Fusion proteins comprising an anti-cd3 single domain antibody, exosome protein cd63, and an rna binding protein and uses thereof

Abstract

The present application provides a fusion protein comprising an anti-CD3 single-domain antibody, an exosome protein CD63 and an RNA binding protein, and uses thereof. The fusion protein of the present application achieves the effects of treating cancer, immune regulation and activating immune cells through various efficacy experiments.

Description

Technical Field

[0001] This invention relates to a fusion protein comprising an anti-CD3 single-domain antibody, exosomal protein CD63, and an RNA-binding protein, and its uses. Background Technology

[0002] Cancer, also known as malignant tumor, is a disease caused by the abnormal proliferation of cells, which may invade other parts of the body. It is caused by the dysfunction of the mechanisms controlling cell division and proliferation. The number of people suffering from cancer worldwide is on the rise. Cancer is one of the top ten causes of death in Taiwan, and has been the leading cause of death for many years.

[0003] Conventional cancer treatments include surgery, radiation therapy, chemotherapy, and targeted therapy. Cancer immunotherapy is another approach to treating cancer besides these methods. It works by activating the patient's own immune system, using tumor cells or tumor antigens to induce specific cellular and humoral immune responses, enhancing the body's anti-cancer capabilities, and preventing tumor growth, spread, and recurrence, ultimately aiming to eliminate or control the tumor. However, current cancer treatments still suffer from limited efficacy and severe side effects, and may even lead to other immune-related diseases.

[0004] CD3ε (CD3 epsilon) is a transmembrane protein found on T cells and has been shown to be associated with tumors and immune function regulation. Therefore, researchers are dedicated to developing CD3ε as a target molecule for tumor identification and immune function regulation, and to identifying the potential of these target molecules as anticancer or immunomodulatory drugs. In addition, CD63 is a protein antigen encoded by the CD63 gene in the human body. CD63 is mainly found on the surface of extracellular vesicles, but can also be found on the surface of ordinary cell membranes; its encoding gene is related to tumor development.

[0005] To address the aforementioned issues, those skilled in the art urgently need to develop novel and more effective pharmaceuticals for treating cancer, modulating the immune system, and activating immune cells to benefit the vast population in need. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a fusion protein comprising an anti-CD3 single-domain antibody, an exosomal protein, and an RNA-binding protein, wherein the amino acid sequence of the anti-CD3 single-domain antibody is a heavy chain variable domain (VHH), and the exosomal protein is CD63.

[0007] In one embodiment of the present invention, the anti-CD3 single-domain antibody specifically binds to CD3 ε (CD3 epsilon).

[0008] In one embodiment of the present invention, the anti-CD3 single-domain antibody is an anti-T cell nanobody.

[0009] In one embodiment of the present invention, the anti-CD3 single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0010] In one embodiment of the present invention, the amino acid sequence at the N-terminus of CD63 is shown in SEQ ID NO: 3, and the amino acid sequence at the C-terminus of CD63 is shown in SEQ ID NO: 4.

[0011] In one embodiment of the present invention, the RNA-binding protein is heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1.

[0012] In one embodiment of the present invention, the hnRNPA2B1 comprises an amino acid sequence as shown in SEQ ID NO: 5.

[0013] In one embodiment of the present invention, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1.

[0014] In one embodiment of the invention, the fusion protein further comprises an extracellular vesicle (EV) sorting motif.

[0015] In one embodiment of the present invention, the nucleotide sequence encoding the amino acid sequence of the extracellular vesicle sorting motif is shown in SEQ ID NO: 6.

[0016] In one embodiment of the present invention, the fusion protein further comprises an HLA-G chimeric antigen receptor (CAR).

[0017] Another object of the present invention is to provide a pharmaceutical composition comprising the fusion protein as described above and a pharmaceutically acceptable carrier.

[0018] Another object of the present invention is to provide the use of the fusion protein as described above in the preparation of pharmaceuticals for treating cancer, modulating immunity and activating immune cells.

[0019] In one embodiment of the present invention, the fusion protein enhances the antitumor activity of peripheral blood mononuclear cells (PBMCs).

[0020] In one embodiment of the present invention, the cancer is colorectal cancer, lung adenocarcinoma, glioblastoma, or pancreatic cancer.

[0021] In summary, the fusion protein of the present invention achieves therapeutic effects on cancer, immune regulation, and activation of immune cells through the results illustrated in the following examples.

[0022] The following will further illustrate the embodiments of the present invention. The examples listed below are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone skilled in the art can make some modifications and refinements 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 defined in the appended claims. Attached Figure Description

[0023] Figures 1A-1E Characterization of the fusion protein is shown, which is also known as preloaded CD3ε-Nb nanobody (Nb) extracellular vesicle (EV). Figure 1AThe diagram shows the preloaded CD3ε-Nb EVs construct and preloaded CD3ε-Nb EVs. In the diagram, the EF-1α promoter represents the elongation factor-1α promoter, the CMV promoter represents the cytomegalovirus promoter, chimeric represents chimera, the RNP motif represents the ribonucleoprotein motif, the EV sorting motif represents the extracellular vesicle sorting motif, CAR represents the chimeric antigen receptor, CD3εNb represents the anti-CD3 single-domain antibody, and the RNA binding motif represents the RNA binding motif. The first elongation factor-1α (EF-1α) promoter drives a CD3ε nanobody (Nb)-CD63 chimeric construct composed of the exosomal tetraspanmembrane protein CD63. CD3ε Nb is inserted into the extracellular loop between the third and fourth transmembrane domains, followed by an RNA recognition motif (RRM) from heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1. This is then fused with the secondary EF-1α promoter, driving an HLA-G Nb-CAR construct containing an EV sorting motif derived from miR198. Transfection of the vector into HEK-293T cells produces CD3ε-Nb extracellular vesicles rich in HLA-G Nb-CAR mRNA (pre-loaded CD3ε-Nb EVs are short). Figure 1B , 1C The particle size distribution of parental and preloaded CD3ε-Nb EVs was determined by nanoparticle tracking analysis (NTA). Figure 1B The expression levels of the heavy chain variable domain (VHH), CD63, TSG101, and β-actin were detected by dot plotting using specific antibodies. Figure 1C ). Figure 1D , 1EThe procedure for determining the HLA-G Nb-CAR mRNA content in HEK-293T-derived EVs was as follows: Parental and pre-loaded CD3ε-Nb EVs were incubated with bead-conjugated anti-VHH antibody, and then EV fractions were separated using a magnetic system. Subsequently, qPCR analysis of the EV fractions was performed using HLA-G Nb-CAR mRNA-specific Taqman primers. Figure 1D ).

[0024] Figure 2A and Figure 2B The in vitro transfection efficiency of the fusion protein was demonstrated, which is also known as preloaded CD3ε-NbEVs. Figure 2A The diagram illustrates the transfection efficiency of the preloaded CD3ε-Nb EVs construct. After one day of treatment with both control and preloaded CD3ε-Nb EVs (2 x 10^9 particles), peripheral blood mononuclear cells (PBMCs) were obtained, and CD3 levels were determined by flow cytometry using specific antibodies against VHH and CD3. + and CD3 - Nb-CAR expression level on cells ( Figure 2B ).

[0025] Figure 3 This study demonstrated that in vitro treatment with the fusion protein, also known as preloaded CD3ε-Nb EVs, enhanced the antitumor activity of PBMCs. After one day of treatment with both control and preloaded CD3ε-Nb EVs (2 x 10^9 particles), PBMCs were co-cultured with COLO205 (human colon cancer cell line), H1975 (human lung adenocarcinoma cell line), U87 (human glioblastoma cell line), and Panc-1 (human pancreatic cancer cell line) at effector cell / target cell (E:T) ratios of 1:1, 3:1, 6:1, and 10:1. Induced cytotoxicity was analyzed by flow cytometry using the LIVE / DEAD Cell-Mediated Cytotoxicity Assay Kit, according to the user's instructions.

[0026] Figure 4A and Figure 4B The in vivo transfection efficiency of the fusion protein, also known as preloaded CD3ε-Nb extracellular vesicles, was demonstrated. Figure 4ASchematic diagram of transfection efficiency of preloaded CD3ε-Nb EVs in humanized NSG mice (huNSG) with COLO 205 tumor PBMCs. After 3 days of infusion of either control or preloaded CD3ε-Nb EVs (1 x 10^10 particles), leukocyte layers were collected from mice, and CD3 was determined by flow cytometry using specific antibodies against VHH and CD3. + and CD3 - Nb-CAR expression level on cells ( Figure 4B ).

[0027] Figures 5A-5D The in vivo antitumor efficiency of the fusion protein was demonstrated; this fusion protein is also known as preloaded CD3ε-Nb extracellular vesicles. Figure 5A A schematic diagram evaluating the antitumor efficacy of preloaded CD3ε-Nb EVs in humanized NSG mice (huNSG) with COLO205 tumor PBMCs. Seven days after implantation of COLO 205 tumor cells (1 x 10^6 cells / intraperitoneal injection (IP)), mice were injected via the tail vein with 5 x 10^6 PBMCs. The next day, mice were injected weekly with or without preloaded CD3ε-Nb EVs (1 x 10^10 particles) for four weeks. The efficacy was assessed using a non-invasive in vivo molecular imaging system (IVIS) via bioluminescent channels (…). Figure 5B , 5C Monitor tumor growth rate and record survival rate. Figure 5D ).

[0028] Figure 6A and Figure 6B This demonstrates the in vivo production of fusion proteins, also known as preloaded CD3ε-Nb extracellular vesicles. Figure 6A This diagram illustrates the in vivo generation of pre-loaded CD3ε-Nb EVs in PBMC humanized NSG mice (huNSG) with COLO 205 tumors. Plasma was collected from mice 7 days after intramuscular injection of either the vector control group or the pre-loaded CD3ε-Nb EV DNA transgene (1 mg / kg) in the right hind leg. The expression level of the CD3ε-Nb moiety on the EV particles was determined by flow cytometry using specific antibodies against VHH and CD3εNb, and the size of the identified beads was measured. Figure 6B ).

[0029] Figures 7A-7DThis demonstrates the in vivo production of a fusion protein from the hind leg via intramuscular injection, where the fusion protein is also known as preloaded CD3ε-Nb extracellular vesicles. Figure 7A Seven days after intramuscular injection of either the vector control group or pre-loaded CD3ε-Nb EV DNA transgene (1 mg / kg) into the right hind leg, mice were sacrificed and muscle tissue from both hind legs was collected. These hind leg tissues were then placed in 6-well plates supplemented with 1 ml of serum-free RPMI 1640 medium. The supernatant was collected the next day and filtered through a 0.22 μm filter membrane and a 30 kDa cutoff column. The tissue was then analyzed by dot plotting (…). Figure 7B The purified supernatant was analyzed by flow cytometry using specific antibodies against VHH and CD3ε Nb, and the size of the beads was determined using specific bead measurements. Figure 7C ), or by using specific Taqman primers and probes via qPCR to detect the level of HLA-G Nb-CAR mRNA ( Figure 7D ).

[0030] Figure 8A and Figure 8B The diagram shows the in vivo generation of Nb-CAR-expressing T cells via intramuscular injection of a fusion protein transgene, also known as preloaded CD3ε-Nb EVs. (8A) Schematic diagram of in vivo generation of Nb-CAR-expressing T cells via injection of preloaded CD3ε-Nb EVs into PBMC-humanized NSG mice (huNSG) with COLO 205 tumors. Seven days after intramuscular injection of either the vector control group or the preloaded CD3ε-Nb EV DNA transgene (1 mg / kg) in the right hind leg, the frequency of Nb-CAR-expressing cells in the leukocyte layer was determined by flow cytometry using antibodies specific to VHH and CD3. Figure 8B ).

[0031] Figures 9A to 9D The study demonstrated the antitumor effect induced by intramuscular injection of the fusion protein transgene, which is also known as preloaded CD3ε-Nb extracellular vesicles. Figure 9AA schematic diagram evaluating the antitumor activity of intramuscular injection of pre-loaded CD3ε-Nb EV transgene in PBMC-humanized NSG mice (huNSG) with COLO 205 tumors. Seven days after intraperitoneal injection of COLO 205 tumor cells (1 x 10^6 cells), mice were injected via tail vein with 5 x 10^6 PBMCs. The next day, pre-loaded CD3ε-Nb EV DNA transgene (1 mg / kg) was injected intramuscularly into the right hind leg of the mice, with or without injection. Tumor growth rate was monitored using an IVIS imaging system via a bioluminescent channel. Figure 9B , 9C ), and record their survival rate ( Figure 9D ). Detailed Implementation

[0032] definition

[0033] The values ​​used in this paper are approximate. All experimental data are expressed within ±20%, the better range is within ±10%, and the optimal range is within ±5%.

[0034] Unless otherwise stated herein, the terms “a,” “the,” and similar terms used in this specification (especially in the appended claims) shall be understood to include both singular and plural forms.

[0035] As used in this article, the terms “CD3e” and “CD3 ε” are used interchangeably.

[0036] As used in this article, the terms “CD3e nanobody”, “CD3e nb”, “CD3ε Nb”, “CD3enanobody”, “anti-CD3 ε nanobody”, “anti-CD3 single-domain antibody” and “anti-T cell nanobody” can be used interchangeably.

[0037] As used in this article, “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 that is being treated.

[0038] According to the present invention, pharmaceutical products can be manufactured in dosage forms suitable for parenterally administration using techniques known to those skilled in the art. These include, but are not limited to, injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0039] The pharmaceutical products according to the present invention can be administered via parenteral routes selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

[0040] The pharmaceutical products according to the present invention may comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of: solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these agents fall within the scope of professional competence and routine practice of those skilled in the art.

[0041] 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), sugar solution, aqueous solution containing alcohol, and combinations thereof.

[0042] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” or “nucleic acid fragment” refer to a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide sequence that contains known naturally occurring nucleotides or man-made chemical imitators. As used herein, the term “nucleic acid” may be used interchangeably with “gene,” “cDNA,” “mRNA,” “oligonucleotide,” and “polynucleotide.”

[0043] According to the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 1. The fusion protein contains an anti-CD3 single-domain antibody, the amino acid sequence of which is shown in SEQ ID NO: 2. The amino acid sequence of the anti-CD3 single-domain antibody is a heavy chain variable domain (VHH).

[0044] According to the present invention, the fusion protein comprises exosomal protein CD63, the amino acid sequence of the N-terminus of exosomal protein CD63 is shown in SEQ ID NO: 3, and the amino acid sequence of the C-terminus of exosomal protein CD63 is shown in SEQ ID NO: 4.

[0045] According to the present invention, the fusion protein comprises an RNA-binding protein, wherein the RNA-binding protein is a heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1. The amino acid sequence of hnRNPA2B1 is shown in SEQ ID NO: 5.

[0046] According to the present invention, the fusion protein may contain an extracellular vesicle (EV) sorting motif. The nucleotide sequence encoding the amino acid sequence of the extracellular vesicle sorting motif is shown in SEQ ID NO: 6.

[0047] According to the present invention, the fusion protein may contain an HLA-G chimeric antigen receptor (CAR).

[0048] According to the present invention, the fusion protein may include a linker, the amino acid sequence of which is shown in SEQ ID NO: 7.

[0049] According to the present invention, the anti-CD3 single-domain antibody specifically binds to CD3 ε (CD3 epsilon).

[0050] According to the present invention, the anti-CD3 single-domain antibody is an anti-T cell nanobody.

[0051] The present invention will now be further illustrated by the following embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of protection of the invention. The scope of protection of the present invention is as indicated by the appended claims.

[0052] Example 1. Characterization of the fusion protein of the present invention

[0053] Figures 1A-1E Characterization of the fusion protein was demonstrated, which is also known as preloaded CD3ε-Nb nanobody (Nb) extracellular vesicle (EV). Figure 1AThe diagram shows the preloaded CD3ε-Nb EVs construct and preloaded CD3ε-Nb EVs. In the diagram, EF-1α promoter represents the elongation factor-1α promoter, CMV promoter represents the cytomegalovirus promoter, chimeric represents chimera, RNP motif represents the ribonucleoprotein motif, EV sorting motif represents the extracellular vesicle sorting motif, CAR represents the chimeric antigen receptor, CD3εNb represents the anti-CD3 single-domain antibody, and RNA binding motif represents the RNA binding motif. The first elongation factor-1α (EF-1α) promoter drives a CD3ε nanobody (Nb)-CD63 chimeric construct composed of the exosomal tetraspanmembrane protein CD63. CD3ε Nb is inserted into the extracellular loop between the third and fourth transmembrane domains, followed by an RNA recognition motif (RRM) from heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1. This is then fused with the secondary EF-1α promoter, driving an HLA-G Nb-CAR construct containing an EV sorting motif derived from miR198. Transfection of the vector into HEK-293T cells produces CD3ε-Nb extracellular vesicles rich in HLA-G Nb-CAR mRNA (pre-loaded CD3ε-Nb EVs are short). Figure 1B , 1C The particle size distribution of parental and preloaded CD3ε-Nb EVs was determined by nanoparticle tracking analysis (NTA). Figure 1B The expression levels of the heavy chain variable domain (VHH), CD63, TSG101, and β-actin were detected by dot plotting using specific antibodies. Figure 1C ). Figure 1D , 1EThe procedure for determining the HLA-G Nb-CAR mRNA content in HEK-293T-derived EVs was as follows: Parental and pre-loaded CD3ε-Nb EVs were incubated with bead-conjugated anti-VHH antibody, and then EV fractions were separated using a magnetic system. Subsequently, qPCR analysis of the EV fractions was performed using HLA-G Nb-CAR mRNA-specific Taqman primers. Figure 1D ).

[0054] via Figures 1A-1E The results yielded the fusion protein of the present invention and determined its relevant characterization. The fusion protein was then used for the following experiments.

[0055] Example 2. In vitro transfection efficiency of the fusion protein of the present invention

[0056] Figure 2A and Figure 2B This demonstrates the in vitro transfection efficiency of the fusion protein, also known as preloaded CD3ε-NbEVs. Figure 2A The diagram illustrates the transfection efficiency of the preloaded CD3ε-Nb EVs construct. After one day of treatment with both control and preloaded CD3ε-Nb EVs (2 x 10^9 particles), peripheral blood mononuclear cells (PBMCs) were obtained, and CD3 levels were determined by flow cytometry using specific antibodies against VHH and CD3. + and CD3 - Nb-CAR expression level on cells (2B).

[0057] Example 3. In vitro treatment of the fusion protein can enhance the antitumor activity of PBMCs.

[0058] Figure 3In vitro treatment with the fusion protein, also known as preloaded CD3ε-Nb EVs, enhanced the antitumor activity of PBMCs. After one day of treatment with both control and preloaded CD3ε-Nb EVs (2 x 10^9 particles), PBMCs were co-cultured with COLO205 (human colon cancer cell line), H1975 (human lung adenocarcinoma cell line), U87 (human glioblastoma cell line), and Panc-1 (human pancreatic cancer cell line) at effector cell / target cell (E:T) ratios of 1:1, 3:1, 6:1, and 10:1. Induced cytotoxicity was analyzed by flow cytometry using the LIVE / DEAD Cell-Mediated Cytotoxicity Assay Kit, according to the user's instructions.

[0059] The results of this embodiment confirm that in vitro treatment of the fusion protein can enhance the antitumor activity of PBMCs.

[0060] Example 4. In vivo transfection efficiency of the fusion protein of the present invention

[0061] Figure 4A and Figure 4B This demonstrates the in vivo transfection efficiency of the fusion protein, also known as preloaded CD3ε-Nb extracellular vesicles. Figure 4A Schematic diagram of transfection efficiency of preloaded CD3ε-Nb EVs in humanized NSG mice (huNSG) with COLO 205 tumor PBMCs. After 3 days of infusion of either control or preloaded CD3ε-Nb EVs (1 x 10^10 particles), leukocyte layers were collected from mice, and CD3 was determined by flow cytometry using specific antibodies against VHH and CD3. + and CD3 - Nb-CAR expression levels on cells (4B).

[0062] Example 5. Fusion protein therapy can enhance in vivo antitumor activity against solid tumors.

[0063] Figures 5A-5D This study demonstrates the in vivo antitumor efficiency of the fusion protein, also known as preloaded CD3ε-Nb extracellular vesicles. Figure 5AA schematic diagram evaluating the antitumor efficacy of preloaded CD3ε-Nb EVs in humanized NSG mice (huNSG) with COLO 205 tumor PBMCs. Seven days after implantation of COLO 205 tumor cells (1 x 10^6 cells / intraperitoneal injection (IP)), mice were injected via the tail vein with 5 x 10^6 PBMCs. The next day, mice were injected weekly with or without preloaded CD3ε-Nb EVs (1 x 10^10 particles) for four weeks. The efficacy was assessed using a non-invasive in vivo molecular imaging system (IVIS) via bioluminescent channels (…). Figure 5B , 5C Monitor tumor growth rate and record survival rate. Figure 5D ).

[0064] The results of this embodiment confirm that fusion protein therapy can enhance the anti-tumor activity against solid tumors in vivo.

[0065] Example 6. Intramuscular injection of in vivo fusion protein particles to induce in vivo fusion protein secretion

[0066] Figure 6A and Figure 6B This illustrates the in vivo production of fusion proteins, also known as preloaded CD3ε-Nb extracellular vesicles. (6A) Schematic diagram of in vivo production of preloaded CD3ε-Nb EVs in PBMC humanized NSG mice (huNSG) with COLO 205 tumors. Plasma was collected from mice 7 days after intramuscular injection of either the vector control group or the preloaded CD3ε-Nb EV DNA transgene (1 mg / kg) in the right hind leg. The expression level of the CD3ε Nb moiety on the EV particles was determined by flow cytometry analysis using specific antibodies against VHH and CD3εNb, and the size of the identified beads was determined (6B).

[0067] The results of this embodiment confirm that intramuscular injection of in vivo fusion protein particles induces in vivo secretion of fusion proteins.

[0068] Example 7. The hind leg treated with in vivo fusion protein particles was able to secrete fusion protein.

[0069] Figures 7A-7DDemonstrates in vivo generation of a fusion protein, also known as preloaded CD3ε-Nb EVs, from the hind leg via intramuscular injection. (7A) Seven days after intramuscular injection of either the vector control group or preloaded CD3ε-Nb EV DNA transgene (1 mg / kg) into the right hind leg, mice were sacrificed and muscle tissue from both hind legs was collected. These hind leg tissues were then placed in 6-well plates supplemented with 1 ml of serum-free RPMI 1640 medium. The supernatant was collected the next day and filtered through a 0.22 μm filter membrane and a 30 kDa cutoff column. The supernatant was then analyzed by dot plotting (…). Figure 7B The purified supernatant was analyzed by flow cytometry using specific antibodies against VHH and CD3ε Nb, and the size of the beads was determined using specific bead measurements. Figure 7C ), or by using specific Taqman primers and probes via qPCR to detect the level of HLA-G Nb-CAR mRNA ( Figure 7D ).

[0070] The results of this embodiment confirm that the hind leg treated with in vivo fusion protein particles can secrete fusion proteins.

[0071] Example 8. Intramuscular injection of in vivo fusion protein particles can increase the frequency of Nb-CAR expression cells in vivo.

[0072] Figure 8A and Figure 8B This study demonstrates the in vivo generation of Nb-CAR-expressing T cells via intramuscular injection of a fusion protein transgene, where the fusion protein is also known as preloaded CD3ε-Nb EVs. Figure 8A This diagram illustrates the in vivo generation of Nb-CAR-expressing T cells by injecting pre-loaded CD3ε-Nb EV transgene into PBMC-humanized NSG mice (huNSG) with COLO 205 tumors. Seven days after intramuscular injection of either the vector control group or the pre-loaded CD3ε-NbEV DNA transgene (1 mg / kg) into the right hind leg, the frequency of Nb-CAR-expressing cells in the leukocyte layer was determined by flow cytometry using specific antibodies against VHH and CD3. Figure 8B ).

[0073] The results of this embodiment confirm that intramuscular injection of in vivo fusion protein particles can increase the frequency of T cells expressing Nb-CAR in vivo.

[0074] Example 9. Intramuscular injection of in vivo fusion protein particles to enhance in vivo anti-solid tumor activity

[0075] Figures 9A to 9DThis study demonstrates the antitumor effect induced by intramuscular injection of a fusion protein transgene, which is also known as preloaded CD3ε-Nb extracellular vesicles. Figure 9A A schematic diagram evaluating the antitumor activity of intramuscular injection of pre-loaded CD3ε-Nb EV transgene in PBMC-humanized NSG mice (huNSG) with COLO 205 tumors. Seven days after intraperitoneal injection of COLO 205 tumor cells (1 x 10^6 cells), mice were injected via tail vein with 5 x 10^6 PBMCs. The next day, pre-loaded CD3ε-Nb EV DNA transgene (1 mg / kg) was injected intramuscularly into the right hind leg of the mice, with or without injection. Tumor growth rate was monitored using an IVIS imaging system via a bioluminescent channel. Figure 9B , 9C ), and record their survival rate ( Figure 9D ).

[0076] The results of this embodiment confirm that intramuscular injection of in vivo fusion protein particles enhances in vivo anti-solid tumor activity.

[0077] In summary, the fusion protein of the present invention achieves the effects of treating cancer, regulating immunity, and activating immune cells, as illustrated in the above embodiments.

[0078] The above description is merely illustrative and not restrictive. Any equivalent modifications or alterations made without departing from the spirit and scope of this invention should be included within the scope of the appended claims.

Claims

1. A fusion protein comprising an anti-CD3 single-domain antibody, an exosomal protein, and an RNA-binding protein, wherein, The amino acid sequence of the anti-CD3 single-domain antibody is a heavy chain variable domain (VHH), and the exosomal protein is CD63.

2. The fusion protein according to claim 1, wherein, The anti-CD3 single-domain antibody binds specifically to CD3ε.

3. The fusion protein according to claim 1, wherein, The anti-CD3 single-domain antibody is an anti-T cell nanobody.

4. The fusion protein according to claim 3, wherein, The anti-CD3 single-domain antibody contains the amino acid sequence shown in SEQ ID NO:

2.

5. The fusion protein according to claim 1, wherein, The N-terminal amino acid sequence of CD63 is shown in SEQ ID NO: 3, and the C-terminal amino acid sequence of CD63 is shown in SEQ ID NO:

4.

6. The fusion protein according to claim 1, wherein, The RNA-binding protein is a heterogeneous nucleonucleotide protein (hnRNP) A2B1.

7. The fusion protein according to claim 6, wherein, The hnRNPA2B1 contains the amino acid sequence shown in SEQ ID NO:

5.

8. The fusion protein according to claim 1, comprising the amino acid sequence shown in SEQ ID NO:

1.

9. The fusion protein of claim 1, further comprising an extracellular vesicle (EV) sorting motif.

10. The fusion protein according to claim 9, wherein, The nucleotide sequence encoding the amino acid sequence of the extracellular vesicle sorting motif is shown in SEQ ID NO:

6.

11. The fusion protein of claim 9, further comprising an HLA-G chimeric antigen receptor.

12. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. Use of a fusion protein according to any one of claims 1 to 11 in the preparation of pharmaceuticals for treating cancer, modulating immunity and activating immune cells.

14. The use according to claim 13, wherein, The fusion protein enhances the antitumor activity of peripheral blood mononuclear cells.

15. The use according to claim 13, wherein, The cancer in question is colorectal cancer, lung adenocarcinoma, glioblastoma, or pancreatic cancer.

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