HER3 nano antibody composition for tumor targeted therapy and preparation method of HER3 nano antibody composition

Abstract

The invention relates to the technical field of biological medicines, and discloses an HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof, according to an HER3 nano-antibody sequence, a gene synthesis technology is adopted to synthesize an HER3 nano-antibody gene, and the synthesized gene is subjected to sequencing verification; cloning the HER3 nano antibody gene subjected to sequencing verification into an expression vector, and carrying out enzyme digestion and sequencing verification on the constructed expression vector; the preparation method comprises the following steps: converting an expression vector into an expression system, expressing under a certain condition, purifying an expression product to obtain a high-purity HER3 nano antibody, and mixing the high-purity HER3 nano antibody with pharmaceutic adjuvants to prepare the HER3 nano antibody composition. The invention has the advantages of specific binding of HER3, small molecular weight, good stability, low immunogenicity and the like, and has wide application prospects in tumor targeted therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof. Background Art

[0002] Tumor is one of the diseases that seriously threaten human health. Traditional tumor treatment methods such as surgery, radiotherapy and chemotherapy have certain limitations, such as damage to normal tissues and large side effects. In recent years, tumor targeted therapy has become a research hotspot. It acts specifically on targets on tumor cells to improve the treatment effect while reducing side effects on normal tissues. HER3 (human epidermal growth factor receptor 3) is highly expressed in many tumors and is closely related to the occurrence and development of tumors. Therefore, HER3 has become an important target for tumor targeted therapy. Nanoantibodies are a new type of antibody molecules with the advantages of small molecular weight, high specificity and good stability. They have broad application prospects in tumor targeted therapy. Summary of the invention

[0003] The purpose of the present invention is to solve the above problems and to design a HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof.

[0004] The present invention provides a HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof, wherein the HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof comprise the following steps:

[0005] S1. Gene synthesis: According to the HER3 nanoantibody sequence, the HER3 nanoantibody gene is synthesized using gene synthesis technology, and the synthesized gene is sequenced and verified;

[0006] S2. Construction of expression vector: Clone the HER3 nanobody gene verified by sequencing into the expression vector, and perform restriction digestion and sequencing verification on the constructed expression vector;

[0007] S3. Expression and purification: The expression vector is transformed into an expression system, and expression is performed under certain conditions. The expression product is purified to obtain a high-purity HER3 nanobody, and the high-purity HER3 nanobody is mixed with pharmaceutical excipients to prepare a HER3 nanobody composition, wherein the pharmaceutical excipients include at least a buffer, a stabilizer, and a preservative.

[0008] Optionally, in a first implementation of the present invention, step S1 specifically includes the following process:

[0009] Collect the sequence information of nanoantibodies with high affinity and specificity binding to HER3, compare and analyze different HER3 nanoantibody sequences, and screen out HER3 nanoantibody sequences for gene synthesis;

[0010] According to the selected HER3 nanoantibody sequence, the HER3 nanoantibody gene is synthesized by a solid phase phosphoramidite triester method. During the gene synthesis process, multiple short oligonucleotide fragments are synthesized, and these fragments cover the entire HER3 nanoantibody gene sequence and have overlapping regions at both ends;

[0011] The synthesized oligonucleotide fragments were assembled using PCR technology to obtain the complete HER3 nanoantibody gene, and the synthesized HER3 nanoantibody gene was sequenced and verified using the Sanger sequencing method.

[0012] Optionally, in a second implementation of the present invention, the synthesis of HER3 nanoantibody gene using the solid phase phosphoramidite triester method specifically includes the following process: selecting controlled pore glass as a solid phase carrier, connecting the first nucleotide to the solid phase carrier through a chemical reaction, removing the protecting group of the nucleotide to expose the hydroxyl group at its 5' end, chemically reacting a new nucleotide monomer with a protecting group with the exposed hydroxyl group, connecting it to the DNA chain being synthesized, blocking the unreacted hydroxyl group, oxidizing the newly formed phosphate bond to a phosphotriester bond, repeating the nucleotide addition cycle until the complete HER3 nanoantibody gene sequence is synthesized, the gene is cut from the solid phase carrier, and purified.

[0013] Optionally, in a third implementation of the present invention, the synthesised HER3 nanoantibody gene is sequenced and verified by the Sanger sequencing method, which specifically includes the following processes: template preparation: the synthesized HER3 nanoantibody gene is used as a template, and the template, primers, DNA polymerase, dNTPs and fluorescently labeled dideoxynucleotides are added to the sequencing reaction system, and a PCR reaction is carried out to allow the DNA polymerase to synthesize a DNA chain complementary to the template, and to incorporate dideoxynucleotides at random positions to terminate the synthesis, and the sequencing reaction products are separated by electrophoresis, and the DNA sequence is determined according to the size of the fragment and the fluorescent label, and the sequence of the HER3 nanoantibody gene is determined by analyzing the electrophoresis results.

[0014] Optionally, in a fourth implementation of the present invention, step S2 specifically includes the following process:

[0015] Select an expression vector with a T7 promoter, a suitable terminator, an ampicillin resistance gene, and multiple restriction sites;

[0016] Performing enzyme digestion reaction on the HER3 nanobody gene verified by sequencing, and selecting a restriction endonuclease that can produce the same sticky ends or blunt ends on the expression vector and the HER3 nanobody gene according to the sequences of the expression vector and the HER3 nanobody gene;

[0017] Then, a ligation reaction is performed to connect the HER3 nanobody gene after enzyme digestion with the expression vector to construct a recombinant expression vector;

[0018] The recombinant expression vector was transformed into the host cell and converted. The expression vector was digested with the same restriction endonuclease as in the cloning process. The size and quantity of the digestion products were analyzed by gel electrophoresis to verify whether the HER3 nanobody gene was correctly inserted into the expression vector. If the size of the digestion product was consistent with the expectation, it indicated that the insertion was correct. At the same time, the constructed expression vector was sequenced by Sanger sequencing, and the sequencing results were compared with the original sequence of the HER3 nanobody gene to analyze whether there were mutations, deletions or abnormal insertions.

[0019] Optionally, in the fifth implementation of the present invention, the amount of the enzyme cleavage reaction vector is 0.1-1 μg, the molar ratio of gene to vector is 2-10, the enzyme cleavage reaction time is 1-4 hours, and the reaction temperature is 37°C; the molar ratio of the ligation reaction vector and gene is 1:3-1:10, 0.5-2 units of T4 DNA ligase are used per microliter of reaction system, and the buffer is ATP or MgCl2; the host cell of the conversion process is an Escherichia coli strain.

[0020] Optionally, in a sixth implementation of the present invention, step S3 specifically includes the following process:

[0021] Select an E. coli expression system, a yeast expression system or a mammalian cell expression system. During the expression process, analyze the molecular weight and purity of the expression product by SDS-PAGE, and compare it with the standard molecular weight to determine whether the size of the expression product meets expectations;

[0022] Affinity chromatography was selected to purify the expression product, and the purified HER3 nanoantibody was stored in phosphate buffer to finally obtain a high-purity HER3 nanoantibody.

[0023] Optionally, in the seventh implementation of the present invention, when an Escherichia coli expression system is selected, the heat shock transformation method is used to transform Escherichia coli, and the expression vector is mixed with the competent Escherichia coli cells, and the mixture is heat-shocked at 42°C for 45-90 seconds, and then rapidly cooled to allow the vector to enter the cells; when a yeast expression system is selected, a lithium acetate transformation method is used, and the expression vector is mixed with the competent yeast cells, a lithium acetate solution with a concentration of 100-200mM and a single-stranded DNA vector, and the mixture is incubated at 30°C for 30-60 minutes to promote the vector to enter the cells; when a mammalian cell culture system is selected, a liposome transfection method is used, and the expression vector and liposome are mixed in a ratio of 2:1-5:1 to form a complex, and then the complex is added to the mammalian cell culture system, and the transfection time is 2-6 hours.

[0024] The technical solution provided by the present invention provides a HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof. The composition has high tumor targeting and therapeutic effect, and provides a new and effective means for the treatment of tumors. A nano-antibody expression vector is constructed by genetic engineering technology, and the nano-antibody is expressed and purified in a suitable expression system to prepare a high-purity HER3 nano-antibody. The nano-antibody is mixed with pharmaceutical excipients to prepare a nano-antibody composition. The HER3 nano-antibody composition of the present invention has high tumor targeting, can specifically bind to the HER3 target on tumor cells, and improve the therapeutic effect. The nano-antibody has a small molecular weight, can penetrate tumor tissue, reach the inside of the tumor to exert its effect, has good stability, is not easily degraded in the body, can maintain the efficacy for a long time, has low immunogenicity, reduces the occurrence of side effects, and improves the tolerance of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0026] Figure 1 The present invention is a flow chart of the method for preparing the HER3 nano-antibody composition for tumor targeted therapy. DETAILED DESCRIPTION

[0027] The terms "first", "second", "third", "fourth", etc. (if any) in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "including" or "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, device, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0028] For ease of understanding, the specific process of the embodiment of the present invention is described below. Figure 1 The present invention provides a flow chart of a method for preparing a HER3 nano-antibody composition for tumor targeted therapy, which specifically comprises the following steps:

[0029] S1. Gene synthesis: According to the HER3 nanoantibody sequence, the HER3 nanoantibody gene is synthesized using gene synthesis technology, and the synthesized gene is sequenced and verified;

[0030] S2. Construction of expression vector: Clone the HER3 nanobody gene verified by sequencing into the expression vector, and perform restriction digestion and sequencing verification on the constructed expression vector;

[0031] S3. Expression and purification: The expression vector is transformed into an expression system, and expression is performed under certain conditions. The expression product is purified to obtain a high-purity HER3 nanobody, and the high-purity HER3 nanobody is mixed with pharmaceutical excipients to prepare a HER3 nanobody composition, wherein the pharmaceutical excipients include at least a buffer, a stabilizer, and a preservative.

[0032] In this embodiment, the buffer is used to maintain the pH value of the composition stable, ensuring that the nanobody remains active in a suitable environment. For example, a phosphate buffer can be used, which has good buffering capacity and biocompatibility, helps to adjust the ionic strength of the solution, and affects the stability and solubility of the nanobody; the stabilizer is used to protect the nanobody from degradation or inactivation during storage and transportation. Common stabilizers include sugars (such as sucrose, trehalose), polyols (such as glycerol), etc., which can interact with the nanobody to prevent its aggregation, precipitation or denaturation.

[0033] Preservatives are used to prevent the composition from being contaminated by microorganisms during use. Suitable preservatives should have low toxicity, high antibacterial properties and good stability. For example, benzyl alcohol, phenol, etc. can be added to the composition as preservatives, but the concentration used must be strictly controlled within a safe range.

[0034] In this embodiment, step S1 specifically includes the following process:

[0035] Collect the sequence information of nanoantibodies with high affinity and specificity binding to HER3, compare and analyze different HER3 nanoantibody sequences, and screen out HER3 nanoantibody sequences for gene synthesis;

[0036] According to the selected HER3 nanoantibody sequence, the HER3 nanoantibody gene is synthesized by a solid phase phosphoramidite triester method. During the gene synthesis process, multiple short oligonucleotide fragments are synthesized, and these fragments cover the entire HER3 nanoantibody gene sequence and have overlapping regions at both ends;

[0037] The synthesized oligonucleotide fragments were assembled using PCR technology to obtain the complete HER3 nanoantibody gene, and the synthesized HER3 nanoantibody gene was sequenced and verified using the Sanger sequencing method.

[0038] In this embodiment, the synthesis of HER3 nanobody gene by solid phase phosphoramidite triester method specifically includes the following process: selecting controlled pore glass as a solid phase carrier, connecting the first nucleotide to the solid phase carrier by chemical reaction, removing the protecting group of the nucleotide to expose the hydroxyl group at its 5' end, chemically reacting a new nucleotide monomer with a protecting group with the exposed hydroxyl group and connecting it to the DNA chain being synthesized, blocking the unreacted hydroxyl group, oxidizing the newly formed phosphate bond to a phosphotriester bond, repeating the nucleotide addition cycle until the complete HER3 nanobody gene sequence is synthesized, the gene is cut from the solid phase carrier, and purified.

[0039] In this embodiment, the Sanger sequencing method is used to sequence and verify the synthesized HER3 nanobody gene, which specifically includes the following processes: template preparation: the synthesized HER3 nanobody gene is used as a template, and the template, primers, DNA polymerase, dNTPs and fluorescently labeled dideoxynucleotides are added to the sequencing reaction system, and a PCR reaction is performed, so that the DNA polymerase synthesizes a DNA chain complementary to the template, and incorporates dideoxynucleotides at random positions to terminate the synthesis, and the sequencing reaction products are separated by electrophoresis, and the DNA sequence is determined according to the size of the fragment and the fluorescent label, and the sequence of the HER3 nanobody gene is determined by analyzing the electrophoresis results.

[0040] In this embodiment, step S2 specifically includes the following process:

[0041] Select an expression vector with a T7 promoter, a suitable terminator, an ampicillin resistance gene, and multiple restriction sites;

[0042] Performing enzyme digestion reaction on the HER3 nanobody gene verified by sequencing, and selecting a restriction endonuclease that can produce the same sticky ends or blunt ends on the expression vector and the HER3 nanobody gene according to the sequences of the expression vector and the HER3 nanobody gene;

[0043] Then, a ligation reaction is performed to connect the HER3 nanobody gene after enzyme digestion with the expression vector to construct a recombinant expression vector;

[0044] The recombinant expression vector was transformed into the host cell and converted. The expression vector was digested with the same restriction endonuclease as in the cloning process. The size and quantity of the digestion products were analyzed by gel electrophoresis to verify whether the HER3 nanobody gene was correctly inserted into the expression vector. If the size of the digestion product was consistent with the expectation, it indicated that the insertion was correct. At the same time, the constructed expression vector was sequenced by Sanger sequencing, and the sequencing results were compared with the original sequence of the HER3 nanobody gene to analyze whether there were mutations, deletions or abnormal insertions.

[0045] In this embodiment, the amount of the enzyme digestion reaction vector is 0.1-1 μg, the molar ratio of the gene to the vector is 2-10, the enzyme digestion reaction time is 1-4 hours, and the reaction temperature is 37°C; the molar ratio of the ligation reaction vector to the gene is 1:3-1:10, 0.5-2 units of T4 DNA ligase are used per microliter of reaction system, and the buffer is ATP or MgCl2; the host cell of the conversion process is an Escherichia coli strain.

[0046] In this embodiment, step S3 specifically includes the following process:

[0047] Select an E. coli expression system, a yeast expression system or a mammalian cell expression system. During the expression process, analyze the molecular weight and purity of the expression product by SDS-PAGE, and compare it with the standard molecular weight to determine whether the size of the expression product meets expectations;

[0048] Affinity chromatography was selected to purify the expression product, and the purified HER3 nanoantibody was stored in phosphate buffer to finally obtain a high-purity HER3 nanoantibody.

[0049] In this embodiment, for E. coli transformation, a heat shock transformation method can be used, in which the expression vector is mixed with competent E. coli cells, subjected to heat shock treatment at 42°C for 45-90 seconds, and then rapidly cooled to allow the vector to enter the cells; an electrical transformation method can also be used, in which a high voltage electric field is used to reversibly perforate the cell membrane in a short period of time, wherein the electric field strength is generally between 12-25 kV / cm, and the pulse time is between 2-10 ms; for yeast transformation, a lithium acetate transformation method can be used, in which the expression vector is mixed with competent yeast cells, a lithium acetate solution with a concentration of 100-200 mM, and a single-stranded DNA vector, and the mixture is kept warm at 30°C for 30-60 minutes to promote the entry of the vector into the cells. For mammalian cell transformation, liposome transfection can be used, in which the expression vector and liposome are mixed in a ratio of 2:1-5:1 to form a complex, and then the complex is added to the mammalian cell culture system. The transfection time is 2-6 hours. Electroporation can also be used to perforate the mammalian cell membrane using a high-voltage electric field. The electric field strength and pulse time are adjusted according to different cell types and experimental conditions.

[0050] The expression is carried out under certain conditions. In the E. coli expression system, IPTG is used as an inducer, and the concentration is generally between 0.1-1mM, and the expression is induced at 25-37°C for 2-8 hours; for the yeast expression system, methanol is used as an inducer, and the concentration is generally between 0.5-2%, and the expression is induced at 25-30°C for 24-72 hours; for the mammalian cell expression system, different inducers, such as dexamethasone, are used according to different expression vectors and cell lines, and culture is carried out at 37°C and 5% CO2. The induction time is adjusted according to the expression of the protein and the growth state of the cells. During the expression process, the molecular weight and purity of the expression product can be analyzed by SDS-PAGE, and compared with the standard molecular weight marker to determine whether the size of the expression product meets expectations; Western blot can be used to detect the expression product with specific antibodies to determine whether it is the target protein and to detect the expression amount and purity; for proteins with enzyme activity, the activity of the expression product can be evaluated by measuring the enzyme activity;

[0051] Finally, the expression product is purified. Affinity chromatography can be selected. For example, for HER3 nanoantibodies with His-tag, a nickel column is used for affinity chromatography purification. According to the properties of the target protein and the purification requirements, a suitable affinity chromatography column is selected, the composition and conditions of the binding buffer, such as pH value, ionic strength, etc., are optimized to improve the binding efficiency of the target protein with the ligand, and a suitable elution buffer, such as imidazole solution of different concentrations, is selected to elute the target protein from the column by competitive binding or changing the pH value. Ion exchange chromatography can also be selected. According to the isoelectric point of the target protein, a suitable ion exchange column, such as a cation exchange column or anion exchange column, is selected. The pH value and ionic strength of the binding buffer are adjusted to bind the target protein to the column. The target protein is eluted from the column by gradient elution by changing the salt concentration or pH value. Gel filtration chromatography can also be selected. According to the molecular weight range of the target protein, a suitable gel filtration column is selected, the exclusion limit of the column is determined, and the appropriate buffer is used for elution. The elution time in the column is different according to the molecular weight of the protein. During the purification process, it is necessary to monitor the purification process by testing the samples after each step of purification, such as measuring the protein concentration, purity and activity, to monitor the effect of the purification process; to remove possible impurities, such as host cell proteins, nucleic acids, endotoxins, etc., the purity can be improved by optimizing the purification method, adding purification steps or using specific reagents for removing impurities; the purified HER3 nanoantibody is stored under appropriate conditions, such as low temperature, in an appropriate buffer, and stabilizers are added to maintain the stability and activity of the protein, and finally a high-purity HER3 nanoantibody is obtained.

[0052] Example 1: Escherichia coli expression system

[0053] 1. Experimental steps

[0054] Gene synthesis: Through literature research and bioinformatics analysis, the sequence of nanoantibodies with high affinity and specificity for binding to HER3 was determined, and the HER3 nanoantibody gene was synthesized using the solid-phase phosphoramidite triester method in chemical synthesis, and verified by Sanger sequencing;

[0055] Construction of expression vector: Select an E. coli expression vector with a T7 promoter, a suitable terminator, an ampicillin resistance gene, and a multiple cloning site, digest the vector and the HER3 nanobody gene verified by sequencing with BamHI and HindIII restriction endonucleases, connect the gene to the expression vector with T4 DNA ligase, and verify the constructed expression vector by digestion and sequencing;

[0056] Expression and purification: The expression vector was transformed into E. coli DH5α competent cells by heat shock transformation. Transformants were selected in a medium containing ampicillin, and IPTG was used as an inducer to induce expression at 37°C for 4 hours. The bacteria were collected and the expression products were released by ultrasonic cell disruption. The HER3 nanobody with His-tag was purified by nickel column affinity chromatography and eluted with different concentrations of imidazole solution.

[0057] 2. Experimental Test

[0058] 1. Affinity and specificity test:

[0059] Using surface plasmon resonance (SPR) technology, the HER3 protein was fixed on the sensor chip, and different concentrations of HER3 nanoantibodies expressed by E. coli were allowed to flow through the chip. By monitoring the changes in the resonance signal, the affinity constant of the nanoantibody to HER3 was calculated. At the same time, a control experiment was set up to use other proteins with similar structures to HER3 for detection to verify the specificity of the nanoantibody.

[0060] An enzyme-linked immunosorbent assay (ELISA) was performed, HER3 protein was coated on an ELISA plate, nanoantibodies of different dilutions were added, and then secondary antibodies and colorimetric substrates were added. The binding of nanoantibodies to HER3 was determined by measuring the absorbance value, and then the affinity and specificity were evaluated.

[0061] 1. Penetration test:

[0062] In vitro cell experiment: Tumor cells are cultured in a Transwell chamber, and the nanobody is added to the upper chamber. After a certain period of time, the content of the nanobody in the lower chamber is detected. The penetration ability of the nanobody in the tumor cell model is evaluated by comparing the penetration rate at different time points.

[0063] Animal experiment: A tumor-bearing mouse model was constructed, and fluorescently labeled nanobodies were injected into the mice through the tail vein. Tumor tissues and other major organs were collected at different time points, and the distribution of nanobodies in tumor tissues was observed using fluorescence imaging technology, and compared with other organs to evaluate their penetration.

[0064] 3. Stability test:

[0065] The purified nanobody composition is stored for a certain period of time under high temperature (such as 45°C), high humidity (relative humidity 75%) and light (light box with a certain intensity). Samples are taken regularly, protein degradation is analyzed by SDS-PAGE, protein integrity is detected by Western blot, and activity changes of nanobodies are detected by activity assay methods (if applicable), and the stability of nanobodies under different conditions is comprehensively evaluated.

[0066] 4. Immunogenicity test:

[0067] Animal experiments: Choose a suitable animal model, such as mice or rats, and give the nanobody to the animal through different routes (such as subcutaneous injection, intravenous injection, etc.). Collect blood from the animal regularly to detect whether there are antibodies against the nanobody in the serum. Use ELISA or other immunoassay methods for analysis. At the same time, observe the general conditions of the animal, such as behavior, weight, diet, and pathological changes in important organs, and evaluate the impact of the immunogenicity of the nanobody on the health of the animal.

[0068] Example 2: Yeast expression system

[0069] 1. Experimental steps

[0070] Gene synthesis: Through literature research and bioinformatics analysis, the sequence of nanoantibodies with high affinity and specificity for binding to HER3 was determined, and the HER3 nanoantibody gene was synthesized using the solid-phase phosphoramidite triester method in chemical synthesis, and verified by Sanger sequencing;

[0071] Expression vector construction: Select a Pichia pastoris expression vector with a GAL1 promoter, an AOX1 promoter, a suitable terminator, and a selection marker, use specific restriction endonucleases to digest and connect the vector and the HER3 nanobody gene, construct a recombinant expression vector, and perform restriction digestion and sequencing verification on the constructed expression vector;

[0072] Expression and purification: The expression vector was transformed into Pichia pastoris competent cells by lithium acetate transformation. Transformants were selected in a medium containing the corresponding selection marker, and methanol was used as an inducer to induce expression at 28°C for 48 hours. The cells were collected and the supernatant was separated by centrifugation. The HER3 nanobody was purified by ion exchange chromatography. The appropriate ion exchange column was selected according to the isoelectric point of the protein, and gradient elution was performed by changing the salt concentration.

[0073] 2. Experimental Test

[0074] 1. Affinity and specificity test:

[0075] SPR technology: same as in Example 1.

[0076] ELISA: Same as Example 1. In addition, a competitive ELISA method was used to simultaneously add nanobodies and known HER3 binding ligands to an ELISA plate coated with HER3 protein, and the competitive binding between the nanobodies and the ligands was observed to further verify the specificity of the nanobodies.

[0077] 2. Penetration test:

[0078] In vitro cell experiments: Experiments were conducted using different types of tumor cell lines to observe the penetration ability of nanobodies in different tumor cells. The distribution of nanobodies in cells was observed by fluorescently labeling the nanobodies using a confocal microscope to evaluate their penetration depth and speed.

[0079] Animal experiments: Construct different types of tumor animal models, such as breast cancer, lung cancer and other tumor-bearing mouse models. After the nanoantibodies are injected into the animals, the accumulation and distribution of the nanoantibodies in the tumor tissues are monitored by in vivo imaging techniques (such as bioluminescence imaging, fluorescence imaging, etc.). At the same time, the tumor targeting and penetration of the nanoantibodies are analyzed by comparison with normal tissues.

[0080] 3. Stability test:

[0081] In addition to stability testing under high temperature, high humidity and light conditions, the nanobody composition was stored in buffers with different pH values ​​to simulate different physiological environments in the body. The stability of the nanobody under different pH conditions was evaluated by measuring the solubility, aggregation state and activity changes of the protein.

[0082] An accelerated stability test is performed, in which the Nanobody is stored at a higher temperature (such as 37°C) for a short period of time, and then compared with the Nanobody stored at room temperature. The long-term stability of the Nanobody is predicted by analyzing the degree of protein degradation and activity changes.

[0083] 4. Immunogenicity test:

[0084] Animal experiment: Same as Example 1. Further detect the immune cell response in the animal, such as the activation of T cells and B cells, and the secretion level of cytokines. Analyze by flow cytometry, ELISA and other methods to evaluate the type and intensity of the immune response caused by the nanobody.

[0085] Example 3: Mammalian cell expression system

[0086] 1. Experimental steps

[0087] Gene synthesis: Through literature research and bioinformatics analysis, the sequence of nanoantibodies with high affinity and specificity for binding to HER3 was determined, and the HER3 nanoantibody gene was synthesized using the solid-phase phosphoramidite triester method in chemical synthesis, and verified by Sanger sequencing;

[0088] Expression vector construction: select a mammalian cell expression vector with a CMV promoter, SV40 promoter, a suitable terminator and a resistance gene, use appropriate restriction endonucleases to digest and connect the vector and the HER3 nanobody gene, construct a recombinant expression vector, and perform enzyme digestion and sequencing verification on the constructed expression vector;

[0089] Expression and purification: The expression vector was transfected into HEK293 cells by liposome transfection. Stable expression cell lines were screened in a medium containing the screening drug, and the cells were cultured at 37°C and 5% CO2. The cell culture supernatant was collected and the HER3 nanobody was purified by gel filtration chromatography. The appropriate gel filtration column was selected according to the molecular weight of the protein and eluted with an appropriate buffer.

[0090] 2. Experimental Test

[0091] 1. Affinity and specificity test:

[0092] SPR technology: Same as Example 1. HER3 proteins from different sources were used for detection, including recombinant proteins and natural proteins, to verify the affinity and specificity of the nanobody to different forms of HER3.

[0093] ELISA: Same as Example 1. A sandwich ELISA method was also performed, using the nanobody as the capture antibody and another known HER3 binding antibody as the detection antibody, to improve specificity and accuracy through double detection.

[0094] 2. Penetration test:

[0095] In vitro cell experiments: Use three-dimensional tumor cell models, such as tumor spheres or organoids, to simulate the three-dimensional structure of tumor tissue. Add nanoantibodies to the model and observe the penetration of nanoantibodies in the three-dimensional structure using confocal microscopy or multiphoton microscopy. Analyze the distribution and concentration changes of nanoantibodies at different depths to evaluate their penetration ability.

[0096] Animal experiments: Construct animal models with different tumor microenvironment characteristics, such as matrix-rich tumor models or immunosuppressive tumor models. After the nanoantibodies are injected into the animals, high-resolution imaging techniques (such as magnetic resonance imaging, positron emission tomography, etc.) are used to observe the distribution and penetration of the nanoantibodies in tumor tissues. At the same time, analyze the effect of the tumor microenvironment on the penetration of the nanoantibodies.

[0097] 3. Stability test:

[0098] The stability of the nanobody composition is tested under simulated in vivo physiological conditions, such as storage in a culture medium containing serum, cytokines, etc. The stability of the nanobody in a complex physiological environment is evaluated by measuring the activity, conformational changes and degradation of the protein.

[0099] To conduct a long-term stability test, the nanobody is stored at a low temperature (such as -20°C or -80°C) for a long time, and samples are taken regularly for analysis. The long-term storage stability of the nanobody is evaluated by comparing the protein amount and activity at different time points.

[0100] 4. Immunogenicity test:

[0101] Animal experiment: Same as Example 1. Combined with immunohistochemistry, detect the infiltration of immune cells in animal tumor tissues and important organs, as well as the interaction between nanobodies and immune cells. By analyzing the type, distribution and activation state of immune cells, evaluate the local and systemic immune response caused by nanobodies.

[0102] Through the implementation of the above scheme, the present invention provides a HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof. The composition has high tumor targeting and therapeutic effect, and provides a new and effective means for the treatment of tumors. A nano-antibody expression vector is constructed by genetic engineering technology, and the nano-antibody is expressed and purified in a suitable expression system to prepare a high-purity HER3 nano-antibody. The nano-antibody is mixed with pharmaceutical excipients to prepare a nano-antibody composition. The composition has the advantages of specific binding to HER3, small molecular weight, good stability, low immunogenicity, etc., and has broad application prospects in tumor targeted therapy.

[0103] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A HER3 nanoantibody composition for tumor targeted therapy and a preparation method thereof, characterized in that: The HER3 nanoantibody composition for tumor targeted therapy and the preparation method thereof comprise the following steps: S1. Gene synthesis: According to the HER3 nanoantibody sequence, the HER3 nanoantibody gene is synthesized using gene synthesis technology, and the synthesized gene is sequenced and verified; S2. Construction of expression vector: Clone the sequencing-verified HER3 nanobody gene into the expression vector, and perform enzyme digestion and sequencing verification on the constructed expression vector; S3. Expression and purification: The expression vector is transformed into an expression system, and expression is performed under certain conditions. The expression product is purified to obtain a high-purity HER3 nanobody, and the high-purity HER3 nanobody is mixed with pharmaceutical excipients to prepare a HER3 nanobody composition, wherein the pharmaceutical excipients include at least a buffer, a stabilizer, and a preservative.

2. A HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof according to claim 1, characterized in that: Step S1 specifically includes the following process: Collect the sequence information of nanoantibodies with high affinity and specificity binding to HER3, compare and analyze different HER3 nanoantibody sequences, and screen out HER3 nanoantibody sequences for gene synthesis; According to the selected HER3 nanoantibody sequence, the HER3 nanoantibody gene is synthesized by a solid phase phosphoramidite triester method. During the gene synthesis process, multiple short oligonucleotide fragments are synthesized, and these fragments cover the entire HER3 nanoantibody gene sequence and have overlapping regions at both ends; The synthesized oligonucleotide fragments were assembled using PCR technology to obtain the complete HER3 nanoantibody gene, and the synthesized HER3 nanoantibody gene was sequenced and verified using the Sanger sequencing method.

3. A HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof according to claim 2, characterized in that: The synthesis of HER3 nanoantibody gene using solid phase phosphoramidite triester method specifically includes the following process: selecting controlled pore glass as a solid phase carrier, connecting the first nucleotide to the solid phase carrier through chemical reaction, removing the protecting group of the nucleotide to expose the hydroxyl group at its 5' end, chemically reacting the new nucleotide monomer with the protecting group with the exposed hydroxyl group and connecting it to the DNA chain being synthesized, blocking the unreacted hydroxyl group, oxidizing the newly formed phosphate bond to a phosphotriester bond, repeating the nucleotide addition cycle until the complete HER3 nanoantibody gene sequence is synthesized, the gene is cut from the solid phase carrier, and purified.

4. A HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof according to claim 2, characterized in that: The synthetic HER3 nanoantibody gene was sequenced and verified by Sanger sequencing. The process includes the following: template preparation: using the synthesized HER3 nano antibody gene as a template, adding the template, primers, DNA polymerase, dNTPs and fluorescently labeled dideoxynucleotides into the sequencing reaction system, carrying out a PCR reaction, allowing the DNA polymerase to synthesize a DNA chain complementary to the template, and incorporating dideoxynucleotides at random positions to terminate the synthesis, separating the sequencing reaction products by electrophoresis, determining the DNA sequence based on the size of the fragments and the fluorescent labels, and determining the sequence of the HER3 nano antibody gene by analyzing the electrophoresis results.

5. A HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof according to claim 1, characterized in that: Step S2 specifically The process includes: Select an expression vector with a T7 promoter, a suitable terminator, an ampicillin resistance gene, and multiple restriction sites; Performing enzyme digestion reaction on the HER3 nanobody gene verified by sequencing, and selecting a restriction endonuclease that can produce the same sticky ends or blunt ends on the expression vector and the HER3 nanobody gene according to the sequences of the expression vector and the HER3 nanobody gene; Then, a ligation reaction is performed to connect the HER3 nanobody gene after enzyme digestion with the expression vector to construct a recombinant expression vector; The recombinant expression vector was transformed into the host cell and converted. The expression vector was digested with the same restriction endonuclease as in the cloning process. The size and quantity of the digestion products were analyzed by gel electrophoresis to verify whether the HER3 nanobody gene was correctly inserted into the expression vector. If the size of the digestion product was consistent with the expectation, it indicated that the insertion was correct. At the same time, the constructed expression vector was sequenced by Sanger sequencing, and the sequencing results were compared with the original sequence of the HER3 nanobody gene to analyze whether there were mutations, deletions or abnormal insertions.

6. A HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof according to claim 5, characterized in that: The amount of the enzyme digestion reaction vector is 0.1-1 μg, the molar ratio of the gene to the vector is 2-10, the enzyme digestion reaction time is 1-4 hours, and the reaction temperature is 37°C; the molar ratio of the ligation reaction vector to the gene is 1:3-1:10, 0.5-2 units of T4 DNA ligase are used per microliter reaction system, and the buffer is ATP or MgCl2; the host cell of the conversion process is an Escherichia coli strain.

7. A HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof according to claim 1, characterized in that: Step S3 specifically The process includes: Select an E. coli expression system, a yeast expression system or a mammalian cell expression system. During the expression process, analyze the molecular weight and purity of the expression product by SDS-PAGE, and compare it with the standard molecular weight to determine whether the size of the expression product meets expectations; Affinity chromatography was selected to purify the expression product, and the purified HER3 nanoantibody was stored in phosphate buffer to finally obtain a high-purity HER3 nanoantibody.

8. A HER3 nano-antibody composition for tumor targeted therapy and a preparation method thereof according to claim 7, characterized in that: When an E. coli expression system is selected, the heat shock transformation method is used to transform E. coli, the expression vector is mixed with the competent E. coli cells, and the mixture is heat-shocked at 42°C for 45-90 seconds, and then rapidly cooled to allow the vector to enter the cells; when a yeast expression system is selected, the lithium acetate transformation method is used, the expression vector is mixed with the competent yeast cells, a lithium acetate solution with a concentration of 100-200 mM and a single-stranded DNA vector, and the mixture is kept warm at 30°C for 30-60 minutes to promote the vector to enter the cells; when a mammalian cell culture system is selected, the liposome transfection method is used, the expression vector and the liposome are mixed at a ratio of 2:1-5:1 to form a complex, and then the complex is added to the mammalian cell culture system, and the transfection time is 2-6 hours.

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