Engineered exosome for expressing complete antibody and preparation method thereof

Abstract

The invention discloses an engineered exosome for expressing a complete antibody and a preparation method of the engineered exosome, and belongs to the field of biotechnology and drug delivery. Aiming at the problem that a complete antibody is difficult to efficiently and stably express on the surface of an exosome membrane in the prior art, the engineering exosome with the complete antibody displayed on the surface is extracted from a culture supernatant after a target antibody and a natural antibody membrane-bound transmembrane region (TMD) sequence are fused, a recombinant lentiviral expression vector is constructed and a host cell is transfected. According to the method, TMD is used as an anchoring element, accurate positioning and efficient expression of an antibody on an exosome membrane are achieved, and the targeting recognition capacity of the exosome and the application potential of the exosome in the fields of targeting delivery, disease treatment and the like are remarkably improved. Experimental results show that the obtained exosome is typical in form, uniform in particle size and high in antibody expression efficiency, and a new technical scheme is provided for a targeting vector system based on the exosome.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and drug delivery, specifically relating to an engineered exosome expressing a complete antibody and its preparation method. Background Technology

[0002] Exosomes are nanoscale vesicles actively secreted by cells. They naturally carry bioactive molecules such as proteins and nucleic acids, exhibiting good biocompatibility and low immunogenicity, making them a promising new carrier for drug delivery, disease diagnosis, and immune modulation. To achieve targeted delivery, exosomes often need to be engineered to display proteins or peptides with targeted recognition functions, such as antibodies and ligands, on their membrane surface.

[0003] Currently, a common engineering strategy involves fusing target molecules (such as single-chain antibodies, nanobodies, or peptides) with certain transmembrane anchoring proteins (such as lysosome-associated membrane protein Lamp2, four-transmembrane protein CD63, or the C1C2 domain of lactobacin), thereby "displaying" these molecules on the exosome surface. However, these general transmembrane regions exhibit significant limitations when faced with structurally complex, large-molecule complete antibodies (i.e., functional full-length antibodies composed of heavy and light chains linked by disulfide bonds): 1) Low expression efficiency: antibodies struggle to integrate efficiently and completely into the exosome membrane; 2) Inaccurate localization: antibodies are prone to misfolding, breakage, or remaining only inside the exosome, failing to stably anchor on the membrane surface; 3) Impaired functional integrity: the antigen-binding function (Fab region) and effector function (Fc region) of the antibody may be lost due to improper fusion or steric hindrance.

[0004] Therefore, existing technologies lack a reliable method for efficiently, stably, and directionally displaying fully functional antibodies on the surface of exosome membranes, which severely limits the development of exosomes as antibody-mediated targeting carriers in high-end applications such as precision medicine and high-sensitivity detection. Summary of the Invention

[0005] This invention provides an engineered exosome expressing a complete antibody and its preparation method. By fusing the target antibody with the membrane-bound transmembrane region (TMD) sequence of a natural antibody and stably producing it in host cells using a lentiviral expression system, engineered exosomes that efficiently and stably display a complete functional antibody on the membrane surface are prepared.

[0006] On the one hand, the present invention provides an engineered exosome expressing a complete antibody, using the following technical solution: An engineered exosome expressing a complete antibody, wherein the membrane surface of the exosome displays a functional complete antibody, which is anchored to the membrane of the exosome via a membrane-binding transmembrane region of the natural antibody; The antibody is a full-length IgG antibody; The nucleotide sequence of the membrane-bound transmembrane region of the natural antibody is SEQ ID NO: 1, or a functional variant thereof having at least 90% sequence identity.

[0007] Preferably, the exosomes have a particle size of 50 nm to 200 nm.

[0008] Preferably, engineered exosomes are obtained by isolating cells after recombinant lentivirus has stabilized.

[0009] Preferably, the recombinant lentivirus is obtained by transfecting packaging cells with a lentiviral expression vector.

[0010] Preferably, the lentiviral expression vector comprises, in sequence, an antibody heavy chain coding sequence, a natural antibody membrane-bound transmembrane region coding sequence, an internal ribosome entry site, and an antibody light chain coding sequence.

[0011] Preferably, the nucleotide sequence encoding the antibody heavy chain is SEQ ID NO: 2; The nucleotide sequence of the internal ribosome entry site is SEQ ID NO: 3; The nucleotide sequence encoding the antibody light chain is SEQ ID NO: 4.

[0012] Preferably, the C-terminus of the antibody light chain coding sequence is connected to a tag sequence coding region; The nucleotide sequence of the tag sequence coding region is SEQ ID NO: 5.

[0013] On the other hand, the present invention provides a method for expressing engineered exosomes with complete antibodies, employing the following technical solution: A method for engineered exosomes expressing complete antibodies includes the following steps: S1. Construct a fusion gene encoding the membrane-binding transmembrane region of the target antibody heavy chain and the natural antibody; S2. The fusion gene is cloned into a lentiviral expression vector to obtain a recombinant vector; S3. Recombinant lentiviruses are produced by transfecting packaging cells with recombinant vectors. S4. Infect host cells with recombinant lentivirus and screen to obtain cell lines that stably express the fusion protein; S5. Culture stable expression cell lines and isolate and purify engineered exosomes with intact antibodies on the surface from the culture supernatant.

[0014] Preferably, the fusion gene constructed in step S1 also includes an internal ribosome entry site and an antibody light chain coding sequence located downstream of the natural antibody membrane-binding transmembrane region coding sequence, forming a tandem expression cassette.

[0015] Preferably, the separation and purification in step S5 includes differential centrifugation, membrane filtration, and size exclusion chromatography performed sequentially.

[0016] In summary, the beneficial effects of the present invention are as follows: This invention achieves, for the first time, the efficient and stable expression of intact antibodies on the exosome membrane surface. By innovatively employing the membrane-bound transmembrane region (TMD) of the natural antibody itself as an anchoring element and combining it with a lentiviral expression system, the technical bottleneck of traditional universal transmembrane regions being unable to adapt to intact large molecular antibodies is successfully solved. This ensures the integrity of the antibody structure, precise localization, and complete functionality, providing exosomes with a powerful specific "navigation" capability.

[0017] Experiments have confirmed that the engineered exosomes prepared in this manner not only maintain the classic morphology and particle size characteristics, but also exhibit significantly higher antibody display efficiency on their membrane surface compared to traditional methods. This fundamentally enhances the active targeting performance of exosomes, greatly expanding their application potential and reliability in biomedical fields such as tumor targeted therapy, precision drug delivery, and high-sensitivity diagnostic detection. Attached Figure Description

[0018] Figure 1 This is a structural diagram of a lentiviral expression vector in the membrane-bound form of an exosome antibody expressing a complete antibody. Figure 2 Figure showing the results of RT-qPCR detection of the expression level of anti-TROP2 gene mRNA in HEK293T and HEK293T-aTROP2 cells; Figure 3 The figure shows the results of flow cytometry analysis using anti-human IgG antibody to detect the expression level of TROP2 antibody on the surface of HEK293T and HEK293T-aTROP2 cells. Figure 4 Figure showing the results of DLS analysis of the hydrated particle size of aTROP2-EVs exosomes extracted from HEK293T-aTROP2 cells; Figure 5 The image shows the results of TEM detection of aTROP2-EVs exosomes extracted from HEK293T-aTROP2 cells; Figure 6 Figure showing the results of Western blot detection of intact antibody and CD63 expression levels of exosomes aTROP2-EVs and Con-EVs extracted from HEK293T cells and HEK293T cells; Figure 7 Image showing the uptake of aTROP2-EVs and Con-EVs by TROP2-positive cells (BxPC-3) and TROP2-negative cells (Panc-1) using immunofluorescence microscopy. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the embodiments.

[0020] Example Example 1 The specific steps for constructing a recombinant lentiviral expression vector expressing a membrane-bound intact antibody are as follows: S1, Gene Design and Synthesis Sequence Design: A tandem expression cassette was designed based on the gene sequence of the anti-TROP2 antibody. 5'-[Antibody heavy chain variable and constant region gene (SEQ ID NO.2)]-[Natural antibody membrane-bound transmembrane region gene (SEQ ID NO.1)]-[Internal ribosome entry site sequence] (SEQ ID NO.3)-[Antibody light chain gene (SEQ ID NO.4)]-[HA tag coding sequence (SEQ ID NO.5)]-3'. Rare restriction enzyme sites (such as NotI) matching the vector were introduced at both ends. The full-length DNA sequence was chemically synthesized by Hunan Fenghui Biotechnology Co., Ltd., and cloned into a universal intermediate plasmid. The delivered product was a high-purity plasmid (named pUC57-HC-TMD-IRES-LC-Flag).

[0021] S2, Enzymatic digestion preparation of vector and insert fragment Take approximately 5 μg of the lentiviral backbone vector pLV-CMV-MCS-EF1-Hygro plasmid.

[0022] Establish the enzyme digestion reaction system (50 μL): plasmid DNA 5 μg, 10×FastDigest Green Buffer 5 μL, NotI restriction endonuclease 1 μL, and sterile deionized water to make up to 50 μL.

[0023] After mixing, place in a 37°C water bath or metal bath and react for 2 hours.

[0024] Take about 3 μg of intermediate plasmid pUC57-HC-TMD-IRES-LC-Flag and establish the same NotI digestion system (50 μL) as in step 1. React at 37℃ for 2 hours.

[0025] Perform 1% agarose gel electrophoresis on a small amount of the enzyme digestion product for verification. The vector should show a band migration change from circular to linear; the insert should release a single target band of the expected size. Using an agarose gel DNA recovery kit, separately excise the gel to recover the linearized vector fragment and the target gene fragment, and dissolve them in 30 μL of elution buffer. Determine the DNA concentration and purity using Nanodrop.

[0026] S4, Connection Reaction Calculate the molar ratio of the vector to the insert based on the length of the purified vector (approximately 8-9 kb) and the length of the insert fragment (approximately 2-3 kb).

[0027] Establish the ligation system (20 μL): 2 μL linearized vector, 6 μL target gene fragment, 1 μL T4 DNA ligase, 2 μL 10×T4 DNA ligase buffer, and sterile deionized water to a final volume of 20 μL.

[0028] After mixing, briefly centrifuge. Incubate at 16°C in a water bath or PCR instrument for 12-16 h for ligation. Simultaneously set up a self-ligation control with only the vector (without the insert fragment).

[0029] S4, Conversion and Plate Screening Remove E. coli DH5α competent cells from the -80℃ freezer and thaw them on ice. Add 5-10 μL of ligation product to 50 μL of competent cells, mix gently, and incubate on ice for 30 min. Then heat shock at 42℃ for 45-60 s, and immediately transfer to ice to cool for 2 min.

[0030] Add 500 μL of antibiotic-free LB liquid medium and incubate with shaking for 1 h (37℃, 220 rpm). After centrifuging and concentrating the revived bacterial culture, spread it evenly onto LB solid plates containing ampicillin. Invert the plates and incubate at 37℃ for 12–16 h.

[0031] S5. Positive clone identification Five well-grown single colonies were randomly picked from the plate using a sterile toothpick and inoculated into LB liquid medium containing ampicillin resistance. The colonies were then incubated at 37°C with shaking for 8-12 hours. Using the bacterial culture as a template, PCR amplification was performed using primers specific to the target insert fragment.

[0032] Establish the PCR reaction system (25 μL): DNA 2 μL, forward primer 1 μL, reverse primer 1 μL, 2×Taq PCR premix (containing staining agent) 12.5 μL, enzyme-free water 8.5 μL.

[0033] PCR reaction procedure: Perform agarose gel electrophoresis on the PCR products. Colonies showing a specific band at the size and position of the insert fragment are considered initial positive clones. Select 2-3 PCR-positive clones and culture them overnight. Extract plasmids using a plasmid miniprep kit. Verify the extracted plasmids by single-restriction digestion with NotI. A correct recombinant plasmid should yield two bands: a vector backbone band and a target insert fragment band. Send the plasmid samples that have been verified by restriction enzyme digestion to BGI Genomics for full-length sequencing. Sequencing primers should include the universal sequencing primers CMV-F (CMV promoter forward primer) / EF1-R (EF1 promoter reverse primer) on the vector, as well as internal specific primers to ensure coverage of the entire expression cassette.

[0034] The sequencing results were compared with the expected design sequence using the sequence alignment software SnapGene. When the sequence identity reached 100% and there were no frameshifts, mutations, or deletions, the recombinant lentiviral expression vector pLV-HC-TMD-IRES-LC was confirmed to have been successfully constructed.

[0035] like Figure 1 The diagram shown is a structural diagram of a lentiviral expression vector in the form of an exosomal antibody membrane binding to express a complete antibody.

[0036] Example 2 The specific steps for lentiviral expression, packaging, and transfection of exosome membrane-bound intact antibodies are as follows: S1, HEK293T Packaging Cell Preparation Remove the frozen HEK293T cells from the liquid nitrogen tank and thaw them rapidly in a 37°C water bath. Transfer the cell suspension to a 15 mL centrifuge tube containing 10 mL of preheated complete medium (DMEM high glucose + 10% high-quality fetal bovine serum + 1% penicillin antibiotics), and gently mix. Centrifuge (800 rpm, 5 min), discard the supernatant, resuspend the cells in 5 mL of complete medium, and seed them into T25 culture flasks. Incubate at 37°C in a 5% CO2 incubator, observing and changing the medium every other day.

[0037] When cell confluence reaches 80%-90%, perform digestion and passage, discard the old culture medium, and wash once with PBS. Add 1 mL of 0.25% trypsin (containing EDTA) and digest at 37°C for 1 min. When cells are observed to become rounded and intercellular spaces increase under a microscope, immediately add 2 mL of complete culture medium to stop digestion, and gently pipette to form a single-cell suspension. Perform cell counting. (The cell count is 2 × 10⁶ cells / year.) 6 At a density of 1,000 cells, seed the cell suspension into 10 cm cell culture dishes and add complete culture medium to a total volume of 10 mL. Gently shake to distribute the cells evenly, and return to the incubator to culture for 48-72 hours, until the cell confluence reaches 70%-80% at the time of transfection.

[0038] S2, three-plasmid co-transfection Using packaging plasmid pMD2.G (expressing VSV-G envelope glycoprotein) and packaging plasmid psPAX2 (expressing structural proteins such as gag / pol), Example 1 constructed the target gene plasmid pLV-HC-TMD-IRES-LC carrying the antibody-TMD gene to be packaged. The total plasmid mass was 20 μg, with a target plasmid:psPAX2:pMD2.G mass ratio of 4:3:1. That is: pLV-HC-TMD-IRES-LC 10 μg, psPAX2 7.5 μg, pMD2.G 2.5 μg.

[0039] Take a sterile 1.5 mL centrifuge tube and add the calculated weights of the three plasmids to 500 μL of serum-free DMEM medium. Mix gently and label this solution A. In another centrifuge tube, according to the PEI reagent instructions, add the appropriate volume of PEI to 500 μL of serum-free DMEM medium. Mix gently and incubate at room temperature for 5 minutes. Label this solution B. Slowly add solution B dropwise to solution A, gently mixing with a pipette as you add. Incubate at room temperature for 5-10 minutes to form the DNA-PEI transfection complex.

[0040] Remove the culture dish and discard the old culture medium. Gently wash the cells twice with 5 mL of PBS to remove residual serum. Add 5 mL of fresh serum-free DMEM medium. Add 1 mL of the prepared transfection complex dropwise and evenly to the culture dish, gently shaking to mix. Return the culture dish to a 37°C, 5% CO2 incubator and incubate for 6-8 hours.

[0041] S3, Fluid Change and Virus Collection Six to eight hours after transfection, carefully aspirate the culture medium containing the transfection complex. Add 10 mL of fresh maintenance medium (DMEM containing 2% fetal bovine serum).

[0042] After changing the medium, continue culturing for 48 hours. Collect the cell supernatant into a 50 mL sterile centrifuge tube using a sterile pipette and temporarily store it at 4°C. Add 10 mL of fresh maintenance medium to the culture dish and continue culturing for 24 hours. Collect the second supernatant and combine it with the first supernatant collection.

[0043] S4. Clarification and Concentration of Virus Supernatant Centrifuge the combined viral supernatant at 4°C and 3000 rpm for 10 min. Carefully aspirate the supernatant and filter it through a 0.45 μm PES membrane filter to obtain a clear viral solution. Select an ultrafiltration concentration tube with an appropriate molecular weight cutoff. After pre-equilibrating the concentration tube with PBS, add the clear viral supernatant. Centrifuge at 4°C and 4000 g for 30 min until the volume of the concentrate is reduced to 1 mL.

[0044] Invert the concentration tube and place it into a new collection tube. Centrifuge at 4°C and 1000 g for 2 minutes to collect the concentrated virus stock solution. Aliquot the virus stock solution and store it immediately at -80°C. Record the initial supernatant volume and the final concentration volume for subsequent titer conversion.

[0045] S5, Virus titer determination HEK293T cells in the logarithmic growth phase were digested and counted at a ratio of 1×10⁻⁶. 4 Seeds were placed at a density of 100 μL / well into 96-well plates and cultured overnight to allow cell adhesion. The concentrated virus stock solution was serially diluted 10-fold with complete culture medium, each subsequent 10-fold dilution... 3 10 4 10 5 10 6 10 7 times.

[0046] Discard the old culture medium from the 96-well plate, and set up three replicates for each dilution. Add 100 μL of virus solution at different dilutions to each well. Also set up an uninfected control group (culture medium only). Incubate in an incubator. 72 hours after infection, replace the culture medium in all wells (including the control) with complete culture medium containing 200 μg / mL hygromycin for screening. Continue incubation for 7–14 days, changing the drug-containing medium every 2–3 days. During this period, all cells in the control group should die. Observe each well under a microscope and count the number of wells containing positive cell colonies (surviving and proliferating cell clusters).

[0047] For the most accurate counting, choose a dilution with 10-100 positive cell colonies per well.

[0048] Virus titer (TU / mL) = (number of positive wells × number of cells inoculated × dilution factor) / (virus volume (mL) × number of wells).

[0049] The final titer is the geometric mean of the calculated values ​​of each effective dilution, as shown in Table 1.

[0050] Table 1 Lentiviral titer determination

[0051] Example 3 S1. Preparation and culture of stable transfected cell lines Forty-eight hours after lentiviral infection of HEK293T cells, the virus-containing culture medium was discarded. Complete culture medium containing 500 μg / mL hygromycin was added, and the medium was changed every 2-3 days. Screening continued for 7-14 days until all cells in the uninfected control group died, while the infected group showed obvious resistant cell colonies.

[0052] The surviving resistant cells were digested and collected, then seeded into new culture dishes and maintained in culture medium containing an appropriate concentration of hygromycin, serving as the experimental group. Simultaneously, uninfected wild-type HEK293T cells were cultured as the control group. Once both groups of cells reached the logarithmic growth phase (approximately 80-90% confluence), they were used for subsequent experiments.

[0053] S2. Extraction of total RNA from cells Discard the culture medium and gently wash the cells twice with pre-cooled PBS. Add 1 mL of TRIzolReagent to each culture dish and incubate at room temperature for 5 minutes to allow for complete cell lysis. Transfer the lysate to RNase-free 1.5 mL centrifuge tubes by pipetting. Add 0.2 mL of chloroform to the lysate, tighten the cap, vortex vigorously for 15 seconds, and incubate at room temperature for 2 minutes. Centrifuge at 12,000 g for 15 minutes at 4°C. Sample separation: bottom layer red phenol-chloroform phase, intermediate phase, and top colorless aqueous phase (containing RNA).

[0054] Carefully aspirate the upper aqueous phase (approximately 400-500 μL) into a new RNase-free centrifuge tube, being careful not to aspirate the middle layer. Add an equal volume of isopropanol, gently invert to mix, and incubate at room temperature for 10 minutes. Centrifuge at 12,000 g for 10 minutes at 4°C; a white RNA precipitate should be visible at the bottom of the tube.

[0055] Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) to wash the precipitate, and vortex to mix. Centrifuge at 7500 g for 5 minutes at 4°C, and carefully discard the ethanol. Let the precipitate dry at room temperature for 5 minutes without the cap open. Add 20-50 μL of RNase-free water to dissolve the RNA precipitate, and incubate at 60°C for 5 minutes.

[0056] S3. RNA quality detection and concentration determination Use a Nanodrop 2000 micro-volume nucleic acid analyzer. Take 1 μL of RNA solution and measure its absorbance at 260 nm and 280 nm.

[0057] RNA concentration (ng / μL) = A260 × 40 × dilution factor.

[0058] The A260 / A280 ratio should be between 1.8 and 2.0, indicating less protein contamination.

[0059] Take 2 μL of RNA sample and perform 1% agarose gel electrophoresis. The 28S and 18S rRNA bands should be clearly visible.

[0060] S4. Removal of genomic DNA and reverse transcription to synthesize cDNA Genomic DNA removal (using gDNA Eraser): Prepare the following reaction mixture (10 μL) on ice: 1 μg total RNA, 2 μL 5× gDNA Eraser Buffer, 1 μL gDNA Eraser, and 6 μL RNase-Free dH2O. Mix well and briefly centrifuge. Incubate at 42°C for 2 minutes. Then immediately place on ice and label this solution C.

[0061] Add 10 μL of solution C, 4 μL of 5× PrimeScript Buffer 2, 1 μL of PrimeScript RTEnzyme Mix I, 1 μL of RT Primer Mix, and 4 μL of RNase-Free dH2O to the reaction tube. Mix well and centrifuge briefly. Perform reverse transcription according to the following program: 37°C, 15 min → 85°C, 5 s → 4°C. The obtained cDNA can be used immediately for qPCR or stored at -20°C.

[0062] S5, Real-time Quantitative PCR Primer preparation: Use the primers designed in Table 2. Target gene (IRES): h-IRES-145-F / h-IRES-145-R; Internal reference gene (GAPDH): h-GAPDH-203-F / h-GAPDH-203-R; Dilute the primers to a working concentration of 10 μM with sterile deionized water.

[0063] Prepare qPCR reaction systems on ice in 96-well plates with 3 replicates per sample per gene: 10 μL of 2× SYBRPremix Ex Taq, 0.8 μL of forward primer (10 μM), 0.8 μL of reverse primer (10 μM), 2 μL of cDNA template, and 6.4 μL of RNase-free dH2O.

[0064] Set up a template-free control (NTC) using water instead of cDNA template to test for reagent contamination; set up a reverse transcription-free control (-RT) using RNA samples that have not undergone reverse transcription as templates to test for residual genomic DNA.

[0065] qPCR program settings: Pre-denaturation: 95℃, 30 seconds. Denaturation: 95℃, 5 seconds; Annealing / Extension / Fluorescence signal acquisition: 60℃, 30 seconds, 40 cycles.

[0066] Melting curve analysis: 95℃, 15 s; 60℃, 60 s. The temperature was slowly increased to 95℃, and fluorescence signals were collected every 0.3℃.

[0067] Table 2 Primers for target gene and internal reference gene

[0068] S6, Data Analysis The analysis software automatically generates amplification and melting curves. The melting curve is confirmed to be a single peak, indicating no nonspecific amplification. The Ct value (cycle threshold) for each reaction well is recorded.

[0069] Relative quantitative analysis was performed using the ΔΔCt method: Calculate the Ct difference between the target gene and the internal reference gene for each sample: ΔCt=Ct(IRES)-Ct(GAPDH) Calculate the ΔΔCt value of the experimental group relative to the control group: ΔCt = ΔCt(experimental group) - ΔCt(control group) Calculate the relative expression level of the target gene mRNA in the experimental group compared to the control group: 2^(-ΔΔCt) The relative expression level of the control group was set to 1, and the fold change in the experimental group was calculated. A t-test was performed using statistical software to compare the differences in ΔCt values ​​between the experimental and control groups.

[0070] like Figure 2 As shown, if the relative expression level of IRES in the HEK293T-aTROP2 group is significantly higher than that in the control group, it confirms that the recombinant lentivirus containing IRES has been successfully integrated into the cell genome and transcribed efficiently.

[0071] Example 4 The flow cytometry detection of surface antibody expression in stably transfected cell lines follows these steps: S1, Cell Preparation HEK293T-aTROP2 stable cell line (experimental group) and wild-type HEK293T cells (negative control group) were cultured to the logarithmic growth phase. The culture medium in the culture dishes was aspirated. The cells were gently washed twice with pre-chilled PBS to remove residual serum. 1 mL of 0.25% EDTA-trypsin was added to digest the cells, and the mixture was incubated at 37°C for approximately 2 min. 2 mL of complete culture medium containing serum was added to terminate the digestion. The cells were gently pipetted to prepare a single-cell suspension. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 300 g for 5 min at 4°C. The supernatant was carefully discarded, and the cells were resuspended in pre-chilled PBS (containing 1% BSA), and washed again by centrifugation. The cell pellet was resuspended in an appropriate amount of PBS, and a small amount was counted using a cell counting chamber or automated cell counter. Based on the counting results, the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 6 Cells / mL. Prepare at least 5 × 10⁶ cells / mL for each sample (experimental group and control group). 5 One cell was used for subsequent staining.

[0072] S2, cell surface antibody staining Add 100 μL PBS (containing 1% BSA) to the cell pellet for resuspending and incubate at room temperature for 15 minutes to block non-specific binding sites.

[0073] After incubation, without washing, add the fluorescently labeled primary antibody (FITC-labeled anti-human IgG (Fc-specific) monoclonal antibody) directly to the target concentration of 10 μg / mL.

[0074] Take 50 μL of cell suspension (approximately 5 × 10⁻⁶ cells) 5 Add an appropriate amount of FITC-anti-human IgG antibody to each cell, and bring the total volume to 100 μL with PBS (containing 1% BSA). Ensure the final antibody concentration is within the working range.

[0075] Experimental group: HEK293T-aTROP2 cells + FITC-anti-human IgG antibody; Isotype control / negative control group: HEK293T-aTROP2 cells + FITC-labeled isotype-independent antibody, used to set background fluorescence and gating; Blank cell control: wild-type HEK293T cells + FITC-anti-human IgG antibody, used to confirm antibody specificity. After gently mixing each tube, incubate at 4°C in the dark for 30 min.

[0076] After incubation, add 2 mL of pre-chilled PBS (containing 1% BSA) to each tube and gently mix. Centrifuge at 300 g for 5 minutes at 4°C, and carefully discard the supernatant. Repeat this washing step 2-3 times to ensure complete removal of unbound fluorescent antibody.

[0077] After washing, resuspend the cells in 300-500 μL of pre-chilled PBS (containing 1% BSA). Add 100 μL of 1%-4% paraformaldehyde or cell fixative, fix at 4°C in the dark for 15 minutes, then wash once with PBS and resuspend in PBS (containing 1% BSA). Filter the cell suspension through a 200-mesh or 40 μm cell filter to remove cell clumps that may clog the instrument.

[0078] S3, flow cytometry detection Power on the instrument (Thermo Fisher Attune NxT) according to the standard operating procedure, run the quality control microspheres (CST beads) to adjust the instrument, ensuring stable fluid flow, normal laser power, and fluorescence signal within the standard range. Load samples for analysis in the following order: blank cell control → isotype control → experimental group samples. Collect at least 10,000 live cell events (based on cells within the FSC / SSC gate) for each sample, and save all FCS data files.

[0079] S4. Data Analysis and Results Interpretation On the FSC-A / SSC-A scatter plot, circle the main single-cell populations, excluding debris and cell clumps. On the FSC-A / FSC-H scatter plot, circle the single cells from P1, excluding adherent cells.

[0080] On the FITC fluorescence histogram of the isotype control samples, a positive / negative cutoff marker (Marker M1) was set with 99% of cells considered negative. The same M1 position was applied to the histograms of the experimental group and all control groups.

[0081] The percentage of cells to the right of gate M1 (where fluorescence is stronger) in the experimental group sample is the antibody-positive cell rate. Record all data. Results are typically expressed as mean ± standard deviation (SD). Using statistical software, compare the positivity rates of the experimental group and the negative control group using an unpaired t-test.

[0082] like Figure 3 As shown, the antibody positivity rate of the HEK293T-aTROP2 group (85.2%) was significantly higher than the background value of the control group (2.1%), which strongly confirms that the exosome membrane-bound intact antibody was successfully expressed and localized on the cell membrane in the transfected cells.

[0083] Example 5 The extraction, concentration, and physical characterization of engineered exosomes (aTROP2-EVs) are carried out through the following steps: S1. Production and initial collection of engineered exosomes Take a well-grown, stable HEK293T-aTROP2 cell line and culture it routinely in complete medium containing hygromycin. When the cell confluence reaches 50%-60%, discard the original medium. Gently wash the cells twice with pre-warmed PBS to thoroughly remove residual fetal bovine serum. Add serum-free DMEM medium. Return the cells to a 37°C, 5% CO2 incubator and continue culturing for 48 hours. During this period, the cells will secrete exosomes into the medium.

[0084] S4. Preparation of crude exosome extract After 48 hours of incubation, transfer all cell culture supernatant to a 50 mL conical centrifuge tube using a sterile pipette. Proceed immediately with further processing to prevent vesicle degradation or aggregation. Centrifuge the supernatant at 500 g for 10 minutes at 4°C. Carefully transfer the supernatant to a new centrifuge tube, discarding the precipitate at the bottom. Centrifuge the supernatant at 2,000 g for 15 minutes at 4°C. Carefully transfer the supernatant to a new tube again, discarding the precipitate. Filter the supernatant through a 0.22 μm PES membrane filter using a syringe to further remove any remaining micron-sized particles and microorganisms, yielding a clear, cell-free supernatant.

[0085] S3. Exosome Concentration and Purification To reduce the loading volume of subsequent purification steps, ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 kDa can be used for concentration. Add the clarified supernatant to the pre-equilibrated ultrafiltration tube and centrifuge at 4°C, 4000 g for 30 min until the volume is concentrated to 1-2 mL. Use a commercially available exosome purification SEC column (Enzekangtai Company). Equilibrate the column thoroughly with 1×PBS (filtered sterile) until the eluent reaches the specified volume. Accurately load the concentrated sample (no more than 2% of the column volume) and elute isocratically with PBS. Collect the eluent in fixed volumes (e.g., 0.5 mL / tube) according to the instructions or preliminary experiments, and combine the exosome-rich fractions.

[0086] S4. Exosome Concentration and Storage The combined SEC fractions were concentrated again to a final volume of 100-200 μL using new 100 kDa ultrafiltration centrifuge tubes at 4°C and 4,000 g, yielding a high-purity aTROP2-EVs concentrate. The concentrate was immediately aliquoted (e.g., 20 μL per tube) and stored long-term at -80°C. Samples were then sent to CAS Pharma for dynamic light scattering (DLS) particle size analysis and electron microscopy observation.

[0087] like Figure 4 As shown, the main peak of the hydration kinetics of aTROP2-EVs should be located at about 200 nm and have a narrow distribution, which is consistent with the typical characteristics of exosomes.

[0088] like Figure 5 As shown, aTROP2-EVs should exhibit a typical "crescent disk" morphology under an electron microscope, with a diameter of about 100 nm, clear boundaries, good dispersion, and no obvious aggregation or breakage.

[0089] Example 6 The specific steps for identifying the target protein and exosome marker proteins in engineered exosomes are as follows: S1. Sample, Reagent and Equipment Preparation Experimental group: aTROP2-EVs (concentrated solution prepared in Example 5); negative control group: exosomes derived from wild-type HEK293T cells; positive control group: cell lysates expressing HA-tagged proteins. All samples were stored at -80°C and thawed on ice before loading.

[0090] Take an appropriate amount of sample and lyse it thoroughly for 30 minutes (on ice) with RIPA lysis buffer (containing protease inhibitor). Centrifuge at 12,000 g for 15 minutes at 4°C, and collect the supernatant for assay. Follow the instructions of the BCA protein assay kit to plot a standard curve. Measure the protein concentration of each sample, adjusting all samples to the same concentration with 1×PBS or RIPA lysis buffer, and record the concentration and volume. Based on the measured concentration, take a sample volume equivalent to 10-30 μg of total protein and add an equal volume of 2× loading buffer (containing SDS and β-mercaptoethanol). After mixing, heat in a metal bath or boiling water bath at 95°C for 5-10 minutes to fully denature the protein. After brief centrifugation, place on ice for later use.

[0091] S2, SDS-polyacrylamide gel electrophoresis Prepare a 12% separating gel (suitable for 10-70 kDa proteins, CD63 approximately 53 kDa, antibody light chains approximately 25 kDa, and heavy chains approximately 50 kDa) and a 5% stacking gel. Pour the gels, carefully insert the comb, and polymerize at room temperature for 30-60 minutes.

[0092] Mount the gel plate into the electrophoresis tank and add 1×Tris-Glycine-SDS electrophoresis buffer. Gently remove the comb. Add the following to the sample wells in sequence: pre-stained protein molecular weight standard marker, experimental group sample, negative control sample, and positive control. Connect the power supply and use a constant voltage of 80-100 V during the stacking gel stage. After the samples enter the separating gel, switch to a constant voltage of 120-150 V and continue electrophoresis. According to the pre-stained marker indication, stop electrophoresis once the target protein bands (target antibody bands are approximately 25 kDa and 50 kDa, CD63 is approximately 53 kDa) are fully separated.

[0093] S3, Protein Transfer Prepare the PVDF membrane by immersing it in methanol for 15 seconds to activate it, then equilibrate it with filter paper and a sponge in 1×TransferBuffer for 10-15 minutes. Assemble the membrane in the transfer holder in the following order from cathode (black) to anode (white): sponge → 3 layers of filter paper → gel → PVDF membrane → 3 layers of filter paper → sponge. Ensure there are no air bubbles between the layers and that the gel is in close contact with the membrane. Air bubbles can be gently removed by rolling with a glass rod.

[0094] Place the transfer clamp into the transfer tank, add pre-cooled transfer buffer, and cool the entire device in an ice bath or with a cold circulation system. Turn on the power, using a constant current of 300 mA, for a transfer time of 90 minutes. The transfer process generates heat, so a low temperature must be maintained.

[0095] S4, Immune Detection After the transfer is complete, remove the PVDF membrane, rinse it quickly once with 1×TBST buffer, and immerse the membrane in 5% (w / v) skim milk (prepared with TBST) or 5% BSA (usually used for phosphorylated antibodies, but can also be used here) blocking solution. Shake gently on a shaker at room temperature for 2 hours (or overnight at 4°C for better results).

[0096] For the detection of the target protein, anti-HA-tagged mouse monoclonal antibody (because the antibody's light chain C-terminus has an HA tag) was diluted with TBST containing 2% BSA at the manufacturer's recommended ratio (usually 1:1000 to 1:5000). For the detection of exosome marker proteins, anti-CD63 mouse monoclonal antibody was diluted at the manufacturer's recommended ratio (usually 1:1000). The blocked membrane was quickly washed once with TBST and placed in an incubator containing the primary antibody dilution buffer. Incubate overnight at 4°C (or at room temperature for 2 hours), gently shaking on a shaker. After incubation, the primary antibody was recovered. The PVDF membrane was washed with TBST buffer for 10 minutes each time, gently shaking on a shaker, for a total of 3 washes.

[0097] Prepare the appropriate HRP (horseradish peroxidase)-labeled goat anti-mouse IgG secondary antibody, diluted with TBST containing 2% skim milk at a ratio of 1:5000 to 1:10000. Immerse the membrane in the secondary antibody dilution solution and incubate at room temperature for 1 hour, gently shaking on a shaker. After incubation, wash the membrane three times with TBST as described above, 10 minutes each time, to completely remove unbound secondary antibody.

[0098] S5, Chemiluminescence Developing and Imaging Prepare the ECL working solution according to the chemiluminescence detection kit instructions. Mix equal volumes of solutions A and B, prepare fresh, and store in the dark. Blot away excess TBST from the membrane with filter paper (do not allow the membrane to dry). Evenly drop the mixed ECL working solution onto the protein side of the membrane and incubate at room temperature for 1-3 minutes. Transfer the membrane to the sample plate of the imaging system using tweezers. Acquire images using the chemiluminescence imaging system. Image the HA and CD63 signals on the same membrane separately.

[0099] S6. Results Analysis and Conclusions Target protein verification: In the aTROP2-EVs sample lanes, a specific band should be detected by the anti-HA antibody, and its position should correspond to the expected molecular weight of the antibody heavy chain (~50 kDa) and / or light chain (~25 kDa). This band should be absent (or very weak) in the Con-EVs lanes. Exosome property verification: In both the aTROP2-EVs and Con-EVs lanes, a characteristic band of approximately 53 kDa should be detected by the anti-CD63 antibody, confirming that the extracted vesicles possess the basic molecular characteristics of exosomes.

[0100] The effectiveness of the HA antibody was verified by a positive control, the correct molecular weight of the band was verified by a marker, and non-specific binding was excluded by a negative control (Con-EVs).

[0101] like Figure 6 As shown, the Western Blot results satisfy both of the above conditions, strongly demonstrating that the target antibody (TROP2 antibody) has been successfully integrated and expressed on engineered exosomes (aTROP2-EVs), and the prepared aTROP2-EVs possess the molecular properties of classical exosomes.

[0102] Example 7 Targeted uptake of engineered exosomes by cells Exosome staining procedure: 40 μg of Con-EVs and aTROP2-EVs were resuspended in 1*PBS, with a total volume of 50 μL. 2 μL of Fluorescenece Dye Red (Shanghai Huaying Biomedical Technology Co., Ltd.) was added to 48 μL of buffer to obtain 50 μL of staining working solution. The exosome suspension and staining working solution were mixed at a 1:1 volume ratio and incubated at room temperature in the dark for 5 min. 100 μL of the completed fluorescent labeling reaction solution was slowly added to the top of the fluorescent adsorption column (Shanghai Huaying). The column was centrifuged at 1000 g for 4 min at room temperature to obtain 100 μL of purified fluorescently labeled exosomes Con-EVs and aTROP2-EVs.

[0103] TROP2-positive cells (BxPC-3) and TROP2-negative cells (PANC-1) were seeded at 20,000 per well in 96-well plates. After 24 hours of adhesion, the medium was replaced with exosome-free medium, and 5 μg of Fluorescenece DyeRed-stained Con-EVs and aTROP2-EVs were added to each well. The cells were cultured at 37°C and 5% CO2 for 8 hours. The old medium was then discarded, and the cells were washed three times with PBS. 80 μL of 1 μg / mL Hoechst 33342 was added to each well for staining for 15 minutes, after which the staining was discarded. The cells were washed three times with PBS to remove free dye, and then 100 μL of PBS buffer was added. The cells were then photographed using an Agilent BioTek Lionheart imaging system.

[0104] like Figure 7As shown, in TROP2-positive BxPC-3 cells, a large amount of uptake of aTROP2-EVs was observed, while the uptake of Con-EVs was extremely low; however, in TROP2-negative PANC-1 cells, no significant cell uptake was detected in either the aTROP2-EVs group or the Con-EVs group. These results indicate that aTROP2-EVs have significant targeting ability for TROP2-highly expressing cells.

[0105] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An engineered exosome expressing a complete antibody, characterized in that, The membrane surface of the exosome displays a functionally intact antibody, which is anchored to the membrane of the exosome by a native antibody membrane-bound transmembrane region; The antibody is a full-length IgG antibody. The nucleotide sequence of the native antibody membrane-bound transmembrane region is SEQ ID NO: 1, or a functional variant having at least 90% sequence identity thereto.

2. The engineered exosome expressing a whole antibody according to claim 1, characterized in that, The particle size of the exosome is 50 nm to 200 nm.

3. The engineered exosome expressing a complete antibody of claim 1, wherein, The engineered exosome is obtained by isolating after stable transfection of a recombinant lentivirus.

4. The engineered exosome expressing a whole antibody according to claim 3, characterized in that, The recombinant lentivirus is obtained by transfecting packaging cells with a lentiviral expression vector.

5. The engineered exosome expressing a whole antibody according to claim 4, characterized in that, The lentiviral expression vector comprises, in sequence, an antibody heavy chain coding sequence, a native antibody membrane-bound transmembrane region coding sequence, an internal ribosome entry site, and an antibody light chain coding sequence.

6. The engineered exosome expressing a whole antibody according to claim 5, characterized in that, The nucleotide sequence of the antibody heavy chain coding sequence is SEQ ID NO: 2; The nucleotide sequence of the internal ribosome entry site is SEQ ID NO: 3; The nucleotide sequence of the antibody light chain coding sequence is SEQ ID NO:

4.

7. The engineered exosome expressing a whole antibody according to claim 5, wherein, The C-terminal of the antibody light chain coding sequence is connected with a tag sequence coding region; The nucleotide sequence of the tag sequence coding region is SEQ ID NO:

5.

8. A method of making the engineered exosome expressing a complete antibody of any one of claims 1-7, characterized in that, The steps include: S1, constructing a fusion gene encoding a target antibody heavy chain and a native antibody membrane-bound transmembrane region; S2, cloning the fusion gene into a lentiviral expression vector to obtain a recombinant vector; S3, transfecting packaging cells with the recombinant vector to produce a recombinant lentivirus; S4, infecting host cells with the recombinant lentivirus to obtain a cell strain stably expressing the fusion protein; S5, culturing the stable expression cell strain to isolate and purify engineered exosomes displaying the intact antibody on the surface from the culture supernatant.

9. The method of claim 8, wherein, The fusion gene constructed in step S1 further comprises an internal ribosome entry site and an antibody light chain coding sequence downstream of the native antibody membrane-bound transmembrane region coding sequence, forming a tandem expression cassette.

10. The method of claim 8, wherein, The isolation and purification in step S5 includes differential centrifugation, membrane filtration, and size exclusion chromatography steps in sequence.

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