CN117959454A

CN117959454A - Synthesis and application of novel antibody coupling drug

Info

Publication number: CN117959454A
Application number: CN202311331244.3A
Authority: CN
Inventors: 夏祖光; 李成勋; 李圣杰
Original assignee: Shanghai Mednova Biopharmaceutical Technology Co ltd
Current assignee: Shanghai Mednova Biopharmaceutical Technology Co ltd
Priority date: 2023-10-13
Filing date: 2023-10-13
Publication date: 2024-05-03

Abstract

The invention relates to the field of pharmaceutical chemistry, in particular to synthesis and application of a novel antibody coupled drug, which comprises a nucleic acid nanostructure formed by assembling nucleic acid and a chemotherapeutic drug, and a targeting drug coupled with the nucleic acid nanostructure. The invention utilizes the structural characteristics of nucleotide analogues, combines the nucleic acid nanotechnology, carries a plurality of nucleotide analogue medicaments on a nucleic acid nanostructure through a base complementary pairing principle, and has the in vivo targeting effect through coupling with an antibody. More importantly, this coupling in the present invention is generic independent of the specific groups on the antibody. Based on the high specificity of the antibody and the flexible modifiable nano structure, the invention not only provides a novel method for preparing the antibody coupled medicine, but also improves the pharmacodynamic action of the medicine.

Description

Synthesis and application of novel antibody coupling drug

Technical Field

The invention relates to the field of pharmaceutical chemistry, in particular to synthesis and application of a novel antibody coupled drug.

Background

An Antibody-conjugated drug (ADC) is a therapeutic agent consisting of an Antibody, a linker and a small molecule by covalent linkage. The antibody coupling medicine combines the high specificity of the antibody and the high efficiency of small molecules, so that the antibody coupling medicine has great breakthrough in the treatment of cancers. However, due to the limitations of small molecule modification, the small molecules linked to antibody-conjugated drugs are limited to a few molecules. This limits not only the kind of conjugated small molecules, but also the therapeutic range of antibody conjugated drugs. Nucleic acid analogs are derivatives of a class of natural nucleotides that result in chemical structural similarity by bioisostere substitution of some of the atoms or groups therein. These nucleotide analogs can participate in the synthesis and metabolic processes of natural nucleotides in the living body, thereby exerting antiviral or antitumor effects. In addition, many chemotherapeutic agents used in clinic can generate firm binding with double-stranded nucleic acids, mainly comprising two modes of insertion or covalent binding, but nucleotide analogs and small molecule drugs bound with nucleic acids often cause serious toxic and side effects due to lack of targeting. Because nucleotide analogs and small molecule drugs that bind to nucleic acids lack groups suitable for antibody coupling, they cannot be linked to antibodies to enhance their targeting and therapeutic effects.

Disclosure of Invention

The present invention aims to solve the problems that the long-standing in the field, nucleotide analogues and small molecule drugs combined with nucleic acid generally lack a group suitable for antibody coupling, so that the targeting is lacking, the therapeutic effect is further weak and more side effects are caused, for example, the nucleotide analogues represented by gemcitabine usually cause serious blood toxicity and bone marrow suppression due to the lack of the targeting, and chemotherapy fails; chemotherapy agents such as doxorubicin, however, are generally cardiotoxic in addition to myelosuppression. Improving the targeting of these drugs by means of antibody-conjugated drugs is an effective means of improving therapeutic effects and reducing toxic side effects. However, these drugs lack conjugated chemical groups, the ratio of drug to antibody (Drug Antibody Ratio, DAR) to be prepared into antibody conjugated drugs is low, usually 3-4, and the manufacturing cost is high.

In order to solve the technical problems, the invention adopts the following technical scheme:

A novel antibody-conjugated drug, comprising a nucleic acid nanostructure formed by assembling a nucleic acid and a chemotherapeutic agent, and a targeting agent conjugated to the nucleic acid nanostructure.

The novel antibody coupling drug designed by the invention can be carried without depending on coupled chemical groups, the DAR value can at least reach 20 (the limit DAR value in the prior art is 8), the drug effect is greatly improved, and the manufacturing cost is reduced.

DNA nanotechnology utilizes Watson-Crick base pairing to assemble different single strands of nucleic acid into nanostructures. This can provide a broad platform for the mounting of nucleotide analogs and small molecule drugs that bind to nucleic acids. The nanostructure can connect a plurality of nucleotide analogues into the nanostructure through phosphodiester bonds, can also be directly combined with each other through small molecules capable of combining nucleic acid, and is coupled with an antibody by utilizing the characteristic that the nanostructure is easy to modify, so that a novel antibody coupling drug is prepared, and the problems that the nucleotide analogues and the small molecule drug combined with the nucleic acid are not easy to modify and have toxic or side effects are solved.

Preferably, the assembling of the nucleic acid with the chemotherapeutic agent to form the nucleic acid nanostructure comprises one or more of intercalation, covalent binding.

Preferably, the chemotherapeutic agent comprises a nucleotide analogue, and/or an agent having an intercalating double stranded nucleic acid structure; the targeting agent includes a specific antibody.

Preferably, the chemotherapeutic agent comprises one or more of cytarabine, gemcitabine, trifluoretoside, iodoside, fluorouracil, fludarabine, 6-thioguanine, clofarabine, cladribine, etoposide, doxorubicin, mitoxantrone, cisplatin, mitomycin, vincristine, bleomycin; the targeting agent comprises one or more of cetuximab, trastuzumab, moruzumab, rituximab, atilizumab, avistuzumab, sha Tuo ximab, tafasitamab, polatuzumab, and gemtuzumab.

Wherein the nucleotide analog comprises one or more of cytarabine, gemcitabine, trifluoretoside, iodoside, fluorouracil, fludarabine, 6-thioguanine, clofarabine, cladribine; the medicine with the structure of inserting and combining double-chain nucleic acid comprises one or more of etoposide, doxorubicin, mitoxantrone, cisplatin, mitomycin, vincristine and bleomycin.

In a preferred embodiment of the invention, the substitution of cytosine in the oligonucleotide strand with gemcitabine is performed by phosphoramidite synthesis, taking advantage of the fact that gemcitabine is a natural cytosine derivative.

Further, gemcitabine is covalently linked to other natural nucleotides with phosphodiester linkages and assembled into regular tetrahedral nanostructures.

Further, the nanostructure is conjugated to cetuximab, which results in gemcitabine producing anti-EGFR positive cell lines that proliferate better than conventional gemcitabine and antibody mixtures.

Further, the antibody-conjugated drug is resistant to a cell model of gemcitabine resistance.

Further, the antibody-conjugated drug can obviously target EGFR-positive tumor sites.

In another preferred embodiment of the invention, cytosine in the oligonucleotide strand is replaced with cytarabine by phosphoramidite synthesis using the feature that cytarabine is a natural cytosine derivative.

Further, cytarabine is covalently linked to other natural nucleotides via phosphodiester bonds and assembled into regular tetrahedral nanostructures.

Further, the nanostructure is coupled with trastuzumab, so that the proliferation capacity of an anti-HER 2 positive cell line generated by cytarabine is better than that of a common mixture of cytarabine and an antibody.

In a preferred embodiment of the invention, the characteristic that doxorubicin has insertion binding double-stranded nucleic acid is utilized, and the doxorubicin is carried into the synthesized regular tetrahedron nucleic acid nanostructure by normal temperature incubation.

Further, coupling the conjugate to rituximab enhances the ability of doxorubicin to inhibit CD20 positive tumor cells.

In another preferred embodiment of the present invention, mitoxantrone is loaded into a synthetic regular tetrahedron nucleic acid nanostructure by incubation at ambient temperature, using the feature of mitoxantrone that it has intercalating binding double stranded nucleic acids.

Further, coupling the conjugate to gemtuzumab, enhances the ability of mitoxantrone to inhibit CD33 positive tumor cells.

Preferably, the nucleic acid nanostructure comprises DNA tetrahedra comprising azide groups.

The oligonucleotide strands are assembled by base pairing to obtain a regular tetrahedral structure having a diameter of 50nm or less.

A method of preparing the novel antibody-conjugated drug of claim 1, comprising the steps of:

A. purification and coupling of the targeting agent: ultrafiltration eluting and purifying the targeting drug, and then incubating the targeting drug with a coupling agent to obtain a product A;

B. Synthesis of nucleic acid nanostructures: when covalent bonding is carried out, the chemotherapeutic medicine and the nucleic acid are put into an assembly liquid, and a product B is obtained through a heating annealing reaction; when the nucleic acid is combined through insertion, the nucleic acid is put into an assembly liquid, a product B1 is obtained through a heating annealing reaction, and then the product B1 and the chemotherapeutic drug are incubated together to obtain a product B;

C. Synthesis of novel antibody-conjugated drugs: and mixing the product A with the product B in equimolar concentration, and incubating to obtain the novel antibody coupling drug.

Preferably, the method comprises the following steps:

A. Purification and coupling of the targeting agent: eluting and purifying impurities in the ultrafiltration tube with the target medicine of 50kd, then incubating the ultrafiltration tube with a coupling agent DBCO-NHS for 2 hours at 37 ℃, concentrating the reaction solution through the ultrafiltration tube with the target medicine of 50kd, removing redundant reagents, and washing with PBS to obtain a product A;

B. Synthesis of nucleic acid nanostructures: metering the chemotherapeutic drug and the nucleic acid according to the nucleic acid sequence of the nucleic acid nanostructure during covalent bonding, putting the chemotherapeutic drug and the nucleic acid into an assembly liquid, and respectively maintaining at 95 ℃, 65 ℃, 50 ℃, 42 ℃, 37 ℃, 22 ℃ and 4 ℃ for 5 minutes for heating and annealing reaction to obtain a product B; when the nucleic acid is inserted and combined, the nucleic acid is put into an assembly liquid, and is respectively kept at 95 ℃, 65 ℃, 50 ℃, 42 ℃, 37 ℃, 22 ℃ and 4 ℃ for 5 minutes for a heating annealing reaction to obtain a product B1, then the product B1 and the chemotherapeutic drug are incubated for 2 hours at 37 ℃ in a molar ratio of 1:20, the reaction liquid is concentrated through an ultrafiltration tube of 50kd, and the PBS is used for cleaning to obtain the product B;

C. Synthesis of novel antibody-conjugated drugs: mixing the equimolar concentration of the product A and the product B, incubating for 24 hours at 4 ℃, and reutilizing And the pure protein purification system uses a size exclusion chromatography column as a separation column and PBS as a mobile phase to separate and collect reaction products, and uses a 50kD ultrafiltration tube for concentration to obtain the novel antibody coupling drug.

Preferably, in step B, the assembly liquid includes the following components in concentration: 25mM HEPES,140mM potassium chloride, 10mM magnesium chloride; ph=7 of the assembly liquid.

The application of the novel antibody coupled medicine is used for preparing medicines for treating cancers.

Preferably, the cancer comprises one or more of non-small cell lung cancer, diffuse large B lymphoma, pancreatic cancer, colorectal cancer.

The implementation of the invention has the following beneficial effects:

The invention utilizes the structural characteristics of nucleotide analogues, combines the nucleic acid nanotechnology, carries a plurality of nucleotide analogue medicaments on a nucleic acid nanostructure through a base complementary pairing principle, and has the in vivo targeting effect through coupling with an antibody. More importantly, this coupling in the present invention is generic independent of the specific groups on the antibody. Based on the high specificity of the antibody and the flexible modifiable nano structure, the invention not only provides a novel method for preparing the antibody coupled medicine, but also improves the pharmacodynamic action of the medicine.

Drawings

FIG. 1 is a schematic illustration of the process flow of the present invention.

FIG. 2 is a non-denaturing PAGE gel electrophoresis to characterize the synthesis of antibody-conjugated drugs of gemcitabine.

FIG. 3 is a particle size analysis of the synthesis of an antibody conjugated drug characterizing gemcitabine.

FIG. 4 is an ATP luminecent cell viability assay for assessing anti-tumor cell activity of an antibody conjugated drug of gemcitabine.

FIG. 5 is a graph showing inhibition of gemcitabine-resistant models by gemcitabine-conjugated drugs.

FIG. 6 is a graph of fluorescence signal of gemcitabine antibody conjugated drug for targeted enrichment of EGFR positive tumor mass

FIG. 7 is a non-denaturing PAGE gel electrophoresis to characterize synthesis of cytarabine antibody-conjugated drug.

FIG. 8 is a particle size analysis of the synthesis of antibody conjugated drugs characterizing cytarabine.

FIG. 9 is a CCK8 cell viability assay for assessing anti-tumor cell activity of an antibody conjugated drug of cytarabine.

Figure 10 is a non-denaturing PAGE gel electrophoresis characterization of the synthesis of antibody-conjugated drugs to doxorubicin.

Figure 11 is a particle size analysis characterizing the synthesis of antibody-conjugated drugs to doxorubicin.

FIG. 12 is a graph showing the evaluation of anti-tumor cell activity of mitoxantrone antibody-conjugated drugs by CCK 8.

FIG. 13 is a non-denaturing PAGE gel electrophoresis to characterize synthesis of mitoxantrone antibody-conjugated drugs.

Figure 14 is a particle size analysis characterizing the synthesis of an antibody-conjugated drug of mitoxantrone.

FIG. 15 is a graph showing the evaluation of anti-tumor cell activity of mitoxantrone antibody-conjugated drugs by CCK 8.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.

In the following examples, the tetrahedral sequence information of the constituent nucleic acids is shown in SEQ ID No: 1-SEQ ID No:7 and table 1:

TABLE 1

Example 1

Experimental material source

Cetuximab was purchased from Merck, DBCO-NHS from sirench, 50kd ultrafiltration tube from Sartorius,0.22 μm pore size CA filter from corning, ATP luminecent cell viability assay kit from Uelandy, C57 mice from collectable drug, DNA sequence Ai Kerui bioengineering company Biology synthesis.

As shown in FIG. 1, preparation of novel antibody-conjugated drugs from cetuximab and gemcitabine-containing DNA tetrahedron

1. Purification and coupling of cetuximab: cetuximab was eluted and purified with a 50kd ultrafiltration tube and then co-incubated with DBCO-NHS for 2h at 37 ℃. The reaction solution was concentrated and the excess DBCO-NHS reagent was removed by a 50kd ultrafiltration tube, washed with PBS and the concentration of the product was determined by a microplate reader (Synergy 2 multifunctional).

2. Synthesis of gemcitabine-containing DNA tetrahedra (TDN-GEM): as shown in Table 1, the synthesized DNA sequences (T1-N3, T2, T3, T4-GEM) were assembled in an equistoichiometric ratio in an assembly solution (25 mM HEPES,140mM potassium chloride, 10mM magnesium chloride, pH=7) by a thermal annealing reaction (5 min at 95 ℃, 65 ℃, 50 ℃, 42 ℃, 37 ℃, 22 ℃ and 4 ℃), to produce DNA tetrahedra containing azide groups and gemcitabine modification. The reaction solution was concentrated through a 50kd ultrafiltration tube, washed with PBS, and the concentration of the product was measured by a microplate reader (Synergy 2 multifunctional microplate reader).

3. Synthesis of gemcitabine-antibody-coupled drug (CTM-TDN-GEM): the products of the two steps are mixed according to equimolar concentration and incubated for 24 hours at 4 ℃. By means ofAnd in the pure protein purification system, a size exclusion chromatography column is used as a separation column, PBS is used as a mobile phase, and reaction products are separated. The isolated product was collected and concentrated using a 50kD ultrafiltration tube and the product was quantified for concentration as DBCO concentration. The concentrated product was sterilized by filtration using a 0.22 μm pore size CA filter and stored at 4 ℃ for subsequent biological experiments.

4. Characterization of gemcitabine-containing antibody-conjugated drug: the products were characterized by non-denaturing PAGE gel electrophoresis and particle size analysis (Malvern Zetasizer Nano ZS a) as shown in FIGS. 2 and 3.

As can be seen from FIG. 2, the gemcitabine-containing antibody-coupled drug is larger in size, the bands are accumulated at the upper portion, and the result can be confirmed by the particle size analysis of FIG. 3, and the size of the gemcitabine-containing antibody-coupled drug is about 20 nm.

ATP luminecent cell viability assay to assess anti-tumor cell activity of antibody conjugated drugs of gemcitabine

1. When the growth confluence of the A549 cells reaches 85%, the cells are digested, centrifuged, resuspended and counted by using a complete culture medium, 2000 cells per well are inoculated into a 96-well plate, and cultured overnight, and the cells are attached to the wall.

2. Design of experimental group: blank, i.e. empty holes without any material; solvent control, i.e., cells cultured in complete medium with corresponding solvent; ctm+gem group, i.e. cells co-incubated with different concentrations of complete medium mixed with CTM: gem=1:18; cetuximab was conjugated to gemcitabine-loaded DNA tetrahedron (CTM-TDN-GEM) groups, i.e., cells co-incubated with complete media at different concentrations of CTM-TDN-GEM. All experimental groups were added to well-attached 96-well plates and incubated for 72h.

3.ATP Luminescent cell viability assay: the kit was thawed to room temperature, the cell culture solution was removed, 100. Mu.l of the detection solution was added to each experimental group, and the mixture was allowed to stand at room temperature for incubation for 10min. The multifunctional enzyme-labeled instrument detects the chemiluminescent value of each well. And (3) carrying out data processing on the measured chemiluminescence values, deducting a blank group, and calculating the cell survival rate of other experimental groups by taking a cell control group as a reference. The results are shown in FIG. 4.

The results of fig. 4 demonstrate that the anti-tumor activity of gemcitabine-conjugated drugs is at least six-fold greater than that of gemcitabine alone in combination with cetuximab.

Inhibition of CTM-TDN-GEM on tumor cell gemcitabine resistance model

1. When the growth confluence of tumor cells reaches 85%, the cells are digested, centrifuged, resuspended and counted by using a complete culture medium, 2000 cells per well are inoculated into a 96-well plate, and cultured overnight, and the cells are attached to the wall.

2. Construction of a tumor cell gemcitabine drug resistance model: dipyridamole are inhibitors of nucleotide transport receptors that significantly inhibit the uptake of gemcitabine by cells. Tumor cells typically develop gemcitabine resistance by down-regulating the expression of nucleotide transport receptor proteins. The medium of the 96-well plate described above was replaced with complete medium containing Dipyridamole (10. Mu.M, available from MCE) and incubated in an incubator for 30min at 50. Mu.l per well.

3. Each experimental group was formulated with a 2-fold concentration of drug solution using complete medium containing Dipyridamole (10 μm). Mu.l of each well was added to the incubated 96-well plate and incubated for 72 hours.

4.ATP Luminescent cell viability assay: as described in example 1. The results are shown in FIG. 5.

CTM-TDN-GEM can enter cells by endocytosis, thereby avoiding drug resistance caused by inhibition of nucleotide transport receptors. The result shows that CTM-TDN-GEM can obviously resist a gemcitabine drug resistance model which is constructed by Dipyridamole, and the antitumor activity of the CTM-TDN-GEM is 20 times that of a common gemcitabine and cetuximab mixture.

Targeted enrichment of EGFR positive tumor masses with gemcitabine antibody-conjugated drugs

1. Dehairing was performed at the pre-inoculation site of the mice, EGFR negative MC38 cells were inoculated subcutaneously on the left side of the mice, EGFR positive MC38 cells were inoculated on the right side of the mice, and the tumor mass was grown to about 200mm ³.

Synthesis of cy5 modified CTM-TDN-GEM: as shown in Table 1, the synthesized DNA sequences (T1-N3, T2-Cy5, T3, T4-GEM) were assembled in equistoichiometric ratios in an assembly solution (25 mM HEPES,140mM potassium chloride, 10mM magnesium chloride, pH=7) by a thermal annealing reaction (maintained at 95℃at 65℃at 50℃at 42℃at 37℃at 22℃at 4℃for 5 minutes, respectively) to produce DNA tetrahedra containing azide groups and gemcitabine modification. The reaction solution was concentrated through a 50kd ultrafiltration tube, washed with PBS, and the concentration of the product was measured by a microplate reader (Synergy 2 multifunctional microplate reader). The Cy 5-labeled tetrahedron described above was conjugated to cetuximab as described previously.

3. The Cy 5-labeled antibody-conjugated drug was diluted to 5. Mu.M with PBS, injected into mice via tail vein in an amount of 5. Mu.l/g, and imaged in an IVIS select fluorescence system after about 30 min.

As shown in fig. 6, CTM-TDN-GEM specifically targeted to EGFR-positive tumor mass tissue enriched in mice through blood circulation, while negative tumor mass tissue signals were low, indicating good targeting.

Example 2

Experimental material source

Trastuzumab was purchased from roche, other materials were synthesized as described in example 1, DNA sequence Ai Kerui bioengineering company Biology.

As shown in FIG. 1, trastuzumab and cytarabine-containing DNA tetrahedron are used for preparing novel antibody-coupled drugs

1. Purification and coupling of trastuzumab: trastuzumab was eluted and purified of impurities with a 50kd ultrafiltration tube followed by co-incubation with DBCO-NHS for 2h at 37 ℃. The reaction solution was concentrated and the excess DBCO-NHS reagent was removed by a 50kd ultrafiltration tube, washed with PBS and the concentration of the product was determined by a microplate reader (Synergy 2 multifunctional).

2. Synthesis of cytarabine-containing DNA tetrahedron (TDN-CTB): as shown in Table 1, the synthesized DNA sequences (T1-N3, T2, T3, T4-CTB) were assembled in an equistoichiometric ratio in an assembly solution (25 mM HEPES,140mM potassium chloride, 10mM magnesium chloride, pH=7) by a thermal annealing reaction (kept at 95℃at 65℃at 50℃at 42℃at 37℃at 22℃and at 4℃for 5 minutes, respectively) to produce DNA tetrahedra containing azide groups and cytarabine modifications. The reaction solution was concentrated through a 50kd ultrafiltration tube, washed with PBS, and the concentration of the product was measured by a microplate reader (Synergy 2 multifunctional microplate reader).

3. Synthesis of cytarabine-containing antibody-conjugated drug (TSM-TDN-CTB): the products of the two steps are mixed according to equimolar concentration and incubated for 24 hours at 4 ℃. By means ofAnd in the pure protein purification system, a size exclusion chromatography column is used as a separation column, PBS is used as a mobile phase, and reaction products are separated. The isolated product was collected and concentrated using a 50kD ultrafiltration tube and the product was quantified for concentration as DBCO concentration. Filtering and sterilizing the concentrated product with 0.22 μm aperture CA filter membrane, and standing at 4deg.C

And storing for subsequent biological experiments.

4. Characterization of cytarabine-containing antibody-conjugated drug: the products were characterized by non-denaturing PAGE gel electrophoresis and particle size analysis (Malvern Zetasizer Nano ZS a) as shown in FIGS. 7 and 8.

As can be seen from FIG. 7, the size of the cytarabine-containing antibody-coupled drug is large, the bands are accumulated at the upper part, and the result can be confirmed by the particle size analysis of FIG. 8, and the size of the cytarabine-containing antibody-coupled drug is about 20 nm.

CCK8 cell viability assay to assess antitumor cell activity of antibody-conjugated drugs of cytarabine

1. When the SKBR3 cell density reaches 85%, the cells are digested, centrifuged, resuspended and counted in complete medium, 2000 cells per well are inoculated into 96 well plates, cultured overnight, and the cells are allowed to adhere to the wall.

2. Design of experimental group: blank, i.e. empty holes without any material; solvent control, i.e., cells cultured in complete medium with corresponding solvent; tsm+ctb group, i.e. cells co-incubated with different concentrations of complete medium mixed with TSM: ctb=1:18; trastuzumab was conjugated to cytarabine-loaded DNA tetrahedron (TSM-TDN-CTB) groups, i.e. cells co-incubated with complete medium of different concentrations of TSM-TDN-CTB. All experimental groups were added to well-attached 96-well plates and incubated for 72h.

Cck8 cell viability assay: to each experimental group, 10. Mu.l of the detection solution was added, and the mixture was allowed to stand at room temperature for incubation for 10min. The multifunctional enzyme-labeled instrument detects the chemiluminescent value of each well. And (3) carrying out data processing on the measured chemiluminescence values, deducting a blank group, and calculating the cell survival rate of other experimental groups by taking a cell control group as a reference. The results are shown in FIG. 9.

The results in fig. 9 show that the antitumor activity of the cytarabine-containing antibody-conjugated drug is at least 10-fold stronger than that of the mixture of cytarabine alone and trastuzumab.

Example 3

CCK8 was purchased from MCE and rituximab was purchased from Roche. Other experimental materials were obtained as in example 1, and the following experiments were conducted with OCI-LY3 as the subject of the present invention. Cells of other tumor types, although not specifically tested, are known to those skilled in the art to be able to obtain similar test results.

As shown in fig. 1, rituximab and doxorubicin-containing DNA tetrahedron prepared into novel antibody-conjugated drugs

1. Purification and coupling of rituximab: rituximab was first eluted and purified of impurities using a 50kd ultrafiltration tube and then co-incubated with DBCO-NHS for 2h at 37 ℃. The reaction solution was concentrated and the excess DBCO-NHS reagent was removed by a 50kd ultrafiltration tube, washed with PBS and the concentration of the product was determined by a microplate reader (Synergy 2 multifunctional).

2. Synthesis of DNA tetrahedron containing doxorubicin (TDN-DOX): as shown in Table 1, the synthesized DNA sequences (T1-N3, T2, T3, T4) were assembled in an equistoichiometric ratio in an assembly solution (25 mM HEPES,140mM potassium chloride, 10mM magnesium chloride, pH=7) by a thermal annealing reaction (kept at 95℃at 65℃at 50℃at 42℃at 37℃at 22℃and at 4℃for 5 minutes, respectively) to produce azide group-containing DNA tetrahedra. Tetrahedra were incubated with doxorubicin at a molar ratio of 1:20, the reaction was concentrated through a 50kd ultrafiltration tube, washed with PBS, and purified by a microplate reader (Synergy

2 Multifunctional microplate reader) to determine the concentration of the product.

3. Synthesis of antibody-conjugated drug containing doxorubicin (RTM-TDN-DOX): the products of the two steps are mixed according to equimolar concentration and incubated for 24 hours at 4 ℃. By means ofAnd in the pure protein purification system, a size exclusion chromatography column is used as a separation column, PBS is used as a mobile phase, and reaction products are separated.

The isolated product was collected and concentrated using a50 kD ultrafiltration tube and the product was quantified for concentration as DBCO concentration. Filtering and sterilizing the concentrated product with 0.22 μm aperture CA filter membrane, and standing at 4deg.C

And storing for subsequent biological experiments.

4. Characterization of antibody-conjugated drug containing doxorubicin: the products were characterized by non-denaturing PAGE gel electrophoresis and particle size analysis (Malvern Zetasizer Nano ZS a) as shown in FIGS. 10 and 11.

As can be seen from fig. 10, the antibody-conjugated drug of doxorubicin is larger in size, the band is gathered at the upper part, and the result can be confirmed by the particle size analysis of fig. 11, and the size of the antibody-conjugated drug containing doxorubicin is about 25 nm.

CCK8 assay to assess anti-tumor cell activity of antibody-conjugated drugs of doxorubicin

1. When the OCI-LY3 cell growth density reached 85%, the cells were digested, centrifuged, resuspended in complete medium and counted, and plated into 96-well plates at 2000 cells per well.

2. Design of experimental group: blank, i.e. empty holes without any material; solvent control, i.e., cells cultured in complete medium with corresponding solvent; rtm+gem groups, i.e. cells co-incubated with different concentrations of complete medium mixed according to RTM: gem=1:20; rituximab was conjugated to doxorubicin-loaded DNA tetrahedron (RTM-TDN-DOX) groups, i.e. cells co-incubated with complete medium at different concentrations of RTM-TDN-DOX. All experimental groups were added to well-attached 96-well plates and incubated for 72h.

Cck8 cell viability assay: the kit was thawed to room temperature, 10. Mu.l of the detection solution was added to each experimental group, and the incubator was incubated for 4 hours. And detecting by a multifunctional enzyme-labeled instrument, deducting a blank group, and calculating the cell survival rate of other experimental groups by taking a cell control group as a reference. The results are shown in FIG. 12.

The results in FIG. 12 show that RTM-TDN-DOX has at least 30-fold greater antitumor activity than gemcitabine alone in combination with cetuximab.

Example 4

CCK8 and gemtuzumab were purchased from MCE. Other experimental material sources were the same as in example 1, and experimental tests were performed using Reh as the content of the study of the present invention. Cells of other tumor types, although not specifically tested, are known to those skilled in the art to be able to obtain similar test results.

1. Purification and coupling of gemtuzumab: the impurities were eluted and purified with a 50kd ultrafiltration tube followed by co-incubation with DBCO-NHS for 2h at 37 ℃. The reaction solution was concentrated and the excess DBCO-NHS reagent was removed by a 50kd ultrafiltration tube, washed with PBS and the concentration of the product was determined by a microplate reader (Synergy 2 multifunctional).

2. Synthesis of mitoxantrone-containing DNA tetrahedra (TDN-MTN): as shown in Table 1, the synthesized DNA sequences (T1-N3, T2, T3, T4) were assembled in an equistoichiometric ratio in an assembly solution (25 mM HEPES,140mM potassium chloride, 10mM magnesium chloride, pH=7) by a thermal annealing reaction (kept at 95℃at 65℃at 50℃at 42℃at 37℃at 22℃and at 4℃for 5 minutes, respectively) to produce azide group-containing DNA tetrahedra. Tetrahedra were co-incubated with mitoxantrone at a molar ratio of 1:20, the reaction was concentrated through a 50kd ultrafiltration tube, washed with PBS, and passed through a microplate reader (Synergy

3. Synthesis of mitoxantrone-containing antibody-conjugated drug (GTM-TDN-MTN): the products of the two steps are mixed according to equimolar concentration and incubated for 24 hours at 4 ℃. By means ofAnd in the pure protein purification system, a size exclusion chromatography column is used as a separation column, PBS is used as a mobile phase, and reaction products are separated.

And storing for subsequent biological experiments.

4. Characterization of mitoxantrone-containing antibody-conjugated drugs: the products were characterized by non-denaturing PAGE gel electrophoresis and particle size analysis (Malvern Zetasizer Nano ZS a) as shown in FIGS. 13 and 14.

As can be seen from FIG. 13, the size of the mitoxantrone-containing antibody-conjugated drug is larger, the bands are concentrated in the upper portion, and the results can be confirmed by the particle size analysis of FIG. 14, and the size of the mitoxantrone-containing antibody-conjugated drug is about 25 nm.

CCK8 assay to assess anti-tumor cell activity of mitoxantrone antibody-conjugated drugs

4. When the Reh cell growth density reached 85%, cells were digested, centrifuged, resuspended in complete medium and counted, and plated into 96-well plates at 2000 cells per well.

5. Design of experimental group: blank, i.e. empty holes without any material; solvent control, i.e., cells cultured in complete medium with corresponding solvent; gtm+mtn group, i.e. cells co-incubated with different concentrations of complete medium mixed with GTM: mtn=1:20; gift bead mab was conjugated to mitoxantrone-loaded DNA tetrahedron (GTM-TDN-MTN) groups, i.e., cells co-incubated with complete media of different concentrations of GTM-TDN-MTN. All experimental groups were added to well-attached 96-well plates and incubated for 72h.

Cck8 cell viability assay: the kit was thawed to room temperature, 10. Mu.l of the detection solution was added to each experimental group, and the incubator was incubated for 4 hours. And detecting by a multifunctional enzyme-labeled instrument, deducting a blank group, and calculating the cell survival rate of other experimental groups by taking a cell control group as a reference. The results are shown in FIG. 15.

The results in FIG. 15 show that GTM-TDN-MTN has at least 10-fold greater antitumor activity than the mixture of mitoxantrone and gemtuzumab alone.

The foregoing description of the preferred embodiments of the invention is merely illustrative of the invention and is not intended to limit the scope of the invention, which is defined by the claims and their equivalents.

Claims

1. A novel antibody-conjugated drug, comprising a nucleic acid nanostructure formed by assembling a nucleic acid and a chemotherapeutic agent, and a targeting agent conjugated to the nucleic acid nanostructure.

2. The novel antibody conjugated drug of claim 1, wherein the assembling of the nucleic acid with the chemotherapeutic agent to form the nucleic acid nanostructure comprises one or more of insertion, covalent binding.

3. The novel antibody conjugated drug of claim 1, wherein the chemotherapeutic agent comprises a nucleotide analogue, and/or a drug having an intercalating double-stranded nucleic acid binding structure; the targeting agent includes a specific antibody.

4. The novel antibody conjugated drug of claim 1, wherein the chemotherapeutic comprises one or more of cytarabine, gemcitabine, trifluoretoside, iodoside, fluorouracil, fludarabine, 6-thioguanine, clofarabine, cladribine, etoposide, doxorubicin, mitoxantrone, cisplatin, mitomycin, vincristine, bleomycin; the targeting agent comprises one or more of cetuximab, trastuzumab, moruzumab, rituximab, atilizumab, avistuzumab, sha Tuo ximab, tafasitamab, polatuzumab, and gemtuzumab.

5. The novel antibody conjugated drug of claim 1, wherein the nucleic acid nanostructure comprises DNA tetrahedra comprising azide groups.

6. A method of preparing the novel antibody conjugated drug of claim 1, comprising the steps of:

7. The method for preparing the novel antibody-conjugated drug according to claim 6, comprising the steps of:

8. The method of preparing a novel antibody-conjugated drug according to claim 6, wherein in step B, the assembly solution comprises the following components in concentration: 25mM HEPES,140mM potassium chloride, 10mM magnesium chloride; ph=7 of the assembly liquid.

9. Use of a novel antibody conjugated drug according to claim 1 for the preparation of a medicament for the treatment of cancer.

10. The use of the novel antibody conjugated drug of claim 9, wherein said cancer comprises one or more of non-small cell lung cancer, diffuse large B lymphoma, pancreatic cancer, colorectal cancer.