Exosome modification methods

Abstract

The present invention relates to the field of bioengineering, and discloses a fusion protein connecting exosomes and targets and its application in constructing and / or transforming exosomes, as well as a method for transforming exosomes. The fusion protein is a protein αCD9-x-αFc, whose sequence is shown in SEQ ID NO.2; a method for constructing and / or transforming exosomes, which includes using the fusion protein to connect the target to the exosome. Using the fusion protein, by constructing or transforming exosomes, different targeted targets can be efficiently displayed on the surface of the exosomes, thereby enhancing the targeted delivery effect of the exosomes.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a linker molecule for connecting exosomes and antibodies and an application thereof, as well as a method for modifying exosomes. Background Art

[0002] Exosomes are membrane vesicles that are released into the extracellular matrix after the fusion of multivesicular bodies (MVBs) with cell membranes. They are about 30-100 nanometers in diameter. They were first discovered in the early 1980s and named "exosomes" in 1987. Exosomes exist in body fluids such as blood, saliva, urine, and breast milk. They can reach other cells and tissues through the circulatory system and participate in long-distance signal transmission. Exosomes play an important role in intercellular communication, and have a key role in transmitting information and regulating cell function, providing a new perspective for disease diagnosis, treatment, and drug delivery.

[0003] Exosomes have broad application prospects in the medical field, mainly including disease diagnosis, disease treatment and drug delivery. Its widespread presence and stability in body fluids make it an ideal tool for disease diagnosis, and it can provide a window into the disease state by analyzing nucleic acids, proteins and other substances in exosomes. Exosomes also play an important role in disease treatment. Modifying exosomes or inhibiting their production can effectively promote the treatment of diseases. As a drug delivery carrier, exosomes are widely used to deliver proteins, nucleic acids and small molecule drugs due to their low toxicity, low immunogenicity and good biomembrane penetration.

[0004] In order to identify and analyze exosomes, scientists often rely on their specific markers. These specific markers not only help distinguish exosomes from other extracellular vesicles, but also provide important information about the cell type and biological function of the exosomes. Exosome markers are mainly divided into the following categories:

[0005] Membrane proteins: such as tetraspanins (CD9, CD63, CD81), etc. These proteins are widely present on the membrane of exosomes and are the most commonly used exosome markers.

[0006] Membrane trafficking and fusion proteins: such as the Rabs protein family (Rab27a, Rab5, Rab11), these small GTPases regulate the formation and secretion of exosomes.

[0007] Tight junction proteins: such as TSG101 and ALIX, these proteins are involved in the important process of exosome formation.

[0008] Internal proteins: such as heat shock proteins (HSP70, HSP90), which are usually present inside exosomes or attached to the surface of exosomes, reflecting the state of cell stress.

[0009] Nucleic acids: Exosomes contain a variety of microRNAs (miR-21, miR-150), mRNA, lncRNA, etc. These nucleic acids regulate gene expression in recipient cells.

[0010] Lipids: Such as sphingomyelin, phosphatidic acid ceramide and cholesterol, these lipids constitute the membrane structure of exosomes and play a role in their function.

[0011] CD9 is a tetraspanin protein that has an important influence on the biological functions of exosomes. During the formation and release of exosomes, CD9 helps regulate the generation of exosomes by promoting the fusion of multivesicular bodies with cell membranes. It is also involved in regulating the loading of specific molecules in exosomes and affecting the abundance of miRNAs and proteins in exosomes. This regulation may be carried out through direct interactions or affecting related cellular pathways. In addition, CD9-enriched exosomes play a specific role in intercellular communication and can specifically interact with other cells, transmit signals or regulate cell behavior. Therefore, CD9 is an important marker of exosomes.

[0012] In order to achieve the functional diversification of exosomes, engineering modification is a key approach, and its main goal is to achieve targeting and drug loading. In order to achieve targeted delivery of exosomes, scientists have adopted a variety of engineering modification strategies. For example, gene editing technology is used to modify the exosome skeleton protein to display specific targeting molecules, such as LAMP-2B protein, and achieve targeting by specifically binding to target cells. In addition, by displaying specific targeting peptides on the surface of exosomes, such as iRGD peptides, exosomes can specifically deliver drugs to tumor cells. Artificial synthesis of exosomes is also a new attempt to construct exosomes with specific targeting through de novo synthesis to more effectively deliver drugs.

[0013] The drug loading methods of exosomes mainly include exogenous loading and endogenous loading. Exogenous loading is to actively or passively load the drug into the exosomes through electroporation, ultrasound, extrusion, freeze-thaw and other methods after isolating and purifying the exosomes. These methods can maintain the morphology of the exosomes, but the loading efficiency may be low. Endogenous loading is to genetically modify the cells that produce exosomes to produce the required drugs at the same time, so that the drugs are naturally contained in the secreted exosomes. This method can ensure the effectiveness and stability of the drug. In addition, exosomes can also be used to deliver proteins, nucleic acids and small molecule drugs, showing excellent results.

[0014] Therefore, new and more efficient methods for constructing or modifying exosomes are needed to achieve therapeutic uses and other applications based on exosome targeting technology. Summary of the invention

[0015] All the existing technical means to achieve exosome targeting require the help of genetic modification, and each design requires the fusion expression of the targeting molecule and the display skeleton molecule. This process consumes a lot of time and cost. In this patent, the purpose of targeted modification of different exosomes is achieved through the expression of a fusion protein (αCD9-x-αFc). αCD9-x-αFc can bind to the CD9 molecules on the exosomes, so that the surface of the exosomes can bind to anti-Fc antibodies or antibody drugs, so that different targeted targets (antibodies or antibody drugs) can be efficiently displayed on the surface of the exosomes, enhancing the targeted delivery effect of the exosomes. Through this method, the application prospects of exosomes in the medical field are broader, especially in targeted therapy and drug delivery. It shows great potential, and multifunctional nanocarriers are expected to become an important tool for disease treatment and drug delivery in the future.

[0016] To achieve the above object, the present invention provides the following technical solutions:

[0017] ① A fusion protein comprising:

[0018] (A) a first antigen binding moiety specific for an exosomal membrane protein, and

[0019] (B) The second antigen binding moiety is specific for Fc, which is human Fc.

[0020] ② According to the fusion protein described in ①, the exosomal membrane protein is CD9, CD63, CD81 or a combination thereof, preferably the exosomal membrane protein is CD9.

[0021] ③ The fusion protein according to any one of ①-②, wherein the first antigen binding portion comprises a light chain variable region and a heavy chain variable region;

[0022] The light chain variable region includes L1-CDR1, L1-CDR2, and L1-CDR3, wherein the amino acid sequence of L1-CDR1 is shown in SEQ ID NO.4, the amino acid sequence of L1-CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of L1-CDR3 is shown in SEQ ID NO.6;

[0023] The heavy chain variable region includes H1-CDR1, H1-CDR2, and H1-CDR3. The amino acid sequence of H1-CDR1 is shown in SEQ ID NO.7, the amino acid sequence of H1-CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of H1-CDR3 is shown in SEQ ID NO.9.

[0024] ④ The fusion protein according to any one of ①-②, wherein the second antigen binding portion comprises a light chain variable region and a heavy chain variable region;

[0025] The light chain variable region L2-CDR1, L2-CDR2, L2-CDR3, the amino acid sequence of L2-CDR1 is shown in SEQ ID NO.10, the amino acid sequence of L2-CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of L2-CDR3 is shown in SEQ ID NO.12;

[0026] The heavy chain variable region includes H2-CDR1, H2-CDR2, and H2-CDR3, the amino acid sequence of H2-CDR1 is shown in SEQ ID NO.13, the amino acid sequence of H2-CDR2 is shown in SEQ ID NO.14, and the amino acid sequence of H2-CDR3 is shown in SEQ ID NO.15.

[0027] ⑤ The fusion protein according to any one of ①-②, which is:

[0028] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO.2; or

[0029] (b) A protein derived from protein (a) in which the amino acid sequence of protein (a) is substituted and / or deleted and / or added with one or more amino acid residues and has the activity of protein (a).

[0030] ⑥ A polynucleotide encoding a fusion protein as described in any one of ①-⑤.

[0031] ⑦ The polynucleotide according to ⑥, comprising the following nucleotide sequence:

[0032] (1) the nucleotide sequence as shown in SEQ ID NO: 2; or

[0033] (2) a nucleotide sequence that hybridizes under stringent conditions to the DNA sequence defined in (1); or

[0034] (3) A nucleotide sequence having a sequence identity of 90% or more with the DNA sequence defined in (1) or (2).

[0035] ⑧A recombinant vector, wherein the recombinant vector comprises the polynucleotide described in ⑥ or ⑦.

[0036] ⑨ A method for constructing and / or modifying exosomes, comprising connecting a target with an exosome using the fusion protein described in any one of ①-⑤ or the polynucleotide described in ⑥ or ⑦, wherein the target is an antibody or an antibody drug.

[0037] ⑩ Use of the fusion protein described in any one of ①-⑤ or the polynucleotide described in ⑥ or ⑦ in constructing and / or transforming exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the absorbance of anti-CD9 antibody at 450nm at different dilution multiples. No. 1-3 are the serum of mice immunized with CD9 protein, and the control is the serum of mice without immunization.

[0039] Figure 2 It is the absorbance of human Fc antibody at 450nm at different dilution multiples. No. 1-3 are the sera of mice immunized with human Fc protein, and the control is the sera of mice without immunization.

[0040] Figure 3 Schematic diagram of connecting CD9 molecules on exosomes and anti-Fc antibodies through αCD9-x-αFc.

[0041] Figure 4 Nanoflow cytometry was used to detect the control group and experimental group. The control group was 293T exosomes, and the experimental group was 293T exosomes + αCD9-x-αFc protein + FITC-Fc protein.

[0042] Figure 5 The control group and experimental group were detected by ordinary flow cytometry. The control group was 293T exosomes + CHO-K1 cells overexpressing PDL1, and the experimental group was 293T exosomes + αCD9-x-αFc protein + αPDL1-Fc protein + CHO-K1 cells overexpressing PDL1.

[0043] Figure 6 The control group and experimental group were detected by ordinary flow cytometry. The control group was 293T exosomes + MDA-MB-231 cells overexpressing PDL1, and the experimental group was 293T exosomes + αCD9-x-αFc protein + αPDL1-Fc protein + MDA-MB-231 cells overexpressing PDL1. DETAILED DESCRIPTION

[0044] Example 1 Screening of anti-CD9 specific antibodies

[0045] Hybridoma antibody screening technology provides a platform for screening monoclonal antibodies with target specificity and high affinity. This technology mainly fuses plasma B cells that can secrete antibodies but cannot proliferate indefinitely with myeloma cells that can proliferate in vitro and culture for a long time, and screens hybridoma cells that can secrete specific antibodies and can be cultured for a long time and proliferate indefinitely. The production and preparation of monoclonal antibodies is achieved through the expansion of the hybridoma, and the hybridoma cells can be frozen and preserved by liquid nitrogen to facilitate the later antibody production. Considering the instability of some hybridoma clones, the antibody variable region is obtained by PCR to preserve the required hybridoma variable region sequence, and the antibody is produced by other cells that highly express antibodies (preferably CHO cells).

[0046] 1. Target immunization - immunization of mice and serological titer determination

[0047] In order to obtain higher mouse immune titer, 6-8 week old C57 / BL6 mice (Shanghai Model Organisms Science Co., Ltd.) were used. Four days before immunization, blood was collected from the eye sockets and centrifuged at 3000g for 5 min to obtain about 20ul of serum, which was frozen at -20°C as a negative control. Freund's adjuvant (Sigma) was used as an immune adjuvant, and the antigen was CD9 protein (protein amino acid sequence: MPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTGVYILIGAGALMMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMV). The vaccine is injected subcutaneously at multiple points. After the initial immunization, booster immunization is performed every two weeks, with 100ug each time, for a total of three immunizations.

[0048] Afterwards, protein ELISA was used to detect the antibody titer in mouse serum. Protein ELISA detection steps: 96-well ELISA plates were coated with CD9 protein (1ug / ml) and incubated at 37°C for 2 hours. Then the liquid in the wells was discarded, rinsed three times with PBST washing buffer (Solarbio), and blocked overnight at 4°C with PBS (Solarbio) solution containing 2% BSA (Solarbio). The next day, the blocked plate was rinsed with PBST washing buffer, and mouse serum samples with different dilutions were added, incubated at 37°C for 1 hour, and after rinsing with PBST washing buffer, HPR-labeled goatanti-mouse IgG (H+L) (Sigma) diluted 1:10000 with 2% BSA-PBST was added and incubated at 37°C for 1 hour. After rinsing with PBST washing buffer, TMB substrate solution (Beyotime) was added for color development, and the reaction was stopped with 2M hydrochloric acid solution after 15 minutes of reaction at room temperature in the dark. The absorbance was read at 450 nm using a microplate reader (Synergy HTX, Agilent BioTek). Figure 1 This immunization method is simple to operate and can detect high antibody titers in mouse serum.

[0049] 2. Cell fusion, ELISA detection and antibody characterization

[0050] Three days after the last booster immunization, a mouse with the highest antibody titer in the mouse serum was selected, the spleen was removed and ground in RPMI-1640 basal medium (Gibco, cat#11875-093) to obtain a suspension rich in lymphocytes. In order to obtain hybridoma cells for screening, we used efficient electroporation to fuse mouse spleen cells with mouse myeloma cells (SP2 / 0) (Wuhan Sean Bio, catalog number SNL-121), with a fusion ratio of 10:1.

[0051] After 7 days of fusion, the supernatant was collected and tested by protein ELISA to determine whether the fused cells produced antibodies.

[0052] Protein ELISA detection steps: Coat a 96-well ELISA plate with CD9 protein (abcam, #ab174040) (1μg / ml) and incubate at 37°C for 2 hours. Then discard the liquid in the wells, rinse three times with PBST washing buffer, add PBS solution containing 2% BSA at 4°C to block overnight. The next day, rinse the blocked plate with PBST washing buffer, add mouse serum samples with different dilutions, incubate at 37°C for 1 hour, and after rinsing with PBST washing buffer, add HPR-labeled goat anti-mouse IgG (H+L) diluted 1:10000 with 2% BSA-PBST, and incubate at 37°C for 1 hour. After rinsing with PBST washing buffer, add TMB substrate solution for color development, react at room temperature in the dark for 15 minutes, terminate the reaction with 2M hydrochloric acid solution and read the absorbance at 450nm. Select the wells with high OD values ​​for subcloning.

[0053] In the verification of this project, we used the above method to screen out multiple antibody-expressing clones that can bind to CD9 protein in 53 screening plates. Several clones have the ability to stably express high-affinity antibodies against CD9 protein, among which clone 17B2G3 has the largest Elisa value.

[0054] Table 1 Elisa values ​​of subcloned hybridomas

[0055]

[0056] The affinity of the antibody was measured using the biomolecular interaction instrument Biocore T200. The antibody affinity activity is as follows:

[0057] Table 2 Antibody affinity

[0058]

[0059] 3. Obtain antibody light and heavy chain variable region sequences

[0060] The specific operation of obtaining the antibody light and heavy chain variable region sequence is as follows: Trizol is used to lyse the candidate hybridoma clone 17B2G3, and the total RNA is extracted. After the first-chain cDNA is synthesized using this as a template, the first-chain cDNA is used as a template, and the antibody variable region specific primers are used for subsequent PCR amplification to obtain the nucleic acid of the antibody light and heavy chain variable region corresponding to the hybridoma cell. Agarose gel electrophoresis is performed, and after gel excision and recovery for TA cloning, Sanger sequencing is performed to obtain the antibody variable region sequence. The anti-CD9 antibody heavy chain variable region CDR sequence is shown in SEQ ID NO.7-9, and the anti-CD9 antibody light chain variable region CDR sequence is shown in SEQ ID NO.4-6.

[0061] The amino acid sequence of the heavy chain variable region of the anti-CD9 specific antibody is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQISSLKSEDTAMYYCARRWDEYFDVWGAGTTVTVSS

[0062] The amino acid sequence of the light chain variable region of the anti-CD9 specific antibody is: DIQMTQSPSSLSASVGDRVTITCRASQGISNWLNWYQQKPGKAPKLLIYAASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNSWPFTFGSGTKLEIK

[0063] Example 2 Screening of human Fc-specific antibodies

[0064] The Fc (Fragment crystallizable) region of an antibody is located at the end of the constant part of the antibody molecule and is not involved in antigen binding, but is mainly responsible for the biological function of the antibody. Therefore, antibodies targeting Fc have little effect on the variable region.

[0065] Hybridoma antibody screening technology provides a platform for screening monoclonal antibodies with target specificity and high affinity. This technology mainly fuses plasma B cells that can secrete antibodies but cannot proliferate indefinitely with myeloma cells that can proliferate in vitro and culture for a long time, and screens hybridoma cells that can secrete specific antibodies and can be cultured for a long time and proliferate indefinitely. The production and preparation of monoclonal antibodies is achieved through the expansion of the hybridoma, and the hybridoma cells can be frozen and preserved by liquid nitrogen to facilitate the later antibody production. Considering the instability of some hybridoma clones, the antibody variable region is obtained by PCR to preserve the required hybridoma variable region sequence, and the antibody is produced by other cells that highly express antibodies (preferably CHO cells).

[0066] 1. Target immunization - immunization of mice and serological titer determination

[0067] In order to obtain higher mouse immune titer, 6-8 week old C57 / BL6 mice were used. Four days before immunization, blood was collected from the eye sockets and centrifuged at 3000g for 5 min to obtain about 20ul of serum, which was frozen at -20°C as a negative control. Freund's adjuvant (Sigma) was used as an immune adjuvant, and the antigen was human IgG1-Fc protein (protein amino acid sequence: MPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTGVYILIGAGALMMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMV). The vaccine is injected subcutaneously at multiple points. After the initial immunization, booster immunization is performed every two weeks, with 100ug each time, for a total of three immunizations.

[0068] Protein ELISA was used to detect the antibody titer in mouse serum.

[0069] Protein ELISA detection steps: Coat a 96-well ELISA plate with human Fc protein (1ug / ml) and incubate at 37°C for 2 hours. Then discard the liquid in the wells, rinse three times with PBST washing buffer (Solarbio), add PBS (Solarbio) solution containing 2% BSA (Solarbio) and block overnight at 4°C. The next day, rinse the blocked plate with PBST washing buffer, add mouse serum samples with different dilutions, incubate at 37°C for 1 hour, and after rinsing with PBST washing buffer, add HPR-labeled goat anti-mouse IgG (H+L) (Sigma) diluted 1:10000 with 2% BSA-PBST, and incubate at 37°C for 1 hour. After rinsing with PBST washing buffer, add TMB substrate solution (Beyotime) for color development, react at room temperature in the dark for 15 minutes, and terminate the reaction with 2M hydrochloric acid solution. Use an enzyme reader (Synergy HTX, Agilent BioTek) to read the absorbance at 450nm, and the results are as follows: Figure 2 This immunization method is simple to operate and detects good antibody titers in mouse serum.

[0070] 2. Cell fusion, ELISA detection and antibody characterization

[0071] Three days after the last booster immunization, a mouse with the highest antibody titer in the mouse serum was selected, the spleen was removed and ground in RPMI-1640 basal medium to obtain a suspension rich in lymphocytes. In order to obtain hybridoma cells for screening, we used efficient electroporation to fuse mouse spleen cells with mouse myeloma cells (SP2 / 0) at a fusion ratio of 10:1. Seven days after fusion, the supernatant was taken and tested by protein ELISA to determine whether the fused cells produced antibodies.

[0072] Protein ELISA detection steps: Coat a 96-well ELISA plate with human Fc protein (1ug / ml) and incubate at 37°C for 2 hours. Then discard the liquid in the wells, rinse three times with PBST washing buffer, add PBS solution containing 2% BSA at 4°C to block overnight. The next day, rinse the blocked plate with PBST washing buffer, add mouse serum samples with different dilutions, incubate at 37°C for 1 hour, and after rinsing with PBST washing buffer, add HPR-labeled goat anti-mouse IgG (H+L) diluted 1:10000 with 2% BSA-PBST, and incubate at 37°C for 1 hour. After rinsing with PBST washing buffer, add TMB substrate solution for color development, react at room temperature in the dark for 15 minutes, terminate the reaction with 2M hydrochloric acid solution and read the absorbance at 450nm. Select the wells with high OD values ​​for subcloning.

[0073] In the validation of this project, we used the above method to screen out clone 21C2H6 in 30 screening plates, and found that it can efficiently bind to human Fc protein.

[0074] Table 3 Elisa values ​​of subcloned hybridomas

[0075]

[0076] The affinity of the antibody was measured using the biomolecular interaction instrument Biocore T200. The antibody affinity activity is as follows

[0077] Table 4 Antibody affinity

[0078]

[0079] 3. Obtain antibody light and heavy chain variable region sequences

[0080] The specific operation of obtaining the antibody light and heavy chain variable region sequence is as follows: Trizol is used to lyse the candidate hybridoma clone, and total RNA is extracted. After the first-chain cDNA is synthesized using this as a template, the first-chain cDNA is used as a template, and the antibody variable region specific primers are used for subsequent PCR amplification to obtain the nucleic acid of the antibody light and heavy chain variable region corresponding to the hybridoma cell. Agarose gel electrophoresis is performed, and after gel excision and recovery, TA cloning is performed, and then Sanger sequencing is performed to obtain the antibody variable region sequence. The heavy chain CDR region sequence of the anti-human Fc antibody is shown in SEQ ID NO.13-15, and the light chain CDR region sequence of the anti-human Fc antibody is shown in SEQ ID NO.10-12.

[0081] Anti-human Fc antibody heavy chain variable region amino acid sequence: QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSGNYGALDYWGQGTSVTVSS

[0082] Anti-human Fc antibody light chain variable region amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSLAPVTFGQGTKVEIK

[0083] Example 3 Construction, expression and purification of αCD9-x-αFc protein

[0084] 1. Construction of expression vector

[0085] Using the pcDNA3.1 plasmid (nucleotide sequence as shown in SEQ ID NO.1) as a template, double restriction enzyme digestion was performed with HindⅢ and XbaI under the following conditions:

[0086] Table 5 Double enzyme digestion reaction system

[0087]

[0088] The αCD9-x-αFc gene sequence with double restriction sites was synthesized by Genewise (nucleotide sequence as shown in SEQ ID NO.2), and then double-digested with HindⅢ and XbaI to obtain the target plasmid (pcDNA3.1-αCD9-x-αFc) under the following conditions:

[0089] Table 6 Double enzyme digestion reaction system

[0090]

[0091] After gel recovery, T4 ligase was used for ligation and then competent bacteria were transformed.

[0092] 1) Thaw Fast-T1 chemically competent cells for cloning (Novozyme, Cat. No. C505-02 / 03) on ice.

[0093] 2) Take 5 μl of the target plasmid (pcDNA3.1-αCD9-x-αFc) and add it to 50 μl of the competent cells in step 1), gently tap the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30 min.

[0094] 3) After heat shock in a 42°C water bath for 45 s, immediately cool on ice for 3 min.

[0095] 4) Add 1 ml of SOC medium (without antibiotics) (Sigma catalog number #S1797) and incubate at 37°C for 1 h.

[0096] 5) Preheat an ampicillin-resistant LB plate solid culture dish (ampicillin concentration is 100 μg / ml) in a 37°C incubator, then take 200 μL of the bacterial solution obtained in step 4) and spread it on the ampicillin-resistant plate, and culture it upside down in a 37°C incubator.

[0097] 6) After sequencing by Suzhou Jinweizhi Biotechnology Co., Ltd., the culture was expanded. The nucleotide sequence was verified by sequencing as shown in SEQ ID NO.3.

[0098] 2. Protein Expression and Purification

[0099] 1) Culture: CHO cells (from ATCC cell bank) were cultured in an incubator at 37°C and 5% CO2. F-12K medium (Gibco, cat#21127022) was used, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0100] 2) Passaging: When the cells are close to 80-90% of the culture dish area, passaging is performed to ensure that the cells are in good growth condition. One day before transfection, CHO cells are seeded in 6 wells so that the cells reach 70-90% of the culture dish area on the day of transfection. Prepare 2-3 µg DNA per well.

[0101] 3) Mix the reaction solutions: Under sterile conditions, prepare Opti-MEM™ I Reduced Serum Medium containing plasmid DNA and Opti-MEM™ I Reduced Serum Medium containing Lipofectamine 3000. Mix according to the manufacturer's ratio (1 µg DNA corresponds to 3 µL Lipofectamine 3000).

[0102] 4) Incubation: After the mixture is allowed to stand for 5-10 minutes, add it to the corresponding cell culture wells. Culture: Continue to culture the cells at 37°C, 5% CO2.

[0103] 5) Protein expression and extraction: 72 hours after transfection, observe protein expression. Use a cell scraper to collect cells for subsequent lysis and protein extraction. Add 1X lysis buffer containing protease inhibitors to lyse the cells.

[0104] 6) Purification: Use the His-tag protein purification kit (IDA-Ni) to purify the protein. The specific steps are as follows:

[0105] ① Add an equal amount of binding buffer to the cell lysate for dilution;

[0106] ② Magnetic bead pretreatment: The target protein is estimated to be 1-2 mg. Take 1 ml of magnetic bead suspension for the purification of the target protein. Place the metal ion chelating magnetic beads on a vortex mixer and mix thoroughly. Take 1 ml to a 15 ml centrifuge tube, add 5 mL of binding buffer, turn the centrifuge tube upside down several times to resuspend the magnetic beads, perform magnetic separation, remove the supernatant, and repeat washing twice;

[0107] ③ Add 12 ml (10 mL) of protein sample to the magnetic beads, incubate with rotation at room temperature for 90 min, perform magnetic separation, and keep the supernatant;

[0108] ④ Add 10 mL of washing buffer to the centrifuge tube containing magnetic beads, gently flip the centrifuge tube several times to resuspend the magnetic beads, magnetically separate, and keep the washing solution. Repeat this step once;

[0109] ⑤ Add 10mL of washing buffer to the centrifuge tube containing magnetic beads to resuspend the magnetic beads, transfer the magnetic bead suspension to a new centrifuge tube (to avoid non-specific adsorption of proteins on the wall of the original centrifuge tube from contaminating the target protein), perform magnetic separation, and remove the supernatant to the washing solution collection tube;

[0110] ⑥ Add 2 to 10 mL of elution buffer, gently flip the centrifuge tube several times to suspend the magnetic beads, separate them magnetically, and collect the eluate into a new centrifuge tube, which is the purified αCD9-x-αFc protein sample.

[0111] Example 4 αCD9-x-αFc protein determination and characterization

[0112] 1. ELISA assay of αCD9-x-αFc protein

[0113] Protein ELISA detection steps: Coat the 96-well ELISA plate with human Fc protein (1ug / ml) and incubate at 37℃ for 2 hours. Then discard the liquid in the well, rinse three times with PBST washing buffer (Solarbio), add PBS (Solarbio) solution containing 2% BSA (Solarbio) and block overnight at 4℃. The next day, rinse the blocked plate with PBST washing buffer, add αCD9-x-αFc protein with different dilutions, incubate at 37℃ for 1 hour, and after rinsing with PBST washing buffer, add HPR-labeled CD9 protein diluted 1:10000 with 2% BSA-PBST, and incubate at 37℃ for 1 hour. After rinsing with PBST washing buffer, add TMB substrate solution (Beyotime) for color development, react at room temperature in the dark for 15 minutes, terminate the reaction with 2M hydrochloric acid solution and read the absorbance at 450nm. The results are shown below.

[0114] Table 7 Elisa values ​​of αCD9-x-αFc protein

[0115]

[0116] 2. Affinity determination of αCD9-x-αFc protein

[0117] The affinity of αCD9-x-αFc protein was measured using Biocore T200, and the antibody affinity activity is as follows:

[0118] Table 8 Antibody affinity (human Fc protein immobilized on chip)

[0119]

[0120] Table 9 Antibody affinity (CD9 protein fixed on chip)

[0121]

[0122] 3. Characterization of the ability of αCD9-x-αFc protein to bind to exosomes

[0123] (1) Take 10 μL of frozen 293T exosomes, add 90 μL of PBS, and mix well; then add 1 μL of αCD9-x-αFc protein constructed in Example 3, and incubate at 4°C for 30 min.

[0124] (2) Add 10 ml of PBS to wash away excess protein, centrifuge at 120,000 g for 1 h, remove the supernatant, and resuspend the precipitate in 100 μL PBS.

[0125] (3) Add 1 μL of FITC fluorescently labeled Fc protein (for the labeling of Fc protein, refer to the instructions of the FITC fluorescent coupling kit), mix well, and incubate at 4°C for 30 min.

[0126] (4) Add 10 ml of PBS to wash away excess protein, centrifuge at 120,000 g for 1 h, remove the supernatant, and resuspend the precipitate in 100 μL PBS.

[0127] (3) The Exoplorer nano-flow cytometer (Weidu Biotechnology) was used for detection, in which 293T exosomes were used as the control group, and 293T exosomes + αCD9-x-αFc protein + FITC-Fc protein were used as the experimental group. The results are shown in Figure 4 , the data are shown in the following table:

[0128] Table 10 Results and statistics

[0129]

[0130] From the results in the above table, it can be seen that the use of αCD9-x-αFc protein can connect the Fc protein to the exosomes, and the target molecules can be efficiently displayed on the surface of the exosomes.

[0131] 4. Characterization of the ability of αCD9-x-αFc protein to modify targeting antibodies on the surface of exosomes

[0132] (1) CHO-K1 cells overexpressing PDL1 and MDA-MB-231 cells overexpressing PDL1 were inoculated into 6-well plates 24 h in advance.

[0133] (2) Take the 293T exosomes previously frozen at -80°C, incubate the exosomes with αCD9-x-αFc protein at 4°C for 30 min, make up the volume to 10 ml with PBS, centrifuge at 120,000 g for 60 min, discard the supernatant, and resuspend the precipitate in 100 μL PBS.

[0134] (3) Add 1 μL of αPDL1-Fc protein, mix well, incubate at 4°C for 30 min, then make up the volume to 10 ml with PBS, centrifuge at 120,000 g for 1 h, remove the supernatant, and resuspend the precipitate in 100 μL PBS.

[0135] (4) Blank 293T exosomes (control group) and 293T exosomes + αCD9-x-αFc protein + αPDL1-Fc protein prepared in the above steps (2 and 3) were incubated with Dio dye at room temperature for 20 min, then filled to a volume of 10 ml with PBS, centrifuged at 120,000 g for 90 min, and the supernatant was discarded; finally, the precipitate was resuspended with 100 μL PBS.

[0136] (5) After NTA determination of exosome concentrations of stained exosomes from different groups, 1×10 9 Exosomes were added to CHO-K1 cells overexpressing PDL1 (overexpressing PDL1 after modification). After incubation for a period of time, CHO-K1 cells were collected and analyzed by flow cytometry using a CytoFLEX S flow cytometer (Beckman). Figure 5 ).

[0137] (6) After NTA determination of exosome concentrations of stained exosomes from different groups, 1×10 9 Exosomes were added to MDA-MB-231 cells overexpressing PDL1 (which overexpress PDL1). After a period of incubation, MDA-MB-231 cells were collected and analyzed by flow cytometry using a CytoFLEX S flow cytometer (Beckman). Figure 6 ).

[0138] pass Figure 5 and Figure 6 It can be seen that the exosomes connected with αPDL1-Fc through αCD9-x-αFc can be more targeted to cells expressing PD-L1, indicating that this modification method significantly enhances the targeted delivery effect of exosomes.

[0139] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A fusion protein comprising: (A) a first antigen binding portion specific for an exosomal membrane protein, wherein the exosomal membrane protein is CD9, and (B) a second antigen binding moiety specific for Fc, wherein the Fc is a human Fc; wherein the first antigen binding portion comprises a light chain variable region and a heavy chain variable region; The light chain variable region includes L1-CDR1, L1-CDR2, and L1-CDR3, wherein the amino acid sequence of L1-CDR1 is shown in SEQ ID NO.4, the amino acid sequence of L1-CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of L1-CDR3 is shown in SEQ ID NO.6; The heavy chain variable region includes H1-CDR1, H1-CDR2, and H1-CDR3. The amino acid sequence of H1-CDR1 is shown in SEQ ID NO.7, the amino acid sequence of H1-CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of H1-CDR3 is shown in SEQ ID NO.

9.

2. The fusion protein of claim 1, wherein the second antigen binding portion comprises a light chain variable region and a heavy chain variable region; The light chain variable region includes L2-CDR1, L2-CDR2, and L2-CDR3, wherein the amino acid sequence of L2-CDR1 is shown in SEQ ID NO.10, the amino acid sequence of L2-CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of L2-CDR3 is shown in SEQ ID NO.12; The heavy chain variable region includes H2-CDR1, H2-CDR2, and H2-CDR3, the amino acid sequence of H2-CDR1 is shown in SEQ ID NO.13, the amino acid sequence of H2-CDR2 is shown in SEQ ID NO.14, and the amino acid sequence of H2-CDR3 is shown in SEQ ID NO.

15.

3. The fusion protein according to claim 1, which is a protein consisting of the amino acid sequence encoded by SEQ ID NO.

2.

4. A polynucleotide encoding the fusion protein according to any one of claims 1 to 3. The polynucleotide according to claim 4 , comprising the nucleotide sequence as shown in SEQ ID NO:

2.

6. A recombinant vector comprising the polynucleotide according to claim 4 or 5.

7. A method for constructing and / or modifying exosomes, comprising using the fusion protein of any one of claims 1 to 3 or the polynucleotide of claim 4 or 5 to connect a target to the exosomes, wherein the target is an antibody or an antibody drug.

8. Use of the fusion protein according to any one of claims 1 to 3 or the polynucleotide according to claim 4 or 5 in constructing and / or modifying exosomes.

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