A signal switching receptor targeting il-10, engineered macrophage and application thereof

Abstract

The present application relates to the technical fields of biological medicine and cellular immunotherapy, and particularly relates to a signal conversion receptor targeting IL-10, an engineered macrophage and application thereof. The signal conversion receptor is composed of an extracellular domain and a transmembrane domain and an intracellular domain derived from TLR9, and the extracellular domain sequentially comprises a signal peptide, a HA tag and a specific binding domain of an IL-10 receptor alpha subunit from N-terminal to C-terminal. The present application further prepares an engineered macrophage SR CAR-M capable of specifically recognizing IL-10 and converting it into a TLR9 activation signal, which can induce macrophages to polarize to M1 type and has excellent phagocytosis and killing capacity for bladder cancer, breast cancer, lung cancer and melanoma cells, and can be used for preparing related tumor treatment drugs, overcoming the common problems of existing cell therapy, such as easy exhaustion, difficult infiltration and easy inhibition in solid tumors, and having significant clinical transformation potential.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cell immunotherapy, specifically to a signal transduction receptor targeting IL-10, engineered macrophages, and their applications. Background Technology

[0002] In recent years, immunotherapy has become a significant breakthrough in cancer treatment, with immune checkpoint inhibitors (ICIs, such as anti-PD-1 / PD-L1 antibodies) and adoptive cell therapy (ACT) demonstrating significant clinical benefits in malignant tumors. However, compared to hematologic malignancies, immunotherapy for solid tumors still faces enormous challenges, as their unique tumor microenvironment (TME) constitutes the main physical and biochemical barriers. Tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) form a potent immunosuppressive network. Tumor cells and these suppressor cells secrete high concentrations of anti-inflammatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which inhibit the function of effector immune cells (such as cytotoxic T cells and NK cells) and induce them to polarize towards a pro-tumorigenic phenotype, ultimately leading to immune escape.

[0003] To overcome the challenges of treating solid tumors, various immunocellular therapies have been developed. Among them, chimeric antigen receptor T-cell (CAR-T) therapy has shown remarkable efficacy in hematological malignancies and has seen the most rapid development. However, CAR-T cells struggle to effectively infiltrate the core of solid tumors and are easily depleted by inhibitory factors in the tumor microenvironment (TME). The toxicity associated with CAR-T therapy, such as cytokine release syndrome (CRS), also limits its widespread application. While CAR-NK cell therapy offers advantages such as "off-the-shelf" potential, relatively high safety profile, and multiple killing mechanisms, NK cells have relatively short persistence in vivo and also face challenges in functional suppression and infiltration within the solid tumor microenvironment, as well as high toxicity.

[0004] Macrophage-based therapies have become a new hot topic in drug development. This is because macrophages possess natural tumor tropism and strong tissue infiltration capabilities, enabling them to engulf tumor cells and present antigens, serving as a crucial bridge between innate and adaptive immunity. Taking CAR-M therapy as an example, it possesses a similar ability to specifically recognize specific antigens as CAR-T therapy, precisely engulfing tumor cells and presenting antigens to activate adaptive immune responses. However, due to the high plasticity of macrophages, they are easily influenced by high concentrations of anti-inflammatory cytokines such as IL-10 in the tumor microenvironment, and can be reprogrammed into M2-type macrophages that promote tumor progression. This makes it difficult to maintain the long-term efficacy of CAR-M therapy, becoming one of the bottlenecks in its development.

[0005] IL-10 is one of the most critical immunosuppressive factors in the tumor microenvironment (TME). After binding to the IL-10 receptor (IL-10R) on the surface of macrophages, it activates JAK1 / TYK2 kinases, which then phosphorylate STAT3 (p-STAT3). Subsequently, p-STAT3 enters the nucleus, initiating the transcription of M2-related genes, inhibiting the secretion of inflammatory factors, and promoting tumor growth and angiogenesis. Current technologies primarily "block" IL-10 signaling through IL-10 antibodies or JAK inhibitors. However, simple blocking strategies only eliminate the inhibitory signal and cannot actively stimulate a potent anti-tumor immune response. If the high concentration of IL-10 in the TME could be converted into an activating signal, rather than simply blocked, it is hoped that the immunosuppressive TME can be reshaped, enhancing the efficacy of macrophage therapy.

[0006] Therefore, developing a novel molecular tool to convert the high-abundance IL-10 signaling in the TME from "inhibitory" to "activating" may break through the bottleneck of macrophage therapy and improve the treatment effect of solid tumors. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a signal transduction receptor that targets IL-10, which addresses the shortcomings of the prior art.

[0008] Another technical problem that this invention aims to solve is to provide a nucleic acid molecule.

[0009] Another technical problem that this invention aims to solve is to provide a lentivirus vector.

[0010] Another technical problem that this invention aims to solve is to provide an engineered macrophage that targets IL-10.

[0011] Another technical problem to be solved by the present invention is to provide the application of the engineered macrophages in the preparation of drugs for treating solid tumors.

[0012] The final technical problem to be solved by the present invention is to provide a pharmaceutical composition for treating solid tumors.

[0013] The design concept of this invention is as follows: Based on an in-depth analysis of the advantages and disadvantages of existing technologies, this invention does not simply knock out the IL-10 receptor or block its downstream processes. Instead, it proposes an innovative strategy for engineered macrophages using a "cytokine-switching receptor" (i.e., signal transduction receptor). This signal transduction receptor uses the extracellular domain of the IL-10 receptor α subunit (IL-10Rα) as a sensor to recognize IL-10; its intracellular domain is the intracellular activation domain of Toll-like receptor 9 (TLR9), which can activate pathways such as NF-κB to promote macrophage M1 polarization. The core design concept of this invention is that when these engineered macrophages infiltrate tumors, they can convert the IL-10 signal in the tumor microenvironment into an activation signal that promotes macrophage M1 polarization and anti-tumor responses, thereby achieving the goal of reshaping the tumor microenvironment.

[0014] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0015] In a first aspect, the present invention provides a signal transduction receptor that targets IL-10, the signal transduction receptor being composed of three functional domains: an extracellular domain, a transmembrane domain, and an intracellular domain.

[0016] The extracellular domain consists of a signal peptide (SP), a HA tag sequence (HA), and a specific binding domain of the IL-10 receptor α subunit (IL-10Rα) from the N-terminus to the C-terminus, which are used to capture IL-10 in the microenvironment.

[0017] Specifically, the amino acid sequences of the signal peptide, HA tag, and IL-10 receptor α subunit specific binding domain are shown in SEQ ID NO. 1~3.

[0018] The transmembrane domain (TM) is derived from the transmembrane signaling domain of TLR9, and the intracellular domain is derived from the signal transduction domain of TLR9 (TLR9).

[0019] Specifically, the amino acid sequence of the transmembrane domain is shown in SEQ ID NO.4, and the amino acid sequence of the intracellular domain is shown in SEQ ID NO.5.

[0020] Secondly, the present invention provides a nucleic acid molecule encoding the aforementioned signal transduction receptor.

[0021] Specifically, the nucleic acid molecule encoding the signal transduction receptor has the nucleotide sequence shown in SEQ ID NO.6.

[0022] Thirdly, the present invention provides a lentiviral vector containing the nucleic acid molecules described in the second aspect.

[0023] Fourthly, the present invention provides an engineered macrophage that targets IL-10, wherein the engineered macrophage expresses the signal transduction receptor described in the first aspect.

[0024] The engineered macrophages are prepared by transfecting macrophages with the lentiviral vector described in the third aspect.

[0025] The macrophages are either Raw264.7 macrophages or immortalized bone marrow-derived macrophages iBMDM.

[0026] Fifthly, the present invention provides the application of the engineered macrophages described above in the preparation of drugs for treating solid tumors.

[0027] The solid tumor is any one of bladder cancer, breast cancer, lung cancer, or melanoma.

[0028] In a sixth aspect, the present invention provides a pharmaceutical composition for treating solid tumors, the pharmaceutical composition comprising the engineered macrophages described in the fourth aspect.

[0029] Beneficial effects:

[0030] (1) This invention designs an IL-10R / TLR9 signal transduction receptor that can target IL-10, and further prepares an engineered macrophage SR CAR-M that can specifically recognize the immunosuppressive factor IL-10 in the tumor microenvironment and convert it into a TLR9 activation signal through genetic engineering. On the one hand, SR CAR-M reduces the inhibition of surrounding innate immune cells by competitively binding to IL-10; on the other hand, it activates itself by transducing the receptor, secreting a large number of pro-inflammatory factors (IL-12, TNF-α, etc.), recruiting and activating T cells and NK cells, and reshaping the entire immune microenvironment. In addition, the more obvious the tumor suppressor microenvironment and the higher the IL-10 concentration, the stronger the activation of SR CAR-MS, effectively avoiding the drawback of conventional CAR-M and other macrophage drugs being easily reshaped by the immunosuppressive microenvironment.

[0031] (2) The engineered macrophages prepared in this invention express specific signal transduction receptors, which can specifically bind to the immunosuppressive factor IL-10 in the tumor microenvironment, block its downstream inhibitory signal (STAT3 phosphorylation), and activate the intracellular innate immune activation signal (TLR9 / NF-κB pathway), induce macrophages to polarize to the M1 type, and enhance their ability to phagocytose and kill tumor cells and activate adaptive immunity.

[0032] (3) This invention provides a novel macrophage engineering strategy that cleverly utilizes the inhibitory characteristics of the tumor microenvironment (IL-10) as a response switch, overcoming the common problems of existing cell therapies in solid tumors, such as easy depletion, difficulty in infiltration, and easy inhibition, and has significant clinical translation potential. Attached Figure Description

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0034] Figure 1 This is a schematic diagram of the structure of the IL-10R / TLR9 signal transduction receptor and the results of its signal transduction mode. In this diagram, A shows a schematic of the IL-10R / TLR9 signal transduction receptor; B shows representative results of the transduction efficiency of Raw264.7 macrophages detected by flow cytometry two days after transduction of the IL-10R / TLR9 signal transduction receptor (based on HA tag expression); C shows the expression levels of STAT3, a key protein in the IL-10 signaling pathway, and its phosphorylated form (p-STAT3) in the UTD of SR CAR-M cells and untransduced macrophages under different concentrations of IL-10 stimulation, analyzed by Western blot, with the corresponding grayscale statistical analysis results on the right; D shows the changes in the expression of TLR9 signaling pathway proteins (IKBα and p-IKBα) and IL-10 signaling pathway proteins (IRF1 and SOCS3) in the UTD of SR CAR-M cells and untransduced macrophages after IL-10 stimulation, detected by Western blot, with the corresponding grayscale statistical analysis results below; E shows the immunofluorescence detection of SR CAR-M at 100 ng / mL Intracellular distribution of NF-κB subunit P65 at 40× magnification after 6 hours of IL-10 stimulation.

[0035] Figure 2 This diagram illustrates the correlation between SR CAR-M reversing IL-10 signaling and promoting its own M1 polarization. In Figure A, Western blotting shows the expression levels of JAK1, a key protein in the IL-10 signaling pathway, and its phosphorylated form (p-JAK1) in SR CAR-M and untransduced macrophage UTDs after IL-10 stimulation; the right side shows the corresponding grayscale statistical analysis results. Figures B and C show the transcription levels of the pro-inflammatory M1 gene (B) and the anti-inflammatory M2 gene (C) in SR CAR-M and UTDs after 1 day of 50 ng / mL IL-10 stimulation, detected by qPCR; Figures D and E show representative flow cytometry analyses of the expression of M1-type markers (CD86) and M2-type markers (CD163 and CD206).

[0036] Figure 3This is a graph showing the high tumor phagocytic efficiency of SR CAR-M. In the graph, A represents the phagocytosis of latex beads by UTD, UTD+IL10, SR CAR-M, or SRCAR-M+IL10 cells; B and C represent the phagocytosis of UTD, UTD+IL10, SR CAR-M, and SR CAR-M+IL10 cells as detected by flow cytometry.

[0037] Figure 4 This diagram illustrates the high tumor-killing efficacy of SR CAR-M. A shows the experimental design for detecting the killing ability of UTD and SR CAR-M cells; B shows the experimental results of UTD and SR CAR-M cells against various tumor cells (including bladder cancer, breast cancer, lung cancer, and melanoma) at different effector-target ratios (E:T = 1:1, 3:1, 5:1, 8:1).

[0038] Figure 5 This is an experimental diagram demonstrating the effective infiltration of SR CAR-M into mouse tumor lesions and inhibition of tumor growth. A shows the experimental timeline for tumor inoculation and macrophage infusion; B shows representative imaging images of DiR-labeled SR CAR-M accumulation in different tissues; C shows tumor growth curves analyzed in mice in the PBS control group (Ctrl), Raw264.7 group (UTD), and SR CAR-M treatment group, with tumor volume measured every 3 days; D shows a statistical comparison of tumor weight in BALB / c mice in the PBS control group (Ctrl), Raw264.7 group (UTD), and SR CAR-M treatment group.

[0039] Figure 6 To assess tumor burden results by performing bioluminescence imaging (BLI) every 7 days. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0042] The macrophages Raw264.7, HEK293T cells, bladder cancer MB49 cells, breast cancer 4T1 cells, lung cancer LLC cells, and melanoma B16 cells (B16F10) described in the following examples were purchased from the Chinese Academy of Sciences Type Culture Collection Committee; the immortalized bone marrow-derived macrophages iBMDM were donated by Professor Shao Feng; the complete culture medium was RPMI1640 or DMEM medium (Gbico) containing 10% fetal bovine serum (FBS, Gbico); and the penicillin-streptomycin was purchased from Beyotime.

[0043] Example 1: Design of IL-10R / TLR9 signal transduction receptor targeting IL-10 and construction of its recombinant plasmid

[0044] 1. Design of IL-10R / TLR9 signal transduction receptor

[0045] The IL-10R / TLR9 signal transduction receptor is designed with three functional domains connected in series: an extracellular domain, a transmembrane domain, and an intracellular domain. The structure of this signal transduction receptor is as follows: Figure 1 As shown in A in the diagram.

[0046] The extracellular domain, from N-terminus to C-terminus, comprises a signal peptide (SP), an HA tag sequence (HA), and a specific binding domain of the IL-10 receptor α subunit (IL-10Rα), for capturing IL-10 in the microenvironment. The amino acid sequence of the signal peptide (SP) is shown in SEQ ID NO.1, the amino acid sequence of the HA tag sequence is shown in SEQ ID NO.2, and the amino acid sequence of the specific binding domain of the IL-10 receptor α subunit (IL-10Rα) is shown in SEQ ID NO.3.

[0047] The transmembrane domain (TM) is derived from the transmembrane signal domain of TLR9, and its amino acid sequence is shown in SEQ ID NO.4.

[0048] The intracellular domain is derived from the signal transduction domain of TLR9 (TLR9), and its amino acid sequence is shown in SEQ ID NO. 5.

[0049] Based on the above amino acid sequence, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to artificially synthesize the complete nucleotide sequence encoding the IL-10R / TLR9 signal transduction receptor (nucleotide sequence as shown in SEQ ID NO. 6), and integrated it into the lentiviral plasmid vector pLenti6 / v5 through BamHI and Xho I restriction sites. After verification by PCR and sequencing, the pLenti6 / v5-IL-10R-TLR9 plasmid was obtained.

[0050] Example 2: Construction of engineered macrophage SR CAR-M

[0051] 1. Virus Packaging

[0052] HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and passaged to 70-80% confluence for subsequent transfection.

[0053] The pLenti6 / v5-IL-10R-TLR9 plasmid obtained in Example 1 was mixed with the packaging plasmids psPAX2 and pMD2G at a mass ratio of 6:3:1 in serum-free Opti-MEM medium and gently pipetted. Lipofectamine 3000 was mixed with Opti-MEM at a volume ratio of 1:1 and allowed to stand at room temperature for 5 minutes to obtain the transfection reagent solution. The transfection reagent solution was mixed with the mixed DNA solution and allowed to stand at room temperature for 15 minutes to form a complex. Finally, the complex was slowly added dropwise to HEK293T cell culture dishes, gently shaken to mix, and cultured at 37°C and 5% CO2.

[0054] Collect the supernatant at 48 h and 72 h after transfection, centrifuge at 2000 g for 15 minutes, filter through a 0.45 μm filter to remove cell debris, mix 5× virus concentrate (Lentivirus Concentration Solution kit, Yisheng) with the filtered virus supernatant at a volume ratio of 1:4, mix well, and allow to settle naturally at 4℃ for 12 hours. Centrifuge at 2000 g for 30 minutes, discard the supernatant, and resuspend the white precipitate at the bottom of the centrifuge tube with 1 / 10 volume of DMEM medium to obtain the target lentivirus suspension, which can be used immediately or aliquoted and stored at -80℃.

[0055] 2. Macrophage transduction and construction of engineered macrophage SR CAR-M

[0056] Next, Raw264.7 macrophage cell lines or immortalized bone marrow-derived macrophages (iBMDM) were transfected using the centrifugation transfection method. Taking Raw264.7 macrophage cell line transfection as an example, the specific procedure is as follows: Raw264.7 macrophages in logarithmic growth phase were transfected at a rate of 1×10⁻⁶ cells / cells. 6Cells were seeded in 6-well plates (1 mL culture medium per well). 1 mL of culture medium containing the target lentivirus (1 / 10 volume) and polybrene at a final concentration of 8 μg / mL was slowly added to each well, and the mixture was gently stirred. After centrifugation at 32°C and 1900 rpm for 60 minutes, the cells were incubated at 37°C and 5% CO2 for 1 hour. The centrifugation and transfection process was repeated once. After 24 hours, the culture medium was changed, and the cells were incubated statically for 48 hours to obtain Raw264.7 cells transfected with the IL-10R / TLR9 signal transduction receptor (denoted as SR CAR-M). The SR CAR-M cells were then cultured with 5 μg / mL blastidin. After 48 hours, the cells were centrifuged at 700 g and resuspended in complete culture medium containing 10% serum, resulting in engineered macrophages SR CAR-M cells with high transfection and membrane-forming efficiency. Figure 1 (B in the middle).

[0057] Example 3: Verification of Signal Transduction Mechanism

[0058] This embodiment evaluates the blocking effect of engineered macrophage SR CAR-M on IL-10 signaling by detecting the phosphorylation levels of key downstream transcription factors activated by IL-10: Janus kinase (p-JAK1) and signal transduction and transcription activator 3 (p-STAT3), and also verifies the activation signal of TLR9 under IL-10 stimulation.

[0059] 1. Validation of IL-10 signal inhibition: SR CAR-M cells and control cells were stimulated with different concentrations of IL-10 (0 ng / ml, 50 ng / ml, 100 ng / ml) under UTD. Proteins were then extracted and subjected to Western blotting to detect the expression levels of STAT3, a key protein in the IL-10 signaling pathway, and its phosphorylated form (p-STAT3), as well as the expression levels of JAK1, a key protein in the IL-10 signaling pathway, and its phosphorylated form (p-JAK1).

[0060] 2. TLR9 Activation Signal Activation Verification: To assess NF-κB activation, SR CAR-M cells, control cells (UTD), and SR CAR-M cells and control cells (UTD) stimulated with 100 ng / mL IL-10 were seeded onto cell slides in 12-well plates. Cells were fixed with 4% paraformaldehyde for 30 minutes, washed twice with phosphate-buffered saline (PBS), and blocked for 1 hour at room temperature with 5% bovine serum albumin (BSA) containing 0.3% Triton X-100 (GenScript). The slides were incubated overnight at 4°C with anti-p65 antibody (1:100, Bioworld, catalog number: BS1560), washed with PBS, and incubated for 2 hours at room temperature with 546-labeled goat anti-rabbit IgG secondary antibody (1:400, Beyotime). Cell nuclei were stained with DAPI for 20 minutes, mounted, and the nuclear translocation of p65 was observed under a fluorescence microscope.

[0061] The results showed that the signal transduction receptor IL-10R / TLR9 constructed in this invention targets IL-10 and achieves macrophage phenotype retargeting. Under IL-10 stimulation, compared with untransduced macrophages (UTDs), the expression levels of p-JAK1 and p-STAT3 in engineered macrophage SR CAR-MS were significantly reduced, while the negative regulator of cytokine signaling, SOCS3, was downregulated. Figure 1 C, D, Figure 2 (A in the text). Conversely, IL-10 stimulation successfully activated the downstream TLR9 signaling pathway in SR CAR-MS, manifested as increased IKBa phosphorylation, IRF1 upregulation, and nuclear translocation of the NF-κB subunit p65. Figure 1 (D and E in the text).

[0062] Example 4: In vitro inflammatory polarization phenotype analysis of SR CAR-M

[0063] This embodiment further investigates the inflammatory polarization phenotype of SR CAR-M cells. Specifically, IL-10-stimulated SR CAR-M cells were collected, blocked with anti-mouse CD16 / CD32, and then incubated with antibodies or isotype controls of CD80, CD86, and MHC-II. After 1 hour, the cells were washed with PBS and then subjected to phenotypic analysis by flow cytometry. Simultaneously, mRNA was collected using the Trizol method, and qRT-PCR was used to detect the transcriptional levels of pro-inflammatory M1 and anti-inflammatory M2 genes in SR CAR-M cells and UTDs one day after stimulation with 50 ng / mL IL-10.

[0064] The results are as follows Figure 2Figures B through E show that, under IL-10 stimulation, M1-related genes and markers of SR CAR-M cells were upregulated, including COX2, NOS2, IL-1β, IL-6, GM-CSF, CD80, and CD86; while M2-related genes and markers were significantly downregulated, including Arg1, VEGFA, TGF-β, CD163, and CD206. Therefore, it is concluded that IL-10, which normally promotes immunosuppression, can in this system reversely drive SR CAR-M cells to polarize towards an immunostimulatory M1-like state.

[0065] Example 5: In vitro antitumor function assessment

[0066] 1. Microbead phagocytosis assay: Phagocytic capacity was assessed using fluorescent red latex microbeads (1 µm in diameter, L-2778, Sigma-Aldrich). Macrophages were pre-stimulated with or without 100 ng / mL IL-10 for 24 hours. The microbeads were incubated in RPMI 1640 complete medium containing 10% FBS at 37°C for 1 hour, and then added to macrophages at a 10:1 ratio and incubated at 37°C for 4 hours. After incubation, phagocytosis was terminated by adding 1 mL of ice-cold PBS. Cells were washed three times and collected for flow cytometry analysis of microbead uptake.

[0067] 2. Flow cytometry-based tumor cell phagocytosis assay: To measure tumor cell phagocytosis, macrophages were pre-stimulated with or without 100 ng / mL IL-10 for 24 hours, and then reacted with 2×10⁻⁶ IL-10. 5 DiR-labeled breast cancer 4T1 cells were co-cultured for 6 hours at effector-to-target ratios (E:T) of 1:1, 2:1, and 4:1. Cells were collected and thoroughly washed, and macrophages were identified by staining with PE-labeled anti-CD11b antibody. Phagocytosis was quantified by flow cytometry, and the phagocytic index was calculated as CD11b. + Mean fluorescence intensity (MFI) of intracellular DiR.

[0068] 3. Cytotoxicity assay: A luciferase-based cytotoxicity assay was used to evaluate cytotoxicity, with bladder cancer MB49 cells, breast cancer 4T1 cells, lung cancer LLC cells, and melanoma B16 cells as target cells. 3 × 10⁻⁶ cells were used as target cells. 5Raw264.7 macrophages or SR CAR-M cells were pre-stimulated with 100 ng / mL IL-10 for 24 hours. Subsequently, macrophages and corresponding cells were co-cultured in 96-well plates containing 10 ng / mL IL-10 at effector-to-target ratios (E:T) of 0:1, 1:1, 3:1, 5:1, and 8:1, with three replicates for each condition. After 24–30 hours of culture, the supernatant was discarded, and 100 µL PBS containing 5 µL of luciferase substrate was added to each well. Bioluminescent signals were quantified using a Tecan Spark microplate reader, and the percentage of specific lysis was calculated relative to cultured tumor cells alone.

[0069] Results of the high tumor phagocytic efficacy of SR CAR-M are as follows: Figure 3 As shown, in the fluorescent latex microbead phagocytosis model, IL-10-stimulated SR CAR-Ms exhibited significantly higher microbead uptake capacity than UTD-Ms or unstimulated SR CAR-Ms. Figure 3 (A) In the phagocytic model of the mouse tumor cell line 4T1 with a high IL-10 secretion phenotype, both SR CAR-M and SR CAR-M groups with additional IL-10 stimulation showed enhanced phagocytic activity against 4T1 cells, and cytotoxicity increased proportionally with the effector-to-target ratio. Figure 3 (B~C in the middle).

[0070] Results of the high tumor-killing efficacy of SR CAR-M are as follows: Figure 4 As shown, SR CAR-M mediated potent killing in all tested cell lines, and the cytotoxicity increased proportionally with the effector-to-target ratio.

[0071] This demonstrates that SR CAR-M exhibits potent in vitro antitumor effects.

[0072] Example 6: In vivo pharmacodynamic analysis of SR CAR-M

[0073] After demonstrating the multifaceted in vitro antitumor effects of SR CAR-M in Example 5, this example further evaluates its in vivo efficacy in a 4T1 orthotopic breast cancer mouse model.

[0074] The specific procedure is as follows: 6-8 week old female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in a specific pathogen-free (SPF) environment at Nanjing University School of Medicine. To establish an orthotopic breast cancer model, 4T1-luc cells were suspended in RPMI 1640 medium (concentration 1×10⁻⁶). 7 (cells / mL), take 20 µL (containing 2×10 cells / mL) 5100 µL of 1×10⁻⁶ cells were injected into the inguinal mammary fat pad of syngeneic mice. Thirty mice were randomly assigned to three groups (n=10 per group): the PBS control group (Ctrl), the Raw264.7 group (UTD), and the SR CAR-M treatment group. Treatment began on day 7 post-inoculation, when the tumor was palpable, by tail vein injection of 100 µL containing 1×10⁻⁶ cells. 6 DiR-labeled macrophages were placed in PBS buffer. Tumor volume was measured every three days using calipers and calculated as 0.5 × length × width × width. The grouping was kept blinded during tumor measurement and subsequent analysis to minimize bias.

[0075] The results are as follows Figure 5 As shown, SR CAR-M successfully infiltrated and accumulated in tumor tissue, as well as the liver, spleen, and lungs. Figure 5 In the B group, compared with the PBS control group (Ctrl) and the Raw264.7 group (UTD), SR CAR-M treatment significantly inhibited tumor growth. Figure 5 C~D、 Figure 6 The above demonstrates that SR CAR-M possesses highly effective anti-triple-negative breast cancer efficacy, effectively infiltrating mouse tumor lesions and inhibiting tumor growth.

[0076] This invention provides a target IL-10 signal transduction receptor, engineered macrophages, and their applications, along with a method and approach. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A signal transduction receptor targeting IL-10, characterized in that, The signal transduction receptor consists of three functional domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain consists of a signal peptide, an HA tag, and a specific binding domain of the IL-10 receptor α subunit, from the N-terminus to the C-terminus. The amino acid sequences of the specific binding domain of the signal peptide, HA tag, and IL-10 receptor α subunit are shown in SEQ ID NO. 1~3. The amino acid sequence of the transmembrane domain is shown in SEQ ID NO.4; The amino acid sequence of the intracellular domain is shown in SEQ ID NO.

5.

2. The signal transduction receptor according to claim 1, characterized in that, The extracellular domain, transmembrane domain, and intracellular domain of the signal transduction receptor are sequentially connected.

3. A nucleic acid molecule, characterized in that, The signal transduction receptor is encoded according to any one of claims 1 to 2.

4. A lentiviral vector, characterized in that, The lentiviral vector contains the nucleic acid molecule as described in claim 3.

5. An engineered macrophage targeting IL-10, characterized in that, The engineered macrophages express the signal transduction receptor as described in any one of claims 1 to 2.

6. The engineered macrophage according to claim 5, characterized in that, The engineered macrophages are prepared by transfecting macrophages with the lentiviral vector described in claim 4.

7. The engineered macrophage according to claim 6, characterized in that, The macrophages are either Raw264.7 macrophages or immortalized bone marrow-derived macrophages iBMDM.

8. The use of the engineered macrophages according to claim 6 or 7 in the preparation of drugs for treating solid tumors.

9. The application according to claim 8, characterized in that, The solid tumor is any one of bladder cancer, breast cancer, lung cancer, or melanoma.

10. A pharmaceutical composition for treating solid tumors, characterized in that, The pharmaceutical composition contains the engineered macrophages as described in claim 6 or 7.

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