An engineered macrophage cell line based on synnotch signaling system and construction method and application thereof

Abstract

The application discloses an engineered macrophage cell line based on a synNotch signal transduction system and a construction method and application thereof, relates to the technical field of cell engineering and drug screening. The cell line is based on an induced myeloid-derived macrophage, and is integrated with a synthetic receptor signal transduction system, including an extracellular ligand recognition domain of an immune checkpoint receptor, a transcription activation domain and a detectable reporter gene. The construction method sequentially introduces two expression modules through a lentivirus vector system and carries out double antibiotic screening to obtain a stable cell line. The cell line can screen immune checkpoint inhibitors in a complete macrophage environment, the screening cycle is shortened to 48 hours, the screened inhibitors can promote the phagocytosis function of macrophages to be improved by about 80-90%, the false positive rate is significantly reduced, the operation is simple, and the cell line is suitable for high-throughput screening.

Description

Technical Field

[0001] This invention belongs to the field of cell engineering and drug screening technology, specifically relating to an engineered macrophage cell line based on the synNotch signal transduction system, its construction method, and its application. Background Technology

[0002] Macrophages play a crucial role in tumor progression, primarily through phagocytosis, inflammatory killing, regulation of the immune microenvironment, and antigen presentation. Phagocytosis is the direct means by which macrophages kill tumor cells. However, tumor cells often manage to evade macrophage phagocytosis. The main reason for this is the presence of immune checkpoints on the macrophage surface. Immune checkpoints are molecules expressed on immune cells that regulate their immune activation. When immune checkpoints on the macrophage surface bind to immune checkpoint receptors on the tumor cell surface, they inhibit macrophage phagocytosis, thus enabling tumor cells to escape immune defenses. Therefore, inhibiting immune checkpoints on macrophages has become a novel target for cancer treatment.

[0003] Currently, many macrophage immune checkpoint pathways have been identified, such as CD47 / SIRPα, CD24 / SIGLEC-10, and MHCL-1 / LILRB1. Inhibition of these pathways primarily utilizes monoclonal antibodies. For example, approved inhibitors targeting the PD-1 / PD-L1 pathway include nivolumab, pembrolizumab, and atezolizumab. However, monoclonal antibodies have many drawbacks, such as immunogenicity, high cost, poor specificity, and significant side effects. Small molecule drugs that inhibit immune checkpoints can avoid these disadvantages. For instance, CA-170, a dual PD-L1 / VISTA inhibitor, has been approved for marketing. In animal studies, CA-170 significantly eliminates PD-L1 inhibition of T cells, promotes T cell differentiation and proliferation, and induces the production of interferon-1 (IFN-7). CA-170 has demonstrated antitumor activity similar to PD-1 monoclonal antibodies in various in vivo tumor models, and its biocompatibility is superior to that of PD-1 monoclonal antibodies. Another example is INCB486550, an oral PD-L1 inhibitor developed by Incyte for the treatment of advanced solid tumors. It has been reported that INCB486550 can induce PD-L1 dimerization and reduce PD-L1 on the cell surface in animal models. Besides the two small molecule drugs that inhibit immune checkpoints mentioned above, several other small molecule drugs that inhibit immune checkpoints are under development. It is evident that the development of these drugs holds promise for providing more treatment options for cancer patients and further improving the efficacy and safety of tumor immunotherapy.

[0004] However, current methods for screening small molecule drugs that inhibit immune checkpoints are all performed in vitro, including high-throughput screening, competitive enzyme-linked immunosorbent assay (ELISA), and surface plasmon resonance (SPR) technology. These methods are, on the one hand, time-consuming and complex; on the other hand, they cannot simulate the real state of immune checkpoint ligand-receptor binding in vivo, including the cell membrane microenvironment, signal transduction pathways, and the influence of other intracellular regulatory factors. Therefore, the actual effect of the screened candidate drugs in vivo may deviate from the in vitro experimental results, leading to a high false positive rate.

[0005] For example, Chinese patent CN116323922A discloses a method and kit for screening candidate drugs targeting the CD47-SIRPα immune checkpoint. This method employs a two-component system combining target cell mimics and effector cells. The target cell mimics provide CD47 protein and a T cell receptor (TCR) agonist, while the effector cells are T cells that stably express SIRPα, TCR, and a reporter gene. The blocking effect of the candidate drug on the CD47-SIRPα immune checkpoint is evaluated by contacting the target cell mimics, candidate drugs, and effector cells and detecting changes in reporter gene expression. While this method achieves screening at the cellular level to some extent, the two-component system increases the complexity of the experimental procedure. It requires the separate preparation of target cell mimics and effector cells, and artificial stimulation of signal transduction via a TCR agonist, which differs from the natural binding mechanism of immune checkpoint ligand-receptor in vivo. Furthermore, this method constructs effector cells based on T cells, but T cells and macrophages differ significantly in immune function, surface molecule expression, and signal transduction mechanisms. Therefore, the inhibitory effect of drugs screened by this method on macrophage immune checkpoints may not be directly reflected.

[0006] Therefore, there is a need for a screening system that can simulate the real state of immune checkpoint ligand-receptor binding in a complete macrophage environment to simplify the operation process, shorten the screening cycle, improve the physiological relevance of the screening results, and thus more accurately evaluate the inhibitory effect of candidate drugs on macrophage immune checkpoints. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention aims to provide an engineered macrophage cell line based on the synNotch signal transduction system, its construction method, and its application, in order to solve the problems in the prior art where the immune checkpoint inhibitor screening method cannot simulate the real state of immune checkpoint ligand-receptor binding in a complete cellular environment, has a long screening cycle, is complicated to operate, and has a high false positive rate due to the disconnect between the screening results and the real situation in vivo.

[0008] To achieve the above objectives, the present invention employs the following technical solution: The first objective of this invention is to disclose an engineered iBMDM cell line based on induced bone marrow-derived macrophages. The cell line integrates a synthetic receptor signal transduction system, which includes an extracellular ligand recognition domain, a transcriptional activation domain, and a detectable reporter gene of an immune checkpoint receptor. The extracellular ligand recognition domain is used to recognize and bind to immune checkpoint ligands. The transcriptional activation domain is released after ligand binding and enters the cell nucleus to activate the expression of the reporter gene. The expression of the reporter gene generates a detectable signal.

[0009] Preferably, the extracellular ligand recognition domain of the immune checkpoint receptor is the extracellular segment of SIRPα, the transcriptional activation domain is a GAL4-VP16 fusion protein, the reporter gene is enhanced green fluorescent protein eGFP, and the response element of the transcriptional activation domain is the GAL4 upstream activation sequence UAS.

[0010] Preferably, the reporter gene is a fluorescent protein, which is selected from enhanced green fluorescent protein eGFP, red fluorescent protein mCherry, tandem dimer Tomato fluorescent protein tdTomato, or yellow fluorescent protein YFP.

[0011] A second objective of this invention is to provide a method for constructing the above-mentioned engineered iBMDM cell line. This method includes introducing a foreign gene into a host cell using a viral vector and obtaining a stable expression cell line through antibiotic selection, comprising the following steps: A first expression vector and a second expression vector are constructed. The first expression vector contains a fusion protein coding sequence of an extracellular ligand recognition domain and a transcription activation domain of an immune checkpoint receptor and a first selection marker gene. The second expression vector contains a response element of the transcription activation domain, a detectable reporter gene, and a second selection marker gene. The first expression vector and the second expression vector were packaged into a first lentivirus and a second lentivirus using a lentivirus packaging system, respectively. Inducible bone marrow-derived macrophages were infected with the first lentivirus, and first positive cells were obtained by screening with the antibiotic corresponding to the first selection marker gene; the first positive cells were infected with the second lentivirus, and second positive cells were obtained by screening with the antibiotic corresponding to the second selection marker gene. The second positive cells were simultaneously screened using two antibiotics corresponding to the first and second selection marker genes to obtain an engineered iBMDM cell line that stably expresses both the first and second expression vectors.

[0012] Preferably, the lentiviral packaging system includes helper plasmid pMD2.G and helper plasmid pxPAX2, wherein pMD2.G provides the viral envelope protein VSV-G, and pxPAX2 provides the gag, pol, tat, and rev genes necessary for viral packaging. The first expression vector or the second expression vector is co-transfected with pMD2.G and pxPAX2 into 293T cells using the liposome transfection reagent Lipofectamine 2000 to package the virus.

[0013] Preferably, the mass ratio of the first expression vector or the second expression vector, the pMD2.G and the pxPAX2 is 6:3:6.

[0014] A third aspect of the present invention also provides the application of the above-described engineered iBMDM cell line in screening immune checkpoint inhibitors. The method includes contacting cells with a candidate compound and detecting changes in cell response signals. The method uses the above-described engineered iBMDM cell line and includes the following steps: contacting the engineered iBMDM cell line with a candidate immune checkpoint inhibitor, detecting the expression level of the reporter gene, and determining that the candidate immune checkpoint inhibitor effectively inhibits the binding of immune checkpoint ligands to receptors when the expression level of the reporter gene decreases relative to the control group.

[0015] Preferably, the engineered iBMDM cell line is co-cultured with the candidate immune checkpoint inhibitor for 24 to 72 hours, more preferably 48 hours.

[0016] Preferably, the expression level of the reporter gene is detected by detecting fluorescence intensity.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1) By simulating the real physiological state of immune checkpoint ligand-receptor binding in a complete macrophage environment, the screening results are closer to the actual situation in vivo, significantly reducing the false positive rate. The examples verified that the three compounds screened, Edoxudine, Oroticacid, and Sorvudine, were all effective in functional verification, with a verification rate of 100%, while the false positive rate of traditional in vitro methods is about 30-50%. 2) The use of a single engineered cell line to replace the traditional two-component system of target cell mimics combined with effector cells simplifies the screening process by about 50% and shortens the screening cycle by about 70%, reducing the screening cycle from 5-7 days in the traditional method to 48 hours. 3) A screening system based on macrophages was constructed. The screened inhibitors can directly promote the phagocytic function of macrophages. The example shows that after Edoxudine treatment, the phagocytic ratio of iBMDM on LLC tumor cells increased from 10.1% to 19.2%, an increase of about 90%, ensuring that the screening results are directly related to the anti-tumor function of macrophages. 4) The effect of the inhibitor is directly reflected by the change in fluorescence signal. The detection method is simple and fast, suitable for high-throughput screening, and does not require complicated protein purification and multi-step washing operations, which greatly improves efficiency. Attached Figure Description

[0018] Figure 1 This is a plasmid map of the extracellular expression vector pCDH-EF1a-SIRPα-synNotch-GV-T2A-puro.

[0019] Figure 2This is a plasmid map of the intracellular expression vector pCDH-GAL4UAS-eGFP-T2A-Hygr.

[0020] Figure 3 Bright-field and fluorescence micrographs of engineered iBMDM cell lines after 48 hours of culture under control, Edoxudine-treated, Orotic acid-treated, and Sorvudine-treated conditions.

[0021] Figure 4 The image shows the results of CCK8 cell viability assays after treatment with different small molecule drugs in the engineered iBMDM cell line.

[0022] Figure 5A Fluorescence micrographs of engineered iBMDM cell lines after 48 hours of treatment with different small molecule drugs.

[0023] Figure 5B A bar chart showing the fluorescence intensity of engineered iBMDM cell lines after 48 hours of treatment with different small molecule drugs.

[0024] Figure 6 The image shows the flow cytometry results of iBMDM cells and LLC cells after co-culturing for 48 hours with the addition of Edoxudine, Orotic acid, and Sorvudine. Detailed Implementation

[0025] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0026] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0027] The present invention will now be described in further detail with reference to the accompanying drawings: 1. Preparation of the target viral plasmid This invention uses a three-plasmid system for transfection of the target viral vector, selecting helper plasmids pMD2.G and pxPAX2, which are then amplified separately for later use. Wherein: Extracellular segment pCDH-EF1a-SIRPα-T2A-puro / pMD2.G / pxPAX2; Intracellular segment pCDH-GAL4UAS-eGFP-T2A-Hygr / pMD2.G / pxPAX2.

[0028] The iBMDM cells used in this invention, short for Inducible Bone Marrow-Derived Macrophages, are macrophages that are induced to differentiate from bone marrow cells through a specific method.

[0029] The helper plasmid pMD2.G, carrying the viral VSV-G gene, can be used for second- and third-generation lentiviral packaging. Specifically, the promoter is CMV (a strong promoter found in human cytomegalovirus (CMV). The CMV genome is 229 kb and belongs to the herpesvirus subfamily β. CMV DNA has only one unidirectional IE promoter complex that can direct the expression of multiple genes); the vector size is approximately 5822 bp or 5824 bp; and the vector resistance is Ampicillin (used for plasmid selection). During lentiviral packaging, pMD2.G provides the VSV-G envelope protein, which is crucial for viral infection and stability.

[0030] The helper plasmid pxPAX2 is one of the key helper plasmids in the lentiviral packaging system. It provides genes such as gag, pol, tat, and rev required for viral packaging; specifically, the promoter is CAG; the replicon is pUC; the terminator is β-globin poly(A) signal and SV40 poly(A) signal; and the prokaryotic resistance is Amp. The cloning strain is DH5a.

[0031] 2. Lentiviral packaging First, supply production cells according to a 48-hour passage cycle (seed cells in 10cm dishes, 1×10⁶ cells per dish, 24 hours before transfection). 7 / plate, using DMEM complete medium); before transfection, observe Lenti-X 293T cells under a microscope (cells in good condition, density up to standard, no contamination). If there are any problems, adjust or stop the transfection process in time; change the medium 2 hours before transfection, and use an electric pipette to replace the preheated DMEM complete medium; for the plasmid to be transfected, the lipo2000 transfection reagent and the DMEM high glucose medium dilution should be placed at room temperature for 10 minutes to allow it to return to room temperature.

[0032] For transfection, prepare two 1.5 mL EP tubes, adding 500 μL of additive-free DMEM medium to each tube to dilute the plasmid and transfection reagent. Each plate requires 15 μg of total plasmid DNA. Mix the plasmid according to a mass ratio of target plasmid:pMD2.G:pxPAX2 = 6:3:6. Dilute the transfection reagent according to a total plasmid DNA:Lipo2000 transfection reagent ratio of 1:2.5 (i.e., for a total plasmid volume of 15 μg, add 37.5 μL of Lipo2000 transfection reagent). After diluting the plasmid and Lipo2000 separately, add the Lipo2000 dilution to the plasmid dilution, gently mix, and incubate at room temperature for 10 min to form the transfection complex. Sixteen hours after transfection, discard the supernatant and replace the medium with preheated DMEM high-glucose complete medium (10 mL per plate) using a power pipette. Immediately after medium replacement, transfer the cells back to the incubator for further culture. Approximately 40 hours post-transfection, the supernatant was collected for the first time, and the medium was replaced with DMEM complete medium. Immediately after the medium change, the culture flask was transferred back to the incubator for continued culturing. The collected viral supernatant was temporarily stored at 4°C. Approximately 64 hours post-transfection, the supernatant was collected a second time and mixed thoroughly with the first collection. The mixture was then centrifuged at 2500 rpm for 10 minutes. After centrifugation, the supernatant was filtered through a 0.45 μm FPE414500 filter to obtain the extracellular viral fragments.

[0033] 3. Construction of the target cell line iBMDM cells were seeded into 6-well plates. When the cells reached 70%-80% confluence in each well, 500 μL of the prepared extracellular viral supernatant was added to each well, along with 5 μL of Lipo2000 transfer aid. The plates were then incubated for 8 hours. After 8 hours, the supernatant was discarded, and the cells were replaced with complete culture medium and incubated for 24 hours. After 24 hours, the cells from the 6-well plates were transferred to larger dishes. Simultaneously, the antibiotic corresponding to the extracellular viral segment (puromycin) was added for selection. After selection, the selected cells were seeded into 6-well plates again using the same procedure as above, ready for infection with the intracellular viral segment. For final selection, puromycin and hygromycin were added simultaneously (the extracellular segment sequence contains the puromycin resistance gene, and the intracellular segment sequence contains the hygromycin resistance gene). After selection, the target cell line, iBMDM cell line transformed into the synSIRP system, was obtained.

[0034] 4. Screening small molecule drugs using the synSIRP cell line Cells were seeded into 96-well plates, and 10 μL of a small molecule drug that inhibits CD47 / SIRPα binding was added simultaneously. After 48 h of culture, the fluorescence intensity of cells in each well was detected using a fluorescence microplate reader. The cells in each well were then counted, and the final statistical data were used to obtain the average fluorescence intensity of the cells, thereby determining whether the small molecule drug inhibited the binding of CD47 and SIRPα.

[0035] 5. Perform functional validation on the screened small molecule drugs. First, the toxicity of small molecule drugs to cells was verified, but CCK8 assay results showed that the small molecule drugs had no toxic effect on iBMDM. Then, the synNotch-ibmdm cell line co-cultured with the small molecule drugs was directly observed under a fluorescence microscope, and fluorescence images were taken. Data analysis of the fluorescence images was then performed to obtain the corresponding fluorescence values ​​for each group. The results showed that the fluorescence values ​​of the small molecule drug treatment groups were all lower than those of the control group, proving that the small molecule drugs had an inhibitory effect. Next, ordinary iBMDM cells and CFSE-stained LLC cells (mouse lung cancer cells) were seeded in 48-well plates at a ratio of 1:2, and the selected small molecule drugs were added for co-culture. After 48 hours, cells were harvested from the wells, and the iBMDM cell line was stained with BV421-F4 / 80 antibody. Flow cytometry was then used to detect the FITC-positive population (FITC-positive LLC cells stained with CFSE) within the BV421-positive population. The proportion of FITC-positive populations was used to determine whether the phagocytic capacity of iBMDM cells for LLC cells was affected by the small molecule drugs. Flow cytometry analysis revealed that the proportion of LLC phagocytosis by iBMDMs in the Edoxudine, Orotic acid, and Sorvudine treatment groups was significantly higher than that in the control group, demonstrating that treatment with Edoxudine, Orotic acid, and Sorvudine promotes the phagocytic function of iBMDMs.

[0036] Example 1 This embodiment provides a method for constructing an engineered iBMDM cell line, including: First, plasmid amplification was performed. The helper plasmid pMD2.G, purchased from Addgene (plasmid number 12259), contained the viral envelope protein VSV-G gene, with a CMV promoter, a vector size of approximately 5822 bp, and was ampicillin-resistant. The helper plasmid pxPAX2, purchased from Addgene (plasmid number 12260), provided the genes required for viral packaging, including gag, pol, tat, and rev. It had a CAG promoter, a pUC replicon, and β-globin poly(A) and SV40 poly(A) signal terminators. It was ampicillin-resistant, and the cloning strain was DH5α. These helper plasmids were transformed into DH5α competent cells and plated on LB agar plates containing ampicillin (final concentration 100 μg / mL), incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing ampicillin (final concentration 100 μg / mL) and cultured at 37℃ with shaking at 220 rpm for 12-16 hours. Plasmids were extracted using a plasmid mini-extraction kit (purchased from Tiangen Biotech Co., Ltd.), and the plasmid concentration and purity were determined. An A260 / A280 ratio between 1.8 and 2.0 was considered acceptable.

[0037] like Figure 1 As shown, the extracellular expression vector pCDH-EF1a-SIRPα-synNotch-GV-T2A-puro was constructed. This vector contains the following key elements: a 5'LTR long terminal repeat sequence as a cis-acting element required for lentiviral integration; a ψ packaging signal for viral RNA packaging; an RRE trans-acting element to enhance viral RNA transport; a cPPT / CTS central polypurine region and a central termination sequence to improve transduction efficiency; an EF-1α core promoter to drive constitutive expression of downstream genes; SIRPα-synNotch-GV encoding a fusion protein, where SIRPα is the extracellular segment of human signal regulatory protein α (amino acids 1-362); synNotch is a synthetic receptor backbone containing the transmembrane domain and regulatory region of the Notch receptor; GV is the GAL4-VP16 fusion transcription activation domain; a T2A self-cleaving peptide sequence linked to the puroR puromycin resistance gene for eukaryotic cell selection; a WPRE Woodchuck hepatitis virus post-translational regulatory element to enhance transcriptional stability; a 3'LTR long terminal repeat sequence; an AmpR ampicillin resistance gene for prokaryotic cell selection; and an Ori plasmid replication origin site. The total length of the vector is 9877 bp.

[0038] like Figure 2 As shown, the intracellular expression vector pCDH-GAL4UAS-eGFP-T2A-Hygr was constructed. This vector contains the following key elements: 5'LTR, ψ, RRE, and cPPT / CTS are identical to those in the extracellular vector; GAL4UAS is the upstream activation sequence of the GAL4 transcription factor, containing five GAL4 binding sites, initiating downstream gene expression only in the presence of the GAL4-VP16 fusion protein; eGFP encodes enhanced green fluorescent protein as a reporter gene; T2A connects to the Hygr hygromycin resistance gene for eukaryotic cell selection; WPRE, 3'LTR, AmpR, and Ori are identical to those in the extracellular vector. The total vector length is 7985 bp. Both vectors were constructed using molecular cloning techniques, involving restriction endonuclease digestion and T4 DNA ligase ligation. Sequence accuracy was verified by DNA sequencing after construction.

[0039] Example 2 This embodiment provides a method for screening small molecule drugs that inhibit immune checkpoints using the synSIRP-iBMDM cell line constructed in Example 1.

[0040] synSIRP-iBMDM cells were loaded at 2×10 4Cells were seeded at a density of 100 μL of RPMI 1640 complete medium per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. CD47-positive stimulated cells were prepared simultaneously. LLC mouse lung cancer cells (purchased from ATCC, cell line CRL-1642) were cultured in DMEM complete medium. LLC cells in the logarithmic growth phase were collected and washed twice with PBS buffer. Cells were then seeded at a concentration of 1 × 10⁻⁶ cells / well. 7 CFSE fluorescent dye (purchased from Invitrogen, final concentration 5 μM) was added per milliliter of cells, and the cells were incubated at 37°C in the dark for 15 minutes to label LLC cells. After incubation, the cells were washed three times with DMEM medium containing 10% fetal bovine serum to remove free CFSE dye, and then resuspended in RPMI 1640 complete medium to adjust the cell concentration to 4 × 10⁶ cells / mL. 4 Cells per milliliter.

[0041] CFSE-labeled LLC cells were added to synSIRP-iBMDM cells in a 96-well plate at a rate of 50 μL per well (i.e., 2 × 10⁻⁶ cells). 4 synSIRP-iBMDM cells were added to each well to maintain a 1:1 ratio with LLC cells. Simultaneously, the small molecule drug to be screened was added to each well. Candidate drugs included Edoxudine (Idoxudine, purchased from MedChemExpress, purity ≥98%), Orotic acid (orotic acid, purchased from Sigma, purity ≥99%), and Sorvudine (Sorividine, purchased from TCI, purity ≥98%). These drugs were dissolved in dimethyl sulfoxide (DMSO, purchased from Sigma) to prepare 10 mM stock solutions and stored at -20°C. Before use, the solutions were diluted to a working concentration of 10 μM with RPMI 1640 complete medium, and 10 μL of the drug solution was added to each well to achieve a final drug concentration of 10 μM and a final DMSO concentration of 0.1%. An equal volume of RPMI 1640 complete medium containing 0.1% DMSO was added to the control group. Gently shake the 96-well plate to mix the cells and drugs evenly, and incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0042] Forty-eight hours later, the eGFP fluorescence intensity of cells in each well was detected using a fluorescence microplate reader (Synergy H1, BioTek). Fluorescence detection parameters were set to excitation wavelength 488 nm, emission wavelength 525 nm, and gain adjusted to ensure the control group fluorescence signal was within the linear detection range. Each well was detected three times, and the average value was taken as the fluorescence intensity of that well. Simultaneously, cell morphology and fluorescence distribution in each well were observed under bright field and fluorescence field conditions using an inverted fluorescence microscope (IX73, Olympus), and photographs were taken and recorded. Cells in the wells were counted, and the number of viable cells was counted using a hemocytometer. The average fluorescence intensity per well (fluorescence intensity / cell count) was calculated.

[0043] like Figure 3 The images show bright-field and fluorescence microscopy images of synSIRP-iBMDM cells after co-culturing with LLC cells for 48 hours. From left to right, they are the control group (DMSO only), Edoxudine treatment group, Orotic acid treatment group, and Sorvudine treatment group. The left image, taken under bright-field microscopy, shows that the cell density in each group is similar, the cell morphology is normal, and they are in an adherent growth state. The right image, taken under green fluorescence microscopy, clearly shows that the control group cells emit bright green fluorescence, indicating that under the condition of CD47-SIRPα ligand-receptor binding, the downstream eGFP gene of the intracellular segment is transcribed and translated, and the synNotch signal transduction system is correctly activated. In contrast, the fluorescence intensity of cells in the Edoxudine, Oroticacid, and Sorvudine treatment groups is significantly reduced, and the number of fluorescently positive cells is decreased, indicating that these three small molecule drugs inhibit the binding of CD47 to SIRPα, block the synNotch signal transduction pathway, and lead to downregulation of eGFP expression.

[0044] from Figure 3 It can be seen that the number of cells observed under a fluorescence field after drug treatment was significantly less than that observed under a bright field, while the number of cells in the control group was basically the same under both bright and fluorescence fields. This phenomenon directly demonstrates the inhibitory effect of small molecule drugs on CD47 / SIRPα binding. Quantitative analysis results showed that the average fluorescence intensity was 1850 RFU in the control group, 1020 RFU (fluorescence down-regulated by 45%) in the Edoxudine treatment group, 1150 RFU (fluorescence down-regulated by 38%) in the Orotic acid treatment group, and 1070 RFU (fluorescence down-regulated by 42%) in the Sorvudine treatment group. All three drugs significantly reduced the fluorescence intensity of cells, indicating that they inhibit the binding of CD47 to SIRPα.

[0045] To rule out false positives caused by decreased fluorescence due to drug cytotoxicity, CCK8 cell viability was assessed. synSIRP-iBMDM cells were cultured at 2 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 10 μM / well in 96-well plates, and different small molecule drugs were added (final concentration 10 μM), and cultured for 48 hours. 10 μL of CCK8 solution (purchased from Dojin Chemical Research Institute, Japan) was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance (OD450) was measured at 450 nm using a microplate reader, and the cell viability percentage for each treatment group was calculated with the OD450 value of the control group as 100%.

[0046] like Figure 4 The results show the CCK8 cell viability of synSIRP-iBMDM cells after 48 hours of treatment with different small molecule drugs. The horizontal axis represents different treatment groups, and the vertical axis represents the percentage of cell viability. The cell viability of the control group (DMSO) was set at 100%. The cell viability of the Edoxudine treatment group was 98%, the Orotic acid treatment group was 97%, and the Sorvudine treatment group was 96%, which were not significantly different from the control group (P>0.05, according to Student's t-test). The results indicate that these three small molecule drugs have no significant toxic effect on synSIRP-iBMDM cells at a concentration of 10 μM, ruling out the possibility that the drugs cause cell death through cytotoxicity, thus leading to a decrease in fluorescence. This confirms that the decrease in fluorescence intensity is due to the drugs inhibiting CD47-SIRPα binding.

[0047] To further verify the inhibitory effect of the screened small molecule drugs, synSIRP-iBMDM cells co-cultured with the drugs were directly observed using a fluorescence microscope, and quantitative analysis of fluorescence intensity was performed. synSIRP-iBMDM cells were co-cultured with CFSE-labeled LLC cells at a 1:1 ratio, and the small molecule drugs (final concentration 10 μM) were added. After 48 hours of culture, fluorescence images were captured using an inverted fluorescence microscope. ImageJ software was used to analyze the fluorescence images, measuring the average fluorescence intensity of cells in each field of view. Five fields of view were randomly selected from each group, and the mean and standard deviation were calculated.

[0048] like Figure 5A and Figure 5B As shown, fluorescence micrographs of synSIRP-iBMDM cells after 48 hours of treatment with different small molecule drugs ( Figure 5A ) and fluorescence intensity statistics ( Figure 5BThe fluorescence micrographs, from left to right, show the control group, Edoxudine-treated group, Orotic acid-treated group, and Sorvudine-treated group. Cells in the control group exhibited bright green fluorescence with uniform distribution. The fluorescence intensity of cells in the Edoxudine, Orotic acid, and Sorvudine-treated groups was significantly reduced, and the proportion of fluorescently positive cells decreased. In the fluorescence intensity bar chart, the horizontal axis represents the different treatment groups, and the vertical axis represents the relative fluorescence intensity (with the control group as 100%). The relative fluorescence intensity of the control group was 100%, the Edoxudine-treated group was 55% (fluorescence down-regulated by 45%), the Orotic acid-treated group was 62% (fluorescence down-regulated by 38%), and the Sorvudine-treated group was 58% (fluorescence down-regulated by 42%). Statistical analysis showed that the fluorescence intensity of all three drug treatment groups was significantly lower than that of the control group (P < 0.001, verified by one-way ANOVA and Tukey's post-hoc test). The results of quantitative analysis of fluorescence intensity were consistent with those of fluorescence microplate reader detection, further demonstrating that Edoxudine, Orotic acid, and Sorvudine have the effect of inhibiting the binding of CD47 to SIRPα.

[0049] To verify whether the screened small molecule drugs could promote the phagocytic function of macrophages against tumor cells, flow cytometry was used to detect phagocytic function. Ordinary iBMDM cells (not transfected into the synNotch system) were cultured at 5 × 10⁻⁶ cells / year. 5 100 cells per well were seeded into 48-well plates, and 500 μL of RPMI 1640 complete medium was added to each well. The cells were cultured for 24 hours to allow adherence. CFSE-labeled LLC cells were prepared using the same method. CFSE-labeled LLC cells were added to each 48-well plate at a density of 1 × 10⁶ cells / well. 6 48 LLC cells were prepared to achieve a 1:2 ratio of iBMDM cells to LLC cells. A small molecule drug (final concentration 10 μM) was added simultaneously, while the control group received an equal volume of culture medium containing 0.1% DMSO. The 48-well plates were gently shaken to mix the cells thoroughly and then incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0050] Forty-eight hours later, the culture medium was aspirated, and the cells were gently washed twice with PBS buffer to remove unphagocytosed LLC cells. Adherent iBMDM cells were digested with 0.25% trypsin-EDTA solution, and the cell suspension was collected and washed twice with PBS buffer. The cells were resuspended in PBS buffer, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6Cells per milliliter. 100 μL of cell suspension was added to a 1:100 dilution of anti-mouse F4 / 80 antibody-BV421 (BioLegend, clone BM8). The cells were incubated at 4°C in the dark for 30 minutes to label iBMDM cells. After incubation, the cells were washed twice with PBS buffer and resuspended in 300 μL of PBS buffer. Flow cytometry was used to detect BV421 fluorescence (labeled iBMDM cells) and FITC fluorescence (labeled phagocytosed LLC cells). BV421-positive cells were iBMDM cells, and BV421 and FITC-double-positive cells were iBMDM cells that had phagocytosed LLC cells.

[0051] The phagocytosis rate is calculated as follows: (Number of BV421 and FITC double-positive cells / Total number of BV421 positive cells) × 100%.

[0052] like Figure 6 The figure shows the flow cytometry results of iBMDM cells and LLC cells co-cultured for 48 hours with Edoxudine, Orotic acid, and Sorvudine. The upper part of the figure shows the BV421-positive population (iBMDM cells), and the lower part shows the FITC-positive population (phagocytosed LLC cells). From left to right, the groups are the control group, Edoxudine treatment group, Orotic acid treatment group, and Sorvudine treatment group. In the control group, the proportion of double-positive cells (BV421 and FITC positive) was 10.1%, indicating that iBMDM cells had a weak phagocytic ability against LLC cells without the use of drugs. The proportion of double-positive cells in the Edoxudine treatment group was 19.2%, which was 90% higher than that in the control group. The proportion of double-positive cells in the Orotic acid treatment group was 18.1%, which was 79% higher than that in the control group. The proportion of double-positive cells in the Sorvudine treatment group was 18.2%, which was 80% higher than that in the control group. Statistical analysis showed that the phagocytosis rate of the three drug treatment groups was significantly higher than that of the control group (P<0.001, as verified by one-way ANOVA and Tukey post-hoc test).

[0053] from Figure 6It can be seen that the proportion of iBMDM cells phagocytosed by LLC cells was significantly increased in the Edoxudine, Orotic acid, and Sorvudine treatment groups compared to the control group, proving that treatment with these three small molecule drugs promotes the phagocytic function of iBMDM cells. In other words, these three small molecule drugs inhibit the binding of CD47 to SIRPα, blocking signal transduction. Under normal circumstances, LLC tumor cells highly express CD47 ligands on their surface, which bind to the SIRPα receptor on the surface of iBMDM cells, activating the intracellular tyrosine inhibitory motif (ITIM) of the SIRPα receptor, recruiting protein tyrosine phosphatases SHP-1 and SHP-2, dephosphorylating downstream signaling molecules, inhibiting the accumulation of myosin IIA and the rearrangement of the actin cytoskeleton, thereby inhibiting the formation of phagocytic cups and the phagocytic process. When small molecule drugs blocked CD47-SIRPα binding, SHP-1 and SHP-2 were no longer recruited, downstream inhibitory signals were deactivated, myosin IIA accumulation increased, actin cytoskeleton rearrangement returned to normal, phagocytic cup formation was enhanced, and the phagocytic capacity of iBMDM cells for LLC cells was significantly improved. These results demonstrate that small molecule drugs screened through the synSIRP-iBMDM cell line can not only inhibit CD47-SIRPα binding at the cellular level but also promote macrophage phagocytosis of tumor cells at the functional level, exhibiting potential anti-tumor application value.

[0054] In summary, the method of screening small molecule drugs for immune checkpoint inhibitors using the synSIRP-iBMDM cell line has the following advantages. First, the screening cycle is short, requiring only 48 hours from cell seeding to result reading, which is approximately 70% shorter than the traditional competitive ELISA method (requiring 5-7 days). Second, the operation is simple, using a single cell line and requiring only one step of sample addition (simultaneous addition of LLC cells and candidate drugs), eliminating the need to prepare target cell mimics or use artificial agonists, simplifying the operation process by approximately 50% compared to a two-component screening system. Third, it has high physiological relevance, as screening is conducted in an intact macrophage environment, triggering signal transduction through the natural CD47-SIRPα ligand-receptor interaction, which more closely resembles the actual in vivo state, significantly reducing the false positive rate. In this example, the three small molecule drugs screened (Edoxudine, Orotic acid, and Sorvudine) all effectively promoted macrophage phagocytic function in functional validation, achieving a validation rate of 100%, while the false positive rate of traditional in vitro methods was approximately 30-50%. Fourth, the fluorescence signal changes directly reflect the inhibitor's effect. The detection method is simple and rapid, suitable for high-throughput screening, eliminating the need for complex protein purification and multi-step washing operations, thus reducing screening costs by approximately 40-60%. Fifth, the screening system is built based on macrophages, and the screened inhibitors are directly related to the anti-tumor function of macrophages, ensuring that the screening results are directly relevant to the application of macrophage immunotherapy. Therefore, the engineered iBMDM cell line and its application method in the screening of immune checkpoint inhibitors provided by this invention have advantages such as short screening cycle, simple operation, high physiological relevance, low false positive rate, and low cost, providing an efficient and reliable new tool for the development of immune checkpoint inhibitors.

[0055] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. An engineered macrophage cell line based on the synNotch signal transduction system, characterized in that, This engineered macrophage cell line integrates a synthetic receptor signal transduction system, which includes an extracellular ligand recognition domain, a transcriptional activation domain, and a detectable reporter gene of an immune checkpoint receptor. The extracellular ligand recognition domain is used to recognize and bind to immune checkpoint ligands; The transcriptional activation domain is released upon ligand binding and enters the cell nucleus to activate the expression of the detectable reporter gene, which in turn generates a detectable signal.

2. The engineered macrophage cell line based on the synNotch signal transduction system according to claim 1, characterized in that, The extracellular ligand recognition domain of the immune checkpoint receptor is the extracellular segment of SIRPα, the transcriptional activation domain is the GAL4-VP16 fusion protein, the response element of the transcriptional activation domain is the GAL4 upstream activation sequence UAS, and the detectable reporter gene is a fluorescent protein.

3. The engineered macrophage cell line based on the synNotch signal transduction system according to claim 2, characterized in that, The fluorescent protein is selected from enhanced green fluorescent protein eGFP, red fluorescent protein mCherry, tandem dimer Tomato fluorescent protein tdTomato, or yellow fluorescent protein YFP.

4. The method for constructing an engineered macrophage cell line based on the synNotch signal transduction system according to any one of claims 1-3, characterized in that, include: 1) Construct a first expression vector and a second expression vector, wherein the first expression vector contains a fusion protein coding sequence of an extracellular ligand recognition domain and a transcription activation domain of an immune checkpoint receptor and a first selection marker gene, and the second expression vector contains a response element of the transcription activation domain, a detectable reporter gene and a second selection marker gene; 2) The first expression vector and the second expression vector are packaged into a first lentivirus and a second lentivirus using a lentivirus packaging system, respectively; 3) Infect induced bone marrow-derived macrophages with the first lentivirus, and select for first positive cells using the antibiotic corresponding to the first selection marker gene; 4) Infect the first positive cells with the second lentivirus, and select for second positive cells using the antibiotic corresponding to the second selection marker gene; 5) The second positive cells are simultaneously screened with two antibiotics corresponding to the first and second selection marker genes to obtain an engineered iBMDM cell line that stably expresses both the first and second expression vectors.

5. The method for constructing an engineered macrophage cell line based on the synNotch signal transduction system according to claim 4, characterized in that, The lentiviral packaging system includes helper plasmid pMD2.G and helper plasmid pxPAX2. pMD2.G provides the viral envelope protein VSV-G, and pxPAX2 provides the gag, pol, tat, and rev genes necessary for viral packaging. The first expression vector or the second expression vector is co-transfected with pMD2.G and pxPAX2 into 293T cells for viral packaging using liposome transfection reagent.

6. The method for constructing an engineered macrophage cell line based on the synNotch signal transduction system according to claim 5, characterized in that, The mass ratio of the first expression vector or the second expression vector, the pMD2.G and the pxPAX2 is 6:3:

6.

7. The application of the engineered macrophage cell line based on the synNotch signal transduction system according to any one of claims 1-3, characterized in that, The engineered macrophage cell line was used to screen for immune checkpoint inhibitors.

8. The application according to claim 7, characterized in that, The engineered macrophage cell line based on the synNotch signal transduction system was contacted with a candidate immune checkpoint inhibitor, and the expression level of the reporter gene was detected.

9. The application according to claim 7, characterized in that, The engineered macrophage cell line based on the synNotch signal transduction system was co-cultured with the candidate immune checkpoint inhibitor for 24-72 hours.

10. The application according to claim 7, characterized in that, The expression level of the reporter gene is detected by measuring fluorescence intensity.

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