Chemokine CXCL12 fusion protein and use thereof

Abstract

Provided are a chemokine CXCL12 fusion protein and a use thereof. The chemokine CXCL12 fusion protein comprises the chemokine CXCL12 and an antibody against a tumor-associated antigen. The chemokine CXCL12 and the antibody against the tumor-associated antigen are directly fused or fused by means of a linker. On the one hand, the provided chemokine CXCL12 fusion protein can promote T cell infiltration and improve targeting. On the other hand, T cell killing signals are highly dependent on target cells, and T cells generate immune signals to kill the target cells only upon coming into contact with the target cells under the mediation of the fusion protein, thereby reducing toxic side effects.

Description

A fusion protein of the chemokine CXCL12 and its applications Technical Field

[0001] This invention belongs to the field of fusion protein technology, specifically relating to a fusion protein of the chemokine CXCL12 and its uses. Background Technology

[0002] The fusion of cytokines with antibodies to form immunocytokines is a common design, including IL-2 fusion with anti-EpCAM, IL-2 fusion with anti-GD2, IL-10 fusion with anti-EGFR, and IL-12 fusion with anti-EGFR. Because one end of the fusion protein contains an antibody targeting the tumor, it binds to the surface of target cells and accumulates around them, thereby better stimulating immune cells that bind to or are near the target cells.

[0003] Most immune cell engagers are bispecific antibodies (Bivariate antibodies), which bind two types of cells together to perform cytotoxic or regulatory functions. Some Bivariate antibodies target cytotoxic immune cells (T cells, NK cells, etc.) at one end and tumor cells at the other; others target cytotoxic immune cells at one end and regulatory cells (DC cells, etc.) at the other. There are also trispecific antibody forms of immune cell engagers, such as CD3 / CD28 / HER2, where one antibody targets the tumor and the other two target T cells. There are also Bivariate antibodies fused with cytokines, such as anti-CD16+IL15+anti-CD19, which can bind CD19-positive tumor cells to CD16-positive NK cells, while IL15 can maintain the continuous division, development, and survival of NK cells, improving their efficiency.

[0004] Chemokine CXCL12 (stromal cell-derived factor 1, SDF-1) belongs to the CXC chemokine family. Mature CXCL12 has a molecular weight of approximately 8 kDa, and its receptors include CXCR4 and CXCR7. As a chemokine, CXCL12 can induce immune cells to migrate towards areas with high CXCL12 concentrations by activating CXCR4 / CXCR7 receptors on immune cells.

[0005] T-cell killing of solid tumors requires T-cell infiltration of tumor cells, but current research shows that T-cells often struggle to infiltrate tumor tissue. Existing technologies for promoting T-cell tumor infiltration focus on engineered T-cells or chimeric antigen receptor T-cells (CAR-T) to express chemokines or chemokine receptors, thus achieving T-cell infiltration of tumors. However, this approach lacks targeting specificity. Existing bispecific T-cell connectors activate T-cells to kill tumors via CD3. The activated T-cells are primarily located on the periphery of solid tumors, continuously releasing cytokines, but they struggle to reach target cells, resulting in limited killing and significant toxicity. Regarding T-cell tumor killing through protein drugs, current technologies focus on the design of T-cell connectors, but the challenge of T-cell infiltration of tumor tissue remains unresolved.

[0006] Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide a fusion protein of the chemokine CXCL12 and its uses. The fusion protein of CXCL12 provided by this invention can be used as an immunotherapeutic agent in tumor treatment, achieving tumor killing and inhibition both in vitro and in vivo by activating immune cell signaling pathways.

[0008] The specific technical solution is as follows:

[0009] The present invention provides a fusion protein of chemokine CXCL12, comprising chemokine CXCL12 or a mutant thereof and an antibody against a tumor-associated antigen, wherein the chemokine CXCL12 mutant has the ability to activate CXCR4 and / or CXCR7 receptors on immune cells, and the chemokine CXCL12 or its mutant thereof and the antibody against the tumor-associated antigen are fused directly or through a linker.

[0010] 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 through CXCR4 and / or CXCR7.

[0011] Preferably, the chemokine CXCL12 has the amino acid sequence shown in SEQ ID NO.1, or has a mutant sequence based on the amino acid sequence shown in SEQ ID NO.1 by substitution, deletion, addition and / or replacement of one or more amino acids, and the mutant sequence has the ability to activate CXCR4 and / or CXCR7 receptors on immune cells.

[0012] Furthermore, the antibody against the antitumor-associated antigen is a single-chain antibody, a complete antibody, or an antibody fragment against any antitumor-associated antigen;

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

[0014] Preferably, the antibody against the tumor-associated antigen is a single-chain antibody against EpCAM, a single-chain antibody against DR5, or a single-chain antibody against CEA.

[0015] Preferably, the amino acid sequence of the anti-EpCAM single-chain antibody is shown in SEQ ID NO.4;

[0016] Preferably, the amino acid sequence of the anti-DR5 single-chain antibody is shown in SEQ ID NO.5;

[0017] Preferably, the amino acid sequence of the anti-CEA single-chain antibody is shown in SEQ ID NO.6.

[0018] Furthermore, the chemokine CXCL12 is located at the N-terminus of the fusion protein, or the antibody against the anti-tumor-associated antigen is located at the N-terminus of the fusion protein;

[0019] The linker is a flexible amino acid sequence;

[0020] Preferably, the amino acid sequence of the linker is shown in SEQ ID NO.3.

[0021] The present invention also provides a nucleic acid molecule that encodes the fusion protein described above.

[0022] The present invention also provides a plasmid containing the aforementioned nucleic acid molecule.

[0023] The present invention also provides a host cell containing the aforementioned nucleic acid.

[0024] Furthermore, in preparing the fusion protein of chemokine CXCL12, the present invention allows for the fusion expression of chemokine CXCL12 or its mutants and antibodies against tumor-associated antigens in the form of a fusion protein within an expression plasmid; alternatively, chemokine CXCL12 or its mutants and antibodies against tumor-associated antigens can be expressed independently and then chemically covalently linked; or chemokine CXCL12 or its mutants and antibodies against tumor-associated antigens can be expressed separately after adding interacting units, and the added units can spontaneously or inducibly link chemokine CXCL12 or its mutants and antibodies against tumor-associated antigens together.

[0025] The present invention also provides the use of the fusion protein in the preparation of antitumor drugs.

[0026] Furthermore, the tumor is a solid tumor;

[0027] Preferably, the tumor is epidermoid carcinoma or breast cancer.

[0028] The present invention also provides an antitumor pharmaceutical composition comprising the fusion protein;

[0029] Preferably, the pharmaceutical composition further comprises other antitumor drugs.

[0030] The beneficial effects of this invention are as follows:

[0031] The fusion protein of chemokine CXCL12 provided by this invention can promote T cell infiltration on the one hand and activate T cell immunity to kill tumors on the other hand, as shown in Figure 9.

[0032] On the one hand, the fusion protein of the chemokine CXCL12 provided by this invention can promote T cell infiltration. This invention fuses the chemokine CXCL12 with an antibody against a tumor-associated antigen (TAA) to form a typical immune cytokine. CXCL12 can be enriched around tumor cells positive for TAAs, forming a concentration gradient that induces CXCR4-positive T cells to migrate towards the tumor, thus promoting T cell infiltration.

[0033] On the other hand, the fusion protein of chemokine CXCL12 provided by this invention can activate T cell immunity and kill tumor cells. In this invention, the fusion protein of chemokine CXCL12 can act as a bispecific connector to link target cells and T cells together. By anchoring CXCL12 to tumor cells, it activates T cell signaling, thereby killing tumor cells. This killing function has never been reported before. Simultaneously, in this invention, the T cell killing signal is dependent on the target cell; T cells only generate immune signals to kill target cells after contact with them under the mediation of the fusion protein, thus reducing toxic side effects. Attached Figure Description

[0034] Figure 1 is a schematic diagram of the expression structure of ChTE and BiTE in Embodiment 1 of the present invention, with ChTE at the top and BiTE at the bottom.

[0035] Figure 2 shows the Coomassie Brilliant Blue staining results of ChTE-aEpCAM protein purification in Example 1.

[0036] Figure 3 shows the results of the small chamber invasion experiment in Example 2.

[0037] Figure 4 shows the target cell killing results of different ChTEs in Example 3.

[0038] Figure 5 shows the expression levels of various cytokines when ChTE-aEpCAM activates T cells to kill target cells in Example 4.

[0039] Figure 6 shows the experimental results of ChTE-aEpCAM activating T cells to kill 3D target cell spheres in Example 5.

[0040] Figure 7 shows the experimental results of ChTE-aEpCAM activating T cells to kill target cells at different concentrations and effector-to-target ratios in Example 6.

[0041] Figure 8 shows the results of the ChTE-aEpCAM animal experiment in Example 7.

[0042] Figure 9 is a schematic diagram illustrating how ChTE promotes T cell infiltration and tumor killing in this invention. Detailed Implementation

[0043] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0044] Example 1

[0045] I. Structural Design of the Fusion Protein of Chemokine CXCL12

[0046] This invention provides a fusion protein of the chemokine CXCL12. In this invention, the chemokine CXCL12 is fused with an antibody against a tumor-associated antigen (TAA) to form a typical immune cytokine. This invention names this fusion protein as the chemotactic T cell engager (ChTE), hereinafter referred to as ChTE. To distinguish the different target scFv involved, the corresponding proteins are named in the format of ChTE-aTAA, where aTAA stands for anti-TAA.

[0047] ChTEs consist of two structural units: the first unit is the chemokine CXCL12, and the second unit is an anti-TAA antibody. These two units are fused directly or via a linker. The anti-TAA antibody can be a single-chain antibody (scFv) against any TAA, a complete antibody, or an antibody fragment. In a ChTE, the first unit can be located at the N-terminus, or the second unit can be located at the N-terminus.

[0048] In one specific implementation, the chemokine CXCL12 can be either natural CXCL12 or de novo engineered artificial CXCL12, which is an artificial protein that can induce cell chemotaxis through CXCR4 and / or CXCR7.

[0049] In one specific embodiment, the amino acid sequence of the chemokine CXCL12 is shown in SEQ ID NO.1.

[0050] In one specific embodiment, the anti-TAA antibody is selected from anti-TAA scFv, and CXCL12 and scFv are linked by any reasonable amino acid fragment. Preferably, CXCL12 and scFv are linked by a flexible amino acid linker, the amino acid sequence of which is shown in SEQ ID NO.3.

[0051] In one specific embodiment, the anti-TAA antibody is scFv anti-EpCAM, whose amino acid sequence is shown in SEQ ID NO.4.

[0052] In one specific embodiment, the anti-TAA antibody is scFv anti-DR5, whose amino acid sequence is shown in SEQ ID NO.5.

[0053] In one specific embodiment, the anti-TAA antibody is scFv anti-CEA, whose amino acid sequence is shown in SEQ ID NO.6.

[0054] Chemokine CXCL12 (SEQ ID NO.1):

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

[0056] anti-EpCAM (SEQ ID NO.4):

[0057] anti-DR5 (SEQ ID NO.5):

[0058] anti-CEA (SEQ ID NO.6):

[0059] II. Expression and purification of the fusion protein of chemokine CXCL12

[0060] When preparing ChTE, the two structural units of ChTE can be expressed as a fusion protein in an expression plasmid, 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 interacting units. The added units can spontaneously or inducibly link the two main structural units together.

[0061] In one specific embodiment, ChTE is expressed by fusing it into an expression plasmid, and ChTE is obtained through protein expression and purification. Preferably, the N-terminus of ChTE contains a signal peptide SP with the amino acid sequence MNAKVVVVLVLVLTALCLSDG, as shown in SEQ ID NO.2. The signal peptide can be replaced with a signal peptide derived from any other secretory protein or membrane protein.

[0062] The specific steps are as follows:

[0063] 1. Construction of ChTE expression plasmid

[0064] To facilitate protein purification, a His-tag sequence (HHHHHH) is added to the C-terminus of ChTE. The DNA sequence encoding ChTE is obtained through gene synthesis. The ChTE-encoding DNA sequence is then cloned into a mammalian expression plasmid. In one specific embodiment, the mammalian expression plasmid is selected from pCMV. The basic expression structure of ChTE is shown in Figure 1.

[0065] 2. Expression of ChTE

[0066] ChTE can be expressed via transient transfection into 293T cells, and its basic characteristics can be tested using culture supernatant as a sample. The specific steps are as follows:

[0067] Cell preparation: HEK 293T cells were seeded in 24-well plates at a density of 1×10^5 cells / well, 500 μL of LMEM medium was added, and transient transfection was performed 6 h later;

[0068] Cell transfection: Take 25 μL of serum-free opti-MEM medium, add 550 ng of target plasmid, add 1.7 μL of FuGENE transfection reagent, mix by pipetting and aspiration, incubate at room temperature for 8 min, and add dropwise to the prepared cells.

[0069] Supernatant collection: Collect cell culture supernatant 36 h after transfection, centrifuge at 2000 g × 5 min, and store at 4 ℃ for 24 h.

[0070] 3. Purification of ChTE

[0071] ChTE can be obtained by purification using 293F cells, and the specific steps are as follows:

[0072] Cell preparation: Transfer cells (1×10⁶ cells) from one cryovial. 7 -1.5×10 7Resuscitate cells using 25 mL of serum-free medium, incubate at 100 rpm, 8% CO2, and 37°C for 2 days. Then, add 25 mL of medium to expand to a 50 mL volume. Incubate under the same conditions for 2 days, then add another 50 mL of medium to expand to a 100 mL volume. Continue incubating for 2 days, then count the cells and adjust the cell concentration to 0.8 × 10⁻⁶. 6 After reaching 1 mL, expand to a 200 mL system and continue culturing for 2 days before transfection.

[0073] Transfection procedure: Prepare 400 μg of target plasmid; prepare PEI at a concentration of 1 mg / mL, filter through a 0.22 μm filter and set aside; add 400 μg of plasmid to 10 mL of serum-free culture medium and vortex for 30 s; add 1.2 mL of PEI (1 mg / mL) to 10 mL of serum-free culture medium and vortex for 30 s; mix the two, vortex for 30 s, let stand for 30 min, and then add 200 mL of a solution with a concentration of [missing information - likely a specific concentration] cells / mL (generally 1-1.5 × 10⁻⁶ cells / mL). 6 After adding 2 mL of penicillin-streptomycin mixture (100x) to the cells, incubate at 80 rpm, 8% CO2, and 37°C for 5 days. Collect the supernatant, centrifuge at 8000g, and filter the supernatant through a 0.22 μm filter membrane for protein purification.

[0074] Protein purification: The Ni-filled purification column (GE) was removed and the protein was purified using an AKTA-pure protein purifier. 3–4 column volumes of ultrapure water were added to elute the column. 3–4 column volumes of binding buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0) were added to equilibrate the column. 3–4 column volumes of washing buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) were added to wash away contaminating proteins. Then, 3–4 column volumes of elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0) were added to elute the target protein. Finally, the buffer was replaced with 1×PBS using a desalting column for storage. Protein concentration was determined using the BSA method, and the purified protein was aliquoted and stored at -80°C.

[0075] III. Expression and purification of ChTE-aEpCAM, ChTE-aDR5 and ChTE-aCEA

[0076] 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 Figure 1. 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 the linker is shown in SEQ ID NO.3. The expression and purification of ChTE-aEpCAM, ChTE-aDR5, and ChTE-aCEA were performed using the method described in Example 1. Simultaneously, after transient transfection with HEK 293T for 36 hours, the supernatants of the ChTE-aEpCAM, ChTE-aDR5, and ChTE-aCEA expression plasmids were collected for subsequent experiments. The purified protein (ChTE-aEpCAM) was subjected to electrophoresis and Coomassie Brilliant Blue staining, and the results are shown in Figure 2.

[0077] IV. Expression and purification of ChTE-aGFP, BiTE-aEpCAM, and BiTE-aDR5

[0078] In the following embodiments of the present invention, ChTE-aGFP is obtained by replacing the anti-EpCAM scFv in ChTE-aEpCAM with the nanobody anti-GFP. The amino acid sequence of anti-GFP is shown in SEQ ID NO.7. After constructing the plasmid, ChTE-aGFP was expressed and purified according to the ChTE expression procedure in Example 1. Simultaneously, after transient transfection of the ChTE-aGFP expression plasmid with HEK 293T for 36 hours, the supernatant was collected for subsequent experiments.

[0079] BiTE is a traditional bispecific T cell engager (BiTE). The BiTE expression structure is shown in Figure 1. The BiTE-aEpCAM and BiTE-aDR5 involved in the following embodiments of the present invention have the following amino acid sequences: anti-EpCAM scFv, anti-DR5 scFv, 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 the linker is shown in SEQ ID NO.3. After constructing the plasmids, BiTE-aEpCAM and BiTE-aDR5 were expressed and purified according to the ChTE expression procedure in Example 1. Simultaneously, after transient transfection of the BiTE-aEpCAM and BiTE-aDR5 expression plasmids with HEK 293T for 36 hours, the supernatant was collected for subsequent experiments.

[0080] anti-GFP (SEQ ID NO.7):

[0081] Anti-CD3 (SEQ ID NO.8):

[0082] Example 2

[0083] This embodiment verifies through a trans-well assay that the ChTE designed in this invention still possesses the chemotactic function of natural CXCL12.

[0084] This embodiment verifies the chemotactic activity of ChTE-aEpCAM, ChTE-aDR5, and ChTE-aCEA on T cells described in Example 1. ChTE-aGFP from Example 1 is used as a control.

[0085] The trans-well invasion assay was performed as follows: Cell culture supernatant was collected 36 hours after HEK 293T transfection, centrifuged at 2000g for 5 minutes, and the supernatant was collected for later use. 400 μL of the above culture supernatant and 300 μL of fresh 1640 medium were added to the lower chamber of a 24-well Trans-well plate; 200 μL of resting PBMCs (peripheral blood mononuclear cells) at a concentration of 1*10^6 were added to the upper chamber of the plate, and the upper chamber was gently placed inside the lower chamber. After culturing at 37°C with 5% CO2 for 4 hours, the upper chamber of the Trans-well plate was removed, the culture medium from the lower chamber was collected, and the cells were centrifuged at 500g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 200 μL of PBS. Cell counting was performed using flow cytometry. The negative control (Blank) supernatant was the same as the blank control culture supernatant, and the positive control (CXCL12) supernatant was the same as the blank control culture supernatant, with the addition of commercial human CXCL12 protein to a final concentration of 50 nM.

[0086] The experimental results are shown in Figure 3. The results show that the ChTE designed in this invention still has the chemotactic function of natural CXCL12.

[0087] Example 3

[0088] This embodiment verifies that the ChTE designed in this invention can activate T cells to kill different target cells through various surface antigens.

[0089] This embodiment verifies the ChTE-aEpCAM and ChTE-aDR5 described in Example 1, using A431 and MCF7 cells as target cells. BiTE-aEpCAM, BiTE-aDR5, and ChTE-aGFP described in Example 1 are used as controls.

[0090] The specific procedure is as follows: 24 hours in advance, prepare target cells A431 and MCF7 expressing luciferase. Seed the target cells at 1*10^4 / well in opaque 96-well plates with a white background, adding fresh DMEM medium to a total volume of 100 μL. Collect the cell culture supernatant 36 hours after transfection with HEK 293T, centrifuge at 2000g for 5 min, and collect the supernatant for later use. Take 50 μL of the prepared supernatant and add it to the prepared target cell wells, then add 50 μL of resting PBMC at a concentration of 4*10^5 / mL, and incubate at 37℃ with 5% CO2 for 24 h. Add 10 μL of 5 mg / mL D-luciferin to each well, incubate at room temperature for 10 min, and detect using a Luminometer. The negative control (Blank) supernatant is the blank control medium supernatant, and the positive control supernatant is BiTE. The lethality can be roughly calculated using the formula 100*(1-experimental group reading / blank control group reading).

[0091] The experimental results are shown in Figure 4. The results show that the target cells that ChTE can target in this invention are broad, the antigen targets of the target cells are adjustable, and T cells can be activated to kill different target cells through multiple surface antigens.

[0092] Example 4

[0093] The ChTE-aEpCAM designed in Example 1 of this invention can activate T cells to express various immune-related cytokines.

[0094] Specifically, target cells A431 were prepared 24 hours in advance and seeded in 24-well plates at a rate of 5*10^4 cells / well. Fresh DMEM medium was added to a total volume of 500 μL. The cell culture supernatant was collected 36 hours after transfection of HEK 293T cells with ChTE-aEpCAM plasmid. The cells were centrifuged at 2000g for 5 minutes, and the supernatant was used for later use. The culture medium was aspirated from the target cell wells, and 125 μL of the prepared supernatant was added to prepared target cell plates (Target+) or blank plates (Target-). 5*10^4 resting T cells were then added, and fresh DMEM medium was added to a total volume of 500 μL. The cells were incubated at 37°C with 5% CO2 for 12 hours. The culture medium after completion was transferred to an EP tube and centrifuged at 2000g for 5 minutes. The supernatant was used to detect IL2, TNF-α, IFN-γ, and GM-CSF. The supernatant was quantified according to the ELISA kit instructions. The positive control group was BiTE-aEpCAM as described in Example 1, and the negative control group was blank culture medium. The experimental results are shown in Figure 5.

[0095] Example 5

[0096] The ChTE-aEpCAM designed in Embodiment 1 of this invention has a better killing effect when facing a 3D spherical solid tumor model compared with the BiTE-aEpCAM described in Embodiment 1.

[0097] Specifically, A431 target cells expressing green fluorescent protein (GFP) were prepared and seeded at 5000 cells / well in 96-well round-bottom ultra-low cell adhesion plates (e.g., Corning catalog number 7007). Fresh DMEM medium was added to a total volume of 100 μL. After culturing at 37°C with 5% CO2 for 72 h, resting T cells were seeded at 1*10^4 cells / well. ChTE-aEpCAM or BiTE-aEpCAM was added at a working concentration of 10 nM or 30 nM, with a final volume of 200 μL / well. The control group was treated with PBS, with a final volume of 200 μL / well. The cells were cultured for another 24 h, and the target cell status was observed and recorded. The experimental results are shown in Figure 6.

[0098] Example 6

[0099] The ChTE-aEpCAM activated T cells to kill tumor cells designed in Example 1 of this invention is concentration-dependent.

[0100] Specifically, Luciferase-expressing target cells (A431) were prepared 24 hours in advance and seeded in opaque 96-well plates at a rate of 1*10^4 cells / well, with a total volume of 100 μL after adding fresh DMEM medium. Concentration gradients were set up from 1*10^(-5) to 1*10^2 nM, with a total volume of 200 μL. Different effector-to-target ratios (E:T = 1:2, 1:1, and 2:1) were set, and resting T cells were added. The cells were cultured at 37°C with 5% CO2 for 24 h and 48 h, and the results were measured. 10 μL of 5 mg / mL D-luciferin was added to each well, and the cells were incubated at room temperature for 10 min, followed by detection using a Luminometer. In this experiment, the blank control group did not contain ChTE protein and contained the same number of T cells as the experimental group. The killing efficiency was calculated using the formula 100*(1 - experimental group reading / blank control group reading). The experimental results are shown in Figure 7.

[0101] Example 7

[0102] The ChTE-aEpCAM designed in Example 1 of this invention has an in vivo antitumor effect. The BiTE-aEpCAM described in Example 1 was used as a control.

[0103] Specifically, A431 target cells were prepared and cultured using standard methods before being used in in vivo experiments. The prepared A431 target cells were collected, resuspended in PBS to obtain a cell suspension with a concentration of 1*10^7 cells / mL, and subcutaneously inoculated into the right axilla of approximately 5-week-old (19-21g) female NSG mice on day 0 to establish an A431 tumor-bearing mouse model. On day 6, resting T cells were injected via the tail vein at a concentration of 1*10^7 cells per mouse. On day 7, mice were randomly divided into 3 groups of 5 mice each. Starting from day 7, subcutaneous peritumoral administration was performed every other day. Each group was administered ChTE-aEpCAM, BiTE-aEpCAM, or a blank control PBS, respectively. Each administration was 100 μg per mouse (approximately 5 mg / kg, dissolved in 100 μL PBS), with the PBS group receiving 100 μL PBS. Tumor size was measured 2-3 times per week. Tumor volume determination: The maximum major axis (L) and maximum minor axis (W) of the tumor were measured using vernier calipers, and the volume was calculated using the formula V = 1 / 2 * L * W^2. Mouse body weight was measured using an electronic balance. Experimental results are shown in Figure 8.

[0104] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A fusion protein of the chemokine CXCL12, characterized in that, It includes chemokine CXCL12 or its mutant 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 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 through CXCR4 and / or CXCR7.

3. The fusion protein according to claim 1, characterized in that, The chemokine CXCL12 has the amino acid sequence shown in SEQ ID NO.1, or has a mutant sequence based on the amino acid sequence shown in SEQ ID NO.1 by substitution, deletion, addition and / or replacement of one or more amino acids, and the mutant sequence has the ability to activate CXCR4 and / or CXCR7 receptors on immune cells.

4. The fusion protein according to claim 1, characterized in that, The antibody against the antitumor-associated antigen is a single-chain antibody, a complete antibody, or an antibody fragment against any antitumor-associated antigen.

5. The fusion protein according to claim 1, characterized in that, The antibody against the tumor-associated antigen is an antibody against EpCAM, DR5, or CEA.

6. The fusion protein according to claim 1, characterized in that, The antibody against the tumor-associated antigen is a single-chain antibody against EpCAM, a single-chain antibody against DR5, or a single-chain antibody against CEA.

7. The fusion protein according to claim 6, characterized in that, The amino acid sequence of the anti-EpCAM single-chain antibody is shown in SEQ ID NO.4; The amino acid sequence of the anti-DR5 single-chain antibody is shown in SEQ ID NO.5; The amino acid sequence of the anti-CEA single-chain antibody is shown in SEQ ID NO.

6.

8. 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 antibody against the antitumor-associated antigen is located at the N-terminus of the fusion protein.

9. The fusion protein according to claim 1, characterized in that, The linker is a flexible amino acid sequence; The amino acid sequence of the linker is shown in SEQ ID NO.

3.

10. A nucleic acid molecule, characterized in that, Encodes the fusion protein of claim 1.

11. A host cell, characterized in that, It contains the nucleic acid as described in claim 10.

12. Use of the fusion protein of claim 1 in the preparation of an antitumor medicament.

13. The use according to claim 12, characterized in that, The tumor is a solid tumor; 14. The use according to claim 12, characterized in that, The tumor is either epidermoid carcinoma or breast cancer.

15. An antitumor pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fusion protein of claim 1.

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