ZP3-targeted chimeric antigen receptor macrophage and application thereof

Abstract

The invention discloses a ZP3-targeting chimeric antigen receptor macrophage and application thereof, and relates to the technical field of biological medicine, the macrophage expresses a chimeric antigen receptor, and the chimeric antigen receptor comprises a signal peptide, an anti-ZP3 single-chain antibody, a hinge region, a transmembrane domain and an intracellular signal region; the anti-ZP3 single-chain antibody comprises an antibody light chain variable region, a GS linker and an antibody heavy chain variable region, the amino acid sequence of the antibody light chain variable region is as shown in SEQ ID NO: 1, and the amino acid variable region sequence of the antibody heavy chain variable region is as shown in SEQ ID NO: 2. The chimeric antigen receptor macrophage constructed based on the chimeric antigen receptor shows remarkable anti-tumor activity in vitro and in vivo, including enhanced tumor cell phagocytic ability and killing efficiency, solves the problems of insufficient target specificity, difficult cell infiltration and tumor microenvironment inhibition in liver cancer immunotherapy, and has a broad application prospect. And a treatment strategy with high targeting property, strong wettability and good safety is provided for patients with hepatocellular carcinoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a chimeric antigen receptor macrophage targeting ZP3 and its applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide, with extremely poor prognosis for patients in advanced stages. Despite advances in systemic therapies, represented by immune checkpoint inhibitors and targeted drugs, overall efficacy remains limited, necessitating the development of novel treatments. Adoptive cellular immunotherapy, particularly chimeric antigen receptor T-cell (CAR-T) therapy, has shown significant efficacy in hematologic malignancies, but its application in solid tumors faces substantial challenges. The dense microenvironment of solid tumors severely hinders T-cell infiltration, and their immunosuppressive properties easily lead to T-cell depletion. Furthermore, the lack of ideal target antigens that are specifically highly expressed on tumor cells but strictly restricted in key normal tissues also limits the safe application of this technology.

[0003] In recent years, macrophages have received widespread attention as carriers for cell therapy. Macrophages possess the natural ability to penetrate tissues, engulf large target cells (such as whole tumor cells), and initiate adaptive immune responses through antigen presentation. Chimeric antigen receptor macrophage technology, by endowing macrophages with specific targeting capabilities, holds promise for overcoming many bottlenecks in CAR-T therapy for solid tumors. This technology can not only directly kill tumors but may also reprogram pro-tumor M2 macrophages into anti-tumor M1 macrophages, fundamentally reshaping the tumor immune microenvironment.

[0004] However, the success of chimeric antigen receptor macrophage (CAR-M) therapy heavily relies on the discovery of highly specific and ideal targets. Current research largely focuses on known targets such as GPC3 and MSLN, but the expression of these targets in some normal tissues poses a potential risk of off-target toxicity. Therefore, finding novel targets that are specifically highly expressed in liver cancer is crucial.

[0005] Zona pellucida glycoprotein 3 (ZP3) is a promising potential therapeutic target. Traditionally, ZP3 is primarily expressed in the zona pellucida of oocytes. However, recent studies have shown that ZP3 is abnormally activated and expressed in various solid tumors, including hepatocellular carcinoma, and is closely associated with malignant tumor progression and poor prognosis. Conversely, in key normal tissues such as the liver, kidney, heart, and lungs in adults, ZP3 expression levels are extremely low or undetectable. This unique expression profile makes it a highly specific and potentially promising therapeutic target. However, to date, no studies have applied ZP3 to CAR-M therapy, leaving this area unexplored. Summary of the Invention

[0006] The technical problem to be solved by this invention is the lack of target specificity, difficulty in cell infiltration, and inhibition of the tumor microenvironment in existing technologies for liver cancer immunotherapy. The purpose is to provide a chimeric antigen receptor macrophage targeting ZP3 and its application, which solves the problems of insufficient target specificity, difficulty in cell infiltration, and inhibition of the tumor microenvironment in existing technologies for liver cancer immunotherapy, and provides a new treatment strategy for hepatocellular carcinoma patients that has high targeting, strong infiltration, and good safety.

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, this application provides a chimeric antigen receptor macrophage that targets ZP3, wherein the macrophage expresses a chimeric antigen receptor, and the chimeric antigen receptor includes a signal peptide, an anti-ZP3 single-chain antibody, a hinge region, a transmembrane domain, and an intracellular signaling region.

[0009] The anti-ZP3 single-chain antibody includes a variable region of the antibody light chain, a GS linker, and a variable region of the antibody heavy chain. The amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO:1, and the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO:2.

[0010] In one specific embodiment, the signal peptide is CD8α, whose amino acid sequence is shown in SEQ ID NO:3;

[0011] And / or the hinge region is CD8α, whose amino acid sequence is shown in SEQ ID NO:4;

[0012] And / or the transmembrane domain is CD8α, whose amino acid sequence is shown in SEQ ID NO:5.

[0013] In one specific embodiment, the intracellular signaling region is CD3ζ, whose amino acid sequence is shown in SEQ ID NO:6.

[0014] In one specific embodiment, the variable region of the antibody light chain includes CDR1, CDR2 and CDR3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO:7; the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:8; and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:9.

[0015] In one specific embodiment, the variable region of the antibody heavy chain includes CDR1, CDR2, and CDR3; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO:10; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:11; and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:12.

[0016] Secondly, this application provides an expression vector that encodes nucleotides of chimeric antigen receptor macrophages as described above.

[0017] In one specific embodiment, the expression vector is a lentiviral expression vector or an adenovirus expression vector.

[0018] Thirdly, this application provides a nucleic acid molecule capable of expressing the chimeric antigen receptor macrophage as described above.

[0019] Fourthly, this application provides a pharmaceutical composition comprising the chimeric antigen receptor macrophages described above, the pharmaceutical composition being used to treat tumors.

[0020] In one specific embodiment, the tumor includes any one of liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, lung cancer, and kidney cancer.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] (1) This invention utilizes the high tumor specificity of ZP3 and combines the inherent advantages of macrophages in solid tumors. Based on this chimeric antigen receptor, the chimeric antigen receptor macrophages constructed exhibit significant anti-tumor activity in vitro and in vivo, including enhanced tumor cell phagocytosis and killing efficacy. This solves the problems of insufficient target specificity, difficulty in cell infiltration and inhibition of tumor microenvironment in the existing technology for liver cancer immunotherapy, and provides a new treatment strategy for hepatocellular carcinoma patients with high targeting, strong infiltration and good safety.

[0023] (2) In the manufacture of biopharmaceuticals, the chimeric antigen receptor macrophages of the present invention can be used not only to prepare drugs for treating liver cancer, but also to prepare drugs for gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, kidney cancer, etc. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0025] Figure 1 Example 3: The transfection effect of CAR was observed using a fluorescence microscope;

[0026] Figure 2 Example 3 describes the detection of CAR transfection efficacy by flow cytometry;

[0027] Figure 3 Example 4: Observation of CAR-M phagocytosis on Huh7 target cells using fluorescence microscopy;

[0028] Figure 4 Example 4: Flow cytometry was used to detect the phagocytic efficiency of CAR-M on Huh7 target cells at different time points;

[0029] Figure 5 Example 4: Flow cytometry was used to detect the killing efficiency of CAR-Ms with different effector-to-target ratios on Huh7 target cells.

[0030] Figure 6 Example 4 uses Luciferase to detect the killing efficiency of CAR-Ms with different effector-to-target ratios on Huh7-luc target cells;

[0031] Figure 7 Example 4 illustrates the therapeutic effect of CAR-M in an in vitro xenograft tumor model; Figure 8 This is a statistical chart of tumor volume in the CON-M and CAR-M groups in Example 4. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0033] Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions may be omitted. This is to avoid making the following description unnecessarily lengthy and to facilitate understanding by those skilled in the art.

[0034] The "scope" disclosed in this invention is defined in the form of a lower limit and an upper limit. A given scope is defined by selecting a lower limit and an upper limit, which define the boundaries of the specific scope. The scope defined in this way can include or exclude end values, and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a scope.

[0035] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0036] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0037] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other substances not listed may also be included, or that only the listed substances may be included.

[0038] Throughout this specification, references to "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases "an embodiment," "an example," "an example," or "an example" appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described herein, as well as the features of those different embodiments or examples.

[0039] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0040] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0041] It should be noted that, unless otherwise specified, the experimental methods used in the embodiments are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0042] Example 1

[0043] Preparation of monoclonal antibodies targeting the ZP3 chimeric antigen receptor.

[0044] 1. Preparation of anti-ZP3 monoclonal antibody

[0045] (1) Synthesis of ZP3 recombinant antigen: The extracellular domain of human ZP3 protein (UniProt P21754) was optimized by codons and cloned into a vector plasmid with an N-terminal 6×His tag. Subsequently, it was transfected into HEK293-F cells for protein expression. The expressed protein was purified by Ni-NTA column affinity chromatography to obtain high-purity recombinant His-ZP3 protein.

[0046] (2) ELISA determination of the titer of ZP3 recombinant antigen in mice: Three 6-week-old female BALB / c mice were selected for antigen, two of which were used for immunization and one as a control. 50 µg of the ZP3 recombinant antigen prepared in (1) was mixed with an equal volume of adjuvant and injected subcutaneously at multiple sites to immunize the mice. A total of four immunizations were performed, with an interval of two weeks between each immunization. Serum was collected one week after the last immunization, and the antibody titer was determined by indirect ELISA. An ELISA plate was coated with 1 µg / mL antigen, blocked, and then serially diluted (1:5,000 to 1:200,000) of the test serum was added as the primary antibody. HRP-labeled secondary antibody was used for detection, and finally, the color development was stopped by TMB and the reading was taken at OD450 nm. The results showed that the ZP3 recombinant antigen had a strong immunogenicity.

[0047] (3) CD138 plasma cell magnetic bead sorting: Mice with the best immunogenic titer were selected, and spleens were aseptically obtained and ground to prepare cell suspensions. Subsequently, CD138 positive plasma cells were sorted from the cell suspensions using a Stemcell CD138 magnetic bead sorting kit. The sorted cells were resuspended and counted, and then added together with AF488-labeled goat anti-mouse IgG secondary antibody to antigen-coated U40 nanoplatelets (Sartorius) and incubated overnight at 37°C. Finally, the cells before and after sorting were stained with CD138 flow cytometry antibody, and the sorting purity was analyzed by flow cytometry. The results showed that the positive rate of sorted CD138 plasma cells reached over 90%.

[0048] (4) Single plasma cell selection: Using the Sartorius CellCelector Flex system, target cells with positive fluorescence signals were identified by high-resolution fluorescence microscopy. In the software interface, the morphology, size, and fluorescence intensity of each identified cell were checked individually, and single plasma cells with intact morphology and strong fluorescence were selected for sorting. After confirmation, the system controlled a sterile capillary tube to aspirate the target cells non-destructively using a "one cell per well" principle through gentle negative pressure, and precisely transferred them to a 96-well PCR plate pre-filled with 10 µL of lysis buffer (Thermo Fisher Scientific SuperScript™ IV reagent). 10 µL lysis buffer: 1 µL 10X lysis buffer, 1 µL capture oligonucleotide, 0.4 µL RNase inhibitor, and 7.6 µL enzyme-free water.

[0049] (5) Reverse transcription system: containing 10 µL of lysis mixture from (4), 4 µL of nuclease-free water, 5 µL of 4×cDNA synthesis premix and 1 µL of template switching oligonucleotide, and reacting at 50°C for 30 min and 85°C for 5 min to synthesize 20 µL of cDNA. Then, 80 µL of pre-amplification mixture (containing 29 µL of nuclease-free water, 50 µL of 2× pre-amplification premix and 1 µL of pre-amplification primer) was added to the 20 µL of cDNA. After denaturation at 98°C for 30 sec, the reaction was cycled at 98°C for 10 sec, 65°C for 10 sec and 67°C for 3 min, and finally extended at 67°C for 5 min to complete the pre-amplification of cDNA. The product can be stored at -80°C. Finally, using the pre-amplified products as templates, nested PCR was performed using a mixture of mouse IgG VH and Vκ primers: the heavy chain program was 95°C for 3 min, 35 cycles (95°C 15 s, 56°C 15 s, 72°C 55 s), annealing at 60°C, and extension at 72°C for 5 min; the light chain program was 95°C for 3 min, 35 cycles (94°C 15 s, 50°C 15 s, 72°C 55 s), annealing at 45°C, and extension at 72°C for 5 min. After verification by agarose gel electrophoresis, the PCR products were purified by gel excision, TOPO cloning, transformation, plasmid extraction, and Sanger sequencing to obtain the light and heavy chain sequences of the monoclonal antibody.

[0050] 2. The light chain variable region (VL) and heavy chain variable region (VH) of the anti-ZP3 monoclonal antibody.

[0051] (1) The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO:1:

[0052] DIVLTQSPASLAVSLGQRATISCRASESVDNYGINFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPRTFGGGTKLEIK.

[0053] The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO:7: RASESVDNYGINFMN.

[0054] The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:8: AASNQGS.

[0055] The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:9: QQSKEVPRT.

[0056] (2) The amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:2:

[0057] EVQLQESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSQSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRAYGNYWGQGTTLTVSS.

[0058] The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO:10: TYAMN.

[0059] The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:11: RIRSQSNNYATYYADSVKD.

[0060] The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:12: AYGNY.

[0061] (3) The amino acid sequence of the single-chain variable fragment (scFv) targeting the ZP3 chimeric antigen receptor is shown in SEQ ID NO:13:

[0062] EVQLQESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSQSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRAYGNYWGQGTTLTVSSGGGGS GGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVDNYGINFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPRTFGGGTKLEIK.

[0063] The sequence GGGGSGGGGSGGGGS is the Linker.

[0064] (4) The nucleotide sequence of the single-stranded variable fragment (scFv) targeting the ZP3 chimeric antigen receptor is shown in SEQ ID NO:14:

[0065] GAAGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGCCGAAAGGCAGCCTGAAACTGAGCTGCGCGGCGAGCGGCTTTACCTTTAACACCTATGCGATGAACTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTCGCAGCCAGAGCAACAACTATGCGACCTATTATGCGGATAGCGTGAAAGATCGCTTTACCATTAGCCGCGATGATAGCCAGAGCATGCTGTATCTGCAGATGAACAACCTGAAAACCGAAGATACCGCGATGTATTATTGCGTGCGCGCGTATGGCAACTATTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGATATTGTGCTGACCCAGAGCCCGGCGAGCCTGGCGGTGAGCCTGGGCCAGCGCGCGACCATTAGCTGCCGCGCGAGCGAAAGCGTGGATAACTATGGCATTAACTTTATGAACTGGTTTCAGCAGAAACCGGGCCAGCCGCCGAAACTGCTGATTTATGCGGCGAGCAACCAGGGCAGCGGCGTGCCGGCGCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTAGCCTGAACATTCATCCGATGGAAGAAGATGATACCGCGATGTATTTTTGCCAGCAGAGCAAAGAAGTGCCGCGCACCTTTGGCGGCGGCACCAAACTGGAAATTAAA。

[0066] Example 2

[0067] Construction of a chimeric antigen receptor (CAR) targeting ZP3.

[0068] The structure of a CAR molecule is assembled from multiple functional modules. CAR is a modularly synthesized membrane protein, and its standard structure, from the N-terminus to the C-terminus, includes: a signal peptide, an antigen recognition domain (usually scFv), a hinge region, a transmembrane region, and an intracellular signaling domain. The signal peptide, located at the leader position, guides the nascent polypeptide chain into the endoplasmic reticulum during translation, ultimately ensuring the accurate delivery of the CAR molecule and its display on the cell membrane surface. The following scFv fragment acts as the molecule's "probe," recognizing target antigens (such as ZP3) on the surface of tumor cells with high affinity and specificity. The hinge region, connecting the scFv to the transmembrane region, provides the necessary spatial conformational flexibility for the antigen-binding domain, allowing it to more flexibly approach and bind to the target site. The transmembrane region, typically composed of hydrophobic amino acids, acts as a "membrane anchor," stably embedding the entire CAR structure within the cell membrane lipid bilayer. The characteristics of this region may also affect the receptor's aggregation state on the membrane and the efficiency of signal transduction. The cytoplasmic signal transduction domain is the "powerhouse" of CAR function. The intracellular portion of the human CD3 ζ chain is commonly used, containing the tyrosine activation motif of the immune receptor. When scFv binds to the antigen, this region is phosphorylated, which initiates a strong downstream signaling cascade that ultimately drives the activation of immune cells (such as macrophages) and enables them to perform killing or clearance functions.

[0069] (1) The connection sequence of the CAR molecular structure is CD8α signal peptide, anti ZP3 scFv, CD8α hinge domain, CD8α transmembrane domain and CD3ζ intracellular activation domain.

[0070] (2) The CD8α signal peptide, the amino acid sequence of which is shown in SEQ ID NO:3:

[0071] MALPVTALLLPLALLLHAARP.

[0072] (3) The CD8α hinge domain has the amino acid sequence shown in SEQ ID NO:4:

[0073] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0074] (4) The amino acid sequence of the CD8α transmembrane domain is shown in SEQ ID NO:5:

[0075] IYIWAPLAGTCGVLLLSLVITLYC.

[0076] (5) The intracellular activation domain of CD3ζ, the amino acid sequence of which is shown in SEQ ID NO:6:

[0077] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0078] (6) The amino acid sequence of the whole CAR is shown in SEQ ID NO.15:

[0079] MALPVTALLLPLALLLHAARPEVQLQESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSQSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRAYGNYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVDNYGINFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPRTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0080] (7) The nucleotide sequence of the whole CAR is shown in SEQ ID NO.16:

[0081]

[0082] Example 3

[0083] Construction of ZP3CAR-M target.

[0084] Based on the CAR gene sequence obtained in Example 2, a lentiviral expression vector was constructed by molecular cloning and transfected and validated in human monocytic leukemia cells THP-1. The specific steps are as follows:

[0085] (1) Vector construction: The synthesized CAR gene sequence was directionally cloned into the lentiviral vector VP098 (General Biosciences) through double enzyme digestion and ligation. In this expression cassette, the CAR molecule and enhanced green fluorescent protein (eGFP) are tandemly linked through the P2A peptide sequence to achieve co-expression of the two.

[0086] (2) Lentiviral packaging: The successfully constructed recombinant expression plasmid and the helper packaging plasmid were co-transfected into HEK293T cells, and lentiviral particles were packaged using a three-plasmid system. The cell supernatant was collected and concentrated by ultracentrifugation to obtain a high-titer stock solution of lentivirus carrying the CAR gene.

[0087] (3) Cell transfection: Take THP-1 cells in good growth condition and add them to the above lentivirus solution at the optimal multiplicity of infection (MOI) of 50, along with an appropriate amount of infection enhancer. 24 hours after transfection, centrifuge at 100×g to remove the viral supernatant, and replace it with RPMI-1640 complete medium containing 10% fetal bovine serum for continued culture.

[0088] (4) Transfection efficiency verification: eGFP fluorescence expression was observed under a fluorescence microscope to preliminarily assess the transfection status (results are shown in the figure). Figure 1 Subsequently, cells were collected, and the proportion of eGFP-positive cells was quantitatively detected using flow cytometry to determine the transfection efficiency of the CAR molecule. According to the flow cytometry results, the eGFP positivity rate was consistently above 90%, which can be used for subsequent experiments (results are shown in Figure 1). Figure 2 ).

[0089] Example 4

[0090] ZP3-targeted CAR-M cells engulf and kill liver cancer cells.

[0091] To evaluate the function of the constructed CAR-M against liver cancer, it was first induced to differentiate into M0, and its phagocytic and killing abilities against target tumor cells were examined.

[0092] 1. Induction of differentiation of CAR-M0 macrophages

[0093] THP-1 cells stably expressing CAR were stimulated with 100 ng / mL phorbol ester (PMA) for 24 h to induce differentiation into adherent M0 macrophages. The culture medium was then replaced with fresh complete medium and cultured for another 24 h to obtain CAR-M0 cells suitable for functional experiments. The control group (CON-M) was obtained by inducing THP-1 cells with an untransfected empty vector virus using the same procedure.

[0094] 2. In vitro phagocytic function test

[0095] (1) Transfect ZP3-overexpressing liver cancer cells Huh7 with mCherry red fluorescent protein to construct a stable cell line.

[0096] (2) Induced CAR-M or CON-M cells were co-cultured with Huh7 liver cancer cells stably expressing mCherry at a 1:1 cell ratio. Cells were collected after 1, 2, 4, and 6 hours of co-culture, and the proportion of cells showing both eGFP (M0) and mCherry (Huh7) signals was detected by flow cytometry. This proportion directly reflects the phagocytosis of tumor cells by macrophages. Results are as follows: Figure 3 As shown, the CAR-M targeting ZP3 exhibited significantly higher phagocytosis rates than the control group at all time points.

[0097] (3) The co-culture system was observed using a fluorescence microscope to evaluate the phagocytosis and killing process of CAR-M on tumor cells. After co-culturing CAR-M or CON-M with Huh7-mCherry cells at a 1:1 ratio for 24 h, cell morphology and fluorescence distribution were observed under a fluorescence microscope. The results are as follows: Figure 4 As shown, a large number of mCherry⁺ tumor cells were observed attached to the surface of CAR-M cells, and red fluorescent signals were visible inside the cells, indicating that the tumor cells had been endocytosed. In contrast, the tumor cells in the CON-M group mostly maintained their intact morphology and had less contact with macrophages.

[0098] 3. In vitro tumor cell killing detection

[0099] (1) After co-culturing CAR-M or CON-M cells with Huh7-mCherry cells at a ratio of 5:1 or 10:1 for 24 h, cells were collected, and the proportion of cells with mCherry (Huh7) signal was detected by flow cytometry. This proportion directly reflects the phagocytic and killing effect of macrophages on tumor cells. The results are as follows: Figure 5 As shown, in the case of 5:1 or 10:1, the number of remaining mCherry (Huh7) in the CAR-M group was less than that in the control group CON-M, and the kill rate of the CAR-M group was significantly higher than that of the control group.

[0100] (2) To evaluate the killing efficacy of CAR-M against targeted tumor cells, the ZP3-overexpressing hepatocellular carcinoma cell line Huh7 was transfected with Luciferase to construct a stable cell line (Huh7-luc). Differentiated CAR-M cells or control CON-M cells were used as effector cells and seeded with Huh7-luc target cells at effector-to-target ratios (E:T) of 1:1, 5:1, and 10:1, respectively, in 96-well plates, with 5 replicates per group. The co-culture system was maintained at 37°C and 5% CO2 for 24 h. The fluorescence intensity in the 96-well plates was detected using a fluorescence imaging system, and the results are as follows: Figure 6 As shown, the fluorescence intensity of the CAR-M group was lower than that of the control group, indicating that there were fewer remaining tumor cells. The specific killing rate of the CAR-M group was significantly higher than that of the control group, and it showed a dose-dependent effect (i.e., the higher the proportion of effector cells, the stronger the killing ability).

[0101] 4. In vivo antitumor effect of ZP3CAR-M in a liver cancer xenograft model

[0102] Five-week-old female BALB / c nude mice were selected, with each mouse receiving 1×10 6 A dose of 10 Huh7 cells was administered to induce subcutaneous tumor formation. Once the tumor was palpable (approximately 50 mm³), the tumor-bearing mice were randomly divided into two groups (n=3 / group). The CON-M control group received a tail vein injection of 1×10⁻⁶ cells. 7 (10 control macrophages); CAR-M treatment group (tail vein injection of 1×10) 7 (ZP3 CAR-M cells). Treatment was administered weekly for a total of 3 weeks. Tumor length and short diameter were measured every 3 days using calipers, and tumor volume was calculated using the formula V = (length × short diameter²) / 2. Mouse body weight was monitored simultaneously. (Tumor anatomical photographs are shown.) Figure 7 ) and tumor growth curve ( Figure 8 The results showed that the CAR-M treatment group exhibited a significant tumor growth inhibition effect, with both the final tumor volume and weight being significantly smaller than those of the control group.

[0103] Conclusion: The above results indicate that ZP3 CAR-M can not only significantly enhance the recognition and phagocytosis efficiency of macrophages on targeted tumor cells, but also promote their killing and clearance of endocytic tumor cells, thus exhibiting stronger anti-tumor activity.

[0104] The above specific embodiments are intended to illustrate the purpose, technical solutions, and beneficial effects of the present invention in detail, but should not be construed as limiting the scope of protection of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications, equivalent substitutions, or improvements can be made to the described technical solutions; all such modifications, substitutions, or improvements, as long as they do not depart from the essential scope of the technical solutions of the embodiments of the present invention, should be included within the scope of protection defined by the claims and specification of the present invention.

Claims

1. A chimeric antigen receptor macrophage targeting ZP3, characterized in that, The macrophages express a chimeric antigen receptor, which includes a signal peptide, an anti-ZP3 single-chain antibody, a hinge region, a transmembrane domain, and an intracellular signaling region. The anti-ZP3 single-chain antibody includes a variable region of the antibody light chain, a GS linker, and a variable region of the antibody heavy chain. The amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO:1, and the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO:

2.

2. The chimeric antigen receptor macrophage targeting ZP3 according to claim 1, characterized in that, The signal peptide is CD8α, and its amino acid sequence is shown in SEQ ID NO:3; And / or the hinge region is CD8α, whose amino acid sequence is shown in SEQ ID NO:4; And / or the transmembrane domain is CD8α, whose amino acid sequence is shown in SEQ ID NO:

5.

3. A chimeric antigen receptor macrophage targeting ZP3 according to claim 1, characterized in that, The intracellular signaling region is CD3ζ, and its amino acid sequence is shown in SEQ ID NO:

6.

4. A chimeric antigen receptor macrophage targeting ZP3 according to claim 1, characterized in that, The variable region of the antibody light chain includes CDR1, CDR2 and CDR3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO:7; the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:8; and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

9.

5. A chimeric antigen receptor macrophage targeting ZP3 according to claim 1, characterized in that, The variable region of the antibody heavy chain includes CDR1, CDR2, and CDR3; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO:10; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:11; and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:

12.

6. An expression carrier, characterized in that, The expression vector encodes nucleotides of the chimeric antigen receptor macrophage as described in any one of claims 1 to 5.

7. An expression vector according to claim 6, characterized in that, The expression vector is a lentiviral expression vector or an adenovirus expression vector.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule is capable of expressing the chimeric antigen receptor macrophage as described in any one of claims 1 to 5.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes chimeric antigen receptor macrophages as described in any one of claims 1 to 5, and is used to treat tumors.

10. A pharmaceutical composition according to claim 9, characterized in that, The tumor includes any one of liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, lung cancer, and kidney cancer.

Images