HER2 nano antibody, antibody conjugate and preparation method thereof

Abstract

The invention relates to an HER2 nano antibody, an antibody conjugate and a preparation method of the HER2 nano antibody and the antibody conjugate, and belongs to the technical field of medicine preparation, the HER2 nano antibody is marked as MAH, and the amino acid sequence of the HER2 nano antibody is shown as SEQIDNO.1. The antibody MAH is connected with ginsenoside to form an MAH-Rh2 conjugate, the molecular weight of the MAH-Rh2 conjugate is as small as 33ka, the MAH-Rh2 conjugate can more easily penetrate blood vessels to directly reach the surfaces of tumor cells, the MAH-Rh2 conjugate is used in the growth inhibition process of the tumor cells, the strong inhibition effect on multiple tumor cells with positive HER2 expression is achieved, the tumor growth inhibition effect is achieved within 24 hours, and the application prospect is wide. Compared with the prior art, the method has the advantages that the effect time is prolonged under the same dosage, the better tumor cell growth inhibiting capacity can be obtained, the use dosage of components such as Rh2 is reduced, the use range of MAH-Rh2 is widened, and ginsenoside is precisely delivered in a targeted mode through MAH-Rh2 which plays a role in double ways.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparation, and in particular to a HER2 nano antibody, an antibody conjugate and a preparation method thereof. Background Art

[0002] HER2 is an immunohistochemical indicator and a member of the human epidermal growth factor receptor family. It plays an important role in signal transduction. HER2 protein is usually expressed during fetal development, but is expressed at low levels or not at all in normal adult tissues. However, HER2 may be amplified or overexpressed in a variety of tumors such as lung cancer, breast cancer, colorectal cancer, endometrial cancer, gastric cancer, and prostate cancer. HER2 mutations in tumors usually involve overexpression and amplification, the latter of which refers to increased replication of the HER2 gene sequence, which may lead to increased HER2 protein synthesis or enhanced protein function, thereby triggering overactivation of downstream signaling pathways and excessive cell proliferation, ultimately promoting tumorigenesis.

[0003] Antibody-drug conjugates (ADCs) are made by coupling monoclonal antibodies targeting tumor-specific antigens or tumor-associated antigens with a specific number of small molecule cytotoxins through linkers. Due to its high targeting, high specificity, high activity, and low systemic toxicity compared to traditional chemotherapy drugs, it has become one of the popular drugs for targeted anti-tumor therapy and is called a "biological missile" by scholars. Antibody-drug conjugates use the cell targeting and high specificity of monoclonal antibodies to selectively act on tumor cells, and use the cytotoxicity of small molecule toxins to efficiently kill tumor cells; through the coupling effect of linkers, the off-target toxicity of small molecule toxins is effectively reduced, thereby distinguishing it from general monoclonal antibodies and chemotherapy drugs, and achieving the purpose of targeted therapy, increased efficacy and reduced toxicity.

[0004] In the field of targeted therapy, single-chain antibody (scFv) has shown great application prospects in the fields of medicine and biotechnology due to its tiny molecular size, excellent tissue penetration ability and precise targeting characteristics. Therefore, single-chain antibody and small molecule toxins can be prepared into conjugates to achieve the purpose of inhibiting tumor growth. Summary of the invention

[0005] The present invention aims to provide a HER2 single-chain nanobody.

[0006] Another object of the present invention is to provide applications of the above-mentioned single-chain antibody.

[0007] The third object of the present invention is to provide an antibody conjugate based on HER2 antibody. The conjugate formed by coupling HER2 antibody MAH with ginsenoside Rg3 or Rh2 achieves a strong inhibitory effect on a variety of HER2-positive tumor cells.

[0008] The fourth object of the present invention is to provide a method for preparing the above-mentioned antibody conjugate. The method solves the problem that ginsenosides Rg3 and Rh2 cannot attach to the surface of MAH and are difficult to form a conjugate, and successfully forms an effective conjugate.

[0009] The fourth object of the present invention is to provide the application of the above conjugate.

[0010] The purpose of the present invention is achieved through the following technical solutions: A HER2 single-chain nano antibody, characterized in that: the amino acid sequence of the antibody is MTMITPSFGAFFLEIFNVKKLLFAIPLVVPFYAAQPAMAQVKLQQSGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALKSRLNISKDNSKSQVFLKMNSLQTDDTAMYYCARNWGSYWYFDVWGQGHGHRLLSGGGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCRASSSISSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGAGTKLEIKRAAAGAPVPYPDPLEPRAA (SEQIDNO.1), denoted as MAH.

[0011] The nucleotide sequence of MAH nanoantibodies consists of a light chain variable region (VL) and a heavy chain variable region (VH), each of which consists of four framework regions (FR) and three complementarity determining regions (CDR).

[0012] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0013] The nucleotide sequence of CDR1 in the heavy chain variable region is shown in SEQ ID NO.3; The nucleotide sequence of CDR2 in the heavy chain variable region is shown in SEQ ID NO.4; The nucleotide sequence of CDR3 in the heavy chain variable region is shown as SEQ ID NO.5.

[0014] The nucleotide sequence of the light chain variable region is shown as SEQ ID NO.10.

[0015] The nucleotide sequence of CDR1 of the light chain variable region is shown as SEQ ID NO.11; The nucleotide sequence of CDR2 of the light chain variable region is AGGACATCC; The nucleotide sequence of CDR3 of the light chain variable region is shown as SEQ ID NO.12.

[0016] The use of the above-mentioned HER2 single-chain nanoantibody in the preparation of antibody conjugates.

[0017] A small molecule antibody conjugate based on the above HER2 antibody, characterized in that: the conjugate MAH-Rg3 or MAH-Rh2 is formed by coupling the single-chain antibody MAH with ginsenoside Rg3 or Rh2, and the molecular weight of the antibody conjugate is about 33kDa.

[0018] Furthermore, the structural formula of the antibody conjugate MAH-Rg3 is: .

[0019] Furthermore, the structural formula of the antibody conjugate MAH-Rh2 is: .

[0020] The preparation method of the above-mentioned antibody conjugate is characterized in that: ginsenoside is dissolved in an anhydrous toluene solution of (3-aminopropyl)trimethoxysilane (APTMS), stirred for reaction to obtain aminosilane-modified ginsenoside, centrifuged to collect the precipitate, washed to obtain aminosilane-activated ginsenoside, MAH is dissolved in ultrapure water to prepare an MAH solution, mixed with an activation buffer to obtain an activated MAH solution, EDC solution and Sulfo-NHS aqueous solution are added to the activation buffer, and then aminosilane-activated ginsenoside is added to form a mixed solution, the mixed solution is added to the activated MAH solution for mixing reaction, and then separated by protein L affinity chromatography filler to obtain an antibody conjugate.

[0021] Furthermore, when the ginsenoside is Rg3, the mass volume ratio of the anhydrous toluene solution of Rg3 and APTMS is 1.5-2.5 g:100 mL, and the mass volume percentage concentration of the anhydrous toluene solution of APTMS is 0.8-1.2%.

[0022] Furthermore, when the ginsenoside is Rh2, the mass volume ratio of Rh2 and APTMS in anhydrous toluene solution is 0.8-1.2 g:100 mL, and the mass volume percentage concentration of APTMS in anhydrous toluene solution is 0.8-1.2%.

[0023] Furthermore, the volume ratio of the MAH solution to the activation buffer in the activated MAH solution is 1:1, the activation buffer is a MES solution with a pH of 6.0 and a concentration of 50 mM, and the mass volume percentage concentration of the MAH solution is 8-12%.

[0024] Furthermore, the concentration of aminosilane-activated ginsenosides in the mixed solution is 1.5-2.5 mg / mL, and the volume ratio of the activation buffer, EDC solution, and Sulfo-NHS aqueous solution is 100:2-2.5:22-25. The activation buffer is a MES solution with a pH of 6.0 and a concentration of 50 mM, the concentration of the EDC solution is 200 mmol / L (19.2 mg EDC is weighed and dissolved in 500 µL ultrapure water), and the concentration of Sulfo-NHS is 200 mmol / L (21.7 mg Sulfo-NHS is added to 500 µL of activation buffer).

[0025] Furthermore, the volume ratio of the activated MAH solution to the mixed solution is 10:1.

[0026] Furthermore, the specific steps of separating the protein L and the chromatography filler are as follows: (1) Equilibration: Use 5 column volumes of equilibration buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) to equilibrate the column so that the filler is in the same buffer system as the target protein to protect the protein. (2) Sample loading: Add the sample to the well-balanced filler to ensure that the target protein is fully in contact with the resin; (3) Washing: Wash with 10-15 column volumes of washing buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0).

[0027] (4) Elution: Use 5-10 column volumes of elution buffer (0.1 M glycine, pH 3.0) and collect the eluate, which is the target protein component.

[0028] The conjugates used in the past were mainly cytotoxic molecules, which delivered toxic molecules into cells to kill them, but this often led to the proximity effect, thus affecting the growth of normal cells and changes in the tumor microenvironment. Secondly, antibody-drug conjugates mainly use complete antibodies (about 150-180kDa) as targeted delivery carriers. This type of antibody-conjugated biological toxin molecule with a large molecular weight is difficult to pass through the microvessels in the tumor infiltration area and is difficult to exert its full effect.

[0029] In the present invention, MAH is used as a carrier, and is coupled with ginsenoside Rg3 and Rh2, respectively, to form complete conjugates MAH-Rg3 and MAH-Rh2. However, in the process of preparing the conjugate, since ginsenoside Rg3 and Rh2 do not have functional groups that can be coupled with single-chain antibody MAH, Rg3 and Rh2 cannot be coupled with MAH. Therefore, it is necessary to modify ginsenosides, and the coupling sites on the surfaces of different antibodies are different, which determines the type of groups modified by saponins. In the present invention, the method of covalently connecting aminocarboxyl groups to form amide bonds is used to couple MAH with Rg3 or Rh2, and then the covalent conjugate is purified by affinity chromatography. The molecular weight of the obtained conjugate is about 33kDa, and the small molecular weight is easy to penetrate microvessels and directly reach the surface of tumor cells, saving medical resources to a greater extent. In addition, since the conjugated Rg3 and Rh2 are almost harmless to ordinary cells, and have a positive effect on the tumor microenvironment and the differentiation and activation of tumor-infiltrating lymphocytes, MAH-Rg3 and MAH-Rh2 do not have the proximity effect of traditional antibody-drug conjugates. Therefore, MAH-Rg3 and MAH-Rh2 have a positive regulatory effect on the tumor microenvironment.

[0030] The antibody conjugate prepared by the above method is used in the preparation of targeted tumor growth inhibition drugs.

[0031] The present invention has the following technical effects: The antibody MAH of the present invention is connected with ginsenoside to form MAH-Rg3 and MAH-Rh2 conjugates, with a molecular weight as low as 33ka, which can more easily penetrate blood vessels and directly reach the surface of tumor cells. It is used in the process of inhibiting tumor cell growth to achieve a strong inhibitory effect on a variety of HER2-positive tumor cells, and can inhibit tumor growth within 24 hours. Prolonging the action time at the same dose can also obtain a better ability to inhibit tumor cell growth, which not only reduces the dosage of ingredients such as Rg3 and Rh2, but also expands the scope of use of MAH-Rg3 and MAH-Rh2. Secondly, MAH-Rg3 and MAH-Rh2, which exert their effects through dual pathways, deliver ginsenosides in a targeted and precise manner, thereby achieving excellent anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Flowchart for constructing a cDNA library containing single-chain antibodies encoding HER2 protein-specific genes.

[0033] Figure 2 :The results of serum titer determination after mouse immunization.

[0034] Figure 3 : Electrophoresis results of VH and VL gene fragments amplified from cDNA templates of mouse lymphocytes.

[0035] Figure 4 :Construction and transformation of scFv-pCANTEB-5E recombinant vector involving electrophoresis results; A. Agarose gel electrophoresis results of connecting VL to Linker by SOE-PCR; B. Electrophoresis results of scFv gene generated by connecting VH and VL chains by SOE-PCR; C. Electrophoresis results of clones containing insert fragments of expected size identified by PCR amplification.

[0036] Figure 5 : Single-chain antibody gene sequencing and homology analysis.

[0037] Figure 6 : Three-dimensional modeling of single-chain antibodies and analysis of antibody amino acid sequences.

[0038] Figure 7 : SDS-PAGE analysis of purified single-chain antibody electrophoresis results.

[0039] Figure 8 :Western Blot was used to verify that the single-chain antibody could bind to HER2 protein electrophoresis.

[0040] Fig. 9 :Effects of different concentrations of single-chain antibody on the survival rate of HEK-293T cells.

[0041] Fig.10 : Detection of changes in HER2 protein content on the NCI-N87 cell membrane treated with gradient concentrations of single-chain antibody and trastuzumab for 24 hours; a. NCI-N87 treated with 100μg / mL single-chain antibody; b. NCI-N87 treated with 0.01μg / ml single-chain antibody; c. NCI-N87 treated with 100μg / mL trastuzumab; d. NCI-N87 treated with 0.01μg / mL trastuzumab.

[0042] Fig.11 : Cell cycle changes of NCI-N87 cells treated with gradient concentrations of single-chain antibody and trastuzumab; a. NCI-N87 treated with 100μg / mL single-chain antibody; b. NCI-N87 treated with 0.01μg / ml single-chain antibody; c. NCI-N87 treated with 100μg / mL trastuzumab; d. NCI-N87 treated with 0.01μg / mL trastuzumab.

[0043] Fig.12 : Inhibitory effect of single-chain antibody treatment on NCI-N87 cells; a. The proportion of G2 phase cells of NCI-N87 cells treated with different concentrations of single-chain antibody; b. Changes in ATP levels in NCI-N87 cells treated with different concentrations of single-chain antibody and trastuzumab.

[0044] Fig.13 : H NMR spectrum of antibody conjugate MAH-Rg3.

[0045] Fig.14 : H NMR spectrum of antibody conjugate MAH-Rh2.

[0046] Fig.15 :Effects of ginsenosides Rg3 and Rh2 on the viability of A549, HCT-116 and NCI-N87 cells at different times; a. 24-hour cell viability; b. 48-hour cell viability; c. 72-hour cell viability.

[0047] Fig.16 : Bright field images of A549, HCT-116, and NCI-N87 cells treated with 100 μmol Rg3 and Rh2 for 24 h; a, b, c: control group, d, e, f: Rg3 group; g, h, i: Rh2 group.

[0048] Fig.17 : Western blot results of total proteins extracted from A549, HCT-116, and NCI-N87 cells treated with 100 μmol Rg3 and Rh2 for 24 h, and q-PCR results of HER2 gene extracted from total RNA.

[0049] Fig.18 : Bright field photographs of A549, HCT-116 and NCI-N87 cells treated with 100 μg / mL MAH-Rg3 / MAH-Rh2 for 24 h; a, b, c: control group, d, e, f: MAH-Rg3 group; g, h, i: MAH-Rh2 group.

[0050] Fig.19 : Cell viability assay of NCI-N87 cells treated with graded concentrations of trastuzumab and single-chain antibody for 24 hours.

[0051] Fig. 20 : Cell viability assay of NCI-N87 cells treated with gradient concentrations of MAH-Rg3 and MAH-Rh2 for 24 h.

[0052] Fig.21 : IC50 of NCI-87 cells treated with Rg3, Rh2, MAH-Rg3 and MAH-Rh2 for 24 hours; a. IC50 of Rg3 treatment group; b. IC50 of Rh2 treatment group; c. IC50 of MAH-Rg3 treatment group; d. IC50 of MAH-Rh2 treatment group.

[0053] Fig. 22: IC50 curves of different organoids for ginsenoside conjugates, Rg3, Rh2, single-chain antibodies and trastuzumab, where P1, P2, and P3 represent organoids from patients 1, 2, and 3, respectively. DETAILED DESCRIPTION

[0054] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0055] Example 1 Preparation of anti-HER2 protein nanoantibody MAH: 1. Mouse Immunity and Antibody Variable Region Gene Amplification Two BALB / C mice (Changchun Yisi Experimental Animal Co., Ltd., quality certificate number: 01021684285444659) were immunized with recombinant human HER2 protein (Novoprotein, CX68). Another healthy BALB / C mouse was selected as the control group and 150 μL of blood was collected by tail cut off according to the immunization schedule. The plasma was centrifuged at 13000 rpm for 20 min at 4°C. The upper serum was taken and diluted into eight gradients of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000 for mouse serum antibody titer detection. The serum titer of mice was determined by ELISA, and the serum titer ≥1:64000 was considered to be successfully immunized. The mice that were successfully immunized were killed by dislocating the neck and disinfected with alcohol, and then quickly placed in a biosafety cabinet and fixed. The abdomen was opened with surgical scissors and forceps to separate the spleen. After the spleen was separated, it was placed in pre-cooled PBS without RNAse, and the spleen was cut open with scissors. The lymphocytes were flushed out by repeated washing with pre-cooled PBS without RNAse, and the lymphocyte RNA was extracted by Trizol method. The cDNA was synthesized using Revert Aid First Strand cDNA Synthesis Kit (K1621, Thermo Scientific), and then the antibody variable region gene was amplified. The above mouse immunization program is shown in Table 1.

[0056] Table 1:

[0057] Test results see Figure 2The titers of the sera of the two mice were both over 1:64000, which met the library construction standards. The spleen lymphocytes of the two successfully immunized mice were isolated to extract total RNA. The total RNA was detected by agarose gel electrophoresis and showed good integrity. Then cDNA synthesis and antibody variable region sequence amplification were performed. The introduction used for amplification is shown in Table 2.

[0058] Table 2:

[0059] Note: VH1BACK, VH1FOR, VK2BACK, MJk1FONX, MJk2FONX , MJk3FONX, MJk4FONX are the upstream and downstream primers for amplifying the heavy chain variable region and the light chain variable region respectively; VH1BACK-Sfi1, JK1-NOT1, JK2-NOT1, JK3-NOT1, JK4-NOT1 are restriction sites for adding to the amplified products. The bold italic positions in the table are Sfi1 restriction sites, and the bold positions are Not1 restriction sites; Linker is a DNA sequence that connects the heavy chain variable region and the light chain variable region, and the amino acid sequence in bold italics and underline is the 3xGGGGS structure; the merged bases R=A / G; Y=C / T; M=A / C; K=G / T; S=C / G; W=A / T; H=A / C / T; B=C / G / T; V=A / C / G; D=A / G / T.

[0060] The results of variable region amplification are shown in Figure 3 , is to amplify VH and VL gene fragments from the cDNA template of mouse lymphocytes, among which channels 1-3 are antibody heavy chain variable region fragments of about 350bp, and channels 4-6 are antibody light chain variable region fragments of about 320bp, which are the same as the predicted results and are classified and recovered.

[0061] 2. Construction and transformation of scFv-pCANTEB-5E recombinant vector The amplified heavy chain and light chain variable region genes were connected to the linker by two-step overlap extension PCR. First, VL was connected to the linker, and then VH was connected to VL-Linker by overlap extension PCR. The scFv gene purified by gel recovery was double-digested with restriction endonucleases Sfi1 (1244S, Takara) and Not1 (1166A, Takara), and then connected to the pCANTEB-5E vector by T4-DNA ligase (2011A, Takara) to construct a recombinant vector, which was then electroporated into Escherichia coli TG1. The results are shown in Figure 2. Figure 4As shown in A and B. A is the scFv gene generated by connecting VL and Linker by SOE-PCR, and the agarose gel electrophoresis result is about 420bp; B is the scFv gene generated by connecting VH and VL chains by SOE-PCR, and the electrophoresis result is about 750bp. TG1 with successful electroporation was selected and 20 single colonies with good morphology were used for bacterial liquid PCR. The primers used are shown in Table 3.

[0062] Table 3:

[0063] The results are as follows Figure 4 As shown in C, clones containing inserts of expected sizes were identified by PCR amplification. The colony PCR results showed that except for single colony No. 6, which was negative, all others were positive. The PCR target band was around 750 bp, which was consistent with the expected result. The initial positive conversion rate was 95%.

[0064] 3. Single-chain antibody library preparation and phage panning The successfully electroporated TG1 was cultured in a shaking flask at 37°C and 180 rpm for 2 h. 5 pfu of M13KO7 helper phage was rescued, and the phage was precipitated by PEG / NaCl solution to obtain the primary phage library. The recombinant human HER2 protein was coated on the ELISA plate, and the primary phage was repeatedly washed four times to obtain the mouse anti-human HER2 single-chain antibody phage library. The single-chain antibody phage library was subjected to polyclonal ELISA according to the procedure to obtain high-affinity HER2 protein phage, as follows: (1) Dilute the recombinant human HER2 protein to 20 μg / mL using the highly absorbent coating solution in the ELISA auxiliary kit. Add 100 μL of the diluted HER2 solution to each well of a 96-well ELISA plate and incubate at 4°C overnight. (2) After overnight, wash five times with 200 μL of universal sandwich ELISA washing buffer per well for 5 minutes each time. After complete washing, block with 200 μL of ready-to-use ELISA blocking buffer per well at 4°C overnight.

[0065] (3) After overnight, wash the cells five times with 200 μL of universal sandwich ELISA washing buffer per well for 5 minutes each time.

[0066] (4) Take a total of 1×1010 pfu of the primary mouse single-chain antibody phage library, evenly add it to the ELISA plate, and incubate at 4°C for 10 h.

[0067] (5) After incubation, wash five times with 200 μL of universal sandwich ELISA wash buffer per well for 10 minutes each time.

[0068] (6) Add 0.2 M glycine-HCl buffer, 100 μL per well. After incubation for 5 min, immediately add 2 M Tris-HCl solution, 10 μL per well, for neutralization.

[0069] (7) Collect the neutralized solution and add it to TG1 Escherichia coli with an OD600 of 0.6. Incubate at 37°C and 180 rpm for 1 h.

[0070] (8) Take 10 μL of the cultured bacterial liquid and perform a gradient dilution with 2×YT liquid medium to the dilution levels of 102, 104, 106, and 108. Take 100 μL of each dilution and add it to 5 mL of top agar, mix well, and immediately pour it onto the prepared plates and culture at 37°C overnight.

[0071] (9) Calculate the number of spots on the plate and multiply it by the corresponding dilution factor to obtain the titer of the phage eluted that time.

[0072] (10) The remaining cells from step (7) were cultured overnight at 37°C, 180 rpm. After overnight culture, the culture solution was transferred to a centrifuge tube and centrifuged at 4°C, 10,000 g for 20 min. The supernatant was transferred to a new centrifuge tube and 1 / 5 volume of PEG / NaCl solution was added to mix. The tube was then allowed to stand at 4°C for 4 h.

[0073] (11) When the solution becomes turbid, centrifuge at 10,000 g for 20 min at 4°C. Discard the supernatant, add 1 mL of pre-cooled PBS to resuspend the precipitate and filter through a 0.22 μm filter membrane. The resulting phages are the mouse single-chain antibody phage primary panning library, which should be stored at 4°C.

[0074] (12) The phage obtained in step (11) is panned again, and the panning steps are (4) → (12). After repeating three times, a high-affinity HER2 protein phage is obtained.

[0075] The selected phages were re-infected into TG1, and ten single colonies were randomly selected for monoclonal phage ELISA to screen positive phages with high P / N values ​​for protein expression; The selected phage infected TG1 and 10 single colonies were selected for monoclonal ELISA. After the phage showed obvious strong positive, it was infected with TG1 and the plasmid was extracted and sequenced. The sequencing results are shown in Figure 5As shown, using Snap Gene analysis, the sequencing results show that it contains Sfi1 and Not1 restriction sites at both ends. Then the gene sequence was compared and analyzed using IgBLAST, and it can be seen that the heavy chain variable region and light chain variable region in this gene segment are complete, among which the homology rate between the heavy chain variable region and the gene IGHV2-6*03 is 96.2% (IMGT Accession numbers: BN000872), and the homology rate between the light chain variable region and the gene IGKV4-58*01 is 95.9% (IMGT Accession numbers: K00884), as follows: MAH nanobodies are mainly composed of light chain variable regions (VL) and heavy chain variable regions (VH). Each variable region consists of four framework regions (FR) and three complementarity determining regions (CDR). The framework region plays the following roles: (1) Structural support: FR1-FR4 constitute the β-folded structure of the variable region, which maintains the three-dimensional conformation of the antibody variable region and ensures the correct spatial arrangement of the CDR region.

[0076] (2) Stability maintenance: The conserved FR sequence stabilizes the antibody structure through hydrophobic interactions and hydrogen bonds, preventing conformational destruction caused by mutations or environmental changes.

[0077] (3) Auxiliary function: Some FR residues may indirectly affect antigen binding, such as regulating the flexibility of CDR or participating in the interaction with other molecules (such as T cell receptors, MHC molecules).

[0078] (4) Immunogenicity regulation: During the antibody humanization process, replacing non-human FRs with human sequences can reduce immunogenicity, indicating that FRs may contain species-specific epitopes.

[0079] The complementarity determining regions play the following roles: (1) Antigen binding core: CDRs (especially CDR3) directly bind to the antigen epitope and determine the specificity and affinity of the antibody. CDR3 has the highest diversity due to gene recombination and high-frequency mutation and is the key region for antigen recognition.

[0080] (2) Conformational diversity: CDR1 and CDR2 are mainly involved in binding to the conserved regions of the antigen, while CDR3 adapts to the complex shape of the antigen surface through its flexible structure.

[0081] (3) Specificity determining factors: The differences in CDR sequences of different antibodies determine the uniqueness of their antigen binding and are the molecular basis of antibody diversity.

[0082] That is, in the entire gene sequence, the backbone region is usually certain depending on the source, while the specificity of the antibody is determined by the complementary determining region, which is the factor affecting the function of the antibody.

[0083] The heavy chain and light chain work together to: (1) The CDRs of VH and VL together form a continuous antigen-binding interface (antigen-binding pocket), in which the heavy chain CDR3 generally contributes more binding energy.

[0084] (2) FRs provide structural support in both chains, ensuring the correct pairing of VH and VL, thereby maintaining the integrity of the binding site.

[0085] The nucleotide sequence of the heavy chain of the nanobody is (as shown in SEQ ID NO.2): ATGGCCCAGGTGAAGCTGCAGCAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTTTCATTAACCAGCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAACTAT AATTCAGCTCTCAAATCCAGACTGAACATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGATGACACAGCCATGTACTACTGTGCCAGAAACTGGGGCAGCTACTGGTACTTCGATGTCTGGGGCCAAGGCCACGGTCACCGTCTCCTCAGTGGAGGC The nucleotide sequence of the complementarity determining region in the heavy chain variable region is: CDR1 (SEQIDNO.3): GGGTTTTCATTAACCAGCTATGGT CDR2(SEQIDNO.4):ATATGGGCTGGTGGAAGCACA CDR3 (SEQ ID NO.5): GCCAGAAACTGGGGCAGCTACTGGTACTTCGATGTC The nucleotide sequence of the heavy chain framework region is: FR1 (SEQ ID NO. 6): ATGGCCCAGGTGAAGCTGCAGCAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCA FR2 (SEQ ID NO. 7): GTACACTGGGTTCCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTA FR3 (SEQ ID NO. 8): AACTATAATTCAGCTCTCAAATCCAGACTGAACATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGATGACACAGCCATGTACTACTGT FR4 (SEQ ID NO.9): TGGGGCCAAGGCCACGGTCACCGTCTCCTCAGTGGAGGC The total nucleotide sequence of the heavy chain was compared and analyzed by IgBLAST, and the results showed that the homology between the heavy chain variable region and the gene IGHV2-6*03 was 96.2% (IMGT Accession numbers: BN000872), and no consistent sequence was detected in the variable region (complementarity determining region) sequence.

[0086] The nucleotide sequence of the light chain of the nanobody is (as shown in SEQ ID NO.10): GACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGCCAGCTCAAGTATAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGCGCTTCCCCCAAACCCTTGATTCATAGGACATCCAACC TGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGATGCAACTTATTACTGCCAGCAGTGGAGTGGTTACCCATTCACGTTCGGTGCTGGGACCAAGCTCGAGATCAAACGG The nucleotide sequence of the complementarity determining region of the light chain variable region is: CDR1 (SEQ ID NO.11): TCAAGTATAAGTTCCAGTTAC CDR2:AGGACATCC CDR3 (SEQ ID NO.12): CAGCAGTGGAGTGGTTACCCATTCACG The nucleotide sequence of the light chain framework region is: FR1 (SEQ ID NO. 13): GACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGC FR2 (SEQ ID NO. 14): TTGCACTGGTACCAGCAGAAGTCAGGCCGTCCCCCAAACCCTTGATTCAT FR3 (SEQ ID NO. 15): AACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGATGCAACTTATTACTGC FR4 (SEQ ID NO. 16): TTCGGTGCTGGGACCAAGCTCGAGATCAAACGG The homology between the light chain variable region and the gene IGKV4-58*01 was 95.9% (IMGT Accession numbers: K00884), and no consensus sequence was detected in the variable region (complementarity determining region) sequence.

[0087] 4. Acquisition of anti-HER2 protein nanoantibody MAH The high P / N value phage was used to infect TG1, and the plasmid was extracted and sequenced. The sequencing results were identified by Snap Gene. Homologous molecular modeling was performed using SWISS-MODEL based on the sequencing results. The single-chain antibody PDB model obtained by modeling was then molecularly docked with the HER2 protein model in the PDB database to predict its binding site.

[0088] The plasmid was electrotransformed into Escherichia coli HB2151. A single HB2151 colony with good condition and clear edges was selected and cultured in a 37°C incubator overnight after electrotransformation. The colony was placed in 1L of 2×YT medium containing ampicillin and cultured with shaking until OD600 was 0.6. IPTG was added to make its final concentration 0.5mM and cultured with shaking at 37°C and 180rpm for 6h. After the culture was completed, the colony was centrifuged at 4°C and 5000rpm for 15min, the supernatant was discarded, and the precipitate was washed with 200ml of pre-cooled Ice-Cold Buffer (17.1g sucrose, 0.0146g ethylenediaminetetraacetic acid, 2.42g Tris, completely dissolve the above drugs in 90 ml ultrapure water, adjust the pH to 8.0 with hydrochloric acid, and place at 4°C for use) resuspend and incubate on ice for 1 hour. After incubation, centrifuge at 4°C and 10,000g for 15 minutes. The supernatant is the periplasmic space, which is purified by protein L affinity chromatography filler. The purified single-chain antibody solution is concentrated through an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The ultrafiltration tube centrifugation parameter is 5,000g. Centrifuge for 30 minutes, recover the concentrated product, and store it at -20°C with 200 μl per EP tube, labeled as MAH.

[0089] Characterization of scFv MAH: The gene sequence of the single-chain antibody obtained above was converted into the corresponding amino acid sequence. The amino acid sequence of the MAH is as follows (as shown in SEQ ID NO.1): MTMITPSFGAFFLEIFNVKKLLFAIPLVVPFYAAQPAMAQVKLQQSGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALKSRLNISKDNSKSQVFLKMNSLQTDDTAMYYCARNWGSYWYFDVWGQG HGHRLLSGGGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCRASSSISSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGAGTKLEIKRAAAGAPVPYPDPLEPRAA The SWISS-MODEL modeling tool was used to predict the protein structure of the sequence based on the homology molecular model. The prediction results are as follows Figure 6As shown, from the results it can be seen that the single-chain antibody molecule based on homologous molecular modeling contains a heavy chain variable region, a linker and a light chain variable region, and the three-dimensional structure is claw-shaped, which is consistent with the three-dimensional structure of a single-chain antibody. The PDB model is compared with the human HER2 protein in the PDB database for molecular docking model prediction based on the CoDock database. From the prediction results, it can be seen that the antibody molecule has affinity for HER2 mainly in its extracellular domain, which is consistent with experimental expectations.

[0090] Characterization of MAH by SDS-PAGE and western blot The strong positive scFv-pCANTEB-5E gene was electroporated into HB2151. After IPTG induction to express single-chain antibody, the periplasmic space of bacterial suspension was extracted for affinity chromatography and ultrafiltration purification. The purified product was qualitatively detected by SDS-PAGE, and the molecular weight (MWs) of MAH was estimated by SDS-PAGE (12.5% ​​acrylamide gel). For Western Blot analysis, recombinant human HER2 protein was transferred to nitrocellulose membrane (Whatman, Kent, UK). After blocking with 3% skim milk for 1 hour, anti-E-Tag labeled monoclonal antibody (1:1000 dilution, Beijing Sino Biological Technology Co., Ltd., China) was bound at 4°C for 12 hours. The membrane was then washed 5 times with TBST buffer and bound with HRP-labeled goat anti-rabbit IgG (H+L) (Biyuntian Biotechnology, China) at 4°C for 4 hours.

[0091] According to Snap Gene, the molecular weight of the single-chain antibody MAH is about 32 kDa, which is consistent with SDS-PAGE ( Figure 7 ) results were consistent with those of the antibody, and the antibody was expressed in the periplasmic space. The protein purified and concentrated from the periplasmic space was subjected to Western-Blot detection, using rabbit anti-E-Tag antibody as the primary antibody and HRP-labeled goat anti-rabbit IgG antibody as the secondary antibody. The test results were as follows Figure 8 As shown, the apparent molecular weight of HER2 protein is 120 kDa, which is consistent with experimental expectations. The single-chain antibody molecule also shows a positive signal in this area, which is consistent with the expected results.

[0092] Safety and anti-tumor growth ability verification of single-chain antibody MAH (1) Security: In order to ensure the safety of single-chain antibody, we chose HEK-293T cells for safety assessment. Fig. 9As shown, after treatment with the single-chain antibody MAH concentration range of 2.5-200 μg / mL for 24 hours, 48 ​​hours and 72 hours, the cell activity in 293T cells was not significantly affected. After 72 hours, the survival rate of HEK-293T cells exceeded 85%, so the single-chain antibody can be considered safe.

[0093] (2) Anti-tumor growth ability In order to evaluate the anti-tumor growth ability of the single-chain antibody, the present invention first detected the ability of the single-chain antibody to target HER2. We selected trastuzumab as a control and co-incubated NCI-N87 cells for 24 hours and then used flow cytometry to detect the remaining HER2 targets on the cell membrane. Fig.10 As shown in the figure, in the experimental group, the single-chain antibody MAH treatment groups at concentrations of 100 μg / mL and 0.01 μg / ml showed obvious FITC peak migration, which was consistent with the results of the trastuzumab group. At the highest concentration of 100 μg / mL, the single-chain antibody MAH and trastuzumab groups showed the same HER2 target affinity. Unlike trastuzumab, the degree of FITC peak shift in the single-chain antibody group was significantly weaker than that in the trastuzumab group as the action concentration decreased, indicating that the single-chain antibody MAH has a stronger affinity for the target, and the affinity attenuation is less than that of trastuzumab in the range of 100-0.01 μg / mL. This may be due to the small molecular weight of the single-chain antibody, which makes it easier to affinity with the target protein.

[0094] HER2 protein has the function of regulating cell proliferation. Therefore, when the amount of HER2 target is reduced, the cell cycle should change accordingly. Therefore, we detected the cell cycle of cells treated with single-chain antibodies and trastuzumab to describe this change. The experimental results showed that trastuzumab did not affect the cell cycle of NCI-N87 cells. In contrast, single-chain antibody MAH blocked the NCI-N87 cell cycle in the G2 phase. The reason may be that the single-chain antibody MAH has a low molecular weight and can bind to HER2 protein at a faster rate, resulting in a large amount of affinity to the target site on the cell surface in the early stage of drug action. In addition, the blocking effect of the single-chain antibody is also dose-dependent, which corresponds to the above-mentioned results of targeting HER2. Fig.11 and Fig.12 As shown in A.

[0095] Cancer cells rely on high ATP levels to maintain rapid proliferation. Reducing ATP can inhibit cancer cell growth or induce their death. We evaluated the effects of single-chain antibody MAH and trastuzumab on cell viability. The results are as follows Fig.12 As shown in B, both substances can significantly reduce the content of intracellular ATP, and compared with trastuzumab, the single-chain antibody MAH reduces the ATP content more at the same treatment concentration and time. That is, the ability of the single-chain antibody MAH prepared by the present invention to inhibit tumor cell activity is better than that of trastuzumab.

[0096] Example 2 An antibody conjugate, specifically a conjugate of a single-chain antibody MAH and ginsenoside Rg3, wherein the preparation method thereof is as follows: (1) 0.2 g of ginsenoside Rg3 was added to 100 mL of anhydrous toluene solution containing 1% (3-aminopropyl)trimethoxysilane (APTMS) (w / v) to obtain a mixture, and the mixture was stirred at room temperature for 24 h to obtain aminosilane-modified ginsenosides; the aminosilane-modified ginsenosides were centrifuged at a centrifugal force of 16099 (×g) for 10 min, the supernatant was removed, and the precipitate was collected to obtain aminosilane-modified ginsenosides; the aminosilane-modified ginsenosides were suspended in toluene, washed three times, and then washed twice with ultrapure water to obtain aminosilane-activated ginsenosides; (2) Prepare 1 mL (w / v) of 10% MAH solution with ultrapure water, add 1 mL of activation buffer (50 mM MES, pH 6.0) and mix thoroughly, add 24 µL of 200 mM EDC solution (weigh 19.2 mg EDC and dissolve it in 500 µL ultrapure water) and 240 µL of 200 mM Sulfo-NHS solution (weigh 21.7 mg Sulfo-NHS and add it to 500 µL activation buffer) to 1 mL of activation buffer, vortex mix at room temperature and incubate on a turntable for 30 min, prepare a 2 mg / mL aminosilane-activated ginsenoside solution with activation buffer, add 0.2 mL of aminosilane-activated ginsenoside solution to the activated MAH, and mix on a rotating mixer at room temperature for 2.5 h. After the reaction is completed, separate the conjugate MAH-Rg3 using protein L affinity chromatography and store it at 4°C.

[0097] Example 3 An antibody conjugate, specifically a conjugate of a single-chain antibody MAH and ginsenoside Rh2, wherein the preparation method thereof is as follows: (1) Add 0.2 g of ginsenoside Rh2 to 100 mL of anhydrous toluene solution containing 1% (3-aminopropyl)trimethoxysilane (APTMS) (w / v) to obtain a mixture, and stir the mixture at room temperature for 24 h to obtain aminosilane-modified ginsenosides; centrifuge the aminosilane-modified ginsenosides at a centrifugal force of 16099 (×g) for 10 min, remove the supernatant, collect the precipitate, and obtain aminosilane-modified ginsenosides; suspend the aminosilane-modified ginsenosides in toluene, wash three times, and then wash twice with ultrapure water to obtain aminosilane-activated ginsenosides; (2) Prepare 1 mL (w / v) of 10% MAH solution with ultrapure water, add 1 mL of activation buffer (50 mM MES, pH 6.0) and mix thoroughly, add 24 µL of 200 mM EDC solution (weigh 19.2 mg EDC dissolved in 500 µL ultrapure water) and 240 µL of 200 mM Sulfo-NHS solution (weigh 21.7 mg Sulfo-NHS added to 500 µL activation buffer) to 1 mL of activation buffer, vortex mix at room temperature and incubate on a turntable for 30 min, prepare a 2 mg / mL aminosilane-activated ginsenoside solution with activation buffer, add 0.2 mL of aminosilane-activated ginsenoside solution to the activated MAH, and mix on a rotating mixer at room temperature for 2.5 h. After the reaction is completed, separate the conjugate using protein L affinity chromatography medium and store at 4°C. The prepared conjugate is recorded as MAH-Rh2.

[0098] Characterization of the conjugates: 1H NMR measurements were performed on a BRUKER AVANCE III 600 MHz (Bruker) spectrometer in dimethyl sulfoxide (DMSO) at 25 °C. Chemical shifts are expressed in ppm (δ units) and referenced to the residual solvent signal. Analytical processing was performed using Mnova software (Mestrelab Research).

[0099] Since the ginsenosides Rg3 and Rh2 used in the present invention do not have functional groups that can be coupled to the single-chain antibody MAH, Rg3 and Rh2 cannot be coupled to MAH. In the present invention, the method of covalently linking aminocarboxyl groups to form amide bonds is used to couple MAH and Rg3, and then the covalent conjugate is purified by affinity chromatography. The 1H NMR of MAH-Rg3 confirms the success of the above coupling method. The characteristic peaks of the polymer are shown in the final 1H NMR. The methyl group carried by the aminosilane appears in the amino-activated Rg3 (ppm: 3.51), and additional amide bonds appear in MAH-Rg3 (ppm: 6.13, 7.16). The 1H NMR of MAH-Rh2 confirms the success of the above coupling method. The characteristic peaks of the polymer are shown in the final 1H NMR. The methyl group carried by the aminosilane appears in the amino-activated Rg3 (ppm: 3.58), and the amide bond appears in MAH-Rg3 (ppm: 6.35). Specifically, Fig.13 and Fig.14 shown.

[0100] Regarding the effects of ginsenosides and antibody conjugates on tumor metabolic activities: Tumor cells and tumor organoids were cultured for 3 generations and then cultured for 4-5 days for drug administration test after the passage was stable. The concentrations of MAH-Rg3, MAH-Rh2, Rg3 and Rh2 were prepared in the culture medium of the corresponding cells. A549, HCT-116, HEK-293T and NCI-N87 cells (4×10 4 ) were inoculated into 96-well plates and cultured overnight at 37 °C, followed by replacement of the culture medium containing MAH-Rg3, MAH-Rh2, Rg3, and Rh2, respectively. After incubation for 48 h, the tumor inhibition ability of each drug was detected by CCK-8 method, and the data were analyzed using GraphPad Prism 8.0 software to calculate the relative cell viability.

[0101] The cell lines and cell culture medium-related reagents used above are as follows: All cell lines were obtained from the American Type Culture Collection (ATCC). HCT-116 and NCI-N87 epithelial cell lines were cultured in RPMI1640 medium (11875119, Gibco) supplemented with FBS (10% v / v) (A5669701, Gbico) and penicillin-streptomycin (1% v / v) (15140148, Gibco). A549 cell lines were cultured in F-12K medium (21127022, Gibco) supplemented with FBS and penicillin-streptomycin (1% v / v). HEK-293T and A549 cells were cultured in DMEM medium (10566016, Gibco) supplemented with FBS (10% v / v) and penicillin-streptomycin. BT-474 cell line was cultured in RPMI1640 medium supplemented with FBS (10% v / v) and penicillin-streptomycin (1% v / v). The cell culture medium was changed every 2-3 days, and the cell line was subcultured after treatment with trypsin (25200114, Gbico). The cell culture conditions were 37 °C, 5% CO2 and 95% relative humidity in an incubator (Heracell Vios 160i Cr, Thermo Scientific). Mycoplasma detection was performed as usual.

[0102] Establishing organoids (1) Human gastric cancer tissue used: Three patients with GC who underwent surgical resection were obtained at the China-Japan Friendship Hospital of Jilin University, Changchun, Jilin Province, China from August to October 2024. This study was approved by the Ethics Committee of the China-Japan Friendship Hospital of Jilin University, review number: 2023103015.

[0103] (2) Establishment of organoid culture Freshly collected tissues were immediately minced in HBSS solution containing amphotericin, penicillin, and streptomycin. The digested tumor tissues were filtered through a 100 μm filter and centrifuged at 350 × g. After washing, the cell pellets were resuspended in Matrigel and 25 μL of Matrigel-cell mixture per well were seeded on preheated 24-well plates. After the matrigel solidified, 500 μL of complete GC organoid medium was added, which included advanced DMEM / F12 (Gibco, 12634010), 1x GlutaMax, 1x HEPES, 40% Wnt3a, 10% RSPO-1, 100 ng / mL Noggin (PeproTech, 120-10C), 1x B27 (Thermo Fisher Scientific, 17504044), 50 ng / mL EGF (PeproTech, AF-100-15) and 100 ng / mL FGF10 (PeproTech, 100-26), 1 mM N-acetylcysteine, 1 nM gastrin, 1 μM A83-01, 10 μM Y-27632, and 1 μg / ul primidiomycin. The culture medium was replenished every 3–4 days, and organoids were passaged every 1–2 weeks using TrypLEExpress (Gibco).

[0104] Histological staining and immunofluorescence Tissues and organoids were fixed in 4% paraformaldehyde and sectioned at 5 μm thickness after processing. Hematoxylin and eosin staining was performed using standard histological protocols. Immunofluorescence staining was performed using anti-CK7 antibody (1:1000, Abcam, ab9021), anti-HER2 antibody (1:20000, Abcam, ab207718), and anti-Ki67 antibody (1:400, Cell Signaling Technology, 12202S).

[0105] Statistical analysis The results are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Comparisons between two independent groups were analyzed using Student's t test in GraphPad Prism 8.0 software (GraphPad, USA). The significance level was set at * < 0.05; ** < 0.01 and **** < 0.001.

[0106] (1) Effects of ginsenosides Rg3 and Rh2 on the metabolic activity of tumor cells Effects of different concentrations of ginsenosides Rg3 and Rh2 on the survival rates of A549, HCT-116 and NCI-N87 cells at different times Fig.15 As shown in the figure, it can be seen that ginsenoside Rg3 or Rh2 has a significant inhibitory effect on the above-mentioned cells, and the effect of Rh2 is stronger than that of Rg3 at the same time and concentration, which can also be seen from the bright field image (see Fig.16 ). In order to explore whether ginsenosides affect the expression of HER2 gene and protein, we performed tests by q-PCR and Western-Blot. The results showed that both ginsenosides reduced the expression of HER2 gene and protein. Fig.17 This may be due to the fact that ginsenosides arrest the cell cycle at the G0 / G1 phase.

[0107] (2) Effects of MAH-Rg3 and MAH-Rh2 on the metabolic activity of tumor cells We treated A549, HCT-116 and NCI-N87 cells with different concentrations of MAH-Rg3 and MAH-Rh2 to verify their effects on tumor cells. The bright field images of the experiment are shown in Figure 2. Fig.18 As shown in the figure, after 24 hours of treatment, A549, HCT-116 and NCI-N87 cells all underwent obvious morphological changes. Compared with the control group, the three cells all showed cell shrinkage and incomplete cell morphology after treatment with MAH-Rg3 and MAH-Rh2. These morphological changes indicate that the drugs have a significant effect on the cells, which may be achieved by inducing cell apoptosis or necrosis. The cell viability of NCI-N87 cells treated with gradient concentrations of trastuzumab and single-chain antibody for 24 hours using the CCK-8 method is shown in the figure. Fig.19 As shown in the figure, the single-chain antibody and trastuzumab in the concentration range of 100-0.01μg / ml had no significant effect on the activity of NCI-N87. The tumor killing ability of MAH-Rg3 and MNAH-Rh2 was further verified in gastric cancer cells NCI-N87. The experimental results are shown in the figure. Fig. 20 As shown in the figure, after 24 hours of treatment, MAH-Rg3 and MAH-Rh2 effectively induced cell death in the experimental group. Compared with the use of Rg3 or Rh2 alone, the IC 50 significantly reduced, 10.34 times that of Rg3 and 18.66 times that of Rh2, respectively. Fig.21 This may be due to the fact that MAH-Rg3 and MAH-Rh2 enhanced the growth inhibitory effect by targeting HER2 and delivering ginsenosides, which was consistent with our expected results.

[0108] (3) Effects of MAH-Rg3 and MAH-Rh2 on metabolic activity of gastric cancer organoids In order to evaluate whether ginsenoside conjugates can effectively induce apoptosis of human gastric cancer organoids, we incubated organoids with ginsenoside conjugates, Rg3, Rh2, single-chain antibody and trastuzumab at gradient concentrations, and detected cell viability with CCK-8 after 24 hours of incubation, and drew IC50 to evaluate its effect, such as Fig. 22 As shown, due to different patient sources, the three gastric cancer organoids had different sensitivities to these substances.

[0109] Compared with the experimental results at the cellular level, the half-inhibitory concentrations of drugs were all improved because the organoids were more complex and had richer cell types. For ginsenosides, the three organoids were all sensitive to Rg3 and Rh2, and the sensitivity to Rh2 was greater than Rg3. For trastuzumab, organoid P2 produced a significant antagonistic effect, while ginsenoside conjugates had a good inhibitory effect on the three organoids.

Claims

1. A HER2 nanobody, characterized in that: The antibody is denoted as MAH, and its amino acid sequence is shown as SEQ ID NO.

1.

2. An antibody conjugate based on the HER2 nanobody described in claim 1, characterized in that: The single-chain antibody MAH is coupled to ginsenoside Rh2 to form a coupled MAH-Rh2, and the molecular weight of the antibody conjugate is about 33 kDa.

3. The antibody conjugate according to claim 2, characterized in that: The structural formula of the antibody conjugate MAH-Rh2 is: 。 4. A method for preparing an antibody conjugate according to claim 3, characterized in that: The method comprises dissolving ginsenosides in an anhydrous toluene solution of (3-aminopropyl)trimethoxysilane (APTMS), stirring the reaction to obtain aminosilane-modified ginsenosides, collecting the precipitate by centrifugation, washing to obtain aminosilane-activated ginsenosides, dissolving MAH in ultrapure water to prepare an MAH solution, and mixing the solution with an activation buffer to obtain an activated MAH solution; Re-take the activation buffer, add EDC solution and Sulfo-NHS aqueous solution, then add aminosilane-activated ginsenoside to form a mixed solution, add the mixed solution to the activated MAH solution for mixed reaction, and then separate it through protein L affinity chromatography filler to obtain the antibody conjugate.

5. The method for preparing an antibody conjugate according to claim 4, characterized in that: The ginsenoside is Rh2, the mass volume ratio of Rh2 and APTMS in anhydrous toluene solution is 0.8-1.2:100, and the mass volume percentage concentration of APTMS in anhydrous toluene solution is 0.8-1.2%.

6. The method for preparing an antibody conjugate according to claim 4 or 5, characterized in that: The volume ratio of the MAH solution to the activation buffer in the activated MAH solution is 1:1, the activation buffer is a MES solution with a pH of 6.0 and a concentration of 50 mM, and the mass volume percentage concentration of the MAH solution is 8-12%.

7. The method for preparing an antibody conjugate according to claim 6, characterized in that: The concentration of aminosilane-activated ginsenoside in the mixed solution is 1.5-2.5 mg / mL, the volume ratio of the activation buffer, the EDC solution and the Sulfo-NHS aqueous solution is 100:2-2.5:22-25, the activation buffer is a MES solution with a pH of 6.0 and a concentration of 50 mM, the concentration of the EDC solution is 200 mmol / L, and the concentration of the Sulfo-NHS is 200 mmol / L.

8. The method for preparing an antibody conjugate according to claim 7, characterized in that: The volume ratio of the activated MAH solution to the mixed solution is 10:

1.

9. Use of the antibody conjugate prepared by the method of claim 8 in the preparation of targeted drugs for inhibiting tumor growth.

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