A dna tetrahedron-based multivalent lysosome-targeting antibody platform

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a kind of multivalent lysosome targeting antibody platform based on DNA tetrahedron.The application constructs a kind of lysosome targeting chimera based on DNA tetrahedron, uses general-purpose secondary antibody as general-purpose binding site, is connected with commercial primary antibody, solves two major core defects of lack of aptamer resources in the prior art and the need to redesign DNA assembly sequence for new target, realizes the efficient degradation of different target proteins "plug and play";The chimera can still maintain structural integrity in complex physiological environment, and after connecting the commercial primary antibody, the target protein can be mediated by the lysosome pathway to realize non-consumption and continuous degradation, which greatly reduces the research and development cost and cycle of targeted protein degradation drugs, significantly improves the application flexibility and degradation efficiency, and is especially suitable for the preparation of antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. More specifically, it relates to a multivalent lysosomal targeting antibody platform based on DNA tetrahedrons. Background Technology

[0002] Abnormal expression of transmembrane and extracellular proteins is closely related to the occurrence and development of various major human diseases, including tumors and autoimmune diseases. In recent years, lysosome-targeting chimeras (LYTACs), through their endogenous lysosomal degradation pathway, have become a research hotspot in the biomedical field, efficiently clearing pathogenic transmembrane proteins on the cell membrane. Traditional LYTACs employ a linear coupling strategy: directly linking the target protein binding domain (such as antibodies or peptides) to the lysosomal receptor ligand (such as DNA aptamers). This linear structure easily induces severe steric hindrance, leading to ligand masking and significantly reducing targeting binding efficiency.

[0003] To address the steric hindrance effect of traditional LYTAC, existing research has introduced DNA tetrahedrons (TDNs) as rigid frameworks, modifying lysosomal receptor ligands and target protein nucleic acid aptamers at their vertices to achieve membrane protein degradation. Although the introduction of TDNs alleviates steric hindrance to some extent, this technology still has significant drawbacks in clinical translation: First, the scarcity of aptamer resources leads to poor universality. This technology relies entirely on nucleic acid aptamers screened by exponential enrichment screening (SELEX) to recognize target proteins; however, currently, there are no available aptamers for most novel transmembrane proteins. Second, the lack of modular universal interfaces results in high R&D costs. For each new target, aptamers need to be screened from scratch and the DNA assembly sequence redesigned. The customized "one target, one aptamer" development model has a long development cycle and high costs, making it difficult to meet the needs of multi-target combination therapy.

[0004] Therefore, the development of novel targeted degradation platforms that combine modularity and versatility with high efficiency in target protein degradation has become an urgent need in the field. Summary of the Invention

[0005] This invention addresses the shortcomings of existing lysosome-targeting chimeras, such as the lack of modular design and the difficulty in development, by providing a lysosome-targeting chimera based on DNA tetrahedrons.

[0006] A second objective of this invention is to provide a complex based on a DNA tetrahedron-targeted lysosomal chimera.

[0007] A third object of the present invention is to provide a pharmaceutical composition.

[0008] A fourth objective of this invention is to provide a method for preparing the DNA tetrahedron-based lysosomal targeted chimera.

[0009] A fifth objective of this invention is to provide the use of the DNA tetrahedral-based lysosomal targeting chimera or the pharmaceutical composition in the preparation of antitumor drugs.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a DNA tetrahedron-based lysosomal targeting chimera, comprising a DNA tetrahedron and a universal secondary antibody; The DNA tetrahedron is loaded with single-stranded DNA with azide-modified ends, and the single-stranded DNA with azide-modified ends is linked to the DNA tetrahedron through complementary base pairing. The DNA tetrahedron is loaded with 1 to 3 lysosomal receptor nucleic acid aptamer sequences, which are linked to the DNA tetrahedron through complementary base pairing. The universal second antibody promotes the linkage of the single-stranded DNA with the terminal modified azide group to the azide-alkyne cycloaddition reaction by strain.

[0011] Furthermore, the universal secondary antibody refers to a secondary antibody capable of specifically binding to crystallizable fragments (Fc segments) of all types of primary antibodies from the same species.

[0012] Preferably, the DNA tetrahedron comprises four nucleotide single strands: nucleotide A strand, nucleotide B strand, nucleotide C strand, and nucleotide D strand; The nucleotide A chain, nucleotide B chain, nucleotide C chain, and nucleotide D chain self-assemble to form the DNA tetrahedron through complementary base pairing.

[0013] More preferably, the sequence of the nucleotide A chain is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0014] More preferably, the sequence of the nucleotide B chain is shown in SEQ ID NO.3.

[0015] More preferably, the sequence of the C-chain of the nucleotide is shown in SEQ ID NO.4.

[0016] More preferably, the sequence of the nucleotide D chain is as shown in SEQ ID NO.5 or SEQ ID NO.6.

[0017] Preferably, the sequence of the single-stranded DNA is shown in SEQ ID NO.7.

[0018] Preferably, the lysosomal receptor nucleic acid aptamer sequence is shown in SEQ ID NO.8.

[0019] The present invention also provides a complex of a DNA tetrahedral-based lysosomal targeting chimera, comprising the DNA tetrahedral-based lysosomal targeting chimera and a first antibody; The first antibody is linked to the antigen-binding fragment of the universal second antibody, which is a DNA tetrahedral-based lysosomal targeting chimera, via a non-covalent affinity interaction between its crystallizable fragment and the antigen-binding fragment.

[0020] Optionally, the first antibody is selected from the epidermal growth factor receptor first antibody and / or the mesenchymal-epithelial transformation factor first antibody.

[0021] Preferably, the complex based on the DNA tetrahedron lysosome-targeting chimera is prepared by the following steps: The DNA tetrahedron-based lysosome-targeting chimera and the first antibody were incubated to obtain the complex of the DNA tetrahedron-based lysosome-targeting chimera.

[0022] More preferably, the molar ratio of the DNA tetrahedral-based lysosomal targeting chimera to the first antibody is (0.8-1.2):(0.8-1.2), specifically 1:1.

[0023] More preferably, the incubation temperature is 20–30°C, specifically 25°C.

[0024] More preferably, the incubation time is 20 to 40 minutes, specifically 30 minutes.

[0025] The present invention also provides a pharmaceutical composition comprising the aforementioned DNA tetrahedral-based lysosomal targeting chimera complex.

[0026] This invention also provides a method for preparing the DNA tetrahedron-based lysosomal targeting chimera, comprising the following steps: S1: Mix single-stranded DNA with azide-modified ends, nucleotide A chain, nucleotide B chain, nucleotide C chain, nucleotide D chain that make up the DNA tetrahedron, and lysosomal receptor nucleic acid aptamer, and form the DNA tetrahedron by self-assembly through complementary base pairing; S2: Mix and incubate the universal second antibody with the DNA tetrahedron obtained in step S1 to obtain the DNA tetrahedron-based lysosomal targeting chimera.

[0027] Preferably, in step S1, the molar ratio of the single-stranded DNA with azide-modified ends, the nucleotide A chain, nucleotide B chain, nucleotide C chain, nucleotide D chain that make up the DNA tetrahedron, and the lysosomal receptor nucleic acid aptamer is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2), specifically 1:1:1:1:1.

[0028] Preferably, in step S1, the self-assembly conditions are to maintain at 90-98°C for 3-10 min, followed by maintaining at 2-8°C for 5-15 min, specifically, to maintain at 95°C for 10 min, followed by maintaining at 4°C for 10 min.

[0029] Preferably, in step S2, the molar ratio of the universal second antibody to the DNA tetrahedron is 1:(1-10), more preferably 1:(8-10).

[0030] Preferably, in step S2, the incubation temperature is 20–40°C, more preferably 25°C.

[0031] Preferably, in step S2, the incubation time is 18 to 30 hours, more preferably 24 hours.

[0032] Optionally, the lysosomal receptor nucleic acid aptamer is a mannose-6-phosphate receptor (M6PR) nucleic acid aptamer.

[0033] Optionally, the universal second antibody and the first antibody are derived from at least one of rabbit, sheep, mouse, and human sources.

[0034] The present invention also provides the use of the DNA tetrahedral-based lysosomal targeting chimera complex or the pharmaceutical composition in the preparation of antitumor drugs.

[0035] The present invention has the following beneficial effects: This invention provides a lysosomal targeting chimera based on DNA tetrahedrons. This chimera uses a DNA tetrahedron as a rigid framework and incorporates a universal second antibody as a modular interface. Through the non-covalent affinity interaction between the second and first antibodies, rapid "plug-and-play" assembly of different target protein binding domains can be achieved, fundamentally solving two core shortcomings of existing lysosomal targeting chimera technologies: first, the lack of universality due to the scarcity of aptamer resources—it eliminates the need for nucleic acid aptamers screened by SELEX, directly utilizing mature commercial antibody libraries to cover the vast majority of known targets; second, the shortcomings of the "one target, one build" customized development model—for new targets, there is no need to redesign the DNA assembly sequence; only the corresponding first antibody needs to be replaced to complete functional reconstruction. This chimera maintains stable structural integrity in complex physiological environments and, after assembly with a commercial first antibody, can mediate non-consumable continuous degradation of target proteins through the endogenous lysosomal pathway. The chimera provided by this invention significantly reduces the development cost and cycle of targeted protein degradation drugs, significantly improves application flexibility and degradation efficiency, and is suitable for the preparation of anti-tumor drugs. Attached Figure Description

[0036] Figure 1 This is a non-denaturing polyacrylamide gel electrophoresis image of the conjugation product (Ab-ssDNA) obtained by conjugating a universal secondary antibody modified with dibenzocyclooctylene (DBCO-Ab) with azide-modified single-stranded DNA (Azide-ssDNA).

[0037] Figure 2 The graph shows the effect of coupling process parameters on the coupling efficiency of dibenzocyclooctylene-modified universal secondary antibody (DBCO-Ab) and azide-modified single-stranded DNA (Azide-ssDNA). Figure 2 In the figure, A represents the effect of different feed ratios on coupling efficiency. Figure 2 B in the figure represents the effect of different reaction times on coupling efficiency.

[0038] Figure 3 The image shows the agarose gel electrophoresis analysis of the stepwise assembly of the trivalent DNA tetrahedral (TDN-3) scaffold. In the image, "+" indicates that the corresponding lane contains the sequence, and "-" indicates that the corresponding lane does not contain the sequence. The number of "+" or "-" indicates the molar number of the sequence added.

[0039] Figure 4 This is a non-denaturing polyacrylamide gel image of DNA tetrahedral multivalent lysosome-targeting chimera (TDN-MLYTAB).

[0040] Figure 5 Here are atomic force microscopy characterization images of TDN-MLYTAB, in which Figure 5 In the figure, A represents the atomic force microscopy height diagram of Ab. Figure 5 B in the image is a high-resolution atomic force microscope image of Ab. Figure 5 In the image, C represents the atomic force microscope height diagram of TDN-3. Figure 5 D in the image is a high-resolution atomic force microscope image of TDN-3. Figure 5 In the figure, E represents the atomic force microscopy height diagram of Ab-TDN-3. Figure 5 F in the figure is a high-resolution atomic force microscopy image of Ab-TDN-3.

[0041] Figure 6 This is a statistical diagram of the size distribution of TDN-MLYTAB using atomic force microscopy.

[0042] Figure 7 The diagram shows the verification of the physicochemical properties of TDN-MLYTAB. Figure 7 In the figure, A represents the zeta potential analysis results of TDN-MLYTAB. Figure 7 B in the image represents the UV-Vis absorption spectrum of TDN-MLYTAB.

[0043] Figure 8 This is a stability evaluation diagram of TDN-MLYTAB in a weakly acidic endosome environment (pH 6.5) simulating early post-endocytosis. Figure 8 In the image, A represents a non-denaturing polyacrylamide gel image of TDN-MLYTAB incubated in pH 6.5 buffer for 0–24 h. Figure 8 B in the graph represents the change in the structural integrity ratio of TDN-MLYTAB over time.

[0044] Figure 9 This is a stability evaluation diagram of TDN-MLYTAB in a simulated in vitro blood circulation environment (10 vol% fetal bovine serum), where... Figure 9 In the image, A represents a non-denaturing polyacrylamide gel image of TDN-MLYTAB incubated in 10 vol% fetal bovine serum for 0–24 h. Figure 9 B in the graph represents the change in the structural integrity ratio of TDN-MLYTAB over time.

[0045] Figure 10 This is a characterization diagram of the binding ability of TDN-MLYTAB loaded with anti-epidermal growth factor receptor (EGFR) primary antibody to the surface of MHCC97H cells, in which... Figure 10 In the figure, A represents the flow cytometry results of MHCC97H cells after incubation with TDN-MLYTAB loaded with anti-EGFR primary antibody for 1 h. Figure 10 B in the figure represents the flow cytometry results of MHCC97H cells after incubation with TDN-MLYTAB loaded with anti-EGFR primary antibody for 1 h.

[0046] Figure 11 To evaluate the degradation effect of TDN-MLYTAB loaded with anti-EGFR primary antibody on EGFR in MHCC97H cells using Western blotting (Wb).

[0047] Figure 12 Confocal imaging characterization of EGFR degradation on the surface of MHCC97H cells induced by TDN-MLYTAB loaded with anti-EGFR primary antibody. Figure 12 In the image, A represents a confocal image of EGFR distribution on the surface of MHCC97H cells after treatment with TDN-MLYTAB loaded with anti-EGFR primary antibody at 0 h, 6 h, and 24 h. Figure 12 B in the figure is a quantitative statistical graph of the average fluorescence intensity on the surface of MHCC97H cells based on confocal imaging.

[0048] Figure 13 The graph shows the effect of TDN-MLYTAB loaded with anti-EGFR primary antibody on EGFR degradation in MCF-7 cells using Western blot analysis.

[0049] Figure 14 The image shows the residual MET on the cell surface of MHCC97H cells after treatment with TDN-MLYTAB loaded with anti-mesenchymal-epithelial transforming factor (MET) primary antibody. Figure 14 In the figure, A represents the flow cytometry result of residual MET in MHCC97H cells after treatment with TDN-MLYTAB loaded with anti-MET primary antibody for 24 h. Figure 14 B in the figure is a quantitative statistical graph of average fluorescence intensity based on the flow cytometry results.

[0050] Figure 15 To evaluate the degradation effect of TDN-MLYTAB loaded with anti-mesenchymal-epithelial transforming factor (MET) primary antibody on MET in MHCC97H cells using Western blot analysis.

[0051] Figure 16 The figure shows the effect of different pathway inhibitors on the TDN-MLYTAB complex-mediated EGFR degradation. Figure 16 In the figure, A represents the Wb results of the proteasome inhibitor (MG132) treatment group. Figure 16 B in the figure represents the Wb result of the lysosomal inhibitor (CQ).

[0052] Figure 17 The figure shows the verification results of the non-consumption cycling mechanism of the TDN-MLYTAB complex receptor provided by this invention. Figure 17 In the image, A represents a confocal imaging pattern used to detect the distribution of M6PR on the cell surface. Figure 17 In the diagram, B represents the relative fluorescence intensity change of M6PR based on confocal imaging. Figure 17 In the figure, C represents the Wb result of the relative expression abundance of M6PR in cells.

[0053] Figure 18 This is a schematic diagram illustrating the use of the TDN-MLYTAB complex provided by this invention for targeted protein degradation. Detailed Implementation

[0054] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0055] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0056] The dibenzylcyclooctyl-sulfonyl-N-hydroxysuccinimide used in this invention was purchased from Sigma-Aldrich Reagents, catalog number 762040 (1 mg). The universal secondary antibody used in this invention was purchased from Wuhan Aiboteke Biotechnology Co., Ltd., catalog number AS070NF; The epidermal growth factor receptor (EGFR) primary antibody (hereinafter referred to as "anti-EGFR primary antibody") used in this invention was purchased from Abogen (Shanghai) Trading Co., Ltd., with catalog number ab52894 (40 μL). The mesenchymal-epithelial transition factor (MET) primary antibody (hereinafter referred to as "anti-MET primary antibody") used in this invention was purchased from Sangon Biotech (Shanghai) Co., Ltd., with catalog number D155219-0025. The nucleotide sequences used in this invention were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0057] Example 1: Preparation of Tetrahedral DNA Nanostructure-Mediated Multivalent Lysosome-Targeting Antibody Chimera (TDN-MLYTAB) 1. Establishment of coupling process parameters Single-stranded deoxyribonucleic acid (ssDNA-S1') was used as the model molecule in the study of coupling process parameters. Its sequence is shown in Table 1 (SEQ ID NO.7). An azide group was introduced into the 5' end of the ssDNA to obtain azide-modified ssDNA-S1', named "Azide-ssDNA". Dibenzylcyclooctyl-sulfonyl-N-hydroxysuccinimide ester was reacted with a universal secondary antibody (Ab) to obtain a universal secondary antibody modified with dibenzocyclooctyne (DBCO), named "DBCO-Ab". DBCO-Ab and Azide-ssDNA were subjected to a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction at room temperature (25℃) to obtain the secondary antibody (Ab)-ssDNA conjugate, named Ab-ssDNA.

[0058] The reaction time was kept at 24 h to explore the effect of different feed ratios (DBCO-Ab to Azide-ssDNA molar ratios of 1:1, 1:2, 1:4, 1:8, and 1:10) on the coupling efficiency. The 3' end of Azide-ssDNA was labeled with a 6-carboxyfluorescein (FAM) fluorescent tag, and the average fluorescence intensity was observed to determine the coupling efficiency.

[0059] The molar ratio of DBCO-Ab to Azide-ssDNA was kept at 1:8 to explore the effect of different reaction times (1 h, 6 h, 12 h, 24 h, 48 h) on coupling efficiency.

[0060] The coupling products were preliminarily identified using non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), and changes in protein bands were observed using Coomassie brilliant blue staining. The results are as follows: Figure 1 As shown, compared to the antibody (Ab), the conjugate (Ab-ssDNA) band showed a significant downward shift. This is because the introduction of the DNA strand significantly increased the negative charge density on the surface of the complex, resulting in a higher mass-to-charge ratio in the electric field and thus exhibiting a faster electrophoretic migration rate, indicating that the single-stranded DNA (ssDNA) has been successfully covalently anchored to the antibody surface.

[0061] Quantitative analysis of gel grayscale (the imaging results of the gel were analyzed using ImageJ software for grayscale quantification) as follows: Figure 2As shown, when the molar ratio of DBCO-Ab to Azide-ssDNA reaches 1:8 and the reaction time is extended to 24 h, the fluorescence signal intensity enters a stable plateau phase, indicating that the coupling reaction has become nearly complete. Based on this, a molar ratio of DBCO-Ab to Azide-ssDNA of 1:8 and a reaction time of 24 h were determined as the coupling process parameters for subsequent experiments.

[0062] Table 1 Sequence List

[0063] Note: In the single-stranded nucleotide name, TDN represents the TDN scaffold, and the uppercase letters A, B, C, and D in the suffix represent the four different single-stranded nucleotides that make up the TDN scaffold; M6PR represents the mannose-6-phosphate receptor, and M6PR-S2' is the M6PR aptamer sequence; the bolded and underlined parts in the sequence are the base-complementary pairing regions; the numbers S1, S2, S1', S2', etc. in the single-stranded nucleotide name suffix correspond to the bolded and underlined parts, which are the base-complementary pairing regions. S1 and S1' are complementary strands, and S2 and S2' are complementary strands, which can undergo specific base-complementary binding.

[0064] 2. Preparation of TDN-MLYTAB (1) Preparation of multivalent DNA tetrahedral (TDN) scaffolds TDN scaffolds with different numbers of M6PR aptamers (M6PR aptamers can bind to M6PR with high affinity and high specificity through a specific three-dimensional structure) were prepared and named TDN-1, TDN-2, and TDN-3, respectively. The Arabic numerals in the naming suffix represent the number of M6PR aptamers modified on the surface of the TDN scaffold; that is, TDN-1 is a monovalent TDN scaffold modified with one M6PR aptamer, TDN-2 is a bivalent TDN scaffold modified with two M6PR aptamers, and TDN-3 is a trivalent TDN scaffold modified with three M6PR aptamers. The single-stranded nucleotide sequences used to prepare the TDN scaffolds are shown in Table 1, and the specific preparation methods are as follows: TDN-1: The four single-stranded nucleotides used to prepare the TDN scaffold are TDN-B-S1 (SEQ ID NO.3), TDN-A (SEQ ID NO.1), TDN-C-S2 (SEQ ID NO.4), and TDN-D (SEQ ID NO.5). The above four single-stranded nucleotides and Azide-ssDNA are mixed in an equimolar ratio, and one M6PR nucleic acid aptamer sequence (SEQ ID NO.8) is introduced at the same time. After mixing, the mixture is kept at 95°C for 10 min, and then kept at 4°C for 10 min. The mixture self-assembles to form a monovalent TDN scaffold with one M6PR nucleic acid aptamer on its surface, namely TDN-1.

[0065] TDN-2: The four single-stranded nucleotides used to prepare the TDN scaffold are TDN-B-S1 (SEQ ID NO.3), TDN-A (SEQ ID NO.1), TDN-C-S2 (SEQ ID NO.4), and TDN-D-S2 (SEQ ID NO.6). The above four single-stranded nucleotides and Azide-ssDNA are mixed in an equimolar ratio, and two M6PR nucleic acid aptamer sequences (SEQ ID NO.8) are introduced at the same time. After mixing, the mixture is kept at 95°C for 10 min and then at 4°C for 10 min. The mixture self-assembles to form a bivalent TDN scaffold with two M6PR nucleic acid aptamers on its surface, namely TDN-2.

[0066] TDN-3: The four single-stranded nucleotides used to prepare the TDN scaffold are TDN-B-S1 (SEQ ID NO.3), TDN-A-S2 (SEQ ID NO.2), TDN-C-S2 (SEQ ID NO.4), and TDN-D-S2 (SEQ ID NO.6). The above four single-stranded nucleotides and Azide-ssDNA are mixed in an equimolar ratio, and three M6PR nucleic acid aptamer sequences (SEQ ID NO.8) are introduced at the same time. After mixing, the mixture is kept at 95°C for 10 min and then at 4°C for 10 min. The mixture self-assembles to form a trivalent TDN scaffold with three M6PR nucleic acid aptamers on its surface, namely TDN-3.

[0067] The TDN-1, TDN-2 and TDN-3 obtained above are multivalent TDN stents.

[0068] Using TDN-3 as an example, four single-stranded nucleotides, Azide-ssDNA, and the M6PR aptamer were added sequentially. Agarose gel electrophoresis was used to illustrate the specific process of preparing the multivalent TDN scaffold. The materials added to each lane are shown below: Lane 1: Add TDN-A-S2; Lane 2: Add TDN-A-S2 and TDN-B-S1; Lane 3: Add TDN-A-S2, TDN-B-S1 and TDN-C-S2; Lane 4: Add TDN-A-S2, TDN-B-S1, TDN-C-S2 and TDN-D-S2; Lane 5: Add TDN-A-S2, TDN-B-S1, TDN-C-S2, TDN-D-S2 and Azide-ssDNA; Lane 6: Add TDN-A-S2, TDN-B-S1, TDN-C-S2, TDN-D-S2, Azide-ssDNA and one M6PR aptamer sequence (M6PR-S2'). Lane 7: Add TDN-A-S2, TDN-B-S1, TDN-C-S2, TDN-D-S2, Azide-ssDNA and two M6PR aptamer sequences (M6PR-S2'). Lane 8: Add TDN-A-S2, TDN-B-S1, TDN-C-S2, TDN-D-S2, Azide-ssDNA and three M6PR aptamer sequences (M6PR-S2').

[0069] The results are as follows Figure 3 As shown, the migration changes of the bands in each lane are illustrated, characterizing the entire self-assembly process. During the basic scaffold assembly stage (lanes 1 to 4), single-stranded nucleotides that make up the TDN scaffold are added sequentially. With the increase of single-stranded nucleotides, the molecular weight of the product increases, and the hydrodynamic radius also increases, so the band position gradually shifts upwards. When four single-stranded nucleotides are present simultaneously (lane 4), a clear main band is observed at the predetermined position. Although there are some slight background signals or stray bands in the lanes, which may be due to residual single strands or base mismatches from the assembly process, the target product band clearly dominates in signal intensity, indicating that the three-dimensional scaffold has been successfully formed. Lane 5 shows that after the introduction of Azide-ssDNA, the band has moved slightly more than in the three-dimensional scaffold (lane 4). With the subsequent increase in the loading of M6PR aptamers (lanes 6 to 8), the migration speed of the product bands slows down to varying degrees.

[0070] (2) Assembly of TDN-MLYTAB DBCO-Ab was mixed with TDN-1, TDN-2, and TDN-3 at a molar ratio of 1:8 and incubated at room temperature for 24 h. DBCO-Ab was then coupled to the TDN scaffold via a SPAAC reaction. The resulting compound was purified (using an ultrafiltration tube with a molecular weight cutoff of 100 kDa, followed by centrifugation and washing, then adding 0.01 M PBS to a volume of 500 μL, centrifuging at 14000 g for 10 min at 4 °C, and repeating the process twice) to obtain the secondary antibody-TDN covalent conjugate (i.e., TDN-MLYTAB), named Ab-TDN-1, Ab-TDN-2, and Ab-TDN-3, respectively. The TDN-MLYTAB was analyzed by non-denaturing polyacrylamide gel electrophoresis, and the following control group was set up: ① Free secondary antibody (Ab) control group; ② Linear coupling control group: DBCO-Ab was directly coupled with M6PR nucleic acid aptamer (molar ratio of 1:8, incubated at room temperature for 24 h), and named Ab-M6PR.

[0071] The results of non-denaturing polyacrylamide gel electrophoresis analysis are as follows: Figure 4 As shown.

[0072] 3. Confirmation of physical structure The physical structure of TDN-MLYTAB was confirmed using Ab-TDN-3 as a representative.

[0073] The morphology and dimensions of Ab, TDN-3, and Ab-TDN-3 were verified using atomic force microscopy (AFM), and the results are as follows: Figure 5 As shown, the characteristic topological morphology of the TDN scaffold and the secondary antibody can be clearly observed at the single-molecule scale, confirming that they achieve true physical cross-linking in space. The statistical results of the horizontal size distribution are as follows: Figure 6 As shown, the average size of the assembled Ab-TDN-3 is approximately 25 nm, which is significantly larger than that of a single TDN scaffold.

[0074] Physicochemical property verification: Zeta potential analysis showed ( Figure 7 In Figure A), after the secondary antibody was coupled to the TDN scaffold, a significant negative shift in surface charge occurred, confirming the introduction of the high charge density TDN scaffold; UV-Vis absorption spectroscopy showed that ( Figure 7 The characteristic absorption peak of protein B in Ab-TDN-3 showed a characteristic shift, which further confirmed from a spectroscopic perspective that the secondary antibody and TDN scaffold formed a stable spatial chimera structure.

[0075] Example 2: Biological stability assessment of TDN-MLYTAB The stability of TDN-MLYTAB (Ab-TDN-1, Ab-TDN-2, Ab-TDN-3) obtained in Example 1 was tested to evaluate its resistance to nuclease degradation and its tolerance to complex environments.

[0076] 1. Experimental Method: 10 vol% fetal bovine serum (FBS) was used to simulate the in vitro blood circulation environment, and pH 6.5 buffer (1 mol / L tris(hydroxymethyl)aminomethane solution, adjusted to pH 6.5 with 1 M hydrochloric acid solution) was used to simulate the weakly acidic environment of the early post-endocytic endosome. TDN-MLYTAB (Ab-TDN-1, Ab-TDN-2, Ab-TDN-3) obtained in Example 1 were placed in the above two environments and incubated at 37°C for 0–24 h, with Ab-M6PR from Example 1 used as a control.

[0077] 2. Experimental Results: TDN-MLYTAB samples incubated for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h were separated by non-denaturing polyacrylamide gel electrophoresis. Representative gel bands at each time point were selected for relative gray-scale quantitative analysis (the imaging results of the gel were analyzed for gray-scale quantitative analysis using ImageJ software), and the stability trend curve of TDN-MLYTAB with incubation time was plotted.

[0078] The results in the weakly acidic environment of the early endosome after endocytosis are as follows: Figure 8 As shown, after incubation for 24 h in a weakly acidic environment at pH 6.5, the structural integrity of TDN-MLYTAB (Ab-TDN-1, Ab-TDN-2, Ab-TDN-3) remained above 65%.

[0079] Results in an extracorporeal blood circulation environment, such as Figure 9 As shown, after incubation for 24 h in 10 vol% FBS containing complex nucleases, the structural integrity of TDN-MLYTAB (Ab-TDN-1, Ab-TDN-2, Ab-TDN-3) remained above 75%.

[0080] The above results confirm that the high-density rigid framework provided by the TDN scaffold endows TDN-MLYTAB with excellent physical barrier and anti-degradation ability, enabling it to maintain structural integrity in complex extracellular blood circulation and intracellular acidic transport environments.

[0081] Example 3: Construction of the TDN-MLYTAB complex and evaluation of its efficient targeted degradation of model targets. To verify the ability of TDN-MLYTAB to overcome the steric hindrance of macromolecules and achieve efficient targeted degradation, this embodiment uses epidermal growth factor receptor (EGFR) as a model target for in vitro evaluation.

[0082] 1. Experimental Method: (1) Degradation platform construction: Anti-EGFR primary antibody (molar ratio of 1:1) was added to the TDN-MLYTAB (Ab-TDN-1, Ab-TDN-2, Ab-TDN-3) prepared in Example 1, and the assembly was completed by incubation at room temperature (25℃) for 30 min to obtain TDN-MLYTAB loaded with anti-EGFR primary antibody, which were named EGFR-Ab-TDN-1, EGFR-Ab-TDN-2, and EGFR-Ab-TDN-3, respectively, which are TDN-MLYTAB complexes targeting EGFR.

[0083] (2) Cell model selection: MHCC97H cells and MCF-7 cells that highly express EGFR and M6PR were selected as experimental models.

[0084] (3) Experimental grouping: ① Experimental group 1: EGFR-Ab-TDN-1 treatment group; ② Experimental group 2: EGFR-Ab-TDN-2 treatment group; ③ Experimental group 3: EGFR-Ab-TDN-3 treatment group; ④ Control group 1: Free anti-EGFR primary antibody treatment group; ⑤ Control group 2: The anti-EGFR primary antibody was coupled with the Ab-M6PR prepared in Example 1 (incubated at room temperature 25°C for 30 min) to obtain the Ab-M6PR covalent conjugate loaded with the anti-EGFR primary antibody, which was named EGFR-Ab-M6PR (EGFR-Ab-M6PR treatment group).

[0085] ⑥ Blank group: Cells that have not undergone any treatment are used as blank control.

[0086] (4) Degradation treatment The above treatment groups (working concentration of 100 nM) were co-incubated with the two types of cells for 24 h.

[0087] (5) Evaluation experiment a. The targeting binding ability of EGFR-Ab-TDN-1, EGFR-Ab-TDN-2, EGFR-Ab-TDN-3, and EGFR-Ab-M6PR to MHCC97H cells was evaluated by flow cytometry; 6-Carboxyfluorescein (FAM) was used as a fluorescent label. b. Western blotting (Wb) was used to evaluate the degradation effects of EGFR-Ab-TDN-1, EGFR-Ab-TDN-2, EGFR-Ab-TDNB-3, EGFR-Ab-M6PR, and free anti-EGFR primary antibodies on the target (EGFR); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and cells without any antibodies were used as a blank control. c. Laser confocal scanning microscopy (CLSM) was used to evaluate the effects of EGFR-Ab-TDN-1, EGFR-Ab-TDN-2, EGFR-Ab-TDN-3, and EGFR-Ab-M6PR on EGFR degradation.

[0088] 2. Experimental Results (1) Experimental results using MHCC97H cells as a cell model Flow cytometry statistical analysis, such as Figure 10 As shown, the binding affinity of EGFR-Ab-TDN-1, EGFR-Ab-TDN-2, and EGFR-Ab-TDN-3 to the cell surface (expressed as mean fluorescence intensity MFI, with stronger MFI indicating stronger cell binding affinity) of the TDN scaffolds was higher than that of EGFR-Ab-M6PR. Among them, EGFR-Ab-TDN-3 was significantly higher than EGFR-Ab-M6PR, confirming that the spatial spacing of the TDN scaffolds effectively avoids the steric hindrance of macromolecular antibodies.

[0089] The relative quantitative results of Wb are as follows Figure 11 As shown, when EGFR-Ab-TDN-1, EGFR-Ab-TDN-2, and EGFR-Ab-TDN-3 provided by this invention are treated at a concentration of 100 nM for 24 h, EGFR-Ab-TDN-3 can achieve an EGFR scavenging rate of over 70%, and the degradation effect is better than that of the control group.

[0090] CLSM imaging, such as Figure 12 As shown, after 24 h of treatment, the fluorescence signal on the cell membrane surface faded extensively and even dropped to the background level.

[0091] (2) Experimental results using MCF-7 cells as a cell model The relative quantitative results of Wb are as follows Figure 13 As shown, when EGFR-Ab-TDN-1, EGFR-Ab-TDN-2, and EGFR-Ab-TDN-3 provided by this invention are treated at a concentration of 100 nM for 24 h, EGFR-Ab-TDN-3 can achieve an EGFR scavenging rate of over 70%, and the degradation effect is better than that of the control group.

[0092] Example 4: Verification of the universality of TDN-MLYTAB To verify that the TDN-MLYTAB provided by this invention can break through the limitation of a single target and achieve broad applicability, this embodiment replaces the anti-EGFR primary antibody in Example 3 with an anti-mesenchymal-epithelial transition factor (MET) primary antibody.

[0093] 1. Experimental Method: (1) Degradation platform construction: Keep TDN-MLYTAB (Ab-TDN-1, Ab-TDN-2, Ab-TDN-3) unchanged, and directly replace the anti-EGFR primary antibody in Example 3 with the anti-MET primary antibody. Incubate at room temperature (25°C) to obtain TDN-MLYTAB loaded with anti-MET primary antibody, which are named MET-Ab-TDN-1, MET-Ab-TDN-2, and MET-Ab-TDN-3, respectively, which are TDN-MLYTAB complexes against MET.

[0094] (2) Cell model selection: MHCC97H cells were selected as the experimental model.

[0095] (3) Experimental grouping: ① Experimental group 1: MET-Ab-TDN-1 treatment group; ② Experimental group 2: MET-Ab-TDN-2 treatment group; ③ Experimental group 3: MET-Ab-TDN-3 treatment group; ④ Control group 1: Free anti-MET primary antibody treatment group; ⑤ Control group 2: The anti-MET primary antibody was coupled with the Ab-M6PR prepared in Example 1 (incubated at room temperature 25℃ for 30 min) to obtain the Ab-M6PR covalent conjugate loaded with the anti-MET primary antibody, which was named MET-Ab-M6PR (MET-Ab-M6PR treatment group). ⑥ Blank group: Cells that have not undergone any treatment are used as a blank control; (4) Degradation treatment The above treatment group (working concentration 100 nM) was incubated with cells for 24 h.

[0096] (5) Evaluation of the experiment: a. The cell degradation capabilities of MET-Ab-TDN-1, MET-Ab-TDN-2, MET-Ab-TDN-3, and MET-Ab-M6PR were evaluated by flow cytometry; anthocyanin 5 (Cyanine 5, Cy5) was used as a fluorescent label. b. Western blotting (Wb) was used to evaluate the degradation effects of MET-Ab-TDN-1, MET-Ab-TDN-2, MET-Ab-TDN-3, MET-Ab-M6PR, and free anti-MET primary antibodies on the target (MET); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and cells without any antibodies were used as a blank control.

[0097] 2. Experimental Results: Flow cytometry statistical analysis, such as Figure 14 As shown, the amount of MET binding on the cell surface was significantly reduced; The relative quantification of Wb further confirms ( Figure 15 After 24 h of treatment, the residual amount of MET in the cells had decreased to about 35% of the initial level (i.e., about 65% was cleared).

[0098] The above experimental results show that the present invention does not require re-screening aptamers for new targets or optimizing the conjugation process. It can achieve efficient clearance of different clinically relevant membrane proteins simply by replacing the commercial primary antibody, and has "plug and play" universality.

[0099] Example 5: Confirmation of lysosomal degradation mechanism and non-consumptive cycling tracing of receptors To verify the physical mechanism by which the TDN-MLYTAB complex provided by this invention achieves long-term degradation without disrupting the underlying receptor homeostasis, the mechanism was explored using EGFR-Ab-TDN-3 prepared in Example 3 as an example.

[0100] 1. Degradation pathway inhibition experiment MHCC97H cells were incubated with EGFR-Ab-TDN-3 for 24 h after being pre-treated with lysosomal inhibitors (chloroquine, CQ) and proteasome inhibitors (MG132), respectively. After incubation, Western blotting (Wb) was used to detect the expression level of the target protein (EGFR). The Wb results showed that... Figure 16In the CQ treatment group, the protein degradation effect induced by the present invention was effectively blocked, and the target protein (EGFR) level was restored to near the control group. Biochemically, this confirmed that the present invention relies on the lysosomal degradation pathway.

[0101] 2. Verification of the receptor non-consumption cycling mechanism (low-temperature blocking method) To eliminate experimental artifacts caused by M6PR aptamer self-induced secondary endocytosis, this experiment was conducted under a strict 4°C ice environment to completely block ATP-dependent membrane dynamics. The transient distribution of free M6PR on the cell membrane surface within 0–24 h was dynamically traced using anthocyanin 5 (Cy5) fluorescently labeled M6PR aptamers.

[0102] Relative fluorescence quantitative trend analysis showed that ( Figure 17 A in Figure 17 In the initial stage of treatment (0–3 h), the fluorescence intensity of M6PR on the membrane surface decreased, reflecting the internalization of the receptor; however, during 6–24 h, the fluorescence signal on the membrane surface gradually recovered. Simultaneously, Wb analysis of extracted whole-cell proteins confirmed (…). Figure 17 (C) indicates that the total M6PR protein expression abundance remained relatively constant within the corresponding time point. These results demonstrate that the degradation platform provided by this invention can achieve non-consumptive cycling of M6PR, enabling long-term degradation without disrupting receptor homeostasis.

[0103] The degradation mechanism of the TDN-MLYTAB complex provided by this invention is as follows: Figure 18 As shown.

[0104] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A lysosomal targeting chimera based on DNA tetrahedrons, characterized in that, Including DNA tetrahedrons and universal secondary antibodies; The DNA tetrahedron is loaded with single-stranded DNA with azide-modified ends, and the single-stranded DNA with azide-modified ends is linked to the DNA tetrahedron through complementary base pairing. The DNA tetrahedron is loaded with 1 to 3 lysosomal receptor nucleic acid aptamer sequences, which are linked to the DNA tetrahedron through complementary base pairing. The universal second antibody promotes the linkage of the single-stranded DNA with the terminal modified azide group to the azide-alkyne cycloaddition reaction by strain.

2. The DNA tetrahedron-based lysosomal targeting chimera as described in claim 1, characterized in that, The DNA tetrahedron comprises four nucleotide single strands: nucleotide A strand, nucleotide B strand, nucleotide C strand, and nucleotide D strand; The nucleotide A chain, nucleotide B chain, nucleotide C chain, and nucleotide D chain self-assemble to form the DNA tetrahedron through complementary base pairing.

3. The DNA tetrahedral-based lysosomal targeting chimera as described in claim 2, characterized in that, The single-stranded nucleotide includes at least one of the following: (1) The sequence of the nucleotide A chain is shown in SEQ ID NO.1 or SEQ ID NO.2; (2) The sequence of the B chain of the nucleotide is shown in SEQ ID NO.3; (3) The sequence of the C-chain of the nucleotide is shown in SEQ ID NO.4; (4) The sequence of the D chain of the nucleotide is shown in SEQ ID NO.5 or SEQ ID NO.

6.

4. The DNA tetrahedron-based lysosomal targeting chimera as described in claim 1, characterized in that, The sequence of the single-stranded DNA is shown in SEQ ID NO.

7.

5. The DNA tetrahedral-based lysosomal targeting chimera as described in claim 1, characterized in that, The lysosomal receptor nucleic acid aptamer sequence is shown in SEQ ID NO.

8.

6. A complex based on a DNA tetrahedron-targeting chimera, characterized in that, Includes the DNA tetrahedral-based lysosomal targeting chimera and the first antibody as described in any one of claims 1 to 5; The first antibody is linked by a non-covalent affinity between its crystallizable fragment and the antigen-binding fragment of the universal second antibody in the DNA tetrahedral-based lysosomal targeting chimera.

7. A pharmaceutical composition, characterized in that, A complex comprising the DNA tetrahedral-based lysosomal targeting chimera of claim 6.

8. The method for preparing the DNA tetrahedral-based lysosomal targeted chimera according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1: Mix single-stranded DNA with azide-modified ends, nucleotide A chain, nucleotide B chain, nucleotide C chain, nucleotide D chain that make up the DNA tetrahedron, and lysosomal receptor nucleic acid aptamer, and form the DNA tetrahedron by self-assembly through complementary base pairing; S2: Mix and incubate the universal second antibody with the DNA tetrahedron obtained in step S1 to obtain the DNA tetrahedron-based lysosomal targeting chimera.

9. The preparation method according to claim 8, characterized in that, The molar ratio of the universal second antibody to the DNA tetrahedron is 1:(1-10).

10. The use of the DNA tetrahedral-based lysosomal targeting chimera complex of claim 6 or the pharmaceutical composition of claim 7 in the preparation of an antitumor drug.

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