Fusion protein of chemotactic factor CXCL12 and application thereof

Abstract

The invention discloses a fusion protein of a chemotactic factor CXCL12 and application thereof, the fusion protein of the chemotactic factor CXCL12 comprises the chemotactic factor CXCL12 and an antibody of an anti-tumor related antigen, and the chemotactic factor CXCL12 and the antibody of the anti-tumor related antigen are directly fused or fused through a connexon. On one hand, the fusion protein of the chemotactic factor CXCL12 provided by the invention can promote T cell infiltration and improve the targeting property; and on the other hand, the T cell killing signal is very dependent on the target cell, and the T cell can generate an immune signal to kill the target cell only after being in contact with the target cell under the mediation of the fusion protein, so that the toxic and side effects are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of fusion proteins, and in particular relates to a fusion protein of chemokine CXCL12 and a use thereof. Background Art

[0002] The fusion of cytokines and antibodies to form immunocytokines is a common design, including IL-2 fusion anti-EpCAM, IL-2 fusion anti-GD2, IL-10 fusion anti-EGFR, IL-12 fusion anti-EGFR, etc. Because there is a tumor-targeting antibody at one end, this fusion protein will bind to the surface of the target cell and accumulate around the target cell, thereby better stimulating immune cells that bind to or are adjacent to the target cell.

[0003] Most immune cell engagers are bispecific antibodies (bispecific antibodies), which can connect two cells together to kill or regulate them. Some bispecific antibodies target cytotoxic immune cells (T cells, NK cells, etc.) at one end and tumor cells at the other end; others target cytotoxic immune cells at one end and regulatory cells (DC cells, etc.) at the other end. There are also some trispecific antibody-based immune cell engagers, such as CD3 / CD28 / HER2, in which one antibody targets tumors and the other two antibodies target T cells. There are also bispecific antibodies that fuse cytokines, such as anti-CD16+IL15+anti-CD19, which can connect CD19-positive tumor cells to CD16-positive NK cells. At the same time, IL15 can be used to maintain the continued division, development, and survival of NK cells, thereby improving their efficiency.

[0004] Chemokine CXCL12 (stromal cell-derived factor 1, SDF-1) belongs to the CXC chemokine family. The molecular weight of mature CXCL12 is about 8kD, and its receptors include CXCR4 and CXCR7. As a chemokine, CXCL12 can induce immune cells to migrate in the direction of high CXCL12 concentration by activating CXCR4 / CXCR7 receptors on immune cells.

[0005] T cells need to infiltrate tumor cells to kill solid tumors. Current studies have found that T cells usually have difficulty infiltrating tumor tissues. In terms of promoting T cell infiltration into tumors, existing technologies focus on engineering T cells or chimeric antigen receptor T cells (CAR-T) to enable T cells to express chemokines or chemokine receptors to achieve T cell infiltration into tumors, but this method lacks targeting. Existing bispecific T cell engagers activate T cells to kill tumors through CD3. The activated T cells are basically located on the periphery of solid tumors and continuously release cytokines, but T cells have difficulty contacting target cells, resulting in strong toxic side effects under limited killing. In terms of achieving T cell killing of tumors through protein drugs, existing technologies focus on the design of T cell engagers, but have not yet solved the problem of T cell infiltration into tumor tissues. Summary of the invention

[0006] In order to solve the deficiencies in the prior art, the present invention aims to provide a fusion protein of chemokine CXCL12 and its use. The fusion protein of chemokine CXCL12 provided by the present invention can be used as an immunotherapy drug in tumor treatment, and can kill and inhibit tumors in vitro and in vivo by activating immune cell signaling pathways.

[0007] The specific technical solutions are as follows:

[0008] The present invention provides a fusion protein of chemokine CXCL12, which comprises chemokine CXCL12 or a mutant thereof and an antibody against tumor-associated antigens, wherein the chemokine CXCL12 mutant has the ability to activate CXCR4 and / or CXCR7 receptors on immune cells, and the chemokine CXCL12 or a mutant thereof and the antibody against tumor-associated antigens are fused directly or through a linker.

[0009] Furthermore, the chemokine CXCL12 is natural CXCL12 or a fragment thereof or artificial CXCL12; the artificial CXCL12 is an artificial protein that induces cell chemotaxis via CXCR4 and / or CXCR7;

[0010] Preferably, the chemokine CXCL12 has an amino acid sequence as shown in SEQ ID NO.1, or has a mutant sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO.1, and the mutant sequence has the ability to activate CXCR4 and / or CXCR7 receptors on immune cells.

[0011] Furthermore, the anti-tumor-associated antigen antibody is a single-chain antibody, a complete antibody or an antibody fragment against any anti-tumor-associated antigen;

[0012] Preferably, the anti-tumor-associated antigen antibody is an antibody against EpCAM, DR5 or CEA;

[0013] Preferably, the anti-tumor-associated antigen antibody is an anti-EpCAM single-chain antibody, an anti-DR5 single-chain antibody or an anti-CEA single-chain antibody;

[0014] Preferably, the amino acid sequence of the anti-EpCAM single-chain antibody is as shown in SEQ ID NO.4;

[0015] Preferably, the amino acid sequence of the anti-DR5 single-chain antibody is as shown in SEQ ID NO.5;

[0016] Preferably, the amino acid sequence of the anti-CEA single-chain antibody is as shown in SEQ ID NO.6.

[0017] Furthermore, the chemokine CXCL12 is located at the N-terminus of the fusion protein or the anti-tumor-associated antigen antibody is located at the N-terminus of the fusion protein;

[0018] The linker is a flexible amino acid sequence;

[0019] Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO.3.

[0020] The present invention also provides a nucleic acid molecule encoding the fusion protein.

[0021] The invention also provides a plasmid containing the nucleic acid molecule.

[0022] The present invention also provides a host cell containing the nucleic acid.

[0023] Furthermore, in the present invention, when preparing the fusion protein of chemokine CXCL12, the chemokine CXCL12 or its mutant and the antibody against tumor-associated antigens can be expressed in a fusion protein in the form of a fusion protein in one expression plasmid; or the chemokine CXCL12 or its mutant and the antibody against tumor-associated antigens can be independently expressed and then chemically covalently linked; or the chemokine CXCL12 or its mutant and the antibody against tumor-associated antigens can be separately expressed after adding interactive units, and the added units can spontaneously or inducibly connect the chemokine CXCL12 or its mutant and the antibody against tumor-associated antigens together.

[0024] The present invention also provides use of the fusion protein in preparing anti-tumor drugs.

[0025] Further, the tumor is a solid tumor;

[0026] Preferably, the tumor is epidermoid carcinoma or breast cancer.

[0027] The present invention also provides an anti-tumor pharmaceutical composition, which comprises the fusion protein;

[0028] Preferably, the pharmaceutical composition further comprises other anti-tumor drugs.

[0029] The beneficial effects of the present invention are:

[0030] The fusion protein of chemokine CXCL12 provided by the present invention can promote T cell infiltration on the one hand, and activate T cell immunity on the other hand to kill tumors, such as Fig. 9 shown.

[0031] On the one hand, the fusion protein of chemokine CXCL12 provided by the present invention can promote T cell infiltration. The present invention fuses chemokine CXCL12 with an antibody against tumor-associated antigen (TAA) to form a typical immune cytokine. CXCL12 can be enriched around tumor cells positive for tumor-associated antigens, forming a concentration gradient, inducing CXCR4-positive T cells to migrate toward the tumor, and promoting T cell infiltration.

[0032] On the other hand, the fusion protein of the chemokine CXCL12 provided by the present invention can activate T cell immunity and kill tumors. The fusion protein of the chemokine CXCL12 in the present invention can be used as a bispecific adapter to connect target cells and T cells together, and activate T cell signals by CXCL12 anchored on tumor cells to kill tumor cells. This killing function has never been reported. At the same time, in the present invention, the T cell killing signal depends on the target cell, and the T cell will only produce an immune signal to kill the target cell after contacting the target cell under the mediation of the fusion protein, thereby reducing toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the expression structure of ChTE and BiTE in Example 1 of the present invention, top: ChTE; bottom: BiTE.

[0034] Figure 2 This is the Coomassie Brilliant Blue staining result of ChTE-aEpCAM protein purification in Example 1.

[0035] Figure 3 The results of the chamber invasion experiment in Example 2 are shown in FIG.

[0036] Figure 4 The target cell killing results of different ChTEs in Example 3 are shown.

[0037] Figure 5These are the expression levels of various cytokines when ChTE-aEpCAM activates T cells to kill target cells in Example 4.

[0038] Figure 6 The experimental results of ChTE-aEpCAM activating T cells to kill 3D target cell spheres in Example 5.

[0039] Figure 7 These are the experimental results of ChTE-aEpCAM at different concentrations and effector-target ratios in Example 6 to activate T cells to kill target cells.

[0040] Figure 8 These are the results of the ChTE-aEpCAM animal experiment in Example 7.

[0041] Fig. 9 Schematic diagram of the ChTE of the present invention promoting T cell infiltration and tumor killing. DETAILED DESCRIPTION

[0042] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0043] Example 1

[0044] 1. Structural design of chemokine CXCL12 fusion protein

[0045] The present invention provides a fusion protein of chemokine CXCL12. In the present invention, chemokine CXCL12 is fused with an antibody against tumor-associated antigen (TAA) to form a typical immune cytokine. The present invention names this fusion protein as chemotactic T cell engager (ChTE), hereinafter referred to as ChTE. In order to distinguish the different target scFvs involved, the corresponding proteins are named in the format of ChTE-aTAA, and aTAA is anti-TAA.

[0046] ChTE contains two structural units: the first structural unit is the chemokine CXCL12, and the second structural unit is an anti-TAA antibody, and the two structural units are directly fused or fused through a linker. The anti-TAA antibody can be a single-chain antibody (scFv), a complete antibody or an antibody fragment against any TAA. In ChTE, the first structural unit can be located at the N-terminus, and the second structural unit can also be located at the N-terminus.

[0047] In a specific embodiment, the chemokine CXCL12 may use natural CXCL12 or artificial CXCL12 designed from scratch, that is, artificial protein that can induce cell chemotaxis through CXCR4 and / or CXCR7.

[0048] In a specific embodiment, the amino acid sequence of the chemokine CXCL12 is shown as SEQ ID NO.1.

[0049] In a specific embodiment, the anti-TAA antibody is selected from anti-TAA scFv, and CXCL12 and scFv are connected by any reasonable amino acid fragment. Preferably, CXCL12 and scFv are connected by a flexible amino acid linker, and its amino acid sequence is shown in SEQ ID NO.3.

[0050] In a specific embodiment, the anti-TAA antibody is scFv anti-EpCAM, and its amino acid sequence is shown in SEQ ID NO.4.

[0051] In a specific embodiment, the anti-TAA antibody is scFv anti-DR5, and its amino acid sequence is shown in SEQ ID NO.5.

[0052] In a specific embodiment, the anti-TAA antibody is scFv anti-CEA, and its amino acid sequence is shown in SEQ ID NO.6.

[0053] Chemokine CXCL12 (SEQ ID NO.1):

[0054] MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQ IVARLKNNNRQVCIDPKLKWIQEYLEKALNKRFKM

[0055] Linker (SEQ ID NO.3):

[0056] SAGGSAGGSAGG

[0057] anti-EpCAM (SEQ ID NO.4):

[0058] ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSS

[0059] anti-DR5 (SEQ ID NO.5):

[0060] DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLPFTFGAGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLKLSCAASGFDFSTCWMNWVRQAPGKGLEWIGEINPDSSRINYMPSLKEKFIISRDNAKNTLYLQMSKVRSEDTALYYCARGGTFYAMDYWGQGTSVTVSS

[0061] anti-CEA (SEQ ID NO.6):

[0062] DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS

[0063] II. Expression and purification of the fusion protein of chemokine CXCL12

[0064] When preparing ChTE, the two structural units of ChTE can be fused and expressed in an expression plasmid in the form of a fusion protein, or the two structural units can be expressed independently and then chemically covalently linked, or the two main structural units can be expressed separately after adding interactive units, and the added units can spontaneously or inducibly connect the two main unit structures together.

[0065] In a specific embodiment, ChTE is fused and expressed in an expression plasmid, and ChTE is obtained by protein expression and purification. Preferably, the N-terminus of ChTE contains a signal peptide SP (signal peptides), and its amino acid sequence is MNAKVVVVLVLVLTALCLSDG, as shown in SEQ ID NO.2, and the signal peptide can be replaced by a signal peptide derived from any other secretory protein or membrane protein.

[0066] The specific steps are as follows:

[0067] 1. Construction of ChTE expression plasmid

[0068] In order to facilitate protein purification, a His-tag sequence (HHHHHH) was added to the C-terminus of ChTE. The DNA sequence encoding ChTE was obtained by gene synthesis. The DNA sequence encoding ChTE was cloned into a mammalian expression plasmid. In one embodiment, the mammalian expression plasmid is selected from pCMV. The basic expression structure of ChTE is shown in the attached Figure 1 .

[0069] 2. Expression of ChTE

[0070] ChTE can be expressed by transient transfection of 293T cells, and the culture supernatant is used as a sample to test its basic properties. The specific steps are as follows:

[0071] Cell preparation: HEK 293T cells were seeded at a density of 1×10^5 cells / well in a 24-well culture plate, 500 μL of DMEM medium was added, and transient transfection was performed 6 hours later;

[0072] Cell transfection: Take 25 μL serum-free opti-MEM medium, add 550 ng of target plasmid, add 1.7 μL FuGENE transfection reagent, pipette to mix, incubate at room temperature for 8 minutes, and add dropwise to the prepared cells;

[0073] Supernatant collection: Collect the cell culture supernatant 36 hours after transfection, centrifuge at 2000g×5min, and store at 4℃ for 24 hours.

[0074] 3. Purification of ChTE

[0075] ChTE can be purified from 293F cells. The specific steps are as follows:

[0076] Cell preparation: Cells (1×10 7 -1.5×10 7 ) Use 25 mL of serum-free medium to resuscitate, culture at 100 rpm, 8% carbon dioxide, 37 ° C for 2 days, directly add 25 mL of medium to expand to 50 mL system, culture under the same conditions for 2 days, add 50 mL of medium to expand to 100 mL system, continue to culture for 2 days, count the cells, and adjust the cell concentration to 0.8×10 6 / mL, expand to 200mL system, continue to culture for 2 days, and then transfection can be carried out.

[0077] Transfection steps: prepare 400 μg of target plasmid; prepare PEI at a concentration of 1 mg / mL and filter through a 0.22 μm filter for later use; add 400 μg of plasmid to 10 mL of serum-free medium and vortex for 30 seconds; add 1.2 mL of PEI (1 mg / mL) to 10 mL of serum-free medium and vortex for 30 seconds; mix the two, vortex for 30 seconds, let stand for 30 minutes, and then add 200 mL of PEI at a concentration of 100 μg / mL (generally 1-1.5×10 6 / mL) cells, add 2mL of penicillin-streptomycin mixture (100x), culture at 80rpm, 8% CO2, 37°C for 5 days, collect the supernatant, centrifuge at 8000g, collect the supernatant and filter with 0.22μm filter membrane for protein purification.

[0078] Purify protein: Take out the purification column containing Ni filler (GE Company) and use AKTA-pure protein purifier to purify the protein. Add 3 to 4 column volumes of ultrapure water to elute the column. Add 3 to 4 column volumes of binding buffer (50mM Tris-HCl, 300mMNaCl, 10mM imidazole, pH 8.0) to balance the column. Add 3 to 4 column volumes of washing buffer (50mM Tris-HCl, 300mM NaCl, 20mM imidazole, pH 8.0) to the purification column to wash the impurities. Then add 3 to 4 column volumes of elution buffer (50mM Tris-HCl, 300mM NaCl, 250mM Imidazole, pH 8.0) to the purification column to elute the target protein. Finally, use a desalting column to replace the buffer with 1×PBS for storage. Use the BSA method to determine the protein concentration and store it at -80℃ after aliquoting.

[0079] 3. Expression and purification of ChTE-aEpCAM, ChTE-aDR5 and ChTE-aCEA

[0080] The basic expression structures of the ChTE-aEpCAM, ChTE-aDR5 and ChTE-aCEA expression plasmids involved in the following embodiments of the present invention are shown in the attached Figure 1 As shown, wherein the amino acid sequence of the chemokine CXCL12 is shown in SEQ ID NO.1, the amino acid sequences of anti-EpCAM scFv, anti-DR5 scFv, and anti-CEA scFv are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively, the amino acid sequence of SP is shown in SEQ ID NO.2, and the amino acid sequence of linker is shown in SEQID NO.3, and the method described in Example 1 is used to express and purify ChTE-aEpCAM, ChTE-aDR5, and ChTE-aCEA. At the same time, after 36 hours of transient transfection of HEK 293T, the ChTE-aEpCAM, ChTE-aDR5, and ChTE-aCEA expression plasmids were respectively collected from the supernatants for subsequent experiments. Among them, the purified protein (ChTE-aEpCAM) was subjected to electrophoresis and Coomassie Brilliant Blue staining, and the results are shown in the attached Figure 2 shown.

[0081] IV. Expression and purification of ChTE-aGFP, BiTE-aEpCAM, and BiTE-aDR5

[0082] The ChTE-aGFP involved in the following embodiments of the present invention is that the anti-EpCAM scFv in ChTE-aEpCAM is replaced with the nano antibody anti-GFP, and the amino acid sequence of anti-GFP is shown in SEQ ID NO.7. After constructing the plasmid, the expression and purification of ChTE-aGFP are carried out according to the expression operation of ChTE in Example 1. At the same time, after 36 hours of transient transfection of the ChTE-aGFP expression plasmid in HEK293T, the supernatant is collected for subsequent experiments.

[0083] BiTE is a traditional bispecific T cell engager (BiTE). The expression structure of BiTE is shown in Figure 1, BiTE-aEpCAM and BiTE-aDR5 involved in the following embodiments of the present invention, wherein the amino acid sequences of anti-EpCAMscFv, anti-DR5scFv and anti-CD3 are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.8 respectively, the amino acid sequence of SP is shown in SEQ ID NO.2, and the amino acid sequence of linker is shown in SEQ ID NO.3. After constructing the plasmid, the expression and purification of BiTE-aEpCAM and BiTE-aDR5 were performed according to the expression operation of ChTE in Example 1. At the same time, after transient transfection of HEK 293T for 36 hours, the supernatant of BiTE-aEpCAM and BiTE-aDR5 expression plasmids was collected for subsequent experiments.

[0084] anti-GFP (SEQ ID NO.7):

[0085] MAQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS

[0086] Anti-CD3 (SEQ ID NO.8):

[0087] DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGE GTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK

[0088] Example 2

[0089] This example verifies through a trans-well invasion experiment that the ChTE designed in the present invention still has the chemotactic function of natural CXCL12.

[0090] This example verifies the chemotactic ability of ChTE-aEpCAM, ChTE-aDR5 and ChTE-aCEA on T cells described in Example 1. ChTE-aGFP in Example 1 is used as a control.

[0091] The specific operation of the trans-well invasion experiment is as follows: collect the cell culture supernatant after transfection of HEK 293T for 36 hours, centrifuge at 2000g*5min, and take the supernatant after centrifugation for use. Add 400μL of the above culture medium supernatant and 300μL of fresh 1640 culture medium to the lower chamber of the 24-well Trans-well plate; add 200μL of resting PBMC cells (peripheral blood mononuclear cells) with a concentration of 1*10^6 to the upper chamber of the well plate, and gently place the upper chamber in the lower chamber. After culturing for 4 hours under 5% carbon dioxide and 37℃, remove the upper chamber of the trans-well, collect the culture medium of the lower chamber, centrifuge at 500g*5min, discard the supernatant, resuspend the cells in 200μL PBS, and count the collected cells using a flow cytometer. The supernatant of the negative control group (Blank) is the supernatant of the blank control culture medium, and the supernatant of the positive control group (CXCL12) is the supernatant of the blank control culture medium, and commercial human CXCL12 protein is added to make the final concentration of 50nM.

[0092] The experimental results are attached. Figure 3 The results showed that the ChTE designed by the present invention still has the chemotactic function of natural CXCL12.

[0093] Example 3

[0094] This example verifies that the ChTE designed in the present invention can activate T cells to kill different target cells through a variety of surface antigens.

[0095] This example verifies the ChTE-aEpCAM and ChTE-aDR5 described in Example 1, and the target cells used are A431 and MCF7. The BiTE-aEpCAM, BiTE-aDR5 and ChTE-aGFP described in Example 1 are used as controls.

[0096] The specific operation is as follows: prepare target cells A431 and MCF7 expressing luciferase 24h in advance, inoculate target cells at 1*10^4 / well in a white opaque 96-well plate, and add fresh DMEM medium to a total volume of 100μL. Collect the cell culture supernatant 36h after transfection of HEK 293T, centrifuge at 2000g*5min, and take the supernatant after centrifugation for use. Take 50μL of the prepared supernatant and add it to the prepared target cell well plate, then add 50μL of resting PBMC with a concentration of 4*10^5 / mL, and culture for 24h at 37℃ with 5% carbon dioxide. Add 10μL of 5mg / mL luciferin to each well, incubate at room temperature for 10min, and detect using a luminometer. The supernatant of the negative control group (Blank) is the supernatant of the blank control culture medium, and the supernatant of the positive control group is BiTE. The killing efficiency was roughly calculated according to the formula 100*(1-experimental group reading / blank control group reading).

[0097] The experimental results are attached. Figure 4 The results show that the target cells that ChTE in the present invention can target are extensive, the antigenic targets of the target cells are adjustable, and T cells can be activated to kill different target cells through a variety of surface antigens.

[0098] Example 4

[0099] The ChTE-aEpCAM designed in Example 1 of the present invention can activate T cells to express various immune-related cytokines.

[0100] Specifically, prepare target cells A431 24 hours in advance, inoculate target cells in a 24-well plate at 5*10^4 / well, and add fresh DMEM medium to a total volume of 500μL. Collect the cell culture supernatant after 36 hours of transfection of HEK 293T with ChTE-aEpCAM plasmid, centrifuge at 2000g*5min, and take the supernatant after centrifugation for use. Aspirate the culture medium in the target cell culture well, take 125μL of the prepared supernatant, add it to the prepared target cell well plate (Target+) or blank well plate (Target-), add 5*10^4 resting T cells, add fresh DMEM medium to 500μL, and culture for 12 hours at 37°C with 5% carbon dioxide. The culture medium was taken into an EP tube and centrifuged at 2000g*5min. The supernatant was used to detect IL2, TNF-a, IFN-g and GM-CSF. The supernatant was quantitatively detected according to the instructions of the Elisa kit. The positive control group was the BiTE-aEpCAM described in Example 1, and the negative control group was blank culture medium. The experimental results are shown in the attached Figure 5 .

[0101] Example 5

[0102] The ChTE-aEpCAM designed in Example 1 of the present invention has a better killing effect than the BiTE-aEpCAM described in Example 1 when facing a 3D spheroid solid tumor model.

[0103] Specifically, target cells A431 expressing green fluorescent protein GFP were prepared and inoculated in a 96-well round-bottom ultra-low cell adhesion culture plate (e.g., Corning catalog number 7007) at 5000 / well. After adding fresh DMEM medium, the total volume was 100 μL. After culturing for 72 hours at 37°C with 5% carbon dioxide, resting T cells were inoculated at 1*10^4 / well, and ChTE-aEpCAM or BiTE-aEpCAM were added at a working concentration of 10nM or 30nM, respectively, with a final volume of 200 μL / well. PBS was added to the control group, with a final volume of 200 μL / well. The culture was continued for 24 hours, and the state of the target cells was observed and recorded. The experimental results are shown in the attached. Figure 6 .

[0104] Example 6

[0105] The ChTE-aEpCAM designed in Example 1 of the present invention activates T cells to kill tumor cells in a concentration-dependent manner.

[0106] Specifically, the target cells A431 expressing luciferase were prepared 24 hours in advance, and the target cells were inoculated at 1*10^4 / well in a white-bottom opaque 96-well plate. After adding fresh DMEM medium, the total volume was 100μL. According to the total system of 200μL, the concentration gradient was set from 1*10^(-5) to 1*10^2nM. Different effect-target ratios (E:T=1:2, 1:1 and 2:1) were set, and resting T cells were added. They were cultured at 37°C with 5% carbon dioxide for 24h and 48h, and then tested respectively. 10μL of 5mg / mL luciferin (D-luciferin) was added to each well, incubated at room temperature for 10min, and detected using a fluorescence detector (Luminometer). In this experiment, no ChTE protein was added to the blank control group, and an equal amount of T cells were added according to the design of the experimental group. The killing efficiency was calculated according to the formula 100*(1-experimental group reading / blank control group reading). The experimental results are shown in the attached Figure 7 .

[0107] Example 7

[0108] The ChTE-aEpCAM designed in Example 1 of the present invention has an in vivo anti-tumor effect. The BiTE-aEpCAM described in Example 1 was used as a control.

[0109] Specifically, A431 target cells were prepared and routinely subcultured for in vivo experiments. The prepared A431 target cells were collected and resuspended in PBS to obtain a cell suspension with a concentration of 1*10^7 / mL. On day 0, the cells were subcutaneously inoculated in the right armpit of female NSG mice of about 5 weeks old (19-21g) to establish an A431 tumor-bearing mouse model. On day 6, resting T cells were injected into the tail vein at 1*10^7 per mouse. On day 7, the mice were randomly divided into 3 groups, with 5 mice in each group. From day 7, subcutaneous peritumoral administration was performed every other day, and each group was administered with ChTE-aEpCAM, BiTE-aEpCAM and blank control PBS, respectively. Each mouse was administered 100μg (about 5mg / kg, dissolved in 100μL PBS) each time, and the PBS group was injected with 100μL PBS. Tumor size was measured 2-3 times a week. Tumor volume determination: Use a vernier caliper to measure the maximum long axis (L) and the maximum short axis (W) of the tumor, and calculate the formula V = 1 / 2*L*W^2. Use an electronic balance to measure the weight of the mice. The experimental results are attached. Figure 8 .

[0110] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A fusion protein of chemokine CXCL12, characterized in that: It includes chemokine CXCL12 or its mutant and an antibody against tumor-associated antigens, and the chemokine CXCL12 mutant has the ability to activate CXCR4 and / or CXCR7 receptors on immune cells, and the chemokine CXCL12 or its mutant and the antibody against tumor-associated antigens are directly fused or fused through a linker.

2. The fusion protein according to claim 1, characterized in that The chemokine CXCL12 is natural CXCL12 or a fragment thereof or artificial CXCL12; the artificial CXCL12 is an artificial protein that induces cell chemotaxis via CXCR4 and / or CXCR7; Preferably, the chemokine CXCL12 has an amino acid sequence as shown in SEQ ID NO.1, or has a mutant sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO.1, and the mutant sequence has the ability to activate CXCR4 and / or CXCR7 receptors on immune cells.

3. The fusion protein according to claim 1, characterized in that The anti-tumor-associated antigen antibody is a single-chain antibody, a complete antibody or an antibody fragment against any anti-tumor-associated antigen; Preferably, the anti-tumor-associated antigen antibody is an antibody against EpCAM, DR5 or CEA; Preferably, the anti-tumor-associated antigen antibody is an anti-EpCAM single-chain antibody, an anti-DR5 single-chain antibody or an anti-CEA single-chain antibody; Preferably, the amino acid sequence of the anti-EpCAM single-chain antibody is as shown in SEQ ID NO.4; Preferably, the amino acid sequence of the anti-DR5 single-chain antibody is as shown in SEQ ID NO.5; Preferably, the amino acid sequence of the anti-CEA single-chain antibody is as shown in SEQ ID NO.

6.

4. The fusion protein according to claim 1, characterized in that The chemokine CXCL12 is located at the N-terminus of the fusion protein or the anti-tumor-associated antigen antibody is located at the N-terminus of the fusion protein; The linker is a flexible amino acid sequence; Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO.

3.

5. A nucleic acid molecule, characterized in that Encodes the fusion protein according to any one of claims 1 to 4.

6. A plasmid, characterized in that Containing the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that Containing the nucleic acid according to claim 5.

8. Use of the fusion protein according to claim 1 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that The tumor is a solid tumor; Preferably, the tumor is epidermoid carcinoma or breast cancer.

10. An anti-tumor pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the fusion protein of claim 1; Preferably, the pharmaceutical composition further comprises other anti-tumor drugs.

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