Tetravalent aptamer drug conjugate and application thereof

Abstract

The invention provides a tetravalent nucleic acid aptamer drug conjugate and application thereof.The tetravalent nucleic acid aptamer drug conjugate comprises streptavidin (SA) and four nucleic acid aptamer drug compounds, and each nucleic acid aptamer drug compound comprises a nucleic acid aptamer, a drug molecule and biotin (Biotin), the tetravalent nucleic acid aptamer drug conjugate is constructed and formed through non-covalent interaction between biotin molecules and streptavidin, wherein the biotin molecules are modified on the nucleic acid aptamer drug complex. The stability, affinity, tumor proliferation inhibition capability and the like of the tetravalent aptamer drug conjugate provided by the invention are superior to those of a monovalent aptamer drug conjugate, and the targeting treatment effect of the aptamer drug conjugate on cancers is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a tetravalent nucleic acid aptamer-drug conjugate and an application thereof. Background Art

[0002] Aptamers are a type of single-stranded DNA / RNA sequence composed of 15-60 bases that can be folded into a specific secondary or tertiary structure to specifically recognize target molecules. Compared with antibodies, aptamers have the advantages of small molecular weight, high chemical stability, easy modification, and large-scale preparation, and are called "chemical antibodies." Based on the specific recognition function of aptamers, they are precisely coupled with various toxin molecules to construct aptamer drug conjugates (ApDC), which can achieve precise targeted delivery of drug molecules, improve the accuracy and effectiveness of treatment, and are expected to become a new type of molecular targeted drug with great potential.

[0003] At present, most of the reported ApDCs are mainly focused on coupling a single aptamer with a drug to construct a monovalent ApDC. However, monovalent ApDCs usually have weak affinity for the target, poor specificity, low stability, and short half-life, which seriously restrict the clinical transformation of ApDC. Multivalent ApDC can enhance its binding force with the target by increasing the valence of ApDC, thereby improving the anti-tumor efficacy. Patent document CN114681619B discloses a nucleic acid aptamer complex based on Biotin-SA and its application, which controls the ratio of streptavidin to nucleic acid aptamer to form various nucleic acid aptamer complex nanostructures, such as nanoparticles or nanosheet structures, and then utilizes the passive targeting ability (EPR effect) of nanomaterials to achieve enrichment at the tumor site for targeted treatment of colorectal cancer. However, one or both ends of the nucleic acid aptamer provided in the patent are modified by Biotin, and the constructed nucleic acid aptamer complex is a sheet or mesh structure, and its unstable valence causes inaccurate prediction results in targeted therapy applications.

[0004] Therefore, providing an ApDC with good stability and high affinity for the target to achieve precise targeted release of toxin drugs has important scientific significance and clinical value, and is expected to provide innovative targeted nucleic acid drugs for the precise treatment of cancer. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a tetravalent nucleic acid aptamer drug conjugate (SA-4ApDC), which specifically includes four nucleic acid aptamer drug complexes modified with biotin molecules and a streptavidin (SA). SA-4ApDC is constructed by the non-covalent interaction between the biotin molecules (Biotin) modified on the nucleic acid aptamer drug complex and the streptavidin. The SA-4ApDC constructed by the present invention has the characteristics of high affinity with the target, strong specificity, long circulation time, high stability, etc.

[0006] On the one hand, the present invention provides a tetravalent aptamer-drug conjugate, comprising a streptavidin and four aptamer-drug complexes, wherein the aptamer-drug complex comprises an aptamer, a drug and biotin, wherein the biotin modifies one end of the aptamer and the drug molecule modifies the other end of the aptamer.

[0007] Streptavidin (SA) is a tetrameric protein with a molecular weight of about 64kDa, which has a very strong non-covalent connection with biotin, performs fixed-point, quantitative and efficient binding, and forms a natural tetravalent system. Therefore, the present invention utilizes this effect between streptavidin and biotin, modifies one end of the nucleic acid aptamer with biotin, and introduces a cytotoxic drug at the other end of the nucleic acid aptamer to form a nucleic acid aptamer drug complex, and then connects biotin at the four binding sites of streptavidin to further construct and form SA-4ApDC. The difference from the prior art is that the present invention is creatively modified with biotin only at one end of the nucleic acid aptamer, ensuring that the four nucleic acid aptamers modified with biotin can accurately bind to the four binding sites of streptavidin to form a SA-4ApDC with a stable valence state, avoiding the problem of valence instability caused by the simultaneous modification of biotin at both ends of the nucleic acid aptamer in the prior art, which causes the nucleic acid aptamers to be connected to form a mesh structure.

[0008] Furthermore, the biotin is introduced into one end of the nucleic acid aptamer by solid phase synthesis, and the drug molecule is introduced into the other end of the nucleic acid aptamer by coupling through any one or more of thiol-maleimide, azide-alkyne, and amino-carboxyl.

[0009] Preferably, the drug molecule is introduced into the other end of the nucleic acid aptamer via thiol-maleimide coupling.

[0010] Furthermore, the nucleic acid aptamer targets any one or more of the following antigens: CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, HLD-DR, CEA, TAG-72, EpCAM, MUC1, MUC15, folate-binding protein, A33, G250, PSMA, ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2receptor, tenascin, a metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, EGFRvIII, Her-2 / neu, MAGE A3, p53 nonmutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a Sarcomatranslocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, MSLN, PSCA, MAGE Al, MAGE-A3, sLe, CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TESL Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3. Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, c-Met, HER3, EPCAM, CA6, NAPI2B, TROP2, CLDN18.2. FAP, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PD-L1, CD204, CD206, CD301, VTCN1, VISTA. .

[0011] Preferably, the nucleic acid aptamer targets the antigen PTK7 and / or c-Met.

[0012] Furthermore, the drug includes any one or more of auristatin-type microtubule protein inhibitors, Deruxtecan (Dxd), exotecan, or derivatives having the same or similar core structure as auristatin-type microtubule protein inhibitors, Dxd or exotecan.

[0013] Preferably, the drug comprises an auristatin-type microtubule inhibitor, and the auristatin-type microtubule inhibitor is selected from auristatin E, auristatin F, auristatin-0101 or a derivative thereof.

[0014] Furthermore, the nucleic acid aptamer includes a PTK7-targeting nucleic acid aptamer and / or a c-Met-targeting nucleic acid aptamer.

[0015] PTK7, protein tyrosine kinase 7, is a receptor tyrosine kinase in the Wnt pathway, involved in Wnt signal transduction during hematopoiesis and somatic progenitor cell and stem cell development. It is overexpressed in a variety of tumor types, including advanced triple-negative breast cancer, non-small cell lung cancer, ovarian cancer, colorectal cancer, gastric cancer and esophageal cancer. c-Met is a protein encoded by the c-Met proto-oncogene, known as the hepatocyte growth factor receptor, with tyrosine kinase activity, associated with a variety of oncogene products and regulatory proteins, involved in cell information transduction, cytoskeleton rearrangement regulation, is an important factor in cell proliferation, differentiation and movement. Studies have shown that patients with a variety of tumors such as lung cancer, gastric cancer, liver cancer, breast cancer, skin cancer, colorectal cancer, etc. have c-Met overexpression and gene amplification during the occurrence and metastasis of their tumors. Therefore, the present invention selects nucleic acid aptamers that can effectively target PTK7 and / or c-Met to construct a quadrivalent nucleic acid aptamer drug conjugate.

[0016] Furthermore, the nucleic acid aptamer includes the PTK7 targeting nucleic acid aptamer sgc8c and / or the c-Met targeting nucleic acid aptamer SL1.

[0017] In some embodiments, the nucleic acid aptamer sgc8c has a sequence as shown in SEQ ID NO.1, and the nucleic acid aptamer SL1 has a sequence as shown in SEQ ID NO.2.

[0018] The nucleic acid aptamer sgc8c can recognize and bind to PTK7 on the surface of cancer cells, thereby achieving targeted delivery or labeling of cancer cells. The nucleic acid aptamer SL1 was screened by the cell-SELEX technology, which can specifically bind to the c-Met protein on the cell surface to recognize the target cells.

[0019] The present invention proves through experiments that when two nucleic acid aptamers are assembled with SA respectively, different steric hindrances are generated and different equivalent driving is required. The binding equivalent of nucleic acid aptamer sgc8c when assembled with SA is 4.5 equivalents, and the binding equivalent of nucleic acid aptamer SL1 when assembled with SA is 4 equivalents. At the same time, the research results of the present invention also found that the stability, affinity, endocytosis ability and ability to inhibit tumor cell proliferation of the tetravalent nucleic acid aptamer drug conjugate prepared by the two nucleic acid aptamers are better than the corresponding monovalent nucleic acid aptamer drug conjugate.

[0020] Furthermore, the biotin modifies the 3' end of the nucleic acid aptamer, and the drug molecule modifies the 5' end of the nucleic acid aptamer.

[0021] Studies have shown that when biotin modifies the 5' ends of two nucleic acid aptamers respectively, the yield of the constructed tetravalent nucleic acid aptamer drug conjugate is significantly lower than the tetravalent nucleic acid aptamer drug conjugate constructed when biotin modifies the 3' end of the nucleic acid aptamer. Therefore, the present invention preferably modifies the 3' end of the nucleic acid aptamer with biotin and the 5' end of the nucleic acid aptamer with the drug molecule.

[0022] In another aspect, the present invention provides a drug for targeted tumor therapy, comprising the tetravalent nucleic acid aptamer-drug conjugate as described above.

[0023] In another aspect, the present invention provides a method for preparing a tetravalent nucleic acid aptamer drug conjugate, comprising the following steps:

[0024] (1) Synthesis of nucleic acid aptamers modified with biotin and drug molecules;

[0025] (2) Assembly of nucleic acid aptamers modified with biotin and drug molecules and streptavidin.

[0026] In some embodiments, the reaction temperature of the drug molecule and the nucleic acid aptamer in step (1) is 4 to 40° C., and the reaction time is 2 to 24 hours.

[0027] Preferably, in step (1), the reaction temperature of the drug molecule and the nucleic acid aptamer is 37° C., and the reaction time is 16 h.

[0028] In some embodiments, when the nucleic acid aptamer binds to streptavidin in step (2), the binding equivalent of the nucleic acid aptamer is 1 to 5 equivalents.

[0029] On the other hand, the present invention provides a composition for preparing a reagent for improving the stability and affinity of nucleic acid aptamer drug conjugates, wherein the composition comprises a streptavidin and four nucleic acid aptamer drug complexes, wherein the nucleic acid aptamer drug complexes comprise a nucleic acid aptamer, a drug molecule and biotin, wherein the biotin modifies one end of the nucleic acid aptamer, and the drug molecule modifies the other end of the nucleic acid aptamer.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) SA-4ApDC constructed with SA as a carrier can effectively improve the stability of nucleic acid aptamers and their ability to bind to target cells;

[0032] (2) SA-4ApDC is used for targeted therapy of tumor models. Compared with monovalent ApDC, it can improve the proliferation inhibition ability and tumor inhibition effect of malignant tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 The Biotin-modified nucleic acid aptamer of the present invention is coupled with a drug;

[0035] Figure 2 Mass spectrometry characterization of MMAE-Sgc8-Biotin (a) and mass spectrometry characterization of MMAE-SL1-Biotin (b) of the present invention;

[0036] Figure 3 The nucleic acid aptamer drug complex of the present invention is assembled with SA to form SA-4ApDC;

[0037] Figure 4 The absorption at a wavelength of 500 nm after different equivalents of MMAE-Sgc8-Biotin and SA are combined (a); the absorption at a wavelength of 500 nm after different equivalents of MMAE-SL1-Biotin and SA are combined (b);

[0038] Figure 5 The stability test of SA-4 (Sgc8-MMAE) and MMAE-Sgc8-Biotin of the present invention in a culture medium containing 25% FBS;

[0039] Figure 6The stability test of SA-4 (SL1-MMAE) and MMAE-SL1-Biotin of the present invention in a culture medium containing 50% FBS;

[0040] Figure 7 Comparison of the binding ability of SA-4 (Sgc8-MMAE) of the present invention and MMAE-Sgc8-Biotin on HT-29 (left) and SKOV3 (right) cell lines;

[0041] Figure 8 Comparison of the binding ability of SA-4 (SL1-MMAE) of the present invention and MMAE-SL1-Biotin on HT-29 (left) and SKOV3 (right) cell lines;

[0042] Fig. 9 Confocal imaging of SA-4 (Sgc8-MMAE), MMAE-Sgc8-Biotin and SA-4 (40A-MMAE) of the present invention on HT-29 cell line;

[0043] Fig.10 Confocal imaging of SA-4 (SL1-MMAE), MMAE-SL1-Biotin and SA-4 (40A-MMAE) of the present invention on HT-29 cell line;

[0044] Fig.11 The inhibitory effects of SA-4 (Sgc8-MMAE), MMAE-Sgc8-Biotin and SA-4 (40A-MMAE) of the present invention on HT-29 cell proliferation;

[0045] Fig.12 The inhibitory effects of SA-4 (SL1-MMAE), MMAE-SL1-Biotin and SA-4 (40A-MMAE) of the present invention on HT-29 cell proliferation;

[0046] Fig.13 The inhibition of mouse tumors by SA-4 (Sgc8-MMAE), MMAE-Sgc8-Biotin, SA-4 (40A-MMAE), and Saline groups of the present invention;

[0047] Fig.14 This is the body weight monitoring status of mice in the SA-4 (Sgc8-MMAE), MMAE-Sgc8-Biotin, SA-4 (40A-MMAE), and Saline groups of the present invention. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments are all commercially available unless otherwise specified.

[0049] Example 1: Construction of the tetravalent nucleic acid aptamer drug conjugate (SA-4ApDC) provided by the present invention

[0050] In this embodiment, the nucleic acid aptamers selected are nucleic acid aptamer sgc8c targeting PTK7 and nucleic acid aptamer SL1 targeting c-Met. The 3' ends of these two nucleic acid aptamers are modified with biotin by solid phase synthesis and named SH-Sgc8-Biotin and SH-SL1-Biotin respectively. The cytotoxic drug molecule is auristatin E (MMAE).

[0051] (1) Synthesis of aptamer-drug complexes by combining biotin-modified aptamers with cytotoxic drug molecules

[0052] 1 equivalent of SH-Sgc8-Biotin and SH-SL1-Biotin were added to two centrifuge tubes respectively, followed by 10 equivalents of MMAE at a concentration of 20 mg / mL; finally, DMF was added to make the organic phase ratio of the reaction system 25%. After reacting in a shaker at 37°C for 16 h, the reaction was continued by C 18 The aptamer drug complexes of MMAE-Sgc8-Biotin and MMAE-SL1-Biotin were obtained after purification by chromatographic column and freeze-dried, respectively, with a yield of about 70%. After desalting, they were freeze-dried for standby use. The preparation process is as follows: Figure 1 shown.

[0053] like Figure 2 As shown, MMAE-Sgc8-Biotin (target molecular weight: 14716 Da) and MMAE-SL1-Biotin (target molecular weight: 17764 Da) were successfully synthesized.

[0054] (2) Assembly of aptamer-drug complexes with SA

[0055] Assembly process such as Figure 3 As shown, 1 equivalent of streptavidin aqueous solution was added to each of the two centrifuge tubes, and then 4.5 equivalents of SH-Sgc8-Biotin and 4 equivalents of MMAE-SL1-Biotin were added to each of the two test tubes, and the mixture was shaken and mixed, and then waited for 5 minutes at room temperature. Finally, two SA-4ApDCs were obtained by ultrafiltration purification using a 30K ultrafiltration tube, which were named SA-4 (Sgc8-MMAE) and SA-4 (SL1-MMAE).

[0056] Example 2: Optimization of the binding equivalent of nucleic acid aptamer drug complex and SA

[0057] Because 4'-hydroxyazobenzene-2-carboxylic acid (HABA) has characteristic absorption at a wavelength of 500nm after binding to SA, and Biotin has a stronger affinity for SA, when Biotin is continuously added to the system, HABA bound to SA will be replaced, and the absorption value at 500nm will decrease. This example uses this principle to further compare the binding equivalents of two nucleic acid aptamer drug complexes when assembled with SA.

[0058] In this example, 3 mg / mL HABA solution and 0.5 mg / mL SA solution were first prepared. Figure 4 As shown in a, first, 1 equivalent of MMAE-Sgc8-Biotin was added to 50 μL SA solution, and the absorption value of the system at a wavelength of 500 nm was detected by an ELISA instrument. It was found that compared with the initial 0 equivalents, the absorption value decreased, indicating that SA and MMAE-Sgc8-Biotin began to combine; when 1 equivalent of MMAE-Sgc8-Biotin was continued to be added to the system, the absorption of the system at a wavelength of 500 nm further decreased; when a total of 4.5 equivalents of MMAE-Sgc8-Biotin were added, the absorption of the system at a wavelength of 500 nm dropped to the minimum, and when MMAE-Sgc8-Biotin was continued to be added to the system, the absorption of the system at a wavelength of 500 nm no longer changed, indicating that the system reached a saturated state. Therefore, SA is preferably combined with 4.5 equivalents of MMAE-Sgc8-Biotin.

[0059] Similarly, by Figure 4 b It can be seen that when 4 equivalents of MMAE-SL1-Biotin are added, the absorption of the system at a wavelength of 500nm no longer changes, and a saturated state is reached. Therefore, SA is preferably combined with 4 equivalents of MMAE-SL1-Biotin. Because each nucleic acid aptamer will produce steric hindrance when assembled with SA after folding into a certain conformation, different nucleic acid aptamers produce different steric hindrances when assembled with SA, so when assembled with SA, different nucleic acid aptamers require different equivalents to drive. In summary, compared with the nucleic acid aptamer Sgc8, the nucleic acid aptamer SL1 requires a smaller equivalent when combined with SA, indicating that the steric hindrance of SL1 and SA is smaller, and it is easier to prepare a tetravalent nucleic acid aptamer drug conjugate, so the nucleic acid aptamer SL1 is preferred in this embodiment.

[0060] Example 3. Stability characterization of SA-4ApDC and monovalent ApDC

[0061] Take 100 μL of cell culture medium containing 25% FBS, add SA-4 (Sgc8-MMAE) and MMAE-Sgc8-Biotin to the culture medium to make the final concentration of the solution 4 μM. After shaking and mixing, take 10 μL of the sample as the 0min sample and store it in a -40℃ refrigerator. The subsequent sampling time is 4h, 8h, 12h, 24h, and 48h.

[0062] Same as above, take 100 μL of culture medium containing 50% FBS, add SA-4 (SL1-MMAE) and MMAE-SL1-Biotin to the culture medium respectively, so that the final concentration of the solution is 4 μM. After shaking, mixing and centrifuging, take 10 μL of sample as 0min sample, put it in -40℃ refrigerator for storage, and the subsequent sampling time is 4h, 8h, 12h, 24h, 48h. Then perform agarose gel electrophoresis analysis, and calculate the degradation percentage at different times by analyzing the gray value. Using the normalization method, the band intensity at 0h is set as 100%, and the retention ratio at the following time points is calculated as: (gray value at this time point / gray value at 0h) × 100%.

[0063] like Figure 5 As shown in the figure, the retention rate of SA-4 (Sgc8-MMAE) in the medium containing 25% FBS is higher than that of MMAE-Sgc8-Biotin, indicating that the stability of SA-4 (Sgc8-MMAE) is stronger than that of MMAE-Sgc8-Biotin. Figure 6 As shown, the retention rate of SA-4(SL1-MMAE) in the medium containing 50% FBS is higher than that of MMAE-SL1-Biotin, indicating that the stability of SA-4(SL1-MMAE) is higher than that of MMAE-SL1-Biotin.

[0064] Example 4: Characterization of the affinity between SA-4ApDC and monovalent ApDC

[0065] HT-29 cells were digested with trypsin-free digestion solution, and after counting, the cells were transferred to 4 centrifuge tubes at a number of 200,000 / tube. After the cells were centrifuged at low speed, the supernatant was discarded and 200 μL of pre-cooled Binding Buffer was added to each of the 4 centrifuge tubes, and then SA-4 (Sgc8-MMAE), MMAE-Sgc8-Biotin, SA-4 (SL1-MMAE), and MMAE-SL1-Biotin were added to each of the 4 centrifuge tubes to make the final concentration of the solution 250 nM. After shaking and mixing, the centrifuge tubes were placed in an ice box and incubated on a shaker in the dark for 40 minutes. After the cell incubation, the cells were washed 3-4 times with Washing Buffer and finally analyzed by flow cytometry.

[0066] like Figure 7As shown, SA-4(Sgc8-MMAE) has a higher binding affinity to HT-29 cells (left) and SKOV3 cells (right) than MMAE-Sgc8-Biotin; Figure 8 As shown, SA-4(SL1-MMAE) has higher binding ability to HT-29 cells (left) and SKOV3 cells (right) than MMAE-SL1-Biotin.

[0067] Example 5: Characterization of the endocytic ability of SA-4ApDC and monovalent ApDC

[0068] HT-29 cells were transferred to a four-grid confocal dish at a number of 200,000 / grid. After the cells were completely attached overnight, SA-4 (Sgc8-MMAE), MMAE-Sgc8-Biotin and SA-4 (40A-MMAE) were added at a final concentration of 400nM, and incubated for 6 hours at 37°C and 5% carbon dioxide. Among them, SA-4 (40A-MMAE) has no targeting effect and is the control group. After the cell incubation is completed, the cell nucleus and lysosomes are stained, and the lysosomal dye is diluted into the ready-to-use nuclear dye at a ratio of 1:20000, and then added to the confocal dish (covering the cell surface) after shaking and mixing, and the staining time is 30min. After the staining is completed, the cells are washed 2-3 times with PBS and confocal imaging is performed. The verification operation steps for the characterization of the endocytic ability of SA-4 (SL1-MMAE) and MMAE-SL1-Biotin on HT-29 cells are the same as above.

[0069] like Fig. 9 and Fig.10 As shown, under the same incubation time, the endocytosis of SA-4(Sgc8-MMAE) by HT-29 cells was stronger than that of MMAE-Sgc8-Biotin, and the endocytosis of SA-4(SL1-MMAE) by HT-29 cells was stronger than that of MMAE-SL1-Biotin; moreover, SA-4(Sgc8-MMAE) and SA-4(SL1-MMAE) both had strong co-localization with lysosomes.

[0070] Example 6: Experiment on the inhibition of HT-29 cell proliferation by SA-4ApDC

[0071] HT-29 cells were seeded into a 96-well plate at a number of 3000 / well and cultured at 37°C and 5% carbon dioxide for 24 hours. After the culture, culture medium containing different concentrations of SA-4 (Sgc8-MMAE), MMAE-Sgc8-Biotin and SA-4 (40A-MMAE) was added, and the cells were incubated for another 4 hours under the above cell culture conditions. After the cell incubation, new culture medium was replaced and the cells were incubated for 72 hours. After the incubation, the inhibitory effects of the three groups of drugs on cell proliferation were determined using the CCK-8 kit. The operation of the HT-29 cell proliferation inhibition experiment with SA-4 (SL1-MMAE) and MMAE-SL1-Biotin was the same as above.

[0072] Depend on Fig.11 It can be seen that compared with MMAE-Sgc8-Biotin, the cell activity of HT-29 cells in the corresponding concentrations of SA-4 (Sgc8-MMAE) medium is lower, indicating that SA-4 (Sgc8-MMAE) has a stronger inhibitory effect on HT-29 cell proliferation; similarly, according to Fig.12 It can be seen that SA-4(SL1-MMAE) has a stronger inhibitory effect on HT-29 cell proliferation than MMAE-SL1-Biotin.

[0073] Example 7: SA-4 (Sgc8-MMAE) is used in the experiment of inhibiting malignant tumors

[0074] Take the HT-29CDX model tumor volume between 100-150mm 3 Twelve mice were randomly divided into four groups. The four groups of mice were injected with SA-4 (Sgc8-MMAE), MMAE-Sgc8-Biotin, SA-4 (40A-MMAE), and saline (Saline) through the tail vein, respectively. The dosage was 0.25 μmol / kg, and the drug was administered once every four days for a total of five times. Starting from the first administration, the tumor growth of the mice was recorded every two days, and the length (a) and width (b) of the tumor were recorded, and then the tumor volume was calculated. The calculation formula is V = (a×b 2 ) / 2. On the 35th day, the mice were euthanized and the experiment was terminated.

[0075] like Fig.13 and Fig.14 As shown, under the four treatments, the tumor volume growth change in the SA-4 (Sgc8-MMAE) group was the smallest, indicating that SA-4 (Sgc8-MMAE) showed a good inhibitory effect on tumors, and there was no significant difference in mouse body weight compared with the normal saline group.

[0076] Example 8: Determination of nucleic acid aptamer terminal modification molecules

[0077] Since the yield of the tetravalent aptamer-drug conjugate obtained by biotin-modifying one end of the aptamer and then connecting with streptavidin may be different, in order to reduce the loss of raw materials and reduce the cost, in this embodiment, the 3' end or 5' end of the aptamers sgc8c and SL1 are modified with biotin, and the other end is modified with a drug molecule, and the tetravalent aptamer-drug conjugate is prepared according to the method of Example 1. The results show that when the 3' end of the aptamer is modified with biotin and the 5' end is modified with a drug, the yield of the constructed SA-4 (Sgc8-MMAE) is about 93.6%, and the yield of SA-4 (SL1-MMAE) is about 94.1%; when the 5' end of the aptamer is modified with biotin and the 3' end is modified with a drug, the yield of SA-4 (Sgc8-MMAE) is about 61.8%, and the yield of SA-4 (SL1-MMAE) is about 59.4%. It can be seen that when biotin modifies the 5' end of the nucleic acid aptamer, the yield of the obtained tetravalent nucleic acid aptamer drug conjugate is significantly reduced. Therefore, the present invention preferably modifies the 3' end of the nucleic acid aptamer with biotin and modifies the 5' end with the drug molecule.

[0078] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0079] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

[0080] Sequence Listing

[0081] SEQ ID NO.1:

[0082] sgc8c:

[0083] 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'

[0084] SEQ ID NO.2:

[0085] SL1:

[0086] 5'-ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGAT-3'

Claims

1. A tetravalent nucleic acid aptamer drug conjugate, characterized in that: The invention comprises a streptavidin and four nucleic acid aptamer drug complexes. The nucleic acid aptamer drug complexes comprise nucleic acid aptamers, drugs and biotin. The biotin modifies one end of the nucleic acid aptamer, and the drug molecule modifies the other end of the nucleic acid aptamer.

2. The tetravalent nucleic acid aptamer drug conjugate according to claim 1, characterized in that: The biotin is introduced into one end of the nucleic acid aptamer by solid phase synthesis, and the drug molecule is introduced into the other end of the nucleic acid aptamer by coupling in any one or more ways of thiol-maleimide, azide-alkyne, amino-carboxyl.

3. The tetravalent nucleic acid aptamer drug conjugate according to claim 2, characterized in that: The specific types of cell lines containing the following types of cells are CD5, CD19, CD20, CD25, CD37, CD30, CD33, and CD45. CAMPATH-1, HLD-DR, CEA, TAG-72, EpCAM, MUC1, MUC15, folate-binding protein、A33、G250、PSMA、ferritin、GD2、GD3、GM2、Leg、CA-125、CA19-9、epidermal growth factor、p185HER2、IL-2receptor、tenascin、a metalloproteinase、endosialin、vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, EGFRvIII, Her-2 / neu, MAGE A3, p53 nonmutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, p53 mutant、PR1、bcr-abl、tyrosinase、survivin、PSA、hTERT、a Sarcoma translocation breakpoint fusion protein、EphA2、PAP、ML-IAP、AFP、ERG、NA17、PAX3、ALK、androgen receptor、cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, MSLN, PSCA, MAGE Al, MAGE-A3, sLe, CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX,PAX5, OY-TESLSperm protein 17. LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3 Page4 VEGFR2 MAD-CT-1 PDGFR-B MAD-CT-2 ROR2 c-Met HER3 EPCAM CA6 NAPI2B TROP2 CLDN18.2、FAP、RON、LY6E、FRA、DLL3、PTK7、LIV1、ROR1、Fos-related antigen1、VEGFR、endoglin、PD-L1、CD204、CD206、CD301、VTCN1、VISTA。.

4. The tetravalent nucleic acid aptamer drug conjugate according to claim 3, characterized in that: The drug includes any one or more of auristatin-type microtubule protein inhibitors, Deruxtecan (Dxd), exotecan, or derivatives having the same or similar core structure as auristatin-type microtubule protein inhibitors, Dxd or exotecan.

5. The tetravalent nucleic acid aptamer drug conjugate according to claim 4, characterized in that: The nucleic acid aptamer includes a PTK7 targeting nucleic acid aptamer and / or a c-Met targeting nucleic acid aptamer.

6. The tetravalent nucleic acid aptamer drug conjugate according to claim 5, characterized in that: The biotin modifies the 3' end of the nucleic acid aptamer, and the drug molecule modifies the 5' end of the nucleic acid aptamer.

7. A drug for targeted tumor treatment, characterized in that: It comprises the tetravalent nucleic acid aptamer drug conjugate as described in any one of claims 1 to 6.

8. A method for preparing a tetravalent nucleic acid aptamer drug conjugate, characterized in that: The following steps are involved: (1) Synthesis of a nucleic acid aptamer modified with biotin and a drug molecule, wherein the biotin is modified at one end of the nucleic acid aptamer and the drug molecule is modified at the other end of the nucleic acid aptamer; (2) Assembly of nucleic acid aptamers modified with biotin and drug molecules and streptavidin.

9. The method according to claim 8, characterized in that When the nucleic acid aptamer in step (2) binds to streptavidin, the binding equivalent of the nucleic acid aptamer is 1 to 5 equivalents.

10. A composition for preparing a reagent for improving the stability and affinity of nucleic acid aptamer drug conjugates, characterized in that: The composition comprises a streptavidin and four nucleic acid aptamer drug complexes, wherein the nucleic acid aptamer drug complexes comprise nucleic acid aptamers, drug molecules and biotin, wherein the biotin modifies one end of the nucleic acid aptamer, and the drug molecules modify the other end of the nucleic acid aptamer.

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