Fusion protein, its nucleic acid coding sequence and application

Abstract

The present invention provides a fusion protein, its nucleic acid coding sequence and applications. Through a variety of efficacy experiments, the fusion protein of the present invention achieves the effects of treating cancer, immunomodulation and activating immune cells.

Description

Technical Field

[0001] The present invention relates to a fusion protein, its nucleic acid coding sequence, and applications thereof. Background Art

[0002] Cancer, also known as malignant tumor, is the abnormal proliferation of cells, and these proliferating cells may invade other parts of the body. It is a disease caused by the disorder of the mechanism controlling cell division and proliferation. The number of people suffering from cancer worldwide has been on the rise. Cancer is one of the top ten causes of death and has ranked first among the top ten causes of death for many consecutive years.

[0003] Conventional tumor treatment methods include surgical treatment, radiotherapy, chemotherapy, and targeted therapy, etc. Tumor immunotherapy is another method for treating tumors other than the above-mentioned treatment methods. It activates the patient's own immune system, uses tumor cells or tumor antigen substances to induce specific cellular and humoral immune responses in the body, enhances the body's anti-cancer ability, and prevents the growth, spread, and recurrence of tumors, so as to achieve the purpose of eliminating or controlling tumors. However, the current tumor treatment methods still have problems such as ineffective treatment and strong side effects, and even may give rise to other immune-related diseases.

[0004] CD3ε (CD3 epsilon) is a transmembrane protein found on T cells and has been found to be associated with tumors and the regulation of immune function. Therefore, some researchers have been committed to developing CD3ε as a target molecule for identifying tumors and regulating immune function and finding out whether these target molecules have the potential to become anti-cancer drugs or immunomodulatory drugs. In addition, CD63 is a protein antigen encoded by the CD63 gene in the human body. CD63 mainly appears on the surface of extracellular vesicles and also on the surface of ordinary cell membranes, and its encoding gene is related to tumor development.

[0005] To solve the above problems, those skilled in the art urgently need to develop novel and more effective pharmaceuticals for treating cancer, immunomodulation, and activating immune cells to benefit the vast population in need. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a fusion protein, comprising an anti-CD3 single-domain antibody and an exosome protein, wherein the anti-CD3 single-domain antibody comprises the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0007] In an embodiment of the present invention, the anti-CD3 single-domain antibody specifically binds to a CD3ε (CD3 epsilon).

[0008] In one embodiment of the present invention, the exosomal protein is CD63.

[0009] In one embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:4.

[0010] In one embodiment of the present invention, the amino acid sequence shown in SEQ ID NO:1 is Complementarity Determining Region 1 (CDR1), the amino acid sequence shown in SEQ ID NO:2 is CDR2, and the amino acid sequence shown in SEQ ID NO:3 is CDR3.

[0011] In one embodiment of the present invention, the anti-CD3 single domain antibody is an anti-T cell nanobody.

[0012] Another object of the present invention is to provide an isolated nucleic acid encoding the amino acid sequence of a fusion protein as described above.

[0013] In one embodiment of the present invention, the nucleotide sequence of the isolated nucleic acid is as shown in SEQ ID NO:5.

[0014] Another object of the present invention is to provide a pharmaceutical composition comprising a fusion protein as described above and a pharmaceutically acceptable carrier.

[0015] Another object of the present invention is to provide the use of a fusion protein as described above for the preparation of a medicament for treating cancer, immunomodulation and activating immune cells.

[0016] In one embodiment of the present invention, the cancer is treated by infiltrating the fusion protein into the interior of a solid tumor and secreting a bispecific T-cell engager (BiTE) and activating peripheral immune cells.

[0017] In summary, the efficacy of the fusion protein of the present invention lies in: through surface plasmon resonance (SPR), cytotoxicity analysis, animal experiments, electroporation experiments, transfection efficiency tests, flow cytometry analysis, and in vivo chimeric antigen receptor T-cell therapy (CAR-T), and by penetrating the fusion protein into solid tumors and secreting bispecific T-cell engager (BiTE) and activating peripheral immune cells, the effects of treating cancer, immunomodulation, and activating immune cells are achieved.

[0018] The following will further illustrate the implementation manners of the present invention. The following listed embodiments are used to clarify the present invention and are not used to limit the scope of the present invention. Any person skilled in this art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to what is defined by the claims. Brief Description of the Drawings

[0019] Figure 1 It is a schematic structural diagram of the fusion protein of the present invention.

[0020] Figure 2 It shows the binding position of exosomal protein CD63 and anti-CD3 single-domain antibody, where the arrow is the embedding position of the anti-CD3 single-domain antibody.

[0021] Figure 3 It shows the results of surface plasmon resonance (SPR) analysis of the Exo of the fusion protein.

[0022] Figure 4 It is a schematic diagram of the utility of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where CD3εNb represents anti-CD3 single-domain antibody, HEK-293T represents human embryonic kidney cell, Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, and Exo represents exosome.

[0023] Figure 5Another schematic diagram of the utility of the fusion protein in the treatment of cancer, immune regulation, and activation of immune cells, 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 microscope.

[0024] Figure 6 Results of transmission electron microscope (TEM) showing the fusion protein, where CD3εNb represents anti-CD3 single-domain antibody, Exo represents exosome, CAR represents chimeric antigen receptor, and BiTE represents bispecific T-cell engager.

[0025] Figure 7 Shows the efficiency of linear Nb-CAR.BiTE DNA encapsulation into Exo by electroporation, 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 qPCR represents quantitative polymerase chain reaction.

[0026] Figure 8To show the efficiency of electroporated Exo coated with Nb-CAR.BiTE DNA transfected into peripheral blood mononuclear cells (PBMCs), 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 measurement.

[0027] Figure 9 Another schematic diagram for testing the efficiency of electroporated Exo coated with Nb-CAR.BiTE DNA transfected 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 CD3εNb represents anti-CD3 single domain antibody.

[0028] Figure 10 To show the efficiency of electroporated Exo coated with linear Nb-CAR.BiTE DNA transfected into PBMCs, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody.

[0029] Figure 11 To show the efficiency of electroporated Exo coated with Nb-CAR.BiTE DNA transfected 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 measurement.

[0030] Figure 12Another schematic diagram for testing the efficiency of electroporated Exo coated with Nb-CAR.BiTE DNA transfected 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 measurement.

[0031] Figure 13 Showing the efficiency of electroporated Exo coated with linear Nb-CAR.BiTE DNA transfected 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.

[0032] Figure 14 Another schematic diagram for testing the efficiency of electroporated Exo coated with linear Nb-CAR.BiTE DNA transfected into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single-domain antibody.

[0033] Figures 15A - 15C Showing that Exo loaded with linear Nb-CAR.BiTE DNA transfected into PBMCs induces excellent cytolytic activity against solid tumor cells, where CRC represents colorectal cancer, GBM represents glioblastoma multiforme, NSCLC represents non-small cell lung cancer, and E:T ratio represents effector-to-target ratio.

[0034] Figure 16Show the results of the fusion protein in in vivo chimeric antigen receptor T-cell therapy (CAR-T), where the peripheral blood mononuclear cell (PBMC)-huNSG mouse model was used. huPBMC represents human peripheral blood mononuclear cells, I.P. represents intraperitoneal, I.V. represents intravenous, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3εNb represents anti-CD3 single-domain antibody, luc represents luciferase. The fourth dose has been administered and blood has been collected to test the transfection efficiency.

[0035] Figure 17 Another schematic diagram showing the utility of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3εNb represents anti-CD3 single-domain antibody.

[0036] Figure 18 Another schematic diagram showing the utility of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where Nb represents nanobody and CAR represents chimeric antigen receptor.

[0037] Figure 19 Another schematic diagram showing the utility of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3εNb represents anti-CD3 single-domain antibody, and BiTE represents bispecific T-cell engager.

[0038] Figure 20Another schematic diagram showing the utility of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3εNb represents anti-CD3 single-domain antibody, BiTE represents bispecific T-cell engager, and PBMC represents peripheral blood mononuclear cell. Detailed implementation mode

[0039] Definition

[0040] The numerical values used in this article are approximate values. All experimental data are expressed within the range of ±20%, preferably within the range of ±10%, and most preferably within the range of ±5%.

[0041] Unless otherwise specified in the text, the terms "a", "the", and similar terms used in this specification (especially in the following claims) should be understood to include both singular and plural forms.

[0042] As used herein, the terms "CD3e" and "CD3ε" can be used interchangeably.

[0043] As used herein, the terms "CD3e nanobody", "CD3e nb", "CD3e Nb", "CD3e nanobody", "anti-CD3ε nanobody", "anti-CD3 single-domain antibody", and "anti-T cell nanobody" can be used interchangeably.

[0044] As used herein, "treating" or "treatment" means alleviating, reducing, ameliorating, relieving, or controlling one or more clinical signs of a disease or disorder, and lowering, stopping, or reversing the progression of the severity of a condition or symptom being treated.

[0045] According to the present invention, the pharmaceutical can be manufactured into a dosage form suitable for parenteral administration by techniques well known to those skilled in the art, which includes, but is 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.

[0046] The pharmaceutical according to the present invention can be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

[0047] The pharmaceutical product according to the present invention may contain a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may contain one or more reagents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these reagents fall within the professional competence and routine techniques of those skilled in the art.

[0048] According to the present invention, the pharmaceutically acceptable carrier contains a solvent selected from the group consisting of: water, normal saline, phosphate buffered saline (PBS), sugar solution, aqueous solution containing alcohol, and combinations thereof.

[0049] As used herein, terms such as "nucleic acid", "nucleic acid sequence", or "nucleic acid fragment" mean a deoxyribonucleotide sequence or ribonucleotide sequence in single-stranded or double-stranded form, and include known naturally occurring nucleotides or artificial chemical mimics. As used herein, the term "nucleic acid" may be used interchangeably with "gene", "cDNA", "mRNA", "oligonucleotide", and "polynucleotide".

[0050] The present invention is further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of the present invention. The scope of the present invention is as defined by the scope shown in the claims.

[0051] Example 1. Preparation of the fusion protein of the present invention

[0052] 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 (generations 4-25) and HEK-293T stable cell lines expressing anti-CD3 single-domain antibody-CD63 chimeric proteins were loaded into COL2.5 NGCs and contained 500 mL of DMEM medium (containing 50 mL of exosome-free FBS) (Thermo Fisher Scientific). After culturing for 3 days, the medium was centrifuged at 2000 g for 15 minutes to remove cell debris, and then filtered through a 0.2 μm filter paper. Then, the supernatant was concentrated by ultrafiltration ( Ultra, 30 kDa, Merck Millipore) at 5000 g for 8 minutes. The collected supernatant was processed by tangential flow filtration (MAP.03-plus TFF System, Lefo Science). Subsequently, the supernatant of the parental HEK-293T cells was filtered through a membrane with a cut-off value of 300 kDa and then resuspended in PBS; the supernatant from the HEK-293T stable cells expressing anti-CD3 single-domain antibody-CD63 chimeric proteins 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 analyzed by (Particle Metrix GmbH) to analyze their size distribution and concentration.

[0053] The anti-CD3 single-domain antibody comprises amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein the amino acid sequence shown in SEQ ID NO: 1 is complementarity determining region 1 (CDR1), the amino acid sequence shown in SEQ ID NO: 2 is CDR2, and the amino acid sequence shown in SEQ ID NO: 3 is CDR3. The amino acid sequence of the fusion protein of the present invention is as shown in SEQ ID NO: 4.

[0054] The present invention also provides an isolated nucleic acid encoding the amino acid sequence of the fusion protein as described above. The nucleotide sequence of the isolated nucleic acid is as shown in SEQ ID NO: 5.

[0055] The amino acid sequence of the anti-CD3 single-domain antibody is as shown in SEQ ID NO:6. The nucleotide sequence encoding the amino acid sequence of the anti-CD3 single-domain antibody is as shown in SEQ ID NO:7. The amino acid sequence of the anti-CD3 single-domain antibody is the heavy chain variable domain (VHH).

[0056] A schematic diagram of the structure of the fusion protein of the present invention is shown in Figure 1 , wherein the anti-CD3 single-domain antibody specifically binds to CD3ε (CD3epsilon).

[0057] Figure 2 The binding position of exosome protein CD63 and the anti-CD3 single-domain antibody is shown, where the arrow is the embedding position of the anti-CD3 single-domain antibody.

[0058] Example 2. Surface plasmon resonance (SPR) analysis of the fusion protein of the present invention

[0059] In this example, the experimental procedure for surface plasmon resonance (SPR) analysis of the exosome of the fusion protein is as follows. CM5 and NTA chips (research grade) were used for SPR analysis by BIAcore T200 (Biacore - GE Healthcare, Piscataway, NJ). Briefly, protein (CD3ε recombinant protein) samples were diluted in 10 mM buffer (pH 4.0, 5.5 or 6.0) in a concentration range of 20 μg / mL to provide maximum surface retention on the chip after surface preparation, and conditions with a higher ligand surface concentration on the chip were selected (anti-CD3 single-domain antibody: 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 nM). Then regeneration reconnaissance and surface performance testing were carried out, following regeneration reconnaissance and surface performance testing and then selecting a regeneration method to run the experiment. Then binding analysis (BINDING ANALYSIS) and direct binding (DIRECT BINDING) were selected to study protein binding. Kinetic analysis was selected and mass transfer (MASS TRANSFER) was selected for kinetic analysis along with the binding experiment. Data analysis and determination of kinetic constants.

[0060] The results of the Exo surface plasmon resonance (SPR) analysis of the fusion protein are shown in Figure 3 . It can be seen from Figure 3 that when the fusion protein (1x10 11)Coated on the CM5 chip, and then its binding affinity was measured using recombinant CD3ε protein (700, 350, 175, 87.5, 43.8, 21.9 nM). The KD was determined to be 2.1 nM.

[0061] Example 3. Evaluation of the efficacy of the fusion protein of the present invention in treating cancer, immunomodulation, and activating immune cells

[0062] This example evaluates the efficacy of the fusion protein in treating cancer, immunomodulation, and activating immune cells.

[0063] Figure 4 It is a schematic diagram of the efficacy of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where CD3εNb represents an anti-CD3 single-domain antibody, HEK-293T represents human embryonic kidney cells, Nb represents a nanobody, CAR represents a chimeric antigen receptor, BiTE represents a bispecific T-cell engager, and Exo represents an exosome. As can be seen, Figure 4 the sequence encoding the anti-CD3 single-domain antibody is inserted into the second outer loop of CD63 to form a CD63 chimeric protein with the anti-CD3 single-domain antibody exposed on the cell surface. Then this construct is transfected into HEK-293T to produce exosomes expressing the anti-CD3 single-domain antibody. Then the harvested exosomes are purified using a VHH-capsule column and then loaded with DNA or mRNA encoding Nb-CAR.BiTE. This Nb-CAR.BiTE-CD3εNb-Exo is directly injected into a mouse model. We hypothesize that these exosomes selectively reprogram CD3 + T cells to express Nb-CAR and secrete Nb-BiTE to combat solid tumor cells in vivo.

[0064] Figure 5Another schematic diagram of the utility of the fusion protein in treating cancer, immunomodulation, and activating immune cells, 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, TEM represents transmission electron microscope, IFNγ represents interferon γ, TNFα represents tumor necrosis factor α, PFN represents perforin, GzmB represents Granzyme B, a serine protease that mediates the apoptotic signaling pathway of cytotoxic T lymphocytes and natural killer cells, SSC represents side scatter light, and VHH represents heavy chain variable domain. As can be seen from Figure 5 After electroporating Exo and CAR.BiTE DNA at a ratio of (3x10 8 Exo vs. 2 μg DNA) with the electroporation program code CM137 of LONZA 4D-Nucleofector, recovering at 4°C for one hour, morphological observation was performed using an electron microscope, or transfected into human PBMC or whole blood for 48 hours, and then co-cultured with tumor cells to confirm the change in the cytotoxic ability against tumor cells by LIVE / DEAD Cell-Mediated Cytotoxicity Assay; or the expression levels of CD3, CD4, CD8, VHH, etc. on the cells were analyzed by flow cytometry after labeling with fluorescent antibodies. The above experiments can confirm the transfection effect of the fusion protein-coated CAR.BiTE DNA.

[0065] For transmission electron microscope (TEM) analysis, HEK-293T-derived exosomes were fixed overnight at 4°C with 1% glutaraldehyde after isolation. After washing, the exosomes were loaded onto formvar carbon-coated grids and negatively stained with an aqueous solution of phosphotungstic acid for 1 minute. The ultrastructure of these exosomes was analyzed by TEM (JEOL JEM-1400, Tokyo, Japan).

[0066] Figure 6 Transmission electron microscope (TEM) results showing 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. From Figure 6 It can be seen that CAR.BiTE DNA@CD3εNb-Exo maintains the ultrastructure and morphology of exosomes. TEM was used to evaluate unmodified and CD3εNb-engineered Exo (with or without electroporation with CAR.BiTE) at 100,000X to visualize the DNA expressing CAR.BiTE.

[0067] HEK293-derived Exo can encapsulate DNA expressing CAR through electroporation. Using the LONZA 4D-Nucleofector, unmodified Exo and the fusion protein were electroporated with the CAR.BiTE expression vector at a ratio of 3x10 8 Exo: 2 μg DNA. The Exo was then incubated with DNase (1000 IU) for 30 minutes, and the incorporated CAR.BiTE DNA was quantified by qPCR using specific primers or by spectrophotometry at O.D. 260 and 280 nm.

[0068] Figure 7 Showing the efficiency of encapsulation of linear Nb-CAR.BiTE DNA into Exo by electroporation, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3εNb represents an anti-CD3 single-domain antibody, and qPCR represents quantitative polymerase chain reaction. From Figure 7 It can be seen that from the spectrophotometry and qPCR results, the electroporation method enables the encapsulation of approximately 100 ng of CAR.BiTE DNA per 3x10 8 Exo.

[0069] Unmodified Exo or the fusion protein was electroporated at 3x10 in the presence or absence of the CAR.BiTE expression vector 8Electroporation was performed at a ratio of Exo: 2 μg DNA. After purification, these Exos were added to 5 x 10 5 PBMCs for 48 hours. After staining on ice for 45 minutes, specific antibodies were used to measure the expression levels of Nb-CAR on CD3 + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + 、CD56 + 、TCRγδ + 、CD14 + 、CD19 + and CD66b + cells by flow cytometry.

[0070] Figure 8 Shows the efficiency of transfection of test Nb-CAR.BiTE DNA-coated electroporated Exos into peripheral blood mononuclear cells (PBMCs), 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 measurement. As Figure 8 can be seen, compared with unmodified Exo (293T Exo), CAR.BiTEDNA@CD3e Nb-Exo showed a more efficient transfection rate on CD3 + cells than on CD3 - cells. This result supports the selective delivery of the transgene by the fusion protein into CD3 + cells.

[0071] Unmodified Exo or the fusion protein was electroporated at a ratio of 3x10 8 Exo: 2 μg DNA. After purification, these Exos were added to 5 x 10 5 PBMCs for 48 hours. After staining on ice for 45 minutes, specific antibodies were used to measure the expression levels of Nb-CAR on CD3 + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + cells by flow cytometry.

[0072] Figure 9 Another schematic diagram for testing the transfection efficiency of electroporated Exo coated with Nb-CAR.BiTE DNA 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 CD3εNb represents anti-CD3 single domain antibody. By Figure 9 It can be seen that, compared with unmodified Exo (293T Exo), CAR.BiTE DNA@CD3εNb-Exo shows more efficient transfection rates on CD3 + / CD4 + and CD3 / CD8 + T cells. This result supports that the fusion protein selectively delivers the transgene into CD4 + and CD8 + cells.

[0073] Unmodified Exo or the fusion protein was electroporated at a ratio of 3x10 8 Exo: 2 μg DNA. After purification, these Exo were added to 5x10 5 PBMCs for 48 hours. After staining on ice for 45 minutes, the expression levels of Nb-CAR on CD3 + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + cells were measured by flow cytometry using specific antibodies.

[0074] Figure 10 Shows the efficiency of testing the transfection of electroporated Exo coated with linear Nb-CAR.BiTE DNA into PBMCs, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. By Figure 10 It can be seen that CAR.BiTE DNA@CD3εNb-Exo on CD3 + , CD3 + / CD4 + and CD3 / CD8 +More efficient transfection rates were exhibited on T cells. This result supports that the fusion protein selectively delivers the transgene to CD4 + and CD8 + cells.

[0075] Unmodified Exo or the fusion protein was electroporated at a ratio of 3x10 8 Exo: 2 μg DNA with or without the CAR.BiTE expression vector. After purification, these Exos were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, the expression levels of Nb-CAR on CD3 + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + cells were measured by flow cytometry using specific antibodies.

[0076] Figure 11 Shows the efficiency of electroporated Exos transfected with test Nb-CAR.BiTE DNA 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 measurement. As Figure 11 can be seen, this result supports that the fusion protein selectively delivers the transgene to CD3 + cells in whole blood.

[0077] Unmodified Exo or the fusion protein was electroporated at a ratio of 3x10 8 Exo: 2 μg DNA with or without the CAR.BiTE expression vector. After purification, these Exos were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, the expression levels of Nb-CAR on CD3 + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + 、CD56 + 、TCRγδ + 、CD14 + 、CD19 + and CD66b + cells were measured by flow cytometry using specific antibodies.

[0078] Figure 12 Another schematic diagram for testing the efficiency of electroporated Exo coated with Nb-CAR.BiTE DNA transfected 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 measurement. As Figure 12 can be seen, this result supports that the fusion protein selectively delivers the transgene to CD3 of whole blood + / CD4 and CD3 + / CD8 + cells.

[0079] Unmodified Exo or the fusion protein was electroporated at a ratio of 3x10 8 Exo: 2 μg DNA, and after purification, these Exo were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, the expression level of Nb-CAR on CD3 + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + cells was measured by flow cytometry using specific antibodies.

[0080] Figure 13 Showing the efficiency of testing the transfection of electroporated Exo coated with linear Nb-CAR.BiTE DNA 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. As Figure 13 can be seen, this result supports that the fusion protein selectively delivers the transgene to CD3 of whole blood + 、CD3 + / CD4 and CD3 + / CD8 + cells.

[0081] Unmodified Exo or the fusion protein was electroporated at a ratio of 3x10 8Exo: Electroporation was performed at a ratio of 2 μg DNA. After purification, these Exos were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, specific antibodies were used to measure CD3 by flow cytometry. + CD3 - CD3 + / CD4 + CD3 + / CD8 + The expression level of Nb-CAR on cells.

[0082] Figure 14 Another schematic diagram for testing the efficiency of electroporated Exos transfected with linear Nb-CAR.BiTE DNA into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. As Figure 14 can be seen, this result supports that the fusion protein selectively delivers the transgene to CD3 + CD3 + / CD4 and CD3 + / CD8 + cells.

[0083] Cytotoxic killing assay: CD3 + cells treated with CAR.BiTE-CD3εNb-Exo or unmodified exosomes were used as effector cells. Target cells (tumor cell lines) and effector cells were co-cultured at 37 °C for 24 to 72 hours according to the specified effector cell / target cell (E:T) ratio (1:1 to 50:1). For the viability assay of live / dead cells, 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 label dead cells. Dead tumor cells were determined as green-fluorescent + / red-fluorescent + cells according to the instructions of the manufacturer (Thermo Fisher Scientific). The cell killing rate was expressed as a percentage of the total cell population.

[0084] Figures 15A - 15CExosomes transfected with linear Nb-CAR.BiTE DNA into PBMCs showed excellent cytolytic activity against solid tumor cells, where CRC represents colorectal cancer, GBM represents glioblastoma multiforme, NSCLC represents non-small cell lung cancer, and E:T ratio represents effector-to-target ratio. By Figures 15A - 15C As can be seen, by comparing with unmodified exosomes (293T Exo) loaded or not loaded with CAR.BiTE DNA and CD3e Nb-Exo not loaded with DNA, PBMCs treated with CD3e Nb-Exo loaded with CAR.BiTE DNA exerted more effective cytotoxic killing ability against these tumor cells. In addition, compared with PBMCs treated with exosomes not loaded with CAR.BiTE DNA, PBMCs treated with 293T Exo loaded with CAR.BiTE DNA also showed excellent cytotoxic killing ability.

[0085] Figure 16 Schematic diagram of the in vivo anti-tumor efficacy process of CAR.BiTE DNA@CD3εNb-Exo. Seven days after intraperitoneal implantation of COLO 205-luc cells (1x 10 6 cells), huPBMCs (5x 10 6 ) were injected into the tail veins of mice (n = 5). The next day, the mice were treated with unmodified exosomes or CD3εNb-Exo loaded or not loaded with CAR.BiTE DNA (3x 10 10 / mouse) once a week for four weeks. The IVIS system monitored tumor growth by detecting bioluminescence signals. Seven days after the last injection, the mice were sacrificed, spleen cells were harvested, and the expression levels of Nb-CAR on each type of immune cell were measured by flow cytometry using specific antibodies against VHH, CD3, CD56, TCRγδ, CD14, CD19, and CD66b.

[0086] Figure 16Show the results of the fusion protein in in vivo chimeric antigen receptor T-cell therapy (CAR-T), where a peripheral blood mononuclear cell (PBMC)-huNSG mouse model was used, huPBMC represents human peripheral blood mononuclear cells, I.P. represents intraperitoneal, I.V. represents intravenous, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3εNb represents anti-CD3 single-domain antibody, luc represents luciferase, the fourth dose has been administered, and blood has been collected to test the transfection efficiency.

[0087] Figure 17 Schematic diagram of the in vivo anti-tumor efficacy process of CAR.BiTE DNA@CD3εNb-Exo. Seven days after intraperitoneal implantation of COLO 205-luc cells (1 x 10 6 cells), huPBMC (5 x 10 6 ) were injected into the tail vein of mice (n = 5). The next day, the mice (3 x 10 10 / mouse) were treated with or without unmodified Exo or CD3εNb-Exo loaded or not loaded with CAR.BiTE DNA once a week for four weeks. The IVIS system monitored tumor growth by detecting bioluminescence signals.

[0088] Figure 17 Another schematic diagram showing the utility of the fusion protein in cancer treatment, immunomodulation, and activation of immune cells, where CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3εNb represents anti-CD3 single-domain antibody. As Figure 17 can be seen, CAR.BiTE DNA@CD3e Nb-Exo showed excellent anti-tumor activity compared to other mouse groups.

[0089] Figure 18 Schematic diagram of the in vivo anti-tumor efficacy process of CAR.BiTE DNA@CD3εNb-Exo. Seven days after intraperitoneal implantation of COLO 205-luc cells (1 x 10 6 cells), huPBMC (5 x 106 )。On the second day, mice (3 x 10 10 / mouse) were treated with or without unmodified Exo or CD3εNb-Exo loaded or not loaded with CAR.BiTE DNA once a week for four weeks. Seven days after the last injection, the mice were sacrificed, submandibular blood was harvested, and the expression level of Nb-CAR on each type of immune cell was measured by flow cytometry using specific antibodies against VHH and CD3.

[0090] Figure 18 Another schematic diagram showing the utility of the fusion protein 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 CD3 + 、CD3 + / CD4 and CD3 + / CD8 + cells in the blood cells of PBMC humanized NSG mice.

[0091] Figure 19 Schematic diagram of the in vivo anti-tumor efficacy process of CAR.BiTE DNA@CD3εNb-Exo. Seven days after intraperitoneal implantation of COLO 205-luc cells (1 x 10 6 cells), huPBMC (5 x 10 6 ) was injected into the tail vein of mice (n = 5). On the second day, mice (3 x 10 10 / mouse) were treated with or without unmodified Exo or CD3εNb-Exo loaded or not loaded with CAR.BiTE DNA once a week for four weeks. Seven days after the last injection, the mice were sacrificed, submandibular blood was harvested, and the expression level of Nb-CAR on each type of immune cell was measured by flow cytometry using specific antibodies against VHH and CD3.

[0092] Figure 19 Another schematic diagram showing the utility of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3εNb represents anti-CD3 single domain antibody, and BiTE represents bispecific T-cell engager.

[0093] Figure 20Another schematic diagram showing the utility of the fusion protein in treating cancer, immunomodulation, and activating immune cells, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3εNb represents anti-CD3 single-domain antibody, BiTE represents bispecific T-cell engager, and PBMC represents peripheral blood mononuclear cell. From Figure 19 and Figure 20 it can be seen that the fusion protein selectively delivers the transgene to CD3 + cells in the blood cells of PBMC humanized NSG mice.

[0094] In summary, the fusion protein of the present invention can achieve the effects of treating cancer, immunomodulation, and activating immune cells through surface plasmon resonance, cytotoxicity analysis, animal experiments, electroporation experiments, transfection efficiency tests, flow cytometry analysis, and in vivo chimeric antigen receptor T cell therapy, and by infiltrating the fusion protein into solid tumors and secreting bispecific T-cell engager antibodies and activating peripheral immune cells.

[0095] The above is only illustrative and not restrictive. Any equivalent modifications or changes made without departing from the spirit and scope of the present invention shall be included in the scope defined by the claims.

Claims

1. A fusion protein comprising an anti-CD3 single domain antibody and an exosome protein, wherein: The anti-CD3 single domain antibody comprises the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the amino acid sequence of the fusion protein is SEQ ID NO:

4.

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

3. The fusion protein according to claim 1, wherein The exosomal protein is CD63.

4. The fusion protein according to claim 1, wherein The amino acid sequence of SEQ ID NO: 1 is the complementarity determining region 1 (CDR1), the amino acid sequence of SEQ ID NO: 2 is CDR2, and the amino acid sequence of SEQ ID NO: 3 is CDR3.

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

6. An isolated nucleic acid encoding an amino acid sequence of a fusion protein comprising an anti-CD3 single domain antibody and an exosome protein, wherein: The anti-CD3 single domain antibody comprises the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the nucleotide sequence of the isolated nucleic acid consists of SEQ ID NO:

5.

7. A pharmaceutical composition comprising the fusion protein according to claim 1 and a pharmaceutically acceptable carrier.

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