A trispecific adaptor for regulating phagocytic function of macrophages and applications thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a trispecific adaptor for regulating phagocytosis of macrophages and application thereof. The trispecific adaptor is composed of a target cell specific recognition module, a pro-phagocytosis signal activation module and an anti-phagocytosis signal blocking module through L1 and L2 in a linear structure. The target cell specific recognition module can specifically recognize and combine with a target cell. The pro-phagocytosis signal activation module can activate a pro-phagocytosis signal receptor on the surface of a macrophage and enhance the phagocytosis capacity of the macrophage. The anti-phagocytosis signal blocking module can block an anti-phagocytosis signal receptor on the surface of the macrophage and release the macrophage from phagocytosis inhibition on the target cell. L1 and L2 are both linear peptides, and the linear peptide is formed by connecting at least one flexible peptide segment and at least one rigid peptide segment. The trispecific adaptor provided by the application can simultaneously regulate pro-phagocytosis signals and anti-phagocytosis signals, accurately regulate phagocytosis of macrophages, activate an immune response and improve a disease treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a three-specificity connector for regulating the phagocytic function of macrophages and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Macrophage-mediated phagocytosis has shown great potential in immunotherapy. Its targeted phagocytosis can effectively clear pathogens, apoptotic cells, and tumor-associated antigens, and can also promote the functional coordination of the innate and adaptive immune systems through antigen cross-presentation mechanisms. In recent years, immunotherapy strategies relying on macrophage phagocytosis have demonstrated significant advantages over traditional treatment methods.

[0004] In immunotherapy, regulating the dynamic balance between pro-phagocytic and anti-phagocytic signals is crucial for macrophages to effectively clear target cells. Macrophages can only overcome inhibition and achieve effective clearance when the intensity of pro-phagocytic signals significantly exceeds that of anti-phagocytic signals. However, the expression levels of pro-phagocytic markers and anti-phagocytic molecules vary significantly across different disease states. If pro-phagocytic signals are weak due to insufficient expression of related markers, even targeted interventions to enhance their activity may still suppress them due to high levels of anti-phagocytic signals; conversely, if only anti-phagocytic signals are inhibited without sufficient pro-phagocytic drive, phagocytic function is difficult to activate. Furthermore, diseased cells may evade immunotherapy by dynamically regulating the expression levels of these two types of signals, leading to unstable efficacy of single-therapy treatments. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a three-specificity connector for regulating macrophage phagocytic function and its application. The three-specificity connector provided by the present invention can simultaneously regulate pro-phagocytic and anti-phagocytic signals, thereby achieving precise regulation of macrophage phagocytosis, activating immune responses, and improving the therapeutic effect of diseases.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In the first aspect, a three-specificity connector for regulating the phagocytic function of macrophages is provided, comprising a target cell-specific recognition module, a phagocytic signal activation module, and an anti-phagocytic signal blocking module, which are connected by a first connector (L1) and a second connector (L2) to form a linear structure. The target cell-specific recognition module can specifically recognize and bind to target cells; the phagocytic signal activation module can activate the phagocytic signal receptors on the surface of macrophages and enhance the phagocytic ability of macrophages; the antiphagocytic signal blocking module can block the antiphagocytic signal receptors on the surface of macrophages and relieve the inhibition of macrophages on target cells. Both the first and second linkers are linear peptides, which are formed by linking at least one flexible peptide segment and at least one rigid peptide segment.

[0007] The flexible peptides described in this invention refer to peptides rich in small molecule amino acids or hydrophilic amino acid sequences, which provide good flexibility and solubility and allow the movement of linked functional domains.

[0008] The rigid peptides described in this invention refer to those with an α-helix structure or containing multiple Pro residues that exhibit a relatively rigid amino acid sequence.

[0009] The connector described in this invention enables the phagocytic signal activation module and the antiphagocytic signal blocking module to act simultaneously on the same macrophage by controlling the rigidity and length of the first connector, thus synergistically regulating the phagocytic and antiphagocytic signals and efficiently initiating the macrophage's phagocytic activity. At the same time, the rigid extension structure of the second connector enables the cell-specific recognition module to specifically recognize and bind to the target cell, effectively clearing the target cell.

[0010] On the other hand, a nucleic acid molecule encodes the aforementioned three-specificity connector that regulates the phagocytic function of macrophages.

[0011] Thirdly, a composition comprising the above-mentioned trispecific adaptor for regulating macrophage phagocytosis or the above-mentioned nucleic acid molecule and a carrier.

[0012] Fourthly, the application of the above-mentioned three-specificity connector, nucleic acid molecule, or composition for regulating macrophage phagocytic function in the preparation of drugs.

[0013] Specifically, the drug has the effects of target cell clearance, tumor immunity, infectious diseases, tissue repair, regulation of inflammatory responses and / or regulation of autoimmune diseases.

[0014] The beneficial effects of this invention are as follows: This invention, through the design of the linker, not only connects the target cell-specific recognition module, the pro-phagocytic signal activation module, and the anti-phagocytic signal blocking module, but also, by adjusting the linker, enables the synergistic regulation of each module to control the pro-phagocytic and anti-phagocytic signals, efficiently initiating macrophage phagocytosis and specifically recognizing and binding to target cells, thereby achieving the goal of effectively clearing target cells. Experiments have demonstrated that the three-specificity linker provided by this invention can effectively improve the phagocytic efficiency of macrophages on target cells. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 This is a schematic diagram of the three specific connectives that regulate the phagocytic function of macrophages in Embodiment 1 of the present invention; Figure 2 TrME in Embodiment 2 of the present invention B7H3 Western blot analysis results; Figure 3 TrME in Embodiment 3 of the present invention B7H3 Activity diagram of specific binding with B7H3; Figure 4 TrME in Embodiment 4 of the present invention B7H3 Effect on macrophage phagocytosis; Figure 5 TrME in Embodiment 5 of the present invention B7H3 Effect of macrophage phenotypic polarization; Figure 6 TrME in Embodiment 6 of the present invention B7H3 The effect of intratumoral injection on mouse survival (Figure); Figure 7 TrME in Embodiment 7 of the present invention B7H3 -mLNP transmission electron microscopy image; Figure 8 TrME in Embodiment 8 of the present invention B7H3 Figure showing the effect of intratumoral injection of -mLNP on mouse survival. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] In a typical embodiment of the present invention, a three-specificity connector for regulating the phagocytic function of macrophages is provided, which consists of a target cell specific recognition module, a phagocytic signal activation module, and an anti-phagocytic signal blocking module forming a linear structure through a first connector (L1) and a second connector (L2). The target cell-specific recognition module can specifically recognize and bind to target cells; the phagocytic signal activation module can activate the phagocytic signal receptors on the surface of macrophages and enhance the phagocytic ability of macrophages; the antiphagocytic signal blocking module can block the antiphagocytic signal receptors on the surface of macrophages and relieve the inhibition of macrophages on target cells. Both the first and second linkers are linear peptides, which are formed by linking at least one flexible peptide segment and at least one rigid peptide segment.

[0020] The first connector is represented as -E1-E2-……E n -, E1, E2, ... E n All represent peptide segments, and n represents a positive integer. The second linker is represented as -F1-F2-……F n -, F1, F2, ... F n All represent peptide segments, and n represents a positive integer. The first and second linkers can be the same or different. In some embodiments, the linear peptide consists of 2 to 6 peptide segments. Too many peptide segments will lead to excessive spatial separation of functional modules, making it difficult to achieve simultaneous action on a single cell.

[0021] In some embodiments, the flexible peptide is selected from GS, GGGS (as shown in SEQ ID NO.1), GGGGS (as shown in SEQ ID NO.2), ASTKG (as shown in SEQ ID NO.3), GSTGSGSGKPGSGEGSTKG (as shown in SEQ ID NO.4), GSTGSGSGKSSEGKG (as shown in SEQ ID NO.5), GGSGRGAAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKAGGSG (as shown in SEQ ID NO.6), GGSGAKLAALKAKLAALKGGGGS (as shown in SEQ ID NO.7), and GGGGSELAALEAELAALEAGGS (as shown in SEQ ID NO.8).

[0022] In some embodiments, the rigid peptide is selected from EAAAK (as shown in SEQ ID NO. 9) and XPXPP, wherein X is any amino acid.

[0023] In some embodiments, the first linker is selected from any of the following amino acid sequences: EAAAKGGGGS, as shown in SEQ ID NO.10; EAAAKGGGGSGGGGS, as shown in SEQ ID NO.11; GGGGSEAAAKGGGGS, as shown in SEQ ID NO.12; GGGGSGGGGSEAAAK, as shown in SEQ ID NO.13; GGGGSEAAAKGGGGSEAAAK, as shown in SEQ ID NO.14; GGGGSEAAAKGGGGSEAAAKGGGGS, as shown in SEQ ID NO.15. That is, selected from any of the amino acid sequences shown in SEQ ID NO.10~15. The first linker is preferably the amino acid sequence shown in SEQ ID NO.12.

[0024] In some embodiments, the second linker is selected from any of the amino acid sequences shown in SEQ ID NO. 10 to 15. Preferably, it is the amino acid sequence shown in SEQ ID NO. 10.

[0025] In some embodiments, the target cell specific recognition module can specifically recognize the target cell sites as CD19, mesothelin, CD20, CD22, CD123, BCMA, CEA, Her2, MUC1, B7H3, GPC3, and EGFR; preferably B7H3.

[0026] In some embodiments, the target cell-specific recognition module is an antigen-binding domain capable of specifically recognizing target cell sites. Specifically, the antigen-binding domain is a single-chain antibody (scFv), a single-chain Fab / Fab', a single-domain antibody fragment, a single-chain immune factor, or a nanobody (VHH); preferably a single-chain antibody (scFv); more preferably a single-chain antibody (scFv) of B7H3. The amino acid sequence of the single-chain antibody (scFv) of B7H3 is shown in SEQ ID NO. 16.

[0027] In some embodiments, the phagocytic signal activation module is a molecule capable of activating macrophage phagocytic signals. Specifically, the molecule capable of activating macrophage phagocytic signals is selected from calreticulin (CRT), signal lymphocyte activation molecule family member 7 (SLAMF7), Fc receptor, oxidized low-density lipoprotein, etc. Calreticulin (CRT) is preferred.

[0028] In some embodiments, the molecules in which the anti-phagocytic signal blocking module inhibits macrophage phagocytosis include, but are not limited to, SIRPα, Siglec-10, PD-1, LILRB1, TIGIT / CD96, or combinations thereof. SIRPα and Siglec-10 are preferred, and SIRPα is more preferred.

[0029] In some embodiments, the anti-phagocytic signal blocking module is an antigen-binding domain that inhibits the phagocytic activity of macrophage molecules. Specifically, the antigen-binding domain that inhibits the phagocytic activity of macrophage molecules is a single-chain antibody (scFv), a single-chain Fab / Fab', a single-domain antibody fragment, or a nanobody (VHH). A single-chain antibody (scFv) is preferred. More preferably, a single-chain antibody (scFv) of SIRPα, whose amino acid sequence is shown in SEQ ID NO. 17.

[0030] In this invention, the target cell-specific recognition module, the pro-phagocytic signal activation module, and the anti-phagocytic signal blocking module can be connected in any order. For example, the order from the N-terminus to the C-terminus could be: target cell-specific recognition module - L1 - pro-phagocytic signal activation module - L2 - anti-phagocytic signal blocking module, target cell-specific recognition module - L1 - anti-phagocytic signal blocking module - L2 - pro-phagocytic signal activation module, pro-phagocytic signal activation module - L1 - target cell-specific recognition module - L2 - anti-phagocytic signal blocking module, pro-phagocytic signal activation module - L1 - anti-phagocytic signal blocking module - L2 - target cell-specific recognition module, anti-phagocytic signal blocking module - L1 - target cell-specific recognition module - L2 - pro-phagocytic signal activation module, anti-phagocytic signal blocking module - L1 - pro-phagocytic signal activation module - L2 - target cell-specific recognition module, etc. In some embodiments, the pro-phagocytic signal activation module and the anti-phagocytic signal blocking module are adjacent. When the pro-phagocytic signal activation module and the anti-phagocytic signal blocking module are adjacent, the two modules can act on the same cell by controlling the length and rigidity of the connector, thereby achieving synergistic regulation of pro-phagocytic and anti-phagocytic signals in a single cell.

[0031] In another embodiment of the present invention, a nucleic acid molecule is provided that encodes the above-mentioned three-specificity adaptor for regulating macrophage phagocytic function.

[0032] In some embodiments, the nucleic acid molecule is a plasmid, messenger RNA (mRNA), circular RNA (circRNA), or self-replicating RNA (saRNA); preferably, a plasmid, messenger RNA (mRNA), or circular RNA (circRNA); more preferably, mRNA. Specifically, the sequence of the mRNA is shown in SEQ ID NO. 18.

[0033] A third embodiment of the present invention provides a composition comprising the above-described trispecific adaptor for regulating macrophage phagocytosis or the above-described nucleic acid molecule and a carrier.

[0034] Specifically, the three-specificity adaptor or nucleic acid molecule regulating macrophage phagocytosis is encapsulated in a vector. After delivery of the composition into the cell, it is translated into a functional protein by the host ribosome.

[0035] In some embodiments, the carrier is selected from one or more of lipid nanoparticles (LNPs), inorganic nanoparticles, exosomes, adenoviruses, lentiviruses, retroviruses, oncolytic viruses, and protein-lipid carriers. Lipid nanoparticles (LNPs) are preferred.

[0036] A fourth embodiment of the present invention provides the application of the above-mentioned three-specificity connector, nucleic acid molecule or composition for regulating macrophage phagocytosis in the preparation of a drug.

[0037] In some embodiments, the drug has the effects of target cell clearance, tumor immunity, infectious diseases, tissue repair, regulation of inflammatory responses and / or regulation of autoimmune diseases.

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0039] Example 1: TrME, a three-specificity connector that regulates macrophage phagocytic function B7H3 The synthesis of.

[0040] Step ①: Recombinant protein design and expression The single-chain antibody (scFv) targeting mouse B7H3 and SIRPα was obtained from the publicly available patent databases US20230192861A1 / US20180105600A1. The truncated CRT sequence lacking the ER but retaining the KDEL motif was obtained from UniProt. The final trispecific adaptor TrME... B7H3 Form as Figure 1 As shown, from the N-terminus to the C-terminus, the sequence consists of an anti-SIRPα single-chain antibody (anti-SIRPα single-chain variable region fragment), a first linker, calreticulin, a second linker, and an anti-B7H3 single-chain antibody (anti-B7H3 single-chain variable region fragment). The protein sequence was reverse translated and codon optimized into a DNA sequence, which was then chemically synthesized and sequenced using GENEWIZ.

[0041] The sequence of the single-chain antibody (scFv) targeting B7H3 is as follows: EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKCLEWVSSISSGSGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLIPLDYWGQGTLVTVSSGGGGSGG GGSGGGGSQSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNAVSWYQQLPGTAPKLLIYYNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAYVFGCGTKLTVL, as in SEQ Shown as ID NO.16.

[0042] The sequence of the single-chain antibody (scFv) targeting SIRPα is as follows: DVQLVESGGGVVRPGESLRLSCAASGFTFSSYDMNWVRQAPGEGLEWVSLISGSGEIIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKENNRYRFFDDWGLGTLVTVSSGGGG SGGGGSGGGGSETVLTQSPGTLTLSPGERATLTCRASQSVYTYLAWYQEKPGQAPRLLIYGASSRATGIPDRFSGSGSGTEFTLTISSLQSEDFAVYYCQQYYDRPPLTFGGGTKVEIK, as in SEQ Shown as ID NO.17.

[0043] The sequence of the first connector is: GGGGSEAAAKGGGGS, as shown in SEQ ID NO.12.

[0044] The sequence of the second connector is: EAAAKGGGGS, as shown in SEQ ID NO.10.

[0045] Step 2: Establishment of stable cell lines and protein expression Recombinant virus (titer 1.5 × 10⁻⁶) was packaged in Expi293F cells using a third-generation lentivirus system. 8 ZsGreen cells were infected with TU / mL at MOI=5 and ZsGreen was obtained by flow cytometry sorting. + High-expression monoclonal lines were developed. Cells were expanded in a suspension culture system, and the supernatant was collected for subsequent purification.

[0046] Step 3: Protein purification The target protein was purified by nickel ion affinity chromatography: the cell supernatant was equilibrated with binding buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and loaded onto the plate. The target peak was collected by gradient elution (500 mM imidazole). The buffer was replaced with PBS using a desalting column. The sample was then concentrated to a final concentration of ≥5 mg / mL by ultrafiltration centrifugation.

[0047] Example 2: Western blot verification of the specific connector TrME B7H3 Successfully built.

[0048] Take purified TrME B7H3 Proteins were dissolved in 4×Bolt™ LDS buffer and separated by electrophoresis on a 4-20% Bis-Tris gradient gel (200 V, 35 min). After transfer to a PVDF membrane, the following steps were performed: blocking at room temperature for 1 h, overnight incubation with primary antibody (anti-CRT: 1:1000; anti-His tag: 1:2000) at 4°C, and incubation with HRP-conjugated secondary antibody (1:5000) at room temperature for 1 h. Immunoblot signal was then generated using an enhanced chemiluminescence reagent. Figure 2 The results showed a distinct band at approximately 98 kD, proving that TrME B7H3 Successfully expressed.

[0049] Example 3 Verification of TrME B7H3 Specific binding activity with B7H3 1×10 5 GL261 and GL261 B7H3- Cells were seeded in 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow them to adhere. Cells were then treated with PBS or TrME. B7H3 After 1 hour of treatment, cells were collected into flow cytometry tubes, and His-Tag antibody diluted 1:800 (at room temperature for 30 minutes) and Alexa Fluor® 488 conjugated secondary antibody of the same concentration (at room temperature for 30 minutes) were added sequentially. Cells were washed three times with PBS between each step (centrifuged at 300×g for 5 minutes). Finally, the cells were resuspended in PBS and analyzed by flow cytometry. Figure 3 The results show that TrME B7H3 It binds efficiently to GL261 cells, but not to GL261. B7H3- Cell binding indicates TrME B7H3 By identification B7H3 It specifically binds to target cells.

[0050] Example 4: Verification of TrME B7H3 Effects on macrophage phagocytosis.

[0051] GL261 and GL261 B7H3- Cells were labeled with CFSE and incubated at 37°C for 15 minutes. 1×10⁶ cells were then added. 5 GL261 and GL261 B7H3- Cells were seeded in 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow them to adhere. The next day, 3 × 10⁶ cells were added to each well. 5 BMDMs, using PBS or TrME B7H3 The treatment lasted 6 hours. After terminating the culture, cells from the co-culture system were collected, washed with PBS, stained with the target antibody for surface markers, and CFSE was detected using flow cytometry. + The proportion of BMDMs. Figure 4 The results show TrME B7H3 CFSE produced after phagocytosis of tumor cells in the treatment group + BMDMs significantly improved, indicating that TrME B7H3 It effectively improves the phagocytic efficiency of macrophages on target cells.

[0052] Example 5 Macrophage phenotypic polarization experiment.

[0053] 1×10 5 One GL261 glioma cell line was seeded into each well of a 24-well plate and incubated overnight at 37°C in a 5% CO2 incubator to allow for adhesion. The following day, 3 × 10⁶ cells were added to each well. 5 BMDMs, using either PBS or TrME B7H3 The treatment lasted 6 hours. After terminating the culture, cells from the co-culture system were collected, washed with PBS, and stained with the target antibody for surface markers. Flow cytometry was used for detection, and CD11b was calculated. + F4 / 80 + CD86 + M1 macrophages and CD11b + F4 / 80 + CD206 + The polarization ratio was obtained from the proportion of M2 macrophages. Figure 5 The results show TrME B7H3 The significantly increased M1 / M2 ratio in the treatment group indicates that TrME B7H3 It effectively mediates the polarization of macrophages into anti-tumor M1 macrophages.

[0054] Example 6 Intratumoral injection of TrME B7H3 Protein assessment of in vivo efficacy.

[0055] GL261-Fluc cells (1×10⁻⁶) 5Each mouse cell line (dissolved in 7 μL of 70% PBS and 30% Matrigel) was stereotactically injected into the brain of C57BL / 6 mice (1.8 mm lateral, 0.6 mm anterior; 2 mm depth) to establish an idiogenic orthotopic glioblastoma model. Mice began receiving intratumoral injections of TrME on day 7 post-tumor inoculation. B7H3 Protein (2.5 mg / kg) or an equal volume of physiological saline was injected every 5 days for a total of 3 injections. On day 22 post-inoculation, 6 mice from each group were used for survival monitoring. Figure 6 The results show TrME B7H3 The treatment group significantly prolonged the survival of mice with glioblastoma in situ, demonstrating superior tumor treatment efficacy.

[0056] Example 7 TrME B7H3 Synthesis of -mLNP.

[0057] Step ①: Encode TrME B7H3 mRNA synthesis The codon-optimized TrME coding sequence was cloned into an in vitro transcription template plasmid for mRNA, which included a T7 promoter, an 80 poly(A) tail, and optimized 3' and 5' UTRs. The modified mRNA was generated by completely replacing the UTP with N1-methylpseudouracil-5'-triphosphate during in vitro transcription. The modified mRNA was then enzymatically capped to acquire a 5'Cap1 structure. All mRNAs were stored at -20°C until use.

[0058] Step 2: LNPs encapsulation encoding TrME B7H3 mRNA.

[0059] Lipid nanoparticles were prepared using a microfluidic device. Ionizable cationic lipids, cholesterol, PEG-modified lipids, and cofactor lipids were dissolved in anhydrous ethanol to prepare an organic phase solution. TrME was also used. B7H3 mRNA molecules were dissolved in 10 mM citrate buffer (pH 4.0) to form an aqueous system, with the mass ratio of lipid mixture to mRNA controlled at 10:1. The two phases were then precisely mixed using a microfluidic mixing system at a flow rate of 3:1 (aqueous phase:organic phase) to achieve LNP self-assembly. The resulting lipid nanoparticles were then dialyzed against phosphate-buffered saline (PBS, 1×) for 12 hours to remove residual organic solvent, yielding mRNA-encapsulated lipid nanoparticles (TrME). B7H3 -mLNP), TrME was evaluated by transmission electron microscopy. B7H3 -mLNP mass. Figure 7 The results show TrME B7H3-mLNP particles are uniform in size and well dispersed, with an average particle size of about 127nm.

[0060] Encoding TrME B7H3 The mRNA sequence is as follows:

[0061] Example 8: Intratumoral injection of TrME B7H3 -mLNP assessment of in vivo efficacy GL261-Fluc cells (1 × 10⁶ cells per mouse) were used. 5 TrME cells (dissolved in 7 μL of 70% PBS and 30% Matrigel) were stereotactically injected into the brains of C57BL / 6 mice to establish an idiogenic orthotopic glioblastoma model. Mice began receiving intratumoral injections of TrME on day 7 post-tumor inoculation. B7H3 -mLNP (1.5 mg / kg) or an equivalent volume of physiological saline was administered every 5 days for a total of 3 injections. On day 22 post-inoculation, 6 mice from each group were used for survival monitoring. Figure 8 The results show TrME B7H3 -mLNP treatment group (also known as TrME) B7H3 The LNP group significantly prolonged the survival of mice with glioblastoma in situ, indicating that TrME was generated in vivo. B7H3 The strategy has shown superior efficacy in cancer treatment.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A three-specificity connector for regulating macrophage phagocytic function, characterized in that, From the N-terminus to the C-terminus, the single-chain antibody against SIRPα, the first linker, calreticulin, the second linker, and the single-chain antibody against B7H3 are linked sequentially. The sequence of the first linker is: GGGGSEAAAKGGGGS, and the sequence of the second linker is: EAAAKGGGGS. The sequence of the anti-SIRPα single-chain antibody is shown in SEQ ID NO.17; The sequence of the single-chain antibody against B7H3 is shown in SEQ ID NO.16; Calreticulin is a CRT sequence that retains the KDEL motif but lacks the ER.

2. A composition, characterized in that, It includes the three-specificity connector and vector for regulating macrophage phagocytic function as described in claim 1.

3. The composition according to claim 2, characterized in that, The carrier is selected from one or more of the following: lipid nanoparticles, inorganic nanoparticles, exosomes, adenoviruses, lentiviruses, retroviruses, oncolytic viruses, and protein-lipid carriers.

4. The use of the trispecific connector for regulating macrophage phagocytosis as described in claim 1, or the composition as described in claim 2 or 3, in the preparation of a medicament for treating glioblastoma.

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