DNA-TRAIL complex and application thereof in tumor treatment

Abstract

The invention discloses a DNA-TRAIL compound, a method for preparing the DNA-TRAIL compound, a kit for preparing the DNA-TRAIL compound, a method for treating tumors by using the DNA-TRAIL compound and application of the DNA-TRAIL compound. The DNA-TRAIL compound comprises a DNA origami structure and three TRAIL monomers which are connected with the DNA origami structure and form TRAIL trimers.

Description

DNA-TRAIL complex and its use in treating tumors Technical Field

[0001] The present invention relates to the field of tumor treatment, and in particular, to a TRAIL complex based on a DNA origami structure. Background Art

[0002] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a type II membrane protein belonging to the tumor necrosis factor superfamily [Pitti RM, et al., Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem. 1996; 271(22): 12687-12690]. TRAIL can assemble into homotrimers, and the trimeric structure is crucial for its biological function [Yan J, et al. Engineered adenovirus fiber shaft fusion homotrimer of soluble TRAIL with enhanced stability and antitumor activity. Cell Death Dis. 2016; 7(6): e2274]. By cross-linking TRAIL receptor (TRAIL-R) 1 or TRAIL-R2, also known as death receptors 4 and 5 (DR4 and DR5), TRAIL trimers can induce apoptosis in a variety of tumor cells while leaving normal cells that lack the corresponding receptors unaffected [de Miguel D, et al. Onto better TRAILs for cancer treatment. Cell Death Differ. 2016; 23(5): 733-747].To date, several therapeutic approaches targeting the interaction between TRAIL and DR4 / DR5 have been evaluated in clinical studies, including recombinant human TRAIL (Dulanermin) and an agonistic antibody against DR5 (AMG655) [Cheah CY, et al. Dulanermin with rituximab in patients with relapsed indolent B-cell lymphoma: an open-label phase 1b / 2 randomized study. Lancet Haematol. 2015; 2(4): e166-e174; Soria JC, et al. Phase 1b study of dulanermin (recombinant human Apo2L / TRAIL) in combination with paclitaxel, carboplatin, and bevacizumab in patients with advanced non-squamous non-small-cell lung cancer. J Clin Oncol. 2010; 28(9): 1527-1533; Herbst RS, et al. A first-in-human study of conatumumab in adult patients with advanced solid tumors. Clin Cancer Res. 2010;16(23):5883-5891].Unfortunately, the results of phase II randomized clinical studies failed to demonstrate significant clinical benefits [Kindler HL, et al. A randomized, placebo-controlled phase 2 study of ganitumab (AMG 479) or conatumumab (AMG 655) in combination with gemcitabine in patients with metastatic pancreatic cancer. Ann Oncol. 2012; 23(11): 2834-2842; Graves JD, et al. Apo2L / TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activity. Cancer Cell. 2014; 26(2): 177-189]. This may be due to the poor agonist activity of these therapeutic agents [Dimberg LY, et al. On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene. 2013; 32(11): 1341-1350]. A deeper understanding of the interaction pattern between TRAIL and its receptor is crucial for developing more effective treatments.

[0003] Receptor aggregation on the cell membrane is crucial for the signal activation of many receptors. Taking DR5 as an example, transmembrane helices (TMHs) alone can assemble higher-order structures to drive downstream signals. However, DR5 exists on the cell membrane as a pre-dimer ligand state through extracellular domain (ECD) dimers, while non-ligand ECD dimers inhibit TMH oligomerization [Pan L, et al. Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling. Cell. 2019; 176 (6): 1477-1489.e14; Fu Q, et al. Structural Basis and Functional Role of Intramembrane Trimerization of the Fas / CD95 Death Receptor. Mol Cell. 2016; 61 (4): 602-613]. Once the ECD of DR5 binds to the TRAIL trimer, TMH is released to trimerize. Finally, high-order hexagonal clusters of DR5 with a dimer-trimer network are formed together with TRAIL trimers. The trimeric structure of TRAIL trimers is crucial in this process as it mediates the trimerization of DR5 dimers and the final network formation [Vanamee et al. Structural principles of tumor necrosis factor superfamily signaling. Sci Signal. 2018; 11(511): eaao4910; Li J, et al. Structural basis of signal transduction in the TNF receptor superfamily. Adv Immunol. 2013; 119: 135-153].

[0004] TRAIL mimetic peptides were hexagonally patterned on DNA origami with ligand spacings below 10 nm, revealing that the key ligand spacing for hexagonally patterned peptides that induce DR5 clustering is approximately 5 nm [Wang Y, et al. Clustering of Death Receptor for Apoptosis Using Nanoscale Patterns of Peptides. ACS Nano. 2021; 15(6): 9614-9626]. However, the relationship between the inter-ligand distance in TRAIL protein multimers and the resulting biological effects has not been comprehensively studied, especially in the inter-ligand distance range of 10 to 60 nm.

[0005] Summary of the Invention

[0006] The present invention developed a "carving and printing" strategy to rapidly modify three TRAIL monomers onto a flat rectangular DNA origami (DNA-TRAIL3), and control the monomer distance to 15-60nm. Compared with natural TRAIL monomers, these DNA-TRAIL3 trimers exhibited stronger receptor binding affinity, with KD values ​​2-3 orders of magnitude lower, among which DNA-TRAIL3-40 was the best candidate. DNA-TRAIL3-40 also induced the strongest signaling pathway activation and the highest cancer cell apoptosis rate in vitro. Importantly, compared with natural TRAIL monomers, DNA-TRAIL3-40 showed more favorable pharmacokinetics and anti-tumor effects in in vivo experiments.

[0007] In a first aspect, a DNA-TRAIL complex is provided, which comprises a DNA origami structure and three TRAIL monomers forming a TRAIL trimer, wherein the three TRAIL monomers are respectively connected to three sites on the same side of the same surface of the DNA origami structure and the distance between any two sites is 15-60 nm, preferably 25-40 nm, most preferably 40 nm, and the three sites form an equilateral triangle or an approximately equilateral triangle.

[0008] In the second aspect, a method for preparing the DNA-TRAIL complex of the first aspect is provided, comprising: (a) providing a DNA origami structure, (b) connecting three TRAIL monomers to three sites on the same side of the same surface of the DNA origami structure, respectively, wherein any two sites are 15-60 nm, preferably 25-40 nm, most preferably 40 nm apart, and the three sites form an equilateral triangle or an approximately equilateral triangle, and (c) harvesting the formed DNA-TRAIL complex connected to the three sites on the same side of the same surface of the DNA origami structure.

[0009] In a third aspect, a kit is provided, comprising: (i) a DNA origami structure having complementary adapter molecules attached at at least three sites on the same side of the same face, wherein any two sites are about 15-60 nm, preferably about 25-40 nm, most preferably about 40 nm apart, and the three sites form an equilateral triangle or an approximately equilateral triangle, and (ii) a TRAIL monomer-adapter molecule fusion protein.

[0010] In a fourth aspect, a pharmaceutical composition for treating tumors is provided, comprising the DNA-TRAIL complex of the first aspect, the DNA-TRAIL complex obtained according to the method of the second aspect, or the DNA-TRAIL complex prepared using the kit of the third aspect, and an optional pharmaceutically acceptable carrier.

[0011] In the fifth aspect, the DNA-TRAIL complex of the first aspect, the DNA-TRAIL complex obtained according to the method of the second aspect, or the DNA-TRAIL complex prepared using the kit of the third aspect is provided for use in treating tumors; or, the use of the DNA-TRAIL complex of the first aspect, the DNA-TRAIL complex obtained according to the method of the second aspect, or the DNA-TRAIL complex prepared using the kit of the third aspect in the preparation of a medicament for treating tumors is provided.

[0012] In a sixth aspect, a method for treating a tumor in a subject is provided, comprising administering to the subject a therapeutically effective amount of the DNA-TRAIL complex of the first aspect, the DNA-TRAIL complex obtained according to the method of the second aspect, the DNA-TRAIL complex formulated using the kit of the third aspect, or the pharmaceutical composition of the fourth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1: Design and characterization of DNA-TRAIL3 trimers with different ligand spacings.

[0014] (A) Schematic diagram of the construction process of the DNA-TRAIL3 trimer. Flat rectangular DNA origami was assembled by slowly annealing the genomic DNA of the M13mp18 phage (SEQ ID NO: 6) as a scaffold with multiple staple strands (SEQ ID NO: 7-149) and a capture strand (SEQ ID NO: 150-160). The azide-modified SpyTag peptide (SEQ ID NO: 3) was coupled to the DBCO-containing complementary strand via click chemistry. The SpyTag adapter conjugate was then engraved on the surface of the DNA origami through annealing and DNA hybridization between the complementary and capture strands. Finally, the TRAIL-SpyCatcher (SEQ ID NO: 5) protein was rapidly printed on the DNA origami via isopeptide bond formation between the SpyTag and SpyCatcher (SEQ ID NO: 2).

[0015] (B) Design of DNA-TRAIL3 trimers with different inter-ligand distances. By changing the position of the capture strand, the position of TRAIL monomers on the DNA origami was set to different inter-ligand distances, including approximately 15 nm (DNA-TRAIL3-15), 25 nm (DNA-TRAIL3-25), 40 nm (DNA-TRAIL3-40), 55 nm (DNA-TRAIL3-55), and 60 nm (DNA-TRAIL3-60).

[0016] (C) AFM observation of different DNA-TRAIL3 trimers. Scale bar, 50 nm.

[0017] (D) Height measurements of different DNA-TRAIL3 trimers. The height of DNA origami was measured at the naked position (top row) and the TRAIL-modified position (bottom row).

[0018] (E) TRAIL assembly efficiency. The yield of different DNA-TRAIL3 trimers was calculated as the percentage of DNA origami carrying TRAIL monomers at the desired positions relative to the total amount of DNA origami. In the figure, 0, 1, 2, and 3 represent the number of TRAIL monomers attached to each DNA origami, respectively.

[0019] Figure 2: In vitro cytotoxicity of DNA-TRAIL3 trimers. (A) Group information. (B) Cell viability of COLO205 cells (B), HeLa cells (C), and NCI-H460 cells (D) as measured by CCK-8 assay. Data were processed using GraphPad Prism 7 and are presented as mean ± SD. P values ​​were determined using one-way ANOVA followed by Tukey's post hoc test. *, P < 0.05; ***, P < 0.001.

[0020] Figure 3: DNA-TRAIL3 trimer induces cell apoptosis in vitro. (A) Group information. (B-C) Percentage of early apoptotic cells (Annexin V+ / PI-). Data were processed using GraphPad Prism 7 and presented as mean ± SD. P values ​​were determined using one-way ANOVA followed by Tukey's post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0021] Figure 4: DNA-TRAIL3 trimer-induced signaling pathway activation in vitro. (A) Grouping information. (B-C) Western blot analysis of caspase-8 (B) and caspase-3 (C) proteins in COLO205 cells treated with different TRAIL formulations at a 10 nM concentration for 6 hours. Pro-cass-8 (Pro-cas-8) and Pro-cass-3 (Pro-cas-3) are cleaved into active caspase-8 (P43 / 41-cas-8 and P18-cas-8) and active caspase-3 (P19-cas-3 and P17-cas-3), respectively. Protein levels were determined by measuring the grayscale value of the protein bands using ImageJ software. (D) Cell viability was measured using a CCK-8 assay after 24 hours of treatment with different TRAIL formulations plus anti-DR4 and / or anti-DR5. Data were processed in GraphPad Prism 7 and are presented as mean ± SD. P values ​​were determined using one-way analysis of variance with Tukey's post hoc test. **, P<0.01; ***, P<0.001.

[0022] Figure 5: In vivo antitumor effect, pharmacokinetics and biodistribution of DNA-TRAIL3 trimer.

[0023] (A) Group information and tumor growth curves of subcutaneous COLO205 tumor-bearing female BALB / c nu / nu mice treated with 30 mg / kg TRAIL of different TRAIL formulations.

[0024] (B) Tumor images at the end of the experiment. Scale bar, 1.5 cm.

[0025] (C) Tumor weight at the end point of the experiment (n=6).

[0026] (D) Pharmacokinetic evaluation. BALB / c nu / nu mice received intravenous injections of different TRAIL formulations (30 mg / kg TRAIL). After different time periods, serum TRAIL protein concentrations were determined by ELISA (n=3). For data calculation, relative values ​​of serum concentrations were analyzed, and the first value (5 minutes) was set as 100%.

[0027] (E) Biodistribution assay. Subcutaneous COLO205 tumor-bearing BALB / c nu / nu mice received intravenous injections of different TRAIL formulations (30 mg / kg TRAIL). 12 and 24 hours later, TRAIL protein concentrations in liver, spleen, kidney, and tumor tissues were determined by ELISA (n=3). Data were processed on GraphPad Prism 7 and expressed as mean ± SD. P values ​​were determined using one-way ANOVA followed by Tukey's post hoc test. **, P < 0.01; ***, P < 0.001.

[0028] Figure 6: Functional organ damage induced by TRAIL trimer. Alanine aminotransferase, aspartate aminotransferase, urea nitrogen, serum creatinine, albumin, globulin, and serum total protein levels were measured in treated animals.

[0029] Summary of the Invention

[0030] Unless otherwise noted, the scientific and technical terms used herein should have the meanings commonly known to those skilled in the art. In addition, unless otherwise required, singular terms should include plural terms, and plural terms should include singular terms. The aforementioned techniques and methods are generally carried out according to conventional methods well known in the art and described in the references cited in this specification. See, for example, Sambrook et al.Molecular Cloning:A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)) and Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J.Wiley & Sons (New York, NY 1994), which are incorporated by reference; Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, are hereby incorporated by reference in their entirety.

[0031] As used herein, the terms "protein", "peptide", "polypeptide" and "amino acid sequence" are used interchangeably to refer to polymers of any length, for example, two or more amino acid residues bound by peptide bonds. The term also includes amino acid polymers modified naturally or by human intervention; for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation or any other manipulation and modification, such as conjugation with a tag or a biologically active component. Conventional single-letter or three-letter amino acid residue codes are used herein. Generally, if an amino acid polymer is long (for example, more than 50 amino acid residues), it is preferably referred to as a polypeptide or protein, but if it is 50 amino acids long or shorter, it is preferably referred to as a "peptide".

[0032] Herein, when describing amino acid sequences, unless otherwise specified or clear from the context, the amino acid sequences are referred to in the N-terminal to C-terminal direction.

[0033] As used herein, the terms "nucleic acid," "DNA," "polynucleotide," and "nucleotide sequence" are used interchangeably and refer to deoxyribonucleic acid, a linear or circular polymer of deoxyribonucleotides (deoxyadenine (A), deoxyguanine (G), deoxycytosine (C), and deoxythymidine (T)) linked by 3', 5'-phosphodiester bonds. The DNA may be single-stranded or double-stranded, linear or circular. Furthermore, the DNA may contain suitable modifications known in the art, such as methylation, phosphorothioate, and interruptions by non-nucleotide components.

[0034] When describing nucleic acid sequences herein, reference to nucleic acid sequences is in the 5' to 3' direction unless otherwise specified or the context indicates otherwise. Generally, reference to DNA (e.g., scaffold DNA, short DNA strands, complementary DNA strands, capture DNA strands, etc.) herein refers to single-stranded DNA unless otherwise specified or the context indicates otherwise.

[0035] Sequence identity can be determined by commercially available computer programs that employ any suitable algorithm to calculate the percent identity between two or more sequences, for example, using default parameters. A typical example of such a computer program is CLUSTAL. More advantageously, the BLAST algorithm is employed with the parameters set to default values. A detailed description of the BLAST algorithm is available on the National Center for Biotechnology Information (NCBI) website.

[0036] In a first aspect, a DNA-TRAIL complex is provided, which comprises a DNA origami structure and three TRAIL monomers forming a TRAIL trimer, wherein the C-termini or N-termini of the three TRAIL monomers are respectively connected to three sites on the same side of the same surface of the DNA origami structure and any two sites are 15-60 nm apart, and the three sites form an equilateral triangle or an approximately equilateral triangle.

[0037] As used herein, a DNA origami structure refers to a structure in which a long single-stranded DNA (usually genomic DNA) is base-complemented with a series of designed short DNA (staple) fragments to controllably construct a desired pattern or structure. For different DNA origami structures, for example, DNA nanostructures designed from M13 / template strands can be designed using the open source caDNAno software and annealed using a general program. DNA nanostructures of small nanostructures can be directly designed and annealed based on sequence complementarity pairing, for example, see Paul WK Rothemund, Folding DNA to create nanoscale shapes and patterns, Nature V440: 297-302 (16 March 2006); RP Goodman et al., Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication, Science, V310: 1661-1664 (9 December 2005).

[0038] The DNA origami structure used herein can be any DNA origami structure, as long as it has a single surface on which at least three sites can be present on the same side for attachment to a TRAIL monomer as described herein, wherein any two sites are about 15-60 nm apart. The DNA origami structure used herein can be a two-dimensional structure (e.g., a planar DNA origami structure) or a three-dimensional structure (e.g., a polyhedral DNA origami structure).

[0039] In one embodiment, the DNA origami structure used herein is a two-dimensional DNA origami structure, preferably a planar DNA origami structure, and its shape can be any two-dimensional figure, such as regular figures such as triangles and quadrilaterals, as well as other irregular figures.

[0040] In one embodiment, the DNA origami structure used herein is a planar rectangular DNA origami structure.

[0041] As used herein, the "surface" refers to a two-dimensional structure formed by at least a portion of the DNA chain that forms the DNA origami structure, without considering the thickness of the DNA molecule itself. Herein, the size of the "surface" can be any suitable size, as long as there can be 3 sites on it and the distance between the two sites meets the requirements of this article, for example, the side length can be about 15-100nm, such as about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100nm. In one embodiment, the DNA origami structure used herein is a planar rectangle with a side length of about 60-90nm, for example, about 60×90nm.

[0042] In a further embodiment, the DNA origami structure is a DNA origami structure formed with M13 genomic DNA (e.g., SEQ ID NO: 6) as a scaffold, and is formed by short-chain DNA (i.e., Staple chain) shown in SEQ ID NO: 7-149 and three capture DNAs (i.e., (a) SEQ ID NO: 152, 155, 158; (b) SEQ ID NO: 151, 154, 158; (c) SEQ ID NO: 151, 153, 159; (d) SEQ ID NO: 151, 156, 160; and (e) SEQ ID NO: 150, 157, 160) among the DNAs shown in SEQ ID NO: 150-160 (Capture chain). Preferably, the three capture DNAs shown in SEQ ID NO: 151, 153, 159 are used.

[0043] As used herein, the "site" refers to a nucleotide residue in a DNA molecule that forms a DNA origami structure, which is located in the surface and pairs with the corresponding residue through the Watson-Crick base pairing principle (i.e., A / T, G / C pairing).

[0044] As used herein, "on the same side of the same plane" means that after the three TRAIL monomers are connected to the DNA origami structure, they are located on the same side of the plane where the three sites are located and can form an equilateral triangle or a nearly equilateral triangle, thereby forming a TRAIL trimer. The nearly equilateral triangle herein means that the inner angle of the triangle formed by the three sites is about 60 o ±6 o , 60 o ±5 o , 60 o ±4 o , 60 o ±3 o , 60 o ±2 o or 60 o±1 o .

[0045] As used herein, "any two sites are about 15-60 nm apart" means that each side of the triangle formed by the three sites is about 15-60 nm or any subrange thereof, and the lengths of the sides may be equal or unequal. For example, in one embodiment, the distance between any two sites is about 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, 30-60, 30-55, 30-50, 30-45, or 30-40 nm. In one embodiment, the distance between any two sites is about 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 nm. Preferably, the distance between any two sites is about 40 nm.

[0046] Human TRAIL, also known as tumor necrosis factor ligand superfamily member 10 (TNFSF10), Apo2Ligand or TL2, has the corresponding accession number P50591. As used herein, "TRAIL monomer" refers to a single TRAIL molecule that includes a domain or region (e.g., the extracellular domain of TRAIL) that is capable of forming a trimer and binding to its receptor (particularly DR4 or DR5 receptor) to induce apoptosis.

[0047] In one embodiment, the TRAIL monomer comprises the extracellular domain of TRAIL, for example, comprising the amino acid sequence at positions 39-281 of SEQ ID NO:1.

[0048] In one embodiment, the TRAIL monomer described herein comprises an amino acid sequence of any one of positions 1-39 to 281 (e.g., positions 39-281) of SEQ ID NO: 1 and has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or even 100% sequence identity with SEQ ID NO: 1. Preferably, the TRAIL monomer is of mammalian or human origin.

[0049] In particular, in one embodiment, the TRAIL monomer comprises the amino acid sequence of any one of positions 1-39 to position 281 of SEQ ID NO: 1, that is, it may comprise positions 1-281, 2-281, 3-281, 4-281, 5-281, 6-281, 7-281, 8-281, 9-281, 10-281, 11-281, 12-281, 13-281, 14-281, 15-281, 16-281, 17-281, 18-281, 19-281, 20-281, 21-281, 22-281, 23-281, 24-281, 25-281, 26-281, 27-281, 28-281, 29-281, 30-31 amino acid sequence at positions 1-281, 22-281, 23-281, 24-281, 25-281, 26-281, 27-281, 28-281, 29-281, 30-281, 31-281, 32-281, 33-281, 34-281, 35-281, 36-281, 37-281, 38-281 or 39-281.

[0050] As described herein, the three TRAIL monomers attached to the DNA origami structure can be the same or different monomers. In one embodiment, the three TRAIL monomers attached to the DNA origami structure are identical monomers, i.e., the amino acid sequences of the monomers are identical, thereby forming a homotrimer. In one embodiment, the three TRAIL monomers attached to the DNA origami structure are different monomers, but all contain the extracellular domain of TRAIL (e.g., amino acids 39-281 of SEQ ID NO: 1), thereby also forming a trimer.

[0051] As described herein, TRAIL monomers can be linked to the DNA origami structure via the C-terminal or N-terminal residue of the TRAIL peptide directly (i.e., the group of the amino acid residue at the C-terminus directly interacts with the group of the site of the DNA origami structure) or indirectly (i.e., the amino acid residue at the C-terminus interacts with the nucleotide residue of the site of the DNA origami structure via an intermediate molecule). In some embodiments, the TRAIL monomer is linked to the DNA origami structure via the C-terminal residue. In some embodiments, the TRAIL monomer is linked to the DNA origami structure via the N-terminal residue.

[0052] Known methods for linking nucleic acids and proteins are categorized as non-covalent and covalent binding, depending on the form of connection. Non-covalent binding methods primarily include: the interaction between avidin (or streptavidin) and biotin, where protein-nucleic acid connection is achieved through the interaction between biotin-modified (or fused) proteins and nucleic acids coupled to avidin (modified biotin); the binding of metal ions to protein recognition domains, such as nickel ions and polyhistidine, where oligohistidine is fused to proteins and nitrilotriacetic acid (NTA) is modified on nucleic acids, achieving protein-nucleic acid connection under the chelation of polyhistidine-nickel ion-NTA; antigen-antibody affinity and protein-nucleic acid aptamers are also important non-covalent binding methods. Covalent binding is mainly achieved through chemical cross-linking, such as using amino groups on the surface of proteins and alkylthio-modified nucleic acids to form covalent bonds under the action of cross-linkers (such as SSMCC); by modifying the mkhkgs short peptide on the protein and the z-qg (N-benzyloxycarbonyl-L-glutaminylglycine, N-carbobenzyloxyglutaminylglycine) on the nucleic acid, covalent binding is produced under the action of glutaminase.

[0053] As used herein, site-linking of a TRAIL monomer to the DNA origami structure refers to linking the TRAIL monomer to the DNA origami structure (e.g., via covalent bonds and / or hydrogen bonds), e.g., directly or through any suitable molecule (e.g., a nucleic acid, a peptide, or both) as an intermediate linker molecule, including but not limited to aptamers such as nucleic acid aptamers.

[0054] Aptamers are known in the art and are generally composed of 25-80 bases. The specific recognition of aptamers with target molecules is based on the ability of oligonucleotides to form various spatial configurations. Aptamers can be directly obtained through in vitro screening, without relying on animal tissues, etc. Herein, the aptamer sequence can be single-stranded or double-stranded.

[0055] For example, in one embodiment, a nucleic acid aptamer that specifically binds to a TRAIL monomer or a peptide fused to a TRAIL monomer can be connected at the desired site of the DNA origami structure to connect the TRAIL monomer to the DNA origami structure. Connecting nucleic acid aptamers at a specific site of the DNA origami structure can be carried out using any suitable method known in the art, for example, a specific short DNA sequence can be designed, which includes the nucleic acid aptamer sequence, and after forming the DNA origami structure, the nucleic acid aptamer sequence does not pair with the scaffold DNA to form a double strand, but exists as a single strand, thereby achieving connection of nucleic acid aptamers at the desired site of the DNA origami structure, and optionally, a complementary sequence of the nucleic acid aptamer sequence can be added to form a double-stranded nucleic acid aptamer sequence.

[0056] In one embodiment, the length of the nucleic acid aptamer that can be used in the present invention can be any suitable length, for example, about 10-50, 10-40 or 10-30 nucleotides, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides.

[0057] In one embodiment, a TRAIL monomer can be fused to a peptide, and then the TRAIL monomer is linked to the DNA origami structure by specifically linking the DNA origami domain to the peptide. The peptide that can be used to fuse to the TRAIL monomer can be any suitable peptide, such as those with specific nucleic acid aptamers.

[0058] The TRAIL monomer molecule described herein can be directly fused with the peptide (i.e., there is no other molecular sequence between the two, forming a TRAIL monomer-peptide or reverse order fusion protein), or can be indirectly fused through a spacer molecule (i.e., forming a TRAIL monomer-spacer-peptide or reverse order fusion protein).

[0059] In one embodiment, an adapter molecule can be attached at a site of the DNA origami structure, and the TRAIL monomer (e.g., at its C-terminus or N-terminus) is fused to a complementary adapter molecule that targets the adapter molecule, thereby connecting the TRAIL monomer to the DNA origami structure through the specific interaction between the adapter molecule and the complementary adapter molecule.

[0060] As used herein, "adaptor molecule" and "reciprocal adaptor molecule" (or "tag" and "reciprocal tag") refer to a pair of components in a binding pair system, wherein each of the pair specifically binds to the other. Examples of binding pair systems include, but are not limited to, Spycatcher / SpyTag, biotin / avidin, and O6-alkylguanine-DNA alkyltransferase (SNAP-tag) / haloalkane dehalogenase (Halo-tag). Binding can be covalent or non-covalent. "Adapter molecule" refers to one of a pair, and "reciprocal adapter molecule" refers to the binding partner of the adapter molecule. Thus, in a Spycatcher / SpyTag system, for example, the adapter molecule can be SpyCatcher and the reciprocal adapter molecule is SpyTag, or the adapter molecule can be SpyTag and the reciprocal adapter molecule is SpyCatcher.

[0061] In some embodiments, the adapter molecule is a SpyCatcher and the cooperating adapter molecule is a SpyTag. In some embodiments, the adapter molecule is a SpyTag and the cooperating adapter molecule is a SpyCatcher.

[0062] In some embodiments, the adapter molecule is biotin and the co-matching adapter molecule is avidin. In some embodiments, the adapter molecule is avidin and the co-matching adapter molecule is biotin.

[0063] In some embodiments, the adapter molecule is an O6-alkylguanine-DNA alkyltransferase (SNAP-tag) and the cooperating adapter molecule is a haloalkane dehalogenase (Halo-tag). In some embodiments, the adapter molecule is a haloalkane dehalogenase (Halo-tag) and the cooperating adapter molecule is an O6-alkylguanine-DNA alkyltransferase (SNAP-tag).

[0064] As used herein, a SpyCatcher peptide is any peptide or variant thereof that is capable of reacting with a SpyTag peptide or variant thereof to form an isopeptide bond linkage (Spy reaction). Various SpyCatcher peptides and variants thereof are known in the art, for example, see Zakeri, B., et al. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences, 109(12), E690-E697; Keeble, A Het al. (2017). Evolving accelerated amidation by SpyTag / SpyCatcher to analyze membrane dynamics. Angewandte Chemie International Edition, 56(52), 16521-16525; Keeble, A Het al. (2019). Approaching infinite affinity through engineering of peptide–protein interaction. Proceedings of the National Academy of Sciences, 116(52), 26523-26533.

[0065] The SpyTag and SpyCatcher systems offer highly specific Spy reactions, are relatively small, and can be produced using conventional recombinant expression techniques (e.g., using E. coli as an expression host). The SpyTag / SpyCatcher system is ideal for binding, labeling, or immobilizing proteins because it creates irreversible peptide linkages. SpyTag reacts with SpyCatcher under a wide range of conditions, and the resulting reaction products are highly stable (Zachari et al., 2012, PNAS vol. 109:12, pp. 690-697).

[0066] The TRAIL monomer molecules described herein can be directly fused to the adapter molecule (e.g., Spycatcher peptide) (i.e., there is no other molecular sequence between the two, forming a TRAIL monomer-adapter molecule or a fusion protein in the reverse order), or can be indirectly fused through a spacer molecule (i.e., forming a TRAIL monomer-spacer-adapter molecule or a fusion protein in the reverse order). Therefore, when referring to TRAIL-Spycatcher fusion proteins herein, it covers fusion proteins formed by direct fusion of TRAIL monomer and Spycatcher peptide in any order (i.e., TRAIL monomer-Spycatcher peptide, or Spycatcher peptide-TRAIL monomer) or indirect fusion (i.e., TRAIL monomer-spacer-Spycatcher peptide, or Spycatcher peptide-spacer-TRAIL monomer).

[0067] The "spacer" described herein can be any suitable spacer known in the art for connecting two polypeptides to form a fusion protein, and refers to a peptide of a certain length composed of amino acids with low hydrophobicity and low charge effect, which, when used in a fusion protein, can fully unfold the connected parts and fully fold into their respective natural conformations without interfering with each other. Commonly used spacers in the art include, for example, flexible GS-type linkers rich in glycine (G) and serine (S); and rigid PT-type linkers rich in proline (P) and threonine (T). Since the GS-type linker has a more suitable amino acid length, is hydrophobic and ductile, and can make the functional protein have better stability and biological activity, the GS-type linker is preferably used in the present invention.

[0068] In some embodiments, a spacer connects the TRAIL monomer to the SPycatcher peptide. In some embodiments, typical amino acid residues for spacers are glycine, serine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, among others. In some embodiments, the spacer region is about 4 to 30 (e.g., about 5-30, 5-25, or 5-20) amino acids long, for example, about 15 amino acids long. Exemplary spacers include, but are not limited to (GGGGS) n , wherein n=1, 2, 3, 4, 5, or 6. In some embodiments, the spacer region is GGGGSGGGGSGGGGS (SEQ ID NO: 4).

[0069] In the fusion proteins described herein, the adapter molecule can be located at the C-terminus or N-terminus of the TRAIL monomer, i.e., the N-terminus of the adapter molecule is directly or indirectly connected to the C-terminus of the TRAIL monomer, or the C-terminus of the adapter molecule is directly or indirectly connected to the N-terminus of the TRAIL monomer. In some embodiments, the N-terminus of the adapter molecule is connected to the C-terminus of the TRAIL monomer.

[0070] Attaching an adapter molecule to the site of the DNA origami structure can be performed using any suitable method known in the art, such as using a linker for connecting nucleic acids and proteins. The linker refers to a chemical moiety capable of covalently linking a protein molecule and a DNA molecule, including, for example, but not limited to, a polymer, a functional group, and the like. Various linkers are known in the art that can achieve this purpose, such as those that react with amino groups of a protein and / or amino groups of a DNA molecule to form a covalent bond, thereby linking the protein and DNA molecules together.

[0071] In one embodiment, the linkers described herein include the following types of linkers: N3 / DBCO; SMCC; SPDP; TCO / Tetrazine and HyNic / 4FB. See Beck A et al., Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov. 2017 May; 16(5): 315-337.

[0072] The "N3 / DBCO" described herein refers to a pair of N-hydroxysuccinimide (NHS) ester-modified linkers, wherein the N3 linker (as shown in the following formula) is NHS ester-modified and can react with the NH2 group on the protein, and the DBCO (as shown in the following formula) is also NHS ester-modified and can react with the modified NH2 on the DNA, and N3 can undergo a ligation reaction with DBCO, thereby connecting the protein and DNA molecules together.

[0073] [Corrected 11.05.2023 in accordance with Article 26]

[0074] "SMCC," as described herein, stands for succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (shown below). SMCC is a heterobifunctional crosslinker with NHS ester and maleimide groups, enabling the covalent coupling of amine- and sulfhydryl-containing molecules. The NHS ester reacts with primary amines at pH 7-9 to form amide bonds, while the maleimide reacts with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds.

[0075] [Corrected 11.05.2023 in accordance with Article 26]

[0076] The "SPDP" described herein is: 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester (as shown in the formula below), which refers to a multifunctional cross-linker (containing a 4-unit polyethylene glycol (PEG) group and a reducible (cleavable) disulfide bond) for protein coupling via amine-amine or amine-thiol cross-linking.

[0077] [Corrected 11.05.2023 in accordance with Article 26]

[0078] The "TCO / tetrazine" described herein, similar to "N3 / DBCO", refers to a pair of NHS ester-modified linkers, wherein the NHS ester-modified TCO linker is a trans-cyclooctene (as shown below), which can be connected to the NH2 of the protein, and the tetrazine is also NHS ester-modified (as shown below), which can be connected to the NH2-modified DNA. The TCO can undergo a ligation reaction with the tetrazine, thereby connecting the protein and DNA molecules together.

[0079] [Corrected 11.05.2023 in accordance with Article 26]

[0080] The "HyNic / 4FB" described herein, similar to "N3 / DBCO", refers to a pair of NHS ester-modified linkers, wherein the HyNic linker is an NHS ester-modified succinimidyl-6-hydrazinonicotinamide (as shown in the formula below), which can be connected to the NH2 of the protein, and 4FB is also an NHS ester-modified 4-formylbenzamide (as shown in the formula below), which can be connected to the NH2-modified DNA. HyNic can react with 4FB to connect the protein and DNA molecules together.

[0081] [Corrected 11.05.2023 in accordance with Article 26]

[0082] Herein, the protein can be connected to any suitable site of the DNA via a linker, for example at the 5' or 3' end, or in the middle of the DNA molecule, preferably at the 5' or 3' end of the DNA. These connections are within the knowledge of those skilled in the art.

[0083] In one embodiment, the DNA-TRAIL complex comprises a fusion protein of three TRAIL monomers and a Spycatcher peptide and a DNA origami structure, wherein the DNA origami structure has Spytag peptides connected to it at three sites, and the fusion protein is connected to the DNA origami structure through the Spy interaction between Spytag and Spycatcher peptide.

[0084] In one embodiment, the N-terminus of the Spycatcher peptide is linked directly or indirectly (eg, via a spacer) to the C-terminus of a TRAIL monomer to form a fusion protein.

[0085] In a further embodiment, the Spycatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 2, and / or the TRAIL monomer comprises the amino acid sequence of positions 39-281 of SEQ ID NO: 1 and an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or even 100% sequence identity with SEQ ID NO: 1, preferably comprises the amino acid sequence of positions 39-281 shown in SEQ ID NO: 1, and more preferably comprises the amino acid sequence of any one of positions 1-39 to position 281 shown in SEQ ID NO: 1.

[0086] In a further embodiment, the spacer comprises the sequence shown in SEQ ID NO:4.

[0087] In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:5.

[0088] In one embodiment, the Spytag peptide is linked to the DNA origami structure via a linker described herein.

[0089] In one embodiment, the azide group-modified SpyTag peptide is connected to a first single-stranded DNA containing DBCO via an N3 / DBCO linker, and a second single-stranded DNA is included at the site of the DNA origami structure, wherein the second single-stranded DNA is complementary to at least part of the sequence, preferably the entire sequence, of the first single-stranded DNA, thereby connecting the Spytag peptide to the DNA origami structure through base pairing of the complementary sequences.

[0090] In one embodiment, the length of the first and second single-stranded DNA can be any suitable length, for example, about 10-50, 10-40, or 10-30 nucleotides, for example, about 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides.

[0091] In particular, the first single-stranded DNA at the three sites may be the same or different, preferably the same. In a further embodiment, the first single-stranded DNA comprises the nucleotide sequence shown in SEQ ID NO: 161, and / or the second single-stranded DNA comprises the nucleotide sequence shown in nucleotides 49-64 of SEQ ID NO: 160.

[0092] In a second aspect, a method for preparing the DNA-TRAIL complex of the first aspect is provided, comprising:

[0093] (a) Provide a DNA origami structure,

[0094] (b) attaching three TRAIL monomers to three sites on the same side of the same plane of the DNA origami structure, wherein the distance between any two sites is about 15-60 nm, preferably about 25-40 nm, and most preferably about 40 nm, and the three sites form an equilateral triangle or a nearly equilateral triangle, and

[0095] (c) The formed DNA-TRAIL complex was harvested.

[0096] The structural features of the DNA origami structure are as described above. Those skilled in the art can prepare DNA origami structures with desired features (e.g., shape, specific sites, or containing specific linkers or nucleotide sequences) according to methods known in the art. See, for example, Paul WK Rothemund, Folding DNA to create nanoscale shapes and patterns, Nature V440: 297-302 (16 March 2006); RP Goodman et al., Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication, Science, V310: 1661-1664 (9 December 2005). For example, a corresponding short DNA sequence can be designed based on the scaffold DNA sequence to generate three or more sites with a desired distance, and TRAIL monomers can be connected to the sites.

[0097] Linking the TRAIL monomer to the desired site of the DNA origami structure can be performed by any suitable means known in the art.

[0098] Alternatively, the TRAIL monomer can be linked to a short DNA strand containing the desired site before forming the DNA origami structure, and then the scaffold DNA and the short DNA strand can be mixed and annealed to link the TRAIL monomer to the desired site of the DNA origami structure. Therefore, the linking of the TRAIL monomer to the desired site of the DNA origami structure can be performed before or after forming the DNA origami structure.

[0099] In one embodiment, the method may include:

[0100] (a) linking a TRAIL monomer to short DNA strands respectively comprising three sites on the same side of a DNA origami structure, wherein the distance between any two sites is about 15-60 nm, preferably about 25-40 nm, most preferably about 40 nm, and the three sites form an equilateral triangle or a nearly equilateral triangle,

[0101] (b) annealing the ligation product of (a), the scaffold DNA, and the short-chain DNA to form a DNA-TRAIL complex, wherein the short-chain DNA is complementary to a portion of the sequence of the scaffold DNA, and

[0102] (c) The DNA-TRAIL complex formed by ligating TRAIL monomers at three sites on the same side of the same face of the DNA origami structure was harvested.

[0103] In one embodiment, linking the TRAIL monomer to the short strand of DNA can be performed by any suitable means known in the art.

[0104] The TRAIL monomer can be linked to the DNA origami structure via the C-terminal or N-terminal residue, i.e., the N-terminus of the TRAIL monomer is directly or indirectly linked to the site of the DNA origami structure, or the C-terminus is directly or indirectly linked to the site of the DNA origami structure. In some embodiments, the C-terminus of the TRAIL monomer is linked to the site of the DNA origami structure.

[0105] In one embodiment, the TRAIL monomer is linked to the desired site of the DNA origami structure through specific binding of an adapter molecule and a cooperating adapter molecule. In one embodiment, the adapter molecule and the cooperating adapter molecule are Spycatcher / SpyTag.

[0106] In one embodiment, a method for preparing the DNA-TRAIL complex of the first aspect is provided, comprising:

[0107] (i) forming a fusion protein between a TRAIL monomer and a Spycatcher peptide, preferably linking the N-terminus of the Spycatcher peptide to the C-terminus of the TRAIL monomer to form a fusion protein, more preferably linking the N-terminus of the Spycatcher peptide to the C-terminus of the TRAIL monomer via a spacer to form a fusion protein,

[0108] (ii) connecting the Spytag peptide to the first single-stranded DNA (ssDNA) via a linker to obtain Spytag-ssDNA, wherein the linker is as described above, preferably, the linker is an N3 / DBCO linker,

[0109] (iii) comprising a second single-stranded DNA at a desired site of the DNA origami structure, wherein the first single-stranded DNA and the second single-stranded DNA are at least partially complementary in sequence,

[0110] (iv) mixing the DNA origami structure of (iii) and the Spytag-ssDNA of (ii) and annealing them to obtain a Spytag-DNA origami structure, and optionally screening and isolating Spytag-DNA origami structures in which Spytag is attached to three sites on the same side of the same surface of the DNA origami structure, and

[0111] (v) mixing the fusion protein of (i) with the Spytag-DNA origami structure of (iv) to obtain a DNA-TRAIL complex, and optionally screening and isolating the DNA-TRAIL complex in which the fusion protein is linked to three sites on the same side of the same surface of the DNA origami structure.

[0112] In one embodiment, the DNA origami structure is a planar DNA origami structure, which can be in any two-dimensional shape, such as a regular shape such as a triangle or a quadrilateral, or other irregular shapes. In one embodiment, the DNA origami structure is a planar rectangular DNA origami structure, with a side length of, for example, about 60-90 nm, such as about 60×90 nm.

[0113] In one embodiment, the TRAIL monomer comprises the amino acid sequence of positions 39-281 of SEQ ID NO: 1 and an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or even 100% sequence identity with SEQ ID NO: 1, preferably comprises the amino acid sequence of positions 39-281 as shown in SEQ ID NO: 1, and more preferably comprises the amino acid sequence of any one of positions 1-39 to position 281 as shown in SEQ ID NO: 1.

[0114] In one embodiment, the three TRAIL monomers attached to the DNA origami structure are identical monomers, ie, the amino acid sequences of the monomers are identical.

[0115] In one embodiment, the Spytag comprises the amino acid sequence of SEQ ID NO: 3, and / or the Spycatcher peptide comprises the amino acid sequence of SEQ ID NO: 2.

[0116] In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:5.

[0117] In one embodiment, the length of the first and second single-stranded DNA can be any suitable length, such as about 10-50, 10-40, or 10-30 nucleotides, such as about 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides.

[0118] The first DNA strands may be identical or different, preferably identical. In one embodiment, the first DNA strand comprises the nucleotide sequence shown in SEQ ID NO: 161; and / or the second DNA strand comprises the nucleotide sequence shown in nucleotides 49-64 of SEQ ID NO: 160.

[0119] In a specific embodiment, there is provided a method for preparing the DNA-TRAIL complex of the first aspect, comprising:

[0120] (i) connecting the Spytag peptide to the complementary DNA chain via a linker to obtain Spytag-ssDNA, wherein the linker is as described above, preferably, the linker is an N3 / DBCO linker,

[0121] (ii) mixing and annealing the scaffold DNA, the staple DNA, and the three capture DNA chains to form a DNA origami structure, wherein the staple DNA is complementary to a partial sequence of the scaffold DNA, the capture DNA chain comprises a partial sequence complementary to the scaffold DNA and a capture sequence at least partially complementary to the complementary DNA chain, wherein the three capture DNA chains are on the same surface of the DNA origami structure, and the three sites connected to the DNA origami structure are about 15-60 nm, preferably about 25-40 nm, and most preferably about 40 nm apart, forming an equilateral triangle or a nearly equilateral triangle.

[0122] (iii) mixing the DNA origami structure obtained in (ii) with the Spytag-ssDNA obtained in (i), and connecting the Spytag peptide to the DNA origami structure through base pairing between the capture DNA chain and the complementary DNA chain, thereby forming a Spytag-DNA origami structure.

[0123] (iv) forming a fusion protein between the TRAIL monomer and the Spycatcher peptide, preferably linking the N-terminus of the Spycatcher peptide to the C-terminus of the TRAIL monomer to form the fusion protein, more preferably linking the N-terminus of the Spycatcher peptide to the C-terminus of the TRAIL monomer via a spacer to form the fusion protein, and

[0124] (v) mixing the fusion protein obtained in (iv) with the Spytag-DNA origami structure in (iii) to obtain a DNA-TRAIL complex, and optionally screening and isolating the DNA-TRAIL complex in which the fusion protein is linked to three sites on the same side of the same surface of the DNA origami structure.

[0125] In one embodiment, the scaffold DNA comprises the genome sequence of M13p18 phage, such as shown in SEQ ID NO:6.

[0126] In one embodiment, the short DNA strand (Staple strand) comprises the sequence shown in SEQ ID NO: 7-149.

[0127] In one embodiment, the three capture DNA strands comprise a nucleic acid sequence selected from any one of the following (a)-(e), preferably (c):

[0128] (a) SEQ ID NO: 152, 155, 158;

[0129] (b) SEQ ID NO: 151, 154, 158;

[0130] (c) SEQ ID NO: 151, 153, 159;

[0131] (d) SEQ ID NO: 151, 156, 160; and

[0132] (e) SEQ ID NO: 150, 157, 160.

[0133] As described herein, the site where the capture DNA strand is connected to the DNA origami structure refers to the site where the sequence portion of the capture DNA strand that is complementary to the scaffold DNA strand and the sequence portion that is not complementary to the scaffold DNA strand are connected. This site is a residue in the sequence that is complementary to the scaffold DNA strand. For example, if the sequence of the capture DNA strand is N1N2N3N4N5N6N7N8N9N 10 , where N1N2N3N4N5 is complementary to the scaffold DNA, and N6N7N8N9N 10 If it is not complementary to the scaffold DNA, the site where the capture DNA chain is connected to the DNA origami structure refers to residue N5.

[0134] In the methods provided herein, unless it is explicitly stated or it can be determined from the context that a certain step needs to be performed after another step (for example, one step needs to be performed based on the completion of another step, such as using the product or result produced by another step), the order of the steps can be changed or performed simultaneously, such as providing a DNA origami structure and preparing a TRAIL fusion protein.

[0135] In a third aspect, a kit is provided, comprising:

[0136] a DNA origami structure having cooperating adaptor molecules attached at three sites on the same side of the same face, wherein the distance between any two sites is about 15-60 nm, preferably about 25-40 nm, most preferably about 40 nm, and the three sites form an equilateral triangle or a nearly equilateral triangle, and

[0137] -TRAIL monomer and adapter molecule fusion protein.

[0138] In one embodiment, the cooperating adaptor molecule is connected to the DNA origami structure via a linker as described herein.

[0139] In one embodiment, the mutual matching adapter molecule is connected to the first DNA single strand through a linker, and the three sites of the DNA origami structure have a second DNA single strand, and the second DNA single strand has a complementary sequence to the first DNA single strand, so that the mutual matching adapter molecule is connected to the DNA origami structure through base pairing of the complementary sequences of the first DNA single strand and the second DNA single strand.

[0140] In one embodiment, the length of the first and second single-stranded DNA can be any suitable length, for example, about 10-50, 10-40, or 10-30 nucleotides, for example, about 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides.

[0141] In one embodiment, the DNA origami structure is a planar DNA origami structure, which can be in any two-dimensional shape, such as a regular shape such as a triangle or a quadrilateral, or other irregular shapes. In one embodiment, the DNA origami structure is a planar rectangular DNA origami structure, with a side length of, for example, about 60-90 nm, such as about 60×90 nm.

[0142] In one embodiment, the TRAIL monomer comprises the amino acid sequence of positions 39-281 of SEQ ID NO: 1 and an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or even 100% sequence identity with SEQ ID NO: 1, preferably comprises the amino acid sequence of positions 39-281 as shown in SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1, more preferably comprises the amino acid sequence of any one of positions 1-39 to position 281 as shown in SEQ ID NO: 1.

[0143] In one embodiment, the three TRAIL monomers attached to the DNA origami structure are identical monomers, ie, the amino acid sequences of the monomers are identical.

[0144] In one embodiment, the Spytag comprises the amino acid sequence of SEQ ID NO: 3, and / or the Spycatcher peptide comprises the amino acid sequence of SEQ ID NO: 2.

[0145] In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:5.

[0146] The first DNA strands may be identical or different, preferably identical. In one embodiment, the first DNA strand comprises the nucleotide sequence set forth in SEQ ID NO: 161; and / or the second DNA strand comprises the nucleotide sequence set forth in nucleotides 49-64 of SEQ ID NO: 160.

[0147] In one embodiment, a kit is provided, comprising:

[0148] A DNA origami structure formed by a DNA sequence comprising SEQ ID NO: 6, a short DNA strand comprising SEQ ID NOs: 7-149, and three capture DNA strands selected from the following, preferably (c), and a Spytag peptide linked to the DNA sequence represented by SEQ ID NO: 161 via a linker (as described above), preferably an N3 / DBCO linker, wherein the Spytag peptide is linked to the DNA origami structure through base complementarity:

[0149] (a) SEQ ID NO: 152, 155, 158;

[0150] (b) SEQ ID NO: 151, 154, 158;

[0151] (c) SEQ ID NO: 151, 153, 159;

[0152] (d) SEQ ID NO: 151, 156, 160; and

[0153] (e) SEQ ID NO: 150, 157, 160,

[0154] - A fusion protein of a TRAIL monomer and a Spycatcher peptide, preferably comprising the amino acid sequence of SEQ ID NO: 5.

[0155] In one embodiment, the kit is for use in treating a tumor in a mammal, such as a human.

[0156] The kit of the present invention may also include a container, label, and / or instructions. Suitable containers include, for example, bottles, vials, syringes, and the like. The label or instructions indicate that the DNA-TRAIL complex of the present invention is prepared by mixing the active ingredients in the kit for use in treating a specific condition and contain information regarding the indications, usage, dosage, administration, contraindications, and / or precautions for use of the product. In one embodiment, the instructions indicate that the DNA-TRAIL complex or pharmaceutical composition is used to treat tumors (e.g., tumors targeting TRAIL), such as solid tumors.

[0157] In a fourth aspect, a pharmaceutical composition is provided, which comprises the DNA-TRAIL complex described in the present invention, the DNA-TRAIL complex obtained according to the method for preparing the DNA-TRAIL complex described in the present invention, or the DNA-TRAIL complex prepared using the kit of the third aspect, and a pharmaceutically acceptable carrier.

[0158] A "pharmaceutically acceptable carrier" refers to a substance that facilitates administration and absorption of an active substance into a subject and can be included in the compositions of the present invention without causing significant toxic side effects in the patient. Pharmaceutically acceptable carriers (vehicles) suitable for use in the present invention are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, ed. Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st edition (2005) describes compositions and formulations suitable for drug delivery.

[0159] Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and coloring agents, etc. Those skilled in the art will appreciate that other pharmaceutical carriers may be used in the present invention.

[0160] As used herein, a "pharmaceutical composition" refers to a pharmaceutical product formulated to meet specific dosage requirements for the treatment or prevention of a disease and intended for use by a subject. The pharmaceutical composition may contain the DNA-TRAIL complex described herein, as well as other pharmaceutical excipients and tools.

[0161] The "excipients" mentioned in this article are pharmaceutical excipients, which refer to substances other than active ingredients used in the production of drugs and the preparation of prescriptions, which have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. In addition to their purpose of excipients, serving as carriers or improving stability, they can also have important functions such as solubilization, dissolution assistance, and sustained-release.

[0162] The medicament or pharmaceutical composition herein can be formulated into a dosage form suitable for any suitable administration route, such as intravenous, subcutaneous, parenteral, oral, intraperitoneal, etc., such as tablets, powders, solutions, etc. In one embodiment, the medicament or pharmaceutical composition of the present invention can be formulated into a form suitable for intravenous administration.

[0163] In the fifth aspect, provided is the use of the DNA-TRAIL complex of the present invention, the DNA-TRAIL complex obtained according to the method for preparing a DNA-TRAIL complex of the present invention, or the DNA-TRAIL complex prepared using the kit of the third aspect in the preparation of a medicament for treating tumors (e.g., tumors with TRAIL as a target), preferably solid tumors; or the DNA-TRAIL complex of the present invention or the DNA-TRAIL complex obtained according to the method for preparing a DNA-TRAIL complex of the present invention for treating tumors (e.g., tumors with TRAIL as a target), preferably solid tumors.

[0164] In the sixth aspect, a method for treating a tumor in a subject (e.g., a tumor targeting TRAIL) is provided, comprising administering to the subject a therapeutically effective amount of the DNA-TRAIL complex of the first aspect, the DNA-TRAIL complex obtained according to the method of the second aspect, the DNA-TRAIL complex formulated using the kit of the third aspect, or the pharmaceutical composition of the fourth aspect.

[0165] As used herein, a "patient" or "subject" refers to an organism that has or is susceptible to a disease or condition that can be treated by administering a DNA-TRAIL complex or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient or subject is a human.

[0166] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a medicament, compound, or material in a dosage formulation that is at least sufficient to produce a therapeutic effect in a subject. The exact amount depends on the purpose of the treatment and can be determined by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0167] The DNA-TRAIL complexes, kits, pharmaceutical compositions, uses and methods provided herein are suitable for treating all types of tumors targeting TRAIL, including cancers, particularly solid tumors. In one embodiment, the solid tumor is selected from, for example, gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, renal cell carcinoma, esophageal cancer and prostate cancer. In one embodiment, the tumor is selected from breast cancer, colon cancer, liver cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer or esophageal cancer.

[0168] The present invention provides a DNA origami-based trimeric TRAIL monomer. Combining the "engraving printing" strategy of click chemistry and peptide glue technology, three TRAIL monomers are patterned on the surface of a planar rectangular DNA origami, and the spacing is controlled between 15 and 60 nanometers. Compared with other DNA-TRAIL3 trimers, they produce superior effects in terms of receptor affinity, agonist activity, and cytotoxicity against tumor cells. In particular, the DNA-TRAIL3 trimer with a 40nm spacing exhibits the best behavior and demonstrates excellent antitumor activity and safety in animal experiments. Finally, a hypothetical "active unit" model is proposed to explain the DR aggregation induced by DNA-TRAIL3 trimers. This study provides a new approach for constructing TRAIL trimers to enhance their agonist activity, which may promote the clinical application of TRAIL-based therapies.

[0169] The word "or" is intended to include "and" unless the context indicates otherwise.

[0170] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist.

[0171] As used herein, the term "about" refers to a range of values ​​that include the specific value and that one skilled in the art would reasonably consider to be similar to the specific value. In certain embodiments, the term "about" refers to within the standard error of measurement using commonly accepted methods in the art. For example, in certain embodiments, about refers to + / - 10% or 5% of the specific value.

[0172] As used herein, when a specific value or ratio is listed for a feature in the specification, a range consisting of any two of the values ​​or ratios is also included. For example, when the values ​​1, 2, 3, and 4 are listed, 1-2, 1-3, 1-4, 2-3, 2-4, and 3-4 are also included. DETAILED DESCRIPTION

[0173] The following examples further illustrate the specific embodiments of the present invention, but the embodiments of the present invention are not limited thereto. The methods used in the following examples are conventional methods unless otherwise specified.

[0174] Example 1: Preparation of DNA-TRAIL3 trimer

[0175] (1) Preparation of TRAIL-SpyCatcher protein

[0176] The extracellular domain of TRAIL (amino acids 39-281 of SEQ ID NO: 1) was fused to the SpyCatcher peptide (SEQ ID NO: 2) via a spacer (SEQ ID NO: 4) to form a TRAIL-SpyCatcher fusion protein (SEQ ID NO: 5). The DNA fragment encoding the TRAIL-SpyCatcher fusion protein was inserted into the expression plasmid pET32a (Wuhan Jinkairui Biotechnology Co., Ltd.) with an N-terminal 6×His tag, and protein expression was induced in Escherichia coli BL21(DE3) by adding 0.5 mM iptgispropyl-β-d-thiogalactopyranoside. The TRAIL-SpyCatcher protein was first purified using a Ni-NTA column (GE Healthcare), and the eluted target protein was then treated with HRV 3C protease to remove the His tag. The mixture was then subjected to a second Ni-NTA column to remove the His tag, and the untagged protein was eluted in an imidazole-free buffer, followed by further purification by Hitrap Q HP (GE Healthcare) ion exchange chromatography. The purified protein was quality controlled for concentration and UV degradation and then stored at -80°C until further use.

[0177] (2) Preparation of DNA-TRAIL3 trimer

[0178] Rectangular DNA origami was assembled using the Rothemund method. Briefly, the genomic DNA of M13p18 phage (SEQ ID NO: 6), multiple staple chains (SEQ ID NO: 7-149) and capture chains (SEQ ID NO: 150-160) were mixed at a molar ratio of 1:5:5. 2+ DNA origami were assembled by annealing the DNA mixture from 95°C to 25°C in a buffer (Tris, 40 mM; acetic acid, 20 mM; EDTA, 2 mM; magnesium acetate, 12.5 mM; pH 8.0). The assembled DNA origami were purified using a filtration device (100 kDa MWCO, Amicon, Millipore, Germany) to remove excess short / capture strands.

[0179] The SpyTag peptide (SEQ ID NO: 3) with an azide group modification at the N-terminus was coupled to a complementary chain (SEQ ID NO: 161) containing DBCO by click chemistry reaction. The azide group-modified SpyTag peptide and the complementary chain containing DBCO were dissolved in phosphate buffered saline (PBS, pH 7.4), respectively. Then, 50 μl of SpyTag solution (100 μM) was mixed with 50 μl of complementary chain solution (100 μM). The mixed solution was gently stirred overnight at room temperature, and the product was purified using a 3 KDa centrifugal filter. The obtained SpyTag complementary chain conjugate was stored at -20 ° C for further use.

[0180] The SpyTag complementary strand conjugate was mixed with a rectangular DNA origami (with the corresponding capture strand) in TAE-Mg2+ buffer at a molar ratio of 5:1. The mixture was annealed from 45°C to 25°C for six cycles. The SpyTag-engraved DNA origami was purified by passing through a 100 kDa centrifugal filter to remove excess material. The SpyTag-engraved DNA origami and TRAIL-SpyCatcher protein were then mixed in a molar ratio of 1:5 in TAE-Mg2+ buffer for 2 hours, and the TRAIL-SpyCatcher protein was printed on the DNA origami.

[0181] Single-stranded genomic DNA of the M13mp18 phage was purchased from New England Biolabs (cat. no. 4040S, Beijing, China). Short and capture strands were synthesized by Rui Biotech (Beijing, China). Azide-modified SpyTag peptides and DBCO-containing complementary strands were purchased from Qiangyao Biotech (Shanghai, China). All reagents were kept at −20°C until use.

[0182] (3) AFM characterization

[0183] For DNA origami characterization using AFM, 10 μl of sample (5 nM) was deposited on freshly cleaved mica and allowed to adsorb on the surface for 5 minutes. AFM imaging of DNA origami was performed in ScanAsyst mode (Multimode-8, Bruker, Germany). Images were collected and processed using Bruker NanoScope Analysis 1.9 software. At least three independent preparations of each sample were observed by AFM, and several images were collected from different areas of the mica surface (typically 3 μm × 3 μm). The yield of DNA-TRAIL3 trimers was calculated as the percentage of DNA origami that carried different numbers of TRAIL monomers at the expected positions.

[0184] (4) SPR analysis

[0185] The sensitivity of the optimized SPR sensor was checked by analyzing solutions of TRAIL preparations of different concentrations in 10mM PBS at pH 7.4. The surface of DR4 was exposed to 10mM PBS at pH 7.4 until a stable SPR angle was established, and then different TRAIL formulations were injected into the SPR reaction cup measurement channel. The formation of the DR4 / TRAIL immune complex resulted in an increase in the SPR angle. PBS buffer was injected at the end of the binding phase, resulting in partial dissociation of the generated immune complex. Finally, DR4 / TRAIL was treated with a regeneration solution of 10mM glycine / HCl (pH 2.0) and 10mM PBS (pH 7.4) to restore the baseline. A Biacore T200 instrument (GE Healthcare, USA) was used for the SPR program at 25°C. KD values ​​were determined using Biacore T200 evaluation software 3.2.0.5 (GE Healthcare, USA).

[0186] result

[0187] Design and characterization of DNA-TRAIL3 trimers with different ligand spacing

[0188] We used Cadnano software to help design rectangular DNA origami as a display scaffold for TRAIL trimers [Douglas SM, et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 2009; 37(15): 5001-5006]. By slowly annealing the genomic DNA of M13mp18 phage as a template chain with multiple short chains and capture chains, rectangular DNA origami was assembled (Figure 1A). Then, a "sculpture printing" strategy combining click chemistry and peptide glue technology was used to decorate the three TRAIL monomers onto the DNA origami (Figure 1A).

[0189] In short, the click chemistry reaction is responsible for the connection between the labeled SpyTag peptide modified with an azide group and the complementary chain containing dibenzocyclopentane (DBCO). The complementary chain is complementary to the capture chain, and then the position-specific engraving of the SpyTag peptide is achieved on the DNA origami through DNA complementary hybridization between the complementary chain and the pre-designed capture chain. The peptide glue technology is responsible for the connection between the TRAIL monomer and the engraved SpyTag peptide. SpyCatcher is fused to the C-terminus of the extracellular domain of the TRAIL (TRAIL-SpyCatcher) monomer, and three TRAIL-SpyCatcher monomers are printed on the DNA origami, which is pre-engraved with SpyTag peptides to form a DNA-TRAIL3 trimer (Figure 1A). Site-specific binding based on peptide glue technology ensures the uniform outward orientation of all TRAIL monomers on the DNA origami.

[0190] By varying the position of the capture strands, the TRAIL monomers were positioned on the DNA origami at varying interligand distances, including approximately 15 nm (DNA-TRAIL3-15), 25 nm (DNA-TRAIL3-25), 40 nm (DNA_TRAIL3-40), 55 nm (DNA.TRAIL3-55), and 60 nm (DNA / TRAIL3-60) ( Figure 1B ). As shown in Figure 1C , atomic force microscopy (AFM) observation of these DNA-TRAIL3 trimers revealed three distinct bright spots at the pre-designed locations on the DNA origami. The height of these spots was approximately 1.5 nm higher than that of the exposed locations on the DNA origami, approaching the diameter of the 42 kDa TRAIL-SpyCatcher protein ( Figure 1D ), indicating successful protein modification. Due to the mild conditions and rapid reaction characteristics of click chemistry and peptide gel technology, the assembly efficiency of these DNA-TRAIL3 trimers reached over 85% (Figure 1E), with the assembly efficiency of DNA-TRAIL3-15, DNA-TRAIL2-25, DNA-TRAIL3-40, DNA-TRAIL3-55 and DNA-TRAIL3-60 being 86.03%, 91.61%, 94.81%, 86.81% and 93.49%, respectively, and the process was limited to 2 hours. The DNA-TRAIL3 trimers prepared in Example 1 were used in the following examples.

[0191] Example 2: In vitro experiments

[0192] (1) Cell survival assay

[0193] The following cell lines were purchased from Co., Ltd. (Guangzhou, China): COLO205, HeLa, and NCI-H460. Cells were used only after authentication and tested negative for mycoplasma contamination. All cell lines were cultured in Dulbecco's modified Eagle's medium (cat. no. #06-1055-57-1A, Biological Industries, Israel) or RPMI1640 medium (cat. no. #01-100-1A, Biological Industries, Israel) containing 10% heat-inactivated fetal bovine serum (cat. no. 10270-106, FBS, Gibco, USA). 100 U / ml penicillin and 100 mg / ml streptomycin were added and incubated at 37°C in a humidified atmosphere with 5% CO2. Experimental cells (1×10 4 TRAIL was then treated with different TRAIL preparations at 10 nM TRAIL concentration for 24 hours. Cell viability was assessed by CCK-8 assay (Cat. No. CK04-500, Dojindo Laboratories, Japan) according to the manufacturer's instructions. For DR4 / DR5 blocking, 10 μg / ml soluble DR4 neutralizing antibody (Cat. No. AF347, R&D Systems, USA) and / or 10 μg / ml soluble DR5 neutralizing antibody (Cat. No. NBP2-80066, Novus Biologicals, USA) were added to the cells 1 hour before TRAIL treatment.

[0194] (2) Apoptosis assay

[0195] 1×10 6 The cells were collected and treated with different TRAIL preparations of 10 nM TRAIL concentration for 6 hours. Then, according to the manufacturer's instructions, cells were harvested and dyed with Annexin V-FITC apoptosis detection kit (catalog number (Cat. No.) 556547, BD biosciences, Germany), and then analyzed by flow cytometry. The percentage of early apoptotic cells (Annexin V+ / PI-) in the total cells was calculated.

[0196] (3) Western blotting

[0197] The cells were collected and washed three times with cold PBS, followed by addition of appropriate RIPA lysis buffer (Catalog #R0010, Solarbio, China), protease inhibitors, and PMSF (Catalog #ST507, Beyotime, China) on ice for 30 minutes. The supernatant was then collected by centrifugation at 12,000 rpm for 10 minutes at 4°C. 15 μg of total protein from each sample was loaded into each well, separated by SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% bovine serum albumin for 1 hour at room temperature and then incubated with the primary antibody overnight at 4°C. The membrane was washed four times and incubated with the secondary antibody for 1 hour at room temperature. Signals were detected using Omni ECL™ (Catalog #SQ101L, Epizyme, USA), and images were acquired using Image Lab software. The following antibodies were used: Caspase-8 antibody (Catalog #T55750S) and Caspase-3 antibody (Catalog #TD6879S) from Abmart (Shanghai, China).

[0198] result

[0199] Cytotoxicity and apoptosis induced by DNA-TRAIL3 trimer in vitro

[0200] After 24 hours of treatment, the cytotoxicity of naked DNA origami, native TRAIL monomer, DNA origami modified with a TRAIL monomer (DNA-TRAIL), and these DNA-TRAIL3 trimers were evaluated in COLO205 (human colon carcinoma), HeLa (human cervical carcinoma), and NCI-H460 (human hepatocellular carcinoma) cells (Figure 2A). Naked DNA origami showed no significant cytotoxicity, while DNA-TRAIL monomer showed cytotoxicity comparable to that of native TRAIL monomer (Figure 2B-D). Compared to native TRAIL monomer, DNA-TRAIL3-25 and DNA-TRAIL3-40 showed greater cytotoxicity in these cancer cells, with DNA-TRAIL3-40 being the best candidate (Figure 2B-D).

[0201] TRAIL induces cytotoxicity primarily through apoptosis [Ashkenazi A. Targeting death and decoy receptors of the tumor-necrosis factor superfamily. Nat Rev Cancer. 2002; 2(6): 420-430], and thus apoptosis was detected in these cancer cells treated with the above-mentioned drugs (Figure 3A). Cells were stained with the Annexin V-FITC apoptosis detection kit, and Annexin V+PI- cells were defined as early apoptotic cells by flow cytometry. Compared with treatment with native TRAIL monomer or DNA-TRAIL monomer, treatment with these DNA-TRAIL3 trimers for 6 hours induced more early apoptotic cells in the three cell lines (Figure 3B). More importantly, these trimers behaved differently, with DNA-TRAIL3-40 inducing the most apoptosis (Figure 3B).

[0202] However, these DNA-TRAIL3 trimers still did not induce significant apoptosis in WRL68 cells, which are normal human hepatocytes, indicating that they are specific in killing cancer cells (Figure 3C). The results show that trimerization using DNA origami is a promising approach to enhance the tumor cell-killing ability of TRAIL monomers, and that the inter-ligand distance is an important parameter in pattern design.

[0203] Receptor binding affinity of DNA-TRAIL3 trimer

[0204] DR4 is one of the functional receptors of TRAIL and initiates the extrinsic apoptosis signaling pathway when activated [Greenlee JD, et al. Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization. Elife. 2021; 10: e67750]; Therefore, we used surface plasmon resonance (SPR) to examine the binding kinetics of DNA-TRAIL3 trimers to soluble DR4 protein (see Table 1). It was observed that the native TRAIL monomer showed a binding affinity to DR4 at the nanomolar level (KD = 8.84 × 10 -7 M). DNA-TRAIL monomers showed similar receptor binding affinity to natural TRAIL monomers (KD = 4.25 × 10 -7M), indicating that the decoration on the DNA origami does not affect receptor binding. As expected, the receptor binding affinity of DNA-TRAIL3 trimers is 2-3 orders of magnitude higher than that of DNA-TRAIL monomers, with DNA-TRAIL3-40 having the highest affinity (KD = 2.03 × 10 -10 M). These results suggest that the multivalent assembly of TRAIL trimers on DNA origami can enhance the affinity for receptors, and the inter-ligand distance can significantly affect this binding affinity, which may be the potential reason for the different cytotoxicity of these DNA-TRAIL3 trimers. Interestingly, DNA-TRAIL3-40 (Kon = 1.33 × 10 9 The binding rate of Ms-1 to DR4 is the same as that of natural TRAIL monomer (Kon = 6.18 × 10 3 The dissociation rate of DNA-TRAIL3-40 from DR4 was 21500 times that of Ms-1, while the dissociation rate of DNA-TRAIL3-40 from DR4 was 2.71×10 -1 s -1 ) is also higher than that of natural TRAIL monomer (Koff = 5.47 × 10 -3 s -1 ) was 50 times faster (Table 1), indicating that the affinity between DNA-TRAIL3-40 and DR4 is a “fast association-fast dissociation” mode.

[0205] Table 1: Affinity and rate constants of DNA-TRAIL3 trimer binding to DR4

[0206]

[0207] DNA-TRAIL3 trimer-induced signaling activation in vitro

[0208] As previously mentioned, DR aggregation is crucial for efficient activation of downstream signaling. Binding of TRAIL or agonistic antibodies to death receptors initiates a caspase-mediated apoptotic signaling cascade, leading to cell death. During this process, Pro-casase-8 (Pro-cas-8) and Pro-casase-3 (Pro-cas-3) are cleaved into active caspase-8 (P43 / 41-cas-8, P18-cas-8, and P19-cas-3) and active caspase-3 (P19-cas3 and P17-cas-3), respectively, which can be detected by Western blotting. As shown in Figures 4B-C, different TRAIL preparations all induced increased expression of active caspase-8 or active caspase-3, but the effect of DNA-TRAIL3 trimers was significantly stronger than that of native TRAIL or DNA-TRAIL monomers. Next, soluble DR4-neutralizing antibodies (anti-DR4) and DR5-neutralizing antibodies (anti-DR5) were used to determine the role of DR4 and DR5 in the cytotoxicity of DNA-TRAIL3 trimers. As shown in Figure 4D, the cytotoxicity of native TRAIL monomer in COLO205, Hela, and NCI-H460 cells was partially inhibited by anti-DR4 or anti-DR5, and completely blocked by the combination of the two antagonists. A similar cytotoxicity blockade was also observed in the DNA-TRAIL3-40 group (Figure 4D).

[0209] These results suggest that although trimerization using DNA origami enhances the cytotoxicity of TRAIL monomers, it does not alter the receptor pathways leading to cytotoxicity, with both DR4 and DR5 playing key roles.

[0210] Example 3: Animal Experiment

[0211] All animal experiments were performed in accordance with animal use protocols approved by the Animal Experimentation Ethics Committee and the Institutional Animal Care and Use Committee of the National Center for Nanoscience and Technology. For the evaluation of antitumor effects, female BALB / c nu / nu mice (7 weeks old) were obtained from Vital River Laboratories (Beijing, China).

[0212] Human colon cancer cells (COLO205; 5×10 6 ) were implanted subcutaneously on the flank of each mouse. Tumor growth was monitored every other day, and tumor volume was calculated by the following formula: Tumor volume (mm3) = length × width 2 ×0.5. When the tumor grows to an average size of about 100 mm 3 The mice were randomly divided into 6 groups (6 mice in each group) and injected intravenously with different TRAIL preparations at a dose of 30 mg / kg TRAIL every day for 8 days.

[0213] To perform pharmacokinetic evaluation, female BALB / c nu / nu mice (7 weeks old, 3 mice per group) received intravenous injections of different TRAIL formulations at 30 mg / kg TRAIL. Blood samples were taken from the tail at time intervals of 5, 10, 30, 60, 120, 240, and 360 minutes and incubated on ice. Serum samples were collected by centrifugation at 3000 × g for 20 minutes at 4°C. TRAIL protein concentrations in serum samples were determined by ELISA (Cat. No. AB-J0958A, Abmart, Shanghai, China) according to the manufacturer's instructions. For data calculation, the relative values ​​of serum concentrations were analyzed, and the first value (5 minutes) was set to 100%. For biodistribution determination, subcutaneous COLO205 tumor-bearing female BALB / c nu / nu mice (7 weeks old, n=3 mice per group) received intravenous injections of different TRAIL formulations at 30 mg / kg TRAIL. At 12 and 24 hour intervals, kidney, liver, tumor and spleen tissues were removed from the animals and immediately frozen in liquid nitrogen. All tissues were dissected and weighed, placed in PBS, and 15 mM protease inhibitor PMSF (1 mg in 10 μl) was added on ice. The samples were homogenized and centrifuged at 14,000 rpm for 15 minutes, and the supernatant was aspirated for ELISA analysis of TRAIL protein concentration.

[0214] For safety assessment, female BALB / c nu / nu mice (7 weeks old, 3 per group) received intravenous injection of different TRAIL formulations (30 mg / kg TRAIL). 24 hours later, blood samples were collected for serum biochemistry tests.

[0215] result

[0216] In vivo antitumor effects, pharmacokinetics and biosafety of DNA-TRAIL3 trimer

[0217] To evaluate the antitumor effect of DNA-TRAIL3 trimer in vivo, nude mice bearing COLO205 xenografts were established by subcutaneous inoculation of tumor cells. 3 At the average size of 100 mice, mice were randomly divided into six groups and received daily treatment for 8 days, including saline as a control, naked DNA origami, natural TRAIL monomer, DNA-TRAIL3-25, and DNA-TRAIL3-40 trimers. Naked DNA origami treatment did not inhibit tumor growth, while treatment with natural TRAIL or DNA-TRAIL monomer significantly reduced tumor growth rate (Figure 5A).

[0218] However, consistent with the results of the in vitro cytotoxicity experiments, DNA-TRAIL3-25 and DNA-TRAIL3-40 trimers showed superior anti-tumor efficiency than natural TRAIL or DNA-TRAIL monomers (Figure 5A). At the experimental endpoint, the tumor weight of the DNA-TRAIL3-40 trimer group was the lowest (Figure 5B-C). In order to analyze the pharmacokinetic characteristics in vivo, serum samples were collected 5, 10, 30, 60, 120, 240 and 360 minutes after a single intravenous injection of natural TRAIL monomer, DNA-TRAIL monomer or DNA-TRAIL3-40 trimer in nude mice. The TRAIL protein concentration in the serum samples was measured by enzyme-linked immunosorbent assay (ELISA), and the concentration 5 minutes after injection was set as the baseline. As shown in Figure 5D, the circulation half-life of DNA-TRAIL monomer (24.08 minutes) or DNA-TRAIL3-40 trimer (29.66 minutes) was longer than that of natural TRAIL monomer (9.27 minutes). Thus, decoration on the DNA origami prolonged the retention of TRAIL in the bloodstream, which could positively affect therapeutic activity.

[0219] The biodistribution of native TRAIL monomer, DNA-TRAIL monomer and DNA-TRAIL3-40 trimer in tumor-bearing mice after intravenous injection was also analyzed. The results showed that more DNA-TRAIL3-40 trimer accumulated in tumor tissue compared with native TRAIL monomer and DNA-TRAIL monomer (Figure 5E), which may be due to the long circulation half-life of DNA-TRAIL3-40 trimer and its high binding affinity to DR4 / DR5 on tumor cells. In addition, TRAIL preparations seemed to be concentrated in the liver and kidneys, which is reasonable to expect because they are reported to be the main organs for TRAIL clearance [Kelley SK, et al. Preclinical studies to predict the disposition of Apo2L / tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety. J Pharmacol Exp Ther. 2001; 299(1): 31-38]. However, accumulation of TRAIL preparations in the liver and kidneys did not result in functional organ damage ( FIG. 6 ).

[0220] Statistical analysis

[0221] Data are expressed as mean ± standard deviation (SD). Multiple groups were compared using one-way analysis of variance (ANOVA) and Tukey's post hoc test. P < 0.05 was considered statistically significant.

Claims

1. A DNA-TRAIL complex, comprising a DNA origami structure and three TRAIL monomers forming a TRAIL trimer, wherein the three TRAIL monomers are respectively connected to three sites on the same side of the same surface of the DNA origami structure and any two sites are about 15-60 nm, preferably about 25-40 nm, most preferably about 40 nm apart, and the three sites form an equilateral triangle or a nearly equilateral triangle, wherein the C-terminus or N-terminus of the TRAIL monomer is directly or indirectly connected to the DNA origami structure, and preferably, the TRAIL monomers are the same.

2. The DNA-TRAIL complex of claim 1, comprising: a fusion protein of three TRAIL monomers and an adaptor molecule and a DNA origami structure, wherein there are complementary adaptor molecules connected to the DNA origami structure at three sites, and the fusion protein is connected to the DNA origami structure through the interaction between the adaptor molecule and the complementary adaptor molecule. Preferably, the adaptor molecule / complementary adaptor molecule is selected from: Spycatcher peptide / SpyTag peptide, biotin / antibiotin protein and O6-alkylguanine-DNA alkyltransferase (SNAP-tag) / haloalkane dehalogenase (Halo-tag).

3. The DNA-TRAIL complex of claim 2, wherein: The mutually compatible adaptor molecule is connected to the first DNA single strand through a linker, and the three sites of the DNA origami structure have a second DNA single strand, and the second DNA single strand has a complementary sequence to the first DNA single strand, so that the mutually compatible adaptor molecule is connected to the DNA origami structure through base pairing of the complementary sequences of the first DNA single strand and the second DNA single strand, wherein the second DNA single strands at the three sites can be the same or different, preferably the same, and preferably, the linker is selected from: N3 / DBCO; SMCC; SPDP; TCO / Tetrazine and HyNic / 4FB.

4. The DNA-TRAIL complex according to any one of claims 1 to 3, wherein: (1) The DNA origami structure is a planar DNA origami structure; and / or (2) the TRAIL monomer comprises the amino acid sequence of positions 39 to 281 of SEQ ID NO:1, preferably comprises the amino acid sequence of any one of positions 1 to 39 to 281 of SEQ ID NO:1 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or even 100% sequence identity with SEQ ID NO:1, more preferably comprises the amino acid sequence of any one of positions 1 to 39 to 281 of SEQ ID NO:1; and / or (3) the adaptor molecule is a Spycatcher peptide, and the cooperating adaptor molecule is a Spytag peptide, preferably, the Spytag comprises the amino acid sequence shown in SEQ ID NO: 3, and / or the Spycatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 2; and / or (4) The fusion protein comprises the amino acid sequence shown in SEQ ID NO:

5.

5. The DNA-TRAIL complex according to any one of claims 1 to 4, wherein: The DNA origami structure is formed by a DNA sequence comprising SEQ ID NO: 6, a short-chain DNA comprising SEQ ID NO: 7-149, and three DNA chains selected from any one of the following (a)-(e), preferably (c), and the Spytag peptide is connected to the DNA sequence shown in SEQ ID NO: 161 through a linker, preferably an N3 / DBCO linker, whereby the Spytag peptide is connected to the DNA origami structure through base complementarity, The TRAIL monomer forms a fusion protein with the Spycatcher peptide, preferably comprising the amino acid sequence of SEQ ID NO: 5, whereby the TRAIL monomer is connected to the DNA origami structure through the interaction of Spytag and Spycatcher: (a) SEQ ID NO: 152, 155, 158; (b) SEQ ID NO: 151, 154, 158; (c) SEQ ID NO: 151, 153, 159; (d) SEQ ID NO: 151, 156, 160; and (e) SEQ ID NO: 150, 157, 160.

6. A method for preparing the DNA-TRAIL complex according to any one of claims 1 to 5, comprising: (a) providing a DNA origami structure, wherein the DNA origami structure has at least one surface having three sites on the same side, wherein any two sites are about 15-60 nm, preferably about 25-40 nm, most preferably about 40 nm apart, and the three sites form an equilateral triangle or a nearly equilateral triangle, (b) connecting three TRAIL monomers to the three sites on the same side of the same surface of the DNA origami structure, respectively, wherein the C-terminus or N-terminus of the TRAIL monomer is directly or indirectly connected to the DNA origami structure, and preferably, the TRAIL monomers are the same, and (c) The formed DNA-TRAIL complex connected to three sites on the same side of the same surface of the DNA origami structure was harvested.

7. A method for preparing the DNA-TRAIL complex according to any one of claims 1 to 5, comprising: (a) connecting three TRAIL monomers to three short-chain DNAs respectively, wherein in the DNA origami structure formed by the scaffold DNA and the short-chain DNAs, the three short-chain DNAs are respectively contained in three sites on the same surface of the DNA origami structure, wherein any two sites are about 15-60 nm, preferably about 25-40 nm, most preferably about 40 nm apart in the formed DNA origami structure, and the three sites form an equilateral triangle or a nearly equilateral triangle, wherein the C-terminus or N-terminus of the TRAIL monomer is directly or indirectly connected to the short-chain DNA, and preferably, the TRAIL monomers are the same, (b) annealing the ligation product of (a), the scaffold DNA and the short-chain DNA to form a DNA-TRAIL complex comprising a DNA origami structure, wherein the short-chain DNA is complementary to a portion of the sequence of the scaffold DNA, and (c) A DNA-TRAIL complex is obtained in which three sites are connected on the same side of the same surface of the DNA origami structure.

8. The method of claim 6 or 7, comprising forming a fusion protein with a TRAIL monomer and an adapter molecule, and comprising complementary adapter molecules connected thereto at three sites of the DNA origami structure, wherein the fusion protein is connected to the DNA origami structure through the interaction between the adapter molecule and the complementary adapter molecule, wherein the C-terminus or N-terminus of the TRAIL monomer is directly connected to the adapter molecule or connected through a spacer to form the fusion protein, preferably, the adapter molecule / complementary adapter molecule is selected from: Spycatcher peptide / SpyTag peptide, biotin / antibiotin protein and O6-alkylguanine-DNA alkyltransferase (SNAP-tag) / haloalkane dehalogenase (Halo-tag).

9. The method of claim 8, comprising: The mutually compatible adaptor molecule is connected to the first DNA single strand through a linker, and a second DNA single strand is respectively contained at the three sites of the DNA origami structure, and the second DNA single strand has a complementary sequence to the first DNA single strand, so that the mutually compatible adaptor molecule is connected to the DNA origami structure through base pairing of the complementary sequences of the first DNA single strand and the second DNA single strand, wherein the second DNA single strands at the three sites can be the same or different, preferably the same, and preferably, the linker is selected from: N3 / DBCO; SMCC; SPDP; TCO / Tetrazine and HyNic / 4FB.

10. The method of any one of claims 6 to 9, comprising: (i) connecting the Spytag peptide to a complementary DNA strand via a linker to obtain Spytag-ssDNA, preferably, the linker is a N3 / DBCO linker, (ii) mixing and annealing the scaffold DNA, the short-chain DNA and the three capture DNA chains to form a DNA origami structure, wherein the short-chain DNA is complementary to a partial sequence of the scaffold DNA, the capture DNA chain comprises a partial sequence complementary to the scaffold DNA and a capture sequence at least partially complementary to the complementary DNA chain, wherein the three sites where the three capture DNA chains are connected to the DNA origami structure are on the same surface of the DNA origami structure, are about 15-60 nm apart, preferably about 25-40 nm apart, and most preferably about 40 nm apart, and form an equilateral triangle or a nearly equilateral triangle, (iii) annealing the DNA origami structure obtained in (ii) and the Spytag-ssDNA obtained in (i), connecting the Spytag peptide to the DNA origami structure by capturing base pairing between the DNA chain and the complementary DNA chain to obtain a Spytag-DNA origami structure, (iv) forming a fusion protein between the TRAIL monomer and the Spycatcher peptide, preferably connecting the N-terminus of the Spycatcher peptide to the C-terminus of the TRAIL monomer to form a fusion protein, more preferably connecting the N-terminus of the Spycatcher peptide to the C-terminus of the TRAIL monomer via a spacer to form a fusion protein, and (v) mixing the fusion protein obtained in (iv) with the Spytag-DNA origami structure in (iii) to obtain a DNA-TRAIL complex, and optionally screening and isolating the DNA-TRAIL complex in which the fusion protein is linked to three sites on the same side of the same surface of the DNA origami structure, Preferably, the DNA origami structure is formed by a scaffold DNA sequence comprising SEQ ID NO: 6, a short-chain DNA comprising SEQ ID NO: 7-149, and three capture DNA chains selected from any one of the following (a)-(e), preferably (c), and the Spytag-ssDNA is a Spytag peptide connected to a DNA sequence shown in SEQ ID NO: 161, (a) SEQ ID NO: 152, 155, 158; (b) SEQ ID NO: 151, 154, 158; (c) SEQ ID NO: 151, 153, 159; (d) SEQ ID NO: 151, 156, 160; and (e) SEQ ID NO: 150, 157, 160.

11. The method of any one of claims 6 to 10, wherein (1) The DNA origami structure is a planar DNA origami structure; and / or (2) the TRAIL monomer comprises the amino acid sequence of positions 39 to 281 of SEQ ID NO:1, preferably comprises the amino acid sequence of any one of positions 1 to 39 to 281 of SEQ ID NO:1 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or even 100% sequence identity with SEQ ID NO:1, more preferably comprises the amino acid sequence of any one of positions 1 to 39 to 281 of SEQ ID NO:1; and / or (3) the adaptor molecule is a Spycatcher peptide, and the cooperating adaptor molecule is a Spytag peptide, preferably, the Spytag comprises the amino acid sequence shown in SEQ ID NO: 3, and / or the Spycatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 2; and / or (4) The fusion protein comprises the amino acid sequence shown in SEQ ID NO:

5.

12. A kit comprising: - a DNA origami structure having cooperating adaptor molecules attached at at least three sites on the same side of the same face, wherein any two sites are about 15-60 nm, preferably about 25-40 nm, most preferably about 40 nm apart, and the three sites form an equilateral triangle or a nearly equilateral triangle, and - a TRAIL monomer-adapter molecule fusion protein, wherein the C-terminus or N-terminus of the TRAIL monomer is directly or indirectly linked to the adaptor molecule, preferably, the TRAIL monomers are identical, Preferably, the mutual adaptor molecule is connected to the first DNA single strand through a linker, the three sites of the DNA origami structure have a second DNA single strand, and the second DNA single strand has a complementary sequence to the first DNA single strand, so that the mutual adaptor molecule is connected to the DNA origami structure through base pairing of the complementary sequence of the first DNA single strand and the second DNA single strand, Preferably, the adaptor molecule is a Spycatcher peptide and the co-adaptor molecule is a Spytag peptide.

13. The kit of claim 12, comprising: - A DNA origami structure consisting of a DNA sequence comprising SEQ ID NO: 6, a short-chain DNA comprising SEQ ID NO: 7-149, and three DNA chains selected from any one of the following (a)-(e), preferably (c), and a Spytag peptide connected to a DNA sequence shown in SEQ ID NO: 161 via a linker, preferably an N3 / DBCO linker, wherein the Spytag peptide is connected to the DNA origami structure via base complementarity: (a) SEQ ID NO: 152, 155, 158; (b) SEQ ID NO: 151, 154, 158; (c) SEQ ID NO: 151, 153, 159; (d) SEQ ID NO: 151, 156, 160; and (e) SEQ ID NO: 150, 157, 160, as well as -TRAIL monomer and Spycatcher peptide fusion protein.

14. The kit of claim 12 or 13, wherein (1) The DNA origami structure is a planar DNA origami structure; and / or (2) the TRAIL monomer comprises the amino acid sequence of positions 39 to 281 of SEQ ID NO:1, preferably comprises the amino acid sequence of any one of positions 1 to 39 to 281 of SEQ ID NO:1 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or even 100% sequence identity with SEQ ID NO:1, more preferably comprises the amino acid sequence of any one of positions 1 to 39 to 281 of SEQ ID NO:1; and / or (3) the Spytag comprises the amino acid sequence shown in SEQ ID NO: 3, and / or the Spycatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 2; and / or (4) The fusion protein comprises the amino acid sequence shown in SEQ ID NO:

5.

15. A pharmaceutical composition for treating tumors, comprising: - a DNA-TRAIL complex according to any one of claims 1 to 5, a DNA-TRAIL complex obtained according to the method according to any one of claims 6 to 11 or a DNA-TRAIL complex prepared using a kit according to any one of claims 12 to 14, and - optionally a pharmaceutically acceptable carrier, The tumor is a tumor with TRAIL as a target, preferably, the tumor is a solid tumor, preferably selected from gastric cancer, hepatocellular carcinoma, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, renal cell carcinoma, esophageal cancer and prostate cancer, pancreatic cancer and esophageal cancer.

16. Use of the DNA-TRAIL complex according to any one of claims 1 to 5, the DNA-TRAIL complex obtained according to the method according to any one of claims 6 to 11, or the DNA-TRAIL complex prepared using the kit according to any one of claims 12 to 14 in the preparation of a medicament for treating a tumor, wherein the tumor is a tumor with TRAIL as a target, preferably, the tumor is a solid tumor, preferably selected from gastric cancer, hepatocellular carcinoma, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, renal cell carcinoma, esophageal cancer and prostate cancer.

17. A method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a DNA-TRAIL complex according to any one of claims 1 to 5, a DNA-TRAIL complex obtained according to the method of any one of claims 6 to 11, a DNA-TRAIL complex prepared using the kit of any one of claims 12 to 14, or a pharmaceutical composition of claim 15, wherein the tumor is a tumor targeting TRAIL, preferably, the tumor is a solid tumor, preferably selected from gastric cancer, hepatocellular carcinoma, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, renal cell carcinoma, esophageal cancer, and prostate cancer.