Exosomes displaying CD47 nanoantibodies and preparation method and application thereof

Abstract

The present invention proposes an exosome displaying CD47 nanoantibody and its preparation method and application, which belongs to the field of genetic engineering technology. The surface of the exosome displays the VHH chain of the CD47 nanoantibody, and the VHH chain of the CD47 nanoantibody is connected to the CD28 transmembrane protein to form a CD28-CD47 nanoantibody connector, which is anchored on the membrane surface of the exosome. The CD47 nanoantibody-displaying exosome of the present invention can be modified on the surface of the exosome membrane through the CD28 transmembrane protein connection, bind to the CD47 molecules on the surface of cancer cells, and block the immunosuppression of cancer cells on macrophages.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to an exosome displaying a CD47 nano antibody and a preparation method and application thereof. Background Art

[0002] Exosomes (exo) are nanoscale cell secretions with a double-layer membrane structure, containing a variety of RNAs and proteins derived from maternal cells. With the gradual deepening of research on exosomes, people have found that exosomes have extremely strong tissue penetration and can be used as drug carriers to effectively deliver anti-tumor drugs to the lesion site. However, there are still many difficulties in the practical application of exosomes, such as the non-targeted nature of exosomes.

[0003] CD47, also known as integrin-associated protein (IAP), belongs to the immunoglobulin superfamily. It is a transmembrane glycoprotein with 5 transmembrane domains and is widely expressed on the surface of almost all normal cells. Currently, there are three known natural ligands of CD47: integrin, thrombospondin-1 (TSP-1) and signal-regulatory protein-α (Sirp-α). CD47 and its ligands are mainly involved in cell adhesion, cell migration, phagocytosis, and maintaining immune homeostasis.

[0004] Traditional monoclonal antibodies have a large molecular weight and are difficult to penetrate into tissues. In addition, the production cycle of monoclonal antibodies is long and humanization is difficult. Therefore, it is particularly important to find antibodies with smaller molecular weights. In 1989, Muyldermans et al. discovered a heavy chain antibody for the first time when separating and detecting antibodies in camel serum. The antibody lacked two light chains CL and the constant region CH1, and was only composed of the N-terminal variable region (VHH), the hinge region and two constant regions (CH1, CH2). Its variable region (VHH) is called a nano antibody. The molecular weight of a nano antibody is only about 15kDa. Its nanoscale molecular size and unique structure give it multiple characteristics that are superior to traditional antibodies, such as high stability, good water solubility, simple humanization, high targeting, and strong penetration. Due to its special structural properties, nano antibodies have the advantages of both traditional antibodies and small molecule drugs, and almost perfectly overcome the defects of traditional antibodies such as long development cycle, low stability, and harsh storage conditions, and are used to develop therapeutic antibody drugs.

[0005] However, nanobodies are highly soluble, and during genetic engineering, most nanobodies are released outside the cell and are difficult to fix on the exosome membrane. By using genetic engineering technology to modify the exosome membrane and connecting nanobodies with transmembrane proteins, targeted antibodies can be displayed on the surface of the exosome membrane, thereby solving the above problem. Summary of the invention

[0006] The purpose of the present invention is to propose an exosome displaying CD47 nanoantibody and its preparation method and application, which can be modified on the surface of the exosome membrane through CD28 transmembrane protein connection, bind to the CD47 molecules on the surface of cancer cells, and block the immunosuppression of cancer cells on macrophages.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The present invention provides an exosome displaying a CD47 nano antibody, wherein the surface of the exosome displays a VHH chain of the CD47 nano antibody, and the VHH chain of the CD47 nano antibody is connected to a CD28 transmembrane protein to form a CD28-CD47 nano antibody connector, which is anchored on the membrane surface of the exosome.

[0009] Preferably, the CD47 nanobody is an anti-human CD47 nanobody.

[0010] As a further improvement of the present invention, the amino acid sequence of the CD28-CD47 nanoantibody connector is shown as SEQ ID NO.1; it comprises a CD47 nanoantibody and a CD28 transmembrane protein, the amino acid sequence of the CD47 nanoantibody is shown as SEQ ID NO.2; the amino acid sequence of the CD28 transmembrane protein is shown as SEQ ID NO.3.

[0011] As a further improvement of the present invention, the exosomes are derived from at least one of stem cells, primary cells, cancer cells, and cell lines.

[0012] As a further improvement of the present invention, the exosomes can tightly bind to CD47 on the surface of cancer cells or other cells.

[0013] The present invention further protects a nucleic acid encoding the CD28-CD47 nanobody linker on the exosomes displaying the CD47 nanobody, and the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:4.

[0014] Preferably, the nucleic acid comprises DNA or RNA.

[0015] The present invention further protects an expression vector, which contains the above-mentioned nucleic acid.

[0016] The present invention further protects a host cell, wherein the host cell contains the above expression vector, or the above nucleic acid is integrated into its genome.

[0017] The present invention further protects a method for producing the above-mentioned exosomes displaying CD47 nanoantibodies on the surface, comprising the following steps:

[0018] (1) constructing a vector for the above-mentioned CD28-CD47 nanobody conjugate;

[0019] (2) expressing the above-mentioned vector in a suitable host cell;

[0020] (3) collecting the culture fluid of the host cells and isolating and purifying the exosomes displaying CD47 nanoantibodies on their surfaces.

[0021] The present invention further protects the use of the above-mentioned exosomes displaying CD47 nanoantibodies in the detection of markers, drugs, and cytokines.

[0022] The present invention has the following beneficial effects: the CD47 nanoantibody-displaying exosomes of the present invention can be modified on the surface of the exosome membrane through CD28 transmembrane protein connection, bind to the CD47 molecules on the surface of cancer cells, and block the immunosuppression of cancer cells on macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 It is a schematic diagram of the vector design of the CD28-CD47 nanobody connector in a specific embodiment of the present invention.

[0025] Figure 2 This is a diagram showing the expression results of the vector in 293T cells.

[0026] Figure 3 This is an electron micrograph of exosomes showing CD47 nanoantibodies.

[0027] Figure 4 This is an immunofluorescence image of the residual CD47 molecules on the surface of cancer cells after co-incubation of exosomes displaying CD47 nanoantibodies with MCF-7 cells (CD47 positive cells). DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1: Preparation of exosomes displaying CD47 nanobodies

[0030] like Figure 1 .

[0031] (1) The plasmid of CD47 nanobody and CD28 fusion protein was constructed by homologous recombination, and the single clone with correct sequencing was selected and amplified by shaking at 37°C overnight;

[0032] (2) The amplified bacterial solution was centrifuged (4000 rpm, 10 min), the supernatant was removed and the bacterial cells were collected, and the plasmid was extracted to obtain a fusion plasmid solution of CD47 nanobody and CD28 (CD28-CD47 nanobody conjugate), and the plasmid concentration was measured;

[0033] (3) HEK 293T cells were seeded in a 10 cm culture dish to a confluence of 60-80%;

[0034] (4) 12 μg of plasmid (mass ratio CD28-CD47 nanobody conjugate: Δ8.9: VSVG = 4:3:1) and PEI reagent were mixed with 200 μL serum-free DMEM and allowed to stand for 5 min;

[0035] (5) Mix the two solutions in step 4, let stand for 30 min, add them to the HEK293T cells in step (3), mix well and place in a cell culture incubator for culture;

[0036] (6) 8 h after transfection, remove the cell supernatant and add 8 mL of complete culture medium;

[0037] (7) After 72 h of culture, the cell supernatant was collected, filtered through a 0.45 μm filter, and 5× PEG8000 solution was added and allowed to stand overnight;

[0038] (8) Centrifuge the solution in step 7 (4000×g, 10 min), remove the supernatant, and dissolve the precipitate with PBS to obtain a CD28-CD47 nanobody conjugate virus solution;

[0039] (9) HEK 293T cells were seeded in a 12-well plate and cultured until the confluence reached about 80%;

[0040] (10) Add the CD28-CD47 nanobody conjugate lentiviral vector solution to the HEK 293T cells in step (9), as Figure 2 , indicating that the vector can be stably expressed in cells;

[0041] (11) After incubation for 48 h, the cell supernatant was removed and complete medium containing 2.5 μg / mL puromycin was added for culture;

[0042] (12) Continuously culturing with complete medium containing 2.5 μg / mL puromycin for five days to screen HEK 293T cells that can express CD28-CD47 nanoantibody conjugates, and then replacing with normal complete medium after five days;

[0043] (13) After the cells grow to a confluence of more than 90%, the complete medium is replaced with a serum-free medium. After culturing for 48 h, the cell supernatant is collected, and the cells and cell debris are removed by gradient centrifugation. The exosome precipitate is obtained by ultracentrifugation (100,000 g, 70 min), and the exosome precipitate is resuspended in 1× PBS to obtain exosomes displaying CD47 nanoantibodies on the surface. Figure 3 .

[0044] Example 2: Demonstration of the blocking function of exosomes with CD47 nanobodies

[0045] (1) MCF-7 cells were inoculated on slides and cultured in 24-well plates at a cell density of approximately 5×10 5 cell / well.

[0046] (2) 30 μg of the prepared exosomes were added to the wells, and a well without exosomes was set up as a negative control, and incubated at 37°C for 30 minutes.

[0047] (3) The cells were fixed with 4% paraformaldehyde for 15 min, blocked with 1% BSA for 1 h, stained with Hoechst33342 for 15 min, incubated with anti-CD47 antibody at 4°C overnight, and incubated with cy3-labeled secondary antibody for 1 h. The results were observed under a confocal microscope. Figure 4 As shown, exosomes displaying CD47 nanoantibodies were able to bind to CD47 on the surface of MCF-7, blocking the binding of anti-CD47 antibodies to it.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An exosome displaying a CD47 nanobody, characterized in that: The surface of the exosomes displays the VHH chain of the CD47 nanoantibody, and the VHH chain of the CD47 nanoantibody is connected to the CD28 transmembrane protein to form a CD28-CD47 nanoantibody connector, which is anchored on the membrane surface of the exosomes; the amino acid sequence of the CD28-CD47 nanoantibody connector is shown in SEQ ID NO.1; it contains CD47 nanoantibody and CD28 transmembrane protein, and the amino acid sequence of the CD47 nanoantibody is shown in SEQ ID NO.2; the amino acid sequence of the CD28 transmembrane protein is shown in SEQ ID NO.

3.

2. The exosomes displaying CD47 nanoantibodies according to claim 1, characterized in that: The exosomes are derived from at least one of stem cells and cancer cells.

3. The exosomes displaying CD47 nanoantibodies according to claim 1, characterized in that: The exosomes can tightly bind to CD47 on the surface of cancer cells or other cells.

4. A nucleic acid, characterized in that The nucleic acid encodes the CD28-CD47 nanobody connector on the exosomes displaying the CD47 nanobody as described in any one of claims 1 to 3, and its nucleotide sequence is shown in SEQ ID NO:

4.

5. An expression vector, characterized in that: The expression vector contains the nucleic acid according to claim 4.

6. A host cell, characterized in that The host cell contains the expression vector of claim 5, or the nucleic acid of claim 4 is integrated into its genome.

7. A method for producing exosomes displaying CD47 nanoantibodies on the surface as claimed in claim 1, characterized in that: The following steps are involved: (1) constructing a vector for CD28-CD47 nanobody conjugate; (2) expressing the above-mentioned vector in a suitable host cell; (3) collecting the culture fluid of the host cells and isolating and purifying the exosomes displaying CD47 nanoantibodies on their surfaces.

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