Hydrogel drug delivery system carrying recombinant protein for cancer treatment and preparation method

Abstract

The invention relates to the technical field of genetic engineering and pharmaceutical preparations, in particular to a hydrogel drug delivery system carrying recombinant protein for cancer treatment and a preparation method of the hydrogel drug delivery system, the hydrogel drug delivery system comprises recombinant plasmids, the recombinant plasmids are protein pcDNA3.4-PD1-ECD-CCL5 with an amino acid sequence, and the amino acid sequence of the protein is Pab-CCL5. According to the present invention, the recombinant plasmid can express the fusion protein of PD1-ECD-IgG1 and CCL5-IgG1, and the recombinant protein Pab-CCL5 prepared by using the recombinant plasmid has good anti-tumor effect, can change the immune state of the tumor microenvironment, and can reshape the tumor microenvironment; immune cells expressing CCL5 receptors are recruited, and the anti-tumor immune response of the body is improved; the compound kills tumor cells through multiple mechanisms including innate immunity and adaptive immunity, is a potential candidate drug for treating prostate cancer, breast cancer and PD-L1 expression cancer, and is free of cytotoxicity.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering technology and pharmaceutical formulation technology, specifically to a hydrogel drug delivery system and preparation method for carrying recombinant proteins for cancer treatment. Background Technology

[0002] Traditional cancer treatments include surgery, radiotherapy, and chemotherapy, but these methods have low efficacy and high recurrence rates. Cancer immunotherapy is a rapidly developing anti-tumor approach in recent years. Bispecific T-cell enablers (BiTEs) are a novel immunotherapy designed to simultaneously bind to two different targets, typically tumor cells and T cells. The main principle of this technology is to enhance the immune system's attack on cancer by guiding immune cells, such as T cells, to the vicinity of tumor cells. BiTEs typically consist of two different antibody binding sites: one that recognizes antigens on the surface of tumor cells, and the other that binds to cytokine receptors on the surface of immune cells. This dual binding effectively activates immune cells, prompting them to recognize and kill tumor cells. This treatment strategy has shown promising results in treating certain types of hematologic malignancies, such as acute lymphoblastic leukemia, and its application in various solid tumors is currently under investigation. With further research, bispecific T-cell enablers are expected to provide new options for cancer treatment. PD-L1 (programmed death-ligand 1) is an immune checkpoint molecule widely expressed on the surface of tumor cells. By binding to PD-1 on the surface of T cells, it inhibits T cell activation and killing function, leading to immune escape. This mechanism is the core link of immunosuppression in the tumor microenvironment. Monoclonal antibody drugs targeting PD-L1 (such as atezolizumab and durvalumab) restore T cell activity by blocking the PD-L1 / PD-1 signaling pathway and have achieved clinical breakthroughs in various solid tumors. However, the efficacy rate of monotherapy is only about 20%, and some patients are prone to developing drug resistance. CCL5 (also known as RANTES) is a small chemokine mainly produced by T cells, monocytes and fibroblasts. It plays a key role in the immune response, attracting leukocytes (especially T cells and stromal cells) to sites of infection or inflammation. CCL5 promotes the migration and activation of immune cells by binding to receptors such as CCR5 and CCR1. Therefore, using BiTEs to combine immune checkpoint inhibitors (ICIs) with cytokines to reverse the "cold" tumor microenvironment and recruit effector immune cells to the tumor site to exert innate and adaptive immunity is an important strategy for treating tumors and has become a technical challenge that urgently needs to be solved in this field. However, protein drugs are more prone to degradation and inactivation than small molecules, requiring short-term storage at 4 degrees Celsius or long-term cryopreservation at -20 to -80 degrees Celsius. In addition, proteins are digested by gastric juices and various enzymes, and the most common delivery method is intravenous injection, not oral administration. Using hydrogels as protein delivery carriers allows for simultaneous implantation during surgical resection, uniformly and stably releasing bispecific cell connectives at the tumor site. By enriching immune cells, it inhibits tumor recurrence, and single-dose administration greatly improves patient compliance. Furthermore, hydrogels can be freeze-dried for longer preservation, facilitating transportation and reducing storage difficulties. Summary of the Invention

[0003] The purpose of this invention is to provide a hydrogel drug delivery system and preparation method for carrying recombinant proteins for cancer treatment. This addresses the challenges of protein drugs being more prone to degradation and inactivation than small molecules, requiring short-term storage at 4 degrees Celsius or long-term cryopreservation at -20 to -80 degrees Celsius. Furthermore, proteins are digested by gastric juices and various enzymes, and the most common delivery method is intravenous injection, making oral administration impossible. Using hydrogels as protein delivery carriers allows for simultaneous implantation during surgical resection, uniformly and stably releasing bispecific cell connectives at the tumor site. This enriches immune cells to inhibit tumor recurrence, significantly improving patient compliance with single-dose administration. Moreover, the hydrogels can be freeze-dried for longer preservation, facilitating transportation and reducing storage difficulties.

[0004] To achieve the above objectives, the present invention provides the following technical solution: A hydrogel drug delivery system carrying recombinant protein for cancer treatment and its preparation method, comprising a recombinant plasmid, wherein the recombinant plasmid is a protein pcDNA3.4-PD1-ECD-CCL5 with an amino acid sequence of Pab-CCL5.

[0005] As a further optimization of the present invention, the preparation of the recombinant plasmid includes the following steps: S1. The CH3 region of human IgG1 was mutated at a site to generate button and pore mutants; S2. Human CCL5 and PD1 extracellular domain sequences were integrated into the N-terminus of the button mutant and pore mutant of IgG1, respectively, to obtain heterodimeric sequences PD1-ECD-IgG1 and CCL5-IgG1; S3. PD1-ECD-IgG1 and CCL5-IgG1 were ligated using the T2A autocleavage enzyme sequence to obtain the recombinant plasmid.

[0006] As a further optimization of the present invention, the amino acid sequence of the human IgG1 is as follows: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0007] As a further optimization of the present invention, the amino acid sequence of the button mutant is as follows: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of the pore mutant is as follows: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0008] As a further optimization of the present invention, the amino acid sequence of the PD1-ECD-IgG1 is as follows: LEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMGGGSGGGSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of the CCL5-IgG1 is as follows: SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMSGGGSGGGSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0009] As a further optimization of the present invention, it also includes a recombinant protein, which is prepared by transfecting the recombinant plasmid into a eukaryotic expression system.

[0010] As a further optimization of the present invention, the recombinant plasmid and recombinant protein are used in the preparation of antitumor drugs.

[0011] As a further optimization of the present invention, a temperature-sensitive hydrogel drug delivery system for delivering the recombinant protein is prepared, which is composed of a composite of temperature-sensitive and biocompatible materials.

[0012] As a further optimization of the present invention, the biocompatible material is a natural biomacromolecule modified with polyphenol components, including dopamine-modified hyaluronic acid. The thermosensitive material includes Pronickel, which is modified with thiol groups at both ends.

[0013] As a further optimization of the present invention, the hydrogel is in an injectable form, which can be directly injected into the tumor site for local drug administration and maintain a local drug concentration for a long time.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a recombinant plasmid, its preparation method, and its application. The recombinant plasmid of this invention can express a fusion protein of PD1-ECD-IgG1 and CCL5-IgG1. The recombinant protein Pab-CCL5 prepared using the recombinant plasmid has a good anti-tumor effect, can alter the immune status of the tumor microenvironment, reshape the tumor microenvironment, recruit immune cells expressing the CCL5 receptor, and enhance the body's anti-tumor immune response. Through multiple mechanisms, including innate immunity and adaptive immunity, it kills tumor cells and is a potential candidate drug for treating prostate cancer, breast cancer, and PD-L1-expressing cancers, and is non-cytotoxic. This invention also provides a temperature-sensitive injectable hydrogel for delivering Pab-CCL5, improving patient compliance, drug bioavailability, and reducing off-target effects. The hydrogel has good biocompatibility and will not cause damage or adverse reactions to healthy tissues surrounding the tumor. Attached Figure Description

[0015] Figure 1 A schematic diagram of the pcDNA3.4-PD1-ECD-CCL5 recombinant plasmid; Figure 2 Western blot (WB) validation results for expressed PD1-ECD-IgG1 and CCL5-IgG1; Figure 3 The chemotaxis of recombinant protein Pab-CCL5 on human PBMC cells; Figure 4 The ADCC of recombinant protein Pab-CCL5 on MDA-MB-231 tumor cells; Figure 5 Pab-CCL5 is the ADCP of recombinant protein Pab-CCL5 on MDA-MB-231 tumor cells; Figure 6 The preparation process of HA-DA; Figure 7 The NMR spectrum of HA-DA; Figure 8 The preparation process of Plu-SH; Figure 9 The NMR spectrum of Plu-SH; Figure 10 The images show the morphology of the blank hydrogel at 20 ℃ and 37 ℃, respectively. Figure 11 The rheological properties of the blank hydrogel; Figure 12 The degradation curve of the drug-loaded hydrogel is shown. Figure 13 For the safety of drug-loaded hydrogels; Figure 14 This is a schematic diagram of the pcDNA3.4-PD1-ECD-CCL5 recombinant plasmid. Detailed Implementation

[0016] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0017] Example 1

[0018] Preparation of recombinant plasmids and proteins This invention provides a method for preparing recombinant plasmids, comprising the following steps: Based on the human IgG1 sequence, site mutations were performed on the CH3 region of human IgG1 to generate buttonhole and pore mutants, effectively generating heterodimers. The extracellular sequences of human PD1 and CCL5 were selected and designed at the N-terminus of the buttonhole and pore mutants, respectively. These were then constructed into co-expression vectors via T2A linking: PD1-ECD-IgG1 (Knob) and CCL5-IgG1 (Hole). Specifically, the modification of the CH3 region of human IgG1 involved targeted mutation of the CH region of the Knob arm using Knob-into-hole (KIH) technology, i.e., the T366W mutation, to generate the buttonhole mutant. Similarly, targeted mutations of the CH region of the Hole arm, i.e., the T366S, L368A, and Y407V mutations, generated the pore mutant. The Knob and Hole arms were linked using a T2A (an autoclavicle enzyme) sequence. The target gene fragment was then generated using chemical synthesis and inserted into pcDNA3.4 using a one-step homologous recombination technique after gene sequence synthesis. (From Thermo Fisher Scientific Inc.) The recombinant plasmid pcDNA3.4-PD1-ECD-CCL5 was obtained, with the target gene positioned on the vector between the Sal I and Kpn I restriction sites, as shown below. Figure 1 As shown; The amino acid sequence of human IgG1-Fc (SEQ ID No: 2) is as follows: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of the button mutant (SEQ ID No: 3) is as follows: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of the pore mutant (SEQ ID No: 4) is as follows: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of PD1-ECD-IgG1 (SEQ ID No: 5) is as follows: LEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMGGGSGGGSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of CCL⁃5⁃IgG1 (SEQ ID No: 6) is as follows: SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMSGGGSGGGSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of the protein expressed by the recombinant plasmid pcDNA3.4⁃PD1⁃ECD⁃CCL5 (SEQ ID No: 1) is as follows: SIP-LEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMGGGSGGGSGGGSGGGSEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK-RKREGRGSLLTCGDVEENPGP-SIP-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMSGGGSGGGSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The target gene fragment was inserted between the Sal I and Kpn I restriction sites of pcDNA3.4 (Thermo Fisher Scientific Inc.) using homologous recombination. pcDNA3.4-PD1-ECD-CCL5 was transfected into HEK293T cells using PEI. The expressed protein was secreted into the culture supernatant, and after ultrafiltration concentration, the recombinant protein Pab-CCL5 was purified by Protein A affinity chromatography.

[0019] The specific experimental steps are as follows: (1) Homologous recombination of plasmids: 1) The pcDNA3.4 was linearized by digestion with Sal I and Kpn I restriction endonucleases; 2) Add the target gene fragment and the linearized vector at a ratio of 2:1; the optimal amount of each fragment Y = [0.04 × number of fragment base pairs] ng, and the optimal amount of the linearized vector X = [0.02 × number of fragment base pairs] ng; 3) Mix the linearized vector, target gene fragment, 5×CE buffer, and Exnase II on ice. After mixing thoroughly by pipetting, incubate at 37 °C for 30 min, then cool on ice. 4) Transform the recombinant plasmid into DH5α, plate it overnight, and then sequence the obtained single clones for verification. 5) The obtained plasmids were amplified and extracted using an endotoxin-free plasmid extraction kit.

[0020] (2) Expression of recombinant protein Pab-CCL5: 1) 24 h before transfection, HEK293T cells in logarithmic growth phase were digested with 0.25% trypsin, and the cell density was adjusted to 0%–40% in DMEM medium containing 10% serum. The cells were then reseeded in cell culture flasks and cultured at 37°C in a 5% CO2 incubator. Transfection was possible after approximately 24 h when the cell density reached 50%–60%. 2) Replace the cell culture medium with serum-free Opti-MEM™ I medium 2 hours before transfection; 3) Before transfection, the total plasmid DNA concentration in the transfection culture must be 1.0 μg / ml, which means 200 μg of endotoxin-free plasmid pcDNA3.4-PD1-ECD-CCL5 is required. Dilute 200 μg of plasmid DNA with 10 ml of Opti-MEM™ I medium; then dilute 600 μg of PEI transfection reagent with 10 ml of Opti-MEM™ I medium. 4) Mix the diluted plasmid DNA with the diluted PEI solution and incubate at room temperature for 20 minutes to form a transfection complex of DNA and PEI dilution. 5) Transfer the DNA and PEI mixture to the culture medium of HEK293T cells, mix well, and incubate at 37°C in a 5% CO2 cell culture incubator; 6) After culturing for 6-8 hours, discard the culture medium containing the transfection mixture. Add 2 ml of PBS to each flask of cells, gently shake the flask from side to side to wash away any remaining transfection mixture, then discard the flask. Replace the flask with DMEM medium containing 10% serum and continue culturing for 2-3 days.

[0021] (3) Concentration and purification of recombinant protein Pab-CCL5: 1) Collect cell culture medium, centrifuge at 4500 rpm and 4 ℃ for 1 h, filter the supernatant through a 0.22 μm filter membrane, collect it in a 500 ml glass bottle, add an equal volume of PBS, shake on ice for 30 minutes and degas. 2) Wash and equilibrate the purification column with 10-20 column volumes of PBS, with a flow rate of 1 ml / min; 3) Load the protein to be purified onto the purification column. After the protein passes through the column, wash with 10-20 column volumes of PBS to remove unbound and non-specifically bound proteins. Complete washing can be determined by measuring the absorbance at 280 nm. Add an appropriate amount of neutralizing solution (Tris-HCl, pH 8.8) to the collection tube at a ratio of 100 μl of neutralizing solution per mL of eluent. Then, elute the bound protein with 10 mL of 50 mM glycine (pH 2.7). Collect the eluted protein in separate tubes. 4) Determine the protein content of each tube by measuring the absorbance at 280 nm, determine the kD of the protein bands by running SDS-PAGE, and select samples with the correct bands for merging. 5) Select a 10 kDa ultrafiltration tube and concentrate the protein at 4℃ and 4000 rpm. Resuspend the protein by pipetting for 4-5 minutes each time to prevent precipitation. 6) Replace with 1×PBS and concentrate the protein concentration to 5 mg / ml.

[0022] (1) Validation of recombinant protein Pab-CCL5: Western blotting was used to verify the expression of CCL5 and the extracellular domain of PD-1. Since the principle of Protein A affinity chromatography purification is to bind to IgG1, it is not necessary to verify the expression of IgG1.

[0023] 1) Take 1 μl of sample, dilute it 10 times, mix it with 2.5 μl of loading buffer, heat it at 98 ℃ for 5 min; then load the sample into a 10% SDS-PAGE gel, place it in a vertical electrophoresis tank and fill it with 1×TAE solution; 2) Turn on the power supply. The voltage should be maintained at 90V when running the upper layer of adhesive and at 120V when running the lower layer of adhesive. Turn off the power supply when the strip reaches the bottom. 3) Immerse the SDS-PAGE gel in transfer buffer and activate it at room temperature (80 rpm) for 30 min on a horizontal shaker; activate the PVDF membrane with methanol, and after laying filter paper, sponge, SDS-PAGE and PVDF membrane in sequence, transfer the membrane at 300 mA for 1 hour. 4) Transfer the PVDF membrane to a small box, completely immerse it in 5% BSA (in PBST), and shake at room temperature for 2 hours; 5) Mix the primary antibody Anti-hPD1 and Anti-hCCL5 at a ratio of 1:2000 with 5% BSA, completely immerse the protein-transferred PVDF membrane, and incubate overnight at 4 ℃ with shaking at 80 rpm. 6) Wash with TBST 5 times, 5 min each time. Mix the secondary antibody Goat Anti Rabbit IgG (H+L)-HRP with 5% BSA at a ratio of 1:10000. Completely immerse the protein-transfected PVDF membrane and incubate on a horizontal shaker at room temperature and 80 rpm for 2 hours. 7) Wash 5 times with TBST, 5 min each time; prepare ECL chemiluminescence solution, develop using a chemiluminescence analyzer, and obtain the results as shown below. Figure 2 As shown.

[0024] Example 2

[0025] Cell migration of recombinant protein Pab-CCL5 in human PBMCs This embodiment uses Transwell chambers (corning) to determine the migration ability of human PBMCs. The specific experimental method is as follows: (1) Isolation of PBMCs from peripheral blood of healthy individuals 1) Transfer 10 ml of whole blood into a 50 ml centrifuge tube, add 10 ml of PBS solution to dilute, and mix gently; 2) Take two 15 ml centrifuge tubes and add 5 ml of Ficoll solution. Then gently add the diluted blood to the top layer of Ficoll in both centrifuge tubes, avoiding mixing the two solutions. Add 10 ml of diluted blood to each centrifuge tube. 3) Centrifuge at 2,000 rpm for 20 minutes. Note that the deceleration setting must be set to "no break" or only 10-20% braking. After centrifugation, you will obtain a homogeneous layered cell solution. 4) Remove the white membrane layer containing PBMCs and transfer it to another clean 15 ml centrifuge tube. Add PBS to 10-15 ml, centrifuge at 1,500 rpm for 10 min, remove the supernatant, and then add culture medium to perform the same washing operation. 5) After resuspending the cells in 5-10 ml of special culture medium, incubate them in a 37°C, 5% CO2 incubator.

[0026] (2) PBMCs were starved with serum-free medium 12 h before the experiment. 5E5 serum-starved cells were seeded into the upper chamber.

[0027] (3) Add purified Pab-CCL5, recombinant human CCL5 as a positive control, or pbs and recombinant human IgG1 as a negative control to the lower chamber.

[0028] (4) The upper and lower chambers were incubated in a 37°C, 5% CO2 incubator for 12 h. Cells that migrated to the lower surface of the porous membrane in the lower chamber were counted.

[0029] The results are as follows Figure 3 As shown, when the Pab-CCL5 concentration was between 2-8 μg / ml, the number of PBMCs migrating within 12 h increased with increasing concentration, similar to the trend of the positive control hCCL5. This indicates that the CCL5 portion expressed in Pab-CCL5 possesses the ability to chemotactically attract immune cells.

[0030] Example 3

[0031] Pab-CCL5 promotes ADCC in NK cells against MDA-MB-231 cells. Natural killer (NK) cells possess inherent cytotoxicity, enabling them to directly recognize and kill tumor cells. Furthermore, in the presence of antibodies, NK cells can exert anti-tumor effects through antibody-dependent cytotoxicity (ADCC). To investigate the role of Pab-CCL5 in regulating NK cell ADCC function, we established a co-culture system of human NK cells and MDA-MB-231 cells that highly express PD-L1, and indirectly assessed the cell killing status by detecting the release of LDH in the cells.

[0032] First, mix 9.1 ml of detection assay buffer and 0.9 ml of chromogenic solution evenly to prepare 10 ml of LDH detection working solution; NK cells isolated from PMBCs and MDA-MB-231 cells were added to 96-well plates at target-efficacy ratios of 40:1, 20:1, and 10:1. Untreated control, background control, total LDH, background of total LDH, and sample groups were set up. The prepared systems were incubated at 37 °C for 6 hours, and the absorbance at 450 nm was measured. Cytotoxicity or mortality was calculated using the formula: (Absorbance of sample group – Absorbance of untreated control group) / (Absorbance of total LDH group – Absorbance of untreated control group) × 100%.

[0033] The results are as follows Figure 4As shown, compared with the IgG1 isotype control, Pab-CCL5 significantly induced ADCC in NK cells that simultaneously targeted MDA-MB-231 tumor cells.

[0034] Example 4

[0035] Pab-CCL5 enhances ADCP of human macrophages against MDA-MB-231 cells. Macrophages play a crucial role in the tumor microenvironment (TME) by mediating antibody-dependent phagocytosis (ADCP) and releasing cytokines. Therefore, this embodiment aims to verify the specific efficacy of Pab-CCL5 on macrophages, particularly M1 macrophages, through relevant experiments, and to determine its ability to induce ADCP by measuring the uptake of CFSE by macrophages in CFSE-labeled tumor cell lines.

[0036] First, THP-1 cells were induced to become M0 macrophages using PMA, and then the macrophages were polarized to M1 type using LPS and IFN-gama. The proportion of M1 polarization could be determined by CD86 staining and flow cytometry analysis. MDA-MB-231 cells were labeled with CFDA-SE working solution 12 hours in advance. The polarized M1 macrophages were gently scraped from the culture dish and mixed separately with MDA-MB-231 cells, Pab-CCL5, and control group IgG1 and avelumab, and co-cultured for 6 hours.

[0037] The results are as follows Figure 5 As shown, flow cytometry revealed that, compared with the IgG1 isotype control or positive control avirumab, Pab-CCL5-induced anti-MDA-MB-231 cells had significantly higher ADCP levels.

[0038] Example 5

[0039] Preparation of dopamine-modified hyaluronic acid (HA-DA) The process for synthesizing the HA-DA component in a temperature-sensitive injectable hydrogel is as follows: Figure 6 As shown. The specific steps are as follows: (1) Dissolve 500 mg of hyaluronic acid (HA) in 25 ml of PBS buffer at pH = 5 and stir thoroughly until completely dissolved. Add 242.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 145.7 mg of N-hydroxysuccinimide (NHS) to the solution and stir overnight at room temperature to activate.

[0040] (2) After the carboxyl group activation is complete, add 500 mg of dopamine and 5 ml of PBS buffer at pH = 5, and react for 12 hours under nitrogen protection. After the reaction is complete, transfer the solution to a 3500 d dialysis bag and dialyze with stirring for 72 hours.

[0041] (3) After dialysis, the mixture in the dialysis bag was placed at -80 °C for 24 hours and then freeze-dried to obtain HA-DA solid product.

[0042] The structure of HA-DA was verified by nuclear magnetic resonance (NMR) spectroscopy, and the results are as follows: Figure 7 As shown, its characteristic peaks are consistent with the expected structure, confirming the successful synthesis of HA-DA.

[0043] Example 6

[0044] Preparation of Plu-SH compounds modified with thiol groups at both ends The process for synthesizing the Plu-SH component in a temperature-sensitive injectable hydrogel is as follows: Figure 8 As shown. The specific steps are as follows: (1) Dissolve 5 g of Pronic F127 in 50 ml of dichloromethane, add 1 ml of triethylamine as an acid-binding agent, and slowly add 0.8 g of p-nitrophenyl chloroformate (p-NPC). Stir at 25°C in the dark for 48 hours.

[0045] (2) The organic phase was extracted with saturated NaCl solution (3×50 ml), dried with anhydrous sodium sulfate, and then precipitated with cold diethyl ether to obtain activated Pronnick intermediate.

[0046] (3) The activated product was dissolved in 40 ml of dichloromethane and reacted in the dark for 60 hours. After purification, it was dried under vacuum to obtain Plu-SH.

[0047] The structure of Plu-SH was verified by nuclear magnetic resonance (NMR) spectroscopy, and the results are as follows: Figure 9 As shown, its characteristic peaks are consistent with the expected structure, confirming the successful synthesis of Plu-SH.

[0048] Example 7

[0049] Assembly and physicochemical property testing of temperature-sensitive injectable hydrogels HA-DA and Plu-SH were dissolved in 450 μl of pre-cooled PBS (4-10℃) to make a volume ratio of 1:1, resulting in a HA-DA solution of 100 mg / ml and a Plu-SH solution of 300 mg / ml (mass ratio of 1:3). The resulting HA-DA and Plu-SH solutions were stirred in an ice bath for 6 h, and the mixture was allowed to stand at 4℃ for 24 h to obtain a blank hydrogel.

[0050] The blank hydrogel samples were placed in constant temperature environments of 20℃ and 37℃ respectively to test their phase transition behavior. Figure 10 As shown, at 20°C, the hydrogel system remains in a transparent sol state, while when the temperature rises to 37°C, the system undergoes a rapid phase transition within 3.5 ± 0.5 minutes, forming a non-flowing solid gel.

[0051] The rheological properties of the hydrogels were systematically evaluated using an advanced rotational rheometer (HAAKE™ MARS™, Thermo Fisher Scientific). like Figure 11 As shown, under a constant shear strain of 1%, the elastic modulus (G') and viscous modulus (G'') of the hydrogel were measured as a function of temperature (15-40℃). The results showed that the critical gelation temperature (CGT) of the HA-DA-Plu hydrogel was 28.1℃, which falls between room temperature and body temperature, meeting the application requirements for body temperature-triggered gelation. Further temperature cycling tests at 20℃ and 37℃ were conducted to examine the changes in G' and G''. The results showed that the hydrogel exhibited highly reversible sol-gel transition behavior during multiple temperature cycles, and the gelation kinetic rate and mechanical strength (G' > G'') did not show significant attenuation, confirming that the hydrogel possesses excellent temperature response reversibility and structural stability.

[0052] Example 8

[0053] Bioperformance testing of temperature-sensitive injectable hydrogels To test the biocompatibility of temperature-sensitive injectable hydrogels, it is necessary to prepare hydrogels loaded with Pab-CCL5. The specific procedure is as follows: Take out 1 ml of sol-state blank hydrogel stored at 4 degrees Celsius, add 5 mg of Pab-CCL5 lyophilized powder while stirring, then rinse the drug residue on the container with 100 μl of PBS, stir for 30 min and let stand for 1 h to wait for defoaming; place at 37°C for 30 min to obtain the drug-loaded hydrogel.

[0054] The in vitro drug release performance of the temperature-sensitive injectable hydrogel for recombinant protein Pab-CCL5 was tested using enzyme-linked immunosorbent assay (ELISA). The drug-loaded hydrogel was placed in a transwell chamber and immersed in PBS (pH = 7.4) solution at 37°C. PBS solution was collected at different time points, and an equal volume of fresh PBS was added to compensate for the release medium. The collected samples were analyzed using a human IgG-Fc ELISA kit to determine the drug concentration, and drug release curves were plotted. The results are shown below. Figure 12 As shown, in the initial stage of release, the drug-loaded hydrogel exhibited rapid release of Pab-CCL5, releasing 13.5% of Pab-CCL5 within 1 hour. As time progresses, Pab-CCL5 inside the drug-loaded hydrogel diffuses and releases outward, the drug concentration gradient gradually decreases, and the release rate gradually slows down. At 48 h, the release rate is at a low level, and at 72 h, the drug is basically completely released.

[0055] In addition, the in vitro biocompatibility of the hydrogel was evaluated using human umbilical vein endothelial cells (HUVECs) as a model, seeded in 96-well plates (cell density 1×10⁻⁶). 4 (cells / well), set the final concentration gradient of hydrogel (0%, 20%, 50%, 80% v / v), and after the cells have adhered for 24 hours, add the hydrogel solution to the corresponding well and co-culture with complete culture medium for 48 hours; Cell viability was quantitatively assessed using a CCK-8 assay kit: 10 μl of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was then measured using a microplate reader (n = 6). Figure 13 As shown, the relative cell viability of the experimental group (20-80% concentration) remained above 95% compared with the blank control group, confirming that the thermosensitive hydrogel has no significant cytotoxicity within the experimental concentration range and has excellent biocompatibility.

[0056] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only for the purpose of helping to understand the method and core ideas of the present invention. The above descriptions are only preferred embodiments of the present invention. It should be noted that due to the limitations of textual expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of the present invention.

Claims

1. A hydrogel drug delivery system for carrying recombinant proteins for cancer treatment, characterized in that, Includes a recombinant plasmid, wherein the recombinant plasmid is a protein pcDNA3.4-PD1-ECD-CCL5 with an amino acid sequence of Pab-CCL5.

2. The method for preparing the hydrogel drug delivery system carrying recombinant proteins for cancer treatment according to claim 1, characterized in that: The preparation of the recombinant plasmid includes the following steps: S1. The CH3 region of human IgG1 was mutated at a site to generate button and pore mutants; S2. Human CCL5 and PD1 extracellular domain sequences were integrated into the N-terminus of the button mutant and pore mutant of IgG1, respectively, to obtain heterodimeric sequences PD1-ECD-IgG1 and CCL5-IgG1; S3. PD1-ECD-IgG1 and CCL5-IgG1 were ligated using the T2A autocleavage enzyme sequence to obtain the recombinant plasmid.

3. The method for preparing the hydrogel drug delivery system for cancer treatment carrying recombinant proteins according to claim 2, characterized in that: The amino acid sequence of the human IgG1 is as follows: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

4. The method for preparing the hydrogel drug delivery system for cancer treatment carrying recombinant proteins according to claim 2, characterized in that: The amino acid sequence of the button mutant is as follows: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of the pore mutant is: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

5. The method for preparing the hydrogel drug delivery system for cancer treatment carrying recombinant proteins according to claim 2, characterized in that: The amino acid sequence of the PD1-ECD-IgG1 is as follows: LEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMGGGSGGGSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of the CCL5-IgG1 is as follows: SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMSGGGSGGGSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

6. The hydrogel drug delivery system for cancer treatment carrying recombinant proteins according to claim 1, characterized in that: It also includes recombinant proteins, which are prepared by transfecting the recombinant plasmid into a eukaryotic expression system.

7. The hydrogel drug delivery system for cancer treatment carrying recombinant proteins according to claims 1 and 6, characterized in that: The recombinant plasmids and recombinant proteins described herein are used in the preparation of antitumor drugs.

8. The hydrogel drug delivery system and preparation method for cancer treatment carrying recombinant proteins according to claim 7, characterized in that: A temperature-sensitive hydrogel drug delivery system for delivering the recombinant protein was prepared, comprising a composite of temperature-sensitive and biocompatible materials.

9. The hydrogel drug delivery system and preparation method for cancer treatment carrying recombinant proteins according to claim 8, characterized in that: The biocompatible material is a natural biomacromolecule modified with polyphenol components, including dopamine-modified hyaluronic acid; The thermosensitive material includes Pronickel, which is modified with thiol groups at both ends.

10. The hydrogel drug delivery system and preparation method for cancer treatment carrying recombinant proteins according to claim 8, characterized in that: The hydrogel is injectable and can be directly injected into the tumor site for local drug administration, maintaining a long-term local drug concentration.

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