Fusion protein and nucleic acid encoding sequence thereof, and uses of the same

Abstract

This invention provides a fusion protein, its nucleic acid coding sequence, and its applications. Through various efficacy experiments, the fusion protein of this invention achieves therapeutic effects on cancer, immune regulation, and activation of immune cells.

Description

Fusion protein and its nucleic acid coding sequence and application The present invention relates to a fusion protein and its nucleic acid coding sequence and application. Cancer, also known as a malignant tumor, is an abnormal cell proliferation that can invade other parts of the body. It is caused by a malfunction in the mechanisms that control cell division and proliferation. The number of people suffering from cancer is increasing worldwide. Cancer is one of the top ten causes of death in China and has remained at the top of the list for many years. Conventional cancer treatments include surgery, radiation therapy, chemotherapy, and targeted therapy. Tumor immunotherapy, an alternative to these treatments, activates the patient's own immune system, inducing specific cellular and humoral immune responses using tumor cells or tumor antigens. This enhances the body's anti-cancer capabilities, inhibits tumor growth, spread, and recurrence, and ultimately eliminates or controls the tumor. However, current cancer treatments still suffer from ineffectiveness and significant side effects, and can even lead to other immune-related diseases. CD3ε (CD3 epsilon) is a transmembrane protein found on T cells that has been linked to tumors and immune regulation. Consequently, researchers are working to develop CD3ε as a target molecule for tumor identification and immune regulation, and to determine whether these target molecules have the potential to become anticancer or immunomodulatory drugs. Furthermore, CD63 is a protein antigen encoded by the CD63 gene in the human body. CD63 is primarily found on the surface of extracellular vesicles, but also on normal cell membranes. The gene encoding it is implicated in tumor development. In order to solve the above problems, technicians in this field are in urgent need of developing novel and more effective pharmaceuticals for treating cancer, immunomodulating and activating immune cells to benefit the vast population in need. In view of this, an object of the present invention is to provide a fusion protein comprising an anti-CD3 single domain antibody and an exosomal protein, wherein the anti-CD3 single domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In one embodiment of the present invention, the anti-CD3 single domain antibody specifically binds to CD3 epsilon. In one embodiment of the present invention, the exosomal protein is CD63. In one embodiment of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 4. In one embodiment of the present invention, the amino acid sequence represented by SEQ ID NO: 1 is complementarity determining region 1 (CDR1), the amino acid sequence represented by SEQ ID NO: 2 is CDR2, and the amino acid sequence represented by SEQ ID NO: 3 is CDR3. In one embodiment of the present invention, the anti-CD3 single domain antibody is an anti-T cell nanobody. Another object of the present invention is to provide an isolated nucleic acid encoding the amino acid sequence of the fusion protein as described above. In one embodiment of the present invention, the nucleotide sequence of the isolated nucleic acid is shown in SEQ ID NO: 5. Another object of the present invention is to provide a pharmaceutical composition comprising the fusion protein as described above and a pharmaceutically acceptable carrier. Another object of the present invention is to provide a use of the aforementioned fusion protein for preparing a medicine for treating cancer, immunomodulation, and activating immune cells. In one embodiment of the present invention, the cancer is treated by allowing the fusion protein to penetrate into the interior of the solid tumor and secrete bispecific T-cell engager (BiTE) and activate peripheral immune cells. In summary, the efficacy of the fusion protein of the present invention lies in: achieving cancer treatment, immune regulation, and immune cell activation through surface plasmon resonance (SPR), cytotoxicity assays, animal experiments, electroporation experiments, transfection efficiency testing, flow cytometry analysis, and in vivo chimeric antigen receptor T-cell therapy (CAR-T). The fusion protein penetrates into solid tumors, secretes bispecific T-cell engagers (BiTEs), and activates peripheral immune cells, achieving the following effects: The following will further illustrate the embodiments of the present invention. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone skilled in the art may make slight changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the appended patent applications. definition The values ​​used in this article are approximate, and all experimental data are expressed in 20%, preferably within 10% range, the best is within the range of 5%. Unless otherwise specified herein, the terms "a", "an", "the" and similar terms used in this specification (especially in the scope of the following patent applications) should be understood to include both singular and plural forms. As used herein, the terms "CD3 epsilon" and "CD3 ε" are used interchangeably. As used herein, the terms "CD3e nanobody", "CD3e nb", "CD3e Nb", "CD3e nanobody", "anti-CD3 epsilon nanobody", "anti-CD3 single domain antibody" and "anti-T cell nanobody" are used interchangeably. As used herein, "treating" or "treatment" means alleviating, reducing, ameliorating, relieving, or controlling one or more clinical signs of a disease or disorder, as well as lowering, stopping, or reversing the progression of the severity of a condition or symptom being treated. According to the present invention, the pharmaceutical product can be manufactured into a dosage form suitable for parenteral administration using techniques well known to those skilled in the art, including, but not limited to, injections (e.g., sterile aqueous solutions or dispersions), sterile powders, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like. The medicament according to the present invention can be administered via a parenteral route selected from the group consisting of intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection. The pharmaceutical product according to the present invention may include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of a solvent, an emulsifier, a suspending agent, a decomposer, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and amount of these agents are within the professional knowledge and routine skills of those skilled in the art. According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), a sugar solution, an aqueous solution containing alcohol, and combinations thereof. As used herein, the terms "nucleic acid," "nucleic acid sequence," or "nucleic acid fragment" refer to a deoxyribonucleotide sequence or a ribonucleotide sequence in single- or double-stranded form, including known naturally occurring nucleotides or synthetic chemical mimics. As used herein, the term "nucleic acid" is used interchangeably with "gene," "cDNA," "mRNA," "oligonucleotide," and "polynucleotide." The present invention is further illustrated by the following examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the attached patent application. Embodiment 1. Preparation of the fusion protein of the present invention In this embodiment, the preparation process of the fusion protein comprising an anti-CD3 single domain antibody and an exosome protein is as follows. HEK-293T cell lines (passages 4 to 25) and HEK-293T stable cell lines expressing an anti-CD3 single domain antibody-CD63 chimeric protein were placed in COL2.5 NGCs and cultured with 500 mL of DMEM medium (containing 50 mL of exosome-free FBS) (Thermo Fisher Scientific). After culturing for 3 days, the culture medium was centrifuged at 2000 g for 15 minutes to remove cell debris and then filtered with 0.2 μm filter paper. Then, the culture medium was purified by superfiltration (Amicon ® Ultra, 30 kDa, Merck Millipore) at 5000 g for 8 minutes. The collected supernatants were processed by tangential flow filtration (MAP.03-plus TFF System, Lefo Science). Subsequently, the supernatant of parental HEK-293T cells was filtered through a membrane with a cutoff value of 300 kDa and then resuspended in PBS; the supernatant from HEK-293T stable cells expressing anti-CD3 single domain antibody-CD63 chimeric protein was filtered through a VHH-capture membrane (GenScript) and then resuspended in PBS. All samples were used immediately or stored at -80°C for further use. For NTA analysis, these exosomes were filtered by ZetaView ® (Particle Metrix GmbH) to analyze their size distribution and concentration. The anti-CD3 single-domain antibody comprises the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein the amino acid sequence set forth in SEQ ID NO: 1 represents complementarity determining region 1 (CDR1), the amino acid sequence set forth in SEQ ID NO: 2 represents CDR2, and the amino acid sequence set forth in SEQ ID NO: 3 represents CDR3. The amino acid sequence of the fusion protein of the present invention is set forth in SEQ ID NO: 4. The present invention also provides an isolated nucleic acid encoding an amino acid sequence of the fusion protein described above. The nucleotide sequence of the isolated nucleic acid is shown in SEQ ID NO: 5. The amino acid sequence of the anti-CD3 single domain antibody is shown in SEQ ID NO: 6. The nucleotide sequence encoding the amino acid sequence of the anti-CD3 single domain antibody is shown in SEQ ID NO: 7. The amino acid sequence of the anti-CD3 single domain antibody is the heavy chain variable domain (VHH). A schematic diagram of the structure of the fusion protein of the present invention is shown in FIG1 , wherein the anti-CD3 single domain antibody specifically binds to CD3 ε (CD3 epsilon). FIG2 shows the binding site of the exosomal protein CD63 and the anti-CD3 single domain antibody, wherein the arrow indicates the embedded position of the anti-CD3 single domain antibody. Example 2. Surface plasmon resonance of the fusion protein of the present invention (surface plasmon resonance, SPR) analyze In this example, the experimental workflow for surface plasmon resonance (SPR) analysis of exosomes containing fusion proteins is as follows. SPR analysis was performed on CM5 and NTA chips (research grade) using a BIAcore T200 (Biacore-GE Healthcare, Piscataway, NJ). Briefly, protein samples (CD3ε recombinant protein) were diluted in 10 mM buffer (pH 4.0, 5.5, or 6.0) at a concentration range of 20 μg / mL to maximize surface retention for immobilization on the chip after surface preparation. Conditions were selected to achieve high surface concentrations of ligand on the chip (anti-CD3 single-domain antibody: 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 nM). Regeneration scouting and surface performance testing were then performed, and the regeneration method was selected for the experiment. Binding analysis and direct binding were then selected to investigate protein binding. Select Kinetic Analysis and Mass Transfer to perform kinetic analysis and concomitant binding experiments. Analyze data and determine kinetic constants. The results of Exo surface plasmon resonance (SPR) analysis of the fusion protein are shown in Figure 3. As shown in Figure 3, the fusion protein (1 x 10 11 ) coated on a CM5 chip, and the binding affinity was determined using recombinant CD3ε protein (700, 350, 175, 87.5, 43.8, and 21.9 nM). The KD was determined to be 2.1 nM. Example 3. Evaluation of the efficacy of the fusion protein of the present invention in treating cancer, immunomodulation and activating immune cells This example evaluates the effectiveness of the fusion protein in treating cancer, immunomodulation, and activating immune cells. Figure 4 is a schematic diagram illustrating the utility of fusion proteins in cancer treatment, immunomodulation, and immune cell activation. CD3εNb represents an anti-CD3 single-domain antibody, HEK-293T represents human embryonic kidney cells, Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, and Exo represents exosome. As shown in Figure 4 , the sequence encoding the anti-CD3 single-domain antibody is inserted into the second external loop of CD63, forming a CD63 chimeric protein with the cell-surface-exposed anti-CD3 single-domain antibody. This construct is then transfected into HEK-293T cells to produce exosomes expressing the anti-CD3 single-domain antibody. The harvested exosomes are then purified using a VHH-capsule column and loaded with DNA or mRNA encoding the Nb-CAR.BiTE. This Nb-CAR.BiTE-CD3ɛ Nb-Exo was directly injected into a mouse model. We hypothesized that these exosomes selectively reprogram CD3 + T cells expressing Nb-CAR and secreting Nb-BiTE can fight against solid tumor cells in vivo. FIG5 is another schematic diagram illustrating the effectiveness of fusion proteins in cancer treatment, immune regulation, and immune cell activation, wherein PCR represents polymerase chain reaction, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, PBMC represents peripheral blood mononuclear cell, TEM represents transmission electron microscope, IFN represents immunofluorescence staining, and IFN represents immunofluorescence staining. Interferon (interferon ), TNF Tumor necrosis factor (tumor necrosis factor ), PFN stands for perforin, GzmB stands for granzyme B, a serine protease that mediates the apoptotic signaling pathway of cytotoxic T lymphocytes and natural killer cells, SSC stands for side scattered light, and VHH stands for heavy chain variable domain. As shown in Figure 5, the Exo and CAR.BiTE DNA were mixed at a rate of (3 x 10 8 After electroporation using the LONZA 4D-Nucleofector electroporation code CM137 (ratio of Exo vs 2 μg DNA), the cells were allowed to recover at 4°C for one hour before electron microscopy observation. Alternatively, the cells were transfected into human PBMCs or whole blood for 48 hours and then co-cultured with tumor cells. Live / dead cell-mediated cytotoxicity assays were used to assess changes in tumor cell cytotoxicity. Alternatively, flow cytometry was used to analyze the expression of CD3, CD4, CD8, and VHH proteins on cells using fluorescent antibody labeling. These experiments confirm the transfection efficacy of the fusion protein-encapsulated CAR.BiTE DNA. For transmission electron microscopy (TEM) analysis, HEK-293T-derived exosomes were isolated and fixed with 1% glutaraldehyde overnight at 4°C. After washing, the exosomes were mounted on formvar carbon-coated grids and negatively stained with aqueous phosphotungstic acid for 1 minute. The ultrastructure of these exosomes was analyzed using TEM (JEOL JEM-1400, Tokyo, Japan). Figure 6 shows transmission electron microscopy (TEM) images of the fusion proteins, where CD3ε Nb represents an anti-CD3 single-domain antibody, Exo represents exosomes, CAR represents chimeric antigen receptor, and BiTE represents bispecific T-cell engager. As shown in Figure 6, CAR.BiTE DNA@CD3ε Nb-Exo maintains the ultrastructure and morphology of exosomes. Unmodified and CD3ε Nb-treated Exo (with or without CAR.BiTE electroporation) were evaluated by TEM at 100,000x using DNA expressing the CAR.BiTE. HEK293-derived Exosomes can be electroporated to encapsulate CAR-expressing DNA. Using a LONZA 4D-Nucleofector, unmodified Exosomes and fusion proteins were electroporated with the CAR.BiTE expression vector at a ratio of 3 x 10 8 Exo: 2 μg DNA. Exo are then treated with DNase (1000 IU) for 30 minutes, and the incorporated CAR.BiTE DNA is quantified by qPCR using specific primers or by spectrophotometry at OD 260 and 280 nm. Figure 7 shows the efficiency of linear Nb-CAR.BiTE DNA encapsulation into Exo cells via electroporation, where Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, CD3ε Nb stands for anti-CD3 single domain antibody, and qPCR stands for quantitative polymerase chain reaction. As shown in Figure 7, based on the spectrophotometer and qPCR results, the electroporation method can achieve a high efficiency of 3 x 10 8 Exo was coated with approximately 100 ng of CAR.BiTE DNA. Unmodified Exo or fusion proteins were expressed in the presence or absence of CAR.BiTE expression vectors at a concentration of 3 x 10 8 The ratio of Exo: 2 μg DNA was electroporated and after purification, these Exo were added to 5 x 10 5 After 48 hours in PBMC, the CD3 + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + 、CD56 + , TCRγδ + 、CD14 + 、CD19 + and CD66b + The expression level of Nb-CAR on cells. Figure 8 shows the efficiency of Nb-CAR.BiTE DNA-coated electroporated Exo transfection into peripheral blood mononuclear cells (PBMCs), where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3ε Nb represents anti-CD3 single domain antibody, and WLSM represents weighted least squares measure. As shown in Figure 8, compared with unmodified Exo (293T Exo), CAR.BiTE DNA@CD3e Nb-Exo has a higher transfection efficiency in CD3 + cells showed a higher expression than CD3 - This result supports that the fusion protein selectively delivers the transgene to CD3 + in cells. Unmodified Exo or fusion proteins were expressed in the presence or absence of CAR.BiTE expression vectors at a concentration of 3 x 10 8 The ratio of Exo: 2 μg DNA was electroporated and after purification, these Exo were added to 5 x 10 5 After 48 hours in PBMC, the CD3 + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + The expression level of Nb-CAR on cells. Figure 9 is another schematic diagram of testing the efficiency of Nb-CAR.BiTE DNA-coated electroporated Exo transfection into peripheral blood mononuclear cells (PBMCs), where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3ε Nb represents anti-CD3 single domain antibody. As shown in Figure 9, by comparing with unmodified Exo (293T Exo), CAR.BiTE DNA@CD3e Nb-Exo has a higher transfection efficiency in CD3 + / CD4 + and CD3 / CD8 + This result supports that the fusion protein selectively delivers the transgene to CD4 T cells. + and CD8 + in cells. Unmodified Exo or fusion proteins were expressed in the presence or absence of CAR.BiTE expression vectors at a concentration of 3 x 10 8 The ratio of Exo: 2 μg DNA was electroporated and after purification, these Exo were added to 5 x 10 5 After 48 hours in PBMC, the CD3 + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + The expression level of Nb-CAR on cells. Figure 10 shows the efficiency of electroporated Exo coated with linear Nb-CAR.BiTE DNA in transfection into PBMCs, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3ε Nb represents anti-CD3 single domain antibody. As shown in Figure 10, CAR.BiTE DNA@CD3e Nb-Exo has a high transfection efficiency in CD3 + 、CD3 + / CD4 + and CD3 / CD8 + This result supports that the fusion protein selectively delivers the transgene to CD4 T cells. + and CD8 + in cells. Unmodified Exo or fusion proteins were expressed in the presence or absence of CAR.BiTE expression vectors at a concentration of 3 x 10 8 The ratio of Exo: 2 μg DNA was electroporated A. After purification, these Exo were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, CD3 was measured by flow cytometry using specific antibodies. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + The expression level of Nb-CAR on cells. Figure 11 shows the efficiency of electroporated Exo transfection into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3ε Nb represents anti-CD3 single domain antibody, and WLSM represents weighted least squares measure. As shown in Figure 11, this result supports that the fusion protein selectively delivers the transgene to CD3 in whole blood. + in cells. Unmodified Exo or fusion proteins were expressed in the presence or absence of CAR.BiTE expression vectors at a concentration of 3 x 10 8 The ratio of Exo: 2 μg DNA was electroporated and, after purification, these Exo were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, CD3 was measured by flow cytometry using a specific antibody. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + 、CD56 + , TCRγδ + 、CD14 + 、CD19 + and CD66b + The expression level of Nb-CAR on cells. Figure 12 is another schematic diagram of testing the efficiency of electroporated Exo coated with Nb-CAR.BiTE DNA in transfection into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3ε Nb represents anti-CD3 single domain antibody, and WLSM represents weighted least squares measure. As shown in Figure 12, this result supports that the fusion protein selectively delivers the transgene to CD3 in whole blood. + / CD4 and CD3 + / CD8 + in cells. Unmodified Exo or fusion proteins were expressed in the presence or absence of CAR.BiTE expression vectors at a concentration of 3 x 10 8 The ratio of Exo: 2 μg DNA was electroporated and, after purification, these Exo were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, CD3 was measured by flow cytometry using a specific antibody. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + The expression level of Nb-CAR on cells. Figure 13 shows the efficiency of electroporated Exo transfection into whole blood by testing linear Nb-CAR.BiTE DNA coating, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3ε Nb represents anti-CD3 single domain antibody. As shown in Figure 13, this result supports that the fusion protein selectively delivers the transgene to the CD3 + 、CD3 + / CD4 and CD3 + / CD8 + in cells. Unmodified Exo or fusion proteins were expressed in the presence or absence of CAR.BiTE expression vectors at a concentration of 3 x 10 8 The ratio of Exo: 2 μg DNA was electroporated and, after purification, these Exo were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, CD3 was measured by flow cytometry using a specific antibody. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + The expression level of Nb-CAR on cells. Figure 14 is another schematic diagram of testing the efficiency of electroporated Exo transfection into whole blood by linear Nb-CAR.BiTE DNA coating, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3ε Nb represents anti-CD3 single domain antibody. As shown in Figure 14, this result supports the selective delivery of transgenes by the fusion protein to CD3 in whole blood. + 、CD3 + / CD4 and CD3 + / CD8 + in cells. Cytotoxic killing assay: CD3 treated with CAR.BiTE-CD3ε Nb-Exo or unmodified exosomes +Cells were used as effector cells. Target cells (tumor cell lines) and effector cells were co-cultured at the specified effector cell / target cell (E:T) ratio (1:1 to 50:1) at 37°C for 24 to 72 hours. For live / dead cell viability assays, all tumor cells were stained with green-fluorescent calcein-AM before co-culture and then stained with red-fluorescent ethidium homodimer-1 after co-culture to mark dead cells. Dead tumor cells were identified as green fluorescent cells according to the manufacturer's instructions (Thermo Fisher Scientific). + / Red Fluorescent + Cells. Cell killing rate is expressed as a percentage of the total cell population. Figures 15A to 15C show that linear Nb-CAR.BiTE DNA-loaded Exo transfected PBMCs induced excellent cytolytic activity against solid tumor cells, where CRC represents colorectal cancer, GBM represents glioblastoma multiforme, and NSCLC represents non-small cell lung cancer. E:T ratio represents the effector-to-target ratio. As shown in Figures 15A to 15C, PBMCs treated with CAR.BiTE DNA-loaded CD3e Nb-Exo exerted more effective cytotoxic killing ability against these tumor cells, compared with unmodified Exo (293T Exo) loaded with or without CAR.BiTE DNA and CD3e Nb-Exo without DNA. In addition, PBMCs treated with 293T Exo loaded with CAR.BiTE DNA also exhibited excellent cytotoxic killing ability compared with PBMCs treated with Exo without CAR.BiTE DNA. Figure 16 is a schematic diagram of the in vivo anti-tumor efficacy process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1 x 10 6 7 days later, mice (n = 5) were injected with huPBMC (5 x 10 6The next day, mice (3 x 10 10 / mouse) once weekly for four weeks. Tumor growth was monitored using an IVIS system by detecting bioluminescent signals. Seven days after the final injection, mice were sacrificed, spleen cells were harvested, and Nb-CAR expression on each immune cell type was measured by flow cytometry using specific antibodies against VHH, CD3, CD56, TCRγδ, CD14, CD19, and CD66b. Figure 16 shows the results of in vivo chimeric antigen receptor T-cell therapy (CAR-T) with fusion proteins, using a peripheral blood mononuclear cell (PBMC)-huNSG mouse model. huPBMC denotes human peripheral blood mononuclear cells, IP denotes intraperitoneal, IV denotes intravenous, CAR denotes chimeric antigen receptor, BiTE denotes bispecific T-cell engager, Exo denotes exosome, CD3ε Nb denotes anti-CD3 single domain antibody, and luc denotes luciferase. The fourth dose has been administered, and blood has been collected to test transfection efficiency. Figure 17 is a schematic diagram of the in vivo anti-tumor efficacy process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1 x 10 6 7 days later, mice (n = 5) were injected with huPBMC (5 x 10 6 The next day, mice (3 x 10 10 / mouse) once a week for four weeks. Tumor growth was monitored by the IVIS system using bioluminescent signals. Figure 17 shows another schematic diagram of the effectiveness of fusion proteins in cancer treatment, immunomodulation, and immune cell activation. CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3ε Nb represents anti-CD3 single-domain antibody. As shown in Figure 17 , CAR.BiTE DNA@CD3e Nb-Exo exhibited superior anti-tumor activity compared to other mouse groups. Figure 18 is a schematic diagram of the in vivo anti-tumor efficacy process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1 x 10 6 7 days later, mice (n = 5) were injected with huPBMC (5 x 10 6 The next day, mice (3 x 10 10 / mouse) once a week for four weeks. Seven days after the last injection, the mice were sacrificed, submandibular blood was harvested, and the expression of Nb-CAR on each type of immune cell was measured by flow cytometry using antibodies specific for VHH and CD3. Figure 18 shows another schematic diagram of the effectiveness of fusion proteins in treating cancer, immunomodulation, and activating immune cells, where Nb represents nanobody and CAR represents chimeric antigen receptor. This result supports that the fusion protein selectively delivers the transgene to the CD3 + 、CD3 + / CD4 and CD3 + / CD8 + in cells. Figure 19 is a schematic diagram of the in vivo anti-tumor efficacy process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1 x 10 6 7 days later, mice (n = 5) were injected with huPBMC (5 x 10 6The next day, mice (3 x 10 10 / mouse) once a week for four weeks. Seven days after the last injection, the mice were sacrificed, submandibular blood was harvested, and the expression of Nb-CAR on each type of immune cell was measured by flow cytometry using antibodies specific for VHH and CD3. Figure 19 shows another schematic diagram of the effectiveness of fusion proteins in cancer treatment, immunomodulation, and immune cell activation, wherein Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3ε Nb represents anti-CD3 single-domain antibody, and BiTE represents bispecific T-cell engager. Figure 20 shows another schematic diagram of the effectiveness of fusion proteins in treating cancer, immunomodulation, and activating immune cells, wherein Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3ε Nb represents anti-CD3 single domain antibody, BiTE represents bispecific T-cell engager, and PBMC represents peripheral blood mononuclear cell. As can be seen from Figures 19 and 20, the fusion protein selectively delivers the transgene to the CD3 + in cells. In summary, the fusion proteins of the present invention can be used for cancer treatment, immune regulation, and immune cell activation through surface plasmon resonance, cytotoxicity assays, animal experiments, electroporation experiments, transfection efficiency testing, flow cytometry analysis, and in vivo chimeric antigen receptor T cell therapy. By penetrating the interior of solid tumors and secreting bispecific T-cell-activating antibodies, the fusion proteins can also activate surrounding immune cells. The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or variations that do not depart from the spirit and scope of the present invention should be included in the scope of the appended patent applications. none Figure 1 is a schematic diagram of the structure of the fusion protein of the present invention. Figure 2 shows the binding site of the exosomal protein CD63 and the anti-CD3 single-domain antibody, where the arrow indicates the embedded position of the anti-CD3 single-domain antibody. Figure 3 shows the results of surface plasmon resonance (SPR) analysis of the fusion protein Exo. Figure 4 is a schematic diagram of the effectiveness of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where CD3εNb represents the anti-CD3 single-domain antibody, HEK-293T represents human embryonic kidney cells, Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, and Exo represents exosome. Figure 5 is another schematic diagram illustrating the utility of fusion proteins in cancer treatment, immune regulation, and immune cell activation, where PCR represents polymerase chain reaction, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, PBMC represents peripheral blood mononuclear cell, and TEM represents transmission electron microscopy. Figure 6 shows the results of transmission electron microscopy (TEM) of the fusion protein, where CD3εNb represents an anti-CD3 single-domain antibody, Exo represents exosome, CAR represents chimeric antigen receptor, and BiTE represents bispecific T-cell engager.Figure 7 shows the efficiency of linear Nb-CAR.BiTE DNA encapsulation into Exosomes via electroporation, where Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, CD3εNb stands for anti-CD3 single-domain antibody, and qPCR stands for quantitative polymerase chain reaction. Figure 8 shows the efficiency of electroporated Exosomes encapsulated with Nb-CAR.BiTE DNA in transfection into peripheral blood mononuclear cells (PBMCs), where Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, CD3εNb stands for anti-CD3 single-domain antibody, and WLSM stands for weighted least squares measure. Figure 9 is another schematic diagram testing the efficiency of electroporated Exo coated with Nb-CAR.BiTE DNA in transfection into peripheral blood mononuclear cells (PBMCs), wherein Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. Figure 10 shows the efficiency of electroporated Exo coated with linear Nb-CAR.BiTE DNA in transfection into PBMCs, wherein Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody.Figure 11 shows the efficiency of testing Nb-CAR.BiTE DNA-coated electroporated Exo transfection into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3εNb represents anti-CD3 single domain antibody, and WLSM represents weighted least squares measure. Figure 12 is another schematic diagram of testing the efficiency of testing Nb-CAR.BiTE DNA-coated electroporated Exo transfection into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3εNb represents anti-CD3 single domain antibody, and WLSM represents weighted least squares measure. Figure 13 shows the efficiency of testing the transfection of linear Nb-CAR.BiTE DNA-coated electroporated Exo into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. Figure 14 is another schematic diagram of testing the efficiency of testing the transfection of linear Nb-CAR.BiTE DNA-coated electroporated Exo into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. Figures 15A to 15C show that linear Nb-CAR.BiTE DNA-loaded Exo-transfected PBMCs induced excellent cytolytic activity against solid tumor cells, where CRC represents colorectal cancer, GBM represents glioblastoma multiforme, and NSCLC represents non-small cell lung cancer. E:T ratio represents the effector-to-target ratio.Figure 16 shows the results of in vivo chimeric antigen receptor T-cell therapy (CAR-T) with fusion proteins, using a peripheral blood mononuclear cell (PBMC)-huNSG mouse model. huPBMC denotes human peripheral blood mononuclear cells, IP denotes intraperitoneal, IV denotes intravenous, CAR denotes chimeric antigen receptor, BiTE denotes bispecific T-cell engager, Exo denotes exosome, CD3ε Nb denotes anti-CD3 single domain antibody, and luc denotes luciferase. The fourth dose has been administered, and blood has been collected to test transfection efficiency. Figure 17 shows another schematic diagram of the effectiveness of fusion proteins in treating cancer, immunomodulation, and activating immune cells, wherein CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell activating antibody, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. Figure 18 shows another schematic diagram of the effectiveness of fusion proteins in treating cancer, immunomodulation, and activating immune cells, wherein Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3εNb represents anti-CD3 single domain antibody, and BiTE represents bispecific T-cell activating antibody. Figure 19 shows another schematic diagram of the effectiveness of fusion proteins in treating cancer, immunomodulation, and activating immune cells, wherein Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3εNb represents anti-CD3 single domain antibody, and BiTE represents bispecific T-cell activating antibody. Figure 20 shows another schematic diagram of the effectiveness of fusion proteins in cancer treatment, immunomodulation, and immune cell activation, wherein Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3ε Nb represents anti-CD3 single-domain antibody, BiTE represents bispecific T-cell engager, and PBMC represents peripheral blood mononuclear cell. TW202530258A_113110587_SEQL.xml

Claims

1. A fusion protein comprising a primary anti-CD3 single-domain antibody and an exosomal protein, wherein the anti-CD3 single-domain antibody comprises an amino acid sequence as shown in Sequence Identification Numbers 1, 2 and 3, the exosomal protein is CD63, the amino acid sequence of the fusion protein is as shown in Sequence Identification Number 4, the anti-CD3 single-domain antibody and the exosomal protein are intercalated, and the fusion protein is produced and purified from the HEK-293T cell line.

2. The fusion protein as claimed in claim 1, wherein the anti-CD3 single-domain antibody specifically binds to a CD3 ε (CD3 epsilon).

3. The fusion protein as claimed in claim 1, wherein the amino acid sequence indicated by sequence identifier 1 is complementarity determining region 1 (CDR1), the amino acid sequence indicated by sequence identifier 2 is CDR2, and the amino acid sequence indicated by sequence identifier 3 is CDR3.

4. The fusion protein as claimed in claim 1, wherein the anti-CD3 single-domain antibody is a primary anti-T cell nanoantibody.

5. An isolated nucleic acid encoding an amino acid sequence of a fusion protein as described in any one of claims 1 to 4.

6. The isolated nucleic acid as described in claim 5, the nucleotide sequence of which is shown in sequence identification number 5.

7. A pharmaceutical composition comprising a fusion protein as described in any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

8. Use of a fusion protein as claimed in any one of claims 1 to 4 for the preparation of a pharmaceutical product for treating cancer, modulating the immune system and activating immune cells, wherein the cancer is treated by infiltrating the fusion protein into a solid tumor and secreting a bispecific T-cell engager (BiTE) and activating peripheral immune cells.

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