Fusion proteins and their nucleic acid coding sequences, and uses thereof

Abstract

The present invention provides fusion proteins and their nucleic acid coding sequences, as well as uses thereof. [Solution] Through various efficacy experiments, the fusion protein of the present invention has been shown to have the effects of cancer treatment, immune regulation, and immune cell activation.

Description

[Technical field]

[0001] The present invention relates to fusion proteins and their nucleic acid coding sequences, as well as uses thereof. [Background technology]

[0002] Cancer, also known as malignant tumor, is a disease caused by the dysfunction of the control of cell division and proliferation, which leads to abnormal cell proliferation and the ability of these proliferated cells to invade other parts of the body. The number of cancer patients is increasing worldwide, and it is one of the top 10 causes of death in Taiwan, and has been ranked first for many years.

[0003] Conventional tumor treatment methods include surgery, radiation therapy, chemotherapy, and targeted therapy. Tumor immunotherapy is a tumor treatment method other than the above-mentioned treatment methods, which activates the patient's own immune system, uses tumor cells or tumor antigen substances to induce the body's specific cellular immune and humoral immune responses, enhances the body's anti-cancer ability, and inhibits tumor growth, spread, and recurrence, thereby achieving the purpose of tumor removal or control. However, current tumor treatment methods have problems of low efficacy and strong side effects, and may even cause other immune-related diseases.

[0004] CD3ε (CD3 epsilon) is a type of transmembrane protein expressed in T cells, and has been found to be related to tumors and immune function regulation. Therefore, researchers are researching and developing CD3ε as a target molecule for tumor identification and immune function regulation, and exploring whether these target molecules have the potential to become anticancer drugs or immune regulators. In addition, CD63 is a type of protein antigen encoded by the CD63 gene in the human body. CD63 is mainly present on the surface of extracellular vesicles and also on the surface of normal cell membranes, and its coding gene is related to the development of tumors. Summary of the Invention [Problem to be solved by the invention]

[0005] To solve the above problems, those skilled in the art need to develop new and more effective pharmaceuticals for cancer treatment, immune regulation, and immune cell activation to benefit many patients in such need. [Means for solving the problem]

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

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

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

[0009] In one embodiment of the invention, the amino acid sequence of the fusion protein is the sequence 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 invention, the anti-CD3 single domain antibody is an anti-T cell nanobody.

[0012] It is another object of the present invention to provide an isolated nucleic acid encoding the amino acid sequence of the fusion protein.

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

[0014] Another object of the present invention is to provide a pharmaceutical composition comprising said fusion protein and a pharma- ceutically acceptable carrier.

[0015] Another object of the present invention is to provide a use of said fusion protein for the manufacture of a medicament for the treatment of cancer, immunomodulation and activation of immune cells.

[0016] In one embodiment of the present invention, the cancer is treated by infiltrating the fusion protein inside a solid tumor and secreting a bispecific T-cell engager (BiTE) to activate surrounding immune cells. Effect of the Invention

[0017] In summary, the effects of the fusion protein of the present invention have been demonstrated by surface plasmon resonance (SPR), cytotoxicity analysis, animal experiments, electroporation experiments, transfection efficiency measurement, flow cytometry analysis, and in vivo chimeric antigen receptor T-cell therapy (CAR-T), which shows that the fusion protein can penetrate into solid tumors and secrete bispecific T-cell engagers (BiTEs) to activate surrounding immune cells, thereby achieving the effects of cancer treatment, immune regulation, and immune cell activation. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram of the structure of the fusion protein of the present invention. [Diagram 2] Figure 2 shows the binding site of the exosome protein CD63 and the anti-CD3 single domain antibody. The arrow indicates the embedding site of the anti-CD3 single domain antibody. [Diagram 3]FIG. 3 shows the results of surface plasmon resonance (SPR) analysis of the fusion protein Exo. [Figure 4] Figure 4 is a schematic diagram showing the effects of the fusion protein on cancer treatment, immune regulation, and immune cell activation. 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. [Diagram 5] Figure 5 is another schematic diagram showing the effects of the fusion protein on cancer treatment, immune regulation and immune cell activation. PCR stands for polymerase chain reaction, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, PBMC stands for peripheral blood mononuclear cell, and TEM stands for transmission electron microscope. [Figure 6] Figure 6 shows the results of transmission electron microscopy (TEM) of the fusion protein. CD3εNb indicates anti-CD3 single domain antibody, Exo indicates exosome, CAR indicates chimeric antigen receptor, and BiTE indicates bispecific T-cell engager. [Figure 7]Figure 7 shows the efficiency of encapsulating linear Nb-CAR.BiTE DNA into Exo by electroporation. 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] Figure 8 shows the transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into peripheral blood mononuclear cells (PBMCs). 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 method. [Figure 9] Figure 9 is another schematic diagram showing the measurement of transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into peripheral blood mononuclear cells (PBMCs). Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. [Figure 10]Figure 10 shows the measurement of transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into PBMCs. Nb stands for nanobody, CAR stands for chimeric antigen receptor, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. [Figure 11] Figure 11 shows the measurement of transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. 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 method. [Figure 12] Figure 12 is another schematic diagram of the measurement of transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. 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 measurement. [Figure 13] Figure 13 shows the measurement of transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. [Figure 14]Figure 14 is another schematic diagram of the measurement of transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. Nb stands for nanobody, CAR stands for chimeric antigen receptor, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. [Figure 15A] Figure 15A shows that PBMCs transfected with Linear Nb-CAR.BiTE DNA-transduced Exo induce superior cytolytic activity against solid tumor cells. CRC stands for colorectal cancer, GBM stands for glioblastoma multiforme, NSCLC stands for non-small cell lung cancer, and E:T ratio stands for effector-to-target ratio. [Figure 15B] Figure 15B shows that PBMCs transfected with Linear Nb-CAR.BiTE DNA-transduced Exo induce superior cytolytic activity against solid tumor cells. CRC stands for colorectal cancer, GBM stands for glioblastoma multiforme, NSCLC stands for non-small cell lung cancer, and E:T ratio stands for effector-to-target ratio. [Figure 15C] Figure 15C shows that PBMCs transfected with Linear Nb-CAR.BiTE DNA-transduced Exo induce superior cytolytic activity against solid tumor cells. CRC stands for colorectal cancer, GBM stands for glioblastoma multiforme, NSCLC stands for non-small cell lung cancer, and E:T ratio stands for effector-to-target ratio. [Figure 16] FIG. 16 shows the results of using fusion proteins in in vivo chimeric antigen receptor T-cell therapy (CAR-T). A peripheral blood mononuclear cell (PBMC)-huNSG mouse model is used. huPBMC stands for human peripheral blood mononuclear cells, IP stands for intraperitoneal, IV stands for intravenous, 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 luc stands for luciferase. The transfection efficiency was measured by administering four times and taking blood samples. [Figure 17] Figure 17 is another schematic diagram showing the effect of fusion proteins on cancer treatment, immune regulation and immune cell activation. CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. [Figure 18] Figure 18 is another schematic diagram showing the effect of fusion proteins on cancer treatment, immunomodulation and immune cell activation. Nb stands for nanobody, and CAR stands for chimeric antigen receptor. [Figure 19]Figure 19 is another schematic diagram showing the effects of fusion proteins on cancer treatment, immune regulation and immune cell activation. Nb stands for nanobody, CAR stands for chimeric antigen receptor, Exo stands for exosome, CD3εNb stands for anti-CD3 single domain antibody, and BiTE stands for bispecific T-cell engager. [Figure 20] Figure 20 is another schematic diagram showing the effect of fusion proteins on cancer treatment, immune regulation and immune cell activation. Nb stands for nanobody, CAR stands for chimeric antigen receptor, Exo stands for exosome, CD3εNb stands for anti-CD3 single domain antibody, BiTE stands for bispecific T-cell engager, and PBMC stands for peripheral blood mononuclear cell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The embodiments of the present invention will be further described below. The following examples are merely for the purpose of illustrating the present invention, and are not intended to limit the scope of the present invention. Modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined based on the scope of the appended claims.

[0020] definition The numerical values ​​given herein are approximate values, all experimental data represents a range of ±20%, preferably ±10%, more preferably ±5% of the numerical value.

[0021] In this specification (particularly the claims), unless specifically stated otherwise, the terms "a," "the," "the," and similar terms include both the singular and the plural.

[0022] As used herein, the terms "CD3e" and "CD3ε" are used interchangeably.

[0023] As used herein, the terms "CD3e nanobody", "CD3enb", "CD3eNb", "CD3e nanobody", "anti-CD3ε nanobody", "anti-CD3 single domain antibody", and "anti-T cell nanobody" are used interchangeably.

[0024] As used herein, the term "treating" or "treatment" refers to 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 the condition or symptom being treated.

[0025] The pharmaceutical agent according to the present invention may be prepared into a dosage form for parenteral administration based on common knowledge, such as, but not limited to, an injection (e.g., a sterile aqueous solution or dispersion), a sterile powder, a tablet, a troche, a lozenge, a pill, a capsule, a dispersible powder or granule, a solution, a suspension, an emulsion, a syrup, an elixir, a slurry, and the like.

[0026] The pharmaceutical product according to the present invention may be administered via parenteral routes. The parenteral routes are selected from the group consisting of intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

[0027] The pharmaceutical product according to the present invention may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may contain, for example, one or more reagents 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 quantity of the reagents belong to the expertise and technical scope of those skilled in the art.

[0028] The pharmaceutically acceptable carrier according to the present invention may contain a solvent. The solvent is selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), a sugar-containing solution, an aqueous solution containing alcohol, and combinations thereof.

[0029] As used herein, terms such as "nucleic acid," "nucleic acid sequence," or "nucleic acid fragment" refer to deoxyribonucleotide or ribonucleotide sequences in single- or double-stranded form, and include known naturally occurring nucleotides or artificial chemical mimetics. As used herein, the term "nucleic acid" is used interchangeably with "gene," "cDNA," "mRNA," "oligonucleotide," and "polynucleotide."

[0030] The present invention will be further described below by disclosing examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The scope of protection of the present invention should be based on the appended claims.

[0031] Example 1. Production of the fusion protein of the present invention In this example, the manufacturing process of the fusion protein containing an anti-CD3 single domain antibody and an exosome protein is as follows: HEK-293T cell line (4th to 25th generation) and HEK-293T stable cell line expressing anti-CD3 single domain antibody-CD63 chimeric protein are added to COL2.5 NGCs containing 500 mL of DMEM medium (containing 50 mL of exosome-free FBS) (Thermo Fisher Scientific).

[0032] After 3 days of incubation, the medium was centrifuged at 2000 g for 15 min to remove cell debris and filtered through 0.2 μm filter paper and then ultrafiltered (Amicon (R) The supernatant is concentrated for 8 min at 5000 g using a 300-μL Ultra filter (30 kDa, Merck Millipore). The collected supernatant is processed by tangential flow filtration (MAP.03-plus TFF System, Lefo Science). The parental HEK-293T cell supernatant is then filtered through a film with a cut-off value of 300 kDa and resuspended in PBS.

[0033] Supernatants from HEK-293T stable cells expressing anti-CD3 single domain antibody-CD63 chimeric proteins are filtered through a VHH capture membrane (GenScript) and resuspended in PBS. All samples are either used immediately or stored at -80°C for further use. For NTA analysis, ZetaView (R) The size distribution and concentration of these exosomes are analyzed by Particle Metrix GmbH.

[0034] 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. 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 the sequence shown in SEQ ID NO: 4.

[0035] The present invention also provides an isolated nucleic acid encoding the amino acid sequence of the fusion protein. The nucleotide sequence of the isolated nucleic acid is the sequence shown in SEQ ID NO:5.

[0036] The amino acid sequence of the anti-CD3 single domain antibody is the sequence shown in SEQ ID NO: 6, and the nucleotide sequence encoding the amino acid sequence of the anti-CD3 single domain antibody is the sequence shown in SEQ ID NO: 7, and the amino acid sequence of the anti-CD3 single domain antibody is the heavy chain variable domain (VHH).

[0037] Fig. 1 is a schematic diagram of the structure of the fusion protein of the present invention, in which an anti-CD3 single domain antibody specifically binds to CD3ε (CD3 epsilon).

[0038] Figure 2 shows the binding site of the exosome protein CD63 and the anti-CD3 single domain antibody. In Figure 2, the arrow indicates the embedding position of the anti-CD3 single domain antibody.

[0039] Example 2. Surface plasmon resonance (SPR) analysis of the fusion protein of the present invention In this example, the experimental process of surface plasmon resonance (SPR) analysis of exosomes of fusion proteins is as follows: CM5 and NTA chips (research grade) are used to perform SPR analysis on BIAcore T200 (Biacore-GE Healthcare, Piscataway, NJ).

[0040] That is, in order to provide the maximum surface area when immobilized on the chip after the surface preparation process, the protein (CD3ε recombinant protein) sample is diluted to a concentration range of 20 μg / mL using a 10 mM buffer solution (pH 4.0, 5.5 or 6.0), and conditions with high ligand surface concentration on the chip (anti-CD3 single domain antibody: 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 nM) are selected. Then, regeneration scouting and surface performance testing are performed, and a regeneration method is selected according to the regeneration scouting and surface performance testing to perform the experiment.

[0041] Then, select BINDING ANALYSIS and DIRECT BINDING to analyze protein binding. Select KINETIC ANALYSIS and MASS TRANSFER to perform kinetic analysis to study binding. Analyze the data and determine kinetic constants.

[0042] FIG. 3 shows the results of surface plasmon resonance (SPR) analysis of the Exo of the fusion protein. As can be seen from FIG. 3, the fusion protein (1×10 11 ) was coated onto a CM5 chip and the binding affinity was measured using recombinant CD3ε protein (700, 350, 175, 87.5, 43.8, 21.9 nM). The resulting KD measurement is 2.1 nM.

[0043] Example 3. Evaluation of the effects of the fusion protein of the present invention on cancer treatment, immune regulation, and activation of immune cells In this example, the effects of the fusion protein on cancer treatment, immune regulation, and activation of immune cells are evaluated.

[0044] Figure 4 is a schematic diagram showing the effects of the fusion protein on cancer treatment, immune regulation, and activation of immune cells. CD3εNb represents an anti-CD3 single-domain antibody, HEK-293T represents a human embryonic kidney cell, Nb represents a nanobody, CAR represents a chimeric antigen receptor, BiTE represents a bispecific T-cell engager, and Exo represents an exosome.

[0045] As can be seen from Figure 4, the sequence encoding the anti-CD3 single-domain antibody is inserted into the second extracellular loop of CD63 to form a CD63 chimeric protein together with the anti-CD3 single-domain antibody exposed on the cell surface. Then, by transfecting this construct into HEK-293T, exosomes expressing the anti-CD3 single-domain antibody are generated. Then, the obtained exosomes are purified by a VHH-capsule column, and DNA or mRNA encoding Nb-CAR.BiTE is introduced. This Nb-CAR.BiTE-CD3ε Nb-Exo is directly injected into the mouse model. It is hypothesized that these exosomes selectively reprogram CD3 + T cells to express Nb-CAR and secrete Nb-BiTE to act on solid tumor cells in vivo.

[0046] Figure 5 is another schematic diagram showing the effect of the fusion protein on cancer treatment, immune regulation and immune cell activation. PCR stands for polymerase chain reaction, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, PBMC stands for peripheral blood mononuclear cell, TEM stands for transmission electron microscope, IFNγ stands for interferon γ, TNFα stands for tumor necrosis factor α, and PFN stands for perforin. GzmB stands for Granzyme B, a type of serine protease that mediates the apoptosis signaling pathway of cytotoxic T lymphocytes and natural killer cells.

[0047] SSC indicates side scatter, and VHH indicates heavy chain variable domain. As can be seen from FIG. 5, Exo and CAR.BiTE DNA (3×10 8 The cells were electroporated using the electrotransfection program code CM137 of the LONZA4D-Nucleofector at a ratio of 1 μg Exo vs 2 μg DNA, and allowed to recover at 4°C for 1 hour, after which the morphology was observed using an electron microscope; or the cells were transfected into human PBMCs or whole blood for 48 hours, and then co-cultured with tumor cells to confirm changes in toxicity to tumor cells using a LIVE / DEAD Cell-Mediated Cytotoxicity Assay; or the cells were labeled with fluorescent antibodies and their expression levels of CD3, CD4, CD8, VHH, etc. were analyzed by flow cytometry.

[0048] The above experiments confirm the transfection effect of the CAR.BiTE DNA encapsulated in the fusion protein.

[0049] For transmission electron microscopy (TEM) analysis, exosomes from HEK-293T cells were isolated and fixed overnight at 4°C with 1% glutaraldehyde. After washing, exosomes were transferred to a formvar carbon-covered mesh and negatively stained with aqueous phosphotungstic acid for 1 min. The ultrastructure of these exosomes was analyzed by TEM (JEOL JEM-1400, Tokyo, Japan).

[0050] FIG. 6 shows the results of transmission electron microscopy (TEM) of the fusion proteins. CD3εNb denotes anti-CD3 single domain antibody, Exo denotes exosome, CAR denotes chimeric antigen receptor, and BiTE denotes bispecific T-cell engager. As can be seen from FIG. 6, CAR.BiTE DNA@CD3ε Nb-Exo maintains the ultrastructure and morphology of exosomes. TEM is used to evaluate unmodified and CD3εNb engineered Exo (with or without CAR.BiTE electroporation) with DNA expressing CAR.BiTE at a magnification of 100,000X.

[0051] HEK293-derived Exo can be packaged with CAR expression DNA by electroporation. Unmodified Exo and fusion protein were electroporated with the CAR.BiTE expression carrier using a LONZA 4D-Nucleofector at a ratio of 3 × 10 8Exo: 2 μg DNA. Exo and DNase (1000 IU) are reacted together for 30 minutes, and the introduced CAR.BiTE DNA is quantified at OD260 and 280 nm using specific primers or via spectrophotometer.

[0052] FIG. 7 shows the efficiency of encapsulating linear Nb-CAR.BiTE DNA into Exo by electroporation. 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. As can be seen from FIG. 7, the spectrophotometer and qPCR results show that electrotransfection can encapsulate 3×10 8 Approximately 100 ng of CAR.BiTE DNA can be encapsulated per Exo.

[0053] Unmodified Exo or fusion protein was administered at 3 × 10 8 Electroporation was performed at a ratio of 5 × 10 Exo:2 μg DNA. After purification, these Exos were 5 PBMCs were added at 4 nm and left on ice for 45 min. After staining, CD3+ cells were detected by flow cytometry using specific antibodies. + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + , CD56 + , TCRγδ + , CD14 + , CD19 + and CD66b + Measure the amount of Nb-CAR expression in cells.

[0054] FIG. 8 shows the transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into peripheral blood mononuclear cells (PBMCs). 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 method. As can be seen from FIG. 8, compared to unmodified Exo (293T Exo), CAR.BiTE DNA@CD3e Nb-Exo showed significantly higher transfection efficiency than CD3 - From cells, CD3 + This result indicates that the fusion protein selectively transfects the transgene into CD3 + Supports delivery to cells.

[0055] Unmodified Exo or fusion protein was administered at 3 × 10 8 Electroporation was performed at a ratio of 5 × 10 Exo:2 μg DNA. After purification, these Exos were 5 PBMCs were added at 4 nm and left on ice for 45 min. After staining, CD3+ cells were detected by flow cytometry using specific antibodies. + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + Measure the amount of Nb-CAR expression in cells.

[0056] FIG. 9 is another schematic diagram showing the measurement of transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into peripheral blood mononuclear cells (PBMCs). Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. As can be seen from FIG. 9, compared with unmodified Exo (293T Exo), CAR.BiTE DNA@CD3e Nb-Exo showed a significantly higher transfection efficiency than CD3 + / CD4 + and CD3 / CD8 + This result indicates that the fusion protein selectively transfects the transgene into CD4 T cells. + and CD8 + Supports delivery to cells.

[0057] Unmodified Exo or fusion protein was administered at 3 × 10 8 Electroporation was performed at a ratio of 5 × 10 Exo:2 μg DNA. After purification, these Exos were 5 PBMCs were added at 4 nm and left on ice for 45 min. After staining, CD3+ cells were detected by flow cytometry using specific antibodies. + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + Measure the amount of Nb-CAR expression in cells.

[0058] FIG. 10 shows the measurement of transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into PBMCs. Nb stands for nanobody, CAR stands for chimeric antigen receptor, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. As can be seen from FIG. 10, CAR.BiTE DNA@CD3e Nb-Exo transfected CD3 + , CD3 + / CD4 + and CD3 / CD8 + This result indicates that the fusion protein selectively transfects the transgene into CD4 T cells. + and CD8 + Supports delivery to cells.

[0059] Unmodified Exo or fusion protein was administered at 3 × 10 8 After purification, these Exos were added to 1 ml of whole blood and incubated for 4 h. After staining for 45 min on ice, CD3+ cells were detected by flow cytometry using specific antibodies. + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + Measure the amount of Nb-CAR expression in cells.

[0060] FIG. 11 shows the measurement of transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. 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 method. As can be seen from FIG. 11, the results show that the fusion protein selectively transduced the transgene into the CD3 of whole blood. + Supports delivery to cells.

[0061] Unmodified Exo or fusion protein was administered at 3 × 10 8 After purification, these Exos were added to 1 ml of whole blood and incubated for 4 h. After staining for 45 min on ice, CD3+ / CD4+ / CD5+ / CD6+ / CD7+ / CD8+ / CD9+ / CD10+ / CD11+ / CD12+ / CD13+ / CD14+ / CD15+ / CD16+ / CD17+ / CD18+ / CD19+ / CD11+ / CD12+ / CD13+ / CD + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + , CD56 + , TCRγδ + , CD14 + , CD19 + and CD66b + Measure the amount of Nb-CAR expression in cells.

[0062] Figure 12 is another schematic diagram of the measurement of transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. 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 method. As can be seen from Figure 12, the results show that the fusion protein selectively transduced the transgene into the CD3 of whole blood. + / CD4 and CD3 + / CD8 + Supports delivery to cells.

[0063] Unmodified Exo or fusion protein was administered at 3 × 10 8 After purification, these Exos were added to 1 ml of whole blood and left for 48 h, stained for 45 min on ice, and then detected by flow cytometry using specific antibodies against CD3 + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + Measure the amount of Nb-CAR expression in cells.

[0064] FIG. 13 shows the measurement of transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. As can be seen from FIG. 13, the results show that the fusion protein selectively transduced the transgene into the CD3 of whole blood. + , CD3+ / CD4 and CD3 + / CD8 + Supports delivery to cells.

[0065] Unmodified Exo or fusion protein was administered at 3 × 10 8 After purification, these Exos were added to 1 ml of whole blood and incubated for 4 h. After staining for 45 min on ice, CD3+ / CD4+ / CD5+ / CD6+ / CD7+ / CD8+ / CD9+ / CD10+ / CD11+ / CD12+ / CD13+ / CD14+ / CD15+ / CD16+ / CD17+ / CD18+ / CD19+ / CD11+ / CD12+ / CD13+ / CD + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + Measure the amount of Nb-CAR expression in cells.

[0066] Figure 14 is another schematic diagram of the measurement of transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. Nb stands for nanobody, CAR stands for chimeric antigen receptor, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. As can be seen from Figure 14, the results show that the fusion protein selectively transduced the transgene into the CD3 of whole blood. + , CD3 + / CD4 and CD3 + / CD8 + Supports delivery to cells.

[0067] Cytotoxicity Testing CD3 treated with CAR.BiTE-CD3ε Nb-Exo or unmodified exosomes +The cells are used as effector cells. Target cells (tumor cell lines) and effector cells are co-cultured at specific effector cell / target cell (E:T) ratios (1:1-50:1) for 24-72 h at 37°C. For measurement of live / dead cell viability, all tumor cells are stained with green-fluorescent calcein-AM before co-culture, and dead cells are identified with red-fluorescent ethidium homodimer-1 staining after co-culture, and dead tumor cells are stained with green-fluorescent ethidium homodimer-1 staining according to the manufacturer's procedure (Thermo Fisher Scientific). + / Red fluorescence + The cell death rate is expressed as a percentage of the total cell population.

[0068] Figures 15A-15C show that PBMCs transfected with linear Nb-CAR.BiTE DNA-introduced Exo induce superior cytolytic activity against solid tumor cells. CRC stands for colorectal cancer, GBM stands for glioblastoma multiforme, NSCLC stands for non-small cell lung cancer, and E:T ratio stands for effector-to-target ratio. As can be seen from Figures 15A-15C, when comparing unmodified Exo (293T Exo) with or without CAR.BiTE DNA and CD3e Nb-Exo without DNA, PBMCs treated with CAR.BiTE DNA-introduced CD3e Nb-Exo have higher cytotoxicity against these tumor cells.

[0069] Furthermore, compared with PBMCs treated with non-CAR.BiTE DNA-transfected Exo, PBMCs treated with CAR.BiTE DNA-transfected 293T Exo also have superior cytotoxicity.

[0070] FIG. 16 is a schematic diagram of the in vivo antitumor effect process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1×10 6 cells) were intraperitoneally transplanted into the mouse, and 7 days later, huPBMCs (5 × 10 6 The next day, mice were injected with or without CD3ε Nb-Exo, either unmodified or transfected with CAR.BiTE DNA (3 × 10 10 Treatment will continue once a week for 4 weeks. The IVIS system will monitor tumor growth by detecting bioluminescent signals. Seven days after the last injection, mice will be euthanized and splenocytes will be harvested. The expression of Nb-CAR on each immune cell will be measured by flow cytometry using specific antibodies against VHH, CD3, CD56, TCRγδ, CD14, CD19 and CD66b.

[0071] FIG. 16 shows the results of using the fusion protein in in vivo chimeric antigen receptor T-cell therapy (CAR-T). A peripheral blood mononuclear cell (PBMC)-huNSG mouse model is used. huPBMC stands for human peripheral blood mononuclear cell, IP stands for intraperitoneal, IV stands for intravenous, 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 luc stands for luciferase. The transfection efficiency was measured by administering four times and taking blood samples.

[0072] FIG. 17 is a schematic diagram of the in vivo antitumor effect process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1×10 6 cells) were intraperitoneally transplanted into the mouse, and 7 days later, huPBMCs (5 × 10 6The next day, mice were injected with or without unmodified Exo or CD3ε Nb-Exo transfected with or without CAR.BiTE DNA (3 × 10 10 The mice were treated with IVIS (100 μg / mouse) once a week for 4 weeks. The treatment was continued for 4 weeks. The IVIS system monitored the tumor growth by detecting the bioluminescent signal.

[0073] Figure 17 is another schematic diagram showing the effect of fusion proteins on cancer treatment, immune regulation and immune cell activation. CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, and CD3εNb stands for anti-CD3 single domain antibody. As can be seen from Figure 17, compared with other mouse groups, CAR.BiTE DNA@CD3e Nb-Exo has superior anti-tumor activity.

[0074] FIG. 18 is a schematic diagram of the in vivo antitumor effect process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1×10 6 cells) were intraperitoneally transplanted into the mouse, and 7 days later, huPBMCs (5 × 10 6 The next day, mice were injected with or without unmodified Exo or CD3ε Nb-Exo transfected with or without CAR.BiTE DNA (3 × 10 10 Treatment will continue once a week for 4 weeks. Seven days after the last injection, mice will be euthanized, submandibular blood will be collected, and the expression level of Nb-CAR in each immune cell will be measured by flow cytometry using specific antibodies against VHH and CD3.

[0075] Figure 18 is another schematic diagram showing the effect of the fusion protein on cancer treatment, immune regulation and immune cell activation. Nb stands for nanobody, and CAR stands for chimeric antigen receptor. The results show that the fusion protein selectively upregulates the CD3+ expression level of the transgene in the blood cells of PBMC-humanized NSG mice. + , CD3 + / CD4 and CD3 + / CD8 + Supports delivery to cells.

[0076] FIG. 19 is a schematic diagram of the in vivo antitumor effect process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1×10 6 cells) were intraperitoneally transplanted into the mouse, and 7 days later, huPBMCs (5 × 10 6 The next day, mice were injected with or without unmodified Exo or CD3ε Nb-Exo transfected with or without CAR.BiTE DNA (3 × 10 10 Treatment will continue once a week for 4 weeks. Seven days after the last injection, mice will be euthanized, submandibular blood will be collected, and the expression level of Nb-CAR in each immune cell will be measured by flow cytometry using specific antibodies against VHH and CD3.

[0077] Figure 19 is another schematic diagram showing the effects of fusion proteins on cancer treatment, immune regulation and immune cell activation. Nb stands for nanobody, CAR stands for chimeric antigen receptor, Exo stands for exosome, CD3εNb stands for anti-CD3 single domain antibody, and BiTE stands for bispecific T-cell engager.

[0078] FIG. 20 is another schematic diagram showing the effect of the fusion protein on cancer treatment, immune regulation and immune cell activation. Nb stands for nanobody, CAR stands for chimeric antigen receptor, Exo stands for exosome, CD3εNb stands for anti-CD3 single domain antibody, BiTE stands for bispecific T-cell engager, and PBMC stands for peripheral blood mononuclear cell. As can be seen from FIG. 19 and FIG. 20, the fusion protein selectively upregulates the CD3 of blood cells of PBMC-humanized NSG mice. + Deliver to cells.

[0079] In summary, the fusion protein of the present invention can penetrate into solid tumors and secrete bispecific T cell engagers to activate surrounding immune cells, thereby achieving the effects of cancer treatment, immune regulation and immune cell activation, as demonstrated by surface plasmon resonance, cytotoxicity assay, animal experiments, electroporation experiments, transfection efficiency assay, flow cytometry assay and in vivo chimeric antigen receptor T cell therapy.

[0080] The above description is merely illustrative of the present invention, and is not intended to limit the scope of the present invention. Any equivalent modifications and variations that do not depart from the spirit and scope of the present invention are encompassed within the scope of the appended claims.

Claims

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

4. Fusion proteins.

2. The anti-CD3 single domain antibody specifically binds to CD3 epsilon. The fusion protein of claim 1.

3. The exosome protein is CD63. The fusion protein of claim 1.

4. 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, The amino acid sequence shown in SEQ ID NO: 3 is CDR3. The fusion protein of claim 1.

5. The anti-CD3 single domain antibody is an anti-T cell single domain antibody. The fusion protein of claim 1.

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

5. Isolated nucleic acid.

7. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 5 and a pharma- ceutical acceptable carrier. Pharmaceutical compositions.

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