A HER2 nanobody, an antibody conjugate and a preparation method thereof

Abstract

The present invention relates to a HER2 nanobody, an antibody conjugate and a preparation method thereof, belonging to the technical field of pharmaceutical preparation. The HER2 nanobody is denoted as MAH, and its amino acid sequence is as shown in SEQ ID NO.1. The antibody MAH of the present invention is connected with ginsenoside to form a MAH-Rh2 conjugate with a molecular weight as small as 33 kDa, which is easier to penetrate blood vessels and directly reach the surface of tumor cells. During the inhibition of tumor cell growth, it can achieve a strong inhibitory effect on a variety of tumor cells with positive HER2 expression, achieve the effect of inhibiting tumor growth within 24 hours, and also obtain a better ability to inhibit tumor cell growth by extending the action time at the same dose. This not only reduces the dosage of components such as Rh2, but also expands the scope of use of MAH-Rh2. Secondly, MAH-Rh2 that exerts effects through a dual pathway targets and precisely delivers ginsenoside.

Description

Technical Field

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

[0002] HER2 is an immunohistochemical index and is one of the members of the human epidermal growth factor receptor family, playing an important role in signal transduction. HER2 protein is usually expressed during fetal development, while its expression level is low or absent in normal adult tissues. However, in various tumors such as lung cancer, breast, colorectal cancer, endometrial cancer, gastric cancer, prostate cancer, etc., HER2 may be amplified or overexpressed. HER2 mutations in tumors usually involve overexpression and amplification. The latter refers to the increased replication of the HER2 gene sequence, which may lead to an increase in 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 composed of monoclonal antibodies targeting tumor-specific antigens or tumor-associated antigens conjugated with a specific number of small molecule cytotoxins through a linker. Due to their high targeting ability, high specificity, high activity, and lower systemic toxicity compared with traditional chemotherapy drugs, they have become one of the popular drugs for anti-tumor targeted therapy and are called "biological missiles" by scholars. Antibody-drug conjugates utilize 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 conjugation of the linker, the off-target toxicity of small molecule toxins is effectively reduced, thus differentiating from general monoclonal antibodies and chemotherapy drugs to achieve the purpose of targeted therapy, enhancing efficacy and reducing toxicity.

[0004] In the field of targeted therapy, single-chain antibodies (scFv) show great application prospects in the fields of medicine and biotechnology due to their small molecular size, excellent tissue penetration ability, and precise targeting characteristics. Therefore, a conjugate can be prepared by single-chain antibodies and small molecule toxins to achieve the purpose of inhibiting tumor growth. Summary of the Invention

[0005] The purpose of the present invention is to provide a HER2 single-chain nanobody.

[0006] Another purpose of the present invention is to provide the application of the above single-chain antibody.

[0007] The third purpose of the present invention is to provide an antibody conjugate based on HER2 antibody. By conjugating the HER2 antibody MAH with ginsenoside Rg3 or Rh2, a conjugate is formed, which realizes a strong inhibitory effect on various HER2-expressing positive tumor cells.

[0008] The fourth object of the present invention is to provide a method for preparing the above-mentioned antibody conjugate. This method solves the problem that ginsenoside Rg3 and Rh2 cannot attach to the surface of MAH and it is 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-mentioned conjugate.

[0010] The objects of the present invention are achieved by the following technical solutions:

[0011] A HER2 single-chain nanobody, characterized in that: the amino acid sequence of the antibody is MTMITPSFGAFFLEIFNVKKLLFAIPLVVPFYAAQPAMAQVKLQQSGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALKSRLNISKDNSKSQVFLKMNSLQTDDTAMYYCARNWGSYWYFDVWGQGHGHRLLSGGGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCRASSSISSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGAGTKLEIKRAAAGAPVPYPDPLEPRAA (SEQ ID NO.1), denoted as MAH.

[0012] The nucleotide sequence of the MAH nanobody consists of a light chain variable region (VL) and a heavy chain variable region (VH), and each variable region consists of four framework regions (FR) and three complementarity determining regions (CDR).

[0013] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0014] The nucleotide sequence of CDR1 in the heavy chain variable region is shown in SEQ ID NO.3;

[0015] The nucleotide sequence of CDR2 in the heavy chain variable region is shown in SEQ ID NO.4;

[0016] The nucleotide sequence of CDR3 in the heavy chain variable region is shown in SEQ ID NO.5.

[0017] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.10.

[0018] The nucleotide sequence of CDR1 of the light chain variable region is shown as SEQ ID NO. 11;

[0019] The nucleotide sequence of CDR2 of the light chain variable region is AGGACATCC;

[0020] The nucleotide sequence of CDR3 of the light chain variable region is shown as SEQ ID NO. 12.

[0021] The application of the above-mentioned anti-HER2 single-chain nanobody in the preparation of an antibody conjugate.

[0022] A small molecule antibody conjugate based on the above-mentioned anti-HER2 antibody, characterized in that: a conjugate MAH-Rg3 or MAH-Rh2 formed by conjugating single-chain antibody MAH with ginsenoside Rg3 or Rh2, and the molecular weight of the antibody conjugate is about 33 kDa.

[0023] Furthermore, the structural formula of the antibody conjugate MAH-Rg3 is:

[0024] 。

[0025] Furthermore, the structural formula of the antibody conjugate MAH-Rh2 is:

[0026] 。

[0027] The preparation method of the above-mentioned antibody conjugate, characterized in that: dissolving ginsenoside in an anhydrous toluene solution of (3-aminopropyl)trimethoxysilane (APTMS), stirring and reacting to obtain amino-silane modified ginsenoside, centrifuging to collect the precipitate, washing to obtain amino-silane activated ginsenoside, dissolving MAH in ultrapure water to prepare a MAH solution, mixing with an activation buffer to obtain an activated MAH solution, adding an EDC solution and an aqueous Sulfo-NHS solution to the activation buffer, then adding the amino-silane activated ginsenoside to form a mixed solution, adding the mixed solution to the activated MAH solution for mixing and reacting, and then separating by a Protein L affinity chromatography packing material to obtain the antibody conjugate.

[0028] Furthermore, when the ginsenoside is Rg3, the mass-volume ratio of Rg3 to the anhydrous toluene solution of APTMS is 1.5 - 2.5 g:100 mL, and the concentration of the anhydrous toluene solution of APTMS is 0.8 - 1.2% (mass-volume percentage concentration).

[0029] Furthermore, when the ginsenoside is Rh2, the mass-volume ratio of Rh2 to the anhydrous toluene solution of APTMS is 0.8 - 1.2 g:100 mL, and the concentration of the anhydrous toluene solution of APTMS is 0.8 - 1.2% (mass-volume percentage concentration).

[0030] Further, 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%.

[0031] Further, the concentration of the amino-silane-activated ginsenoside in the mixed solution is 1.5-2.5 mg / mL, and 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 (19.2 mg of EDC is dissolved in 500 µL of ultrapure water), and the concentration of Sulfo-NHS is 200 mmol / L (21.7 mg of Sulfo-NHS is added to 500 µL of the activation buffer).

[0032] Further, the volume ratio of the activated MAH solution to the mixed solution is 10:1.

[0033] Further, the specific steps for separating the protein L and the chromatography packing material are as follows:

[0034] (1) Equilibration: Equilibrate the chromatography column with 5 column volumes of the equilibration Buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) to place the packing material in the same buffer system as the target protein, which serves to protect the protein.

[0035] (2) Loading: Add the sample to the equilibrated packing material to ensure that the target protein is in full contact with the resin.

[0036] (3) Washing of impurities: Wash with 10-15 column volumes of the impurity washing Buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0).

[0037] (4) Elution: Use 5-10 column volumes of the elution Buffer (0.1 M glycine, pH 3.0), and collect the eluate, which is the target protein fraction.

[0038] The conjugates used in the past were mainly cytotoxic molecules that delivered the toxic molecules into cells to kill the cells, but this often led to the bystander effect, thus affecting the growth of normal cells and the change of the tumor microenvironment. Secondly, antibody-drug conjugates mainly used intact antibodies (about 150-180 kDa) as the targeted delivery carriers. The biotoxin molecules conjugated to such large-molecular-weight antibodies were difficult to pass through the microvessels in the tumor infiltration area and difficult to exert their full effects.

[0039] In the present invention, MAH is used as a carrier and coupled with ginsenoside Rg3 and Rh2 respectively to form complete conjugates MAH-Rg3 and MAH-Rh2. However, during the preparation of the conjugates, 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. Different coupling sites on the surface of antibodies determine the types of groups for saponin modification. In the present invention, the method of covalently connecting amino groups and carboxyl groups to form amide bonds is used for the coupling of MAH with Rg3 or Rh2, and then the covalent conjugate is purified by affinity chromatography. The molecular weight of the obtained conjugate is about 33 kDa. The small molecular weight is easy to penetrate microvessels and directly reach the surface of tumor cells, saving more medical resources to a greater extent. In addition, since the coupled Rg3 and Rh2 are almost harmless to normal cells and have a positive effect on the differentiation and activation of the tumor microenvironment and tumor-infiltrating lymphocytes, there is no proximity effect of traditional antibody-drug conjugates in MAH-Rg3 and MAH-Rh2. Therefore, MAH-Rg3 and MAH-Rh2 have a positive regulatory effect on the tumor microenvironment.

[0040] Application of the antibody conjugate prepared by the above method in the preparation of drugs for targeted inhibition of tumor growth.

[0041] The present invention has the following technical effects:

[0042] The antibody MAH of the present invention is connected with ginsenosides to form conjugates MAH-Rg3 and MAH-Rh2. The molecular weight is as small as 33 kDa, which is easier to penetrate blood vessels and directly reach the surface of tumor cells. During the inhibition of tumor cell growth, it can achieve a strong inhibitory effect on a variety of tumor cells with positive HER2 expression. The inhibitory effect on tumor growth can be achieved within 24 hours. Under the same dose, a better ability to inhibit tumor cell growth can also be obtained by extending the action time. This not only reduces the dosage of components such as Rg3 and Rh2, but also expands the application range of MAH-Rg3 and MAH-Rh2. Secondly, MAH-Rg3 and MAH-Rh2 that exert effects through dual pathways targetedly and accurately deliver ginsenosides, thus achieving excellent anti-tumor effects. Description of the Drawings

[0043] Figure 1 : Flow chart for constructing a cDNA library containing a single-chain antibody encoding a HER2 protein-specific gene.

[0044] Figure 2 : Results of serum titer determination after immunization of mice.

[0045] Figure 3 : Electrophoresis results of amplifying VH and VL gene fragments from the cDNA template of mouse lymphocytes.

[0046] Figure 4 : Construction and transformation of the scFv-pCANTEB-5E recombinant vector involving electrophoresis results; A. Agarose gel electrophoresis results of linking VL with Linker by SOE-PCR; B. Electrophoresis results of the scFv gene generated by linking VH and VL chains by SOE-PCR; C. Electrophoresis results of clones identified by PCR amplification containing the insert fragment of the expected size.

[0047] Figure 5 : Sequencing and homology analysis of the single-chain antibody gene.

[0048] Figure 6 : Three-dimensional modeling of the single-chain antibody and analysis of the antibody amino acid sequence.

[0049] Figure 7 : Electrophoresis results of the purified single-chain antibody analyzed by SDS-PAGE.

[0050] Figure 8 : Western Blot for verifying that the single-chain antibody can bind to the HER2 protein electrophoresis pattern.

[0051] Figure 9 : Effects of different concentrations of the single-chain antibody on the viability of HEK-293T cells.

[0052] Figure 10 : Detection of changes in the content of HER2 protein on the membrane of NCI-N87 cells treated with gradient concentrations of the single-chain antibody and trastuzumab for 24 hours; a. Treatment of NCI-N87 with 100 μg / mL single-chain antibody; b. Treatment of NCI-N87 with 0.01 μg / ml single-chain antibody; c. Treatment of NCI-N87 with 100 μg / mL trastuzumab; d. Treatment of NCI-N87 with 0.01 μg / mL trastuzumab.

[0053] Figure 11 : Cell cycle changes of NCI-N87 cells treated with gradient concentrations of the single-chain antibody and trastuzumab; a. Treatment of NCI-N87 with 100 μg / mL single-chain antibody; b. Treatment of NCI-N87 with 0.01 μg / ml single-chain antibody; c. Treatment of NCI-N87 with 100 μg / mL trastuzumab; d. Treatment of NCI-N87 with 0.01 μg / mL trastuzumab.

[0054] Figure 12 : Inhibitory effect of the single-chain antibody on NCI-N87 cells; a. Proportion of G2-phase cells in NCI-N87 cells treated with different concentrations of the single-chain antibody; b. Changes in the ATP level in NCI-N87 cells treated with different concentrations of the single-chain antibody and trastuzumab.

[0055] Figure 13 : 1H NMR spectrum of the antibody conjugate MAH-Rg3.

[0056] Figure 14 : 1H NMR spectrum of the antibody conjugate MAH-Rh2.

[0057] Figure 15 : Effects of ginsenoside Rg3 and Rh2 on the viability of A549, HCT-116 and NCI-N87 cells at different times; a. Cell viability at 24 h; b. Cell viability at 48 h; c. Cell viability at 72 h.

[0058] Figure 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.

[0059] Figure 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.

[0060] Figure 18 : Bright-field photos 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.

[0061] Figure 19 : Determination of cell viability of NCI-N87 cells treated with gradient concentrations of trastuzumab and single-chain antibody for 24 h.

[0062] Figure 20 : Determination of cell viability of NCI-N87 cells treated with gradient concentrations of MAH-Rg3 and MAH-Rh2 for 24 h.

[0063] Figure 21 : IC50 of Rg3, Rh2, MAH-Rg3 and MAH-Rh2 on NCI-87 cells treated for 24 h; 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.

[0064] Figure 22:IC50 curves of different organoids against ginsenoside conjugate, Rg3, Rh2, single-chain antibody and trastuzumab, where P1, P2, and P3 represent organoids from patients 1, 2, and 3 respectively. Detailed implementation mode

[0065] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above content of the present invention.

[0066] Example 1

[0067] Preparation of anti-HER2 protein nanobody MAH:

[0068] 1. Mouse immunization and amplification of antibody variable region genes

[0069] 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 not immunized as a control group. According to the immunization procedure, 150 μL of blood was taken from the tail at intervals, and the plasma was centrifuged at 13,000 rpm for 20 min at 4°C. The upper serum was diluted to eight gradients of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000 for detection of mouse serum antibody titers. The mouse serum titers were determined by ELISA, and a serum titer ≥ 1:64000 was considered successful immunization. The immunized mice were sacrificed by decapitation, disinfected with alcohol, and quickly placed in a biosafety cabinet and fixed. The abdomen was dissected with surgical scissors and forceps, and the spleen was separated. After the spleen was separated, it was placed in pre-cooled RNase-free PBS. The spleen was cut open with scissors, and lymphocytes were flushed out with pre-cooled RNase-free PBS repeatedly, and lymphocyte RNA was extracted by the Trizol method. cDNA synthesis was carried out using Revert Aid First Strand cDNA Synthesis Kit (K1621, Thermo Scientific), and then antibody variable region gene amplification was carried out. The above mouse immunization procedure is shown in Table 1.

[0070] Table 1:

[0071]

[0072] The test results are shown in Figure 2, the serum titers of both mice exceeded 1:64000, meeting the criteria for library construction. Two immunized mice were selected to isolate spleen lymphocytes and extract total RNA. The integrity of the total RNA was detected by agarose gel electrophoresis and showed good integrity. Then, cDNA synthesis and antibody variable region sequence amplification were carried out. The primers used for amplification are shown in Table 2.

[0073] Table 2:

[0074]

[0075] Note: VH1BACK, VH1FOR, VK2BACK, MJk1FONX, MJk2FONX, MJk3FONX, MJk4FONX are the upstream and downstream primers for amplifying the variable regions of the heavy and light chains respectively; VH1BACK-Sfi1, JK1-NOT1, JK2-NOT1, JK3-NOT1, JK4-NOT1 are the enzyme cleavage sites added to the amplified products. The bold and italic positions in the table are the Sfi1 enzyme cleavage sites, and the bold positions are the Not1 enzyme cleavage sites; Linker is a DNA sequence that connects the variable regions of the heavy and light chains. The bold, italic, and underlined amino acid sequence is the 3xGGGGS structure; among them, the degenerate bases are 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.

[0076] The results of variable region amplification are shown in Figure 3 , which amplified the VH and VL gene fragments from the cDNA template of mouse lymphocytes. Among them, lanes 1-3 are antibody heavy chain variable region fragments of about 350 bp, and lanes 4-6 are antibody light chain variable region fragments of about 320 bp, which are the same as the predicted results, and they were classified and gel recovered.

[0077] 2. Construction and transformation of scFv-pCANTEB-5E recombinant vector

[0078] The amplified variable region genes of the heavy and light chains were ligated to the linker (Linker) by two-step overlap extension PCR. First, VL was ligated to the linker, and then VH was ligated to VL-Linker by overlap extension PCR. After the scFv gene purified by gel recovery was double-digested with restriction enzymes Sfi1 (1244S, Takara) and Not1 (1166A, Takara), it was ligated to the pCANTEB-5E vector by T4-DNA ligase (2011A, Takara) to construct a recombinant vector, which was then electrotransformed into Escherichia coli TG1. The results are as shown in Figure 4As shown in A and B. In A, VL was linked to the Linker by SOE-PCR, and the result of agarose gel electrophoresis was about 420 bp; in B, the scFv gene was generated by linking VH and VL chains through SOE-PCR, and the electrophoresis result was about 750 bp. Twenty single colonies with good morphology were picked from the successfully electrotransformed TG1 for colony PCR, and the primers used are shown in Table 3.

[0079] Table 3:

[0080]

[0081] The results are as Figure 4 shown in C. Clones containing the expected-sized inserted fragments were identified by PCR amplification. The colony PCR results showed that all were positive except for the 6th single colony, and the PCR target band was around 750 bp, which was in line with the expected result. The preliminary positive conversion rate was 95%.

[0082] 3. Preparation of single-chain antibody library and phage panning

[0083] The successfully electrotransformed TG1 was cultured by shaking flask at 37 °C and 180 rpm for 2 h, and then 1×10 ,

[0084] , ,

[0085] , ,

[0082] , , ,

[0087] ,

[0083] , ,

[0088] ,

[0081] , 5 , , Figure 4 , , ,

[0086] ,

[0080] pfu of M13KO7 helper phage was added for rescue. The phage was precipitated with 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 added and washed repeatedly four times to obtain the murine 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:

[0084] (1) The recombinant human HER2 protein was diluted to 20 μg / mL with the high-adsorbing coating solution in the ELISA auxiliary kit, and 100 μL of the diluted HER2 solution was added to each well of the 96-well ELISA plate and incubated overnight at 4 °C.

[0085] (2) After overnight incubation, each well was washed 5 times with 200 μL of the universal sandwich ELISA washing solution for 5 minutes each time. After complete washing, 200 μL of the ready-to-use ELISA blocking solution was added to each well and incubated at 4 °C overnight for blocking.

[0086] (3) After overnight incubation, each well was washed 5 times with 200 μL of the universal sandwich ELISA washing solution for 5 minutes each time and reserved for use.

[0087] (4) A total of 1×1010 pfu of the primary murine single-chain antibody phage library was taken and evenly added to the ELISA plate and incubated at 4 °C for 10 h.

[0088] (5)After the incubation, wash each well 5 times with 200 μL of the general sandwich ELISA washing solution for 10 minutes each time.

[0089] (6)Add 100 μL of 0.2 M glycine - hydrochloride buffer to each well, incubate for 5 min, and immediately add 10 μL of 2 M Tris - HCl solution to each well for neutralization.

[0090] (7)Collect the neutralized solution and add it to TG1 Escherichia coli with an OD600 of 0.6, and incubate at 37 °C with shaking at 180 rpm for 1 h.

[0091] (8)Take 10 μL of the cultured bacterial solution and perform serial dilutions with 2×YT liquid medium at dilutions of 102, 104, 106, 108. Take 100 μL of the liquid at each dilution and add it to 5 mL of upper agar, mix well, immediately pour it onto the pre - prepared plate, and culture overnight at 37 °C.

[0092] (9)Count the number of plaques on the plate and multiply by the corresponding dilution factor to obtain the phage titer of the elution for that time.

[0093] (10)For the remaining bacteria, culture the bacteria in step (7) overnight at 37 °C with shaking at 180 rpm. After overnight culture, transfer the bacterial solution to a centrifuge tube, centrifuge at 4 °C and 10000 g for 20 min, transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG / NaCl solution, mix well, and let it stand at 4 °C for 4 h.

[0094] (11)When the solution becomes turbid, centrifuge at 4 °C and 10000 g for 20 min, discard the supernatant, add 1 mL of pre - cooled PBS to resuspend the precipitate and filter it through a 0.22 μm filter membrane. All the obtained phages are the murine single - chain antibody phage primary panning library, and store it at 4 °C.

[0095] (12)Re - pan the phages obtained in step (11), and the panning steps are (4) → (12). Repeat three times in total to obtain high - affinity HER2 - protein phages.

[0096] Re - infect the panned phages into TG1, randomly pick ten single colonies for monoclonal phage ELISA, and screen for positive phages with a high P / N value for protein expression;

[0097] After the panned phages infect TG1 and 10 single colonies are picked for monoclonal ELISA, and the phages show obvious strong positivity, infect them into TG1 and extract the plasmid for sequencing. The sequencing results are shown in Figure 5As shown, analyzed by Snap Gene, the sequencing results show that its two ends contain Sfi1 and Not1 restriction enzyme sites. Then, the gene sequence was compared and analyzed using IgBLAST. It can be seen that the variable region of the heavy chain and the variable region of the light chain in this gene segment are complete. Among them, the homology rate of the variable region of the heavy chain with the gene IGHV2-6*03 is 96.2% (IMGT Accession numbers: BN000872), and the homology rate of the variable region of the light chain with the gene IGKV4-58*01 reaches 95.9% (IMGT Accession numbers: K00884). The details are as follows:

[0098] The MAH nanobody is mainly composed of the variable region of the light chain (VL) and the variable region of the heavy chain (VH). Each variable region consists of four framework regions (FR) and three complementarity determining regions (CDR). The framework regions play the following roles:

[0099] (1) Structural support: FR1-FR4 form the β-sheet structure of the variable region, maintaining the three-dimensional conformation of the antibody variable region and ensuring the correct spatial arrangement of the CDR region.

[0100] (2) Stability maintenance: The conserved FR sequences stabilize the antibody structure through hydrophobic interactions and hydrogen bonds, preventing conformational disruption caused by mutations or environmental changes.

[0101] (3) Auxiliary function: Some FR residues may indirectly affect antigen binding, for example, by regulating the flexibility of the CDR or participating in interactions with other molecules (such as T cell receptors, MHC molecules).

[0102] (4) Immunogenicity regulation: During the humanization of antibodies, replacing non-human FR with human sequences can reduce immunogenicity, indicating that FR may contain species-specific epitopes.

[0103] The complementarity determining regions play the following roles:

[0104] (1) Core of antigen binding: CDRs (especially CDR3) directly bind to the antigen epitope, determining the specificity and affinity of the antibody. CDR3 has the highest diversity due to gene recombination and high-frequency mutations and is the key region for antigen recognition.

[0105] (2) Conformational diversity: CDR1 and CDR2 mainly participate in binding to the conserved regions of the antigen, while CDR3 adapts to the complex shape of the antigen surface through a flexible structure.

[0106] (3) Specificity determining factor: The differences in CDR sequences among different antibodies determine their uniqueness in binding antigens and are the molecular basis for antibody diversity.

[0107] That is, in the entire gene sequence, the framework region is usually constant according to its origin, while the specificity of the antibody is determined by the complementarity-determining regions, which are the factors affecting the function of the antibody.

[0108] The heavy chain and the light chain play the following cooperative roles:

[0109] (1) The CDRs of VH and VL together form a continuous antigen-binding interface (antigen-binding pocket), where CDR3 of the heavy chain usually contributes more binding energy.

[0110] (2) The FRs provide structural support in both chains, ensuring the correct pairing of VH and VL, thereby maintaining the integrity of the binding site.

[0111] The nucleotide sequence of the heavy chain of the nanobody is (as shown in SEQ ID NO.2):

[0112] ATGGCCCAGGTGAAGCTGCAGCAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTTTCATTAACCAGCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAACTATAATTCAGCTCTCAAATCCAGACTGAACATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGATGACACAGCCATGTACTACTGTGCCAGAAACTGGGGCAGCTACTGGTACTTCGATGTCTGGGGCCAAGGCCACGGTCACCGTCTCCTCAGTGGAGGC

[0113] The nucleotide sequence of the complementarity-determining regions in the variable region of the heavy chain is:

[0114] CDR1 (SEQ ID NO.3): GGGTTTTCATTAACCAGCTATGGT

[0115] CDR2 (SEQ ID NO.4): ATATGGGCTGGTGGAAGCACA

[0116] CDR3 (SEQ ID NO.5):

[0117] GCCAGAAACTGGGGCAGCTACTGGTACTTCGATGTC

[0118] The nucleotide sequence of the heavy chain framework region is as follows:

[0119] FR1 (SEQ ID NO.6):

[0120] ATGGCCCAGGTGAAGCTGCAGCAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCA

[0121] FR2 (SEQ ID NO.7):

[0122] GTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTA

[0123] FR3 (SEQ ID NO.8):

[0124] AACTATAATTCAGCTCTCAAATCCAGACTGAACATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGATGACACAGCCATGTACTACTGT

[0125] FR4 (SEQ ID NO.9):

[0126] TGGGGCCAAGGCCACGGTCACCGTCTCCTCAGTGGAGGC

[0127] The total nucleotide sequence of the heavy chain was analyzed by IgBLAST. The result showed that the homology rate of the heavy chain variable region with the gene IGHV2-6*03 was 96.2% (IMGT Accession numbers: BN000872), and no identical sequence was detected in the variable region (complementary determining region) sequence.

[0128] The nucleotide sequence of the light chain of this nanobody is (as shown in SEQ ID NO.10):

[0129] GACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGCTCAAGTATAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGCGCTTCCCCCAAACCCTTGATTCATAGGACATCCAACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGATGCAACTTATTACTGCCAGCAGTGGAGTGGTTACCCATTCACGTTCGGTGCTGGGACCAAGCTCGAGATCAAACGG

[0130] The nucleotide sequence of the complementarity-determining region of the light chain variable region is:

[0131] CDR1 (SEQ ID NO.11): TCAAGTATAAGTTCCAGTTAC

[0132] CDR2: AGGACATCC

[0133] CDR3 (SEQ ID NO.12): CAGCAGTGGAGTGGTTACCCATTCACG

[0134] Among them, the nucleotide sequence of the light chain framework region is:

[0135] FR1 (SEQ ID NO.13):

[0136] GACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGC

[0137] FR2 (SEQ ID NO.14):

[0138] TTGCACTGGTACCAGCAGAAGTCAGGCGCTTCCCCCAAACCCTTGATTCAT

[0139] FR3 (SEQ ID NO.15):

[0140] AACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGATGCAACTTATTACTGC

[0141] FR4 (SEQ ID NO.16):

[0142] TTCGGTGCTGGGACCAAGCTCGAGATCAAACGG

[0143] The homology rate of the light chain variable region with the gene IGKV4-58*01 reaches 95.9% (IMGT Accession numbers: K00884), and no identical sequence was detected in the variable region (complementary determining region) sequence.

[0144] 4. Obtaining the anti-HER2 protein nanobody MAH

[0145] Infect TG1 with phages with a high P / N value, extract the plasmid and sequence it. Use Snap Gene to identify the sequencing results, and perform homology molecular modeling through SWISS-MODEL based on the sequencing results. Then, perform molecular docking prediction on the single-chain antibody PDB model obtained from the modeling and the HER2 protein model in the PDB database to predict its binding site.

[0146] Electrotransform the plasmid into Escherichia coli HB2151. Pick a single colony of HB-2151 with good growth state and clear edges grown overnight in a 37°C incubator after electroporation, and place it in 1 L of 2×YT medium containing ampicillin and shake culture until the OD600 reaches 0.6. Add IPTG to a final concentration of 0.5 mM, and shake culture at 37°C and 180 rpm for 6 h. After the culture is completed, centrifuge at 4°C and 5000 rpm for 15 min, discard the supernatant, resuspend the precipitate with 200 ml of pre-cooled Ice-Cold Buffer (weigh 17.1 g of sucrose, 0.0146 g of ethylenediaminetetraacetic acid, and 2.42 g of Tris, completely dissolve the above drugs in 90 ml of ultrapure water, adjust the pH to 8.0 with hydrochloric acid, and store at 4°C for later use), and incubate on ice for 1 h. After the incubation is completed, centrifuge at 4°C and 10,000 g for 15 min. The supernatant is the periplasmic space, and the periplasmic space is purified with protein L affinity chromatography packing material. The purified single-chain antibody solution is concentrated through an ultrafiltration tube with a molecular weight cut-off of 3 kDa. The centrifugation parameters of the ultrafiltration tube are 5000 g and centrifugation for 30 minutes. Recover the concentrated product and store it at -20°C in 200 μl per EP tube, labeled as MAH.

[0147] Characterization of the single-chain antibody MAH:

[0148] Convert the gene sequence of the obtained single-chain antibody into the corresponding amino acid sequence. The amino acid sequence of the MAH is as follows (shown in SEQ ID NO. 1):

[0149] MTMITPSFGAFFLEIFNVKKLLFAIPLVVPFYAAQPAMAQVKLQQSGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALKSRLNISKDNSKSQVFLKMNSLQTDDTAMYYCARNWGSYWYFDVWGQGHGHRLLSGGGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCRASSSISSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGAGTKLEIKRAAAGAPVPYPDPLEPRAA

[0150] Use the SWISS-MODEL modeling tool to predict the protein structure based on the homologous molecular model for this sequence. The prediction results are as Figure 6 shown. It can be seen from the results 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 its three-dimensional structure is claw-shaped, which conforms to the three-dimensional structure of the single-chain antibody. Perform molecular docking model prediction of this PDB model with the human HER2 protein in the PDB database based on the CoDock database. It can be known from the prediction results that the antibody molecule binds to HER2 mainly in its extracellular domain, which is in line with the experimental expectations.

[0151] Characterize MAH by SDS-PAGE and western blot

[0152] The strongly positive scFv-pCANTEB-5E gene was electrotransferred into HB2151. After inducing the expression of the single-chain antibody by IPTG, the periplasmic space of the bacterial solution was extracted for affinity chromatography and ultrafiltration purification. The purified product was qualitatively detected by SDS-PAGE. The molecular weight (MWs) of MAH was estimated by SDS-PAGE (12.5% acrylamide gel). When performing Western Blot immunoblot analysis, the recombinant human HER2 protein was transferred onto a nitrocellulose membrane (Whatman, Kent, UK). After blocking with 3% skim milk for 1 hour, it was incubated with an anti-E-Tag labeled monoclonal antibody (1:1000 dilution, Beijing Proteintech Group Co., Ltd., China) at 4 °C for 12 hours. Then the membrane was washed 5 times with TBST buffer, and then incubated with HRP-labeled goat anti-rabbit IgG(H+L) (Beyotime Biotechnology, China) at 4 °C for 4 hours.

[0153] According to Snap Gene prediction, the molecular weight of the single-chain antibody MAH is around 32 kDa, which is consistent with the results of SDS-PAGE ( Figure 7 ). Moreover, this antibody is expressed in the periplasmic space. The protein purified and concentrated from the periplasmic space was detected by Western-Blot using rabbit anti-E-Tag tag antibody as the primary antibody and HRP-labeled goat anti-rabbit IgG antibody as the secondary antibody. The detection results are as Figure 8 shown. The apparent molecular weight of the HER2 protein is 120 kDa, which is in line with the experimental expectation. The single-chain antibody molecule also shows a positive signal in this region, consistent with the expected results.

[0154] Verification of the safety and anti-tumor growth ability of the single-chain antibody MAH

[0155] (1) Safety:

[0156] To ensure the safety of the single-chain antibody, we chose HEK-293T cells for safety assessment. The results are as Figure 9 shown. After treating with the single-chain antibody MAH at concentrations ranging from 2.5 - 200 μg / mL for 24 hours, 48 hours, and 72 hours, its cell viability was not significantly affected in 293T cells. After 72 hours, more than 85% of the HEK-293T cells survived. Therefore, the single-chain antibody can be considered safe.

[0157] (2) Anti-tumor growth ability

[0158] 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, asFigure 10 As shown in the figure, in the experimental group, the single-chain antibody MAH treatment groups with 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 state of 100 μg / mL, both the single-chain antibody MAH and the trastuzumab group showed the same HER2 target affinity ability. Different from 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. This indicates that the single-chain antibody MAH has a stronger ability to bind to the target, and the affinity attenuation within the range of 100 - 0.01 μg / mL is less than that of trastuzumab. This may be due to the smaller molecular weight of the single-chain antibody, which makes it easier to bind to the target protein.

[0159] The HER2 protein has the function of regulating cell proliferation. Therefore, when the amount of HER2 target decreases, there should be corresponding changes in the cell cycle. Therefore, we detected the cell cycle of cells treated with the single-chain antibody and trastuzumab to describe this change. The experimental results showed that trastuzumab did not affect the cell cycle of NCI-N87 cells. Different from it, the single-chain antibody MAH blocked the cell cycle of NCI-N87 cells in the G2 phase. The reason may be that the single-chain antibody MAH has a low molecular weight and can bind to the HER2 protein at a faster speed, resulting in a large amount of binding to the target position on the cell surface at the initial stage of drug action. In addition, this blocking effect of the single-chain antibody also showed a dose-dependence, which corresponded to the above results targeting HER2, specifically as shown in Figure 11 and Figure 12 shown in A of

[0160] Cancer cells rely on high ATP levels to maintain rapid proliferation. Reducing ATP can inhibit the growth of cancer cells or induce their death. We evaluated the effects of the single-chain antibody MAH and trastuzumab on cell viability. The results are shown in Figure 12 shown in B of <{

[0161] Example 2

[0162] An antibody conjugate, specifically a conjugate of the single-chain antibody MAH and ginsenoside Rg3, and its preparation method is as follows:

[0163] (1) Add 0.2 g of ginsenoside Rg3 to 100 mL of anhydrous toluene solution containing 1% (3-aminopropyl) trimethoxysilane (APTMS) (w / v) to obtain a mixture. Stir the mixture at room temperature for 24 h to obtain amino-silane modified ginsenoside. Centrifuge the amino-silane modified ginsenoside at a centrifugal force of 16099 (×g) for 10 min, discard the supernatant, collect the precipitate, and obtain amino-silane modified ginsenoside. Suspend the amino-silane modified ginsenoside in toluene, wash it three times, and then wash it twice with ultrapure water to obtain amino-silane activated ginsenoside;

[0164] (2) Prepare a 10% solution of MAH (1 mL, w / v) with ultrapure water, add 1 mL of activation buffer (50 mM MES, pH 6.0) and mix well. Add 24 μL of 200 mM EDC solution (weigh 19.2 mg of EDC and dissolve it in 500 μL of ultrapure water) and 240 μL of 200 mM Sulfo-NHS solution (weigh 21.7 mg of Sulfo-NHS and add it to 500 μL of activation buffer) to 1 mL of activation buffer. Vortex and mix at room temperature and incubate on a turntable for 30 min. Prepare a 2 mg / mL amino-silane activated ginsenoside solution with activation buffer. Add 0.2 mL of amino-silane activated ginsenoside solution to the activated MAH and mix on a rotary mixer at room temperature for 2.5 h. After the reaction, separate the conjugate MAH-Rg3 by protein L affinity chromatography packing and store it at 4°C.

[0165] Example 3

[0166] An antibody conjugate, specifically a conjugate of single-chain antibody MAH and ginsenoside Rh2, and its preparation method is as follows:

[0167] (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. Stir the mixture at room temperature for 24 h to obtain amino-silane modified ginsenoside. Centrifuge the amino-silane modified ginsenoside at a centrifugal force of 16099 (×g) for 10 min, discard the supernatant, collect the precipitate, and obtain amino-silane modified ginsenoside. Suspend the amino-silane modified ginsenoside in toluene, wash it three times, and then wash it twice with ultrapure water to obtain amino-silane activated ginsenoside;

[0168] (2) Prepare 1 mL of a 10% solution (w / v) of MAH with ultrapure water, add 1 mL of activation buffer (50 mM MES, pH 6.0) and mix well. Add 24 μL of 200 mM EDC solution (weigh 19.2 mg of EDC and dissolve it in 500 μL of ultrapure water) and 240 μL of 200 mM Sulfo-NHS solution (weigh 21.7 mg of Sulfo-NHS and add it to 500 μL of activation buffer) to 1 mL of activation buffer. Vortex mix at room temperature and incubate on a rotary shaker for 30 min. Prepare a solution of amino-silane-activated ginsenoside with a concentration of 2 mg / mL using activation buffer. Add 0.2 mL of the amino-silane-activated ginsenoside solution to the activated MAH and mix on a rotary mixer at room temperature for 2.5 h. After the reaction, separate the conjugate by protein L affinity chromatography packing material, store it at 4 °C, and the prepared conjugate is denoted as MAH-Rh2.

[0169] Characterization of the conjugate:

[0170] 1H NMR measurements were carried out using a BRUKER AVANCE III 600 MHz (Bruker Corporation) 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).

[0171] Since the ginsenosides Rg3 and Rh2 used in the present invention do not have functional groups that can be conjugated to the single-chain antibody MAH, Rg3 and Rh2 cannot be conjugated to MAH. In the present invention, a method of covalently connecting amino and carboxyl groups to form an amide bond was used for the conjugation of MAH and Rg3, and the covalent conjugate was purified by affinity chromatography. 1H NMR of MAH-Rg3 confirmed the success of the above conjugation method, showing characteristic peaks of the polymer in the final 1H NMR. The methyl group carried by the amino-silane appeared in the amino-activated Rg3 (ppm: 3.51), and additional amide bonds appeared in MAH-Rg3 (ppm: 6.13, 7.16). 1H NMR of MAH-Rh2 confirmed the success of the above conjugation method, showing characteristic peaks of the polymer in the final 1H NMR. The methyl group carried by the amino-silane appeared in the amino-activated Rg3 (ppm: 3.58), and an amide bond appeared in MAH-Rg3 (ppm: 6.35), as specifically shown in Figure 13 and Figure 14 as shown.

[0172] Regarding the effects of ginsenosides and antibody conjugates on the metabolic activities of tumors:

[0173] The tumor cells and tumor organoids were continuously cultured for 3 passages and, after the passages were stable, cultured for another 4 - 5 days for drug administration tests. Media containing MAH - Rg3, MAH - Rh2, Rg3, and Rh2 at respective concentrations were prepared using the media for the corresponding cells. A549, HCT - 116, HEK - 293T, and NCI - N87 cells (4×10 4 )were seeded into 96 - well plates and cultured overnight at 37 °C, after which the media were replaced with those containing MAH - Rg3, MAH - Rh2, Rg3, and Rh2 respectively. After incubation for 48 h, the tumor - suppressing ability of each drug was detected by the CCK - 8 method. The results were analyzed using GraphPad Prism 8.0 software to calculate the relative cell viability.

[0174] The relevant reagents for the cell lines and cell media used above are as follows:

[0175] All cell lines were from the American Type Culture Collection (ATCC). The HCT - 116 and NCI - N87 epithelial cell lines were cultured in RPMI1640 medium (11875119, Gibco), with FBS (10% v / v) (A5669701, Gbico) and penicillin - streptomycin (1% v / v) (15140148, Gibco) added to the medium. The A549 cell line was cultured in F - 12K medium (21127022, Gibco), with FBS and penicillin - streptomycin (1% v / v) added to the medium. HEK - 293T and A549 cells were cultured in DMEM medium (10566016, Gibco), with FBS (10% v / v) and penicillin - streptomycin added to the medium. The BT - 474 cell line was cultured in RPMI1640 medium, with FBS (10% v / v) and penicillin - streptomycin (1% v / v) added to the medium. The cell medium was changed every 2 - 3 days, and the cell lines were passaged after treatment with trypsin (25200114, Gbico). The cell culture conditions were in an incubator (Heracell Vios 160i Cr, Thermo Scientific) at 37 °C, 5% CO2, and 95% relative humidity. Mycoplasma detection was carried out routinely.

[0176] Establishment of organoids

[0177] (1) Human gastric cancer tissues used:

[0178] Three GC patients who underwent surgical resection were obtained from the China-Japan Union Hospital of Jilin University in Changchun, Jilin Province, China, from August 2024 to October 2024. This study was approved by the Ethics Committee of the China-Japan Union Hospital of Jilin University, review number: 2023103015.

[0179] (2) Establishment of organoid cultures

[0180] The freshly collected tissue was immediately placed in HBSS solution containing amphotericin, penicillin, and streptomycin and minced. The digested tumor tissue was filtered through a 100 μm filter and centrifuged at 350× g. After washing, the cell pellet was resuspended in Matrigel, and 25 μL of the Matrigel-cell mixture per well was seeded on a pre-warmed 24-well plate. 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 primamycin. The medium was replenished every 3 - 4 days, and the organoids were passaged every 1 - 2 weeks using TrypLE Express (Gibco).

[0181] Histological staining and immunofluorescence

[0182] The tissues and organoids were fixed in 4% paraformaldehyde and sectioned at a thickness of 5 μm after processing. Hematoxylin and eosin staining was performed using a standard histological protocol. 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).

[0183] Statistical analysis

[0184] Results are expressed as mean ± standard deviation (SD) of at least three independent experiments. In GraphPad Prism 8.0 software (GraphPad, USA), Student's t-test was used to analyze the comparison between two independent groups. The significance level was set at * < 0.05; ** < 0.01 and **** < 0.001.

[0185] (1) Effects of ginsenoside Rg3 and Rh2 on the metabolic activities of tumor cells

[0186] The effects of different concentrations of ginsenoside Rg3 and Rh2 on the viability of A549, HCT-116, and NCI-N87 cells at different times are shown as Figure 15 follows. It can be seen that both ginsenoside Rg3 and Rh2 have obvious inhibitory ability 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 pictures (see Figure 16 ). To explore whether ginsenosides have an impact on the expression of HER2 gene and protein, we detected them by q-PCR and Western-Blot. The results showed that both ginsenosides reduced the expression of HER2 gene and protein, as Figure 17 shown. This may be due to the cell cycle arrest at the G0 / G1 phase caused by ginsenosides.

[0187] (2) Effects of MAH-Rg3 and MAH-Rh2 on the metabolic activities of tumor cells

[0188] We treated A549, HCT-116, and NCI-N87 cells with different concentrations of MAH-Rg3 and MAH-Rh2 respectively to verify their effects on tumor cells. The bright-field pictures of the experiment are shown as Figure 18 follows. After 24 hours of treatment, obvious morphological changes occurred in A549, HCT-116, and NCI-N87 cells. Compared with the control group, cell shrinkage and incomplete cell morphology were observed in the three types of cells after treatment with MAH-Rg3 and MAH-Rh2. These morphological changes indicate that the drugs have obvious effects on cells, and may achieve their effects by inducing apoptosis or necrosis. The results of the cell viability assay of NCI-N87 cells treated with trastuzumab and single-chain antibody at gradient concentrations for 24 hours by the CCK-8 method are shown as Figure 19 follows. Neither the single-chain antibody nor trastuzumab in the concentration range of 100 - 0.01 μg / ml had an obvious effect on the viability of NCI-N87. Further verification of the tumor-killing ability of MAH-Rg3 and MNAH-Rh2 in gastric cancer cell NCI-N87, the experimental results are shown as Figure 20As shown, 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 was significantly reduced, being 10.34 times that of Rg3 and 18.66 times that of Rh2, respectively. As Figure 21 shown, this may be because MAH-Rg3 and MAH-Rh2 enhanced the growth inhibitory effect by targeting HER2 and delivering ginsenosides in two ways, which was consistent with our expected results.

[0189] (3)Effects of MAH-Rg3 and MAH-Rh2 on the metabolic activities of gastric cancer organoids

[0190] To evaluate that the ginsenoside conjugate can effectively promote the apoptosis of human gastric cancer organoids, we incubated the organoids with the ginsenoside conjugate, Rg3, Rh2, single-chain antibody, and trastuzumab at gradient concentrations, detected the cell viability with CCK-8 after 24 hours of incubation, and plotted the IC50 to evaluate their effects. As Figure 22 shown, due to different patient sources, the sensitivities of three cases of gastric cancer organoids to these substances were different.

[0191] Compared with the experimental results at the cell level, since the organoids were more complex and had a more diverse cell type, the half-maximal inhibitory concentration of the drugs increased. For ginsenosides, all three cases of organoids were sensitive to Rg3 and Rh2, and were more sensitive to Rh2 than to Rg3. For trastuzumab, an obvious antagonistic effect occurred in organoid P2, while the ginsenoside conjugate had a good inhibitory effect on all three cases of organoids.

Claims

1. An antibody conjugate of a HER2 nanobody, characterized in that: The conjugate MAH-Rh2 is formed by coupling the single-chain antibody MAH with ginsenoside Rh2. The amino acid sequence of the single-chain antibody MAH is shown in SEQ ID NO.

1. The structural formula of the antibody conjugate is as follows: 。 2. A method for preparing an antibody conjugate as described in claim 1, characterized in that: Dissolve ginsenoside in an anhydrous toluene solution of (3-aminopropyl)trimethoxysilane (APTMS), stir and react to obtain ginsenoside modified with amino silane, centrifuge to collect the precipitate, wash to obtain ginsenoside activated with amino silane. Take MAH and dissolve it in ultrapure water to prepare a MAH solution, and mix it with an activation buffer to obtain an activated MAH solution; Take the activation buffer again, add an EDC solution and an aqueous solution of Sulfo-NHS, then add ginsenoside activated with amino silane to form a mixed solution. Add the mixed solution to the activated MAH solution for mixing reaction, and then separate it through a Protein L affinity chromatography packing material to obtain the antibody conjugate.

3. The preparation method of an antibody conjugate according to claim 2, wherein: The ginsenoside is Rh2. The mass-volume ratio of Rh2 to the anhydrous toluene solution of APTMS is 0.8-1.2:100, and the concentration of the anhydrous toluene solution of APTMS is 0.8-1.2% (mass-volume percentage concentration).

4. The preparation method of an antibody conjugate according to claim 2 or 3, characterized in that: In the activated MAH solution, the volume ratio of the MAH solution to the activation buffer is 1:

1. The activation buffer is a MES solution with a pH of 6.0 and a concentration of 50 mM. The mass-volume percentage concentration of the MAH solution is 8-12%.

5. The preparation method of an antibody conjugate according to claim 4, wherein: In the mixed solution, the concentration of ginsenoside activated with amino silane is 1.5-2.5 mg / mL, and the volume ratio of the activation buffer, the EDC solution and the aqueous solution of Sulfo-NHS 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 Sulfo-NHS is 200 mmol / L.

6. The preparation method of an antibody conjugate according to claim 5, characterized in that: The volume ratio of the activated MAH solution to the mixed solution is 10:

1.

7. Use of the antibody conjugate prepared by the method according to claim 6 in the preparation of a drug for targeting HER2 to inhibit tumor growth.

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