A spatiotemporal controllable t cell splicer based on DNA nanostructure and construction method and application thereof

Abstract

The application discloses a kind of space-time controllable T cell junction based on DNA nanostructure and its construction method and application, belong to DNA nanotechnology field.T cell junction includes six helix bundle paper structure and assembled internal functional layer and external shielding layer on it;Six helix bundle paper structure is assembled using 63 staple chain and M13;The internal functional layer includes nucleic acid-antibody conjugate combined on the four helix extension sequences of the top and bottom of the six helix bundle paper structure, and hand-in-hand palindromic sequence combined on the two helix extension sequences on left and right sides;External shielding layer includes C chain modified serum albumin binding peptide and chain I, and chain I includes pH response i-motif switch.T cell junction of the application has the space-time control ability of pH response and the potential of inducing TCR cluster to enhance T cell activation, has great potential to improve the safety and effectiveness of T cell immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of DNA nanotechnology, in particular to a spatiotemporal controllable T cell engager based on DNA nanostructure, a construction method and application thereof. BACKGROUND

[0002] Tumor immunotherapy methods represented by bispecific antibody engagers (BiTEs) have made significant progress in clinical practice. In 2023 alone, the FDA approved four types of T cell engagers for the treatment of hematological malignancies. Despite some good results, T cell engager therapy still has some limitations in safety and effectiveness. Systemic toxicity caused by non-spatiotemporal selective activation of T cells is a very serious and urgent problem in T cell engager therapy. Generally, BiTEs have two different binding sites to simultaneously target the T cell receptor (TCR) CD3 moiety and specific antigens on the surface of tumor cells. The direct exposure of the binding site of the engager often leads to non-specific activation of T cells due to the lack of spatial specificity, which can cause systemic adverse reactions including neurotoxicity and cytokine release syndrome. B cell-related antigens CD19 and CD20 have been proven to be ideal targets for treating B cell malignancies. However, in other tumors, tumor-specific antigens are relatively few, especially in solid tumors, where tumor-associated antigens also have some expression levels in healthy tissues, which can lead to serious targeted healthy tissue toxicity. Therefore, the safety of T cell engager therapy still needs to be improved.

[0003] In addition to the lack of safety, poor therapeutic effect is another problem with conventional T cell engagers. Generally, BiTEs only provide the first signal for T cell activation, and the co-stimulatory signal for T cell activation depends on the tumor cells themselves. However, tumor cells may not express or express low levels of co-stimulatory signal molecules, or even express PD-L1, an immune checkpoint inhibitory molecule, which can prevent T cells from recognizing target cells and reduce the therapeutic effect of T cell engagers. Kinetic partitioning models show that TCR clustering plays a key role in T cell activation, and compared to engagers that only bind TCR and tumor-associated antigens, simultaneous induction of receptor clustering assembly and introduction of co-stimulatory signals show higher efficiency in activating T cells against tumors.

[0004] New clinical and preclinical T cell engagers have been developed to address the current shortcomings of T cell immunotherapy in terms of safety and efficacy, such as fusing PD-L1 or CD28 ligands to T cell engagers in order to reduce PD-L1 mediated immunosuppression and enhance T cell activation. In addition, other forms of T cell engagers or inducers based on non-protein scaffolds also provide different solutions. In order to improve the level of T cell activation, multiple pMHC and CD28 ligands are anchored on red blood cells or nanomaterials to induce TCR clustering. Recent studies have shown that DNA nanotechnology with high programmability and parallel computing capability endows T cell engagers with more diverse designs, such as introducing DNA logic computing into DNA-antibody chimeric T cell engagers to enhance specificity. The development of DNA origami-based T cell engagers with stronger modularization and programmability can facilitate the screening and development of multispecific engagers. These more functional engagers have made some progress in strengthening the effectiveness and specificity of immunotherapy. However, these engagers still lack the ability to precisely control the activation of T cells in space and time. SUMMARY

[0005] The purpose of the present application is to provide a DNA nanostructure-based spatiotemporal controllable T cell engager and its construction method and application to solve the problems existing in the prior art, which has the potential of pH-responsive spatiotemporal control and TCR clustering induced T cell activation enhancement, which has great potential to improve the safety and effectiveness of T cell immunotherapy, and can achieve precise mediation of immunotherapy tumors.

[0006] To achieve the above purpose, the present application provides the following solutions:

[0007] The present application provides a DNA nanostructure-based spatiotemporal controllable T cell engager, which comprises a rod-shaped six-helix bundle origami structure and an internal functional layer and an external shielding layer assembled on the six-helix bundle origami structure.

[0008] The six-helix bundle origami structure is co-assembled by 63 staple strands and single-stranded circular DNA M13;

[0009] The internal functional layer comprises nucleic acid-antibody conjugates bound to four helix extension sequences on the top and bottom of the six-helix bundle origami structure, and hand-in-hand palindromic sequences bound to two helix extension sequences on the left and right sides;

[0010] The external shielding layer comprises a C-chain modified serum albumin binding peptide and a chain I, the chain I comprises a pH-responsive i-motif switch, and the external shielding layer is combined on the surface of the internal functional layer; the nucleotide sequence of the C chain is shown as SEQ ID NO. 76, and the nucleotide sequence of the chain I is shown as any one of SEQ ID NO. 71-75.

[0011] Preferably, the nucleotide sequence of the 63 staple chains is shown as SEQ ID NO. 1-63.

[0012] Preferably, the nucleic acid-antibody conjugate is a conjugate of the chain A and the streptavidin-modified antibody, the nucleotide sequence of the chain A is shown as any one of SEQ ID NO. 65, SEQ ID NO. 66 or SEQ ID NO. 68, and the antibody comprises aCD3, aCD28 and / or aPD-L1 antibody.

[0013] Preferably, the molar ratio of the chain A and the streptavidin-modified antibody is 1.5-3:1.

[0014] Preferably, the nucleic acid-antibody conjugate combined on the top two helix extension sequences of the six-helix bundle origami structure is a conjugate of the chain A and aCD3 and aCD28 antibodies respectively; and / or the quantity ratio of the aCD3 and aCD28 antibodies is 3:2.

[0015] Preferably, the 3' end of the chain I comprises a pH-responsive i-motif switch and a sequence complementary to the chain A, so that the external shielding layer is combined on the surface of the internal functional layer, and then fixed on the six-helix bundle origami structure.

[0016] Preferably, when the pH value is greater than the response threshold value, the chain I is combined on the chain A, and is fixed on the six-helix bundle origami extension sequence through the chain A, and forms an external shielding layer on the six-helix bundle origami structure with the C-chain modified serum albumin binding peptide, so as to shield the internal functional layer, avoid activating T cells in healthy tissues, and improve the stability of blood circulation in vivo; when the pH is less than the response threshold value, the chain I forms an i-motif quadruplex structure and separates from the six-helix bundle origami structure, so that the exposed antibody and hand-in-hand palindromic sequence induce TCR clustering, activate T cells, and achieve tumor cell killing.

[0017] The application also provides a construction method of the spatiotemporal controllable T cell engager.

[0018] The nucleic acid-antibody conjugate, the C-chain modified serum albumin binding peptide and the chain I are mixed in equal molar ratio to form a triple-stranded structure, and then the triple-stranded structure is mixed with the six-helix bundle origami structure to obtain the spatiotemporal controllable T cell engager.

[0019] Preferably, the molar ratio of the triple helix structure to the six-helix bundle origami structure is 1.2n:1, n represents the number of six-helix bundle origami structure extension sequence binding sites.

[0020] The application also provides the use of the above-mentioned spatiotemporal controllable T cell engager in the preparation of a drug or a drug carrier for improving the treatment of tumors by immunotherapy.

[0021] The application discloses the following technical effects:

[0022] The application discloses a multivalent antibody T cell engager with an inner-outer double-layer functional structure based on a 6HB origami, which has a series of characteristics such as spatiotemporal specificity, the ability to enhance T cell activation efficiency, reduce systemic toxicity, and good physiological stability and biocompatibility. The programmability and addressability of the origami structure make it possible for antibodies to be orderly and multivalently assembled, and the application proves that the origami structure assembled with CD3 and CD28 antibodies can effectively induce TCR clustering, can significantly improve the T cell activation level compared with monovalent antibodies, and can effectively improve the toxicity of T cells to target cells. The serum albumin coating layer can effectively prevent the engager from exposing CD3 and CD28 antibodies in advance during blood circulation, prevent T cells from mistakenly killing normal tissues, and block the systemic toxicity caused by non-specific T cell activation; the H + The pH-responsive outer layer shedding switch provides a basis for spatiotemporal controllable T cell activation. Considering the T cell activity inhibition mediated by PD-L1, the application fixes PD-L1 antibodies in the origami structure to mediate T cell targeted removal of MC38 tumor cells with high expression of PD-L1. The application proves the effectiveness of the spatiotemporal controllable multivalent T cell engager for tumor treatment through in-vitro and in-vivo experiments, and provides a basis for improving the safety, specificity and effectiveness of T cell immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The application discloses the following technical effects:

[0024] Figure 2Serum albumin binding peptide (ABD) expression analyzed by SDS-PAGE (10%); Marker is standard protein molecules, Purification is purified protein, supernatant is supernatant, Whole protein is whole protein without separation;

[0025] Figure 3 Agarose gel electrophoresis characterization of 6HB; from left to right, Marker is DNA standard molecules, M13 is single-stranded circular DNA M13, Annealed products are annealed products, Purified products are purified products, Filtered staples are ultrafiltered staple chains;

[0026] Figure 4 AFM and TEM characterization of 6HB structure;

[0027] Figure 5 Design of 6HB; the scaffold of ssDNA (2659 nt) is depicted in gray line; the staple chain is shown in black; these dots represent the positions of extended capture strands, which are used to bind DNA-protein conjugates; orange, purple and green dots are the positions of capture strands binding with aCD3, aCD28 and aPD-L1 DNA-protein conjugates, respectively; gray dots are the positions of hand-in-hand;

[0028] Figure 6 Gel electrophoresis verification of the coupling of biotinylated antibody and DNA with streptavidin (A) and the optimization of the binding ratio of Chain A and antibody-streptavidin conjugate (B);

[0029] Figure 7 Agarose gel electrophoresis characterization of 6HB assembled with different spacing aCD3;

[0030] Figure 8 Effect of antibody spacing on T cell activation; (A) Fluo-4AM calcium probe fluorescence vs. time after adding free aCD3 or aCD3@6HB; (B) T cell response to different calcium signals aCD3@6HB was stimulated by monitoring the fluorescence signal using Fluo-4AM calcium probe, and the fluorescence signal was normalized;

[0031] Figure 9 Effect of antibody spacing on T cell proliferation; (A) flow cytometry analysis of T cell CD69 expression after different types of stimulation Ab@6HB has different spacings from 70 nm to 14 nm; (B) quantitative analysis of T cell secreted IFN-γ levels in response to different stimulants; (C) flow cytometry analysis of T cell proliferation in response to different stimulants;

[0032] Figure 10 6HBs assembled with different density of aCD3 were characterized by agarose gel electrophoresis;

[0033] Figure 11 Effect of 6HB antibody density on T cell activation; (A) Fluo-4 AM calcium probe fluorescence versus time after addition of aCD3 aCD3@6HB; (B) T cell response to different calcium signals Ab@6HB stimulated by monitoring fluorescence signals using Fluo-4 AM calcium probe, which were normalized to the maximum response of all samples in the replicate;

[0034] Figure 12 Effect of 6HB antibody density on T cell and proliferation; (A) Flow cytometry analysis of T cell CD69 expression after different types of stimulation Ab@6HB with different antibody density; (B) Quantitative analysis of T cell secreted IFN-γ level after different types of stimulation Ab@6HB; (C) Flow cytometry analysis of T cell proliferation response to different stimulation;

[0035] Figure 13 Optimization of i-motif switch; (A) Design principle of outer shielding layer; (B) Different i-motif switch response to pH from 7.28 to 5.51 folding efficiency;

[0036] Figure 14 Stability of ABD assembly on 6HB structure; (A) Stability of ABD@6HB structure against enzyme digestion, M is marker, lane 1-2 is 6HB, lane 3-4 is No. 0 / 1 / 2 / 3 / 4 / 5 ABD@6HB, lane 5-6 is No. 0 / 2 / 4 ABD@6HB, lane 7-8 is No. 0 / 1 / 2 ABD@6HB, lane 9-10 is No. 0 / 3 ABD@6HB; (B) Stability of No. 0 / 2 / 4 ABD@6HB and No. 0 / 1 / 2 / 3 / 4 / 5 ABD@6HB structure in cell culture medium incubated for different time;

[0037] Figure 15 Agarose gel electrophoresis characterization of ABD@6HBs shielding layer response to outer layer shedding under different pH;

[0038] Figure 16Characterization of SpTCE hierarchical assembly and pH response ability by agarose gel electrophoresis; (A) Antibody hierarchical assembly of 6HB; Lane 1 is 6HB, lanes 2-5 are aCD3, aCD28, aPD-L1 antibodies and their combination mediated by chain A in 6HB hierarchical assembly products; (B) Separation of pH-responsive outer shielding layer and hand-in-hand formation of large structure at low pH; Lanes 1-3 are antibody inside 6HB assembly, SpTCE co-assembly product of antibody outside ABD and HSA / SpTCE, respectively, lane 4 is the product of HSA / SpTCE formed at pH 6.5, and lane 5 is hand-Ab@6HB;

[0039] Figure 17 Stability of SpTCE, Ab@6HB and 6HB in mouse plasma by agarose gel electrophoresis; (A) Stability of SpTCE in mouse plasma; (B) Stability of Ab@6HB in mouse plasma; (C) Stability of 6HB in mouse plasma;

[0040] Figure 18 Flow cytometry to verify the ability of Ab@6HB to target T cells and tumor cells; (A) Flow cytometry verification of Ab@6HB targeting T cells; (B) Flow cytometry verification of Ab@6HB targeting MC38 cells;

[0041] Figure 19 Flow cytometry and confocal microscopy to verify the ability of SpTCE to target T cells in response to pH; (A) Flow cytometry analysis of SpTCE targeting T cells; (B) Confocal analysis of SpTCE targeting T cells, scale bar is 1 μm;

[0042] Figure 20 CCK8 to verify the ability of SpTCE to guide T cells to target and kill tumor cells and cytokine detection; (A) CCK8 to verify the ability of SpTCE to guide T cells to target and kill tumor cells; (B) IFN-γ cytokine detection; The abscissa in the figure from left to right is T+MC38 (pH 7.4), free aCD3, aCD28, aPD-L1, 6HB, aCD3 / aCD28@6HB, aPD-L1@6HB, Ab@6HB, hand-Ab@6HB, SpTCE, HSA / SpTCE (pH 7.4), HSA / SpTCE (pH 6.5), T+MC38 (pH 6.5), MC38;

[0043] Figure 21 In vivo distribution of SpTCE; Cy7-labeled origami fluorescence distribution imaging in MC38 tumor model mice at different time points, three independent experiments were set for each group;

[0044] Figure 22The fluorescence intensity at different time points in mouse tumor sites is represented by the mean ± standard deviation of three independent experiments. The p-value was calculated using a two-tailed t-test, with *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0045] Figure 23 Fluorescence distribution in isolated mouse tissues;

[0046] Figure 24 The following data represent the effects of different drug administration groups on tumor treatment in mice: (A) Flowchart of antitumor treatment drug administration; (B) Tumor volume changes in MC38 tumor-bearing mice treated in different groups; (C) Body weight changes in MC38 tumor-bearing mice treated in different groups; (D) Survival curves of MC38 tumor-bearing mice treated in different groups.

[0047] Figure 25 (A) Photographs of tumor growth in MC38 tumor-bearing mice treated with different drugs, with a scale bar of 1 cm; (B) Tumor mass distribution in mice treated with different drugs.

[0048] Figure 26 The secretion of IFN-γ, IL-6, TNF-α and IL-2 cytokines in the plasma of MC38 tumor-bearing mice under different treatment groups was analyzed. The data are presented as mean ± standard deviation (n=3). P values ​​were calculated using a two-tailed t-test, with *P<0.05, **P<0.01 and ***P<0.001.

[0049] Figure 27 H&E and TUNEL histological images of tumor sections from MC38 tumor-bearing mice treated with different groups; scale bar is 50 μm.

[0050] Figure 28 H&E histological images of the heart, liver, spleen, lung, and kidney of MC38 tumor-bearing mice under different treatment groups; scale bar is 50 μm. Detailed Implementation

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0053] The experimental principle of the technical solution of this invention (e.g.) Figure 1(As shown): To improve the specificity, safety, and efficacy of T-cell immunotherapy, this invention utilizes a six-helix bundle origami substrate (6HB) to develop a spatiotemporally controllable TCR cluster conjugate (SpTCE). Figure 1 (A). SpTCE is designed with a two-layer structure: an inner functional layer and an outer shielding layer. The inner functional layer contains multiple types of antibody binding sites. Four extended binding sites on the top and bottom of the 6HB are used to connect various classes of antibodies (aCD3, aCD28, and aPD-L1), which are anchored to the 6HB at a controllable spacing at the nanoscale to guide T cells to specifically target and eliminate tumor cells. Other binding sites located on the left and right helical bundles are connected to tandem palindromic sequences (red bases in Table 2) to induce the formation of larger TCR clusters. The outer shielding layer, acting as a spatiotemporal control core, contains a serum albumin-binding peptide (ABD) and a pH-responsive i-motif switch, which binds to the inner structure and binds to endogenous serum albumin in the blood. When SpTCE is intravenously injected in vivo, the outer layer can bind serum albumin to form an outer protective layer that shields the inner functional layer, preventing the activation of T cells in healthy tissues and improving the stability of blood circulation in vivo. Once SpTCE enters tumor tissue, the acidic microenvironment triggers i-motif folding, causing the outer layer of the HSA to detach and exposing the inner antibody layer. The exposed antibodies and their linked oligonucleotide chains then induce TCR clustering, activating T cells and leading to tumor cell killing. This invention validates the fundamental concept of SpTCE, demonstrating its ability to achieve spatiotemporal control in response to pH and its potential to induce TCR clustering and enhance T cell activation. These results showcase the system's significant potential to improve the safety and efficacy of T-cell immunotherapy.

[0054] The following examples involve some of the experimental materials:

[0055] Biotin-labeled anti-mouse CD monoclonal antibody, clone number: 17A2; biotin-labeled anti-mouse CD28 monoclonal antibody, clone number: 37.51; biotin-labeled anti-mouse PD-L1 monoclonal antibody, clone number: 10F.9G2; FITC-labeled anti-mouse CD69 flow cytometry antibody, clone number: H1.2F3; all the above antibodies were purchased from BioLegend (Beijing) Biotechnology Co., Ltd.

[0056] CCK-8 kit, purchased from Shanghai Yisheng Biotechnology Co., Ltd.; mouse T cell negative sorting kit, purchased from Shanghai Luoyan Biotechnology Co., Ltd.; mouse T cell calcium flow assay kit and mouse T cell proliferation assay kit (CFSE), purchased from Thermo Fisher Scientific (China) Co., Ltd.; mouse IFN-γ, IL-2, TNF-α, IL-6 (ELISA) kit, purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0057] Mouse colorectal cancer cell MC38 was purchased from the Chinese Academy of Sciences Cell Bank; mouse CD3 + T cells were isolated from the spleen of mice.

[0058] The DNA sequences involved in the following examples are shown in Table 1. The corresponding nucleotide sequences of Table 1 from 0-1 to 3 according to the sequence name are shown in SEQ ID NO. 1-63.

[0059] Table 1 DNA sequences used in the experiment

[0060]

[0061]

[0062]

[0063] * The paper folding staple sequence and the extended sequence are separated by tt.

[0064] Table 2 DNA sequences used in the experiment

[0065]

[0066] Note: i-motif sequence is marked with underlined, and red marked base is palindromic sequence.

[0067] Example 1

[0068] 1. ABD protein expression

[0069] ABD was expressed using an E. coli system, and SNAP and 6xHis tags were added to the C-terminus of the ABD protein. The synthesized gene was amplified by PCR, and the amplification product was subjected to homologous recombination and cloned into the pET-28a vector for protein expression. After the recombinant plasmid was sequenced correctly by Shenguo Bioengineering (Shanghai) Co., Ltd., protein expression was performed (ABD protein amino acid sequence (SEQ ID NO. 79): DKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNAY FHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLAVKEWLLAHEGHRLGKPGLGLAAALEHHHHHH). The specific steps of protein expression are as follows:

[0070] (1) ABD expression strain construction. BL21(DE3) competent cells were taken out from -80°C refrigerator, and placed on ice for 5 min. 2 μL plasmid was added to the competent cell centrifuge tube, and shaken 3 times. The plasmid was added to the competent cell centrifuge tube and placed in a 42°C water bath for 90 s, and then placed on ice for 2-3 min. 500 μL LB medium was added to the centrifuge tube and placed in a 37°C, 220 r / min shaker for 30 min. Centrifugation was performed at 6900 g / min for 1 min, and 400 μL supernatant was removed. The remaining bacterial solution was mixed evenly by blowing, and was evenly coated on a solid medium plate containing kanamycin. The plate was dried in a clean bench for 2 min, and then inverted and cultured in a 37°C incubator for 12 h.

[0071] (2) Bacterial screening, culture and induction expression. Single colonies of the solid medium plate containing kanamycin and cultured for 12 h were selected and transferred to 5 mL liquid LB medium. 5 μL kanamycin (1:1000) was added to the medium, and the culture was incubated in a 37°C incubator at 250 r / min for 12 h.

[0072] (3) The bacterial solution was poured into a flask containing 100 mL sterilized medium, and 100 μL kanamycin (1000x) was added. The culture was incubated at 37°C and 220 r / min for about 4 h, and the OD 600 When the OD value of the bacterial solution was between 0.5 and 0.7, the corresponding dose of IPTG was added, and the protein expression was induced at 18°C for 12-16 h.

[0073] (4) After induction, the bacterial solution was transferred to a 50 mL centrifuge tube and placed in a centrifuge at 4°C and 7200 rpm for 10 min. The supernatant was discarded, and the bacterial solution was washed once with 1x PBS buffer. Centrifugation was performed at 4°C and 7200 rpm for 10 min, and the bacterial solution was collected.

[0074] The specific steps for ABD purification are as follows:

[0075] (1) Ultrasonic disruption of bacterial cells. The collected bacterial cells were resuspended with 1x PBS on ice, and the bacterial cells were disrupted using an ultrasonic disrupter at a power of 45% for 30-40 min. The disrupted bacterial cells were centrifuged at 4°C and 7200 rpm for 10 min, and the supernatant was collected. The sample was retained, and SDS-PAGE was performed to verify the expression of the target protein in the supernatant.

[0076] (2) Protein affinity purification. Prepare ultrapure water and wash the Ni-NTA affinity column multiple times. Equilibrate the Ni-NTA column using binding buffer. Filter the cell lysate supernatant through a 0.22 μm filter to remove impurities. Add the filtered supernatant to the Ni column; the target protein binds to the Ni column. Elute non-specific proteins with wash buffer, retaining the target protein in the Ni column. Finally, elute the target protein with elution buffer and collect the protein eluent.

[0077] (3) Ultrafiltration concentration. The collected target protein was desalted and concentrated using an ultrafiltration tube to remove impurities such as salts. The purity of the target protein was verified using SDS-PAGE gel electrophoresis. Figure 2 Ensure the purity of the target protein meets the requirements. Quantify the obtained target protein using a BCA protein quantification kit. Aliquot the target protein into appropriate volumes as needed for the experiment and store at -80°C.

[0078] 2. T cell isolation and extraction from spleen

[0079] CD3 cells were extracted from mouse spleens according to the instructions using the StemCell EasySep™ Mouse T Cell Sorting Kit. + T cells. Extracted CD3 + T cells were added to RPMI-1640 medium containing 10% FBS, and the cell density was maintained at 5 × 10⁶ cells / mL. 5 -1×10 6 Quantity / mL, use within one week.

[0080] 3. Synthesis and Characterization of Six-Helix Bundle DNA Origami

[0081] 6HB consists of bacteriophage single-stranded circular DNA M13 (2695nt) (BioBio Biotechnology Co., Ltd., catalog number: B3010) and 63 short DNA staple strands (see Table 1) in 1×Tris-EDTA-Mg 2+ Solution (TE-Mg) 2+ The DNA staples were assembled using gradient annealing at pH 7.4. Sixty-three DNA staples were dissolved in ultrapure water to a final concentration of 100 μM. Equal volumes of the DNA staples were mixed and diluted to 100 nM, then stored at 4°C for later use. The specific assembly method is as follows:

[0082] (1) Mix M13 (2695nt, 100nM, 20μL) and DNA staple (100nM, 20μL) at a molar ratio of 1:10, and then add 10×TE-Mg 2+(10 μL), final volume 50 μL, gradient annealing in PCR thermal cycler, annealing program set to 75 °C for 5 min, then ramp down from 65 °C to 4 °C at 15 min / °C, ultrafiltration purification of the formed product.

[0083] (2) Ultrafiltration tube (Ultra-0.5) with 0.5 mL size and 100 kDa molecular cut-off size was used to purify the annealed product, washed with 1 x TE-Mg buffer for 3 times (4700 g / min, 5 min, 4 °C), and the purified sample was recovered by inverting the ultrafiltration tube, quantified by microplate reader, and stored at 4 °C. Ultra-0.5) with 0.5 mL size and 100 kDa molecular cut-off size was used to purify the annealed product, washed with 1 x TE-Mg buffer for 3 times (4700 g / min, 5 min, 4 °C), and the purified sample was recovered by inverting the ultrafiltration tube, quantified by microplate reader, and stored at 4 °C. 2+ Ultra-0.5) with 0.5 mL size and 100 kDa molecular cut-off size was used to purify the annealed product, washed with 1 x TE-Mg buffer for 3 times (4700 g / min, 5 min, 4 °C), and the purified sample was recovered by inverting the ultrafiltration tube, quantified by microplate reader, and stored at 4 °C.

[0084] (3) Transmission electron microscopy was used to characterize 6HB, to determine its specific morphology and size. First, 10 μL of magnesium acetate (2 M) was added to the copper grid, and after 2 min, the magnesium acetate was absorbed with filter paper. Then, 10 μL of purified 6HB origami (10 nM) was added to the copper grid, and after 30 min, 10 μL of uranyl acetate (1%, w:v) was added and quickly absorbed with filter paper. Finally, 6HB was negatively stained with 10 μL of uranyl acetate (1%, w:v) for 2 min. After complete drying at room temperature, 6HB was imaged using a transmission electron microscope (JEM-2100FX, Hitachi) at 800 kV.

[0085] (4) Atomic force microscopy was also used to characterize 6HB origami. For AFM characterization, first, the mica surface was treated with 20 μL of 100 mM NiCl2for 2 min to increase the adsorption of DNA origami structures, then washed with ddH2O and dried with compressed air. Then, 5 μL of 10 nM 6HB was added to the mica surface and left for 5 min, washed slowly with ddH2O, and dried again with compressed air. Then, the Scanasyst gas-phase probe was used for scanning, and the images were collected using a Bruker Dimension Icon atomic force microscope.

[0086] 4. Protein-nucleic acid conjugation

[0087] For aCD3, aCD28, and aPD-L1 functional antibodies, antibody-nucleic acid conjugation was achieved using the biotin-labeled antibody-streptavidin-biotin-labeled nucleic acid connection method. The biotin-labeled antibody was incubated with streptavidin at a molar concentration ratio of 1:10 in PBS solution at 4 °C for 30 min, and the excess streptavidin was removed by washing three times with an ultrafiltration tube with a molecular cut-off size of 100 kDa. Then, the antibody-streptavidin conjugate was incubated with biotin-modified DNA at a molar concentration ratio of 1:1.5 in PBS solution at 4 °C for 30 min, to obtain the antibody-streptavidin-nucleic acid conjugate. ​

[0088] ABD-SNAP and DBCO-modified DNA strands were covalently linked by a bifunctional linking reagent. BG-NH2 [O6-(4-aminomethyl-benzyl) guanine] and NHS-Peg4-Azide reagent were taken out from -20 °C, after equilibrated to room temperature, 100 pL, 100 mM of NHS-Peg4-Azide was mixed with 150 pL, 100 mM of BG-NH2 [O6-(4-aminomethyl-benzyl) guanine], and supplemented with 250 pL of DMSO containing 300 mM triethylamine to a final volume of 500 pL, incubated at 30 °C overnight to get a final concentration of 20 mM BG-Azide. The resulting product BG-Azide was reacted with DBCO-modified DNA at a molar ratio of 20: 1 in aqueous solution at 37 °C for 1 h. The excess BG-Azide complex was washed away using an ultrafiltration tube with a molecular cut-off size of 3 kDa. The ultrafiltration purified DNA-BG-Azide and the expressed and purified SNAP fusion protein ABD were mixed at a molar ratio of 10: 1 and incubated at room temperature for 2 h. The excess DNA was washed away by purifying three times using an ultrafiltration tube with a molecular cut-off size of 10 kDa. The ultrafiltration tube was rinsed with ddH2O three times before use.

[0089] 5. i-motif sequence optimization

[0090] The synthesized three strands I, C and A were reacted under different pH conditions to evaluate the folding ratio of different i-motif. The folding efficiency of i-motif switch was evaluated by recording the change of fluorescence signal intensity, all reactions were carried out in 1xPBS containing 5 mM Mg 2+ The pH response of i-motif core sequences I1-C5(T4C5)3, I2-C5(T3C5)3, I3-C5(T2C5)3, I4-C4(T3C4)3and I5-C4(T3C3)3was compared by mixing C-FAM, A-BHQ1 and I at a molar ratio of 1:4:2 and incubating at 37 °C for 2 h under the condition of pH value ranging from 7.28 to 5.51. The fluorescence signal intensity was recorded during the process. The folding efficiency of i-motif was determined by the following formula:

[0091]

[0092] Where F is the fluorescence intensity of the unquenched fluorescence strand (C), F0and F t are the fluorescence intensity of C / A / I structure at the starting point and the end point of the reaction, respectively.

[0093] 6. SpTCE assembly

[0094] The antibody-nucleic acid conjugate Ab-A was mixed with ABD-C and I chain in equal molar ratios to form a triple-stranded structure. Then, the triple-stranded structure was mixed with purified 6HB in a molar ratio of 1.2n:1 (where n represents the number of binding sites in the 6HB extension sequence) to form SpTCE.

[0095] 7. Confocal imaging

[0096] To observe the T cell recognition and activation process of antibody origami conjugates, the antibody origami conjugates were labeled with FAM fluorescence, and T cells were activated using CellMask. TM The process for deep red membrane dye labeling is as follows:

[0097] (1) Connect the nucleic acid sequences extending from helical bundles 0 and 3 to their complementary Atto-488 fluorescently modified DNA strands;

[0098] (2) After centrifuging the T cell suspension at 400g for 10min, remove the culture medium, reconstitute it in 100μL of working concentration deepred staining solution, stain for 5min, centrifuge to remove the supernatant, and wash 3-5 times with 200μL PBS.

[0099] (3) The experimental group will use 1×10 5 T cells labeled with membrane dye were mixed with 1 nM fluorescently labeled antibody-origami conjugate; T cells in the blank group were not stained or labeled, nor were antibody-origami conjugates added.

[0100] (4) The control groups were either T cell membranes stained and labeled only, without the addition of fluorescently labeled antibody-origami conjugate; or T cells were not stained and labeled, but only fluorescently labeled antibody-origami conjugate was added.

[0101] (5) Mix and co-incubate in a 24-well glass-bottom confocal plate for 2 hours, and take pictures under a 100x oil immersion using a Nikon confocal laser scanning imaging system. The images are processed using ImageJ.

[0102] 8. Calcium flow analysis

[0103] To analyze the effect of antibody-origami conjugates on T cell activation, the real-time calcium flow signal of different antibody-origami conjugates on T cell activation was detected using the Fluo-4AM calcium flow reagent. The specific procedure was as follows:

[0104] (1) Isolated T cells at 10 8 A concentration of cells / mL was incubated with 10 μM Fluo-4AM in a cell culture incubator at 37°C for 30 min.

[0105] (2) After centrifugation to remove the culture medium, use 180 μL of Fluo-4 labeled T cells (100 μL per mL) at 37°C.7 The flux of the flow cytometer was measured in individual cells.

[0106] (3) Baseline fluorescence was then recorded for 30 seconds, and then 20 μL of free antibody or antibody-origami conjugate was added to the T cell suspension until the final antibody concentration reached 100 pM, unless otherwise stated.

[0107] (4) In order to simplify the comparison between different antibody-origami conjugate experimental groups and to evaluate the role of SpTCE rather than the role of antibody concentration, the antibody-origami conjugate addition concentration was adjusted so that the antibody concentration in each experimental group was kept consistent.

[0108] The raw calcium flux signal was normalized, and the calcium flux intensity before the addition of the antibody was determined to be I. min Then the calcium flux intensity I at each time point t Subtract I min The maximum calcium flux value (I) in all experimental groups t ) was determined to be (I max The calcium flow signal (normalized AUC) of these samples in each repetition was calculated as (I... t -I min ) / (I max -I min The repeated results were averaged to determine the height of the bars in the chart. A t-test was performed on the normalized AUC used for this average, and all experiments were conducted in triplicate.

[0109] 9. T-cell killing assay

[0110] The killing effect of T cells on target cells was evaluated using a CCK-8 cell counting kit. First, 8 × 10⁸ cells were seeded in each well of a 96-well plate. 3 MC38 cells. Separate T cells (2 × 10⁻⁶) 5T cell suspensions (cells / mL) and MC38 cells were co-incubated at a 10:1 ratio in RPMI-1640 medium containing 10% FBS at pH 7.4. The control group consisted of MC38 cells alone, while the experimental groups were supplemented with three free antibodies (aCD3, aCD28, aPD-L1), aCD3 / aCD28@6HB, aPD-L1@6HB, Ab@6HB, HSA / SpTCE, and SpTCE, respectively. Additionally, the experimental groups were prepared with HSA / SpTCE at pH 6.5. All cells were co-cultured at 37°C for 24 hours. T cell suspensions were collected, and the supernatant was centrifuged and stored at -20°C for cytokine detection. MC38 cells were washed twice with 100 μL PBS, and RPMI-1640 medium containing 10% CCK-8 reagent was added to each well according to the reagent instructions. After incubation at 37°C for 1-2 hours, the OD of each well was measured using a microplate reader. 450 The absorption value under [condition]. OD 450 This reflects the killing level of activated T cells on MC38 target cells. Three parallel experiments were set up for each group.

[0111] In the above experiment, the control group consisted of target cells and control cells inoculated alone without T cells. The formula for calculating cytotoxicity is:

[0112] {1-[(A expriment -A blank ) / (A control -A blank )]}×100=Cytotoxicity(%)

[0113] Where A expriment Representing the experimental group, A control A represents the control group without T cells. blank This represents the absorbance of 100 μL of 1640 medium containing 10% CCK-8 reagent.

[0114] 10. T cell proliferation analysis

[0115] First, 2×10 7 One isolated T cell was suspended in 1 mL of PBS buffer, and 1 μL of 8 mM CFSE reagent was added to the T cell suspension. The cells were incubated at 37°C in the dark for 10 min. Then, the cells were centrifuged at 250 g for 10 min, the supernatant was removed, and the CFSE-stained T cells were collected and resuspended in 1640 medium. Next, 200 μL of 2×10⁻⁶ cells / mL of PBS buffer was added to the T cell suspension. 5Suspension CFSE stained T cells were added to 96-well plate and co-incubated with SpTCE at 37°C. After 48h incubation, 250g centrifuge for 10min, collect T cells and stain with live / dead cell staining in flow buffer, and analyze by flow cytometry using Beckman flow cytometer. Cell proliferation was analyzed by CFSE fluorescence signal.

[0116] 11. CD69 expression analysis

[0117] The ability of antibody-folding conjugate to activate T cells was further evaluated by analyzing CD69 expression. T cells were negatively selected from mouse spleen using StemCell EasySep T cell negative selection kit according to manufacturer's instruction. 2x10 5 Cells were incubated with free antibody or SpTCE at a concentration of 2x10 5 cells per well in 1640 medium supplemented with 10% FBS for 6h. After washing with PBS, cells were suspended in 200μL of FITC-labeled CD69 flow antibody buffer (phosphate buffer + 1% FBS) and incubated on ice for 30min. Then 250g centrifuge for 10min, wash the stained cells with PBS, resuspend in 200μl of FACS buffer and analyze using flow cytometer.

[0118] 12. Cytokine detection

[0119] T cell activation cytokine secretion was determined using commercial ELISA kit according to the following procedure:

[0120] (1) Sample preparation: 5x10 5 cells were co-cultured with 5x10 4 MC38 cells in 250μL of 10% serum, 100U / mL penicillin, 100μg / mL streptomycin 90% RPMI-1640 medium for 24h. Supernatant was collected by centrifugation, 100μL for IFN-γ quantification and another 100μL for IL-2 quantification.

[0121] (2) Plate preparation: capture antibody was first diluted to 2μg / mL with phosphate buffer, 100μL of diluted capture antibody was added to each well of 96-well plate and incubated at 4°C overnight. Then the incubation solution was poured out, 300μL of washing buffer (phosphate buffer + 2mM EDTA + 0.5% BSA) was added to wash the plate for 2-3 times, and the residual liquid in the plate was removed as much as possible in the last washing. Subsequently, 300μL of blocking buffer (washing buffer + 2% BSA) was added and incubated at room temperature for 1-2h. Washing was repeated for 2-3 times, and the residual liquid in the plate was removed as much as possible in the last washing to prepare for the next test.

[0122] (3) Test procedure: 100 μL of diluted sample and standard were added to each well, and incubated at room temperature for 2-3 h. Then the sample was poured out, and the plate was washed with washing buffer for 4-5 times. The residual liquid in the plate was removed as much as possible in the last washing. Then 100 μL of diluted detection antibody (0.3 μg / mL) was added to each well, and incubated at room temperature for 1 h. The plate was washed for 3-4 times, and the residual liquid in the plate was removed as much as possible in the last washing. 200 μL of substrate working solution was added, and incubated for 30-60 min. Finally, 50 μL of stop solution was added, and the absorbance at 450 nm was detected by a microplate reader.

[0123] The IFN-γ, IL-2, TNF-α and IL-6 cytokines in the serum were also detected according to the above procedure.

[0124] 13. In vivo fluorescence imaging of mice

[0125] (1) The MC38 tumor cells were expanded and cultured, and the MC38 cells were collected and counted in PBS buffer;

[0126] (2) The nude mice were randomly divided into 5 groups, and 1 x 10 6 MC38 cell suspension was inoculated subcutaneously into the hind legs of the nude mice by subcutaneous injection;

[0127] (3) The tumor volume of the mice was recorded, and the volume calculation formula was V (mm 3 ) = 0.5 x L x W 2 , (W represents the short diameter of the tumor, and L represents the long diameter);

[0128] (4) When the tumor volume reached about 200-300 mm 3 , different groups of drugs PBS, 6HB, Ab@6HB, unSpTCE and SpTCE were injected through the tail vein (the injection volume was 100 μL, and the concentration was 10 nM).

[0129] (5) The mice were anesthetized with ether, and the anesthetized mice were placed in the Lago X small animal in vivo fluorescence imaging instrument for in vivo fluorescence imaging. The detection time points were before injection and 2 h, 24 h, 48 h, 72 h, 96 h after injection.

[0130] (6) After completing the 48 h in vivo fluorescence imaging, the mice were sacrificed by cervical dislocation, and the tumor, heart, liver, spleen, lung, kidney and main organs of the digestive tract were removed, arranged and imaged. Then the fluorescence intensity was semi-quantitatively analyzed.

[0131] 14. Anti-tumor treatment

[0132] (1) 1 x 10 6C57B / L6 mice were inoculated with MC38 cell suspension subcutaneously in the hind leg to get tumor-bearing mice, and the tumor size of mice was recorded;

[0133] (2) The tumor grew to about 100 mm 3 The mice were randomly divided into 6 groups, and 100 μL PBS, 6HB, free antibody, unSpTCE, Ab@6HB and SpTCE were injected into the tail vein of the mice, respectively (the concentration of 6HB was 10 nM, the concentrations of aCD3, aCD28 and aPD-L1 were 120 nM, 80 nM and 200 nM, respectively, the concentration of unSpTCE was 10 nM, the concentration of Ab@6HB was 10 nM, and the concentration of SpTCE was 10 nM);

[0134] (3) Injection once every two days, a total of 8 times, and the mouse weight and tumor size were observed and recorded one day after injection;

[0135] (4) On the 16th day, the eyeball blood of the mice was taken, the mice were executed by cervical dislocation, and the tumor was collected.

[0136] 15. Results and analysis

[0137] 15.1 Assembly of internal functional layer and external shielding layer of SpTCE and verification of its function

[0138] 15.1.16HB fold synthesis and characterization

[0139] The 6HB was constructed by the classic Rothemund method with the aid of cadnano software. The designed 63 staple strands and M13 (2695 nt) template were co-assembled under gradient annealing conditions, and 6HB was obtained by ultrafiltration purification. The structure of the annealing product and the purified product was verified by agarose gel electrophoresis. The electrophoresis experiment results show that the mixed chain system can form an assembly structure 6HB with a larger molecular weight under gradient annealing conditions, and the ultrafiltration purified product presents a clear single band, and a higher purity 6HB is obtained. Figure 3 ).

[0140] The structure of the synthesized and purified 6HB was further characterized by more intuitive atomic force and transmission electron microscopy imaging methods. The 6HB presents a uniform and dispersed rod-like structure with a length of about 150 nm ( Figure 4 ), which is consistent with the theoretical design size.

[0141] In order to distinguish each helical bundle, the six helical bundles of 6HB are numbered as 0, 1, 2, 3, 4 and 5, respectively ( Figure 5). Each helical bundle has ten specific oligonucleotide extension sequences for linking nucleic acid conjugated antibodies and designed functional nucleic acids. aCD3 and aCD28 antibodies are assembled on the 1st and 2nd helical bundles as TCR cluster inducers, aPD-L1 antibody is assembled on the 4th and 5th helical bundles to target tumor, and hand-in-hand palindromic sequences are assembled on the 0th and 3rd helical bundles, which together constitute the internal functional layer Figure 5

[0142] 15.1.26 Assembly of HB with antibodies

[0143] For antibody assembly and 6HB assembly, functional antibodies and DNA are first conjugated. Biotin-labeled antibodies are combined with biotin-labeled DNA strands (A) through streptavidin. A1, A2 and A3 strands are designed to bind aCD3, aCD28 and aPD-L1 antibodies, respectively, and are anchored at specific positions of 6HB Figure 5 ) First, 10-fold excess streptavidin and biotinylated antibodies are combined, and antibody-streptavidin conjugates are obtained by 100 kDa ultrafiltration purification. Non-denaturing protein gel electrophoresis experiment results show that the antibody molecules in the experimental group combined with streptavidin produce a lagging band, indicating that the molecular weight of the antibody-streptavidin conjugate has increased significantly Figure 6 A). Then the combination ratio of strand A and antibody-streptavidin conjugate is further optimized. Strand A and antibody-streptavidin conjugate are mixed at a molar ratio of 3:1, 2:1 and 1.5:1, respectively. Agarose gel electrophoresis experiment results show that mixing strand A and antibody-streptavidin conjugate at a ratio of 1.5:1 can obtain a relatively ideal DNA-antibody conjugate Figure 6 B).

[0144] Next, nucleic acid conjugated antibodies are assembled onto the 6HB structure. By truncating the oligonucleotide sequences of the corresponding extension sites of 6HB, Ab-DNA is assembled on the 6HB structure at different arrangement intervals and arrangement densities on the nanoscale.

[0145] 15.1.36 Arrangement of antibodies in the inner layer of 6HB on T cell activation

[0146] TCR signaling dynamics school of thought suggests that ligand affinity and spatial arrangement can have a decisive influence on T cell downstream signal transduction and activation. In order to overcome the dynamics school of thought and enhance T cell activation, the present invention studies the influence of antibody assembly and arrangement on T cell activation from three aspects, including the arrangement interval and density of aCD3 antibodies, and the addition of aCD28 antibodies. First, two pairs of aCD3 antibodies are assembled on the 1st and 2nd helical bundles of 6HB, with arrangement intervals set at 70 nm, 56 nm, 42 nm, 28 nm and 14 nm, and characterized by gel electrophoresis Figure 7 ​), the experimental results show that the molecular weight of the origami structure with different arrangement spacing is not significantly different when combined with the same amount of antibody.

[0147] The T cells isolated from mouse spleen were incubated with these different arrangement spacing aCD3-6HB conjugates for different time to test the corresponding indicators of T cell activation. Ca 2+ As the second messenger of cell signal transduction, intracellular Ca 2+ The concentration change is closely related to cell signal transduction, and by monitoring the dynamic change of intracellular Ca 2+ , the T cell activation mediated by multivalent antibody can be judged. The present application uses Ca 2+ Sensitive fluorescent dye reagent (Fluo-4AM) to monitor the dynamic calcium flow signal in T cells treated with aCD3-6HB conjugates, and a significant increase in calcium flow fluorescence signal is observed within 1min ( Figure 8 A). Compared with the same concentration of free aCD3 antibody, 6HB pair assembly triggers enhanced calcium flow signal, and with the shortening of the spacing, the calcium flow signal also gradually increases, and the T cell calcium flow signal of aCD3 antibody arrangement spacing of 28nm and 14nm tends to be relatively stable ( Figure 8 B).

[0148] Further measurement of other indicators of T cell activation, including 6h CD69 expression level, 24h cytokine secretion (IFN-γ) and 48h T cell proliferation. The up-regulation of CD69 proves that the arrangement of aCD3 antibody with shortened spacing in 6HB can trigger stronger T cell activation ( Figure 9 A). It is worth noting that compared with the blank group, the same concentration of free aCD3 antibody does not promote significant up-regulation of CD69. In addition, no obvious IFN-γ secretion and T cell proliferation was found in all groups ( Figure 9 B-C), which indicates that the lack of costimulatory signal may affect the further activation of T cells.

[0149] Further evaluate the effect of antibody arrangement density on T cell activation, assemble different number of aCD3 antibodies on 6HB No. 1 and No. 2 helical bundles, aCD3@6HB assembly number increases from 2 pairs (4) to 6 pairs (12), and aCD28 costimulatory signal is introduced, the results of agarose gel electrophoresis experiment show that the molecular weight presents increasing trend with the increase of antibody assembly number ( Figure 10 ).

[0150] Under the premise that the total concentration of antibody in experimental group and control group is consistent, it is found that assembling more aCD3 antibodies on 6HB can trigger stronger calcium flow signal in T cells ( Figure 11Figure 6. The 6HB structure contains 6 pairs of aCD3 antibodies. The 6HB structure further assembled 4 pairs of aCD28 antibodies (20Ab@6HB). The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells Figure 11 Figure 6. The 6HB structure contains 6 pairs of aCD3 antibodies. The 6HB structure further assembled 4 pairs of aCD28 antibodies (20Ab@6HB). The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells

[0151] On this basis, the present application further studies other indicators of T cell activation. CD69 expression gradually increases with the increase of the number of aCD3 antibodies on a single 6HB, and reaches the highest level in the case of co-assembly of aCD28 antibodies and aCD3 antibodies Figure 12 Figure 6. The 6HB structure contains 6 pairs of aCD3 antibodies. The 6HB structure further assembled 4 pairs of aCD28 antibodies (20Ab@6HB). The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells Figure 12 Figure 6. The 6HB structure contains 6 pairs of aCD3 antibodies. The 6HB structure further assembled 4 pairs of aCD28 antibodies (20Ab@6HB). The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells

[0152] 15.1.46HB outer shielding layer function verification

[0153] The acidic pH around tumor tissue is a major feature of the tumor microenvironment and an important entry point for intelligent nanodrug targeted therapy. The present application designs a spatiotemporally controllable outer shielding layer by assembling a pH-responsive ABD (I / C-ABD). Chain I contains an i-motif sequence at the 3' end and a sequence complementary to the A chain, thereby being fixed on the origami structure Figure 13 Figure 6. The 6HB structure contains 6 pairs of aCD3 antibodies. The 6HB structure further assembled 4 pairs of aCD28 antibodies (20Ab@6HB). The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells Figure 13 Figure 6. The 6HB structure contains 6 pairs of aCD3 antibodies. The 6HB structure further assembled 4 pairs of aCD28 antibodies (20Ab@6HB). The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells Figure 13 Figure 6. The 6HB structure contains 6 pairs of aCD3 antibodies. The 6HB structure further assembled 4 pairs of aCD28 antibodies (20Ab@6HB). The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells Figure 13 Figure 6. The 6HB structure contains 6 pairs of aCD3 antibodies. The 6HB structure further assembled 4 pairs of aCD28 antibodies (20Ab@6HB). The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells

[0154] To investigate the effect of ABD assembly density on the stability of 6HB structure under physiological conditions, ABDs were assembled on different helices of 6HB, and No. 0 / 3ABD@6HB, No. 0 / 1 / 2ABD@6HB, No. 0 / 2 / 4ABD@6HB and No. 0 / 1 / 2 / 3 / 4 / 5ABD@6HB (fully covered) were constructed. These ABD-assembled 6HBs were incubated in 100 nM HSA and different concentrations of DNase I for 12 h. Agarose gel electrophoresis analysis showed that 6HBs with three or more helix ABD assemblies were sufficiently stable in high concentrations of nuclease (10 U / mL) compared to simple 6HB structures Figure 14 Fig. 2A). In addition, in ordinary cell culture medium (DMEM containing 10% FBS), the fully covered ABD 6HB had higher stability than the partially ABD-assembled 6HB within 36 h Figure 14 Fig. 2B), which confirmed that ABD assembly on the structure enhanced the nuclease resistance and physiological stability of DNA origami chassis. ABD assembly on all six helices was selected as the optimal outer layer configuration.

[0155] Further study of the i-motif switch response to pH-mediated outer layer shedding was carried out by incubating ABD@6HBs under different pH conditions. Using chain I* which does not respond to pH as a control, agarose gel electrophoresis results showed that at pH 7.4, I / C-ABD@6HBs had little difference in migration rate from I* / C-ABD@6HBs under normal conditions, and I / C-ABD@6HBs had higher mobility than I* / C-ABD@6HBs at pH 6.5 and 5.5, indicating the response ability of the outer shielding layer to acidic pH Figure 15 ). In addition, the outer layer of I / C-ABD@6HBs was completely separated at pH 6.5 and 5.5, as indicated by the similar molecular weight to that of simple 6HB.

[0156] 15.2 In vitro performance characteristics of SpTCE

[0157] 15.2.1 In vitro pH response ability and stability of SpTCE

[0158] After completing the assembly and optimization of the two layers, the inner functional layer and the outer shielding layer were assembled into the 6HB structure to construct the SpTCE. Gel electrophoresis results showed the hierarchical assembly of aCD3, aCD28 and aPD-L1 antibodies mediated by chain A in the 6HB structure Figure 16 Fig. 3A). The hierarchical assembly of the inner and outer layers of the 6HB structure and the response of the outer layer structure to pH-induced disassembly were analyzed by gel electrophoresis. Lanes 1 and 2 were added with 6HB assembly of the inner antibody and the co-assembly product SpTCE of the inner antibody and the outer ABD of the 6HB structure, respectivelyFigure 16 Figure 7. pH-dependent unfolding of ABD / SpTCE. (A) Schematic of ABD / SpTCE. (B) Gel images of ABD / SpTCE at different pH values. Lane 1, ABD / SpTCE at pH 7.4; lane 2, ABD / SpTCE at pH 6.5; lane 3, ABD / SpTCE at pH 5.5; lane 4, ABD / SpTCE at pH 4.5; lane 5, ABD / SpTCE at pH 3.5; lane 6, ABD / SpTCE at pH 2.5. The ABD / SpTCE band migrates slower at pH 7.4 (lane 1) than at pH 6.5 (lane 2) and pH 5.5 (lane 3), indicating that the ABD / SpTCE structure is stable at pH 7.4 but not at pH 6.5 and pH 5.5. At pH 4.5 (lane 4), the ABD / SpTCE band migrates faster than at pH 3.5 (lane 5) and pH 2.5 (lane 6), indicating that the ABD / SpTCE structure is stable at pH 4.5 but not at pH 3.5 and pH 2.5. Figure 16 Figure 16

[0159] Further evaluation of the in vitro stability of SpTCE was performed to verify its stability in serum. The low ionic strength and high nuclease activity of serum have a significant impact on the stability of DNA structures. To evaluate the effect of the shielding layer on serum stability, the present application incubated SpTCE in mouse serum for different times and used Image J to measure the remaining amount of structure over time. After incubation in mouse serum for 36 h, 41% of the ABD-carrying SpTCE remained intact, more than both Ab@6HB (13%) and 6HB (20%), indicating that the serum albumin layer wrapping can significantly enhance the stability of SpTCE ( Figure 17 ).

[0160] 15.2.2 SpTCE targeting ability

[0161] The present application further verified the ability of Ab@6HB to target T cells and tumor cells MC38 (derived from C57BL / 6 mouse colon adenocarcinoma, PD-L1 high expression cell line) through flow cytometry experiments. By doxorubicin (Dox) labeling the origami, co-incubation of Ab@6HB with T cells and MC38 cells, respectively, the flow cytometry results showed that Ab@6HB could target both T cells and MC38 cells ( Figure 18 , light blue peak).

[0162] To further verify the response of SpTCE to pH after the antibody is exposed from the shielding to the exposed state, the present application used Dox-labeled 6HB to track the origami base and FAM-labeled strand I to track the outer shielding layer. Through flow cytometry experiments, it was verified that SpTCE could specifically recognize T cells ( Figure 19 , light blue peak). In addition, T cells incubated with SpTCE without HSA showed fluorescence shifts in both FAM / FITC and Dox / PE channels ( Figure 19 , blue peak). In contrast, HSA / SpTCE did not show significant shifts in both FAM / FITC and Dox / PE channels at pH 7.4.​​Figure 19 The green peak (A) indicates that only the HSA attachment layer can shield the binding of internal antibodies to T cells. At pH 6.5, HSA / SpTCE-treated T cells shifted in the Dox / PE channel but not in the FAM channel. Figure 19 The red peak (A) indicates the separation of the outer shielding layer and the restoration of antibody binding capacity. Furthermore, confocal microscopy results also validated the ability of SpTCE to target T cells. Figure 19 (B)

[0163] 15.2.3 SpTCE-guided T cell killing targeting MC38

[0164] Next, this invention evaluated SpTCE-induced T cell targeted killing ability by mixing T cells and MC38 cells at a ratio of 10:1. Intact Ab@6HB significantly enhanced T cell cytotoxicity against MC38 cells compared to Ab-6HB alone or partially assembled. Figure 20 (A), and increased the IFN-γ secretion level of T cells ( Figure 20 (Middle B). hand-Ab@6HB showed the highest cytotoxicity against MC38 cells, confirming that larger TCR clusters enhance T cell activation. This invention also tested the cytotoxicity induced by HSA / SpTCE under different pH conditions. No significant cytotoxicity was observed on MC38 cells at pH 7.4, but at pH 6.5, SpTCE achieved a killing efficiency of nearly 80%, similar to that of hand-Ab@6HB. Figure 20 (B) indicates that acidic pH conditions trigger the shedding of the SpTCE outer layer and demonstrates that functional antibodies guide T cells to target and kill MC38 cells.

[0165] 15.3 Distribution of SpTCE in vivo

[0166] To test the biodistribution of SpTCE in vivo, a tumor model was established by subcutaneously inoculating MC38 cells into immunodeficient NOD-NSG nude mice. Unresponsive pH-dependent I* chains were assembled on 6HB to form unresponsive SpTCE (unSpTCE), and Cy7-modified H1 and H2 chains were immobilized on the structure to trace 6HB. When the tumor volume reached 300 mm², the tumor was targeted for biodistribution. 3 Subsequently, T cells were injected into mice via the tail vein. Two hours later, PBS, 6HB, Ab@6HB, unSpTCE, SpTCE, and other drugs were injected into mice in different groups via the tail vein, and the changes in fluorescence intensity in the mice over time were monitored using an in vivo fluorescence imaging system. Figure 21). At 2h, all Cy7-labeled 6HB-treated mice exhibited systemic fluorescence signal distribution, while the Ab@6HB group and SpTCE group showed significant accumulation at the tumor site. However, unSpTCE-treated mice did not show tumor site accumulation, confirming that the shielding effect of the outer layer of albumin prevented the structure from targeting the tumor site. At 48h and 72h, SpTCE-treated mice maintained stronger tumor fluorescence accumulation signals than the Ab@6HB control group, possibly due to the intact shielding layer enhancing its half-life in vivo. At 96h, the fluorescence signals of all groups of mice were significantly reduced Figure 22 ).

[0167] On this basis, the present application analyzes the fluorescence distribution in the main organs of different experimental groups and control groups after 48h Figure 23 ). The fluorescence signal of the SpTCE group accumulated at the tumor site was higher than that of other treatment groups, and was also much higher than that of other organs Figure 23 ), indicating that SpTCE has excellent tumor-specific targeting ability and higher stability in vivo.

[0168] 15.4 In vivo efficacy study

[0169] 15.4.1 Anti-tumor effect of SpTCE in vivo

[0170] To evaluate the therapeutic potential of SpTCE in vivo, the present application studies the spatiotemporal control of anti-tumor efficacy of SpTCE in vivo. A tumor model was prepared by subcutaneously inoculating MC38 in C57B / L6 mice. After 6 days of tumor growth, the mice were randomly divided into six groups and injected intravenously with PBS, 6HB, free antibody, unSpTCE, Ab@6HB and SpTCE, respectively, every two days for a total of 8 injections Figure 24 A). At the same time, the tumor size and body weight of these experimental group mice were continuously monitored Figure 24 C). The tumor size of the 6HB-treated mice was very close to that of the PBS-treated blank group Figure 24 B), indicating that the empty vector had no significant effect on tumor growth. The mice treated with free antibody may have inhibited tumor growth to some extent through immune checkpoint blockade by the PD-L1 antibody Figure 24 B). The unSpTCE-treated mice showed similar tumor growth rates to the free antibody-treated group Figure 24 B). The tumor growth rate of the Ab@6HB-treated mice was slowed down, and the SpTCE-treated mice showed the lowest tumor growth rate Figure 24 B) and the longest survival rate Figure 25 D).

[0171] After treatment was terminated on day 16, I removed and weighed the tumors from all groups of mice. Figure 25 (A). Experimental results showed that the tumor weight in mice treated with PBS and 6HB was approximately 1.5g, the tumor weight in mice treated with free antibody and unSpTCE was approximately 0.7g, the tumor weight in mice treated with Ab@6HB was approximately 0.3g, and the tumor weight in mice treated with SpTCE was approximately 0.2g. Figure 26 In the six groups (B), the SpTCE treatment group showed the best results in terms of tumor weight in mice.

[0172] At the end of treatment, ocular blood was collected, and the levels of IFN-γ, IL-6, TNF-α, and IL-2 cytokines in mouse plasma were detected using an ELISA kit. Based on the cytokine secretion levels, the Ab@6AB treatment group showed relatively high plasma secretion levels. Figure 27 Therefore, it is believed that naked antibodies are more likely to cause systemic toxic side effects. However, mice treated with SpTCE showed relatively low plasma IFN-γ, IL-6, TNF-α, and IL-2 secretion levels. Combined with its effective anti-tumor efficacy, this indicates that SpTCE has reduced side effects and enhanced specificity and safety.

[0173] Next, this invention analyzed the antitumor effects of mouse tumor tissue sections treated with different groups of drugs by performing H&E and TUNEL staining. Figure 27 As shown, H&E staining results indicated that the Ab@6HB and SpTCE groups showed more tumor area necrosis compared to the blank group and the 6HB control group. The Ab group and the unSpTCE treatment group showed relatively mild tumor tissue damage. TUNEL staining was used to distinguish the apoptosis of tumor cells in different groups. The Ab@6HB and SpTCE groups showed higher levels of green fluorescence (TUNEL) compared to other groups, indicating that Ab@6HB and SpTCE treatment induced more tumor cell apoptosis. Figure 28 The Ab group and the unSpTCE treatment group exhibited moderate apoptosis signals, while the blank group and the 6HB control group showed almost no significant tumor cell apoptosis. These experimental results indicate that the Ab@6HB group and the SpTCE group have good tumor treatment effects.

[0174] Finally, the biosafety of PBS, 6HB, free antibody Ab, unSpTCE, Ab@6HB, and SpTCE in mice was evaluated. Experimental results showed that H&E staining of the heart, liver, spleen, lung, and kidney organs in mice did not cause significant histological damage. ​ This indicates that these multivalent antibody-drug conjugates have good biosafety in important tissues.

[0175] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A DNA nanostructure-based spatiotemporal controllable T cell engager, characterized in that, It comprises a rod-like six-helix bundle origami structure and an internal functional layer and an external shielding layer assembled on the six-helix bundle origami structure; The six-helix bundle origami structure is co-assembled by 63 staple strands and single-stranded circular DNA M13; The internal functional layer comprises nucleic acid-antibody conjugates combined on four helix extension sequences at the top and bottom of the six-helix bundle origami structure, and hand-in-hand palindromic sequences combined on two helix extension sequences at the left and right sides, wherein the hand-in-hand palindromic sequences are shown in SEQ ID NO. 64 or SEQ ID NO. 67; The external shielding layer comprises C-chain modified serum albumin binding peptide and chain I, wherein the chain I comprises a pH-responsive i-motif switch, and the external shielding layer is combined on the internal functional layer, wherein the nucleotide sequence of the C-chain is shown in SEQ ID NO. 76, and the nucleotide sequence of the chain I is shown in any one of SEQ ID NO. 71-75; The nucleic acid-antibody conjugate is obtained by conjugating chain A and streptavidin-modified antibody, wherein the nucleotide sequence of the chain A is shown in any one of SEQ ID NO. 65, SEQ ID NO. 66 or SEQ ID NO. 68, and the antibody comprises aCD3; The 3' end of the chain I comprises a pH-responsive i-motif switch and a sequence complementary to the chain A, so that the external shielding layer is combined on the internal functional layer, and then fixed on the six-helix bundle origami structure; When the pH value is greater than the response threshold, the chain I is combined on the chain A, and is fixed on the six-helix bundle origami structure extension sequence through the chain A, and forms an external shielding layer on the six-helix bundle origami structure with the C-chain modified serum albumin binding peptide, so as to shield the internal functional layer, avoid activating T cells in healthy tissues, and improve the stability of blood circulation in vivo; when the pH is less than the response threshold, the chain I forms an i-motif quadruplex structure and separates from the six-helix bundle origami structure, so that the exposed antibody and hand-in-hand palindromic sequence induce TCR clustering and activate T cells, thereby achieving tumor cell killing.

2. The spatiotemporally controllable T cell engager of claim 1, wherein, The nucleotide sequence of the 63 staple strands is shown in SEQ ID NO. 1-63.

3. The spatiotemporally controllable T cell engager of claim 1, wherein, The molar ratio of the chain A and the streptavidin-modified antibody is 1.5-3:

1.

4. The spatiotemporally controllable T cell engager of claim 1, wherein, The antibody further comprises aCD28 and / or aPD-L1 antibody; The nucleic acid-antibody conjugate combined on the two helix extension sequences at the top of the six-helix bundle origami structure is obtained by conjugating chain A with aCD3 and aCD28 antibodies, respectively; The quantity ratio of the aCD3 and aCD28 antibodies is 3:

2.

5. A method of constructing a spatiotemporally controllable T cell engager as claimed in any one of claims 1-4, characterized in that, The method comprises the following steps: The nucleic acid-antibody conjugate, the C-chain modified serum albumin binding peptide and the chain I are mixed in equal molar ratio to form a triple-stranded structure, and then the triple-stranded structure is mixed with the six-helix bundle origami structure to obtain a spatiotemporal controllable T cell engager.

6. The construction method of claim 5, wherein, The molar ratio of the triple-stranded structure and the six-helix bundle origami structure is 1.2n:1, wherein n represents the number of extension sequence binding sites of the six-helix bundle origami structure.

7. Use of the spatiotemporal controllable T cell engager of any one of claims 1-4 in the preparation of a medicament for improving immunotherapy treatment of tumors in weakly acidic microenvironments.

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