Genetically engineered CD20-targeting microvesicle / exosome as well as preparation method and application thereof

Abstract

The invention discloses a gene engineering modified CD20 targeting microvesicle / exosome as well as a preparation method and application thereof, and belongs to the technical field of biological nano-carrier preparation and targeting delivery. Aiming at the problems of poor targeting property, insufficient modification stability, high immune side reaction risk and the like of an existing extracellular vesicle (EV) delivery system, a CD20 antibody single-chain variable region (scFv) structural domain is fused with EV membrane anchoring structural domains such as Lamp2b, CD63, C1-C2, PDGFR beta and the like to construct a multi-type surface display carrier, so that stable presentation of the CD20 scFv structural domain on an EV membrane is realized. The engineered exosome / vesicle can specifically recognize CD20 positive B cells, improves the delivery efficiency, does not contain an Fc structure, avoids complement activation and related immunotoxicity, keeps the structural integrity of an EV natural membrane, and has large-scale production potential. The invention can be widely applied to the fields of treatment of CD20 positive B cell diseases, drug screening and transformational medicine, and has important clinical value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological nanocarrier preparation and targeted delivery, and specifically relates to a genetically engineered extracellular vesicle (including microvesicle and exosome) which has the ability to target CD20 and can be used for drug delivery, disease treatment and translational medical application. BACKGROUND

[0002] In recent years, the incidence of tumors continues to rise, and there is an urgent need for new drug delivery carriers that are safe, efficient and highly targeted. Existing delivery methods such as viral vectors, liposomes and high molecular weight nanoparticles have the disadvantages of high immunogenicity, poor in vivo stability, obvious toxic side effects, lack of cell-specific targeting ability and other defects, which cannot meet the needs of precision medicine development.

[0003] Extracellular vesicles (EVs) are natural nanovesicles with good biocompatibility and transmembrane delivery capacity, and are ideal candidates for the next generation of drug and gene delivery platforms. However, natural EVs lack targeting ability, surface receptors and functional molecules cannot be artificially controlled, and drug loading efficiency is limited, which results in insufficient controllability and delivery efficiency in clinical application.

[0004] Existing EV surface engineering techniques include chemical modification and genetic engineering, but have many shortcomings: (1) chemical modification may damage the EV membrane structure, resulting in decreased biological activity; (2) genetic engineering has low expression efficiency and is difficult to scale up; (3) lipid or polymer intercalation modification has poor stability and is easily shed in vivo.

[0005] Some B-cell lymphoma patients relapse or are refractory after standard treatment, and have poor prognosis. Existing treatment options such as CAR-T cell therapy and BTK inhibitors have high treatment costs, strong toxic side effects and significant immune escape, etc. CD20 is a transmembrane protein highly expressed by B cells and an important target for lymphoma targeted therapy, but antibody-based drugs based on CD20 have poor tissue penetration and significant immune side effects, and existing CD20-based carriers are mostly in the form of antibodies or fusion proteins, which cannot be efficiently immobilized on the surface of EVs.

[0006] In summary, the existing technology has the problems of lack of targeting ability of natural EVs, insufficient modification stability, complex process, high risk of immune side effects, etc., and there is an urgent need to develop an engineered EV delivery platform targeting CD20. SUMMARY

[0007] The present application aims to solve the problems of poor targeting, insufficient modification stability and strong immune side effects in existing extracellular vesicle delivery technology, and provides an engineered microvesicle / exosome specifically targeting CD20, which has high stability, low toxicity and potential for large-scale production.

[0008] In a first aspect, to achieve the above object, the present application adopts the following technical solution: A genetically engineered microvesicle / exosome targeting CD20, the membrane surface of the microvesicle / exosome displaying a CD20 antibody single-chain variable region (scFv), the CD20 scFv being stably anchored on the lipid membrane of the microvesicle / exosome by being fused with an EV membrane anchoring domain selected from at least one of Lamp2b, CD63, C1-C2 and PDGFRβ.

[0009] Lamp2b is a member of the lysosome-associated membrane protein family, and its extracellular region and transmembrane region have been confirmed in many studies to be effective in mediating the directional display of exogenous molecules to the vesicle membrane; CD63 is a classic representative of the four-transmembrane protein superfamily, and is naturally enriched in the vesicle membrane, having stable and repeatable advantages in terms of EV localization and enrichment efficiency; the C1-C2 domain can achieve efficient adsorption to the vesicle membrane through interaction with phosphatidylserine; and the transmembrane region structure of PDGFRβ has also been proven to be a reliable membrane anchoring sequence for anchoring and presenting target proteins on the vesicle membrane. The present application selects from among multiple known EV localization domains, and finally determines Lamp2b, CD63, C1-C2 and PDGFRβ as anchoring modules to ensure that the functionalized exosomes constructed by the present application have high membrane localization efficiency and structural stability.

[0010] Further, the fusion expression vector also comprises a signal peptide for guiding the secretion of the fusion protein and a Myc tag sequence for expression verification and detection.

[0011] In a second aspect, the present application also provides a preparation method of the genetically engineered microvesicle / exosome targeting CD20, comprising the following steps: (1) constructing a fusion expression vector of CD20 scFv and EV membrane anchoring domain; (2) transfecting the fusion expression vector into a host cell; (3) culturing the transfected host cell and collecting the secreted microvesicle / exosome; (4) purifying the engineered microvesicle / exosome targeting CD20.

[0012] In a third aspect, the present application also provides use of the genetically engineered microvesicle / exosome targeting CD20 in the preparation of a medicament for treating a CD20-positive cell disease, the CD20-positive cell being a B-cell malignancy.

[0013] In a fourth aspect, the present application also provides use of the genetically engineered microvesicle / exosome targeting CD20 in drug screening or translational medicine research.

[0014] Further, the genetically engineered CD20-targeting microvesicles / exosomes serve as drug delivery carriers, carrying nucleic acids, proteins, small molecule drugs or any active ingredients for targeted delivery to CD20-overexpressing B cell tumors.

[0015] In a fifth aspect, the present application also provides a pharmaceutical composition comprising the genetically engineered CD20-targeting microvesicles / exosomes and a pharmaceutically acceptable carrier.

[0016] In a sixth aspect, the present application also provides a drug delivery biological agent, which is the genetically engineered CD20-targeting microvesicles / exosomes loaded with drugs for preventing or treating tumors.

[0017] Further, the tumor is a B cell malignancy with CD20-positive cells.

[0018] Compared with the prior art, the present application has the following beneficial effects: Strong targeting: through the specific binding of the CD20 scFv domain to CD20-positive B cells (such as DLBCL cells), the selectivity of EVs for target cells and the uptake efficiency are significantly improved, solving the problem of lack of targeting of natural EVs.

[0019] High stability: based on the fusion modification mode of the membrane protein anchor domain, no chemical cross-linking is required, the integrity of the natural membrane structure of EVs is not damaged, and the functional domain after modification is not easy to fall off, which is superior to the existing chemical modification and lipid intercalation technology.

[0020] Good safety: the fusion protein does not contain the Fc segment of the antibody, which can avoid the immune side reactions such as Fc-mediated complement activation and antibody-dependent cell-mediated cytotoxicity, and reduce the risk of treatment.

[0021] Scalable production: the modification mode is based on cell engineering technology, and the preparation process has repeatability, which is convenient for scale-up production and meets the needs of clinical application.

[0022] Wide application range: it can be used as a general delivery carrier to carry nucleic acids, proteins, small molecule drugs and other active ingredients, and is suitable for the treatment of CD20-positive B cell diseases, drug screening and translational medicine research. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Verification of microvesicle marker protein expression.

[0024] Figure 2 Verification of successful expression of CD20-scFv fusion protein by microvesicles.

[0025] Figure 3: Verification of exosome marker protein expression.

[0026] Figure 4 : Verification of successful expression of CD20-scFv fusion protein by exosomes.

[0027] Figure 5 : Verification fluorescence map of successful uptake of microvesicles by DLBCL cells.

[0028] Figure 6 : Fluorescence statistical map of successful uptake of microvesicles by DLBCL cells.

[0029] Figure 7 : Verification fluorescence map of successful uptake of exosomes by DLBCL cells.

[0030] Figure 8 : Fluorescence statistical map of successful uptake of exosomes by DLBCL cells. DETAILED DESCRIPTION

[0031] Further details are described below through a specific embodiment: I. Construction of fusion expression vector 1. In the vector design, integrate membrane fusion skeleton sequences such as lamp2b, CD63, C1-C2, PDGFRβ domain, etc. to effectively anchor the targeting molecule CD20-scFv to the surface of the vesicle membrane. (The relevant sequence numbers are shown in Table 1 below) Table 1:

[0032] 2. Construct a fusion expression vector containing a signal peptide (select a commonly used signal peptide CSF2RB for secreted proteins) - CD20 transmembrane domain - EV membrane anchoring domain - Myc tag, and obtain four fusion vectors CD20-Lamp2b, CD20-CD63, CD20-C1-C2, CD20-PDGFRβ, respectively. Set up a blank control group (CON). Transfect the fusion expression vector into 293FT cells through retrovirus, and continuously screen with 16 µg / mL tetracycline for 7 days, sub-clone, and obtain a stable expression cell strain.

[0033] II. Expression verification and purity detection 1、Collect 50 mL of cell supernatant, centrifuge at 300 g for 10 min to remove dead cells and cell debris. Centrifuge the supernatant at 2000 g for 20 min to remove apoptotic bodies. Then centrifuge at 15000 g for 30 min, collect the precipitate, resuspend in 500 μL PBS to obtain microvesicles. Detection of microvesicle marker proteins found that positive markers CD9 and CD81 were expressed significantly, while intracellular marker GM130 was not detected, further confirming the purity and success of microvesicle extraction Figure 1 . At the same time, the expression of Myc tag protein in microvesicles was detected. As shown in Figure 2 , Western blot results showed that CD20-scFv expressed by the four methods could be localized in microvesicles.

[0034] 2、After the supernatant of microvesicle separation was centrifuged at 120000 g for 120 min, the precipitate was collected and resuspended in 200 μL PBS to obtain exosomes. Detection of exosome marker proteins found that positive markers CD9 and CD81 were expressed significantly, while intracellular marker GM130 was not detected, further confirming the success of exosome extraction Figure 3 . At the same time, the expression of Myc tag protein in exosomes was detected. As shown in Figure 4 , Western blot results showed that CD20-scFv expressed by the four methods could be localized in exosomes.

[0035] Three, target binding and uptake ability verification 1、By detecting the interaction between microvesicles and DLBCL cells. PKH26 dye was used to label microvesicles, and confocal microscopy was used to observe that, as shown in Figure 5 and Figure 6 , the four kinds of modified microvesicles can be effectively taken up by DLBCL cells, and the red fluorescence signal is obviously distributed around the cell nucleus, indicating that these surface modifications successfully express CD20 scfv and promote the binding and uptake ability of microvesicles to tumor cells.

[0036] 2、By detecting the interaction between exosomes and CD20-positive DLBCL cells (SU-DHL-2). PKH26 dye was used to label exosomes, and confocal microscopy was used to observe that, as shown in Figure 7 and Figure 8 , the four kinds of modified exosomes can be effectively taken up by DLBCL cells, and the red fluorescence signal is obviously distributed around the cell nucleus, indicating that these surface modifications successfully express CD20 scfv and promote the binding and uptake ability of microvesicles to tumor cells.

[0037] The above-mentioned are only embodiments of the present application, and common technical solutions and / or common knowledge of the scheme are not described in detail. It should be pointed out that, for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the patent. The protection scope claimed in the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A genetically engineered microvesicle / exosome targeting CD20, characterized in that, The membrane surface of the microvesicles / exosomes exhibits a CD20 antibody single-chain variable region scFv, which is stably anchored to the lipid membrane of the microvesicles / exosomes by fusing with an EV membrane anchoring domain. The EV membrane anchoring domain is selected from at least one of Lamp2b, CD63, C1-C2, and PDGFRβ.

2. The genetically engineered CD20-targeting microvesicles / exosomes as described in claim 1, characterized in that, The fusion expression vector also contains a signal peptide and a Myc tag sequence. The signal peptide is used to guide the secretion of the fusion protein, and the Myc tag is used for expression verification and detection.

3. The method for preparing genetically engineered CD20-targeting microvesicles / exosomes as described in claim 1, characterized in that, Includes the following steps: (1) Construct a fusion expression vector for CD20scFv and EV membrane anchoring domain; (2) Transfect the fusion expression vector into host cells; (3) Culture the transfected host cells and collect the secreted microvesicles / exosomes; (4) Purification to obtain engineered microvesicles / exosomes targeting CD20.

4. The use of the genetically engineered CD20-targeting microvesicles / exosomes as described in claim 1 in the preparation of a medicament for treating CD20-positive cell diseases, characterized in that... The CD20-positive cells are B-cell malignant tumors.

5. The application of the genetically engineered CD20-targeting microvesicles / exosomes as described in claim 1 in drug screening or translational medicine research.

6. The application according to claim 4, characterized in that: The genetically engineered CD20-targeting microvesicles / exosomes are used as drug delivery carriers to deliver any active ingredient, such as nucleic acid, protein, or small molecule drug, to B-cell tumors that highly express CD20.

7. A pharmaceutical composition, characterized in that: The product comprises a genetically engineered CD20-targeting microvesicle / exosome as described in any one of claims 1 or 2, or a CD20-targeting microvesicle / exosome prepared by the method described in claim 3, and a pharmaceutically acceptable carrier.

8. A drug delivery biological agent, characterized in that: The biological agent is a genetically engineered CD20-targeting microvesicle / exosome as described in claim 1 or 2, or a genetically engineered CD20-targeting microvesicle / exosome prepared by the method described in claim 3, loaded with a drug for the prevention or treatment of tumors.

9. A drug delivery biological agent as described in claim 8, characterized in that, The tumor is a CD20-positive B-cell malignant tumor.

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