Methods for constructing and applying nucleic acid self-assembly-mediated ADC drugs

Abstract

The present invention relates to the field of biotechnological pharmaceuticals. Specifically, the present invention provides an ADC (antibody-drug conjugate) complex based on a complementary paired nucleic acid backbone. The ADC complex is a polymer formed by the complexation of n monomers having a complementary paired nucleic acid backbone. Here, the polymer includes m "targeting monomers", which are antibodies or proteins targeting the cell surface linked to a single-stranded nucleic acid, and k "drug monomers", which are drugs (toxin payloads) 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 paired nucleic acid backbone structure by forming a complementary paired double-stranded structure with the single-stranded nucleic acid of another 1 to 3 monomers through base complementarity.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology pharmaceuticals, and more specifically to methods and applications for constructing nucleic acid polymerization-mediated ADCs (antibody-drug conjugates). [Background technology]

[0002] ADCs are targeted drugs developed for cancer treatment that deliver highly chemotherapeutic drugs specifically to cancer cells, reducing nonspecific cell death in the body. The mechanism of ADCs involves antibodies binding to receptors on the surface of cancer cells, leading to endocytosis. The drug is then released within lysosomes, escapes the lysosomes, enters the cytoplasm, and induces cell death. While the concept of ADCs has been proposed for a long time, the development of related drugs was once slow and complex due to various limitations in knowledge and technical conditions. However, with technological advancements and the accumulation of clinical research experience, ADC drugs have gradually emerged from their stagnation in recent years and have become one of the mainstreams in the field of biopharmaceutical development.

[0003] Currently, in this field, the preparation of ADC molecules focuses on coupling a compound consisting of a chemical linker and a small molecule load (linker-payload) with a monoclonal antibody molecule. Classical methods involve fixed-point coupling of the linker to the lysine or cysteine ​​site on the surface of the antibody molecule via specific chemical reactions. However, antibody molecules typically have multiple lysine or cysteine ​​sites, and the steric hindrance effects near each amino acid are not exactly the same. This leads to heterogeneity in the number of linker payloads actually coupled to the antibody molecule, i.e., the Drug-Antibody Ratio (DAR), which further introduces significant uncertainty into the analysis of various aspects of ADCs, such as efficacy, pharmacokinetics, biodistribution, and toxicology, and also contributes to high production costs of ADCs. However, this DAR heterogeneity is due to inherent flaws in conventional coupling methods, and even FDA-approved ADC molecules and DARs are highly heterogeneous.

[0004] Therefore, there is an urgent need in this field to develop a simple, flexible, efficient, and modular method for preparing ADC drugs that enables ADCs to have a completely uniform DAR. [Overview of the project] [Problems that the invention aims to solve]

[0005] The main objective of the present invention is to provide a simple, flexible, efficient, and modular method for preparing an ADC drug that enables the ADC 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 to form 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 absent or a drug, D1 is absent or a drug, D2 is absent 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 example, 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 shown in SEQ ID NO:1.

[0010] In another preferred example, D1 / D2 are small molecules or polypeptide toxins for killing cells. In another preferred example, the nucleic acid chain is resistant to degradation and is selected from the group consisting of L-type 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 a combination thereof.

[0011] In another preferred example, in the polymer, component A of each targeted monomer is either 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 example, in the polymer, W of each monomer is different. In another preferred example, drug D1 or D2 can be ligated to the sum of both ends and / or the central nucleotide of W by chemical modification.

[0013] In another preferred example, the single-stranded nucleic acid sequence W in the targeted monomer (Formula I) and the drug monomer (Formula II) has the structure shown in Formula III, X1-R1-X2-R2-X3(III) Here, R1 is the base complementary pairing region 1, R2 is the base complementary pairing region 2, X1, X2, and X3 are each independently either none or redundant nucleic acids. The hyphen "-" indicates a conjunction.

[0014] In another preferred example, the lengths of R1 and R2 are 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 its left (or left side), and R2 forms a base complementary pairing structure with R1 of the monomer to its right (or right side).

[0016] A second aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition is (a) an antibody-drug conjugate based on a complementary pairing nucleic acid skeleton as described in the first embodiment, Here, the targeted monomer is selected from the targeted monomer library, the drug monomer is selected from the drug monomer library, and (b) comprising a pharmaceutically acceptable carrier.

[0017] In another preferred example, the pharmaceutical composition comprises a 2-8 mer complex. In another preferred example, the assembly unit within the targeted monomer library holds an antibody or protein that can induce endocytosis by specifically binding to a surface receptor on cancer cells. In another preferred example, the assembly unit within the drug monomer library holds a small molecule or polypeptide drug moiety that kills cells.

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

[0019] A third aspect of the present invention provides a nucleic acid sequence library comprising nucleic acid sequences for forming antibody-drug conjugates based on complementary paired nucleic acid skeletons as described in the first aspect. In another preferred example, the nucleic acid sequence W has the structure shown in formula III, X1-R1-X2-R2-X3(III) Here, R1 is the base complementary pairing region 1, R2 is the base complementary pairing region 2, X1, X2, and X3 are each independently either none or redundant nucleic acids. The hyphen "-" indicates a conjunction.

[0020] A fourth aspect of the present invention provides an application of the nucleic acid sequence library described in the third aspect for use in preparing antibody-drug conjugates of complementary pairing nucleic acid skeletons described in the first aspect.

[0021] A fifth aspect of the present invention is: (1) A step of forming a nucleic acid-drug assembly unit by chemical fixed-point coupling of a single strand of nucleic acid and a cytotoxic drug, (2) The step of forming an antibody-nucleic acid assembly unit by fixing-point coupling of an antibody and a complementary nucleic acid single strand, (3) A method for preparing an antibody-drug conjugate according to the first embodiment, comprising the step of 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, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0023] [Figure 1] This diagram shows the mode of self-assembly of ADC drugs that mediate complementary pairing of nucleic acid chains. [Figure 2] This image shows the electrophoretic gel image of nickel column affinity purified anti-HER2 nanobodies. [Figure 3] Electrophoretic gel images showing the relationship between anti-HER2 nanobodies and L-DNA coupling efficiency are shown. [Figure 4] This shows an ion-exchange purified anti-HER2 nanobody-L-DNA conjugate. [Figure 5] This paper demonstrates the identification of DM4-L-DNA1 coupling efficiency by anion-mode ESI ion trap liquid chromatography-mass spectrometry. [Figure 6] The separation results using a hydrophobic DM4-L-DNA1 phenyl-agarose gel purification column are shown. [Figure 7] This paper demonstrates the identification of MMAE-L-DNA1 coupling efficiency by anion-mode ESI ion trap liquid chromatography-mass spectrometry. [Figure 8] Self-assembling electrophoresis gel images of nanobody drug conjugates are shown. Here, a. SDS-PAGE, b. 2% agarose DNA gel. [Figure 9] This paper demonstrates the in vitro cell-killing activity evaluation of nanobody drug conjugates. [Figure 10] This paper demonstrates the in vitro killing activity evaluation of nanobody drug conjugates in different tumor cell lines. [Figure 11]This shows the changes in tumor volume of BT474 human breast cancer model mice subcutaneously inoculated with BALB / c-nu in different dose groups. Data are expressed as mean ± SEM. N=3-6 mice / group. [Figure 12] This report shows the endpoint tumor weight of BT474 human breast cancer model mice subcutaneously inoculated with BALB / c-nu in different dose groups. Data are expressed as mean ± SEM. An independent sample t-test is used compared to the PBS group. N=3-6 mice / group. [Figure 13] This shows the weight changes of BALB / c-nu mice subcutaneously inoculated with BT474, a human breast cancer model, in different dose groups. Mean weight is expressed as mean ± SEM. N=3-6 mice / group. [Modes for carrying out the invention]

[0024] As a result of extensive and detailed research, the inventors have unexpectedly developed, for the first time, ADC complexes formed based on nucleic acid chain complementary pairing self-assembly, methods for preparing them, and applications. Based on the self-assembly characteristics, the inventors have developed for the first time 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 be rapidly, efficiently, and cost-effectively prepared to meet individualized therapeutic needs, producing ADC drugs with highly specific targeting and high homogeneity DAR. Based on this, the present invention was 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 a different nucleic acid element moiety 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 complementary manner of nucleic acid bases, 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 conjugate 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 conjugate. The ADC conjugate 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 conjugate drug of the present invention can cause endocytosis by specifically targeting cells, thereby killing the cells.

[0027] Term As used herein, “the antibody-drug coupling of the present invention,” “the antibody-drug conjugate of the present invention,” “the coupling of the present invention,” “the antibody-drug conjugate of the present invention,” “polymer ADC drug,” “the ADC complex based on the complementary pairing nucleic acid skeleton of the present invention,” “the ADC of the present invention,” or “the ADC drug of the present invention” means that the ADC complex is interchangeable and has the structure shown in Formula I.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs. As used herein, when used in relation to a specifically stated value, the term “about” means that the value may vary by no more than 1% from the stated value. For example, as used herein, the expression “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 cell surface molecules, a cytotoxic drug, and a linker that connects the antibody and the drug. Typically, the antibodies in the ADC include, but are not limited to, nanobodies, single-chain antibodies, Fabs, monoclonal antibodies, etc.

[0030] Typically, the drugs in the ADC include, but are not limited to, MMAE / MMAF, DM1 / DM4, calicheamicin, duocarmycin, PBD, amanitin, SN38, DXd, and PNU-159682. 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 nucleic acid single-strand.

[0031] Target antibody / protein A In the present invention, the antibody / protein component can specifically target cell surface molecules and induce endocytosis. Typically, the antibodies / proteins include, but are not limited to, nanobodies, single-chain antibodies, Fabs, monoclonal antibodies, cytokines, hormones (e.g., insulin, growth hormone, etc.), and polypeptides.

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

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

[0034] Drug D In the present invention, the drug component is a small molecule or polypeptide drug load commonly used in ADC drugs, and the targeted monomer-drug monomer complex is endocytized by cells, enters lysosomes, and is then released into the cytoplasm.

[0035] Typically, the drug component includes, but is not limited to, microtubule toxins such as auristatins, maytansinoids, epothilone, toxoids, tubulysins, and vinorelbine; DNA toxins such as calicheamycin, duocarmycin and analogs, PBD dimers, doxorubicin, topotecan, blemycin A2, dactinomycin, and mitomycin C; transcription toxins such as amatoxins and thailanstatin A; and inhibitors such as oligomycin and ipatasertib.

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

[0037] Nucleic acid strand W As used herein, “nucleic acid chain,” “single-stranded nucleic acid,” “single-stranded nucleic acid,” and “nucleic acid element moiety” are interchangeable terms and all refer to nucleic acid sequences to which complementary paired nucleic acid backbone structures constituting the antibody-drug conjugate of the present invention are linked to an antibody, protein, or drug (toxin payload).

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

[0039] In a preferred embodiment of the present invention, the nucleic acid element portion consists of four levorotatory DNA molecules of different sequences that can precisely self-assemble into a stable tetrameric nucleic acid backbone. L-type nucleic acids refer to those that exist as a mirror image of naturally occurring dextrorotatory nucleic acids (D-nucleic acids) and are divided into levorotatory DNA (L-DNA) and dextrorotatory RNA (L-RNA). The levorotatory (chiral center) is mainly located in the deoxyribose or ribose portion of the nucleic acid and exhibits 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 pairing nucleic acid skeleton, comprising the following steps:

[0041] 1. Design and preparation of L-nucleic acid chain frameworks According to the present invention, an L-nucleotide chain framework is formed by pairing two or more L-nucleotide single strands. The 5' or 3' end of each L-nucleotide single strand is activated with a subsequently modifiable group (e.g., NH2), and then coupled to the activating group on the L-nucleotide single strand using one end of a linker (e.g., SMCC, SBAP, etc.). The L-nucleotides having linkers can be assembled into a desired L-nucleotide chain framework. In another preferred example, the activating functional group at the 5' or 3' end of the L-nucleotide single strand (e.g., aldehyde, maleimide, etc.) is included in the nucleic acid synthesis. After it is determined that the L-nucleotides having linkers can successfully self-assemble to form a framework, the L-nucleotide single strands having linkers can be coupled to antibodies for subsequent assembly. The L-nucleotide framework of the present invention can be prepared in the following steps:

[0042] 1.1. Design of a rapidly self-assembling single-strand L-nucleotide The required number of nucleotides n (e.g., trimer, tetramer) is determined, the required number of L-nucleotide single strands n is determined according to the number of nucleotides n, the corresponding number of L-nucleotide single-strand sequences are designed, and the stability of the target nucleic acid framework is regulated by optimizing base pairing, thereby reducing the possibility of nonspecific 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, the following L-DNA is prepared. 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 Activation of L-nucleic acids involves modification of the active group at its 5' (X1) or 3' (X3) end and subsequent linker coupling. The modification of the active group can be customized by the nucleic acid synthesis company, 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 their 5' ends with NH2, thereby completing the activation of the L-nucleic acids, which can then be coupled with a sulfhydryl group on the cysteine ​​of the protein.

[0044] 2. Method for preparing 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 to a specific site on the protein (e.g., a cysteine ​​mutation is introduced at the C-terminus of the protein), and then the L-nucleic acid and protein are crosslinked using a double linker linker molecule (e.g., SMCC), and the protein-L-nucleic acid complex is purified by methods such as affinity chromatography or ion exchange chromatography.

[0045] 3. Method for preparing drug-L-nucleic acid complexes An NH2 modification is added to the 5' end of the L-nucleic acid to activate the L-nucleic acid and facilitate subsequent ligation. Drugs generally have an -SuO / -NHS reactive group, which can be coupled with the active group of the nucleic acid.

[0046] The main advantages of this invention are as follows: (1) The present invention enables the flexible and efficient preparation of polymer ADC complex drugs, allowing for the rapid preparation of personalized ADC drugs for combinations of target requirements and drug payloads for personalized therapy. (2) The ADC drug prepared according to the present invention can achieve a highly uniform DAR very easily. (3) The present invention makes it possible to ensure a highly uniform DAR while realizing bispecific, or even triplicate, ADC drugs by utilizing modular and precise self-assembly properties. (4) The present invention can solve the problem of ADC drug resistance by using modular and precise self-assembly properties to easily realize ADC drugs having two different drug payloads while ensuring a highly uniform DAR.

[0047] The present invention will be further described below in conjunction with specific examples. These examples are used solely to illustrate the present invention and should not be used to limit its scope. In the following examples, experimental methods that do not specify conditions typically follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: An Experimental Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions proposed by the manufacturer. Unless otherwise specified, percentages and quantities refer to percentages and quantities by weight.

[0048] Example 1: Preparation of anti-HER2 nanobodies To purify the protein, His tags and double Strep tags are added to the amino group ends of the nanobodies, and cysteine ​​mutations are introduced to the carboxyl group ends of the nanobodies to couple the nucleic acid fixation sites. The gene sequence of the anti-HER2 nanobodies is optimized to codons preferred by E. coli, and then subcloned into the pET-28b(+) plasmid. The amino acid sequence of the anti-HER2 nanobodies is SEQ ID NO:1.

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

[0050] 1 µl of the constructed expression vector is collected and transformed into E. coli shuffle T7. The transformed shuffle T7 single colonies are transferred to LB medium (50 µg / mL kanamycin) and cultured at 37°C until OD600 = 0.6-0.8. Expression is induced by adding 0.1 mM IPTG, and the cells are cultured at 16°C for 18-20 hours. The single-cell protein after expression is collected by centrifugation, resuspended in Tris buffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.4), and a small amount of reducing agent (e.g., 10 μM TCEP) and a protease inhibitor cocktail (Sigma) are added. The bacteria are lysed by sonication, and the bacterial suspension is centrifuged at 17000 rpm for 30 minutes, after which the supernatant is collected. Nanobodies in the supernatant were purified using a His-tagged affinity column. After passing the supernatant through the column, the column was washed with Tris buffer containing 10 mM, 30 mM, and 50 mM imidazole (50 mM Tris-HCl, 200 mM NaCl, 10 μM TCEP, pH 7.4) until no more impurity proteins flowed out (the flow-through solution was collected, and the A280 absorption of the flow-through solution was detected using UV to confirm whether the impurity proteins had been completely removed). The nanobodies bound to the column were then eluted with Tris buffer containing 250 mM imidazole (50 mM Tris-HCl, 200 mM NaCl, 10 μM TCEP, pH 7.4) to obtain nanobodies of higher purity (Figure 2).

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

[0052] Unreacted SMCC-L-DNA single strands are removed using a Strep affinity column, and the nanobodies and nanobodies-L-DNA mixture is collected. This buffer is replaced with the loading buffer for an anion exchange column. Utilizing the negatively charged properties of DNA, nanobodies-L-DNA are 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 20 mM Tris-HCl, 15 mM NaCl, pH 8.5 loading buffer, and 20 mM Tris-HCl, 1 M NaCl, pH 8.5, 0-100% elution buffer, resulting in consecutive peaks for unreacted nanobodies and nanobodies-L-DNA. Because DNA exists in various three-dimensional structures in the elution buffer, three elution peaks for nanobodies-L-DNA conjugates appear during gradient elution (Figure 4). Nanobody-L-DNA is collected and concentrated, then the buffer is replaced 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 conjugate Let's take the coupling of DM4-L-DNA1 as an example. First, SPDB-DM4 powder is dissolved in 100% dimethylacetamide to obtain a 10 mM SPDB-DM4 solution. L-DNA1 is dissolved in 100% phosphate buffer to obtain a 1 mM L-DNA1 solution. Next, the 1 mM L-DNA1 solution, the 10 mM SPDB-DM4 solution, and the phosphate reaction solution are mixed in a volume ratio of 2:5:3 and reacted overnight, achieving a coupling efficiency of approximately 70%. The reaction product can be detected and analyzed by ESI ion trap liquid mass spectrometry in anion mode (Figure 5).

[0054] Example 4: Purification of the toxin DM4-nucleic acid conjugate Let's take the purification of DM4-L-DNA1 as an example. The reaction product of DM4-L-DNA1 is loaded onto a phenyl-agarose gel hydrophobic purification column and eluted in one step with 20% ethanol, allowing for complete separation of L-DNA1 and DM4-L-DNA1 (Figure 6). The separated products can then be identified as purified products by anion-mode ESI ion trap liquid chromatography-mass spectrometry.

[0055] Example 5: Coupling of toxin MMAE-nucleic acid conjugate Let's take the coupling of MMAE-L-DNA1 as an example. First, SuO-vc-PAB-MMAE powder is dissolved in 100% dimethylacetamide to obtain a 10 mM SuO-vc-PAB-MMAE solution. L-DNA1 is dissolved in 100% phosphate buffer to obtain a 1 mM L-DNA1 solution. Next, the 1 mM L-DNA1 solution, the 10 mM SuO-vc-PAB-MMAE solution, and the phosphate reaction solution are mixed in a volume ratio of 2:5:3 and allowed to react overnight. Detection and analysis are performed by anion mode ESI ion trap liquid chromatography-mass spectrometry, and the coupling efficiency can reach 100% (Figure 7). The reaction product can be precipitated with 100% ethanol to completely separate it from the unreacted SuO-vc-PAB-MMAE, and after washing four times with 75% ethanol, the precipitate is 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) The self-assembly process of nanobody drug conjugates is illustrated using molecular structure as an example. DM4 is coupled to DNA numbers 1 and 2, and the anti-HER2 nanobody (prepared in Example 1) is coupled to DNA numbers 3 and 4. Measure the concentrations of DM4-L-DNA1, DM4-L-DNA2, anti-HER2 Nb-L-DNA3 conjugate, and anti-HER2 Nb-L-DNA4 conjugate respectively. Take an appropriate amount of the above components, preheat at 37 °C for 5 minutes, then mix at a molar ratio of 1:1 under the condition of 37 °C, and incubate for 1 minute to obtain NAPPA4-DM4 (1,2) -HER2 (3,4) Complete the self-assembly of the nanobody drug conjugate with the molecular structure (Figure 8).

[0057] Example 7: In vitro cell killing experiment based on L-nucleic acid framework-mediated antibody drug conjugate To further analyze the in vitro cell killing activity based on the antibody drug conjugate of L-nucleic acid framework, four drug molecules 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 respectively, and the HER2-positive cell line BT474 was used as a cell model to evaluate the in vitro killing activity. Here, the anti-HER2 nanobody used was the anti-HER2 nanobody prepared in the example, and the amino acid sequence of the anti-PD-L1 antibody used was as shown 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 inoculated into a 96-well plate with a cell density of 20,000 cells / well, and three duplicate wells were placed. The plates were incubated in a 37°C incubator, and after 24 hours, the medium was changed to fresh medium. A specific concentration gradient of the drug was added, and the plates were continued to incubate in a 37°C incubator. After 48-72 hours, the medium was changed to fresh medium, and a 10% CCK8 solution was added. The plates were then incubated in the dark at 37°C, and after 2-4 hours, the absorbance at 450 nm of each sample was measured using a microplate reader to calculate the drug's ability to kill cells.

[0060] According to the experimental results, the control group NAPPA4-MMAE (1,2) -PD-L1 (3,4) In comparison, the targeted drug NAPPA4-MMAE (1,2) -HER2 (3,4) This demonstrates a more pronounced killing effect against the HER2-positive cell line BT474. NAPPA4-MMAE (1,2) NAPPA4-HER2 (3,4) It has almost no lethal effect (Figure 9).

[0061] To further investigate the in vitro killing effects of antibody-drug conjugates based on the L-nucleic acid framework 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 toxicology was performed on BT474, SK-BR-3, NCI-N87, HCC1954, SKOV-3, and Calu-3 tumor cell lines. The detection kit used was CellTiter-Glo (Promega, G7572). A microplate reader was used to measure the absorbance of each sample at 450 nm and calculate the drug's toxicity to the cells.

[0062] According to experimental results, NAPPA4-MMAE (1,2) -HSA (4)Compared to NAPPA4-MMAE (1,2) -HER2 (3) -HSA (4) It 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 surface of tumor cells (Figure 10).

[0063] Example 8: In vivo tumor inhibition experiment 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, 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, we constructed a nude mouse model carrying BT474 human breast cancer tumors and evaluated its in vivo tumor inhibitory activity. (1,2) NAPPA4-HER2 (3,4) The PBS group is set up as a control.

[0064] BT474 cells were cultured in DMEM medium containing 10% FBS and maintained in a 37°C saturated humidity incubator with 5% CO2. Logarithmic growth phase BT474 cells were collected, resuspended in DMEM base medium, and Matrigel was added in a 1:1 ratio. Under sterile conditions, 0.2 mL of the cell suspension was subcutaneously inoculated into the right back of nude mice, with an inoculation concentration of 1 × 10⁻⁶. 7 The cell volume is 0.2 mL per mouse. The diameter of the transplanted tumor was measured using calipers, and the tumor was 100-300 mm. 3 Once the animals have grown to a certain size, they are randomly divided into groups of six. The day of group division is defined as Day D0, and the mice are given the drug NAPPA4-MMAE. (1,2) -HER2 (3,4) NAPPA4-MMAE (1,2) -HER2 (3) -HSA (4) NAPPA4-MMAE (1,2) NAPPA4-HER2 (3,4)A single intravenous injection of PBS control was administered at a dose of 125 nmol / kg. After the start of administration, on days D0, D3, D5, D7, D10, D12, D14, D17, D19, D21, D24, D26, and D28, the size of the tumor was detected using calipers and the body weight of the mice was weighed. The method for calculating tumor volume is: tumor volume (mm 3 ) = 0.5 × Tumor length × Tumor width 2 That is the case.

[0065] Based on the results of the change in mean tumor volume of mice in different groups during the administration period (Figure 11) and the mean tumor weight of mice in different groups at the end of the study (Figure 12), NAPPA4-MMAE was superior to the PBS control group. (1,2) -HER2 (3,4) and NAPPA4-MMAE (1,2) -HER2 (3) -HSA (4) It has a remarkable in vivo tumor inhibitory effect, and the anti-HSA antibody-containing drug NAPPA4-MMAE (1,2) -HER2 (3) -HSA (4) This is a drug that does not contain anti-HSA antibodies: NAPPA4-MMAE (1,2) -HER2 (3,4) It has superior in vivo tumor-inhibiting effects compared to the drug NAPPA4-MMAE. (1,2) and NAPPA4-HER2 (3,4) This indicates that there is little to no lethal effect. Throughout the entire experimental observation period, the mean body weight changes of the different groups of mice remained within the normal range of variation (Figure 13), which indicates that each test drug does not have a significant effect on the body weight of the mice under a single dose condition of 125 nmol / kg.

[0066] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. An antibody-drug conjugate (ADC) based on a complementary pairing nucleic acid skeleton, The antibody-drug conjugate is a polymer formed by the compounding of n monomers having a complementary pairing nucleic acid skeleton. Here, the polymer is m targeted monomers, the "targeted monomer" is an antibody that targets the cell surface linked to a single strand of nucleic acid, and k drug monomers, the "drug monomers" being drugs linked to a single strand of nucleic acid, Includes, n is a positive integer between 3 and 8, m is a positive integer between 2 and 3 such that m < n, and k is a positive integer between 1 and (n - m). In the polymer described above, each monomer's nucleic acid single strand forms a complementary paired double strand with one to three other monomer's nucleic acid single strands through base complementarity, thereby forming a complementary paired nucleic acid skeletal structure. The targeted 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, W is a single-stranded nucleic acid sequence, D0 is either none or a drug. D1 is a drug, D2 is a drug, "-" is a linker or coupling. Single-stranded nucleic acids are resistant to degradation and are selected from the group consisting of L-type nucleic acids, locked nucleic acids, phosphoromolhodate nucleic acids, thio-modified nucleic acids, 2'-fluoro-modified nucleic acids, 5-hydroxymethylcytosine nucleic acids, or combinations thereof. The drug is a small molecule toxin for killing cells, and the antibody is characterized by being a nanobody or single-chain antibody. The aforementioned antibody-drug conjugate.

2. One of the targeted monomers, A, is an antibody that specifically binds to a cell surface receptor and induces endocytosis, while the other targeted monomer, A, is an anti-HSA antibody. The antibody-drug conjugate according to claim 1.

3. The drug is characterized by being selected from the group consisting of MMAE, MMAF, DM1, DM4, calicheamycin, duocalmycin, PBD, amanitin, SN38, DXd, and PNU-159682. The antibody-drug conjugate according to claim 1.

4. The single strand of nucleic acid is characterized by being an L-type nucleic acid. The antibody-drug conjugate according to claim 1.

5. The single strand of nucleic acid is characterized by being a phosphoromorphonte nucleic acid. The antibody-drug conjugate according to claim 1.

6. In the aforementioned targeted monomer (Formula I) and drug monomer (Formula II), W has the structure shown in Formula III. X1-R1-X2-R2-X3 (III) Here, R1 is the base complementary pairing region 1, R2 is the base complementary pairing region 2, X1, X2, and X3 are each independently redundant nucleic acids, with the length of X1 being 0 to 5 bases, the length of X2 being 0 to 3 bases, and the length of X3 being 0 to 5 bases. "-" indicates a connection, Each monomer's R1 forms a base-complementary pairing structure with the R2 of another monomer, and each monomer's R2 forms a base-complementary pairing structure with the R1 of another monomer. The antibody-drug conjugate according to claim 1.

7. The lengths of R1 and R2 are each independently 10 to 20 base pairs. The antibody-drug conjugate according to claim 6.

8. The lengths of R1 and R2 are each independently 14 to 16 base pairs. The antibody-drug conjugate according to claim 6.

9. Each drug monomer is characterized in that component D1 is the same or different, and component D2 is the same or different. The antibody-drug conjugate according to claim 1.

10. A pharmaceutical composition, The aforementioned pharmaceutical composition, (a) an antibody-drug conjugate based on the complementary pairing nucleic acid skeleton described in claim 1, and (b) comprising a pharmaceutically acceptable carrier, Here, the targeted monomer of the antibody-drug conjugate holds an antibody that specifically binds to a cell surface receptor and causes endocytosis, and the drug monomer of the antibody-drug conjugate holds a small molecule toxin that kills cells. The aforementioned pharmaceutical composition.

11. The use of a nucleic acid sequence library used to prepare an antibody-drug conjugate based on a complementary paired nucleic acid skeleton as described in claim 1, The nucleic acid sequence library comprises nucleic acid sequences used to form antibody-drug conjugates based on complementary paired nucleic acid skeletons as described in claim 1, wherein the nucleic acid sequences have the structure shown in formula III. X1-R1-X2-R2-X3 (III) Here, R1 is the base complementary pairing region 1, R2 is the base complementary pairing region 2, X1, X2, and X3 are each independently redundant nucleic acids, with the length of X1 being 0 to 5 bases, the length of X2 being 0 to 3 bases, and the length of X3 being 0 to 5 bases. "-" indicates a connection, Each monomer's R1 forms a base-complementary pairing structure with the R2 of another monomer, and each monomer's R2 forms a base-complementary pairing structure with the R1 of another monomer. The aforementioned use.

12. A method for preparing the antibody-drug conjugate described in claim 1, (1) A step of forming a nucleic acid-drug assembly unit by chemical fixed-point coupling of a single strand of nucleic acid and a cytotoxic drug, (2) The step of forming an antibody-nucleic acid assembly unit by fixing-point coupling of an antibody and a complementary nucleic acid single strand, (3) The method comprising the step of 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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