Fusion protein as well as nucleic acid coding sequence and application thereof

Abstract

The invention provides a fusion protein as well as a nucleic acid coding sequence and application thereof. The fusion protein disclosed by the invention achieves the effects of treating cancers, regulating immunity and activating immune cells by virtue of a plurality of efficacy experiments.

Description

Technical Field

[0001] The invention relates to a fusion protein and a nucleic acid encoding sequence and application thereof. Background Art

[0002] Cancer, also known as malignant tumor, is an abnormal proliferation of cells that may invade other parts of the body. It is a disease caused by the abnormal control of cell division and proliferation. The number of people suffering from cancer in the world is increasing. Cancer is one of the top ten causes of death and has been at the top of the list for many years.

[0003] Conventional tumor treatment methods include surgery, radiotherapy, chemotherapy and targeted therapy. Tumor immunotherapy is another method of treating tumors besides the above treatment methods. It activates the patient's own immune system, uses tumor cells or tumor antigens to induce the body's specific cellular immunity and humoral immune response, enhances the body's anti-cancer ability, and prevents tumor growth, spread and recurrence, so as to achieve the purpose of eliminating or controlling tumors. However, current tumor treatment methods still have problems of poor effect and strong side effects, and may even lead to other immune-related diseases.

[0004] CD3ε (CD3 epsilon) is a transmembrane protein found on T cells, which has been found to be associated with tumors and immune regulation. Therefore, 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 anticancer drugs or immunomodulatory drugs. In addition, CD63 is a protein antigen that is 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. Its encoding gene is related to tumor development.

[0005] In order to solve the above problems, technicians in this field are in urgent need of developing novel and more effective drugs for treating cancer, immunomodulation and activating immune cells to benefit the vast population in need. Summary of the invention

[0006] In view of this, an object of the present invention is to provide a fusion protein comprising an anti-CD3 single domain antibody and an 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 one embodiment of the present invention, the anti-CD3 single domain antibody specifically binds to 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 the sequence identification number SEQ ID NO:1 is complementarity determining region 1 (CDR1), the amino acid sequence shown in the sequence identification number SEQ ID NO:2 is CDR2, and the amino acid sequence shown in the sequence identification number 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 the fusion protein as described above.

[0013] In one embodiment of the present invention, the nucleotide sequence of the isolated nucleic acid is 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 a use of the fusion protein as described above for preparing a medicine for treating cancer, immunomodulation and activating immune cells.

[0016] In one embodiment of the present invention, the cancer is treated by allowing the fusion protein to penetrate into the solid tumor and secrete bispecific T-cell engager (BiTE) and activate peripheral immune cells.

[0017] In summary, the efficacy of the fusion protein of the present invention is to achieve the efficacy of treating cancer, immunomodulating and activating immune cells by 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 infiltrating the fusion protein into solid tumors and secreting bispecific T-cell engager (BiTE) and activating peripheral immune cells.

[0018] The following will further illustrate the implementation mode of the present invention. The following examples are used to illustrate the present invention, not to limit the scope of the present invention. Anyone skilled in the art may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the definition of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the structure of the fusion protein of the present invention.

[0020] Figure 2 The binding position of the exosomal protein CD63 and the anti-CD3 single domain antibody is shown, wherein the arrow indicates the embedded position of the anti-CD3 single domain antibody.

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

[0022] Figure 4 Schematic diagram of the effectiveness of fusion proteins in treating cancer, immunomodulation and activating immune cells, wherein 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 5is another schematic diagram of the effectiveness of fusion proteins in treating cancer, immunomodulation and activating immune cells, wherein PCR represents polymerase chain reaction, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, PBMC represents peripheral blood mononuclear cell, and TEM represents transmission electron microscope.

[0024] Figure 6 The transmission electron microscope (TEM) results of the fusion protein are shown, 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 Figure 2 shows the efficiency of linear Nb-CAR.BiTE DNA encapsulation into Exo via electroporation, where Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, CD3εNb stands for anti-CD3 single domain antibody, and qPCR stands for quantitative polymerase chain reaction.

[0026] Figure 8The efficiency of testing Nb-CAR.BiTE DNA-coated electroporated Exo transfection into peripheral blood mononuclear cells (PBMCs) is shown, wherein 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] Fig. 9 Another schematic diagram for testing the efficiency of Nb-CAR.BiTE DNA-coated electroporated Exo transfection into peripheral blood mononuclear cells (PBMCs), wherein Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody.

[0028] Fig.10 The efficiency of testing linear Nb-CAR.BiTE DNA-coated electroporated Exo transfection into PBMCs is shown, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody.

[0029] Fig.11 The efficiency of transfection of electroporated Exo coated with Nb-CAR.BiTE DNA into whole blood is shown, 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] Fig.12Another schematic diagram for testing the efficiency of Nb-CAR.BiTE DNA-coated electroporated Exo transfection into whole blood, wherein 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] Fig.13 The efficiency of testing linear Nb-CAR.BiTE DNA-coated electroporated Exo transfection into whole blood is shown, wherein Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody.

[0032] Fig.14 Another schematic diagram for testing the efficiency of linear Nb-CAR.BiTE DNA-coated electroporated Exo transfection 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 to 15C The linear Nb-CAR.BiTE DNA-loaded Exo-transfected PBMCs induced excellent cytolytic activity against solid tumor cells, where CRC represents colorectal cancer, GBM represents glioblastoma multiforme, NSCLC represents non-small cell lung cancer, and E:T ratio represents effector-to-target ratio.

[0034] Fig.16The results of the fusion protein in in vivo chimeric antigen receptor T-cell therapy (CAR-T) are shown, in which a peripheral blood mononuclear cell (PBMC)-huNSG mouse model is used, huPBMC denotes human peripheral blood mononuclear cells, IP denotes intraperitoneal, IV denotes intravenous, CAR denotes chimeric antigen receptor, BiTE denotes bispecific T-cell engager, Exo denotes exosome, CD3εNb denotes anti-CD3 single domain antibody, and luc denotes luciferase. The fourth dose has been administered and blood has been collected to test the transfection efficiency.

[0035] Fig.17 Another schematic diagram showing the effectiveness of fusion proteins in treating cancer, immunomodulating 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] Fig.18 Another schematic diagram showing the effectiveness of fusion proteins in treating cancer, immunomodulating and activating immune cells, where Nb represents nanobody and CAR represents chimeric antigen receptor.

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

[0038] Fig. 20Another schematic diagram showing the effectiveness of fusion proteins in treating cancer, immunomodulation and activating immune cells, wherein Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, CD3εNb represents anti-CD3 single domain antibody, BiTE represents bispecific T-cell engager, and PBMC represents peripheral blood mononuclear cell. DETAILED DESCRIPTION

[0039] definition

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

[0041] Unless otherwise specified herein, the terms "a", "an", "the" and similar terms used in this specification (especially in the scope of the following patent application) should be understood to include both singular and plural forms.

[0042] As used herein, the terms "CD3e" and "CD3epsilon" are used interchangeably.

[0043] As used herein, the terms "CD3e nanobody", "CD3e nb", "CD3e Nb", "CD3enanobody", "anti-CD3ε nanobody", "anti-CD3 single domain antibody" and "anti-T cell nanobody" are 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, as well as lowering, stopping or reversing the progression of the severity of a condition or symptom being treated.

[0045] According to the present invention, the pharmaceutical product can be manufactured into a dosage form suitable for parenteral administration using techniques well known to those skilled in the art, including, but not limited to, injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries and the like.

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

[0047] The pharmaceutical product according to the present invention may include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer, binder, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delaying agent, liposome and the like. The selection and amount of these agents fall within the professional qualities and routine technical scope of those skilled in the art.

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

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

[0050] The present invention is further illustrated by the following examples, which are provided for illustration only and are not intended to limit the scope of the present invention, which is defined by 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 containing anti-CD3 single domain antibody and exosome protein is as follows. HEK-293T cell line (passage 4 to 25) and HEK-293T stable cell line expressing anti-CD3 single domain antibody-CD63 chimeric protein were loaded into COL2.5 NGCs and contained 500mL DMEM culture medium (containing 50mL exosome-free FBS) (Thermo Fisher Scientific). After 3 days of culture, the culture medium was centrifuged at 2000g for 15 minutes to remove cell debris and then filtered with 0.2μm filter paper. Then, by ultrafiltration ( Ultra, 30 kDa, Merck Millipore) at 5000g for 8 minutes. The collected supernatant was processed by tangential flow filtration (MAP.03-plus TFF System, Lefo Science). Subsequently, the supernatant of parental HEK-293T cells was filtered through a membrane with a cutoff value of 300 kDa and then resuspended in PBS; the supernatant from HEK-293T stable cells expressing anti-CD3 single domain antibody-CD63 chimeric protein was filtered through a VHH-capture membrane (GenScript) and then resuspended in PBS. All samples were used immediately or stored at -80°C for further use. For NTA analysis, these exosomes were filtered through a membrane with a cutoff value of 300 kDa and then resuspended in PBS; (Particle Metrix GmbH) to analyze their size distribution and concentration.

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

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

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

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

[0057] Figure 2 The binding position of the exosomal protein CD63 and the anti-CD3 single domain antibody is shown, wherein the arrow indicates the embedded 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 embodiment, the experimental process of the surface plasmon resonance (SPR) analysis of the exosomes of the fusion protein is as follows. CM5 and NTA chips (research grade) were subjected to SPR analysis by BIAcore T200 (Biacore-GE Healthcare, Piscataway, NJ). In brief, the protein (CD3ε recombinant protein) sample was diluted with a concentration range of 20 μg / mL in a 10 mM buffer solution (pH 4.0, 5.5 or 6.0) to provide maximum surface retention for fixation on the chip after the surface preparation process, and select conditions with higher surface concentrations of ligands on the chip (anti-CD3 single domain antibodies: 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 nM). Then regeneration scouting and surface performance testing were performed, and the regeneration method was selected to run the experiment following the regeneration scouting and surface performance testing. Then select BINDING ANALYSIS and DIRECT BINDING to study protein binding. Select KINETICS ANALYSIS and select MASS TRANSFER to perform kinetic analysis and concomitant binding experiments. Analyze the data and determine the kinetic constants.

[0060] The results of Exo surface plasmon resonance (SPR) analysis of the fusion protein are shown in Figure 3 .Depend on Figure 3 It can be seen that the fusion protein (1x 10 11) were coated on a CM5 chip, and then the binding affinity was determined 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 effectiveness of the fusion protein in treating cancer, immunomodulation, and activating immune cells.

[0063] Figure 4 Schematic diagram of the effects of fusion proteins on cancer treatment, immune regulation and activation of 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. Figure 4 As can be seen, the sequence encoding the anti-CD3 single domain antibody is inserted into the second external loop of CD63 to form a CD63 chimeric protein with the anti-CD3 single domain antibody exposed on the cell surface. This construct is then transfected into HEK-293T to produce exosomes expressing the anti-CD3 single domain antibody. The harvested exosomes are then 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, and we assume that these exosomes selectively reprogram CD3 + T cells express Nb-CAR and secrete Nb-BiTE to fight solid tumor cells in vivo.

[0064] Figure 5This is another schematic diagram of the effectiveness of fusion proteins in treating cancer, immunomodulation, and activating immune cells, where 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 interferonγ, TNFα stands for tumor necrosis factorα, PFN stands for perforin, GzmB stands for Granzyme B, which is a serine protease that mediates the apoptosis signaling pathway of cytotoxic T lymphocytes and natural killer cells, SSC stands for side scattered light, and VHH stands for heavy chain variable domain. Figure 5 It can be seen that the Exo and CAR.BiTE DNA were mixed at (3x10 8 Exo vs2μgDNA) ratio was electroporated with the LONZA 4D-Nucleofector electroporation program code CM137, and after one hour of recovery at 4°C, the morphology was observed by electron microscopy, or transfected into human PBMC or whole blood for 48 hours, and then co-cultured with tumor cells, and the changes in tumor cell cytotoxicity were confirmed by live / dead cell-mediated cytotoxicity assay (LIVE / DEAD Cell-Mediated Cytotoxicity Assay); or the expression of CD3, CD4, CD8, VHH, etc. on the cells was analyzed by flow cytometry after fluorescent antibody calibration. The above experiments can confirm the transfection effect of fusion protein-coated CAR.BiTE DNA.

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

[0066] Figure 6 Transmission electron microscope (TEM) results showing fusion proteins, where CD3εNb represents anti-CD3 single domain antibody, Exo represents exosome, CAR represents chimeric antigen receptor, and BiTE represents bispecific T-cell engager. Figure 6 As can be seen, CAR.BiTE DNA@CD3εNb-Exo maintains the super structure and morphology of exosomes. Unmodified and CD3εNb artificially treated Exo (regardless of whether CAR.BiTE was used for electroporation) were evaluated by TEM at 100,000X with DNA expressing CAR.BiTE.

[0067] HEK293-derived Exo can be electroporated with DNA expressing CAR. Using LONZA 4D-Nucleofector, unmodified Exo and fusion protein were electroporated with CAR.BiTE expression vector at a ratio of 3x 10 8 Exo: 2 μg DNA. Exo was then treated with DNase (1000 IU) for 30 minutes, and the incorporated CAR.BiTE DNA was quantified by qPCR using specific primers or by spectrophotometer at OD260 and 280 nm.

[0068] Figure 7 The figure shows the efficiency of linear Nb-CAR.BiTE DNA encapsulation into Exo through electroporation, where Nb stands for nanobody, CAR stands for chimeric antigen receptor, BiTE stands for bispecific T-cell engager, Exo stands for exosome, CD3εNb stands for anti-CD3 single domain antibody, and qPCR stands for quantitative polymerase chain reaction. Figure 7 It can be seen from the spectrophotometer and qPCR results that the electrotransfection method can make every 3x 10 8 Exo coated with about 100ng CAR.BiTE DNA.

[0069] Unmodified Exo or fusion proteins were expressed at 3x10 8Exo: 2 μg DNA was electroporated and after purification, these Exo were added to 5x 10 5 PBMCs were stained for 48 hours on ice for 45 minutes and CD3 was measured by flow cytometry using specific antibodies. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + 、CD56 + , TCRγδ + 、CD14 + 、CD19 + and CD66b + Expression of Nb-CAR on cells.

[0070] Figure 8 The efficiency of electroporated Exo transfection into peripheral blood mononuclear cells (PBMC) coated with Nb-CAR.BiTE DNA is shown, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell activating antibody, Exo represents exosome, CD3εNb represents anti-CD3 single domain antibody, and WLSM represents weighted least squares measurement. Figure 8 It can be seen that by comparing with unmodified Exo (293T Exo), CAR.BiTEDNA@CD3e Nb-Exo has a + CD3 - This result supports that the fusion protein selectively delivers the transgene to CD3 + in cells.

[0071] Unmodified Exo or fusion proteins were expressed at 3x10 8 Exo: 2 μg DNA was electroporated and after purification, these Exo were added to 5x 10 5 PBMCs were stained for 48 hours on ice for 45 minutes and CD3 was measured by flow cytometry using specific antibodies. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + Expression of Nb-CAR on cells.

[0072] Fig. 9 Another schematic diagram for testing the efficiency of Nb-CAR.BiTE DNA-coated electroporated Exo transfection into peripheral blood mononuclear cells (PBMC), 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. Fig. 9 It can be seen that by comparing with unmodified Exo (293T Exo), CAR.BiTE DNA@CD3e Nb-Exo has a + / CD4 + and CD3 / CD8 + This result supports that the fusion protein selectively delivers the transgene to CD4 + and CD8 + in cells.

[0073] Unmodified Exo or fusion proteins were expressed at 3x10 8 Exo: 2 μg DNA was electroporated and after purification, these Exo were added to 5x 10 5 PBMCs were stained for 48 hours on ice for 45 minutes and CD3 was measured by flow cytometry using specific antibodies. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + Expression of Nb-CAR on cells.

[0074] Fig.10 The results show the efficiency of testing the electroporated Exo transfection into PBMCs coated with linear Nb-CAR.BiTE DNA, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. Fig.10 It can be seen that CAR.BiTE DNA@CD3e Nb-Exo in CD3 + 、CD3 + / CD4 + and CD3 / CD8 +This result supports that the fusion protein selectively delivers the transgene to CD4 + and CD8 + in cells.

[0075] Unmodified Exo or fusion proteins were expressed at 3x10 8 Exo: 2 μg DNA ratio was used for electroporation A. After purification, these Exo were added to 1 ml of whole blood for 48 hours. After staining on ice for 45 minutes, CD3 was measured by flow cytometry using specific antibodies. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + Expression of Nb-CAR on cells.

[0076] Fig.11 The efficiency of electroporated Exo transfection into whole blood by testing Nb-CAR.BiTE DNA is shown, 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. Fig.11 This result supports the selective delivery of transgenes by fusion proteins to CD3 + in cells.

[0077] Unmodified Exo or fusion proteins were expressed at 3x10 8 The ratio of Exo:2μg DNA was electroporated and, after purification, these Exo were added to 1ml of whole blood for 48 hours. After staining on ice for 45 minutes, CD3 was measured by flow cytometry using specific antibodies. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + 、CD56 + , TCRγδ + 、CD14 + 、CD19 + CD66b + Expression of Nb-CAR on cells.

[0078] Fig.12 Another schematic diagram for testing the efficiency of Nb-CAR.BiTE DNA-coated electroporated Exo transfection into whole blood, where Nb represents nanobody, CAR represents chimeric antigen receptor, BiTE represents bispecific T-cell engager, Exo represents exosome, CD3εNb represents anti-CD3 single domain antibody, and WLSM represents weighted least squares measurement. Fig.12 This result supports the selective delivery of transgenes by fusion proteins to CD3 + / CD4 and CD3 + / CD8 + in cells.

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

[0080] Fig.13 The results show the efficiency of testing the electroporation of linear Nb-CAR.BiTE DNA-coated Exo transfection 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. Fig.13 This result supports the selective delivery of transgenes by fusion proteins to CD3 + 、CD3 + / CD4 and CD3 + / CD8 + in cells.

[0081] Unmodified Exo or fusion proteins were expressed at 3x10 8The ratio of Exo:2μg DNA was electroporated and, after purification, these Exo were added to 1ml of whole blood for 48 hours. After staining on ice for 45 minutes, CD3 was measured by flow cytometry using specific antibodies. + 、CD3 - 、CD3 + / CD4 + 、CD3 + / CD8 + Expression of Nb-CAR on cells.

[0082] Fig.14 Another schematic diagram for testing the efficiency of electroporated Exo transfection into whole blood coated with linear Nb-CAR.BiTE DNA, where Nb represents nanobody, CAR represents chimeric antigen receptor, Exo represents exosome, and CD3εNb represents anti-CD3 single domain antibody. Fig.14 This result supports the selective delivery of transgenes by fusion proteins to CD3 + 、CD3 + / CD4 and CD3 + / CD8 + in cells.

[0083] Cytotoxic killing assay: CAR.BiTE-CD3εNb-Exo or unmodified exosome-treated CD3 + Cells were used as effector cells. Target cells (tumor cell lines) and effector cells were co-cultured at the specified effector cell / target cell (E:T) ratio (1:1 to 50:1) at 37°C for 24 to 72 hours. For live / dead cell viability assays, all tumor cells were stained with green-fluorescent calcein-AM before co-culture and then stained with red-fluorescent ethidium homodimer-1 after co-culture to mark dead cells. Dead tumor cells were identified as green fluorescent cells according to the manufacturer's instructions (Thermo Fisher Scientific). + / Red Fluorescence + Cells. Cell killing rate is expressed as a percentage of the total cell population.

[0084] Figures 15A to 15CThe linear Nb-CAR.BiTE DNA-loaded Exo-transfected PBMCs induced excellent cytolytic activity against solid tumor cells, where CRC represents colorectal cancer, GBM represents glioblastoma multiforme, NSCLC represents non-small cell lung cancer, and E:T ratio represents effector-to-target ratio. Figures 15A to 15C It can be seen that by comparing with unmodified Exo (293T Exo) loaded with or without CAR.BiTE DNA and CD3e Nb-Exo without DNA loading, PBMCs treated with CD3e Nb-Exo loaded with CAR.BiTE DNA exerted more effective cytotoxic killing ability on these tumor cells. In addition, compared with PBMCs treated with Exo without CAR.BiTE DNA, PBMCs treated with 293T Exo loaded with CAR.BiTE DNA also showed excellent cytotoxic killing ability.

[0085] Fig.16 Schematic diagram of the in vivo anti-tumor efficacy of CAR.BiTE DNA@CD3εNb-Exo. COLO 205-luc cells (1x 10 6 7 days later, mice (n=5) were injected with huPBMC (5×10 6 The next day, mice were treated with or without unmodified Exo or CD3εNb-Exo loaded with or without CAR.BiTE DNA (3x 10 10 / mouse) once a week for four weeks. Tumor growth was monitored by the IVIS system by detecting bioluminescent signals. Seven days after the last injection, the mice were sacrificed, spleen cells were harvested, and the expression of Nb-CAR on each type of immune cell was measured by flow cytometry using specific antibodies against VHH, CD3, CD56, TCRγδ, CD14, CD19, and CD66b.

[0086] Fig.16The results of the fusion protein in in vivo chimeric antigen receptor T-cell therapy (CAR-T) are shown, in which a peripheral blood mononuclear cell (PBMC)-huNSG mouse model is used, huPBMC denotes human peripheral blood mononuclear cells, IP denotes intraperitoneal, IV denotes intravenous, CAR denotes chimeric antigen receptor, BiTE denotes bispecific T-cell engager, Exo denotes exosome, CD3εNb denotes anti-CD3 single domain antibody, and luc denotes luciferase. The fourth dose has been administered and blood has been collected to test the transfection efficiency.

[0087] Fig.17 Schematic diagram of the in vivo anti-tumor efficacy of CAR.BiTE DNA@CD3εNb-Exo. COLO 205-luc cells (1x 10 6 7 days later, mice (n=5) were injected with huPBMC (5×10 6 The next day, mice were treated with or without unmodified Exo or CD3εNb-Exo loaded with or without CAR.BiTE DNA (3x 10 10 / mouse) once a week for four weeks. The IVIS system monitored tumor growth by detecting bioluminescent signals.

[0088] Fig.17 Another schematic diagram showing the effectiveness of fusion proteins in treating cancer, immunomodulation, and activating immune cells, where 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. Fig.17 It can be seen that CAR.BiTE DNA@CD3e Nb-Exo exhibited excellent anti-tumor activity compared with other mouse groups.

[0089] Fig.18 Schematic diagram of the in vivo anti-tumor efficacy of CAR.BiTE DNA@CD3εNb-Exo. COLO 205-luc cells (1x 10 6 7 days later, mice (n=5) were injected with huPBMC (5×106 The next day, mice were treated with or without unmodified Exo or CD3εNb-Exo loaded with or without CAR.BiTE DNA (3x 10 10 / mouse), once a week for four weeks. Seven days after the last injection, the mice were sacrificed, submandibular blood was harvested, and the expression of Nb-CAR on each type of immune cell was measured by flow cytometry using specific antibodies against VHH and CD3.

[0090] Fig.18 Another schematic diagram showing the utility of fusion proteins in cancer treatment, immunomodulation, and immune cell activation, where Nb stands for nanobody and CAR stands for chimeric antigen receptor. This result supports that the fusion protein selectively delivers the transgene to the CD3 + 、CD3 + / CD4 and CD3 + / CD8 + in cells.

[0091] Fig.19 Schematic diagram of the in vivo anti-tumor efficacy of CAR.BiTE DNA@CD3εNb-Exo. COLO 205-luc cells (1x 10 6 7 days later, mice (n=5) were injected with huPBMC (5×10 6 The next day, mice were treated with or without unmodified Exo or CD3εNb-Exo loaded with or without CAR.BiTE DNA (3x 10 10 / mouse), once a week for four weeks. Seven days after the last injection, the mice were sacrificed, submandibular blood was harvested, and the expression of Nb-CAR on each type of immune cell was measured by flow cytometry using specific antibodies against VHH and CD3.

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

[0093] Fig. 20Another schematic diagram showing the effectiveness of fusion proteins 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. Fig.19 and Fig. 20 It can be seen that the fusion protein selectively delivers the transgene to the CD3 + in cells.

[0094] In summary, the fusion protein of the present invention can be used through surface plasmon resonance, cytotoxicity analysis, animal experiments, electroporation experiments, transfection efficiency testing, 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 activating antibodies and activating peripheral immune cells, to achieve the effects of treating cancer, immune regulation, and activating immune cells.

[0095] The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or changes made thereto 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, characterized in that: 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.

2. The fusion protein according to claim 1, characterized in that The anti-CD3 single domain antibody specifically binds to CD3 epsilon (CD3 epsilon).

3. The fusion protein according to claim 1, characterized in that The exosomal protein is CD63.

4. The fusion protein according to claim 1, characterized in that The amino acid sequence is shown in SEQ ID NO:

4.

5. The fusion protein according to claim 1, characterized in that The amino acid sequence shown in the sequence identification number SEQ ID NO:1 is complementarity determining region 1 (CDR1), the amino acid sequence shown in the sequence identification number SEQ ID NO:2 is CDR2, and the amino acid sequence shown in the sequence identification number SEQ ID NO:3 is CDR3.

6. The fusion protein according to claim 1, characterized in that The anti-CD3 single domain antibody is an anti-T cell nanobody.

7. An isolated nucleic acid encoding the amino acid sequence of the fusion protein according to any one of claims 1 to 6.

8. The isolated nucleic acid according to claim 7, characterized in that The nucleotide sequence is shown in SEQ ID NO:

5.

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

10. Use of the fusion protein according to any one of claims 1 to 6 for preparing a medicine for treating cancer, immunomodulation and activating immune cells.

11. The use according to claim 10, characterized in that The cancer is treated by allowing the fusion protein to penetrate into the interior of solid tumors and secrete bispecific T-cell engager (BiTE) and activate peripheral immune cells.

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