank: CAB39168.1
  • nucleotide sequence of NKp44 cut is shown in SEQ ID NO.7
  • amino acid sequence is shown in SEQ ID NO.8, nucleoside of KIRS2 See the patent for acid and amino acid sequences (targeted cytotoxic cells with chimeric receptors for adoptive immunotherapy, publication number: CN
  • pELNS Dap12-T2A-MSLN-KIRS2 is kept by Nanjing Kati Medical Technology Co., Ltd., or according to the literature (Enxiu Wang et al. Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors. 2015, Cancer Immunology Research, 3 (7): 815) was used to construct.
  • the truncated NKp44 cut gene synthesis was synthesized by Nanjing Kingsray Biotechnology Company and provided pUC19-NKp44 cut plasmid.
  • the plasmids pELNS Dap12-T2A-MSLN-KIRS2 and pUC19 -NKp44 cut was double-digested by NheI and SalI (purchased from Takara).
  • the digestion reaction was performed according to the instructions to obtain a pELNS Dap12-T2A-MSLN fragment of about 8900bp, a truncated NKp44 cut fragment of about 483bp of DNA fragment, and then recovered Kit (Takara company) for DNA fragment recovery, the specific method is described in the instruction manual, the obtained pELNS Dap12-T2A-MSLN and NKp44 cut genes were recovered, and then the target fragment NKp44 cut and the vector fragment pELNS Dap12-T2A-MSLN were ligated by T4 ( (Purchased from Takara)), and obtained a lentiviral vector expressing a chimeric antigen receptor, named pELNS Dap12-T2A-MSLN-
  • lentiviral vector MSLN4 5 ⁇ L of the lentiviral vector MSLN4 was transformed into E. coli TOP10 competent cells (purchased from Nanjing Anjieyou Biotechnology Co., Ltd.), and monoclonals were picked after 16 hours of culture at 37 ° C. Plasmids were extracted using a plasmid extraction kit (purchased from Takara). The specific method is described in the instructions.
  • pELNS MSLN-CD8 ⁇ -4-1BB-CD3 ⁇ (MSLN1 for short); pELNS Dap12-T2A-MSLN (for short MSLN2); pELNS Dap12-T2A-MSLN-KIRS2 (for short MSLN3); pELNS Dap12-T2A-MSLN -NKp44 cut (abbreviated MSLN4); pELNS Dap12-T2A-MSLN-NKp44 wt (abbreviated MSLN5) lentiviral vector.
  • the lentivirus is packaged using the calcium phosphate method.
  • the specific steps are as follows:
  • the cell density is about 80% -90%, at this time you can start transfection
  • plasmid DNA and calcium chloride solution taking a T150 bottle as an example, 28ug pRSV.rev (purchased from Invitrogen), 28ug pGAG-Pol (purchased from Invitrogen), 11ug pVSVG (purchased from Invitrogen), 23ug recombinant lentivirus expression plasmid pELNS Dap12-T2A-MSLN-NKp44 cut was added to 1.5 ml of calcium chloride solution and mixed.
  • the two supernatants collected were pooled together and filtered through a 0.45 ⁇ m filter to remove cell debris.
  • 293T cells were plated into 24-well plates before infection, and 200 ⁇ L of purified concentrated virus was added to 293T cells. After 24 hours, the medium was exchanged with DMEM medium containing 10% FBS (wt). After 72 hours of infection, the cells were treated at 1200 r / min. Centrifuge for 5 min to collect cells and extract the genome.
  • the genomic extraction kit was purchased from Takara Company and operated according to the kit instructions
  • the reaction system is as follows: Probe qPCR Mix 12.5 ⁇ L (purchased from Takara), upstream primer 0.5 ⁇ L (synthesized from Nanjing Kingsray), downstream primer 0.5 ⁇ L (synthesized from Nanjing Kingsray), probe 1 ⁇ L (from Nanjing Kingsray) Synthesis), template 2 ⁇ L, sterilized water 8.5 ⁇ L, reaction system is 25 ⁇ L, reaction conditions were set according to the instructions, after the reaction, the data was analyzed by analysis software, and the virus titer was calculated according to the standard curve. The calculation results showed that the virus titer was 1 ⁇ 10 6 TU / ml.
  • lymphocyte separation solution purchased from Tianjin Ouyang Biological Products Technology Co., Ltd.
  • gradient centrifugation 3000r / min, centrifugation for 30min.
  • the centrifuge tube is layered from top to bottom: the first layer is the plasma layer ; The second layer is the white blood cell layer of lymphocytes; the third layer is a transparent separating liquid layer; the fourth layer is a red blood cell layer.
  • the white membrane layer of lymphocytes was aspirated, washed twice with PBS, centrifuged twice at 1500 r / min, centrifuged for 10 min, and the cells were resuspended in PBS.
  • 5% autologous plasma + 300 IU / ml recombinant human IL-2 + KBM581 complete medium was cultured Peripheral blood mononuclear cells.
  • Newly prepared mononuclear cells PBMC were cultured in complete medium containing 5% autologous plasma + 300IU / ml recombinant human IL-2 + KBM581, IL-2 was purchased from R & D Systems, KBM581 was purchased from Corning, and CD3 / CD28 Dynabeads was added on day 0 for immunization. Magnetic beads (purchased from invitrogen) activated T cells. Lentiviral infection was performed in the first 3 days. Lentiviral vectors corresponding to 0.25MOI were added. Uninfected T lymphocytes were used as blank controls. After 48 hours, the medium was replaced with 5% autologous plasma. + 300IU / ml recombinant human IL-2 + KBM581 complete medium was continued for 7-9 days.
  • Virus-infected T cells cultured to day 7 were centrifuged at 1200 r / min for 5 min. The supernatant was discarded to collect the cells, and the cells were resuspended with a PBS solution containing 1% FBS in volume fraction, and the cells were adjusted to a density of 1 ⁇ 10 5 cells / ml, add biotin-labeled goat anti-mouse F (ab) 2 (Jackson ImmunoResearch), then add Streptavidin-PE (BD Biosciences), incubate for 15 min at 4 ° C, wash twice with PBS solution, and perform flow cytometry The results showed that after 7 days of culture, the CAR-T cell CAR positive rate was 41% in the MSLN1 virus infection group, 52% in the MSLN2 virus infection group, 59% in the MSLN3 virus infection group, and MSLN4 virus. The positive rate in the infected group was 61% ( Figure 2), and the positive rate in the MSLN5 virus infected
  • T cells were infected by each group of viruses, the T cells were counted every 1-2 days with 5% autologous plasma + 300 IU / ml recombinant human IL-2 + KBM581 complete medium. Then observe the growth of T lymphocytes, and the results are shown in Figure 3. The results show that cells infected with CAR-expressing virus can still form typical proliferating clones. By counting the cells and plotting the cell proliferation curve, we can see that the proliferation of infected MSLN4CAR-T cells is similar to that of MSLN1, MSLN2, MSLN3, and MSLN5CAR-T. Slightly less proliferative capacity than non-infected T cells (NTD in Figure 3).
  • Example 4 Detection of cytokine secretion by CAR-T cells infected by a virus
  • the cytokine detection was performed using Elisa's method using R & D's kit.
  • Sample loading set blank wells (the blank control wells are replaced with water, and the enzyme-labeled reagents and biotin-labeled antibodies are handled as usual) and the sample wells to be tested. Add 100 ⁇ L of samples to the sample wells on the enzyme-labeled coating plate. Add the sample to the bottom of the well of the microtiter plate, try not to touch the wall of the well, and shake gently to mix
  • Color development Add 100 ⁇ L of color development solution to each well, mix gently by shaking, and incubate at room temperature for 20 min in the dark
  • MSLN4CAR-T Select target cells with different antigen expression levels to co-culture with MSLN4CAR-T, detect the levels of IL-2 and IFN- ⁇ secreted by MSLN4CAR-T in response to antigen stimulation, and select OVCAR3 (MSLN high expression) and SKOV3 (MSLN low) Expression), 293T (MSLN negative), to show that MSLN4CAR-T specifically secretes IL-2 and IFN- ⁇ when stimulated by MSLN antigen, the results reflect that MSLN4CAR produces target cells with different levels of antigen expression Gave a different response.
  • MSLN1, MSLN2, MSLN3, MSLN4, MSLN5CAR-T were co-cultured with OVCAR3, the levels of IL-2 and IFN- ⁇ cytokines secreted by each group were compared.
  • the results are shown in Figure 6.
  • the secretion level of MSLN4CAR-T was significantly increased (since the antitumor effect of MSLN2CAR-T is very weak, so there is no secretion of cytokines), and the secretion of MSLN1 and MSLN3 is to some extent Decline.
  • In vitro cytokine secretion results show that MSLN4CAR-T and MSLN5CAR-T produce lower levels of cytokines, which is likely to improve clinical application safety.
  • OVCAR3 cells MSLN overexpressing cell line
  • SKOV3 cells MSLN underexpressing cell line
  • 293T MSLN negative cell line
  • effector cells MSLN1, MSLN2, MSLN3, MSLN4, and MSLN5 respectively. cell.
  • RTCA real-time cell analysis system

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Abstract

La présente invention concerne un récepteur antigénique chimérique (CAR), comprenant : un domaine structural de liaison à l'antigène (scfv) et un domaine structural de transduction de signal, le domaine structurel de transduction de signal comprenant un premier domaine structurel de transduction et un second domaine structural de transduction, le domaine structurel de liaison à l'antigène étant connecté en tandem entre le premier domaine structurel de transduction et le second domaine structurel de transduction. La structure CAR selon la présente invention possède un très faible taux de cytokines sécrétées lorsqu'elle est stimulée par des antigènes, ce qui permet de mieux garantir la sécurité en utilisation clinique, c'est-à-dire une sécurité d'utilisation clinique élevée ; la capacité de destruction de cellules tumorales positives à l'antigène in vitro est plus forte, et l'activité anti-tumorale est meilleure.
PCT/CN2019/088051 2018-06-08 2019-05-23 Récepteur antigénique chimérique ayant une structure du signal de récepteur cytotoxique naturel tronqué ou non tronqué, et utilisation associée WO2019233281A1 (fr)

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