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WO2021036793A1 - Immunothérapie induite par pyroptose - Google Patents

Immunothérapie induite par pyroptose Download PDF

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WO2021036793A1
WO2021036793A1 PCT/CN2020/108776 CN2020108776W WO2021036793A1 WO 2021036793 A1 WO2021036793 A1 WO 2021036793A1 CN 2020108776 W CN2020108776 W CN 2020108776W WO 2021036793 A1 WO2021036793 A1 WO 2021036793A1
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tumor
ether
phe
gasdermin
cells
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PCT/CN2020/108776
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Feng Shao
Zhibo LIU
Qinyang WANG
Yupeng Wang
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National Institute Of Biological Sciences, Beijing
Peking University
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    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
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Definitions

  • Bioorthogonal chemistry capable of operating in the living system is desirable for dissecting complex biological processes such as cell death and immunity 1, 2 .
  • Recent studies in innate immunity identify a gasdermin family of pore-forming proteins that executes inflammasome-dependent or -independent pyroptosis 3-9 . Pyroptosis is proinflammatory but its exact immunologic effect in disease contexts, particularly cancer immunity, is unclear.
  • a bioorthogonal chemical system e.g., a cell-enterable cancer-imaging probe Phe-BF 3 specifically desilylates and “cleaves” a silyl ether-containing carbamate linker
  • the linker was attached to gold nanoparticle (NP) vehicle
  • NP gold nanoparticle
  • the system achieved tumor-selective targeting for controlled release of a client GFP protein from the endocytosed NPs.
  • Application of the system to a gasdermin triggered “clean” pyroptosis at the death execution step. Tumor cell pyroptosis induced by Phe-BF 3 -released gasdermin causes nearly complete regression of tumorgrafts including the 4T1 mammary carcinoma.
  • the invention provides method and compositions for treating cancer by activating a pore-forming, pyrpotogenic molecule (e.g. a gasdermin) , wherein the activating induces tumor cell pryoptosis and causes tumor regression by T-cell mediated anti-tumor immunity. Moreover, activation transforms the tumor from immunologically cold to hot, and synergizes with anti-PD1 therapy.
  • a pore-forming, pyrpotogenic molecule e.g. a gasdermin
  • the invention provides a method of inducing an antitumor immune response in a person in need thereof, comprising: (a) administering to the person a pyroptosis activator; and (b) detecting a resultant pyroptosis-induced antitumor immune response.
  • the method comprises: administering to the person an anti-PD1 therapeutic wherein the pyroptosis activator and the anti-PD1 therapeutic synergistically induce tumor regression in the person; and detecting a resultant tumor regression in the person;
  • the pryoptosis activator is a gasdermin agonist
  • the pyroptosis activator is a bioothogonal agent that releases an activationally restrained gasdermin in tumor cells of the person;
  • the pyroptosis activator is a bioothogonal agent that releases an activationally restrained gasdermin in tumor cells of the person, wherein the gasdermin is conjugated to a matrix (such as a nanoparticle) in the cells, and the biothorganal agent releases (deconjugates) the gasdermin from the matrix;
  • the gasdermin is activationally restrained though a silyl ether linkage, and the agent is an organotrifluoroborate that cleaves the linkage and thereby releases the gasdermin from activational restraint;
  • the linker comprises an ortho-carbamoylmethylene silyl-phenolic ether, in which the carbamate carbon is linked to the gasdermin;
  • the ortho-carbamoylmethylene silyl-phenolic ether comprises a silyl ether moiety selected from: trimethylsilyl ether (TMS) , triethylsilyl ether (TES) , tert-butyldimethylsilyl ether (TBS/TBDMS) , tert-butyldiphenylsilyl ether (TBDPS) , and triisopropylsilyl ether (TIPS) ;
  • TMS trimethylsilyl ether
  • TES triethylsilyl ether
  • TBS/TBDMS tert-butyldimethylsilyl ether
  • TPS tert-butyldiphenylsilyl ether
  • TIPS triisopropylsilyl ether
  • the organotrifluoroborate comprises a boroamino acid (BAA) , e.g. PheBF3; and/or
  • BAA boroamino acid
  • the activationally restrained gasdermin is conjugated to a nanoparticle (NP) .
  • the invention provides a method for releasing in a cell a client molecule from a silyl ether containing linker, comprising: introducing into the cell an organotrifluoroborate under conditions wherein the organotrifluoroborate reacts with the silyl ether to desilylate and release from the linker the client molecule; and optionally detecting resultant release of the client molecule.
  • the linker comprises an ortho-carbamoylmethylene silyl-phenolic ether, in which the carbamate carbon is linked to the client molecule;
  • the linker comprises a silyl ether that is trimethylsilyl ether (TMS) , triethylsilyl ether (TES) , tert-butyldimethylsilyl ether (TBS/TBDMS) , tert-butyldiphenylsilyl ether (TBDPS) , or triisopropylsilyl ether (TIPS) ;
  • TMS trimethylsilyl ether
  • TES triethylsilyl ether
  • TBS/TBDMS tert-butyldimethylsilyl ether
  • TDPS tert-butyldiphenylsilyl ether
  • TIPS triisopropylsilyl ether
  • the linker is joined to a nanoparticle
  • the organotrifluoroborate comprises a boroamino acid (BAA) , e.g. PheBF3;
  • BAA boroamino acid
  • the client molecule is a label (e.g. GFP) , a cytotoxic drug (e.g. imatinib, pemetrexed) , or a tumor suppressor protein (e.g. gasdermin) ;
  • a label e.g. GFP
  • a cytotoxic drug e.g. imatinib, pemetrexed
  • a tumor suppressor protein e.g. gasdermin
  • the cell is a cancer cell
  • the linker is joined to a nanoparticle, the client molecule is a gasdermin, the cell is a cancer cell of a tumor, and the method activates the gasdermin in the cell to stimulate pyroptosis in the cell and achieve anti-tumor immunotherapy;
  • the introducing step comprises administering the organotrifluoroborate to a person in need of anti-tumor immunotherapy, and optionally detecting a resultant pyroptosis-induced antitumor immune response; and/or
  • the method further comprises administering to the person an anti-PD1 therapeutic wherein the organotrifluoroborate and the anti-PD1 therapeutic synergistically induce tumor regression in the person; and optionally, detecting a resultant tumor regression in the person.
  • compositions, reagents and kits formulated and adapted specifically for the subject methods.
  • Figs. 1a-g A desilylation-based orthogonal chemistry by which Phe-BF 3 efficiently releases a client molecule from a silyl ether-containing carbamate linker.
  • Figs. 2a-h. Phe-BF 3 -mediated desilylation can release GFP from the silyl ether carbamate-linked NP_GFP in vitro and selectively in tumors in mice.
  • c d, Assays of Phe-BF 3 desilylation-induced release of GFP from the NP_GFP conjugates in mammalian cells.
  • HeLa, EMT6, 4T1, or priBMDM (primary bone marrow-derived macrophage) cells were first treated with NP_GFP (1 mg/mL) for 12 h and then for another 24 h with Phe-BF 3 (100 ⁇ M) or NaF as a control.
  • c Cell lysates were centrifuged and subjected to anti-GFP and anti-GAPDH immunoblotting analyses.
  • d Representative confocal fluorescence images of the treated priBMDM cells (scale bar, 20 ⁇ m) .
  • e Representative PET-CT 3D projection images of tumor-bearing mice intravenously injected with [ 89 Zr] GFP, [ 89 Zr] NP_GFP or [ 18 F] Phe-BF 3 .
  • t tumor; l, liver; k, kidney; gb, gallbladder.
  • Assay of Phe-BF 3 desilylation-induced release of mNeonGreen-NLS from the NP_mNeonGreen-NLS conjugates in tumor-bearing mice. Representative confocal fluorescence images of tumor sections.
  • Phe-BF 3 -mediated desilylation can release gasdermin from NP_GA3 to trigger pyroptotic cell death.
  • NP_GA3 Design of Phe-BF 3 -mediated desilylation of the silyl ether carbamate-linked NP_GA3 for releasing the gasdermin from the NP and inducing pyroptosis.
  • Purified gasdermin-N and -C noncovalent complex (N+C) was conjugated to the NP to generate NP_GA3.
  • b-d Pyroptosis assay of cultured mammalian cells treated with the NP_GA3 conjugates and Phe-BF 3 .
  • HeLa, EMT6, 4T1, or priBMDM cells were treated as indicated.
  • NP+GA3 means the unconjugated GSDMA3 protein (N+C) mixed with the NP.
  • GA3 Mut the pore-forming activity-deficient E14K/L184D mutant version of GSDMA3 (N+C) .
  • b Phase-contrast images of HeLa (Left) and EMT6 (Right) cells (arrows, cells with pyroptotic morphology) .
  • c Flow cytometry plots of propidium iodide (PI) and Annexin V-fluorescein isothiocyanate (FITC) -stained cells.
  • d Percentages of PI/Annexin V-positive pyroptotic cells measured by the flow cytometry analyses. Data are shown as mean ⁇ s.d. from three biological replicates; two-tailed unpaired Student’s t-test was performed (***P ⁇ 0.001, ****P ⁇ 0.0001) . All data are representative of three independent experiments.
  • Figs. 4a-h Tumor cell pyroptosis induced by NP_GA3+Phe-BF 3 treatment triggers tumor regression in mice.
  • a Representative PET-CT 3D projection images of 4T1 tumor-bearing mice intravenously injected with [ 89 Zr] GA3, [ 89 Zr] NP_GA3 or [ 18 F] Phe-BF 3 .
  • t tumor; l, liver; k, kidney; gb, gallbladder.
  • b-h Assays of tumor growth in mice treated with NP_GA3+Phe-BF 3 .
  • c-h The tumor-bearing mice were intravenously injected (i.v. ) or intratumorally injected (i.t. ) with NP_GA3 or Phe-BF 3 alone or in combination.
  • GA3 Mut the pore-forming activity-deficient E14K/L184D mutant version of GSDMA3 (N+C) .
  • c, g Tumor volume of individual mouse at indicated time points after implantation.
  • d, h Average tumor volumes of each group of mice (P values are included in Fig. 9c for d) .
  • e, f Photograph (e) and weight (f) of the 4T1 tumors on day 26 after inoculation.
  • a PI staining assay of NP_GA3+Phe-BF 3 -induced tumor cell pyroptosis in mice.
  • Prior to assay PI was intravenously injected into the mice and shown are representative images of the tumor sections (scale bar, 100 ⁇ m) .
  • Assays of tumor growth in Nu/Nu mice treated with NP_GA3+Phe-BF 3 (6-8 mice per group) The treatment scheme is the same as depicted in Fig. 4b.
  • TILs tumor-infiltrating lymphocytes
  • c Numbers of tumor-infiltrating lymphocytes (TILs) per gram of 4T1 tumors and percentages of Treg cells. Data were acquired from 6 (the PBS and NP_GA3+Phe-BF 3 groups) or 7 (the NP_GA3 Mut +Phe-BF 3 group) mice from three independent experiments.
  • d Effect of antibody depletion of T cells on NP_GA3+Phe-BF 3 -induced tumor regression.
  • e, f scRNA-Seq analyses of the effect of NP_GA3+Phe-BF 3 treatment on tumor immune microenvironment.
  • t-SNE stochastic neighbor embedding
  • g-j Assay of the synergistic effect between NP_GA3+Phe-BF 3 treatment and anti-PD1 blockage therapy on tumor growth.
  • g 4T1 tumor growth curve.
  • h i, Photograph (h) and weight (i) of 4T1 tumors at the end of indicated treatments.
  • j Scheme for the combined treatment.
  • b-d g, i, Data (mean ⁇ s.e.m.
  • Figs. 6a-d Phe-BF 3 -mediated desilylation of a designed silyl ether-containing carbamate linker and synthetic routes of related compounds.
  • a Chemical structures of silyl-phenolic ether-conjugated coumarin derivatives.
  • b Proposed mechanism for Phe-BF 3 catalyzing desilylation of the silyl ether that triggers decarboxylation on the carbamate and therefore release of the coumarin.
  • c Synthetic route of TESO-Coumarin. TBSO-Coumarin (TBSO-C) and TIPSO-Coumarin (TIPSO-C) were synthesized via a similar strategy.
  • d Synthetic route of the silyl ether-containing carbamate linker used to conjugate the NP.
  • Figs. 7a-d Assay of Phe-BF 3 desilylation-induced release of GFP from NP_GFP and biodistribution of [ 89 Zr] GFP and [ 89 Zr] NP_GFP in mice.
  • a Workflow of assaying Phe-BF 3 desilylation-induced release of GFP from NP_GFP in vitro. The samples were subjected to immunoblotting and Coomassie blue staining analysis in Fig. 2b.
  • b Expression of LAT1 transporter in the four cells assayed in Fig. 2c.
  • c Representative dynamic PET-CT 3D projection images of 4T1 tumor-bearing mice at 1, 6, 12, 18 h after intravenous injection of [ 89 Zr] GFP or [ 89 Zr] NP_GFP.
  • t tumor; l, liver.
  • d Representative confocal fluorescence images of HeLa cells transfected with a plasmid expressing mNeonGreen-NLS (scale bar, 20 ⁇ m) .
  • the nuclear localization sequence (NLS) is derived from the SV40 protein.
  • Data (b, d) are representative of two independent experiments.
  • Figs. 8a-c Phe-BF 3 -mediated desilylation can release gasdermin from NP_GA3 to trigger cell death.
  • a, b Preparation of the gasdermin-N and -C noncovalent complex of GSDMA3 protein (GA3- (N+C) ) used for conjugation onto the NP.
  • Engineered GSDMA protein containing a PreScisson protease (PPase) cleavage site between the gasdermin-N and -C domain was recombinantly purified and cleaved in vitro to obtain the GA3- (N+C) protein.
  • a Coomassie blue staining of the prepared GSDMA3 proteins.
  • b ATP-based viability analysis of mouse CT26 cells electroporated with the prepared GSDMA3 protein.
  • c ATP-based viability analysis of HeLa, EMT6 and 4T1 cells treated with NP_GA3 or Phe-BF 3 alone or in combination.
  • GA3 Mut the pore-forming activity-deficient E14K/L184D mutant version of GSDMA3 (N+C) .
  • b, c Data are shown as mean ⁇ s.d.; two-tailed unpaired Student’s t-test was performed (****P ⁇ 0.0001) . All data are representative of at least three independent experiments.
  • Figs. 9a-e Biodistribution of [ 89 Zr] GA3 and [ 89 Zr] NP_GA3 and evaluation of the side effect of NP_GA3+Phe-BF 3 treatment in mice.
  • a, b Dynamic PET-CT 3D projection images of 4T1 tumor-bearing mice at 2, 6, 12, 18 h after the intravenous injection of [ 89 Zr] GA3 or [ 89 Zr] NP_GA3.
  • t tumor; l, liver.
  • c P values for tumor regression data in Fig. 4d (two-tailed unpaired Student’s t-test) .
  • d Records of mouse body weight after indicated treatments (8 or 9 mice per group) (data are shown as mean ⁇ s.d.
  • Figs 10a-g Tumor cell pyroptosis induced by NP_GA3+Phe-BF 3 treatment increases the tumor-infiltrating lymphocytes.
  • a PI staining assay of NP_GA3+Phe-BF 3 -induced tumor cell pyroptosis in mice (an independent experiment from that in Fig. 5a) (scale bar, 100 ⁇ m) .
  • b, e, f Gating strategy (b) and representative flow cytometry plots for assessing 4T1 tumor-infiltrating CD3 + T cells (e) or Foxp3 + CD4 + regulatory T cells (f) following indicated treatments.
  • c d, Immunofluorescence staining of CD3 + T cells within the 4T1 tumors.
  • Scale bar 200 ⁇ m (c) , 50 ⁇ m (Left in d) and 20 ⁇ m (Right in d) .
  • f, g Flow cytometry analysis of CD4 + or CD8 + T cells depletion by the corresponding antibody. Data shown are representative of two (a, c, d) or three (e, f) independent experiments.
  • Figs. 11a-d Tumor-infiltrating immune cells subtypes analysis by single-cell RNA-sequencing (scRNA-Seq) .
  • a Gating strategy and representative flow cytometry plots for the enrichment of 4T1 tumor-infiltrating single CD45 + immune cells.
  • b Heatmap of ten immune cell clusters with unique signature genes. Colors on top of the map indicate different immune cell clusters. Three or four selective marker genes are listed alongside.
  • c t-SNE plot of 18, 069 RNA-sequenced 4T1 tumor-infiltrating single CD45 + immune cells.
  • d Expression patterns of signature genes of the corresponding cell clusters on the t-SNE plot. All data shown are representative of two independent experiments.
  • Figs. 12a-c scRNA-Seq analyses indicate that T lymphocytes are attracted and activated in NP_GA3+Phe-BF 3 -treated 4T1 tumors.
  • b, c Expression levels of protumoral and immunosuppressive gens (b) and proinflammatory chemokine, T/NK cell activation or effector genes (b) in immune cells.
  • Phenylalanine trifluoroborate (Phe-BF 3 ) is a representative BAA with a highly similar structure to phenylalanine (Phe) except for the replacement of carboxylate (-COO - ) with trifluoroborate (-BF 3 - ) .
  • FDG F-fluorodeoxylglucose
  • Phe-BF 3 exhibits a comparable sensitivity but a higher specificity in labeling tumors in mice 37 .
  • BAAs also show non-specific uptake in the bone, which is thought to be caused by defluorination of the trifluoroborate group 38-40 .
  • Free fluoride can catalyze a rapid and efficient desilylation reaction and is commonly used to remove the protecting silyl ether group in organic synthesis 41, 42 .
  • Phe-BF 3 could also catalyze efficient desilylation and if so whether the reaction could be developed into a useful bioorthogonal system, therefore making Phe-BF 3 a perturbing probe for tumor-selective protein activation 37 .
  • Desilylation of this system will trigger elimination of the carbamate, leading to breakdown of the amide bond 43, 44 (Fig. 6b) and therefore release of the coumarin.
  • Free coumarin as opposed to the amine-blocked coumarin in the silyl-phenolic ether system, is highly fluorescent, thus providing a simple and quantitative assay for Phe-BF 3 -mediated desilylation.
  • the silyl group was diversified into triethylsilyl (TES) , tert-butyldimethyl silyl (TBS) , or triisopropyl silyl (TIPS) (Fig. 1a and Fig. 6a) ; the corresponding three compounds, referred to as TESO-, TBSO-and TIPSO-Coumarin, respectively, were synthesized (Fig. 6c) .
  • TESO-Coumarin but not TIPSO-Coumarin, released free coumarin extensively as a result of the desilylation reaction while TBSO-Coumarin only reacted weakly (Fig. 1b, c) .
  • Liquid chromatography-mass spectrometry revealed that 98%of TESO-Coumarin and less than 20%of TBSO-Coumarin were desilylated by Phe-BF 3 while TIPSO-Coumarin remained untouched (Fig. 1d, e) .
  • incubation with sodium fluoride led to complete desilylation of TESO-Coumarin and TBSO-Coumarin but had no effect on TIPSO-Coumarin.
  • the decreasing reactivity observed with TESO-, TBSO-and TIPSO-Coumarin suggests a steric hindrance on the silica atom for Phe-BF 3 -mediated desilylation, echoing the situation of sodium fluoride-induced desilylation 45 .
  • organic chemists have used tetrabutylammonium difluorotriphenylsilicate, instead of free fluoride, to remove silyl ether 47 , in which the fluoride is transferred directly from the difluorotriphenylsilicate to the empty d-orbital of the silica atom on the silyl ether 48 .
  • TESO-Coumarin (as well as the other two silyl ether-linked compounds) are highly stable and showed no spontaneous desilylation even after 12-h incubation in phosphate-buffered saline (PBS) (Fig. 6e) . Further, treating TESO-Coumarin with cellular concentrations of H 2 O 2 , GSH or other biologically relevant anions, including Cl - , I - , and NO 3 - , caused no release of free coumarin (Fig. 1f) .
  • Gold nanoparticle is a biocompatible delivery vehicle and has been approved by the FDA for clinical trials in humans 50 .
  • NP has also been widely used in biological applications including drug delivery and bio-imaging 51, 52 .
  • NP has a selectivity for tumors owing to the enhanced permeability and retention (EPR) effect towards the tumor lesion as well as its superior internalization by cancer cells 53, 54 .
  • EPR enhanced permeability and retention
  • NPs were decorated with MeO-PEG-thiol (for better solubility and biocompatibility) mixed with 5%dibenzyl cyclooctyne (DBCO) -PEG-thiol.
  • MeO-PEG-thiol for better solubility and biocompatibility
  • DBCO 5%dibenzyl cyclooctyne
  • the TES silyl-phenolic ether was then conjugated to the decorated NPs through a copper-free “click” reaction between the DBCO and an azide added to the para-dimethylaminoacetamide group (Fig. 6d) .
  • GFP green fluorescence protein
  • Fig. 2a thiol-maleimide linkage between the carbamate and Cys-147 in GFP
  • NP_GFP purified NP_GFP materials were treated with Phe-BF 3 , or sodium fluoride as a positive control, or another fluorine-containing compound in vitro for 4 h, and the reactions were centrifuged to precipitate the NPs (Fig. 7a) . Nearly all GFP molecules were found to be released into the supernatants by Phe-BF 3 or sodium fluoride treatment (Fig. 2b) . In contrast, incubation of NP_GFP in PBS alone did not result in appearance of GFP in the supernatants. Echoing the situation in TESO-Coumarin, treatment with leflunomide, 5-FU, capecitabine or FDG also caused no release of GFP from NP_GFP (Fig. 2b) . Thus, Phe-BF 3 -mediated desilylation of the TES silyl ether is also highly effective in releasing a protein client.
  • NPs can efficiently enter mammalian cells through the endocytic pathway 55 . This allowed us to test whether Phe-BF 3 -mediated desilylation can really function inside living cells. For this, primary mouse bone marrow-derived macrophages (priBMDMs) , human cervical carcinoma cell line HeLa, and murine mammary carcinoma EMT6 and 4T1 cells were treated with NP_GFP (1 mg/mL) and then stimulated with 100 ⁇ M Phe-BF 3 or sodium fluoride. All these four cells expressed the LAT1 transporter that could uptake extracellular Phe-BF 3 (Fig. 7b) .
  • priBMDMs primary mouse bone marrow-derived macrophages
  • HeLa human cervical carcinoma cell line
  • EMT6 and 4T1 cells were treated with NP_GFP (1 mg/mL) and then stimulated with 100 ⁇ M Phe-BF 3 or sodium fluoride. All these four cells expressed the LAT1 transporter that could uptake extracellular Phe-BF 3 (Fig. 7
  • NPs exhibit strong absorption of light between 200 nm to 600 nm, causing fluorescence quenching of the GFP immobilized on the NPs 56 ; thus, cells treated with NP_GFP alone showed no green fluorescence. However, upon stimulation with Phe-BF 3 , strong GFP fluorescence was detected, particularly in the primary BMDMs (Fig. 2d) . This indicates that the desilylation-released GFP remains functional in live mammalian cells.
  • Phe-BF 3 -treated cells maintained normal morphology with intact nuclei and membrane integrity (Fig. 2d) .
  • trifluoroborate-derived imaging probes have already been systematically evaluated in many cancer cells and animal models with no negative effects on cell proliferation and animal viability observed 18, 57 .
  • Phe-BF 3 even when used at the high 25-mM concentration, exhibits little cytotoxicity 37 . This encouraged us to investigate the feasibility of using Phe-BF 3 to achieve controlled protein activation in the tumor in mice.
  • tail-vein injected into mice that had been subcutaneously engrafted with the 4T1 mouse mammary carcinoma cells [ 18 F] Phe-BF 3 was found to accumulate in the tumor within 1 h (Fig.
  • Zirconium-89 ( 89 Zr) was prepared through a Y (p, n) Zr reaction in a 14.6-MeV cyclotron, and the purified isotope (final specific activity > 1 Ci/ ⁇ mol) was used to label the GFP protein as previously described 58 .
  • 89 Zr Zirconium-89
  • NP_ [ 89 Zr] GFP was found to accumulate in the tumor at 6 h post-injection.
  • NP_ [ 89 Zr] GFP also appeared in the liver as expected, but this should not be a concern because liver uptake of Phe-BF 3 was almost negligible 37 (Fig. 2e) .
  • the distribution of NP_GFP and Phe-BF 3 overlaps only in the tumor, indicating that the orthogonal desilylation reaction can achieve tumor-selective targeting in vivo.
  • Dynamic PET imaging scan further revealed that NP_GFP had the highest enrichment in the tumor but the lowest level in the liver at 18 h after injection (Fig. 7c) , indicating that this time point should be the optimal one for administration of Phe-BF 3 .
  • mice injected with NP_mNeonGreen-NLS alone no green fluorescence was detected in the tumor sections (Fig. 2f, g) , agreeing with the high stability of the TES silyl ether linkage.
  • additional injection of Phe-BF 3 into the mice caused evident appearance of nucleus-localized mNeonGreen in the tumor samples (Fig. 2f, g) .
  • Statistical data suggested that about 15%of cells in the tumor were positive for the mNeonGreen signal (Fig. 2h) .
  • the gasdermin-N domain when unleashed from the C-terminal inhibitor gasdermin-C domain, translocates to the plasma membrane and oligomerizes there to form pores, thus executing pyroptotic cell death.
  • the gasdermin-N domain could be an ideal application target of the Phe-BF 3 -mediated desilylation system for two reasons. First, the gasdermin-N domain, when immobilized onto the large-size NPs, is “caged” and completely inactive; it will perforate the plasma membrane only upon being released from the NPs by Phe-BF 3 -mediated desilylation (Fig. 3a) .
  • pyroptosis is known to be highly proinflammatory, but its effect on the tumor has not been investigated due to the lack of approaches capable of inducing tumor cell pyroptosis without affecting other signaling pathways.
  • GSDMA3 a potent gasdermin whose inactive monomer as well as oligomeric pore are both structurally characterized 6, 31 .
  • cysteine residues in the gasdermin-C domain of GSDMA3 so that conjugation to NPs would only take place via the cysteine residues in the gasdermin-N domain.
  • Recombinant GSDMA3 as well as its gasdermin-N and -C noncovalent complex (N+C) was purified to homogeneity (Fig.
  • Pyroptotic cells are positive for both Annexin-V and propidium iodide (PI) staining, which can be quantified by flow cytometry.
  • PI propidium iodide
  • NP_GA3 Mut +Phe-BF 3 showed no differences in Annexin-V and PI staining from the control cells or cells treated with a single agent (Fig. 3c, d) , indicating that the observed pyroptosis owes to the pore-forming activity of GSDMA3 released from the NPs by Phe-BF 3 -mediated desilylation. It is worth mentioning that NP itself is known to weakly inhibit cell proliferation. We did observe a subtle cell viability decrease with NP_GA3 (Fig. 8c) . However, the effect occurred independently of the pore-forming activity of NP_GA3 and did not require Phe-BF 3 , and therefore does not affect our analyses of the effect of pyroptosis on the tumor.
  • NP_ [ 89 Zr] GA3 When injected to the 4T1 tumor-bearing mice, unconjugated [ 89 Zr] GA3, like GFP, was rapidly and predominantly trapped in the liver with little uptake in the tumor even at 18 h after injection (Fig. 9a) .
  • NP_ [ 89 Zr] GA3 showed a better biocompatibility, most of which could stay in the blood for 2 h after injection.
  • NP_GA3 also showed apparent tumor targeting at 12 h after infection and then started to be cleared through the hepatobiliary system (Fig. 9b) .
  • the tumor uptake of NP_ [ 89 Zr] GA3 reached a plateau with the maximal level up to 15 %ID/g.
  • NP_GA3 and Phe-BF 3 desilylation-induced GSDMA3 activation i.e., pyroptosis
  • BALB/c mice engrafted subcutaneously with the 4T1 cancer cells were injected intravenously with NP_GA3 (5 mg/kg) at day 6 followed by two sequential intravenous injections of Phe-BF 3 (50 mg/kg) at day 7 and 8, respectively (Fig. 4b) .
  • the treatment cycle was repeated twice at day 9 and 12 to augment the extent of pyroptosis induction.
  • mice treated for three cycles with NP_GA3 or Phe-BF 3 alone behaved similarly as the PBS-treated mice and showed normal and aggressive tumor growth (Fig. 4b-d and Fig. 9c) .
  • the pore-forming activity-deficient NP_GA3 Mut proteins were used in combination with Phe-BF 3 , there was no tumor shrinkage and the treated mice showed aggressive tumor growth as the PBS-treated mice (Fig. 4c-e and Fig. 9c) . Measuring the tumor weight confirmed the antitumor effect caused by the pyroptotic activity of GSDMA3 released from the NPs (Fig.
  • mice In addition to intravenous injection, we also performed intratumoral injection of NP_GA3+Phe-BF 3 and observed the same regression of the implanted 4T1 tumors in mice (Fig. 4c-f) . This indicates that the therapeutic effect seen with intravenous injection is likely due to GSDMA3 activation at the tumor local site, which agrees with the observation that targeting of NP_GA3 and Phe-BF 3 converges on the tumor tissue in mice. Furthermore, we tail-vein injected PI into mice that had been subjected to three cycles of sequential NP_GA3 and Phe-BF 3 treatments and then examined the tumor slice for possible pyroptotic cells.
  • mice could support the growth of implanted 4T1 tumors similarly as the wild-type BALB/c mice (Fig. 5b) .
  • the same scheme of NP_GA3+Phe-BF 3 treatment did not cause any tumor regression in the Nu/Nu mice (Fig. 5b) .
  • This finding indicates a requirement of T cells for GSDMA3 activation-induced tumor clearance.
  • flow cytometry revealed a drastically increased CD3 + T cell population in tumors from NP_GA3+Phe-BF 3 -treated mice, compared with tumors from PBS-treated mice (Fig. 5c and Fig. 10b) .
  • Anti-CD3 fluorescent imaging of the tumor tissue confirmed the massive T cell infiltration (Fig. 10c, d) .
  • both the CD4 + and the CD8 + subpopulations were elevated while the percentage of CD4 + Foxp3 + T regulatory (Treg) cells, the negative regulator of antitumor immunity, were decreased in tumors from NP_GA3+Phe-BF 3 -treated mice (Fig. 5c and Fig. 10b, e, f) .
  • injection with the pyroptosis-deficient NP_GA3 Mut (plus Phe-BF 3 ) which could not cause tumor regression, also did not induce T cell infiltration into the tumors (Fig. 5c and Fig. 10c, e, f) .
  • scRNA-Seq single-cell RNA sequencing analyses
  • Fig. 11a A total of 18, 069 single immune cells from two control mice (10, 171 cells) and two therapeutic mice (7, 879 cells) were sequenced, which were clustered into 10 subsets on the two-dimensional t-SNE map (Fig. 5e) . These subsets were identified and classified by the high and differential expression of genes marking classical immune cell populations (Fig. 11b-d) .
  • genes that are known to be protumoral or immunosuppressive such as Csf1, Vegfa, Arg1, Cd274 (encode PD1) and Pdcd1lg2 (encode PD-L2) , showed decreased expression upon NP-GA3+Phe-BF 3 treatment (Fig. 12b) .
  • These analyses provide a global view of pyroptosis-induced immunological changes within the TME, which well agrees and collaborates the potent antitumor effect observed in NP-GA3+Phe-BF 3 -treated mice.
  • Immune checkpoint blockade therapy such as anti-PD1/PD-L1 and anti-CTLA4, has been highly successful in clinical treatment of a broad range of cancers.
  • this emerging promising cancer therapy suffers from the low response rate, limiting its application to a wide population of cancer patients 63, 64 .
  • the exact reason for the general resistance of cancer patients to checkpoint blockade therapy is still unclear and probably varies among different cancers 65 .
  • One widely accepted view is that inflammation within the TME is low or ineffective for inducing sufficient infiltration and/or activation of the lymphocytes, and for this reason the checkpoint blockade-resistant tumors are considered to be “cold” 66, 67 .
  • necrotic or lytic cell death in promoting anticancer immune response was considered several decades ago owing to immune-dependent anticancer effect observed with certain cell-killing chemotherapeutic drugs 68, 69 , for which the term “immunogenic cell death” is coined 70, 71 .
  • cancer cell necroptosis another form of programmed necrosis mediated by the death receptor complex-RIPK3-MLKL axis under the context of caspase inhibition, can promote antitumor immunity 72, 73 .
  • a very recent report also suggests that iron overload-induced ferroptosis in cancer cells is activated by IFN ⁇ released from antigen-primed CD8 + T cells during cancer immunotherapy 74 .
  • necroptosis DAMPs-induced maturation of dendritic cells, cross-priming of CD8 + T cells as well as IFN ⁇ production underlie its antitumor immunity 72, 73 , in which the cross-priming appears to rely on activation of the NF- ⁇ B proinflammatory signaling. It has also been noted that fibroblast necroptosis, initiated by the more upstream death-receptor ligand, instead suppresses NF- ⁇ B-mediated production of proinflammatory factors from the dying cells 75 and is unable to stimulate co-cultured macrophages to produce proinflammatory cytokines 76 .
  • 5-Fluorouracil/5-FU (920052) , Leflunomide (448506) , Capecitabine (392078) and Fluoro-2-deoxy-D-glucose/FDG (D234500) were obtained from J&K Scientific. Synthesis and characterization of compounds- (1-6) are shown in Fig. 6c, 1d. Other chemical reagents and solvents used in this study were purchased from Sigma-Aldrich, J&K Scientific, Energy Chemical or Thermo Fisher Scientific. All NMR spectra were recorded at room temperature (RT) on a Bruker Avance 400 MHz or 600 MHz spectrometer.
  • cDNA Complementary DNA for mouse Gsdma3 was synthesized by our in-house gene synthesis facility.
  • cDNAs for eGFP and mNeonGreen were gifts from Dr. P. Xu (Institute of Biophysics, Chinese Academy of Sciences) .
  • the cDNAs were cloned into a modified pET vector with an N-terminal 6 ⁇ His-SUMO tag for recombinant expression in E. coli.
  • cDNA for mNeonGreen-NLS was generated by standard PCR cloning strategy with a reverse primer containing the SV40 nuclear localization signal (NLS) sequence (5′-CCG AAA AAA CGT AAA GTT-3′) .
  • NLS nuclear localization signal
  • the cDNA was inserted into a modified pCS2-3 ⁇ Flag vector for transient expression in HeLa cells.
  • Primers used for generating point mutations were designed using an online program (https: //www. agilent. com/store/primerDesignProgram. jsp) . All plasmids were verified by DNA sequencing.
  • PE-conjugated anti-mouse CD3 (clone 17A2)
  • FITC-conjugated anti-mouse CD4 (clone RM4.5)
  • APC-conjugated anti-mouse CD8 (clone 53-6.7) were purchased from BioLegend.
  • eFluor 450-conjugated anti-mouse Foxp3 antibody (clone FJK-16s) was obtained from Invitrogen.
  • the PD1 antibody used for treating 4T1 tumors was a gift from BeiGene.
  • anti-mouse CD4 (clone GK1.5) and isotype control (clone LTF-2) antibodies were produced by BioXcell, and anti-mouse CD8 was a gift from Dr. J. Sui (National Institute of Biological Sciences, Beijing) .
  • Anti-LAT1 antibody (D-10) was obtained from Santa Cruz Biotechnology.
  • Anti-GFP (11814460001) and anti-GAPDH antibodies were obtained from Roche and Sigma-Aldrich, respectively.
  • coumarin-NCO 500 mg, 2.48 mmol
  • Compound 5 1.0 equiv., 2.37 mmol
  • DBTL dibutyltin dilaurate
  • the solution was stirred under refluxing THF for 24 h. Without washing, the organic layer was removed under reduced pressure. The residue was purified by flash chromatography to obtain silyl ether-conjugated coumarin as the white solid (40%yield) .
  • TESO-Coumarin, TBSO-Coumarin and TIPSO-Coumarin were treated with Phe-BF 3 (150 ⁇ M) in PBS (including 5%DMSO) at 37°C.
  • HPLC analysis was performed at 5 and 240 min after incubation (Fig. 1d, e) .
  • TESO-Coumarin (150 ⁇ M) was treated with H 2 O 2 , GSH or other biologically relevant anions, including Cl - , I - , NO 3 - , PO 4 3- , SO 4 2- (150 ⁇ M) in PBS (including 5%DMSO) at 37°C.
  • HPLC analysis was performed at 240 min after incubation.
  • TESO-Coumarin 150 ⁇ M was treated with Phe-BF 3 or another organofluorine in PBS (including 5%DMSO) at 37°C.
  • HPLC analysis was performed at 240 min after incubation.
  • Solvent A water (0.1%TFA) ; solvent B: MeCN; 0 to 2 min: 5%B, 2 to 10 min: 5%to 95%B, 10 to 15 min: 95%B, 15 to 17 min: 95%to 5%B; flow rate: 0.6 mL/min; column temperature: 19 to 21°C.
  • the reaction yields were determined by HPLC peak integration at the given wavelength (254 nm) . All the measurements were performed in triplicates and the average numbers are shown.
  • [ 18 F] Phe-BF 3 are radiosynthesized via the one-step 18 F- 19 F isotope exchange (IEX) reaction.
  • the labeling method and purification procedure have been described previously 36, 37 .
  • PET scans were obtained and images analysis were performed using a Mediso 122s PET scanner.
  • About 3.7 MBq of [ 18 F] Phe-BF 3 and [ 89 Zr] Zr-labeled protein-NP conjugates were administrated via tail-vein injection under isoflurane anesthesia. Standard data acquisition and image reconstruction of the PET data were performed.
  • NPs water-soluble spherical gold nanoparticles
  • A68095 The water-soluble spherical gold nanoparticles (NPs) (A68095) were generated by 3A Chemicals. PEG-decorated NPs were prepared according to the published methods 77, 78 . Briefly, a solution of NPs (60 nm) containing citric acid was centrifuged at 14,000 g for 5 min, decanted, and resuspended in water to remove excess citric acid. 10 ⁇ L of 8 mM DBCO-PEG3400-SH were added to 1 mL of 1 mg/mL NPs solution. The mixture was stirred for 30 min at RT, and 200 ⁇ L of 10 mM MeO-PEG5000-SH were then added, stirring for 24 h at 4°C. The reaction crudes were centrifuged at 14,000 g for 5 min, decanted, and resuspended in water to remove excess PEG. The size of the DB
  • NP_GFP and NP_mNeonGreen-NLS were prepared by following the previous publications 79, 80 . Briefly, to obtain NP_GFP and NP_mNeonGreen-NLS, 100 ⁇ L of N 3 -OTES-maleimide linker (Compound 7, TESO-Linker) (PBS, 500 ⁇ M) was added to 100 ⁇ L of GFP or mNeonGreen-NLS solution (PBS, 100 ⁇ M) at 4°C for 24 h. Excess linker was removed from the solution by ultrafiltration centrifugation for 4 times and to modified GFP or mNeonGreen-NLS protein were obtained.
  • N 3 -OTES-maleimide linker Compound 7, TESO-Linker
  • NP_GA3 100 ⁇ L of the modified proteins (20 ⁇ M) were reacted with 100 ⁇ L of DBCO-PEG-NPs solutions (200 ⁇ M) with stirring at RT for 30 min. After overnight storage at 4°C, the reaction was centrifuged at 5,000 g for 5 min, decanted, and resuspended in water to remove excess un-conjugated proteins. To prepare NP_GA3, all operations were carried out at 4°C. 100 ⁇ L of N 3 -OTES-maleimide linker (PBS, 500 ⁇ M) was added slowly to 100 ⁇ L of the (N+C) nonvalent complex of GSDMA3 (60 ⁇ M) solution, and the reaction proceeded for 6 h.
  • PBS N 3 -OTES-maleimide linker
  • modified GA3 protein was obtained and resuspended in PBS. 100 ⁇ L of the modified GA3 protein (20 ⁇ M) was incubated with 100 ⁇ L of DBCO-PEG-NPs solutions (200 ⁇ M) for 6 h. The mixture was used directly to treat cells or mice.
  • HeLa and mouse EMT6 and CT26 cells were obtained from the American Type Culture Collection (ATCC) .
  • Mouse mammary carcinoma 4T1 cells were obtained from the China Infrastructure of Cell Line Resources (Chinese Academy of Medical Sciences, Beijing, China) .
  • Primary bone marrow-derived macrophage (priBMDM) cells were prepared and cultured by following a standard protocol as previously described 4 . The cells are frequently checked by virtue of their morphological features and functionalities, but have not been subjected to authentication by short tandem repeat (STR) profiling. All cell lines were tested for mycoplasma regularly by the commonly used PCR strategy.
  • STR short tandem repeat
  • EMT6, CT26 and priBMDM cells were cultured in RIPM 1640 medium and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) . The media were supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 2 mM L-glutamine. All cells were cultured in a 5%CO 2 incubator at 37°C. Transient transfection was performed with the JetPRIME (Polyplus Transfection) by following the manufacturer’s instructions. GA3-FL or the GA3- (N+C) proteins were electroporated into CT26 cells using the Neon Transfection System (Life Technologies) .
  • DMEM Dulbecco’s modified Eagle’s medium
  • Frozen 4T1 tumor sections were used for anti-CD3 immunostaining. At the end of indicated treatments on day 16, 4T1 tumors were dissected from mice and embedded into Cryomold moulds filled with the OCT compound (SAKURA 4583) . The tissue samples were frozen on dry ice for 30 min and stored at -20°C. For immunostaining, the frozen tumor tissues were sectioned into 10- ⁇ m pieces using cryostat (Lecia CM1950) and mounted on slides. After washing out the OCT with PBS, the slices were fixed with 4%paraformaldehyde for 30 min and then blocked and permeabilized with PBS containing 1%FBS and 0.2%Triton X-100 for 1 h. The slides were stained with PE-conjugated anti-mouse CD3 antibody for 1 h and Hoechst for 1 min. Zeiss LSM800 confocal laser scanning microscope was used to acquire images on the 10x or 40x objectives.
  • the 4T1 tumor model was used for quantifying Phe-BF 3 -mediated release of mNeonGreen-NLS from NP_mNeonGreen-NLS.
  • the NP_mNeonGreen-NLS conjugates were administrated into 4T1 tumor-bearing mice through intravenous injections on day 6, 9 and 12 followed by intravenous injections of Phe-BF 3 on day 7, 8, 10, 11, 13, and 14.
  • mice were sacrificed and tumors were dissected from the surrounding fascia, embedded and frozen in the Cryomold moulds filled with the OCT compound.
  • HeLa, EMT6, 4T1, and priBMDM cells were seeded into a 6 or 96-well plate 24 h before subjected to indicated treatments.
  • static bright field images of pyroptotic cells were captured using an Olympus IX71 microscope. The image data shown are representative of at least three randomly selected fields.
  • All cells in each 6-wel plate were collected and washed twice with PBS, stained by using the Annexin V-FITC/PI staining kit (Abmaking) (Annexin V-FITC for 10 min and PI for 5 min) .
  • the sample volume was increased to 500 ⁇ l with adding more PBS and the samples were analyzed on a BD FACS Aria III flow cytometer. Data were processed using FlowJo software. Cell viability was determined by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) .
  • E. coli BL21 (DE3) cells harboring pET28a-6 ⁇ His-SUMO-GSDMA3 or mNeonGreen-NLS were grown in Luria-Bertani (LB) media supplemented with 30 ⁇ g/ml kanamycin. After OD 600 of the culture reached 0.8, 0.4 mM isopropyl-B-D-thiogalactopyranoside (IPTG) was added to induce protein expression at 18°C overnight.
  • IPTG isopropyl-B-D-thiogalactopyranoside
  • Bacteria were harvested and sonicated in the lysis buffer containing 20 mM Tris-HCl (pH 8.0) , 150 mM NaCl, 20 mM imidazole and 10 mM 2-mercaptoethanol.
  • the fusion protein was first affinity-purified by Ni-Sepharose beads (GE Healthcare Life Sciences) .
  • the homemade ULP-1 protease was used to remove the 6 ⁇ His-SUMO tag by overnight cleavage at 4°C.
  • HiTrap Q ion-exchange and Superdex G75 gel-filtration chromatography were then performed sequentially for further purification.
  • the purified engineered GSDMA3 protein was digested overnight with homemade PPase at 4°C.
  • the (N+C) complex of GSDMA3 was further purified by Superdex G75 gel-filtration chromatography in the absence of reductants. 2-mercaptoethanol present in the mNeonGreen-NLS protein was also removed at Superdex G75 gel-filtration chromatography step.
  • HeLa, EMT6, 4T1, and priBMDM cells seeded in 6-well plates, were treated with the NP_GA3 conjugates for 24 h. Subsequently, the NP_GA3-containing media were replaced with Phe-BF 3 -containing media and incubated for another 24 h. The cells were then subjected to flow cytometry analyses.
  • priBMDM cells were seeded onto glass coverslips in 24-well plates and treated sequentially with the NP_GFP conjugate for 24 h and Phe-BF 3 for another 24 h. The treated cells were washed with PBS and fixed with 4%paraformaldehyde. Nucleus was stained with Hoechst. Fluorescence images were captured by using the Zeiss LSM800 confocal microscope on a 20x objective.
  • PI propidium iodide labeling of pyroptotic cells in vivo
  • 4T1 tumor-bearing mice were administrated with PI (2.5 mg/kg) via intravenous injection at 24 h after the last round of NP_GA3+PheBF 3 treatment (Fig. 4b) . 10 min later, the mice were sacrificed and the tumors were harvested, placed and frozen into the OCT-containing Cryomold moulds. After slicing and mounting, the slides were scanned directly and immediately on the Zeiss LSM800 confocal microscope.
  • mice used were purchased from the Vital River Laboratories.
  • 4T1 (1 x 10 6 ) or EMT6 (2 x 10 6 ) cells in 100 ⁇ l of PBS were implanted into the right flank of BALB/c female mice (6-8 weeks old) .
  • Mice were intravenously injected with NP_GA3 or an indicated control on day 6, 9, and 12, and each of the three treatments was followed by two intravenous injections of Phe-BF 3 (on day 7, 8, 10, 11, 13, and 14) , as illustrated in Fig. 4b.
  • Fig. 4b For the combinatory therapy in Fig.
  • the tumors were dissected from the surrounding fascia, weighted, minced into pieces by sterile scissors, and grinded. Cell clumps were removed through a 70- ⁇ m cell strainer to obtain sing-cell suspensions. The suspension was centrifuged and the cell pellets were washed twice with PBS containing 1%BSA (FACS buffer) . Lymphocytes were isolated by Percoll density gradient centrifugation, washed and resuspended in the FACS buffer, blocked with anti-mouse CD16/CD32 (clone 93, BioLegend) for 30 min, and finally stained with the indicated antibodies for another 1 h.
  • FACS buffer FACS buffer
  • the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (L10119, Invitrogen) was used to determine cell viability during FACS analysis.
  • the Foxp3 Fixation/Permeabilization Kit (00-5521-00, Invitrogen) was used to stain the intracellular Foxp3 by following the manufacturer’s instructions.
  • the tumor-infiltrating lymphocytes isolated above were stained with PE-conjugated anti-mouse CD45 antibody (clone 30-F11, Biolegend) .
  • CD45 + immune cells were then enriched by using a BD FACS Aria III flow cytometer. Cell viability was monitored in real time during preparation of the single CD45 + immune cells suspension. 10,000 cells ( ⁇ 600 single cells/ ⁇ l) from each experimental group were barcoded and pooled using the 10x Genomics device. Samples were prepared following the manufacturer’s protocol and sequenced on an Illumina NextSeq sequencer. The Cell Ranger analysis pipeline (v3.0.2) was used for sample de- multiplexing, barcode processing, alignment, filtering, UMI counting, and aggregation of the sequencing runs.
  • Each tumor-bearing mouse was intraperitoneally administrated with 200 ⁇ g of anti-mouse CD4, anti-mouse CD8, or the isotype control antibody at day 5, 8, 11, and 14 after inoculation of the 4T1 cells.
  • the percentage of CD4 + or CD8 + T lymphocytes in the spleen was determined on day 16 by using a BD FACSAria III flow cytometer.
  • the tumor-bearing mice were subjected to indicated treatments and small-animal PET studies when the tumor volume reached 100 mm 3 (about 1 week after inoculation) and 100-300 mm 3 (2-3 weeks after inoculation) , respectively.

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Abstract

L'immunothérapie induite par pyroptose est réalisée par l'activation d'une molécule pyroptogène formant des pores, l'activation induisant la pyroptose cellulaire tumorale et provoquant une régression tumorale par une immunité antitumorale médiée par les lymphocytes T.
PCT/CN2020/108776 2019-08-23 2020-08-13 Immunothérapie induite par pyroptose WO2021036793A1 (fr)

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CN117363652A (zh) * 2023-10-10 2024-01-09 郑州大学 一种双转录因子调控的双启动质粒、纳米材料及其制备方法和应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117363652A (zh) * 2023-10-10 2024-01-09 郑州大学 一种双转录因子调控的双启动质粒、纳米材料及其制备方法和应用
CN117363652B (zh) * 2023-10-10 2024-06-07 郑州大学 一种双转录因子调控的双启动质粒、纳米材料及其制备方法和应用

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