Method for constructing nucleic acid self-assembly mediated ADC drug and application thereof

Abstract

The present invention relates to the field of biotechnological pharmaceuticals.SOLUTION: Specifically, the present invention provides an ADC (antibody-drugconjugate) conjugate based on a complementary paired nucleic acid backbone, wherein the ADC conjugate is a polymer formed by conjugating n monomers having complementary paired nucleic acid backbones, wherein the polymer comprises m "targeting monomers" which are cell surface-targeting antibodies or proteins linked to a single nucleic acid strand, and k "drug monomers" which are drugs (toxin payloads) linked to a single nucleic acid strand; N is a positive integer of 2-8, m is a positive integer of 1-3 and m <n, k is a positive integer of 1 to (n-m), in the polymer, the nucleic acid single strand of each monomer forms a complementarily paired double strand with the nucleic acid single strand of another 1-3 monomers by base complementarity, thereby forming a complementarily paired nucleic acid backbone structure.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology medicine, and in particular to nucleic acid multimerization-mediated ADCs (antibody-drug conjugates), i.e., methods for constructing and applying antibody-drug conjugates. [Background technology]

[0002] ADCs are targeted drugs developed for cancer treatment, specifically delivering highly cytotoxic chemotherapy drugs to cancer cells and reducing nonspecific cell killing within the human body. The ADC mechanism involves endocytosis by antibodies binding to receptors on the surface of cancer cells, followed by drug release into lysosomes. The drug then escapes from the lysosomes, enters the cytoplasm, and triggers cell death. The concept of ADCs has been proposed for a long time, but due to limitations in various knowledge and technical conditions, related drug development was previously slow and complex. However, with technological advances and accumulated clinical research experience, ADC drugs have gradually emerged from their slump in recent years and become a mainstream in the field of biopharmaceutical development.

[0003] Currently, the preparation of ADC molecules focuses on the coupling of a compound (linker-payload) consisting of a chemical linker and a small molecule payload to a monoclonal antibody molecule. The traditional method involves fixed-point coupling of the linker to a lysine or cysteine ​​site on the surface of the antibody molecule via a specific chemical reaction. However, antibody molecules typically contain multiple lysine or cysteine ​​sites, and the steric hindrance effects around each amino acid are not identical. This leads to heterogeneity in the number of linker payloads actually coupled to the antibody molecule, i.e., the drug-antibody ratio (DAR). This further leads to significant uncertainty in the analysis of various aspects of ADCs, such as efficacy, pharmacokinetics, biodistribution, and toxicology, and also leads to increased ADC production costs. However, due to the inherent shortcomings of traditional coupling methods, even FDA-approved ADC molecules exhibit significant heterogeneity in DAR.

[0004] Therefore, there is an urgent need in the art to develop a simple, flexible, efficient, and modular method for preparing ADC drugs that enables the ADCs to have a completely uniform DAR. Summary of the Invention [Problem to be solved by the invention]

[0005] The main objective of the present invention is to provide a simple, flexible, efficient and modular method for preparing ADC drugs, which allows the ADCs to have a completely uniform DAR. [Means for solving the problem]

[0006] The first aspect of the present invention provides an antibody-drug conjugate (ADC) based on a complementary pairing nucleic acid backbone, wherein the antibody-drug conjugate is a polymer formed by the complexation of n monomers having a complementary pairing nucleic acid backbone. Here, the polymer includes m targeting monomers, where the "targeting monomer" is an antibody or protein targeting the cell surface linked to a single-stranded nucleic acid, and k drug monomers, where the "drug monomer" is a drug (toxin payload) linked to a single-stranded nucleic acid. n is a positive integer from 2 to 8, m is a positive integer from 1 to 3 and m < n, k is a positive integer from 1 to (n - m). In the polymer, the single-stranded nucleic acid of each monomer forms a complementary pairing double-stranded structure by complementary pairing with the single-stranded nucleic acids of another 1 to 3 monomers, thereby forming a complementary pairing nucleic acid backbone structure. In another preferred example, n = 3 to 8.

[0007] In another preferred example, the targeting monomer has the structure of formula I, A-W-D0(I) The drug monomer has the structure of formula II, D1-W-D2(II) In the formula, A is an antibody or protein, W is a single-stranded nucleic acid sequence, D0 is none or a drug, D1 is none or a drug, D2 is none or a drug, "-" is a linker or a bond, At least one of D0, D1 and D2 is a drug.

[0008] In another preferred example, "-" is a covalent bond or a linker or a combination of therapies. In another preferred example, A is an antibody or protein that causes endocytosis by specifically binding to a cell surface receptor.

[0009] In another preferred embodiment, the antibody or protein is selected from the group consisting of an anti-HER2 antibody, an anti-HSA antibody, an anti-PD-L1 antibody, or a combination thereof. In another preferred example, the antibody or protein is an anti-HER2 nanobody, the amino acid sequence of which is as shown in SEQ ID NO:1.

[0010] In another preferred embodiment, D1 / D2 is a small molecule or polypeptide toxin for killing cells. In another preferred embodiment, the nucleic acid strand is resistant to degradation and is selected from the group consisting of L-form nucleic acids, peptide nucleic acids, locked nucleic acids, phosphoromorpholidate nucleic acids, thio-modified nucleic acids, 2'-fluoro-modified nucleic acids, 5-hydroxymethylcytosine nucleic acids, or combinations thereof.

[0011] In another preferred embodiment, in the polymer, component A of each targeting monomer is the same or different. In another preferred example, in the polymer, component D1 of each drug monomer is the same or different, and component D2 of each drug monomer is the same or different.

[0012] In another preferred embodiment, in the polymer, W of each monomer is different. In another preferred example, the drug D1 or D2 can be linked to both terminal nucleotides of W and / or the central nucleotide by chemical modification.

[0013] In another preferred embodiment, the nucleic acid single-stranded sequence W in the targeting monomer (Formula I) and the drug monomer (Formula II) has the structure shown in Formula III: X1-R1-X2-R2-X3(III) where: R1 is base-complementary pairing region 1; R2 is base-complementary pairing region 2, X1, X2 and X3 are each independently null or redundant nucleic acids; "-" is a bond.

[0014] In another preferred example, the length of R1 and R2 is independently 10 to 20 bases, preferably 14 to 16 bases. In another preferred example, the length of X1 is 0 to 5 bases. In another preferred example, the length of X3 is 0 to 5 bases. In another preferred example, the length of X2 is 0 to 3 bases. In another preferred example, the sequence of X2 is selected from the group consisting of A, AA, AGA, or AAA.

[0015] In another preferred example, R1 of each monomer forms a base-complementary pairing structure with R2 of the monomer to the left (or left side), and R2 forms a base-complementary pairing structure with R1 of the monomer to the right (or right side).

[0016] A second aspect of the present invention provides a pharmaceutical composition, said pharmaceutical composition comprising: (a) an antibody drug conjugate based on a complementary paired nucleic acid backbone according to the first aspect; wherein the targeting monomer is selected from a targeting monomer library, the drug monomer is selected from a drug monomer library, and (b) a pharmaceutically acceptable carrier.

[0017] In another preferred example, the pharmaceutical composition contains a dimer to octamer complex. In another preferred example, the assembly units in the targeting monomer library carry antibodies or proteins that can be endocytosed by specifically binding to receptors on the surface of cancer cells. In another preferred embodiment, the assembly units in the drug monomer library carry small molecule or polypeptide drug moieties that kill cells.

[0018] In another preferred embodiment, the targeting monomer and drug monomer in the pharmaceutical composition can be selected according to individual therapeutic needs, and polymeric ADC drugs are prepared by real-time self-assembly. In another preferred embodiment, the ratios of A / (D1+D2) and D1 / D2 in the pharmaceutical composition can be selected according to individual treatment needs.

[0019] A third aspect of the present invention provides a nucleic acid sequence library, said nucleic acid library comprising nucleic acid sequences for forming antibody drug conjugates based on the complementary paired nucleic acid backbone according to the first aspect. In another preferred embodiment, the nucleic acid sequence W has the structure shown in formula III: X1-R1-X2-R2-X3(III) where: R1 is base-complementary pairing region 1; R2 is base-complementary pairing region 2, X1, X2 and X3 are each independently null or redundant nucleic acids; "-" is a bond.

[0020] A fourth aspect of the present invention provides the use of a nucleic acid sequence library according to the third aspect, used for preparing an antibody drug conjugate of a complementary paired nucleic acid backbone according to the first aspect.

[0021] A fifth aspect of the present invention is (1) forming a nucleic acid-drug assembly unit by coupling a single stranded nucleic acid and a cytotoxic drug to a chemical fixed point; (2) fixed-point coupling of an antibody with a complementary nucleic acid single strand to form an antibody-nucleic acid assembly unit; (3) rapidly self-assembling an antibody-nucleic acid assembly unit and a plurality of complementary nucleic acid-drug assembly units via complementary nucleic acid sequences to form the antibody-drug conjugate. [Effects of the Invention]

[0022] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows a mode diagram of self-assembly of ADC drugs mediated by complementary pairing of nucleic acid strands. [Figure 2] Electrophoresis gel image of nickel column affinity purified anti-HER2 nanobodies. [Figure 3] Electrophoresis gel images of anti-HER2 nanobodies and L-DNA coupling efficiency. [Figure 4] 1 shows ion-exchange purified anti-HER2 nanobody-L-DNA conjugates. [Figure 5] Figure 1 shows the determination of DM4-L-DNA1 coupling efficiency by negative ion mode ESI ion trap liquid chromatography mass spectrometry. [Figure 6] DM4-L-DNA1 phenyl-agarose gel hydrophobic purification column separation results are shown. [Figure 7] Figure 1 shows the determination of MMAE-L-DNA1 coupling efficiency by negative ion mode ESI ion trap liquid chromatography mass spectrometry. [Figure 8] Electrophoresis gel images of self-assembly of nanobody-drug conjugates are shown, where a. SDS-PAGE, b. 2% agarose DNA gel. [Figure 9] 1 shows in vitro cell killing activity evaluation of nanobody-drug conjugates. [Figure 10] Figure 1 shows the in vitro killing activity evaluation of nanobody-drug conjugates in different tumor cell lines. [Figure 11]Changes in tumor volume in BT474 human breast cancer models subcutaneously inoculated into BALB / c-nu mice in different treatment groups are shown. Data are expressed as mean ± SEM. N = 3–6 mice / group. [Figure 12] Figure 1 shows the endpoint tumor weight of the BT474 human breast cancer model subcutaneously inoculated into BALB / c-nu mice in different treatment groups. Data are expressed as mean ± SEM. Compared to the PBS group, an independent sample t-test was used. N = 3–6 mice / group. [Figure 13] Figure 1 shows the body weight change of BT474 human breast cancer model subcutaneously inoculated into BALB / c-nu mice in different treatment groups. Average body weight is expressed as mean ± SEM. N = 3–6 mice / group. DETAILED DESCRIPTION OF THE INVENTION

[0024] After extensive and detailed research, the present inventors have unexpectedly and for the first time developed an ADC complex formed based on the self-assembly of complementary nucleic acid strands, as well as its preparation method and application. Based on the characteristics of this self-assembly, the present inventors have developed an ADC drug assembly unit library that can be used to prepare ADC complexes in real time. Using this preparation method, the assembly unit library can rapidly, efficiently, and cost-effectively prepare ADC drugs with highly specific targeting and highly uniform DARs according to the needs of personalized treatment. Based on this, the present invention has been completed.

[0025] Specifically, the present invention provides a multivalent protein drug, which comprises n protein drug units, wherein each drug unit comprises the same type of drug element moiety and different nucleic acid element moieties linked to the drug element moiety, n is a positive integer of ≧2, the n different nucleic acid element moieties form an n-mer in a nucleic acid base complementary manner, thereby constituting the multivalent protein drug, and the multivalent protein drug of the present invention forms a stable nucleic acid base complementary pair structure only by rapid assembly (for example, within 1 minute) (not by complex peptide bonds or other chemical modifications, etc.). According to experiments, it is shown that the drug of the present invention can increase the molecular weight by increasing the price, thereby extending the half-life in the body of animals.

[0026] Specifically, the present invention provides an ADC complex drug based on a complementary paired nucleic acid backbone, which comprises n units that can accurately complete self-assembly to form an n-mer complex, and n is a positive integer of 2 or more. Here, m targeting monomers are antibodies or proteins of target cells linked to a single-stranded nucleic acid, k drug monomers are drugs (toxin payloads) linked to a single-stranded nucleic acid, n is a positive integer of 3 to 8, m (<n) is a positive integer of 1 to 4, k is a positive integer of 1 to (n - m), and in the polymer, the single-stranded nucleic acid of each monomer forms a complementary paired double-stranded nucleic acid with the single-stranded nucleic acid of another 1 to 3 monomers by base complementarity, thereby constituting the n-mer ADC complex. The ADC complex drug of the present invention forms a stable nucleic acid base complementary pair structure only by rapid assembly (for example, within 1 minute), and at the same time has a uniform DAR (ADC drug obtained by random chemical modification). According to experiments, it is shown that the ADC complex drug of the present invention can cause endocytosis by specifically targeting cells, thereby killing the cells.

[0027] Term As used herein, the terms "antibody-drug coupling product of the invention," "antibody-drug conjugate of the invention," "coupling product of the invention," "antibody-drug conjugate of the invention," "polymeric ADC drug," "ADC complex based on a complementary nucleic acid backbone of the invention," "ADC of the invention," or "ADC drug of the invention" are used interchangeably and refer to having an ADC complex shown in the structure of Formula I.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in connection with a specifically stated value, indicates that the value may vary by no more than 1% from the stated value. For example, as used herein, the phrase "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0029] ADC In the present invention, the ADC refers to an antibody-drug conjugate comprising an antibody capable of specifically targeting a cell surface molecule, a cytotoxic drug, and a linker connecting the antibody and the drug. Typically, the antibodies in the ADC include, but are not limited to, nanobodies, single chain antibodies, Fabs, monoclonal antibodies, and the like.

[0030] Typically, drugs in the ADC include, but are not limited to, MMAE / MMAF, DM1 / DM4, calicheamicin, duocarmycin, PBD, amanitin, SN38, DXd, PNU-159682, and the like. In a preferred embodiment of the present invention, the antibody is an anti-HER2 single domain antibody, the drug is DM4 or MMAE, and the linker is a complementary single strand of nucleic acid.

[0031] Target antibody / protein A In the present invention, the antibody / protein component portion is capable of specifically targeting a cell surface molecule and inducing endocytosis. Typically, said antibodies / proteins include, but are not limited to, nanobodies, single chain antibodies, Fabs, monoclonal antibodies, cytokines, hormones (e.g., insulin, growth hormone, etc.), polypeptides.

[0032] In a preferred embodiment of the present invention, the antibody moiety is an anti-HER2 single domain antibody for targeting the HER2 receptor on BT474 cells, and the amino acid sequence of the anti-HER2 single domain antibody is as shown in SEQ ID NO:1. In another preferred embodiment of the present invention, the antibody component further comprises an anti-HSA antibody, the amino acid sequence of which is as set forth in SEQ ID NO:2.

[0033] SEQ ID NO:2, amino acid sequence of anti-HSA nanobody mutant: MGSAHHHHHHWSHPQFEKGGGSGGGSGGSAWSHPQFEKENLYFQSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGSC

[0034] Drug D In the present invention, the drug moiety is a small molecule or polypeptide drug load commonly used in ADC drugs, and after the targeting monomer-drug monomer complex is endocytosed by the cell, it enters the lysosome and then is released into the cytoplasm.

[0035] Typically, the drug element moiety includes, but is not limited to, microtubule toxins such as auristatins, maytansinoids, epothilone, toxoids, tubulysins, vinorelbine, etc.; DNA toxins such as calicheamicin, duocarmycins and analogs, PBD-dimers, doxorubicin, topotecan, blemycin A2, dactinomycin, mitomycin C, etc.; transcription toxins such as amatoxins, thailanstatin A, etc.; and inhibitors such as oligomycins, ipatasertib, etc.

[0036] In a preferred embodiment of the present invention, the drug moiety is MMAE, which acts on tubulin to disrupt the cellular microtubule network, causing cell cycle arrest and apoptosis.

[0037] Nucleic acid strand W As used herein, the terms "nucleic acid strand," "single-stranded nucleic acid," "single-stranded nucleic acid," and "nucleic acid element portion" are used interchangeably and all refer to a nucleic acid sequence in which the complementary paired nucleic acid backbone structure that constitutes the antibody-drug conjugate of the present invention is linked to an antibody or protein or drug (toxin payload).

[0038] In the present invention, the nucleic acid strand is a nucleic acid strand that is resistant to degradation in vivo and does not induce a strong innate immune response. Typically, the nucleic acid element portion includes, but is not limited to, an L-form nucleic acid, a peptide nucleic acid, a locked nucleic acid, a phosphoromorpholidate nucleic acid, a thio-modified nucleic acid, a 2'-fluoro-modified nucleic acid, a 5-hydroxymethylcytosine nucleic acid.

[0039] In a preferred embodiment of the present invention, the nucleic acid element portion is four left-handed DNAs of different sequences that can precisely self-assemble into a stable tetrameric nucleic acid backbone. L-type nucleic acids exist as mirror images of naturally occurring right-handed nucleic acids (D-nucleic acids), and are divided into left-handed DNA (L-DNA) and right-handed RNA (L-RNA). The left-handed (chiral center) is mainly present in the deoxyribose or ribose moiety of nucleic acids, exhibiting mirror image inversion. Therefore, L-type nucleic acids are not degraded by nucleases (e.g., exonucleases, endonucleases) that are ubiquitous in plasma.

[0040] Preparation method The present invention provides a method for preparing an ADC (antibody-drug conjugate) complex based on a complementary paired nucleic acid backbone, comprising the steps of:

[0041] 1. Design and Preparation of L-Nucleic Acid Strand Framework According to the present invention, an L-nucleic acid chain framework is formed by pairing two or more L-nucleic acid single strands. The 5' or 3' end of each L-nucleic acid single strand is activated to a group that can be subsequently modified (e.g., NH2, etc.), and then one end of a linker (e.g., SMCC, SBAP, etc.) is used to couple to an activated group on the L-nucleic acid single strand. The L-nucleic acid having a linker can be assembled into the desired L-nucleic acid chain framework. In another preferred example, an activated functional group (e.g., aldehyde, maleimide, etc.) at the 5' or 3' end of the L-nucleic acid single strand is included during nucleic acid synthesis. After it has been determined that the L-nucleic acid having a linker can successfully self-assemble to form a framework, the L-nucleic acid single strand having a linker can be coupled to an antibody, respectively, for subsequent assembly. The L-nucleic acid framework of the present invention can be basically prepared by the following steps:

[0042] 1.1. Design of single stranded L-nucleic acid capable of rapid self-assembly Determine the required number of polyvalents n (e.g., trimers, tetramers), and determine the required number of single-stranded L-nucleic acids n according to the number of polyvalents n. Design the corresponding number of single-stranded L-nucleic acids sequences, and optimize base pairing to adjust the stability of the target nucleic acid framework and reduce the possibility of non-specific pairing between nucleic acid strands. Details of nucleic acid sequence design are specifically described in the summary of the invention and examples. In a preferred embodiment of the present invention, L-DNA is prepared as follows. L-DNA1:5' AGGCGATCACAATCCAAATGAGCGTGTTACGG 3' (SEQ ID NO:4) L-DNA2:5' ACCGTAACACGCTCAAAACCGAAGTGCCAATT 3'(SEQ ID NO:5) L-DNA3:5' AAATTGGCACTTCGGAAAACTATGCGGCTGCT 3' (SEQ ID NO:6) L-DNA4:5' AAGCAGCCGCATAGTAAAGGATTGTGATCGCC 3'(SEQ ID NO:7)

[0043] 1.2. Activation of L-DNA or L-RNA The activation of L-nucleic acid involves modifying the active group at its 5'-end (X1) or 3'-end (X3) and then coupling with a linker. The modification of the active group can be customized by nucleic acid synthesis companies, and the linker generally has a bifunctional group, i.e., one end can be coupled to the active group of the nucleic acid, and the other end can be linked to a specific site on the protein (e.g., NH3, SH). According to a preferred embodiment of the present invention, all L-nucleic acids constituting the framework are modified at the 5' end with NH2, which completes the activation of the L-nucleic acids and allows their subsequent coupling with sulfhydryl groups on cysteines of proteins.

[0044] 2. Preparation of Protein-L-Nucleic Acid Complexes First, the 5' or 3' end of the L-nucleic acid is modified with an active group (e.g., NH2), then another active group is introduced into a specific site of the protein (e.g., introducing a cysteine ​​mutation into the C-terminus of the protein), and then the L-nucleic acid and the protein are crosslinked using a double linker molecule (e.g., SMCC), and the protein-L-nucleic acid complex is purified by methods such as affinity chromatography, ion exchange chromatography, etc.

[0045] 3. Preparation of drug-L-nucleic acid complex The NH2 modification is added to the 5' end of the L-nucleic acid to activate the L-nucleic acid and subsequently link it. The drug generally has an -SuO / -NHS reactive group, which can couple with the active group of the nucleic acid.

[0046] The main advantages of the present invention are: (1) The present invention enables flexible and efficient preparation of polymer-ADC conjugate drugs, enabling personalized ADC drugs to be prepared in a short time to meet the target requirements and drug payload combinations for personalized treatment. (2) The ADC drugs prepared according to the present invention can very easily achieve a highly uniform DAR. (3) The present invention can utilize the properties of modular and precise self-assembly to realize bispecific and even trispecific ADC drugs while ensuring highly uniform DAR. (4) The present invention utilizes the properties of modular and precise self-assembly to easily realize ADC drugs with two different drug payloads while ensuring a highly uniform DAR, thereby solving the problem of ADC drug resistance.

[0047] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods for which no specific conditions are given generally follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are percentages and parts by weight.

[0048] Example 1: Preparation of anti-HER2 nanobodies A His tag and a double Strep tag were added to the amino terminus of the nanobody for protein purification, and a cysteine ​​mutation was introduced at the carboxyl terminus of the nanobody for coupling to a nucleic acid anchoring site. The gene sequence of the anti-HER2 nanobody was optimized for E. coli codon preference and then subcloned into the pET-28b(+) plasmid. The amino acid sequence of the anti-HER2 nanobody is SEQ ID NO:1.

[0049] SEQ ID NO: 1, amino acid sequence of anti-HER2 nanobody mutant: MSAHHHHHHHWSHPQFEKGGGSGGGSGGSAWSHPQFEKENLYFQSEVQLVESGGGLVQAGGSLRLLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTLVTVSSGSC

[0050] Transform 1 μl of the constructed expression vector into E. coli shuffle T7. Transfer a single colony of the transformed shuffle T7 to LB medium (50 μg / mL kanamycin) and culture at 37°C until OD600 reaches 0.6-0.8. Induce expression by adding 0.1 mM IPTG and continue culturing at 16°C for 18-20 hours. Collect the single-cell protein after expression completion by centrifugation and resuspend in Tris buffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.4). Add a small amount of reducing agent (e.g., 10 μM TCEP) and a protease inhibitor cocktail (Sigma). Disrupt the bacteria by sonication, and then centrifuge the bacterial solution at 17,000 rpm for 30 minutes. Collect the supernatant. The nanobodies in the supernatant were purified using a His-tag affinity column. After the supernatant was passed through the column, the column was washed with Tris buffer (50 mM Tris-HCl, 200 mM NaCl, 10 μM TCEP, pH 7.4) containing 10 mM, 30 mM, and 50 mM imidazole until no impurity proteins were removed (the flow-through was collected and the A280 absorbance of the flow-through was detected using UV light to confirm complete removal of impurity proteins). The nanobodies bound to the column were then eluted with Tris buffer (50 mM Tris-HCl, 200 mM NaCl, 10 μM TCEP, pH 7.4) containing 250 mM imidazole, finally obtaining nanobodies of higher purity (Figure 2).

[0051] Example 2: Coupling and purification of nanobody-nucleic acid complexes The anti-HER2 nanobody purified in Example 1 was mixed with a 1-2 fold molar excess of SMCC-L-DNA single strand (the molar ratio can be determined through preliminary experiments) and subjected to a coupling reaction at 4°C overnight, resulting in a coupling efficiency of over 90% (Figure 3).

[0052] The unreacted SMCC-L-DNA single strands are removed using a Strep affinity column, and the nanobody and nanobody-L-DNA mixture is collected. The resulting buffer is then replaced with the loading buffer of an anion exchange column. Taking advantage of the negatively charged nature of DNA, the nanobody-L-DNA is further separated and purified using an anion exchange column (HiTrap Q HP column) to remove unreacted nanobodies. The separation process is achieved by gradient elution, using a loading buffer of 20 mM Tris-HCl, 15 mM NaCl, pH 8.5, and an elution buffer of 20 mM Tris-HCl, 1 M NaCl, pH 8.5, varying from 0 to 100%, resulting in successive peaks representing unreacted nanobody and nanobody-L-DNA. Because DNA exists in various conformations in the elution buffer, the elution peaks of the three nanobody-L-DNA conjugates appear during the gradient elution (Figure 4). Collect the nanobody-L-DNA and after concentration replace the buffer with 50 mM NaH2PO4, 150 mM NaCl, pH 7.4 using a PD-10 desalting column.

[0053] Example 3: Coupling of Toxin DM4-Nucleic Acid Conjugates Take the DM4-L-DNA1 coupling as an example. First, SPDB-DM4 powder is dissolved in 100% dimethylacetamide to obtain a 10 mM SPDB-DM4 solution. L-DNA1 is then dissolved in 100% phosphate buffer to obtain a 1 mM L-DNA1 solution. Next, the 1 mM L-DNA1 solution, 10 mM SPDB-DM4 solution, and phosphate reaction solution are mixed in a volume ratio of 2:5:3 and reacted overnight. The coupling efficiency can reach approximately 70%. The reaction products can be detected and analyzed by ESI ion trap liquid mass spectrometry in negative ion mode (Figure 5).

[0054] Example 4: Purification of Toxin DM4-Nucleic Acid Conjugates For example, the purification of DM4-L-DNA1 was performed by loading the DM4-L-DNA1 reaction mixture onto a phenyl-agarose gel hydrophobic purification column and eluting it in one step with 20% ethanol. This allowed complete separation of L-DNA1 and DM4-L-DNA1 (Figure 6). The purified products were then identified by ESI ion trap liquid chromatography mass spectrometry in the negative ion mode.

[0055] Example 5: Coupling of Toxin MMAE-Nucleic Acid Conjugates We use the coupling of MMAE-L-DNA1 as an example. First, SuO-vc-PAB-MMAE powder was dissolved in 100% dimethylacetamide to obtain a 10 mM SuO-vc-PAB-MMAE solution. L-DNA1 was then dissolved in 100% phosphate buffer to obtain a 1 mM L-DNA1 solution. The 1 mM L-DNA1 solution, 10 mM SuO-vc-PAB-MMAE solution, and phosphate buffer solution were then mixed in a volume ratio of 2:5:3 and reacted overnight. The coupling efficiency was detected and analyzed by ESI ion trap liquid chromatography mass spectrometry in negative ion mode. The coupling efficiency reached 100% (Figure 7). The reaction product was precipitated with 100% ethanol, allowing complete separation of unreacted SuO-vc-PAB-MMAE. After washing four times with 75% ethanol, the precipitate was dissolved in phosphate buffer.

[0056] Example 6: Self-assembly of nanobody-drug conjugates Below, double antibody double toxin NAPPA4-DM4 (1,2) -HER2 (3,4) Taking molecular structures as examples, the self-assembly process of nanobody-drug conjugates is demonstrated: DM4 is coupled to DNAs Nos. 1 and 2, and anti-HER2 nanobody (prepared in Example 1) is coupled to DNAs Nos. 3 and 4. The concentrations of DM4-L-DNA1, DM4-L-DNA2, anti-HER2 Nb-L-DNA3 conjugate, and anti-HER2 Nb-L-DNA4 conjugate were measured. Appropriate amounts of the above components were preheated at 37°C for 5 minutes, then mixed at a 1:1 molar ratio at 37°C and incubated for 1 minute to obtain NAPPA4-DM4. (1,2) -HER2 (3,4) Complete the self-assembly of the molecular structure nanobody-drug conjugate (Figure 8).

[0057] Example 7: In vitro cell killing experiments based on L-nucleic acid framework mediated antibody drug conjugates To further analyze the in vitro cell killing activity of antibody-drug conjugates based on the L-nucleic acid framework, four drug molecules were used: NAPPA4-MMAE (1,2) -HER2 (3,4) , NAPPA4-MMAE (1,2) -PD-L1 (3,4) , NAPPA4-MMAE (1,2) , NAPPA4-HER2 (3,4) were assembled, and their in vitro killing activities were evaluated using the HER2-positive cell line BT474 as a cell model, where the anti-HER2 nanobody used was the anti-HER2 nanobody prepared in the Examples, and the amino acid sequence of the anti-PD-L1 antibody used is as set forth in SEQ ID NO:3.

[0058] SEQ ID NO:3, amino acid sequence of anti-PD-L1 nanobody mutant: MGSAHHHHHHWSHPQFEKGGGSGGGSGGSSAWSHPQFEKENLYFQSEVQLLE SGGGEVQPGGSLRLSCAASGGIFAIKPISWYRQAPGKQREWVSTTTSSGATNYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNVFEYWGQGTLVTVKPGSC

[0059] BT474 cells were seeded into a 96-well plate at a cell density of 20,000 cells / well, and three duplicate wells were placed in a 37°C incubator. After 24 hours, the medium was replaced with fresh medium, and a specific concentration gradient of drugs was added. The plates were then incubated in a 37°C incubator. After 48-72 hours, the medium was replaced with fresh medium, 10% CCK8 solution was added, and the plates were incubated in the dark at 37°C. After 2-4 hours, the absorbance of each sample at 450 nm was measured using a microplate reader to calculate the drug-killing effect on the cells.

[0060] According to the experimental results, the control group NAPPA4-MMAE (1,2) -PD-L1 (3,4) Compared with the targeted drug NAPPA4-MMAE (1,2) -HER2 (3,4) shows that NAPPA4-MMAE has a more pronounced killing effect on the HER2-positive cell line BT474. (1,2) ,NAPPA4-HER2 (3,4) has almost no killing effect (Figure 9).

[0061] To further verify the in vitro killing effect of the L-nucleic acid framework-based antibody-drug conjugate against different tumor cell lines, two drug molecules, NAPPA4-MMAE, were used. (1,2) -HER2 (3) -HSA (4) and NAPPA4-MMAE (1,2) -HSA (4) In vitro cell killing was performed on selected tumor cell lines: BT474, SK-BR-3, NCI-N87, HCC1954, SKOV-3, and Calu-3. The detection kit used was CellTiter-Glo (Promega, G7572). A microplate reader was used to measure the absorbance at 450 nm of each sample and calculate the drug's cell killing activity.

[0062] Experimental results show that NAPPA4-MMAE (1,2) -HSA (4)Compared with NAPPA4-MMAE (1,2) -HER2 (3) -HSA (4) showed a more pronounced killing effect in all six tumor cell lines, and the killing effect was positively correlated with the reported HER2 expression level on the tumor cell surface (FIG. 10).

[0063] Example 8: In vivo tumor inhibition experiments of L-nucleic acid framework-mediated antibody drug conjugates To test the in vivo tumor-inhibitory activity of antibody-drug conjugates based on the L-nucleic acid framework, we used the drug NAPPA4-MMAE. (1,2) -HER2 (3,4) and NAPPA4-MMAE (1,2) -HER2 (3) -HSA (4) To evaluate the in vivo tumor-inhibitory activity of NAPPA4-MMAE, a BT474 human breast cancer tumor-bearing nude mouse model was established. (1,2) , NAPPA4-HER2 (3,4) and PBS group is set as control.

[0064] BT474 cells were cultured in 10% FBS-containing DMEM medium and maintained in a 5% CO2, 37°C, humidity-saturated incubator. Logarithmic growth phase BT474 cells were harvested and resuspended in DMEM basal medium, and Matrigel was added at a 1:1 ratio. Under sterile conditions, 0.2 mL of the cell suspension was subcutaneously inoculated into the right back of nude mice at an inoculation concentration of 1 × 10 7 The cells were 0.2 mL per mouse. The diameter of the transplanted tumor was measured using a caliper. The tumor diameter was determined to be between 100 and 300 mm. 3 At the age of 18, the animals were randomly divided into groups of 6. The day of grouping was defined as day D0, and the mice were administered the test drug, NAPPA4-MMAE. (1,2) -HER2 (3,4) , NAPPA4-MMAE (1,2) -HER2 (3) -HSA (4) , NAPPA4-MMAE (1,2) , NAPPA4-HER2 (3,4)The mice were intravenously injected with 125 nmol / kg of PBS control at doses of 125 nmol / kg. After the start of administration, tumor size was measured using a caliper and the mice were weighed on days D0, D3, D5, D7, D10, D12, D14, D17, D19, D21, D24, D26, and D28. The tumor volume was calculated as follows: 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 is.

[0065] The results of the changes in the average tumor volume of mice in different groups during the administration period (Figure 11) and the average tumor weight of mice in different groups at the end of the study (Figure 12) showed that the NAPPA4-MMAE group was significantly more potent than the PBS control group. (1,2) -HER2 (3,4) and NAPPA4-MMAE (1,2) -HER2 (3) -HSA (4) showed a significant in vivo tumor-inhibiting effect and was confirmed by the anti-HSA antibody-containing drug NAPPA4-MMAE. (1,2) -HER2 (3) -HSA (4) The drug NAPPA4-MMAE, which does not contain anti-HSA antibodies, (1,2) -HER2 (3,4) The drug NAPPA4-MMAE has a superior tumor-inhibiting effect in vivo compared to (1,2) and NAPPA4-HER2 (3,4) The mean body weight changes of mice in different groups throughout the experimental observation period remained within the normal range (Figure 13), indicating that under the single-dose condition of 125 nmol / kg, each test drug had no significant effect on the body weight of the mice.

[0066] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. 1. An antibody-drug conjugate (ADC) based on a complementary paired nucleic acid backbone, comprising: the antibody-drug conjugate is a polymer formed by conjugation of n monomers having complementary nucleic acid backbones, wherein the polymer is m targeting monomers, each of which is a cell surface-targeting antibody or protein linked to a single strand of nucleic acid; and k drug monomers, each of which is a drug (toxin payload) linked to a single strand of nucleic acid; Including, n is a positive integer from 2 to 8, m is a positive integer from 1 to 3, and m<n, and k is a positive integer from 1 to (n-m); In the polymer, a single-stranded nucleic acid of each monomer forms a complementary paired double-stranded nucleic acid with a single-stranded nucleic acid of another 1 to 3 monomers by base complementarity, thereby forming a complementary paired nucleic acid backbone structure. The antibody drug conjugate.

2. The targeting monomer has the structure of Formula I: A-W-D0(I) The drug monomer has the structure of Formula II: D1-W-D2(II) In the formula: A is an antibody or a protein, W is a nucleic acid single-stranded sequence; D0 is none or drug; D1 is none or a drug; D2 is none or a drug; "-" is a linker or bond, At least one of D0, D1 and D2 is a drug; The antibody-drug conjugate of claim 1.

3. A is an antibody or a protein that specifically binds to a cell surface receptor and thereby induces endocytosis; The antibody-drug conjugate of claim 2.

4. D1 / D2 are small molecule or polypeptide toxins used to kill cells; The antibody-drug conjugate of claim 2.

5. The single-stranded nucleic acid is resistant to degradation and is selected from the group consisting of L-form nucleic acids, peptide nucleic acids, locked nucleic acids, phosphoromorpholidate nucleic acids, thio-modified nucleic acids, 2'-fluoro-modified nucleic acids, 5-hydroxymethylcytosine nucleic acids, or combinations thereof. The antibody-drug conjugate of claim 2.

6. The nucleic acid single-stranded sequence W in the targeting monomer (Formula I) and drug monomer (Formula II) has the structure shown in Formula III: X1-R1-X2-R2-X3 (III) where: R1 is base-complementary pairing region 1; R2 is base-complementary pairing region 2; X1, X2 and X3 are each independently null or redundant nucleic acids; "-" is a bond; The antibody-drug conjugate of claim 2.

7. The length of R1 and R2 is independently 10 to 20 bases, preferably 14 to 16 bases; The antibody-drug conjugate of claim 6.

8. R1 of each monomer forms a base-complementary pairing structure with R2 of the left adjacent (or left-side) monomer, and R2 forms a base-complementary pairing structure with R1 of the right adjacent (or right-side) monomer; The antibody-drug conjugate of claim 6.

9. 1. A pharmaceutical composition comprising: The pharmaceutical composition comprises: (a) an antibody-drug conjugate based on the complementary paired nucleic acid backbone of claim 1; wherein the targeting monomer is selected from a targeting monomer library, the drug monomer is selected from a drug monomer library, and (b) a pharmaceutically acceptable carrier; wherein the assembly units in the targeting monomer library carry antibodies or proteins that can be endocytosed by specifically binding to receptors on the surface of cancer cells, and the assembly units in the drug monomer library carry small molecule or polypeptide drug moieties that kill the cells. The pharmaceutical composition.

10. 1. A nucleic acid sequence library, comprising: The nucleic acid library comprises nucleic acid sequences used to form antibody-drug conjugates based on the complementary paired nucleic acid backbone of claim 1. The nucleic acid sequence library.

11. The nucleic acid sequence has the structure shown in Formula III: X1-R1-X2-R2-X3 (III) where: R1 is base-complementary pairing region 1; R2 is base-complementary pairing region 2; X1, X2 and X3 are each independently null or redundant nucleic acids; "-" is a bond The nucleic acid sequence library of claim 10.

12. Use of the nucleic acid sequence library according to claim 10, The complementary paired nucleic acid backbone of claim 1 is used to prepare an antibody-drug conjugate. The above use.

13. 10. A method for preparing the antibody drug conjugate of claim 1, comprising: (1) forming a nucleic acid-drug assembly unit by coupling a single stranded nucleic acid and a cytotoxic drug to a chemical fixed point; (2) fixed-point coupling of an antibody with a complementary nucleic acid single strand to form an antibody-nucleic acid assembly unit; (3) Rapidly self-assembling an antibody-nucleic acid assembly unit and a plurality of complementary nucleic acid-drug assembly units via complementary nucleic acid sequences to form the antibody-drug conjugate.

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