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

Abstract

To provide a fusion protein and a nucleic acid encoding sequence thereof, and uses of the same.SOLUTION: The fusion protein achieves the effect of treating cancer, immunoregulation and activating immune cells through various efficacy experiments.SELECTED DRAWING: Figure 1

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 a malfunction in the control of cell division and proliferation, resulting in 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 in Taiwan it is one of the top 10 causes of death, 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 treatments, which activates the patient's own immune system and uses tumor cells or tumor antigens to induce the body's specific cellular immune and humoral immune responses, thereby enhancing the body's anti-cancer capabilities and inhibiting tumor growth, spread, and recurrence, thereby achieving the goal of tumor removal or control. However, current tumor treatment methods have problems such as low efficacy and severe side effects, and may even cause other immune-related diseases.

[0004] CD3ε (CD3 epsilon) is a transmembrane protein expressed on T cells and has been found to be associated with tumor and immune function regulation. Therefore, researchers are exploring CD3ε as a target molecule for tumor identification and immune function regulation, exploring whether these target molecules have the potential to become anticancer or immunomodulatory agents. Meanwhile, CD63 is a protein antigen encoded by the CD63 gene in the human body. CD63 is primarily present on the surface of extracellular vesicles and also on the surface of normal cell membranes, and its encoding gene is associated with tumor development. 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, immunomodulation, 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 comprising 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 present invention, the anti-CD3 single domain antibody specifically binds to 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 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] Another object of the present invention is 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 the fusion protein and a pharmaceutically 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 into solid tumors and secreting bispecific T-cell engagers (BiTEs) to activate surrounding immune cells. [Effects 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 measurements, flow cytometry analysis, and in vivo chimeric antigen receptor T-cell therapy (CAR-T), demonstrating that the fusion protein penetrates into solid tumors and secretes bispecific T-cell engagers (BiTEs) to activate surrounding immune cells, thereby achieving the effects of cancer treatment, immunomodulation, and immune cell activation. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram of the structure of the fusion protein of the present invention. [Figure 2] Figure 2 shows the binding site of the exosomal protein CD63 and the anti-CD3 single domain antibody. The arrow indicates the embedding site of the anti-CD3 single domain antibody. [Figure 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 fusion proteins on cancer treatment, immunomodulation, and immune cell activation. CD3εNb represents anti-CD3 single domain antibody, HEK-293T represents human embryonic kidney cells, Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, and Exo represents exosome. [Figure 5] Figure 5 is another schematic diagram showing the effects of fusion proteins on cancer treatment, immunomodulation, 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 proteins. 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 Exosomes by electroporation. Nb: nanobody, CAR: chimeric antigen receptor, BiTE: bispecific T-cell engager, Exo: exosome, CD3εNb: anti-CD3 single domain antibody, and qPCR: 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: nanobody, CAR: chimeric antigen receptor, BiTE: bispecific T-cell engager, Exo: exosome, CD3εNb: anti-CD3 single domain antibody, and WLSM: weighted least squares method. [Figure 9] Figure 9 is another schematic diagram showing the measurement of transfection efficiency of electroporated Exo-carried Nb-CAR.BiTE DNA into peripheral blood mononuclear cells (PBMCs). Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. [Figure 10]Figure 10 shows the transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into PBMCs. Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3ε Nb represents anti-CD3 single domain antibody. [Figure 11] Figure 11 shows the 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 shows 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. [Figure 13] Figure 13 shows the transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. [Figure 14]Figure 14 shows another schematic diagram of the measurement of transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into whole blood. Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. [Figure 15A] Figure 15A shows that PBMCs transfected with Linear Nb-CAR.BiTE DNA-transfected Exo induce potent cytolytic activity against solid tumor cells. CRC stands for colorectal cancer, GBM stands for glioblastoma multiforme, and NSCLC stands for non-small cell lung cancer. The E:T ratio indicates the effector-to-target ratio. [Figure 15B] Figure 15B shows that PBMCs transfected with Linear Nb-CAR.BiTE DNA-transfected Exo induce potent cytolytic activity against solid tumor cells. CRC: colorectal cancer, GBM: glioblastoma multiforme, NSCLC: non-small cell lung cancer, and E:T ratio: effector-to-target ratio. [Figure 15C] Figure 15C shows that PBMCs transfected with Linear Nb-CAR.BiTE DNA-transfected Exo induce potent cytolytic activity against solid tumor cells. CRC: colorectal cancer, GBM: glioblastoma multiforme, NSCLC: non-small cell lung cancer, and E:T ratio: effector-to-target ratio. [Figure 16] Figure 16 shows the results of in vivo chimeric antigen receptor T-cell therapy (CAR-T) using fusion proteins. A peripheral blood mononuclear cell (PBMC)-huNSG mouse model was 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. After four doses, blood samples were collected to measure transfection efficiency. [Figure 17] Figure 17 is another schematic diagram showing the effects of fusion proteins on cancer treatment, immunomodulation, 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 effects 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, immunomodulation, 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 effects of fusion proteins on cancer treatment, immunomodulation, 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 INVENTION

[0019] The following provides a further description of the embodiments of the present invention. The following examples are merely for illustrative purposes and are not intended to limit the scope of the present invention. Improvements and modifications 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 by the appended claims.

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

[0021] In this specification (particularly the claims), unless otherwise stated, 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" 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 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 granules, a solution, a suspension, an emulsion, a syrup, an elixir, a slurry, and the like.

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

[0027] The pharmaceutical preparation of the present invention may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include 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 amount of the agents are within the expertise and technical scope of those skilled in the art.

[0028] The pharmaceutically acceptable carrier of the present invention may comprise a solvent 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 a deoxyribonucleotide or ribonucleotide sequence 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 the purpose of 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 process for producing a fusion protein containing an anti-CD3 single domain antibody and an exosome protein was as follows: HEK-293T cell line (4th to 25th generation) and a HEK-293T stable cell line expressing an anti-CD3 single domain antibody-CD63 chimeric protein were 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 minutes to remove cell debris and filtered through 0.2 μm filter paper and then ultrafiltered (Amicon (R) The supernatant was concentrated at 5000 g for 8 minutes using a 300 kDa Ultra filter (Merck Millipore). The collected supernatant was processed by tangential flow filtration (MAP.03-plus TFF System, Lefo Science). The parental HEK-293T cell supernatant was then filtered through a 300 kDa cutoff film and resuspended in PBS.

[0033] Supernatants from HEK-293T stable cells expressing anti-CD3 single domain antibody-CD63 chimeric proteins were filtered through a VHH capture membrane (GenScript) and resuspended in PBS. All samples were 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. The amino acid sequence of the anti-CD3 single domain antibody is the heavy chain variable domain (VHH).

[0037] Figure 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 exosomal protein CD63 and the anti-CD3 single domain antibody. In Figure 2, the arrow indicates the embedding site 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 for surface plasmon resonance (SPR) analysis of the fusion protein exosomes was as follows: CM5 and NTA chips (research grade) were used to perform SPR analysis on a BIAcore T200 (Biacore-GE Healthcare, Piscataway, NJ).

[0040] Specifically, to maximize the surface area when immobilized on the chip after the surface preparation step, the protein (CD3ε recombinant protein) sample was diluted to a concentration range of 20 μg / mL using 10 mM buffer solution (pH 4.0, 5.5, or 6.0), and conditions with high ligand surface concentrations on the chip (anti-CD3 single domain antibody: 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 nM) were selected. Regeneration scouting and surface performance testing were then performed, and a regeneration method was selected based on the results.

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

[0042] Figure 3 shows the results of surface plasmon resonance (SPR) analysis of the Exo of the fusion protein. As can be seen from Figure 3, the fusion protein (1 x 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 KD measurement result was 2.1 nM.

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

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

[0045] As shown in Figure 4, the sequence encoding the anti-CD3 single domain antibody was 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. This construct was then transfected into HEK-293T cells to generate exosomes expressing the anti-CD3 single domain antibody. The resulting exosomes were then purified using a VHH-capsule column, and DNA or mRNA encoding Nb-CAR.BiTE was introduced. This Nb-CAR.BiTE-CD3ε Nb-Exo was then directly injected into a mouse model. These exosomes selectively bind to CD3 + We hypothesize that T cells can be reprogrammed to express Nb-CARs and secrete Nb-BiTEs to act on solid tumor cells in vivo.

[0046] Figure 5 is another schematic diagram showing the effects of fusion proteins on cancer treatment, immunomodulation, 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 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 Figure 5, Exo and CAR.BiTE DNA were added (3 x 10 8 The cells were electroporated using the LONZA4D-Nucleofector electrotransfection program code CM137 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. Alternatively, 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. Alternatively, 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, the exosomes were transferred to a formvar carbon-coated mesh and negatively stained with phosphotungstic acid solution for 1 minute. The ultrastructure of these exosomes was analyzed by TEM (JEOL JEM-1400, Tokyo, Japan).

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

[0051] HEK293-derived Exo can be packaged with CAR-expressing 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 8The Exo and DNase (1000 IU) were then reacted together for 30 minutes, and the introduced CAR.BiTE DNA was quantified using specific primers via qPCR or a spectrophotometer at OD 260 and 280 nm.

[0052] 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. As can be seen from Figure 7, electrotransfection yielded 3 x 10 DNA fragments, as shown by the spectrophotometer and qPCR results. 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 Exo were 5 PBMCs were added to 4 nM IgG1 and left on ice for 45 min. After staining, CD3 was detected by flow cytometry using specific antibodies. + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + , CD56 + , TCRγδ + , CD14 + , CD19 + and CD66b + The amount of Nb-CAR expression in the cells is measured.

[0054] Figure 8 shows the transfection efficiency of Nb-CAR.BiTE DNA-encapsulated electroporated Exo into peripheral blood mononuclear cells (PBMCs). Nb indicates nanobody, CAR indicates chimeric antigen receptor, BiTE indicates bispecific T-cell engager, Exo indicates exosome, CD3ε Nb indicates anti-CD3 single domain antibody, and WLSM indicates weighted least squares method. As can be seen from Figure 8, compared to unmodified Exo (293T Exo), CAR.BiTE DNA@CD3e Nb-Exo significantly increased 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 Exo were 5 PBMCs were added to 4 nM IgG1 and left on ice for 45 min. After staining, CD3 was detected by flow cytometry using specific antibodies. + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + The amount of Nb-CAR expression in the cells is measured.

[0056] 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 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 can be seen from Figure 9, compared to unmodified Exo (293T Exo), CAR.BiTE DNA@CD3e Nb-Exo significantly increased 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 Exo were 5 PBMCs were added to 4 nM IgG1 and left on ice for 45 min. After staining, CD3 was detected by flow cytometry using specific antibodies. + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + The amount of Nb-CAR expression in the cells is measured.

[0058] Figure 10 shows the transfection efficiency of linear Nb-CAR.BiTE DNA-encapsulated electroporated Exo into PBMCs. Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3ε Nb represents anti-CD3 single domain antibody. As can be seen from Figure 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 Exo were added to 1 ml of whole blood and incubated for 4 h. After 45 min on ice, staining was performed using specific antibodies to detect CD3+ / CD ... + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + The amount of Nb-CAR expression in the cells is measured.

[0060] Figure 11 shows the 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 11, the results show that the fusion protein selectively transduced the transgene into CD3 in whole blood. + Supports delivery to cells.

[0061] Unmodified Exo or fusion protein was administered at 3 × 10 8 After purification, these Exo were added to 1 ml of whole blood and incubated for 4 h. After 45 min on ice, staining was performed using specific antibodies to detect CD3+ / CD ... + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + , CD56 + , TCRγδ + , CD14 + , CD19 + and CD66b + The amount of Nb-CAR expression in the cells is measured.

[0062] Figure 12 is another schematic diagram of the measurement of the 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 Exo were added to 1 ml of whole blood and left for 48 hours. After staining for 45 minutes on ice, CD3+ cells were detected by flow cytometry using specific antibodies. + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + The amount of Nb-CAR expression in the cells is measured.

[0064] 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. As can be seen from Figure 13, the results show that the fusion protein selectively transduced the transgene into CD3 in 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 Exo were added to 1 ml of whole blood and incubated for 4 h. After 45 min on ice, staining was performed using specific antibodies to detect CD3+ / CD ... + , CD3 - , CD3 + / CD4 + , CD3 + / CD8 + The amount of Nb-CAR expression in the cells is measured.

[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 represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. As can be seen from Figure 14, the results show that the fusion protein selectively transduced the transgene into CD3 in whole blood. + , CD3 + / CD4 and CD3 + / CD8 + Supports delivery to cells.

[0067] Cytotoxicity test CD3 treated with CAR.BiTE-CD3ε Nb-Exo or unmodified exosomes +The cells were used as effector cells. Target cells (tumor cell lines) and effector cells were co-cultured at specific effector / target (E:T) ratios (1:1 to 50:1) for 24 to 72 hours at 37°C. To measure live / dead cell viability, all tumor cells were stained with green-fluorescent calcein-AM before co-culture, and red-fluorescent ethidium homodimer-1 staining was used to identify dead cells. Dead tumor cells were stained with green-fluorescent ethidium homodimer-1 according to the manufacturer's protocol (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-transfected Exo induce superior cytolytic activity against solid tumor cells. CRC indicates colorectal cancer, GBM indicates glioblastoma multiforme, and NSCLC indicates non-small cell lung cancer. The E:T ratio indicates the effector-to-target ratio. As can be seen from Figures 15A-15C, when comparing unmodified Exo (293T Exo) with or without CAR.BiTE DNA transfection with CD3e Nb-Exo, PBMCs treated with CAR.BiTE DNA-transfected CD3e Nb-Exo exhibited higher cytotoxicity against these tumor cells.

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

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

[0071] Figure 16 shows the results of using the fusion protein for in vivo chimeric antigen receptor T-cell therapy (CAR-T). A peripheral blood mononuclear cell (PBMC)-huNSG mouse model was 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. After four doses, blood samples were collected to measure transfection efficiency.

[0072] Figure 17 is a schematic diagram of the in vivo antitumor effect process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1 x 10 6 cells) were intraperitoneally transplanted into the tail vein of each mouse (n = 5). Seven days later, huPBMCs (5 × 10 6The next day, mice (3 × 10) were injected with or without unmodified Exo or CD3ε Nb-Exo transfected with or without CAR.BiTE DNA. 10 The mice were treated once a week for four weeks. The IVIS system monitored tumor growth by detecting bioluminescent signals.

[0073] Figure 17 is another schematic diagram showing the effects of fusion proteins on cancer treatment, immunomodulation, and immune cell activation. CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, and CD3eNb stands for anti-CD3 single-domain antibody. As can be seen from Figure 17, CAR.BiTE DNA@CD3eNb-Exo has superior antitumor activity compared with other mouse groups.

[0074] Figure 18 is a schematic diagram of the in vivo antitumor effect process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1 x 10 6 cells) were intraperitoneally transplanted into the tail vein of each mouse (n = 5). Seven days later, huPBMCs (5 × 10 6 The next day, mice (3 × 10) were injected with or without unmodified Exo or CD3ε Nb-Exo transfected with or without CAR.BiTE DNA. 10 Treatment will be continued once a week for 4 weeks. Seven days after the last injection, the 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 effects of the fusion protein on cancer treatment, immunomodulation, and immune cell activation. Nb stands for nanobody, and CAR stands for chimeric antigen receptor. The results show that the fusion protein selectively inhibits the CD3 expression of the transgene in the PBMC-humanized NSG mice. + , CD3 + / CD4 and CD3 + / CD8 + Supports delivery to cells.

[0076] Figure 19 is a schematic diagram of the in vivo antitumor effect process of CAR.BiTE DNA@CD3ε Nb-Exo. COLO 205-luc cells (1 x 10 6 cells) were intraperitoneally transplanted into the tail vein of each mouse (n = 5). Seven days later, huPBMCs (5 × 10 6 The next day, mice (3 × 10) were injected with or without unmodified Exo or CD3ε Nb-Exo transfected with or without CAR.BiTE DNA. 10 Treatment will be continued once a week for 4 weeks. Seven days after the last injection, the 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, immunomodulation, 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] Figure 20 is another schematic diagram showing the effects of the fusion protein on cancer treatment, immunomodulation, 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 Figures 19 and 20, the fusion protein selectively induced the CD3 expression of PBMC-humanized NSG mice blood cells. + 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, immunomodulation, and immune cell activation, as demonstrated by surface plasmon resonance, cytotoxicity assays, animal experiments, electroporation experiments, transfection efficiency assays, flow cytometry analysis, and in vivo chimeric antigen receptor T cell therapy.

[0080] The foregoing is merely illustrative of the present invention and is not intended to limit the scope of the present invention. All 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. 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. Fusion proteins.

2. The anti-CD3 single domain antibody specifically binds to CD3ε (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 is the sequence shown in SEQ ID NO:

4. The fusion protein of claim 1.

5. The amino acid sequence shown in SEQ ID NO: 1 is a 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.

6. the anti-CD3 single domain antibody is an anti-T cell nanobody; The fusion protein of claim 1.

7. A gene encoding the amino acid sequence of the fusion protein according to any one of claims 1 to 6. Isolated nucleic acid.

8. The nucleotide sequence is the sequence shown in SEQ ID NO:

5. The isolated nucleic acid of claim 7.

9. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 6 and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

10. For the manufacture of medicines for the treatment of cancer, immunomodulation and activation of immune cells, Use of the fusion protein according to any one of claims 1 to 6.

11. The cancer is treated by infiltrating the fusion protein into solid tumors and secreting bispecific T-cell engagers (BiTEs) to activate surrounding immune cells. The use according to claim 10.

Images