Method for efficiently constructing multivalent antibody complex based on DNA origami

Abstract

The invention discloses a method for efficiently constructing a multivalent antibody complex based on DNA origami, which comprises the following steps: 1) constructing a DNA origami framework through DNA origami, and setting a plurality of specific coupling sites; 2) combining the antibody with a DNA single chain through a chemical method to prepare an antibody-DNA compound; (3) fully mixing a DNA origami framework with a specific coupling site with the antibody-DNA compound, and constructing a multivalent antibody compound based on DNA origami by utilizing complementary base pairing; and 4) purifying the multivalent antibody compound, and verifying the purity, form, stability and coupling efficiency of the multivalent antibody compound through a characterization technology. According to the invention, the multivalent antibody compound can be rapidly constructed, and the high synthesis efficiency of a complete antibody pattern is realized. The method has high operability and controllability, can be applied to the field of biomedicine, and particularly has important significance in research and development of multivalent antibody drugs.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical engineering and nanotechnology, and more particularly to a method for efficiently constructing multivalent antibody complexes based on DNA origami. Background Art

[0002] With the development of monoclonal antibody technology, antibody drugs have shown significant clinical value in the fields of cancer targeted therapy, immunomodulation, and intervention in autoimmune diseases. However, traditional monomeric antibodies are limited by a single antigen-binding site and have problems such as insufficient binding affinity when treating complex diseases, making it difficult to meet the needs of precision medicine. Multivalent antibody complexes integrate multiple antibody molecules or fragments through precisely controlled spatial arrangements, exhibiting enhanced antigen-binding affinity, higher specificity, and extended multifunctionality.

[0003] DNA nanotechnology can shape programmable nanostructures into a multifunctional template capable of precisely organizing functional molecules at the nanoscale. Currently, DNA origami scaffolds can perform addressable spatial patterning of protein arrays with sub-5 nm precision. Multivalent antibody complexes on DNA origami templates can exhibit enhanced therapeutic effects through high-affinity binding to tumor-associated receptors, achieve excellent cancer cell targeting, increase drug payload delivery, and reduce off-target cytotoxicity in mouse models. Given that the biological effects of these multivalent complexes highly depend on protein valency and spatial arrangement, it is crucial to rationally improve the efficiency and uniformity of multivalent protein conjugation on DNA frameworks.

[0004] Patent CN109535257A discloses "Novel Bispecific CD3 / CD19 Polypeptide Complex", and constructs a multivalent antibody complex using a genetically engineered fusion protein, but its process is cumbersome and it is impossible to prepare high-valence (trivalent, tetravalent, etc.) antibody complexes. Patent CN114539422A discloses "Construction Method and Application of Nucleic Acid Polymerization-Mediated Multivalent Protein Drugs and Vaccines", and constructs multivalent protein complexes using DNA polymers by a covalent coupling method, but this method cannot control the spatial position and orientation of antibodies, and the yield will decrease as the valence of the manufactured antibody complexes increases. It can be seen that although the prior art discloses some methods for constructing multivalent antibody complexes, there are usually problems such as low construction efficiency and poor controllability.

[0005] Therefore, there is an urgent need for a method for constructing multivalent antibody complexes that is efficient and controllable. Summary of the Invention

[0006] The object of the present invention is to provide a method for efficiently constructing a multivalent antibody complex based on DNA origami, so as to fill the gap in the prior art regarding the protein coupling efficiency at different positions on the DNA framework and the correlation between the coupling efficiency and the spatial arrangement.

[0007] In view of the above problems, the present invention adopts the following technical solutions:

[0008] According to a first aspect of the present invention, there is provided a method for efficiently constructing a multivalent antibody complex based on DNA origami, the method comprising the following steps: 1) constructing a DNA origami framework by DNA origami technology, and arranging a plurality of specific coupling sites on the DNA origami framework; 2) chemically binding an antibody to a single-stranded DNA to prepare an antibody-DNA complex, and the single-stranded DNA can be base-complementary paired with the specific coupling sites on the DNA framework; 3) fully mixing the DNA origami framework with the specific coupling sites and the antibody-DNA complex, and using base complementary pairing, the antibody can be coupled to a specific position on the DNA origami framework to construct a multivalent antibody complex based on DNA origami; 4) purifying the multivalent antibody complex based on DNA origami, and verifying its purity, morphology, stability and coupling efficiency through characterization techniques.

[0009] According to the method provided by the present invention, its working principle is: by replacing the staple strands at specific positions in the DNA origami with staple strands having additional extended sequences, specific coupling sites are provided for antibody coupling. Among them, the antibody is chemically cross-linked with a chemical cross-linking reagent and a single-stranded DNA, and an antibody-DNA complex is obtained, and the single-stranded DNA can be base-complementary paired with the extended sequence of the staple strand. Therefore, by mixing the DNA origami framework with the extended sequence and the antibody-DNA complex, a DNA framework multivalent antibody complex can be obtained.

[0010] In step 1), the DNA origami framework is a stable structure designed by DNA origami technology, that is, a DNA origami structure assembled by multiple DNA staple strands and a circular DNA template strand through a thermal annealing program, and the staple strands at specific serial numbers in the DNA origami are selected and replaced with staple strands having additional extended sequences to provide specific sites for antibody coupling.

[0011] The optimal extended sequence length can be selected according to the additional extended sequence of the staple strand. Preferably, the extended length is 5-60 bases. Less than 5 bases will affect the efficiency of base complementary pairing, and more than 60 bases will affect the accuracy of the antibody pattern obtained by arrangement. Taking the rectangular origami as an example, the optimal extended sequence length is 20 bases.

[0012] In step 1), the DNA origami framework includes rectangular origami, triangular origami, hexagonal origami and other two-dimensional DNA origami structures. Preferably, the rectangular origami with a length of 100 nanometers and a width of 50 nanometers has the best tunability of the multivalent antibody coupling efficiency.

[0013] In step 1), the specific coupling site is an elongated DNA arm strand, which is set by selecting the staple strand at a specific serial number position in the DNA origami and replacing it with a staple strand having an additional extended sequence.

[0014] Preferably, in step 1), monovalent, divalent, trivalent or tetravalent specific coupling sites are designed by adjusting the coupling site spacing and the position on the DNA origami framework.

[0015] Preferably, in step 1), the center spacing between adjacent coupling sites is 18 nm to 37 nm to ensure that there is enough spacing between the coupling sites so that a single antibody will not be coupled to two specific coupling sites simultaneously.

[0016] Preferably, in step 2), the antibody includes monoclonal antibody, fully human antibody, recombinant antibody or fragments of the aforementioned antibodies.

[0017] Preferably, in step 2), the antibody and the DNA single strand can be coupled by a chemical reaction to form an antibody-DNA complex, including using a Sulfo-SMCC crosslinker to connect the amino group on the antibody and the thiol group on the DNA, or connecting the azide group modified on the antibody and the DBCO modified on the DNA by click chemistry. Preferably, using a Sulfo-SMCC crosslinker does not require additional modification because the antibody itself has an amino group.

[0018] Preferably, in step 3), the base complementary pairing means that the arm strand of the DNA origami framework with specific coupling sites can specifically pair and bind with the DNA single strand in the antibody-DNA complex.

[0019] Preferably, in step 3), sufficient mixing means placing the DNA origami framework with specific coupling sites and the antibody-DNA complex in a shaker at a certain mixing ratio and reacting for a certain period of time.

[0020] Preferably, the reaction time in step 3) is 1 h to 12 h. The optimal reaction time can be selected according to the sequence length of the base complementary pairing.

[0021] Preferably, the reaction temperature in step 3) is 4 to 37 °C. The optimal reaction temperature can be selected according to the formation conditions of the DNA origami framework.

[0022] The method further includes: designing the DNA origami structure by using Cadnano2 or Athena or Tiamat software to make a certain number of arm chains extend at different staple strand positions, so as to realize the regulation of the number and spatial position of the conjugated antibodies.

[0023] Preferably, in step 4), the purification method of the multivalent antibody complex based on the DNA origami framework includes separating by using a liquid chromatography column or centrifuging with an ultrafiltration tube, etc., to remove the unconjugated DNA origami framework and antibody-DNA complex.

[0024] Preferably, in step 4), the characterization techniques include atomic force microscopy imaging (AFM), gel electrophoresis (GE), electron microscopy (EM), etc.

[0025] According to the second aspect of the present invention, there is provided a multivalent antibody complex based on DNA origami prepared by the above method. By regulating the position and number of the arm chains extending from the DNA origami framework, the valence state and spatial position of the antibodies are precisely regulated, and a multivalent antibody complex based on DNA origami is prepared.

[0026] According to the present invention, the monovalent antibody complex based on DNA origami, i.e., DNA origami, has a single antibody conjugation site and conjugates with one antibody through incubation. By analogy, the divalent, trivalent, and tetravalent antibody complexes based on DNA origami are the complex structures of DNA origami conjugated with two, three, and four antibodies respectively.

[0027] According to the third aspect of the present invention, there is also provided a method for regulating the conjugation efficiency of the number and spatial position of antibodies. The adjustable conjugation efficiency of the number and spatial position of antibodies means that the DNA origami structure is designed by using Cadnano2 or Athena or Tiamat software, and the number and spatial position of specific conjugation sites on the DNA origami framework are regulated to make different numbers of arm chains extend at different staple strand positions, so as to realize the regulation of the conjugation efficiency of the number and spatial position of the conjugated antibodies.

[0028] According to the present invention, AFM with single-molecule imaging ability is used to characterize the conjugation efficiency of the multivalent antibodies of the DNA origami framework. By obtaining the specific images of the DNA origami framework-antibody complex, it is analyzed whether the antibodies are conjugated at each designed specific conjugation site, and then the conjugation efficiency of each site and the yield of the overall pattern are obtained. AFM provides a label-free and stain-free method for single-molecule morphology characterization, and can easily realize the visualization of the conjugation of antibodies and the DNA origami framework at the single-molecule level.

[0029] The inventive concept of the present invention lies in replacing some staple strands in the DNA origami framework with segments containing specific extension sequences to create programmable specific coupling sites on the DNA framework, and achieving precise positioning and coupling of antibodies through base complementary pairing, thereby preparing for the first time a monovalent, divalent, trivalent or tetravalent antibody complex based on DNA origami. Further, by designing software to regulate the number and spatial arrangement of specific coupling sites on the DNA origami framework, changing the number of "arm strands" of the staple strand extension sequences, flexibly controlling the antibody loading amount and its spatial distribution, and based on the label-free and stain-free single-molecule imaging technology of AFM, directly observing the morphology and coupling state of the DNA origami framework-antibody complex, and quantitatively analyzing the efficiency of each coupling site and the overall pattern yield.

[0030] In the prior art, coupling antibodies through DNA origami usually improves the efficiency by increasing the number of coupled DNA strands, and does not improve the coupling efficiency through spatial position arrangement. The present invention is the first to improve the antibody coupling efficiency and arrange antibodies through position arrangement. The technical difficulty of the present invention lies in designing multivalent antibody coupling sites on the DNA origami, making the sites evenly distributed while ensuring that each single antibody site has a sufficiently high coupling efficiency, and the distance between sites is greater than the antibody size so that there is no interference between sites.

[0031] The positive and progressive effects of the present invention are as follows: It breaks through the limitations of the research on the correlation between spatial position and coupling efficiency in traditional multivalent antibody coupling technologies. That is, traditional multivalent antibody coupling technologies usually directly connect through covalent molecules, do not have antibody spatial arrangement information, and cannot adjust the antibody coupling efficiency through spatial position. The present invention for the first time establishes a systematic regulation method for the design, number and coupling efficiency of antibody coupling sites on the DNA origami framework. The present invention proves through experiments that the coupling efficiency of antibodies can be systematically regulated by regulating the design of antibody coupling sites on the DNA origami framework, and a multivalent antibody coupling complex with a high yield is obtained through optimization. This method provides a new multivalent antibody construction platform for fields such as targeted drug delivery, immunotherapy, and biosensing, and can guide and enhance its functional activity or targeting by precisely controlling the antibody spatial configuration.

[0032] In summary, the present invention provides a method for efficiently constructing a multivalent antibody complex based on DNA origami. By using the DNA origami framework for special coupling site design, the present invention can quickly construct a multivalent antibody complex and achieve a relatively high synthesis efficiency of complete antibody patterns. This method has high operability and controllability and can be applied to the biomedical field, especially has important significance in the research and development of multivalent antibody drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a diagram of the DNA origami template and antibody coupling sites;

[0034] Figure 2 It is the SDS-PAGE result diagram of the antibody-DNA complex;

[0035] Figure 3 It is the AFM diagram of the DNA origami framework;

[0036] Figure 4 It is the antibody conjugation efficiency diagram;

[0037] Figure 5 It is the AFM diagram of the antibody-DNA complex conjugated with the DNA origami framework;

[0038] Figure 6 It is the yield bar chart of the multivalent antibody pattern;

[0039] Figure 7 It is the yield bar chart of the multivalent antibody pattern. Detailed implementation mode

[0040] The following further describes the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0041] Example 1: Design of specific conjugation sites on the DNA origami framework

[0042] The construction of the multivalent antibody pattern based on the DNA origami framework first requires the design of the DNA origami framework and antibody conjugation sites. In this embodiment, the DNA origami framework consists of a long DNA template strand and 208 DNA staple strands. Each staple strand is a single-stranded DNA with a length between 30 and 60 nucleotides, and the DNA template strand is a circular single-stranded DNA with a length of 7249 nucleotides. The template strand binds to the staple strands and folds into a rectangular planar structure under their action. Each origami structure is a rectangle with a length of 100 nm and a width of 50 nm.

[0043] As Figure 1 shown in the left figure in, it is a complete rectangular DNA origami structure. The antibody conjugation sites are designed on the original DNA origami framework, and the monovalent to tetravalent antibody conjugation sites are designed by adjusting the site spacing and positions on the origami for the efficient production of multivalent antibodies.

[0044] As Figure 1As shown in the middle right figure, it shows the design of antibody conjugation sites with different valences on rectangular DNA origami. Among them, there are two types of monovalent site designs, namely, at the upper left corner and the center position of the rectangular origami. There are two types of divalent site designs. One is the design that includes two corner sites at the upper left corner and the lower right corner, and the other is the design that includes one upper left corner site and one center site. The trivalent site design is to add one more corner site or a right edge site on the divalent site design. The tetravalent site design includes four edge sites, so that the sites do not include the center position. When designing from monovalent to trivalent sites, two groups with more and relatively fewer edge sites are designed respectively to compare the differences in antibody conjugation efficiency under the designs of edge sites and center sites.

[0045] Based on the original DNA origami sequence, the specific 4 DNA staple strands at each original site are replaced with extended strands with a specific 20-nucleotide length sequence added to the 3' end of the original strand. One site requires 4 staple strands to be replaced, and 8 to 16 staple strands need to be replaced for two to four sites. These extended strands are used as antibody conjugation sites to achieve the construction of multivalent antibody patterns. For the existing design, at most 9 antibody conjugation sites can be designed on a rectangular DNA origami. The multivalent antibody conjugation sites designed above are evenly distributed at multiple positions on the rectangular plane of the DNA origami, and the center-to-center distance between adjacent conjugation sites is 18 nm to 37 nm, so as to ensure that there is enough space between the sites so that a single antibody cannot conjugate to two sites simultaneously. The nucleotide compositions of the extended sequences are the same, all being -TTTGCATTCACTCCTAACTACCAC. It should be understood that this extended sequence is only for illustration and not for limitation.

[0046] Example 2: Preparation and Characterization of DNA Origami Framework Multivalent Antibody Complexes

[0047] First, the raw materials and reagents involved in the preparation process of this example are described as follows:

[0048] Tris (Tris(hydroxymethyl)aminomethane), disodium ethylenediaminetetraacetate (EDTA), magnesium acetate, glacial acetic acid, and boric acid were all purchased from Sinopharm Chemical Reagent Co., Ltd.; the experimental water used was all ultrapure water; the 100 kDa ultrafiltration tube was purchased from Thermo Scientific; the M13mp18 phage genomic DNA was purchased from BioBiolink Biotechnology Co., Ltd.; the DNA staple strands and the DNA strands for antibody conjugation were all purchased from Shanghai Sangon Biotech Co., Ltd.; the experimental reactions in this example were carried out in 1×TAE-Mg 2+It is carried out in a buffer solution (40 mM Tris, 20 mM glacial acetic acid, 2 mM EDTA and 12.5 mM magnesium acetate tetrahydrate, pH = 8.0). For those not specifying specific techniques or conditions in the examples, they are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0049] The specific preparation process includes the following steps:

[0050] S1: Prepare the antibody-DNA complex.

[0051] Rituximab (RTX, 100 mg / 10 mL, Roche, Switzerland) is purified and buffer-exchanged through an ultrafiltration membrane. The antibody concentration is measured using the NanoDrop system. Sulfo-SMCC is dissolved in PBS buffer and mixed with the antibody at a molar ratio of 10:1. React at room temperature for 30 minutes. After the reaction, an excess of unreacted Sulfo-SMCC is removed using a desalting column to obtain SMCC-modified antibody. The purchased thiol-modified single-stranded DNA (SH-SH-Oligo) solution reacts with tris(2-carboxyethyl)phosphine (TCEP) at room temperature for 2 hours to break the disulfide bonds therein. Subsequently, the reaction solution is treated with sodium acetate buffer and ice-cold ethanol and centrifuged at 12,000 rpm and 4 °C. Discard the supernatant, and the precipitate is resuspended in 70% ethanol and centrifuged again to purify SH-Oligo. The precipitate is dried at room temperature and redissolved in pure water, and the concentration of SH-Oligo is measured using NanoDrop. SMCC-RTX and SH-Oligo are mixed at a molar ratio of 3:1 and reacted at room temperature for 30 minutes. After the reaction, unreacted Oligo-SH is removed by ultrafiltration. The final product is characterized by 10% SDS-PAGE,

[0052] The results are as Figure 2 shown. A new band appears in the antibody-DNA experimental group, indicating that the antibody has been successfully conjugated with the DNA sequence.

[0053] S2: Prepare the DNA origami template.

[0054] The preparation raw materials include: M13mp18 template strand and DNA staple short chains. The DNA short chains include: 180 kinds of DNA staple strands with sequences and 16 staple strands with extended sequences (antibody-specific conjugation sites). Mix the above DNA strands in 1×TAE / Mg 2+In the buffer, the total volume after mixing is 100 μL, where the concentration of the M13mp18 template strand is 10 nM, equal amounts and in excess of each DNA short strand sequence are added, and the total concentration of the DNA short strands is 100 nM. The mixture of the above-mentioned M13mp18 template strand and DNA short strands was slowly annealed using a gradient PCR instrument, and the annealing conditions were: starting temperature 95 °C, holding for 10 min, ending temperature 25 °C, with a gradient of 1 °C each, staying at each gradient for 120 s, and annealing from 95 °C to 25 °C within two and a half hours. The sample was taken out and centrifuged and separated using a 100 kDa ultrafiltration tube to remove the excess DNA short strands. The centrifugation conditions were: adding 300 μL of 1×TAE-Mg 2+ buffer to 100 μL of the sample, centrifuging at 3000 g / min for 10 min, repeating the centrifugation 3 times, and finally observing the morphology of the lamellar structure of the collected sample using AFM.

[0055] The results are as Figure 3 shown. The constructed DNA framework template is in a single-layer rectangular lamellar structure. The AFM characterization results show that the rectangular two-dimensional DNA nanostructure is about 100 nm long, about 50 nm wide, and about 1.5 nm high, proving the successful synthesis of the rectangular DNA origami framework.

[0056] S3: Construction of multivalent antibody complexes based on DNA origami.

[0057] The preparation materials include: the antibody-DNA complex prepared in S1 and the DNA origami framework prepared in S2. First, we mixed the above-mentioned antibody-DNA complex and the DNA origami framework with a monovalent central coupling site at different concentration ratios in 1×TAE / Mg 2+ buffer, and used an oscillator to oscillate and incubate the mixture of the DNA origami template and the antibody-DNA conjugate at a temperature of 25 °C for 6 h. Then, directly observe the incubated sample using AFM and count the coupling efficiency of individual antibodies on the origami. The results are as Figure 4 shown. The antibody coupling efficiency increases with the increase of the antibody-origami ratio and tends to be stable at about 16:1.

[0058] Mix the above-mentioned antibody-DNA complex and DNA origami framework at a concentration ratio of 16:1 in 1×TAE / Mg 2+In the buffer, the total volume after mixing is 100 μL, where the concentration of the M13mp18 template strand is 10 nM and the total concentration of the antibody-DNA conjugate is 160 nM. The mixture of the above DNA origami template and the antibody-DNA conjugate was incubated with shaking using an oscillator. The incubation conditions were: temperature 25 °C for 6 h. The sample was taken out and centrifuged using a 100 kDa centrifuge tube to remove the excess antibody-DNA conjugate; the centrifugation conditions were: 300 μL of 1×TAE-Mg 2+ buffer was added to 100 μL of the sample, and centrifuged at 3000 rcf / min for 10 min, and the centrifugation was repeated 3 times. The finally collected sample was observed by AFM to observe the morphology of the antibody pattern on the DNA template.

[0059] The results are as Figure 5 shown. The DNA origami template has a rectangular lamellar structure, and the white dots on the origami template represent the conjugated antibodies. The antibody distribution corresponding to each design was obtained from the AFM image, and it was found that antibodies were likely to be conjugated at the designed conjugation sites through height image analysis.

[0060] Example 3: Analysis of the conjugation efficiency of the antibody pattern

[0061] Multiple sample images were obtained using AFM, and the images were processed using analysis software to analyze the antibody conjugation efficiency corresponding to the sites and the synthesis efficiency of the overall antibody pattern under different antibody site designs.

[0062] The results of the conjugation efficiency of different sites in the obtained multivalent antibody patterns are as Figure 6 shown. When designing a monovalent antibody site, the conjugation efficiency of each site is above 85%. In the conjugation of multivalent antibodies, the individual conjugation efficiency of each position shows different distributions. According to the spacing and position of the designed sites, the antibody conjugation efficiency corresponding to the site is higher at the corner or edge, such as the II-a and III-a site designs in the divalent and trivalent sites. In the tetravalent antibody pattern designed with edge sites, the conjugation efficiency of each site can reach above 80%, which is at the same level as the monovalent site.

[0063] The yield results of manufacturing multivalent antibody patterns using DNA origami are as Figure 7As shown, the overall yield of the optimized bivalent and trivalent antibody complexes reaches over 70%. This data indicates that the coupling efficiency at the corner or edge sites is higher, thus obtaining antibodies with higher coupling efficiency through the control of the site position. For the unoptimized designs, i.e., II-b and III-b, the yield of the multivalent antibody complexes is less than 50%. However, the coupling yield of the optimized tetravalent antibody complex can still reach over 60%, which is higher than that of the unoptimized bivalent and trivalent complexes. The results show that the antibody coupling efficiency can be systematically regulated by controlling the antibody coupling site design on the DNA origami framework, and high-yield multivalent antibody coupling complexes are obtained through optimization.

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of the claims of the present invention patent. The content not described in detail in the present invention is all conventional technical content.

Claims

1. An efficient method for constructing multivalent antibody complexes based on DNA origami, characterized in that, The method includes the following steps: 1) Construct a DNA origami framework by DNA origami technology, and set a plurality of specific coupling sites on the DNA origami framework; 2) Antibodies are bound to single-stranded DNA by chemical methods to prepare antibody-DNA complexes, and the single-stranded DNA can be base complementary paired with the specific coupling sites on the DNA framework; 3) The DNA origami framework with specific coupling sites is fully mixed with the antibody-DNA complex. By using base complementary pairing, the antibodies can be coupled to specific positions on the DNA origami framework to construct a multivalent antibody complex based on DNA origami; 4) Purify the multivalent antibody complex based on DNA origami, and verify its purity, morphology, stability and coupling efficiency through characterization techniques.

2. The method according to claim 1, characterized in that, In step 1), the specific coupling site is an extended DNA arm chain, which is set by selecting staple strands at specific serial numbers in the DNA origami and replacing them with staple strands having additional extended sequences.

3. The method according to claim 1, wherein In step 1), a plurality of specific coupling sites are designed by adjusting the spacing between the coupling sites and their positions on the DNA origami framework.

4. The method according to claim 3, characterized in that, In step 1), the center spacing between adjacent coupling sites is 18 nm to 37 nm to ensure that there is enough spacing between the coupling sites so that a single antibody will not be simultaneously coupled to two specific coupling sites.

5. The method according to claim 1, wherein In step 2), the antibody is a monoclonal antibody, a fully human antibody, a recombinant antibody or a fragment of the foregoing antibodies.

6. The method according to claim 1, characterized in that In step 2), the chemical reaction includes using a Sulfo-SMCC crosslinker to connect the amino group on the antibody to the thiol group on the DNA, or connecting the azide group modified on the antibody and the DBCO modified on the DNA through click chemistry.

7. The method according to claim 1, characterized in that, In step 3), the DNA origami framework with specific coupling sites is mixed with the antibody-DNA complex according to a certain mixing ratio, and the mixing molar ratio between the two is between 1:1 and 32:

1.

8. The method according to claim 1, characterized in that, In step 3), the reaction time is 1 h to 12 h, and the reaction temperature is 4 to 37 °C.

9. The method according to claim 1, characterized in that, The method further includes: designing the DNA origami structure by using Cadnano2 or Athena or Tiamat software, and regulating the number and spatial positions of specific coupling sites on the DNA origami framework, so that different numbers of arm chains extend at different staple strand positions, thereby realizing the regulation of the number of coupled antibodies and the coupling efficiency of the spatial positions.

10. A DNA origami-based multivalent antibody complex prepared by the method according to any one of claims 1-9, characterized in that, By regulating the positions and numbers of the arm chains extending from the DNA origami framework, the valence state and spatial positions of the antibodies are precisely regulated to prepare a multivalent antibody complex based on DNA origami.

Images