Space-time controllable T cell adapter based on DNA (deoxyribonucleic acid) nanostructure as well as construction method and application of space-time controllable T cell adapter

Abstract

The invention discloses a time-space controllable T cell adapter based on a DNA nanostructure as well as a construction method and application of the time-space controllable T cell adapter, and belongs to the field of DNA nanotechnology. The T cell adapter comprises a six-spiral-bundle paper folding structure, and an internal functional layer and an external shielding layer which are assembled on the six-spiral-bundle paper folding structure; the six-spiral bundle paper folding structure is formed by assembling 63 staple chains and M13 in total; the internal functional layer comprises nucleic acid-antibody conjugates which are combined on four spiral extension sequences at the top and the bottom of the six-spiral bundle origami structure, and hand-in-hand palindromic sequences which are combined on two spiral extension sequences at the left side and the right side; the outer shielding layer comprises a C-chain modified serum albumin binding peptide and a chain I, and the chain I comprises a pH-responsive i-motif switch. The T cell adapter disclosed by the invention has the space-time control capability of pH response and the potential of inducing TCR clustering to enhance T cell activation, and has huge potential in the aspect of improving the safety and effectiveness of T cell immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of DNA nanotechnology, and in particular to a spatiotemporally controllable T-cell conjugate based on DNA nanostructures, its construction method, and its application. Background Technology

[0002] Tumor immunotherapy, represented by bispecific antibody conjugates (BiTEs), has made significant progress in clinical practice. In 2023 alone, the FDA approved four types of T-cell conjugates for the treatment of hematologic malignancies. Despite these promising results, T-cell conjugate therapies still face limitations in terms of safety and efficacy. Systemic toxicity caused by non-spatiotemporally selective activation of T cells is a serious and pressing issue that needs to be addressed in T-cell conjugate therapy. Typically, BiTEs have two distinct binding sites to simultaneously target the CD3 portion of the T-cell receptor (TCR) and specific antigens on the surface of tumor cells. Directly exposed binding sites often lack spatial specificity, leading to non-specific activation of T cells and potentially causing systemic adverse reactions including neurotoxicity and cytokine release syndrome. B-cell-associated antigens CD19 and CD20 have proven to be ideal targets for treating B-cell malignancies. However, in other tumors, tumor-specific antigens are relatively scarce, especially in solid tumors, where tumor-associated antigens are also expressed at certain levels in healthy tissues, potentially leading to severe targeted toxicity to healthy tissues. Therefore, the safety of T-cell conjugate therapies still needs improvement.

[0003] Besides safety concerns, poor therapeutic efficacy is another problem with conventional T-cell conjugates. Typically, BiTEs only provide the primary signal for T-cell activation; the co-stimulatory signaling for T-cell activation depends on the tumor cells themselves. However, tumor cells may not express or express low levels of co-stimulatory signaling molecules, and may even express immune checkpoint inhibitory molecules like PD-L1, preventing T cells from recognizing target cells and reducing the therapeutic effect of T-cell conjugates. Kinetic separation models indicate that TCR clusters play a crucial role in T-cell activation. Compared to conjugates that bind only TCRs and tumor-associated antigens, simultaneously inducing the assembly of receptor clusters and introducing co-stimulatory signals demonstrates higher efficiency in activating T cells to fight tumors.

[0004] Novel clinical and preclinical T-cell conjugates have been developed to address the shortcomings of current T-cell immunotherapy in terms of safety and efficacy. For example, PD-L1 or CD28 ligands are fused into T-cell conjugates to reduce PD-L1-mediated immunosuppression and enhance T-cell activation. Furthermore, other forms of T-cell conjugates or inducers based on non-protein scaffolds offer different solutions. To enhance T-cell activation levels, multiple pMHC and CD28 ligands are anchored to erythrocytes or nanomaterials to induce TCR clusters. Recent studies have shown that DNA nanotechnology, with its highly programmable and parallel computing capabilities, enables more diverse designs for T-cell conjugates, such as incorporating DNA logic computation into DNA-antibody chimeric T-cell conjugates to enhance specificity. Developing DNA origami-based T-cell conjugates offers greater modularity and programmability, facilitating the screening and development of multi-specific conjugates. These more functionally rich conjugates have made some progress in enhancing the efficacy and specificity of immunotherapy. However, these conjugates still lack the ability to precisely control the spatiotemporal activation of T cells. Summary of the Invention

[0005] The purpose of this invention is to provide a spatiotemporally controllable T-cell conjugate based on DNA nanostructures, its construction method, and its application, in order to solve the problems existing in the prior art. This T-cell conjugate has the ability to control the spatiotemporal response to pH and the potential to induce TCR clusters to enhance T-cell activation. This has great potential to improve the safety and efficacy of T-cell immunotherapy and can achieve precise mediation of immunotherapy for tumors.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a spatiotemporally controllable T-cell conjugate based on DNA nanostructures, which includes a rod-shaped six-helix bundle origami structure and an inner functional layer and an outer shielding layer assembled on the six-helix bundle origami structure.

[0008] The six-helix bundle origami structure is assembled using 63 staple chains and single-stranded circular DNA M13.

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

[0010] The outer shielding layer comprises a C-chain modified serum albumin-binding peptide and chain I, wherein chain I contains a pH-responsive i-motif switch, and the outer shielding layer is bound to the inner functional layer; the nucleotide sequence of the C-chain is as shown in SEQ ID NO. 76, and the nucleotide sequence of chain I is any one of those shown in SEQ ID NO. 71-75.

[0011] Preferably, the nucleotide sequences of the 63 staple chains are as shown in SEQ ID NO.1-63.

[0012] Preferably, the nucleic acid antibody conjugate is formed by conjugating chain A with an antibody modified with streptavidin, wherein the nucleotide sequence of chain A is any one of those shown in SEQ ID NO.65, SEQ ID NO.66 or SEQ ID NO.68, and the antibody includes aCD3, aCD28 and / or aPD-L1 antibodies.

[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 conjugates attached to the top two helical extension sequences of the six-helix bundle origami structure are formed by conjugating chain A with aCD3 and aCD28 antibodies respectively; and / or the ratio of the number of aCD3 and aCD28 antibodies is 3:2.

[0015] Preferably, the 3' end of chain I includes a pH-responsive i-motif switch and a sequence complementary to chain A, so that the outer shielding layer is bonded to the inner functional layer and thus fixed to the six-helix bundle origami structure.

[0016] Preferably, when the pH value is greater than the response threshold, chain I binds to chain A and is fixed to the extended sequence of the six-helix bundle origami structure via chain A. It forms an outer shielding layer with the serum albumin-binding peptide modified by chain C on the six-helix bundle origami structure to shield the inner functional layer, thereby preventing the activation of T cells in healthy tissues and improving the stability of blood circulation in vivo. When the pH value is less than the response threshold, chain I forms an i-motif quadruplex structure and separates from the six-helix bundle origami structure, allowing the exposed antibody and the hand-in-hand palindromic sequence to induce TCR clustering, activate T cells, and achieve the killing of tumor cells.

[0017] The present invention also provides a method for constructing the spatiotemporally controllable T-cell conjugate, comprising the following steps:

[0018] Nucleic acid-antibody conjugate, C-chain modified serum albumin-binding peptide and chain I are mixed in equal molar ratio to form a three-chain structure. Then, the three-chain structure is mixed with a six-helix bundle origami structure to obtain a spatiotemporally controllable T-cell conjugate.

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

[0020] This invention also provides the use of the aforementioned spatiotemporally controllable T-cell connector in the preparation of drugs or drug carriers for enhancing immunotherapy treatment of tumors. The aforementioned tumors include, but are not limited to, colorectal cancer.

[0021] The present invention discloses the following technical effects:

[0022] This invention presents a multivalent antibody T-cell conjugate with an inner and outer double-layered functional structure based on 6HB origami. It exhibits spatiotemporal specificity, enhances T-cell activation efficiency, reduces systemic toxicity, and possesses excellent physiological stability and biocompatibility. The programmability and addressability of the origami structure enable ordered and multivalent antibody assembly. This invention demonstrates that the origami structure assembling CD3 and CD28 antibodies can effectively induce TCR clustering, significantly improving T-cell activation levels compared to monovalent antibodies, and also effectively enhancing T-cell cytotoxicity against target cells. The serum albumin encapsulation layer effectively prevents premature exposure of CD3 and CD28 antibodies during blood circulation, preventing T-cell mis-killing of normal tissues and blocking systemic toxicity caused by non-specific T-cell activation. The invention also utilizes the weakly acidic tumor microenvironment to design an H... + The responsive outer layer shedding switch provides a foundation for spatiotemporally controlled T cell activation. Considering PD-L1-mediated T cell activity inhibition, this invention uses an origami structure to immobilize PD-L1 antibody-mediated T cell targeting and elimination of PD-L1-overexpressing MC38 tumor cells. This invention has demonstrated the effectiveness of spatiotemporally controlled activation of multivalent T cell conjugates in tumor therapy through in vitro and in vivo experiments, providing a basis for improving the safety, specificity, and efficacy of T cell immunotherapy. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the principle of T cell activation for precise tumor targeting using a DNA origami-based spatiotemporally controllable TCR cluster conjugate (SpTCE); (A) Assembly of the six-helix bundle (6HB) base and functionalization of the antibody with albumin-binding peptide (ABD) to construct the SpTCE; (B) The outer layer of the SpTCE detaches via a pH-responsive i-motif switch, exposing the inner layer and forming a macroscopic hand-in-hand structure; (C) Schematic diagram of SpTCE for tail vein injection immunotherapy;

[0024] Figure 2Serum albumin-binding peptide (ABD) expression was analyzed by SDS-PAGE (10%); Marker is the standard protein molecule, Purification is the purified protein, supernatant is the supernatant, and Whole protein is the unseparated whole protein.

[0025] Figure 3 6HB is characterized by agarose gel electrophoresis; from left to right, Marker is the DNA standard molecule, M13 is the single-stranded circular DNA M13, Annealed products are the annealed products, Purified products are the purified products, and Filtered staples are the ultrafiltered staple strands.

[0026] Figure 4 The 6HB structure was characterized by AFM and TEM.

[0027] Figure 5 A schematic diagram of the 6HB design; the scaffold of ssDNA (2659nt) is depicted with gray lines; the staple chains are shown in black; these dots represent the locations of extended capture chains used to bind DNA-protein conjugates; orange, purple, and green dots represent the locations of capture chains that bind to aCD3, aCD28, and aPD-L1 DNA-protein conjugates, respectively; gray dots represent the locations of tethered links.

[0028] Figure 6 To verify the conjugation of biotinylated antibody and DNA with streptavidin by gel electrophoresis (A) and to optimize the binding ratio of streptavidin A and antibody-streptavidin conjugate (B);

[0029] Figure 7 6HB aCD3s with different spacings were assembled for agarose gel electrophoresis characterization.

[0030] Figure 8 The effect of antibody spacing on T cell activation; (A) The relationship between Fluo-4AM calcium probe fluorescence and time after the addition of free aCD3 or aCD3@6HB; (B) The response of T cells to different calcium signals. aCD3@6HB was stimulated by monitoring fluorescence signals using Fluo-4AM calcium probe, and all fluorescence signals were normalized.

[0031] Figure 9 To investigate the effect of antibody spacing on T cell proliferation; (A) Flow cytometry analysis of CD69 expression in T cells after different types of stimulation showed that Ab@6HB had different spacings ranging from 70 nm to 14 nm; (B) Quantitative analysis of IFN-γ secretion levels in response to different stimuli by T cells; (C) Flow cytometry analysis of T cell proliferation in response to different stimuli.

[0032] Figure 10 6HBs assembled with aCD3 at different densities were characterized by agarose gel electrophoresis.

[0033] Figure 11 The effect of 6HB antibody density on T cell activation; (A) Relationship between Fluo-4 AM calcium probe fluorescence and time after adding aCD3 aCD3@6HB; (B) T cell response to different calcium signals Ab@6HB was stimulated by monitoring fluorescence signals using Fluo-4 AM calcium probe, and these fluorescence signals were normalized to the maximum response of all samples in the replicate.

[0034] Figure 12 The effects of 6HB antibody density on T cells and their proliferation were investigated. (A) Flow cytometry analysis of CD69 expression in T cells after different types of stimulation showed that Ab@6HB had different antibody densities. (B) Quantitative analysis of IFN-γ secretion levels in T cells after different types of stimulation showed that Ab@6HB had different antibody densities. (C) Flow cytometry analysis of the response of T cell proliferation to different stimuli was conducted.

[0035] Figure 13 Optimization for i-motif switches; (A) Design principles of the outer shielding layer; (B) Folding efficiency of different i-motif switches at pH 7.28 to 5.51;

[0036] Figure 14 The stability of ABD assembly to the 6HB structure; (A) Stability of ABD@6HB structure against enzymatic digestion, M is the marker, lanes 1-2 are 6HB, lanes 3-4 are No.0 / 1 / 2 / 3 / 4 / 5ABD@6HB, lanes 5-6 are No.0 / 2 / 4ABD@6HB, lanes 7-8 are No.0 / 1 / 2ABD@6HB, lanes 9-10 are No.0 / 3ABD@6HB; (B) Stability of No.0 / 2 / 4ABD@6HB and No.0 / 1 / 2 / 3 / 4 / 5ABD@6HB structures after incubation in cell culture medium for different times;

[0037] Figure 15 The outer layer of the ABD@6HBs shielding layer was characterized by agarose gel electrophoresis in response to different pH values.

[0038] Figure 16To characterize the stepwise assembly and pH-responsiveness of SpTCE by agarose gel electrophoresis; (A) Stepwise assembly of antibodies in 6HB; lane 1 is 6HB, lanes 2-5 are chain A-mediated aCD3, aCD28, aPD-L1 antibodies and their combination in the 6HB structural hierarchy assembly products, respectively; (B) Separation of the pH-responsive outer shielding layer and the formation of large structures by hand-linking at low pH; lanes 1-3 are 6HB assembly of internal antibodies, internal antibody and external ABD co-assembly products SpTCE, HSA / SpTCE, respectively, lane 4 is the product formed by HSA / SpTCE under pH 6.5 conditions, and lane 5 is hand-Ab@6HB;

[0039] Figure 17 The stability of SpTCE, Ab@6HB and 6HB in mouse plasma was characterized 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 To validate the ability of Ab@6HB to target T cells and tumor cells by flow cytometry; (A) Flow cytometry validation of Ab@6HB targeting T cells; (B) Flow cytometry validation of Ab@6HB targeting MC38 cells;

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

[0042] Figure 20 To validate SpTCE-guided T cell-mediated tumor cell killing and to detect cytokines; (A) CCK8 validation of SpTCE-guided T cell-mediated tumor cell killing; (B) IFN-γ cytokine detection; The horizontal axis in the figure, from left to right, represents 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 To illustrate the distribution of SpTCE in vivo; to image the fluorescence distribution of Cy7-labeled origami in MC38 tumor model mice at different time points, with three independent experiments set up 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 cells MC38, purchased from the Cell Bank of the Chinese Academy of Sciences; mouse CD3 + T cells were isolated and extracted from mouse spleen.

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

[0059] Table 1. DNA sequences used in the experiment.

[0060]

[0061]

[0062]

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

[0064] Table 2. DNA sequences used in the experiment.

[0065]

[0066] Note: i-motif sequences are marked with a comma, and red-marked bases are palindromic sequences.

[0067] Example 1

[0068] 1. ABD protein expression

[0069] ABD was expressed using an *E. coli* system. The ABD protein had a SNAP and 6×His tag added to its C-terminus. The synthesized gene was amplified by PCR, and the amplification product underwent homologous recombination. The resulting product was cloned into the pET-28a vector for protein expression. After successful recombination via sequencing by Sangon Biotech (Shanghai) Co., Ltd., the recombinant plasmid was expressed (ABD protein amino acid sequence (SEQ ID NO. 79): DKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNAY FHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLAVKEWLLAHEGHRLGKPGLGLAAALEHHHHHH). The specific steps for protein expression are as follows:

[0070] (1) Construction of ABD expression strain. BL21(DE3) competent cells were removed from a -80℃ freezer and incubated on ice for 5 min. 2 μL of plasmid was added to the competent cell centrifuge tube, gently shaken 3 times, and incubated on ice for 30 min. The plasmid was then added to the competent cell centrifuge tube and placed in a 42℃ water bath for 90 s, followed by incubation on ice for 2-3 min. 500 μL of LB medium was added to the centrifuge tube, and the tube was incubated at 37℃ and 220 rpm for 30 min. The tube was centrifuged at 6900 g / min for 1 min, 400 μL of supernatant was removed, and the remaining bacterial culture was mixed by pipetting and evenly spread onto a solid medium plate containing kanamycin. The plate was air-dried in a clean bench for 2 min, and then incubated upside down at 37℃ for 12 h.

[0071] (2) Screening, culture and induction of bacterial expression. Single colonies of kanamycin on solid agar plates for 12 hours were selected, transferred to 5 mL of liquid LB medium, and 5 μL of kanamycin (1:1000) was added to the medium. The culture was then incubated overnight at 37°C and 250 r / min for 12 hours.

[0072] (3) Pour the bacterial culture into a shake flask containing 100 mL of sterile culture medium, add 100 μL of kanamycin (1000×), and continue culturing at 37℃ and 220 r / min for about 4 h until the bacterial culture OD 600 Add the appropriate dose of IPTG between 0.5 and 0.7, and induce protein expression at 18°C ​​for 12-16 hours.

[0073] (4) After induction, transfer the bacterial culture to a 50 mL centrifuge tube, place it in a centrifuge, and centrifuge at 7200 rpm for 10 min at 4 °C. Discard the supernatant and wash once with 1×PBS buffer. Centrifuge at 7200 rpm for 10 min at 4 °C and collect the bacterial cells.

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

[0075] (1) Ultrasonic disruption of bacterial cells. The collected bacterial cells were resuspended on ice with 1×PBS and disrupted using an ultrasonic disruptor at 45% power for 30-40 min. The disrupted bacterial cells were then centrifuged at 7200 rpm for 10 min at 4℃, and the supernatant was collected. The sample was retained, and SDS-PAGE was used 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), the final total volume is 50 μL, placed in a PCR thermal cycler for gradient annealing, the annealing program is set to 75 °C for 5 min, and then the temperature is reduced from 65 °C to 4 °C at a cooling rate of 15 min / °C, and the product is purified by ultrafiltration.

[0083] (2) Use a 0.5 mL ultrafiltration tube with a molecular cutoff of 100 kDa. The annealing product was purified by ultrafiltration (Ultra-0.5) using 1×TE-Mg. 2+ Wash three times with buffer (4700g / min, 5min, 4℃), invert the purified sample from the ultrafiltration tube to recover it, quantify it with an ELISA reader, and store it at 4℃.

[0084] (3) 6HB was characterized using transmission electron microscopy (TEM) to determine its specific morphology and size. First, 10 μL of magnesium acetate (2M) was added to a copper grid and allowed to stand for 2 min. The magnesium acetate was then removed using filter paper. Next, 10 μL of purified 6HB folded paper (10 nM) was added to the copper grid and allowed to stand for 30 min. Subsequently, 10 μL of uranyl acetate (1%, w:v) was added and quickly removed using 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 an 800 kV TEM (JEM-2100FX, Hitachi).

[0085] (4) Simultaneously, 6HB origami was characterized using atomic force microscopy (AFM). For AFM characterization, the mica sheet surface was first treated with 20 μL of 100 mM NiCl2 for 2 min to increase the adsorption of DNA origami structures, followed by rinsing with ddH2O and drying with compressed air. Next, 5 μL of 10 nM 6HB was dropped onto the mica surface and allowed to stand for 5 min, then slowly washed with ddH2O and dried again with compressed air. Then, scanning was performed using a Scanasyst gas chromatograph probe and a Bruker Dimension microscopy system. Images acquired using an atomic microscope.

[0086] 4. Protein-nucleic acid coupling

[0087] For functional antibodies against aCD3, aCD28, and aPD-L1, antibody-nucleic acid conjugation was achieved using a biotin-labeled antibody-streptavidin-biotin-labeled nucleic acid linkage. The biotin-labeled antibody and streptavidin were incubated in PBS solution at a molar ratio of 1:10 at 4°C for 30 min. The mixture was then washed three times using an ultrafiltration tube with a molecular cutoff of 100 kDa to remove excess streptavidin. The antibody-streptavidin conjugate was then incubated with biotin-modified DNA at a molar ratio of 1:1.5 in PBS solution at 4°C for 30 min to obtain the antibody-streptavidin-nucleic acid conjugate.

[0088] For ABD proteins with a SNAP tag, ABD-SNAP and DBCO-modified DNA strands were covalently linked using a bifunctional ligation reagent. After removing BG-NH2[O6-(4-aminomethyl-benzyl)guanine] and NHS-Peg4-Azide reagents from -20°C and equilibrating to room temperature, 100 μL of 100 mM NHS-Peg4-Azide was mixed with 150 μL of 100 mM BG-NH2[O6-(4-aminomethyl-benzyl)guanine], and 250 μL of DMSO containing 300 mM triethylamine was added to a final volume of 500 μL. The mixture was incubated overnight at 30°C to obtain a final concentration of 20 mM BG-Azide. The resulting BG-Azide product was then reacted with DBCO-modified DNA in an aqueous solution at a molar ratio of 20:1 at 37°C for 1 h. Excess BG-Azide complex was washed away using an ultrafiltration tube with a molecular cutoff of 3 kDa. The ultrafiltration-purified DNA-BG-Azide and the expression-purified SNAP fusion protein ABD were mixed at a molar ratio of 10:1 and incubated at room temperature for 2 h. The mixture was purified three times using an ultrafiltration tube with a molecular cutoff of 10 kDa, washing away excess DNA to obtain the ABD-DNA protein-nucleic acid conjugate. The ultrafiltration tube was rinsed three times with ddH2O before use.

[0089] 5. i-motif sequence optimization

[0090] The folding ratios of different i-motifs were evaluated by reacting the synthesized three chains (I, C, and A) under different pH conditions. The folding efficiency of the i-motif switches was assessed by recording changes in fluorescence signal intensity. All reactions were carried out in a solution containing 5 mM Mg. 2+ The reaction was performed in 1×PBS. C-FAM, A-BHQ1, and I were mixed in a 1:4:2 molar ratio and incubated at 37°C for 2 h at pH 7.28 to 5.51. The pH responses of the i-motif core sequences I1-C5(T4C5)3, I2-C5(T3C5)3, I3-C5(T2C5)3, I4-C4(T3C4)3, and I5-C4(T3C3)3 were compared. Fluorescence signal intensity was recorded during this process. The folding efficiency of the i-motif was determined using the following formula:

[0091]

[0092] Where F is the fluorescence intensity of the unquenched fluorescent chain (C), and F0 and F t These represent the fluorescence intensities of the C / A / I structures at the reaction initiation and endpoint, 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 deep red membrane dye labeling process 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 flux signal of different antibody-origami conjugates on T cell activation was detected using the Fluo-4AM calcium flux 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 2×10⁶ T cells was added to the PBS buffer. 5Suspension CFSE-stained T cells were added to 96-well plates and co-incubated with SpTCE, then cultured in a 37°C cell culture incubator. After 48 h of culture, the cells were centrifuged at 250 g for 10 min to collect them. The cells were then stained with live / dead cells in flow cytometry buffer and analyzed by flow cytometry using a Beckman flow cytometer. Cell proliferation was analyzed by CFSE fluorescence signal.

[0116] 11. CD69 Expression Analysis

[0117] The antibody-origami conjugate's T-cell activation capacity was further evaluated by analyzing CD69 expression. T cells were negatively screened from mouse spleens using the StemCell EasySep T-cell Negative Sorting Kit, following the manufacturer's instructions. 2 × 10⁶ cells were extracted per well. 5 Cells were incubated with free antibody or SpTCE in 1640 medium supplemented with 10% FBS for 6 h. After washing with PBS, the cells were resuspended in 200 μL of FITC-labeled CD69 flow cytometry antibody buffer (phosphate buffer + 1% FBS) and incubated on ice for 30 min. The cells were then centrifuged at 250 g for 10 min, washed with PBS, resuspended in 200 μL of FACS buffer, and analyzed using flow cytometry.

[0118] 12. Cytokine detection

[0119] The secretion of activated cytokines by T cells was measured using a commercially available ELISA kit. The specific steps are as follows:

[0120] (1) Sample preparation: Take 5×10 5 5 × 10 T cells and 5 × 10 4 One MC38 cell was co-cultured for 24 h in 250 μL of 10% serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in 90% RPMI-1640 medium. The supernatant was collected by centrifugation, with 100 μL used for IFN-γ quantification and the other 100 μL used for IL-2 quantification.

[0121] (2) Plate preparation: First, dilute the capture antibody to 2 μg / mL with phosphate buffer. Add 100 μL of the diluted capture antibody to each well of a 96-well plate and incubate overnight at 4°C. Then, pour out the incubation solution and add 300 μL of wash buffer (phosphate buffer + 2 mM EDTA + 0.5% BSA) to wash the plate 2-3 times, removing as much residual liquid as possible from the plate during the last wash. Next, add 300 μL of blocking buffer (wash buffer + 2% BSA) and incubate at room temperature for 1-2 hours. Repeat the washing process 2-3 times, removing as much residual liquid as possible during the last wash, and prepare for the next test.

[0122] (3) Test Procedure: Add 100 μL of diluted sample and standard to each well and incubate at room temperature for 2-3 hours. Then pour out the sample and wash the plate 4-5 times with washing buffer, removing as much residual liquid as possible in the last wash. Next, add 100 μL of diluted detection antibody (0.3 μg / mL) to each well and incubate at room temperature for 1 hour. Continue washing the plate 3-4 times, removing as much residual liquid as possible in the last wash. Add 200 μL of substrate working solution and incubate for 30-60 minutes. Finally, add 50 μL of stop solution and measure the absorbance at 450 nm using a microplate reader.

[0123] Serum cytokines IFN-γ, IL-2, TNF-α, and IL-6 were also detected following the steps described above.

[0124] 13. Mouse in vivo fluorescence imaging

[0125] (1) Expand and culture MC38 tumor cells, collect MC38 cells in PBS buffer and count them;

[0126] (2) Nude mice were randomly divided into 5 groups and injected subcutaneously with 1×10 6 One MC38 cell suspension was subcutaneously injected into the hind leg of a nude mouse;

[0127] (3) Record the size of the mouse tumor. The volume calculation formula is: V(mm) 3 )=0.5×L×W 2 (W represents the short diameter of the tumor, and L represents the long diameter);

[0128] (4) When the tumor volume reaches 200-300 mm 3 On the left and right sides, different groups of drugs, namely PBS, 6HB, Ab@6HB, unSpTCE and SpTCE (injection volume 100μL, concentration 10nM), were injected via the tail vein.

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

[0130] (6) After completing the in vivo fluorescence imaging at 48h, the mice were euthanized by cervical dislocation, and the tumor, heart, liver, spleen, lung, kidney and major digestive organs were removed. These isolated organs were arranged and fluorescence imaging was performed. Subsequently, the fluorescence intensity was semi-quantitatively analyzed.

[0131] 14. Anti-tumor treatment

[0132] (1) 1×10 6MC38 cell suspension was subcutaneously injected into the hind leg of mice to obtain C57B / L6 mice bearing tumors, and the size of the mouse tumors was recorded.

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

[0134] (3) Inject once every two days for a total of two weeks, for a total of 8 injections. Observe and record the mouse's weight and tumor size one day apart;

[0135] (4) On day 16, blood was collected from the eyeballs of mice, the mice were euthanized by cervical dislocation, and the tumors were collected.

[0136] 15. Results and Analysis

[0137] 15.1 Assembly and Functional Verification of SpTCE's Internal Functional Layer and External Shielding Layer

[0138] 15.1.16HB Origami Synthesis and Characterization

[0139] This invention utilizes Cadnano software and the classic Rothemund method to construct 6HB. Sixty-three designed staple chains and an M13 (2695nt) template were co-assembled under gradient annealing conditions. The resulting 6HB was purified by ultrafiltration. The structures of the annealed and purified products were verified by agarose gel electrophoresis. Electrophoresis results showed that under gradient annealing conditions, the mixed-chain system could form an assembled structure with a larger molecular weight, 6HB. The ultrafiltration purified product exhibited a clear single band, yielding high-purity 6HB. Figure 3 ).

[0140] The structure of the synthesized and purified 6HB was further characterized using more intuitive atomic force and transmission electron microscopy imaging methods. TEM and AFM confirmed that 6HB exhibits a uniform and dispersed rod-like structure with a length of approximately 150 nm. Figure 4 ( ), consistent with the theoretical design dimensions.

[0141] To facilitate differentiation of the various helical bundles, the six helical bundles of 6HB are numbered 0, 1, 2, 3, 4, 5, respectively. Figure 5Each helical bundle has ten specific oligonucleotide extension sequences used to link nucleic acid-coupled antibodies and designed functional nucleic acids. aCD3 and aCD28 antibodies are assembled onto helical bundles 1 and 2 as TCR cluster inducers, aPD-L1 antibodies are assembled onto helices 4 and 5 to target tumors, and hand-in-hand palindromic sequences are assembled onto helices 0 and 3. These elements together constitute the inner functional layer. Figure 5 ).

[0142] 15.1.26 HB and antibody assembly

[0143] For antibody assembly and 6HB assembly, the functional antibody and DNA are first conjugated. The biotinylated antibody binds to the biotinylated DNA strand (A) via streptavidin. Strains A1, A2, and A3 are designed to bind aCD3, aCD28, and aPD-L1 antibodies, respectively, and are anchored to specific sites on 6HB. Figure 5 First, a 10-fold excess of streptavidin was combined with a biotinylated antibody, and the antibody-streptavidin conjugate was obtained by ultrafiltration at 100 kDa. Non-denaturing protein gel electrophoresis showed that the antibody molecules bound to streptavidin in the experimental group produced a lag band, indicating that the molecular weight of the streptavidin antibody-conjugate was significantly increased. Figure 6 (A). Then, the binding ratio of strand A and the antibody-streptavidin conjugate was further optimized. Strain A and the antibody-streptavidin conjugate were mixed at molar ratios of 3:1, 2:1, and 1.5:1, respectively. Agarose gel electrophoresis results showed that a 1.5:1 mixture of strand A and the antibody-streptavidin conjugate yielded a relatively ideal DNA-antibody conjugate. Figure 6 (B)

[0144] Next, the antibody coupled with nucleic acid is assembled onto the 6HB structure. By truncating the oligonucleotide sequence corresponding to the extension site of 6HB, the Ab-DNA is assembled onto the 6HB structure at different spacings and densities at the nanoscale.

[0145] 15.1.36 HB Inner Layer Antibody Distribution on T Cell Activation

[0146] The TCR signal transduction dynamic schooling model indicates that ligand affinity and spatial arrangement may have a decisive influence on downstream T cell signal transduction and activation. To overcome the dynamic schooling model and enhance T cell activation, this invention investigated the effects of antibody assembly and arrangement on T cell activation from three aspects: the spacing and density of aCD3 antibodies, and the addition of aCD28 antibodies. First, two pairs of aCD3 antibodies were assembled in helical bundles 1 and 2 of a 6HB gel, with spacings set to 70 nm, 56 nm, 42 nm, 28 nm, and 14 nm, respectively, and characterized by gel electrophoresis. Figure 7The experimental results showed that there was no significant difference in molecular weight between origami structures with different spacing but the same number of antibodies.

[0147] T cells isolated from mouse spleens were incubated with aCD3-6HB conjugates with different spacing for varying durations to test corresponding indicators of T cell activation. 2+ As a second messenger in cell signal transduction, intracellular calcium plays an important role in cellular physiological activities. 2+ Changes in Ca2+ concentration are closely related to cell signaling. Monitoring intracellular Ca2+ in immune cells... 2+ Dynamic changes can be used to determine the activation status of multivalent antibody-mediated T cells. This invention uses Ca... 2+ The dynamic calcium flux signal in T cells treated with aCD3-6HB conjugate was monitored using a sensitive fluorescent dye reagent (Fluo-4AM), and a significant increase in calcium flux fluorescence signal was observed within 1 minute. Figure 8 (A). Compared with the same concentration of free aCD3 antibody, 6HB pair assembly triggered enhanced calcium flux signal, and the calcium flux signal gradually increased with decreasing spacing. The calcium flux signal of T cells with aCD3 antibody spacing of 28 nm and 14 nm tended to be relatively stable. Figure 8 (B)

[0148] Further measurements were taken of other indicators of T cell activation, including CD69 expression levels at 6 h, cytokine secretion (IFN-γ) at 24 h, and T cell proliferation at 48 h. Upregulation of CD69 confirmed that the shortened spacing of aCD3 antibodies in a 6HB arrangement triggered stronger T cell activation. Figure 9 (A). Notably, compared to the control group, the same concentration of free aCD3 antibody did not significantly upregulate CD69. Furthermore, no significant IFN-γ secretion or T cell proliferation was observed in any of the groups. Figure 9 These experimental results (including those from the BC) suggest that the lack of co-stimulatory signals may affect further activation of T cells.

[0149] To further evaluate the effect of antibody alignment density on T cell activation, different numbers of aCD3 antibodies were assembled onto the first and second helical bundles of a 6HB helix. The number of aCD3@6HB assembled pairs increased from 2 pairs (4 cells) to 6 pairs (12 cells), and aCD28 co-stimulatory signals were introduced. Agarose gel electrophoresis results showed that the molecular weight increased with the increase of the number of assembled antibodies. Figure 10 ).

[0150] While maintaining the same total antibody concentration in both the experimental and control groups, it was found that assembling more aCD3 antibody on 6HB cells induced a stronger calcium flux signal within T cells. Figure 11(A). This invention further assembles four pairs of aCD28 antibodies (20Ab@6HB) based on the 6HB structure containing six pairs of aCD3 antibodies. The 20Ab@6HB conjugate induced the highest calcium flux signal in T cells. Figure 11 (B). The above results indicate that immobilizing high-density aCD3 antibody with 6HB and simultaneously immobilizing aCD28 antibody can significantly improve the activation efficiency of T cells.

[0151] Building upon this, the present invention further investigated other indicators of T cell activation. CD69 expression gradually increased with the number of aCD3 antibodies on a single 6HB, reaching its highest level in the case of co-assembly of aCD28 antibody and aCD3. Figure 12 (A). IFN-γ secretion and T cell proliferation levels gradually increased with increasing aCD3 antibody density, and increased sharply with the introduction of aCD28 antibody. Figure 12 (Central BC). The level of IFN-γ secreted by T cells induced by 20Ab@6HB was higher than that induced by exposure to the same concentration of free aCD3 and aCD28 antibodies, indicating that the TCR cluster induced by Ab@6HB can enhance T cell activation.

[0152] 15.1.46HB Outer Shielding Layer Function Verification

[0153] The acidic pH surrounding tumor tissue is a key characteristic of the tumor microenvironment and a crucial entry point for targeted therapy with smart nanomedicines. This invention designs a spatiotemporally controllable outer shielding layer by assembling a pH-responsive ABD (I / C-ABD). Chain I contains an i-motif sequence at its 3' end and a sequence complementary to that of chain A, thereby fixing it onto an origami structure. Figure 13 (A). Notably, when pH > response threshold, chain I binds to chain A and is fixed to the origami extension sequence H via chain A, forming an outer shielding layer with C-ABD on 6HB. When pH < response threshold, I forms an i-motif quadruplex structure and separates from 6HB. Figure 13 (A). Typically, cancer cell metabolism and other factors create an acidic pH environment (5.5-7.0) in solid tumor tissue. This invention uses FAM-labeled chain C and BHQ1-labeled chain A as indicators to screen for the most suitable pH response switch from five i-motif sequences (I1, I2, I3, I4, and I5) to optimize the pH response threshold. Figure 13 (B). The I2 and C5(T3C5)3 sequence showed 100% response efficiency at pH 6.5 and was selected as the most suitable i-motif switch. Figure 13 (B)

[0154] To investigate the effect of ABD assembly density on the stability of 6HB structures under physiological conditions, ABD was assembled onto different helices of 6HB to construct No.0 / 3ABD@6HB, No.0 / 1 / 2ABD@6HB, No.0 / 2 / 4ABD@6HB, and No.0 / 1 / 2 / 3 / 4 / 5ABD@6HB (complete coverage). These ABD-assembled 6HB structures were incubated for 12 h with 100 nM serum albumin (HSA) and different concentrations of DNase I. Agarose gel electrophoresis analysis showed that, compared to the simple 6HB structure, 6HB assemblies with three or more helices of ABD maintained sufficient stability in high concentrations of nuclease (10 U / mL). Figure 14 (A). Furthermore, in standard cell culture medium (DMEM containing 10% FBS), 6HB cells fully covered with ABD exhibited greater stability within 36 hours than 6HB cells assembled with partial ABD. Figure 14 (B) This result confirms that the structural assembly of ABD enhances the nuclease tolerance and physiological stability of the DNA origami chassis. The assembly of ABD on all six helices was selected as the optimal outer layer configuration.

[0155] Further investigation was conducted on the pH-mediated shedding of the outer masking layer mediated by the i-motif switch by incubating ABD@6HBs under different pH conditions. Using the pH-insensitive chain I* as a control, agarose gel electrophoresis results showed that at pH 7.4, the migration rates of I / C-ABD@6HBs and I* / C-ABD@6HBs were not significantly different under normal conditions. However, at pH 6.5 and 5.5, I / C-ABD@6HBs exhibited higher migration rates than I* / C-ABD@6HBs, indicating the responsiveness of the outer masking layer to acidic pH. Figure 15 Furthermore, the outer layer of I / C-ABD@6HBs detached under pH 6.5 and 5.5 conditions with a molecular weight similar to that of pure 6HB, indicating complete separation of the outer shielding layer.

[0156] 15.2 In vitro performance characteristics of SpTCE

[0157] 15.2.1 In vitro pH response 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 a 6HB structure to construct SpTCE. Gel electrophoresis results showed that chain A-mediated aCD3, aCD28, and aPD-L1 antibodies were assembled at the 6HB structural level. Figure 16 (A). The hierarchical assembly of the inner and outer layers of the 6HB structure and the pH-responsive disassembly of the outer layer were analyzed by gel electrophoresis. Lanes 1 and 2 were respectively filled with the 6HB assembly internal antibody and the internal antibody-external ABD co-assembly product SpTCE. Figure 16 (B) After incubation of SpTCE with serum albumin, it was found that at pH 7.4, the migration rate of the albumin-coated SpTCE (HSA / SpTCE) product band was slower. Figure 16 Lane B, lane 3). However, at pH 6.5, the outer layer detached, and it was found that the large structure hand-Ab@6HB had a similar migration rate compared to only assembling the inner functional layer. Figure 16 Lanes B (4 and 5) reflect the changes in the acidic pH response of SpTCE. The pH response process involves two steps: the outer layer detaches in response to acidic pH and the self-assembly of large structures through hand-to-hand palindromic sequences.

[0159] The in vitro stability of SpTCE was further evaluated, and its stability in serum was verified. The low ionic strength and high nuclease activity of serum significantly affect the stability of DNA structures. To assess the effect of the shielding layer on serum stability, SpTCE was incubated in mouse serum for different times, and the residual structure was measured over time using ImageJ. After 36 hours of incubation in mouse serum, SpTCE with ABD retained 41% of its intact structure, exceeding the retention of Ab@6HB (13%) and 6HB (20%), indicating that serum albumin coating can significantly enhance the stability of SpTCE. Figure 17 ).

[0160] 15.2.2 SpTCE Targeting Capability

[0161] This invention further validates the ability of Ab@6HB to target T cells and MC38 tumor cells (derived from C57BL / 6 mouse colon adenocarcinoma, a PD-L1 highly expressing cell line) using flow cytometry experiments. Using doxorubicin (Dox)-labeled origami, Ab@6HB was co-incubated with T cells and MC38 cells, respectively. Flow cytometry results showed that Ab@6HB could target both T cells and MC38 cells. Figure 18 (Light blue peaks).

[0162] To further verify the antibody's transition from shielded to exposed state after responding to pH, this invention used Dox-labeled 6HB to track the origami substrate and FAM-labeled chain I to track the outer shielding layer. Flow cytometry experiments were used to verify that SpTCE specifically recognizes T cells (…). Figure 19 (Middle A, light blue peak). Furthermore, T cells incubated with SpTCE without HSA showed fluorescence shifts in both the FAM / FITC and Dox / PE channels. Figure 19 (Middle A, blue peak). In contrast, HSA / SpTCE did not show a significant shift in either the FAM / FITC or 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 21Two hours later, all mice treated with Cy7-labeled 6HB showed systemic fluorescence signal distribution, while the Ab@6HB and SpTCE groups showed significant accumulation at the tumor site. However, unSpTCE-treated mice did not show tumor accumulation, confirming that the shielding effect of the outer albumin layer prevented the structure from targeting the tumor site. At 48 and 72 hours, SpTCE-treated mice maintained stronger tumor fluorescence accumulation signals than Ab@6HB control mice, possibly due to the intact shielding layer enhancing its half-life in vivo. At 96 hours, the fluorescence signal in all treated mice was significantly reduced. Figure 22 ).

[0167] Based on this, the present invention analyzed the fluorescence distribution in major tissues and organs of different experimental groups and control groups after 48 hours. Figure 23 The SpTCE group showed significantly higher fluorescence signals at the tumor site compared to other treatment groups, and also much higher levels in other tissues and organs. Figure 23 This indicates that SpTCE has excellent tumor-specific targeting ability and high stability in vivo.

[0168] 15.4 In vivo efficacy study

[0169] 15.4.1 In vivo antitumor effects of SpTCE

[0170] To evaluate the therapeutic potential of SpTCE in vivo, this invention investigated the antitumor efficacy of SpTCE in vivo with spatiotemporal control. A tumor model was established in C57B / L6 mice by subcutaneous inoculation with MC38. Six days after tumor growth, the mice were randomly divided into six groups and injected via tail vein with PBS, 6HB, free antibody, unSpTCE, Ab@6HB, and SpTCE, respectively, once every two days for a total of eight injections. Figure 24 (A). Meanwhile, the tumor size and body weight of these experimental group mice were continuously monitored. Figure 24 (C). The tumor size in the 6HB-treated mice was very similar to that in the PBS-treated control group. Figure 24 (B) indicates that the empty vector had no significant effect on tumor growth. Mice treated with the free antibody group may have a certain inhibitory effect on tumor growth through PD-L1 antibody blockade of immune checkpoints. Figure 24 Mice treated with unSpTCE showed similar tumor growth rates to those treated with free antibodies (B). Figure 24 (B). In mice treated with Ab@6HB, tumor growth was slowed, and mice treated with SpTCE showed the lowest tumor growth rate (B). Figure 24 (B) and the longest survival rate ( Figure 24 (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 25 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 26 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 27 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. Figure 28 This indicates that these multivalent antibody-drug conjugates have good biosafety in important tissues.

[0175] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A spatiotemporally controllable T-cell conjugate based on DNA nanostructures, characterized in that, It includes 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; The six-helix bundle origami structure is assembled using 63 staple chains and single-stranded circular DNA M13. The internal functional layer includes nucleic acid-antibody conjugates on four helical extension sequences at the top and bottom of the six-helix bundle origami structure, and hand-in-hand palindromic sequences on two helical extension sequences on the left and right sides. The outer shielding layer comprises a C-chain modified serum albumin-binding peptide and chain I, wherein chain I contains a pH-responsive i-motif switch, and the outer shielding layer is bound to the inner functional layer; the nucleotide sequence of the C-chain is as shown in SEQ ID NO. 76, and the nucleotide sequence of chain I is any one of those shown in SEQ ID NO. 71-75.

2. The spatiotemporally controllable T-cell conjugate as described in claim 1, characterized in that, The nucleotide sequences of the 63 staple chains are shown in SEQ ID NO.1-63.

3. The spatiotemporally controllable T-cell conjugate as described in claim 1, characterized in that, The nucleic acid antibody conjugate is formed by conjugating chain A with an antibody modified with streptavidin, wherein the nucleotide sequence of chain A is any one of those shown in SEQ ID NO. 65, SEQ ID NO. 66 or SEQ ID NO. 68, and the antibody includes aCD3, aCD28 and / or aPD-L1 antibodies.

4. The spatiotemporally controllable T-cell conjugate as described in claim 3, characterized in that, The molar ratio of the chain A and streptavidin-modified antibody is 1.5-3:

1.

5. The spatiotemporally controllable T-cell conjugate as described in claim 3, characterized in that, The nucleic acid-antibody conjugates attached to the top two helical extension sequences of the six-helix bundle origami structure are formed by conjugating chain A with aCD3 and aCD28 antibodies respectively; and / or the ratio of the number of aCD3 and aCD28 antibodies is 3:

2.

6. The spatiotemporally controllable T-cell conjugate as described in claim 1, characterized in that, The 3' end of chain I includes a pH-responsive i-motif switch and a sequence complementary to chain A, so that the outer shielding layer is bonded to the inner functional layer and thus fixed to the six-helix bundle origami structure.

7. The spatiotemporally controllable T-cell conjugate as described in claim 6, characterized in that, When pH > response threshold, chain I binds to chain A and is fixed to the extended sequence of the six-helix bundle origami structure via chain A. It forms an outer shielding layer with the serum albumin-binding peptide modified by chain C on the six-helix bundle origami structure to shield the inner functional layer, preventing the activation of T cells in healthy tissues and improving the stability of blood circulation in vivo. When pH < response threshold, chain I forms an i-motif quadruplex structure and separates from the six-helix bundle origami structure, allowing the exposed antibody and hand-in-hand palindromic sequence to induce TCR clustering, activate T cells, and achieve the killing of tumor cells.

8. A method for constructing a spatiotemporally controllable T-cell conjugate as described in any one of claims 1-7, characterized in that, Includes the following steps: Nucleic acid-antibody conjugate, C-chain modified serum albumin-binding peptide and chain I are mixed in equal molar ratio to form a three-chain structure. Then, the three-chain structure is mixed with a six-helix bundle origami structure to obtain a spatiotemporally controllable T-cell conjugate.

9. The construction method as described in claim 8, characterized in that, The molar ratio of the three-chain structure to the six-helix bundle origami structure is 1.2n:1, where n represents the number of binding sites for the extended sequence of the six-helix bundle origami structure.

10. The use of the spatiotemporally controllable T-cell binder as described in any one of claims 1-7 in the preparation of drugs or drug carriers for enhancing immunotherapy for tumor treatment.

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