Application of anti-PTPRZ1 chimeric antigen receptor macrophages in the treatment of lung cancer

Abstract

The present invention relates to the application of anti-PTPRZ1 chimeric antigen receptor macrophages in the treatment of lung cancer, belonging to the technical field of tumor targeted therapy. The present invention constructs a novel chimeric antigen receptor (CAR), which comprises a coding sequence of a single-chain variable region fragment specifically recognizing PTPRZ1, a CD8TM transmembrane region, and a coding sequence of a CD3zeta intracellular signaling domain. This ingenious design endows the chimeric antigen receptor (CAR) with high specificity, enabling it to accurately recognize and firmly bind to the surface of tumor cells expressing the PTPRZ1 protein. The present invention successfully transfects the chimeric antigen receptor targeting PTPRZ1 into human macrophages to form CAR-M. Through flow cytometry verification, compared with untransfected macrophages (Mφ) or macrophages transfected with a lack of a specific target binding domain (CAR-M-Δ), CAR-M significantly improves the efficiency of phagocytosing PTPRZ1-positive tumor cells. Therefore, the CAR-M constructed by the present invention has the potential to be used as a therapeutic means targeting tumors expressing PTPRZ1.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor targeted therapy, and relates to the application of anti-PTPRZ1 chimeric antigen receptor macrophages in the treatment of lung cancer and lung cancer brain metastasis. Background Art

[0002] Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the main causes of cancer-related deaths worldwide. Early metastasis of tumors and resistance to radiotherapy and chemotherapy are key factors leading to poor prognosis. With the in-depth understanding of the tumor microenvironment, targeted therapy has gradually become a research hotspot, which aims to inhibit tumor growth and metastasis by specifically acting on molecules in tumor cells or tumor stroma.

[0003] PTPRZ1, namely protein tyrosine phosphatase receptor Z1, is a transmembrane receptor protein tyrosine phosphatase that is abnormally expressed in various tumors. In lung cancer, the high expression of PTPRZ1 is closely related to tumor invasiveness, metastatic ability, and poor prognosis. Therefore, PTPRZ1 is considered a potential tumor treatment target.

[0004] Chimeric Antigen Receptor (CAR) technology is an advanced bioengineering technology that endows immune cells with the ability to highly specifically recognize and kill tumor cells by binding single-chain variable region antibody fragments that can specifically recognize tumor-associated antigens to T cells or macrophages. This single-chain variable region antibody fragment is formed by connecting the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of a tumor antigen-reactive antibody through a flexible linker. This design enables CAR-T cells or CAR-M cells to precisely locate and attack tumor cells like precision-guided missiles, providing a new strategy for cancer treatment. CAR-T cell therapy has made significant progress in the treatment of certain hematological tumors, but still faces challenges in the treatment of solid tumors, especially lung cancer.

[0005] Macrophages, as important members of the tumor microenvironment, have the potential to regulate immune responses and directly phagocytose tumor cells. However, due to the immunosuppressive characteristics of the tumor microenvironment, endogenous macrophages are often induced to become the M2 phenotype that promotes tumor growth and metastasis. Genetically engineering macrophages to express CAR targeting PTPRZ1 may provide a new strategy for tumor immunotherapy. Summary of the Invention

[0006] In view of the above, the purpose of the present invention is to construct a chimeric antigen receptor macrophage (CAR-M) that specifically recognizes PTPRZ1, so as to enhance the phagocytosis of tumor cells by macrophages. The present invention aims to provide a new treatment method for lung cancer. By constructing CAR-M targeting PTPRZ1, the immune response to lung cancer, especially tumor cells resistant to conventional treatments, can be enhanced. The present invention introduces the CAR targeting PTPRZ1 into human macrophages through transfection technology, enabling them to specifically recognize and phagocytose tumor cells expressing PTPRZ1, thereby improving the treatment effect without increasing damage to normal tissues. Through the implementation of the present invention, the targeting and effectiveness of lung cancer treatment can be significantly improved, providing new options for clinical treatment. Especially for patients who are ineffective or resistant to conventional treatments, new hope may be brought.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] 1. The present invention provides an antibody or antigen-binding fragment that specifically binds to the PTPRZ1 protein, and the antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region;

[0009] The heavy-chain variable region includes:

[0010] CDR1: GFSLTDYG;

[0011] CDR2: MWGGGNT;

[0012] CDR3: AKQTSYRYDAYAMDY;

[0013] The light-chain variable region includes:

[0014] CDR1: QNVNTN;

[0015] CDR2: SAS;

[0016] CDR3: QQYNNYPLT.

[0017] 2. The present invention provides a nucleic acid molecule encoding the above antibody or antigen-binding fragment that specifically binds to the PTPRZ1 protein.

[0018] 3. The present invention provides an expression vector containing the above nucleic acid molecule.

[0019] 4. The present invention provides a host cell containing the above nucleic acid molecule or expression vector.

[0020] 5. The present invention provides an anti-PTPRZ1 chimeric antigen receptor (CAR), which is designed by molecular engineering means and contains a single-chain variable region fragment (Anti-PTPRZ1 ScFv) capable of specifically binding to the PTPRZ1 protein. This single-chain variable region fragment is an antigen-binding fragment that has been screened and optimized to ensure its high affinity and specificity for PTPRZ1. The single-chain variable region fragment is formed by connecting the above-mentioned heavy-chain variable region and light-chain variable region through a linker (Linker), and its amino acid sequence is shown in SEQ ID NO:20.

[0021] The above chimeric antigen receptor also includes a signal peptide (CD8αLeader) that guides the expression of the CAR protein to the cell membrane, a tag protein (MYC), a linker (CD8αHinge), a transmembrane domain (CD28-TM), an intracellular signal transduction domain (CD3zeta). Additionally, it also includes a cleavage protein (P2A) and a green fluorescent protein (GFP).

[0022] 6. The present invention further provides an engineered macrophage CAR-M cell, which expresses the above-designed CAR through genetic engineering techniques. These CAR-M cells show targeted recognition and phagocytosis ability of tumor cells in in vitro experiments, demonstrating significant anti-tumor properties.

[0023] The CAR of the present invention can be stably expressed on the surface of macrophages. This stable expression is crucial for the function of CAR-M cells, as it ensures the persistence and effectiveness of CAR-M cells in vivo.

[0024] The present invention successfully constructed and expressed a chimeric antigen receptor targeting PTPRZ1 in 293T tool cells. The expression of the CAR confirmed the successful construction of the chimeric antigen receptor vector plasmid targeting PTPRZ1 and provided a reliable vector structure for subsequent research. By using the adenovirus-mediated gene transfection technique, we successfully achieved the expression of the chimeric antigen receptor (CAR) targeting PTPRZ1 on the surface of macrophages. The experimental results showed that when these modified macrophages (CAR-M) were co-cultured with tumor cells expressing PTPRZ1, they showed a significantly increased phagocytosis ratio compared to the control group of macrophages without targeted modification. Therefore, the humanized anti-PTPRZ1 chimeric antigen receptor macrophages of the present inventor can treat patients who are ineffective or resistant to conventional treatments for PTPRZ1 expression.

[0025] 7. Therefore, the present invention provides the use of the above antibody or antigen-binding fragment that specifically binds to the PTPRZ1 protein, nucleic acid molecule, expression vector, host cell, chimeric antigen receptor, and engineered macrophage expressing the chimeric antigen receptor in the preparation of drugs for treating lung cancer or brain metastasis of lung cancer.

[0026] The beneficial effects of the present invention are as follows:

[0027] 1. The present invention successfully constructs a vector of a humanized anti-PTPRZ1 chimeric antigen receptor.

[0028] 2. The present invention realizes the transfection of the humanized anti-PTPRZ1 chimeric antigen receptor in human monocyte-derived macrophages, ensuring that CAR-M cells can stably express CAR in vivo, thereby continuously exerting an anti-tumor effect.

[0029] 3. Through co-culture phagocytosis experiments, the present invention finds that macrophages transfected with anti-PTPRZ1 (CAR-M) have a significantly increased phagocytosis ratio of PTPRZ1-positive tumor cells compared to untransfected macrophages and macrophages without specific targets (CAR-M-Δ). This result indicates that CAR-M cells have stronger targeted phagocytosis ability and can effectively recognize and eliminate tumor cells.

[0030] 4. Generally speaking, the humanized anti-PTPRZ1 chimeric antigen receptor constructed by the present invention significantly enhances the phagocytosis ability of macrophages against PTPRZ1-positive tumor cells. This enhanced phagocytosis helps to inhibit tumor growth, improve the body's immune surveillance and clearance ability against tumors, thereby achieving the purpose of anti-tumor treatment.

[0031] Other advantages, objectives and features of the present invention will be described to some extent in the subsequent description, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following description. Brief Description of the Drawings

[0032] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail preferably with reference to the accompanying drawings, where:

[0033] Figure 1 For monoclonal antibody affinity detection;

[0034] Figure 2 For the pSB50 vector (A) in Example 2, as well as the base sequences (B) of the anti-PTPRZ1 chimeric antigen receptor vector structure and MYC-CD3ZETA (CD8αleader-MYC-CD8α-Hinge+CD28-TM+CD3zeta, PTPRZ1-MYC-CD3ZETA (CD8αleader-PTPRZ1-MYC-CD8α-Hinge+CD28-TM+CD3zeta);

[0035] Figure 3 The anti-PTPRZ1 chimeric antigen receptor structure (A), and verification of its plasmid expression in 293T cells (detection of CAR expression at the RNA level (B) and protein level (C)).

[0036] Figure 4 Detection of stable surface expression in human macrophages transfected with the anti-PTPRZ1 chimeric antigen receptor.

[0037] Figure 5 Enhanced phagocytic ability of PTPRZ1-positive tumor cells in human macrophages transfected with the anti-PTPRZ1 chimeric antigen receptor. Detailed implementation manners

[0038] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in the following examples only illustrate the basic concept of the present invention in a schematic manner. Without conflict, the following examples and the features in the examples can be combined with each other.

[0039] Preservation information of the hybridoma cell line of the present invention:

[0040] Preservation unit: China Center for Type Culture Collection, Address: Wuhan University, Wuhan, China, Preservation date: June 28, 2023, Preservation number: CCTCC NO: C2023175, Name of the culture and noted identification characteristics: Anti-human hybridoma cell line 7B4 (Hybridoma cell line 7B4).

[0041] Example 1. Preparation of PTPRZ1 monoclonal antibody and antibody sequence

[0042] 1. Expression and purification of recombinant PTPRZ1 protein

[0043] A specific region of the human PTPRZ1 gene (UniProtKB: P23471) (the selected fragment is the gene encoding amino acids 26 - 300 of human PTPRZ1) was cloned into the eukaryotic expression vector pSIN-EF-1a-puro (Shanghai Shengbo Biomedical Co., Ltd.) to obtain a eukaryotic expression plasmid. After transfection of the expression plasmid into HEK293 cells, the cells were cultured in suspension for 5 days, and the cell supernatant was collected by centrifugation. The target protein (recombinant PTPRZ1 protein) was obtained through nickel column purification and anion exchange purification.

[0044] 2. Mouse Immunization and Antibody Detection

[0045] Mix Freund's complete adjuvant with antigen protein (recombinant PTPRZ1 protein) at a concentration of 2 mg / mL in equal volume and emulsify. Immunize SPF-grade female Balb / c mice aged 6 - 8 weeks with the emulsified antigen. By intradermal injection into the footpad, inject 100 μg of antigen protein into each mouse. Two weeks after the primary immunization, mix and emulsify the antigen protein with Freund's incomplete adjuvant, and again inject 100 μg of antigen protein into each mouse by intradermal injection into the footpad. Two weeks later, collect blood from the tail vein, centrifuge to collect the supernatant, and detect the serum titer by ELISA. Select mice with a serum titer > 80000 for booster immunization before fusion.

[0046] 3. B Cell Fusion and Screening and Subcloning of Positive Hybridoma Cells

[0047] 1) Preparation of spleen B lymphocyte and SP2 / 0 cell suspensions

[0048] Take female Balb / c mice immunized with recombinant PTPRZ1 protein, kill them by exsanguination after enucleation of the eyeballs, collect the serum after centrifugation of the eye blood as a positive control for ELISA. Under aseptic conditions, remove the inguinal lymph nodes of the mice, place them in a glass dish containing 10 mL of incomplete medium, wash them, carefully remove the surrounding connective tissue and adipose tissue, change to another glass dish, fish out the spleen, place it in a 200-mesh stainless steel mesh, grind it with the inner core of a syringe, and rinse it with incomplete medium from time to time to make the splenocytes pass through the mesh holes into the solution. Transfer the splenocytes to a 10 mL glass centrifuge tube, centrifuge at 1500 rpm horizontally for 10 min, and discard the supernatant. Similarly, wash the cells once with 10 mL of incomplete medium, centrifuge to collect the precipitated cells, resuspend and mix the cells with 10 mL of incomplete medium, and the cell count is about 1×10 8 cells.

[0049] Take out SP2 / 0 cells from liquid nitrogen, quickly put them into a 37 °C water bath, shake constantly until the cell solution is completely dissolved, transfer the cells to a 10 mL centrifuge tube, centrifuge at 1500 rpm horizontally for 10 min, discard the supernatant, resuspend the precipitate with 10 mL of complete culture medium, transfer the cell suspension to a 50 mL culture flask, and culture it in an incubator at 37 °C and 5% CO2. After the cells grow well, screen the cells with a selective medium containing 8-AG for one week; two days before fusion, transfer one flask of cells to four flasks, so that the cells are in the logarithmic growth phase on the day of fusion, with just the right vitality, uniform cell size, round and translucent. On the day of fusion, gently blow down the SP2 / 0 cells from the tube wall with a bent pipette, collect them in a centrifuge tube, centrifuge, discard the supernatant, wash the precipitate with incomplete medium, resuspend it with 10 mL of incomplete medium, and count the cells, which is about 5×10 7 cells.

[0050] 2) Preparation of feeder cells

[0051] Take non-immunized female rats, sacrifice them by exsanguination through the eye socket, soak and disinfect them with 70% ethanol for 5 min, cut open the skin of the rats, lift the peritoneum with forceps, make a small incision with scissors, and aspirate the pre-cooled incomplete medium to rinse the abdominal cavity with a bent-tip pipette. Aspirate the washing liquid into a 50 mL centrifuge tube. Using the same method, rinse the abdominal cavity with incomplete medium three times, collect the washing liquid, centrifuge it horizontally at 1000 rpm for 10 min at room temperature, discard the supernatant, resuspend the cells with 10 mL of incomplete medium and count them.

[0052] 3) Fusion of myeloma cells and spleen B lymphocytes

[0053] Pre-warm PEG 1450 in a 37°C incubator before fusion. Aspirate 1×10 7 suspension of myeloma cells and 1×10 8 suspension of spleen B lymphocytes into a 50 mL sterile centrifuge tube, add 30 mL of RPMI-1640 medium, mix well, centrifuge at 1500 rpm for 10 min, discard the supernatant, gently flick the bottom of the tube to loosen the cell mass into a paste. Place the centrifuge tube in a 37°C water bath, and use a pipette to aspirate 0.8 mL of pre-warmed 50% PEG1450 solution, and slowly add it to the cells along the tube wall about 2 cm from the bottom of the tube. Rotate the centrifuge tube while adding, and finish adding in about 1 min. Then let it stand for 90 s, and gradually add 30 mL of pre-warmed RPMI-1640 medium at 37°C to terminate the fusion, finish adding within 3 min, with the speed starting slow and then fast and the action being gentle. Place the centrifuge tube in a 37°C incubator and let it stand for 5 min. Take out the centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, add 10 mL of HAT medium to resuspend the cells, gently pipette and mix well. Inoculate the fused cells into a 96-well cell culture plate pre-coated with feeder cells, at 100 μL / well. Leave 6 wells on each culture plate to inoculate SP2 / 0 cells as the negative control for HAT selection, and culture them in a 37°C, 5% CO2 incubator.

[0054] On the 4th - 5th day after fusion, the growth of cells can be observed under an inverted microscope, and 100 μL of HAT medium is supplemented. On the 10th - 12th day, the titer of hybridoma can be measured by indirect ELISA. On the 14th - 15th day, change to HAT medium for culture.

[0055] 4) Screening of specific hybridoma cells

[0056] Primary screening: 12 - 15 days after fusion, when the cells grow to cover 1 / 4 - 1 / 2 of the bottom of the culture well, the culture supernatant is detected by free ELISA to screen for positive clones. Coat the ELISA plate with goat anti - mouse polyclonal antibody (0.5 μg / well), incubate overnight at 4°C, wash 5 times with washing buffer for 5 minutes each time, pat dry the liquid, add 50 μL of cell culture supernatant and 50 μL of 0.2% PBST. Select mouse immune serum as the positive control, SP2 / 0 culture supernatant as the negative control, and washing solution as the blank control, incubate at 37°C for 1 h. Use biotin - labeled recombinant PTPRZ1 protein to obtain PTPRZ1 - biotin. After washing the ELISA plate, add 100 μL of PTPRZ1 - biotin dilution (about 0.5 μg / mL) and incubate at 37°C for 1 h. After washing the ELISA plate, add streptavidin - HRP diluted 1:5000 and incubate at 37°C for 30 min. Wash, pat dry the liquid, add 100 μL of freshly prepared TMB solution per well, react at room temperature in the dark for 3 - 5 min, add 50 μL of stop solution per well to terminate the reaction, and measure the absorbance at 450 nm with an ELISA reader.

[0057] 5) Re - screening

[0058] Cell ELISA method is used for re - screening. Select the cell wells with the color development value OD 450 > 0.5 for re - screening, and discard the remaining cell wells. Culture glioma spheroid cells in a 96 - well cell culture plate, about 4×10 4 cells / well, and culture adherently overnight. The next day, aspirate the culture medium, wash twice with PBS, and fix with 4% PFA for 10 min. Aspirate the fixative and wash twice with PBS. Take 50 μL of the cell supernatant to be tested and add it to the cell ELISA well, incubate at room temperature at 300 rpm for 1 h. Aspirate the supernatant, wash twice with washing buffer, pat dry the liquid, add HRP - labeled goat anti - mouse polyclonal antibody diluted 1:5000, and incubate at room temperature at 300 rpm for 1 h. Wash, pat dry the liquid, add 100 μL of freshly prepared TMB solution per well, react at room temperature in the dark for 3 - 5 min, add 50 μL of stop solution per well to terminate the reaction, and measure the absorbance at 450 nm with an ELISA reader. Select the cells with the color development value > 0.3 for subsequent cloning culture.

[0059] The results showed that using recombinant PTPRZ1 protein as an antigen, 6 female Balb / c mice were immunized successively, and the fusion was carried out 2 times. A total of 5 hybridoma cell lines that could secrete PTPRZ1 antibodies with high affinity were screened. After 3 times of cloning and ELISA screening, hybridoma cell lines secreting PTPRZ1 monoclonal antibodies were obtained. These hybridoma cells were cryopreserved several times and could stably secrete PTPRZ1 monoclonal antibodies after in vitro sub - culture for more than 3 months, and were finally cryopreserved in a liquid nitrogen tank.

[0060] 4. Purification and Preparation of Antibodies

[0061] The obtained target hybridoma cells after screening and cloning were expanded in culture and inoculated into a shake flask for serum-free shake flask culture fermentation. Inoculate the shake flask at a concentration of 1×10 6 cells / mL, and continuously culture for 4 days under sterile conditions at 37°C and 65 rpm. After fermentation, collect the culture medium, centrifuge at 15°C and 4000g for 30 min, collect the centrifuged supernatant, and filter the centrifuged supernatant with a 0.45 μm filter membrane to collect the filtrate. Use a Protein G purification column for affinity purification to obtain the target antibody.

[0062] 5. Subtype Identification and Gene Sequence Cloning of Antibodies

[0063] Use the SBA Clonotyping System-HRP kit from Southern Biothech to identify the subtypes of the heavy and light chains of monoclonal antibodies according to the instructions.

[0064] The specific operation is as follows: Dilute the capture antibody to 1 μg / mL with the coating solution (0.05 M carbonate and bicarbonate buffer with pH = 9.5), add 100 μL / well to the enzyme-linked immunosorbent assay (ELISA) plate, and coat overnight at 4°C. Wash the plate 3 times with PBS buffer containing 0.05% Tween-20 (washing solution). Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with the dilution solution (1% BSA, 0.1% PBST), add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 min. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-RP, kappa-HRP, lambda-HRP) 1:3000 with the dilution solution. After washing the plate 3 times, add 100 μL of the diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 min. Wash the plate 3 times again, then add the chromogenic solution. After about 5 min (depending on the strength of the reaction), add 2 M sulfuric acid to terminate the reaction, and read the absorbance at 450 nm. After identification, the heavy chains of the antibodies of the present invention are all IgG2a, and the light chains are all Kappa. According to the antibody subtype results, use a mature technical route to clone the antibody gene sequence. Specifically, collect hybridoma cells in good growth state, extract the total RNA of hybridoma cells using Trizol from Thermo, and reverse transcribe the mRNA into cDNA according to the operation method of the PrimerScript II ReverseTranscriptase instruction manual from Takara, and store it at -20°C for standby.

[0065] The specific operation process of reverse transcription is as follows:

[0066] First, prepare a premix of template RNA / priming DNA with a total volume of 10 μL, which includes 1 μL of reverse transcription primer (Oligo d(T)18 Primer) at a concentration of 10 μM, 4 μL of dNTP mixture at a concentration of 2.5 mM, and 5 μL of RNA (total amount less than 5 μg). After mixing, incubate at 65 °C for 5 min and immediately place on ice. Add 4 μL of 5×PrimeScript II buffer, 20 units of RNase inhibitor, and 1 μL of Primer Script II RTase (200 units) to the premix. After mixing, react at 42 °C for 60 min, then incubate at 70 °C for 15 min, and then place on ice for cooling and standby.

[0067] Then, using the cDNA as a template, independent amplification attempts were carried out respectively with the antibody gene amplification primers reported in the literature (Sequencing and cloning of antigen - specific antibodies from mouse memory B cells, Nature Protocols, 2016 Oct; 11(10):1908 - 1923.doi:10.1038 / nprot.2016.102.) to screen out the primers that can efficiently amplify the antibody gene. PCR was carried out according to the operation protocol of the Phanta Max Super - Fidelity DNA Polymerase instruction manual of Nanjing Novoprotein Co., Ltd.

[0068] The PCR reaction system was: 25 μL of 2×Phanta, 1 μL of dNTP, 4 μL of 10 μM primer pair, 4 μL of hybridoma cell cDNA, 1 μL of DNA polymerase, and 15 μL of dd H2O, with a total reaction volume of 50 μL. The amplification conditions were: pre - denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s; annealing at 56 °C for 30 s; extension at 72 °C for 2 min. The PCR products were recovered by gel extraction according to the instruction manual of the OMEGA Gel Extraction Kit of OMEGA Company. After the amplified products were ligated to the T - vector, the antibody gene sequence was obtained through plasmid sequencing.

[0069] 6. Flow cytometry detection of antibody - binding specificity

[0070] 1) Incubate with the primary antibody at 1 μg / mL on ice for 30 min;

[0071] 2) Wash once with PBS;

[0072] 3) Dilute the secondary antibody 1:1000 and incubate on ice for 20 min;

[0073] 4) Wash twice with PBS and detect by flow cytometry.

[0074] Through the detection and analysis by flow cytometry, we observed significant differences in the specific binding efficiency of the obtained different antibodies. Among them, the 7B4 antibody showed the highest specific binding efficiency, with a positive ratio as high as 72.1%, significantly superior to other antibodies. Followed by the 19H6 antibody, with a positive ratio of 71.8%, which also demonstrated strong specific binding ability. The positive ratios of the 14C9, 25A12, and 18F10 antibodies were 56.1%, 55.2%, and 51.7% respectively. Although they also showed certain specific binding efficiency, they were slightly inferior to the 7B4 and 19H6 antibodies. In summary, the 7B4 antibody stood out with its excellent binding efficiency in this detection and became the best-performing antibody.

[0075] 7. Capture molecule conjugation and affinity determination

[0076] Prepare the running buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05% Tween-20, pH = 7.4), and connect the reagent bottle of the running buffer to the Biacore T200 (GE Healthcare). Place the CM5 chip in the chip chamber, run the Primer program, and rinse the system pipeline. Dilute Anti-Mouse IgG 20-fold with 10mM sodium acetate buffer (pH = 5.0), load it into an EP tube, and load the amino-coupling related reagents into an EP tube. Place them on the reagent rack, put them into the sample chamber of the Biacore T200, run the manual program, and perform the three steps of chip activation, ligand conjugation, and blocking in sequence to covalently conjugate Anti-Mouse IgG to the 4 channels of the CM5 chip.

[0077] Dilute the ligand and analyte to the target concentration with the HEPES running buffer. Load the ligand and analyte into EP tubes and place them on the reagent rack; load the regeneration solution of 10mM glycine (pH = 1.7) into an EP tube and place it on the reagent rack. Place the reagent rack into the sample chamber of the Biacore T200, open the affinity determination program, select the "2-1, 3-1, 4-1" channels for the chip flow path, and use the multi-cycle kinetic method (set parameters: capture channels 2 / 3 / 4, flow rate 10μL / min, capture for 45s; analyte channels 1, 2, 3, 4, flow rate 30μL / min, injection for 120s, dissociation for 900s; regeneration solution flow rate 30μL / min, regeneration time 90s. Select 25°C for the temperature of the chip chamber and the sample chamber to determine the affinity between the analyte and the ligand. Analyze the kinetic data using a 1:1 binding model.

[0078] Clone number Dissociation constant (KD) 7B4 9.57E-10 M 19H6 3.66E-09 M 14C9 1.46E-09 M 25A12 1.95E-09 M 18F10 2.67E-09 M

[0079] The affinity of 5 PTPRZ1 antibodies was detected by SPR experiment. All KD values were within the credible range (KD at the E-08M level), among which the KD value of the murine anti-PTPRZ1 antibody (7B4) was the lowest, at 9.57E-10M. This indicates that the murine anti-PTPRZ1 antibody (7B4) has the best antibody affinity. As Figure 1 shown. Therefore, the anti-human hybridoma cell line 7B4 of this antibody was preserved.

[0080] The nucleotide sequence of the heavy chain of the murine anti-PTPRZ1 antibody (7B4) is shown in SEQ ID NO:1.

[0081] The nucleotide sequence of the light chain of the murine anti-PTPRZ1 antibody (7B4) is shown in SEQ ID NO:2.

[0082] The amino acid sequence of the heavy chain of the murine anti-PTPRZ1 antibody (7B4) is shown in SEQ ID NO:3.

[0083] Among them, the sequence of the heavy chain variable region is as follows (Kabat):

[0084] FR1: QVQLKESGPGLAAPSQSLSITCTVS, as shown in SEQ ID NO:4;

[0085] CDR1: GFSLTDYG, as shown in SEQ ID NO:5;

[0086] FR2: VSWIRQPPGKGLEWLGL, as shown in SEQ ID NO:6;

[0087] CDR2: MWGGGNT, as shown in SEQ ID NO:7;

[0088] FR3: YYNSVLKSRLSISKDNSKSQVFLKMNSVQTDDTARYYC, as shown in SEQ ID NO:8;

[0089] CDR3: AKQTSYRYDAYAMDY, as shown in SEQ ID NO:9;

[0090] FR4: WGQGTSVTVSS, as shown in SEQ ID NO:10.

[0091] The amino acid sequence of the light chain of the murine anti-PTPRZ1 antibody (7B4) is shown in SEQ ID NO:11.

[0092] The sequence of the light chain variable region is as follows:

[0093] FR1: DIVMTQSQKFMSTSVGDRVSVTCKAS, as shown in SEQ ID NO:12;

[0094] CDR1: QNVNTN, as shown in SEQ ID NO:13;

[0095] FR2: VAWYQQKPGQSPKALIY, as shown in SEQ ID NO:14;

[0096] CDR2: SAS;

[0097] FR3: YRYSGVPDRFTGSGSGTDFTLTINNVQSEDLAEYFC, as shown in SEQ ID NO:15;

[0098] CDR3: QQYNNYPLT, as shown in SEQ ID NO:16;

[0099] FR4: FGAGTKLELK, as shown in SEQ ID NO:17.

[0100] Example 2. Expression of the target gene in the chimeric antigen receptor vector plasmid against PTPRZ1

[0101] 1. Transfect 293T cells with the plasmid (pCAR-M) with PTPRZ1 recognition target and the plasmid (pCAR-M-Δ) without PTPRZ1 recognition target

[0102] As Figure 2 shown, pCAR-M is based on the pSB50 vector backbone, and the coding DNA of CD8αLeader - humanized single-chain variable region fragment of anti-PTPRZ1 - MYC - CD8αHinge - CD28 TM - CD3zeta - furin - SGSG - P2A - CopGFP is inserted between the restriction enzyme cleavage sites of EcoRI and XbaI. pCAR-M-Δ is based on the PSB50 vector backbone, and the coding gene of CD8αLeader - MYC - CD8αHinge - CD28 TM - CD3zeta - furin - SGSG - P2A - CopGFP is inserted between the restriction enzyme cleavage sites of EcoRI and XbaI.

[0103] The coding DNA of CD8αLeader (signal peptide that guides the expression of CAR protein to the cell membrane) is as shown in SEQ ID NO:18.

[0104] The coding DNA of the humanized single-chain variable region fragment of anti-PTPRZ1 is as shown in SEQ ID NO:19.

[0105] The amino acid sequence of the humanized anti-PTPRZ1 single-chain variable fragment is: DIVMTQSQKFMSTSVGDRVSVTCKASQNVNTNVAWYQQKPGQSPKALIYSASYRYSGVPD RFTGSGSGTDFTLTINNVQSEDLAEYFCQQYNNYPLTFGAGTKLELK- GGGGSGGGGSGGG GS -QVQLKESGPGLAAPSQSLSITCTVSGFSLTDYGVSWIRQPPGKGLEWLGLMWGGGNTY YNSVLKSRLSISKDNSKSQVFLKMNSVQTDDTARYYCAKQTSYRYDAYAMDYWGQGTSV TVSS, as shown in SEQ ID NO:20. Among them GGGGSGGGGSGGGGS represents the linker.

[0106] The coding DNA of MYC (tag protein) is as shown in SEQ ID NO:21.

[0107] The coding DNA of CD8α Hinge (linker) is as shown in SEQ ID NO:22.

[0108] The coding DNA of CD28 TM (transmembrane domain) is as shown in SEQ ID NO:23.

[0109] The coding DNA of CD3zeta (intracellular signal transduction domain) is as shown in SEQ ID NO:24.

[0110] The coding DNA of P2A (cleavage protein) is as shown in SEQ ID NO:25.

[0111] The coding DNA of CopGFP is as shown in SEQ ID NO:26.

[0112] 1). Inoculate 1×10 6 third-generation 293T cells after resuscitation into a six-well plate, and then place the culture plate in a constant temperature incubator with 5% CO2 for overnight culture;

[0113] 2). After 16h, the cell density in the six-well plate is 60-80%;

[0114] 3). In an EP tube, take 250 μL of OPTI-MEM medium and add 7.5 μL of Lipofectamine 3000 transfection reagent. Gently mix and let stand at room temperature for 5 min to allow the formation of transfection complexes;

[0115] 4). In another EP tube, add 250 μL of OPTI-MEM medium, and then add 4 μL of Lipofectamine 3000 reagent and 2 μg of the target plasmid DNA (pCAR-M containing the PTPRZ1 recognition target or pCAR-M-Δ without the PTPRZ1 recognition target). After gently mixing, also let it stand at room temperature for 5 min.

[0116] 5). Gently mix the liquids prepared in steps 3 and 4, and let it stand at room temperature for 15 min to ensure the stable formation of the transfection complex;

[0117] 6). Before transfection, change the medium in the six-well plate to fresh antibiotic-free medium, adding 2 mL to each well;

[0118] 7). Drop all the transfection complexes formed in step 5 into the cell culture in each well;

[0119] 8) Four hours after transfection, change the medium to complete medium containing serum, and put the culture plate back into the constant temperature incubator containing 5% CO2 for continued culture;

[0120] 9). Forty-eight hours after transfection, observe the transfection efficiency of the cells through a fluorescence microscope, use quantitative polymerase chain reaction (RT-qPCR) to detect the expression of plasmid RNA, and evaluate the expression level of CAR protein by flow cytometry.

[0121] 2. Detection of CAR RNA expression by RT-qPCR

[0122] 1) Rapid extraction of total RNA using the Fastagen kit

[0123] (1). After the cells are digested and centrifuged, discard the supernatant and retain the cell pellet;

[0124] (2). Add 500 μL of RA2 lysis buffer, invert and mix well, and let it stand for 1 minute to lyse the cells;

[0125] (3). Transfer the lysate to the inner tube, centrifuge at 12000 rpm for 1 min, and then discard the supernatant;

[0126] (4). Add 500 μL of wash buffer, and repeat the centrifugation and liquid discard steps for thorough washing;

[0127] (5). Centrifuge again to remove the residual wash buffer;

[0128] (6). Transfer to a nuclease-free EP tube, add 50 μL of elution buffer, let it stand, and then centrifuge to collect the purified RNA;

[0129] (7) Determine the concentration and purity of RNA using a spectrophotometer.

[0130] 2) Reverse transcription of RNA into cDNA

[0131] Specifically, use the PrimeScript RT Master Mix reverse transcription kit.

[0132] The reaction system is as follows:

[0133] 5×PrimeScript RT Master Mix 4 μL RNA 1 μg DEPC water Make up to 20 μL

[0134] The reaction program is as follows:

[0135] 37℃ 15 - 30 min 85℃ 5s 4℃ Forever

[0136] 3) qPCR amplification

[0137] Perform quantitative PCR on cDNA using the SYBR Premix Ex Taq II fluorescence quantitative kit, design specific primers to amplify the target gene, and the primer sequences are as follows:

[0138]

[0139] The reaction system:

[0140] SYBR Green (2×) 10 μL cDNA 1 μL Forward primer (10 μL) 0.4 μL Reverse primer (10 μL) 0.4 μL DEPC water Make up to 20 μL

[0141] Set 3 replicates for each group to ensure the repeatability and reliability of the data.

[0142] 4. Detection of MYC and GFP expression by flow cytometry

[0143] 1). After digestion, collect the cells by centrifugation, remove the supernatant, and retain the cell pellet;

[0144] 2). Blocking: Use FACS buffer (PBS containing 2% FBS) as the diluent, dilute the Fcγ blocking antibody at a ratio of 1:50 to prepare the blocking solution. For every 1×10 6 cells, add 50 μL of the blocking solution and incubate at 4°C for 20 min to reduce non-specific binding;

[0145] 3). Primary antibody staining: Dilute the MYC-APC (MYC can characterize the expression of CAR) and L / D-APC-CY7 antibodies at a ratio of 1:50 in FACS buffer to prepare the working solution. Mix the working solution with the cell suspension at a ratio of 1:1 and incubate at 4°C for 30 min to achieve specific antigen labeling;

[0146] 4). Washing: Add 1 mL of FACS buffer to each tube and mix well. Then centrifuge at 300 g for 5 min to remove the supernatant.

[0147] 5). Fixation: Fix the cells with 1% PFA (Paraformaldehyde) at room temperature for 15 min. Subsequently, add 1 mL of FACS buffer for washing, centrifuge at 300 g for 5 min to remove the supernatant. Resuspend the cells with 100 μL of FACS buffer and store at 4°C for flow cytometry analysis.

[0148] The results are as Figure 3 shown in B and C. Using quantitative polymerase chain reaction (RT-qPCR) technology, we observed that the transfected plasmid showed significant expression activity at the transcriptome level. Further flow cytometry analysis revealed that the proportion of GFP and MYC double-positive cells was approximately 40%, indicating that a considerable number of transfected cells successfully expressed both marker proteins simultaneously. These data provide strong evidence for the successful expression of the anti-PTPRZ1 chimeric antigen receptor (CAR) at the RNA and protein levels. Specifically, the expression of GFP was used to label the transfection efficiency, while the expression of MYC indicated the integration of CAR. Therefore, these results not only confirmed the functionality of the transfected plasmid but also verified the expression and functionality of anti-PTPRZ1 CAR at the cellular level.

[0149] Example 3. Infection of human macrophages with anti-PTPRZ1 chimeric antigen receptor adenovirus and detection of its expression

[0150] 1. Extraction and culture of human macrophages

[0151] 1). Extraction of human monocytes

[0152] (1) Isolate 7.5 mL of buffy coat from peripheral blood, add 22.5 mL of PBS for dilution to form a 30 mL mixture, and gently pipette to ensure uniform mixing.

[0153] (2) Gently add 15 mL of Ficoll along the tube wall to a new 50 mL centrifuge tube, and then slowly add the diluted buffy coat blood on top of the Ficoll to ensure a clear interface is formed.

[0154] (3) Centrifuge at 800 rcf for 20 min in an Eppendorf centrifuge preheated to 22°C.

[0155] (4) After centrifugation, carefully aspirate the monocytes and platelets in the middle cloudy layer using a pipette, avoiding aspirating the red blood cell layer.

[0156] (5) Wash the collected monocytes with PBS, centrifuge at 800 rcf for 5 minutes, and repeat the washing twice to remove residual red blood cells;

[0157] (6) Resuspend the cells with MACS Rinsing Solution Buffer and filter through a 40-μm filter to remove cell clumps.

[0158] 2). CD14 + Sorting of monocytes

[0159] (1) Adjust the cell concentration to 80 μL of Buffer per 1×10 7 cells, add 20 μL of CD14 beads, and incubate at 4 °C for 15 min;

[0160] (2) Perform magnetic sorting through an LS column to collect the target CD14+ monocytes.

[0161] 3). Induction of macrophages

[0162] Induce the sorted monocytes in an ultra-low attachment 6-well plate under the condition of 1640 medium + 10% FBS + 20 ng / mL m-CSF for 5 days to form macrophages;

[0163] 2. Production of CAR adenovirus

[0164] The AdMax adenovirus packaging system was established by Professor Frank L. Graham and is distributed by Microbix Biosystems Inc. in Canada. Its working principle is to co-transfect HEK293 cells with an adenovirus shuttle plasmid carrying an exogenous gene and a helper packaging plasmid carrying most of the adenovirus genome, and achieve recombination through the action of the Cre / loxP (or FLP / frt) recombinase system to produce recombinant adenovirus. This system is easy to operate, has high recombination efficiency, high virus yield, and high expression level of the target gene.

[0165] 3. Infection of chimeric antigen receptor adenovirus against PTPRZ1

[0166] 1). Replace the culture medium of the macrophages induced for 5 days and add 8 μg / mL of polybrene to enhance the transduction efficiency;

[0167] 2). Add the recombinant adenovirus at a ratio of MOI equal to 200, and collect the cells for flow cytometry detection after 48 hours.

[0168] 4. Flow cytometry detection

[0169] The expression analysis of MYC in transfected human macrophages was performed by flow cytometry. The experimental procedures were the same as those in Example 2. In the experiment, macrophages transfected with anti-PTPRZ1 chimeric antigen receptor (CAR-M) were compared with non-transfected or control macrophages (CAR-M-Δ) transfected with a construct lacking the target-binding domain.

[0170] As Figure 4 shown, compared with non-transfected and control macrophages (CAR-M-Δ), macrophages transfected with anti-PTPRZ1 chimeric antigen receptor (CAR-M) showed a positive ratio of approximately 90%, indicating that the Scfv against PTPRZ1 was effectively expressed in the transfected macrophages. In addition, GFP, as a reporter gene, also showed strong expression in the transfected macrophages, further verifying the transfection efficiency and the expression of the chimeric antigen receptor. The experimental results showed that the anti-PTPRZ1 chimeric antigen receptor could be expressed in human macrophages. These findings demonstrated the ability of the constructed anti-PTPRZ1 chimeric antigen receptor to specifically express in human macrophages.

[0171] Example 4. Anti-PTPRZ1 chimeric antigen receptor enhances the phagocytic ability of human macrophages

[0172] 1. Adenovirus infection of human macrophages with anti-PTPRZ1 chimeric antigen receptor

[0173] According to the experimental procedures in Example 3, the extraction and adenovirus infection of human macrophages were carried out.

[0174] 2. Co-culture of human macrophages transfected with anti-PTPRZ1 chimeric antigen receptor and PTPRZ1-positive tumor cells

[0175] 1). Plate the differentiated and adenovirus-infected macrophages: Discard the supernatant, wash once with PBS, digest with Accutase enzyme, centrifuge at 800g for 5 min, and then discard the supernatant;

[0176] 2). Add the medium containing 20 ng / mL m-CSF, count the cells, and seed the cells at 10 5 per well (1×10 5 ) in an ultra-low attachment 96-well plate, 100 μL per well, and culture overnight;

[0177] 3). Digest the tumor cells with high expression of PTPRZ1 and perform staining: Take 500 μL of the starting solvent for the PTPRZ1-high expressing cells, add 2.5 μL of PKH26 dye, and incubate at 37 °C for 15 min;

[0178] 4). Add 5 mL of serum to terminate the staining. After mixing, centrifuge the sample, aspirate and discard the supernatant, resuspend the cells with serum-containing medium, count the cells, and then centrifuge at 300 g for 5 min;

[0179] 5). Resuspend the cells with serum-free RPMI 1640 medium to a density of 2×10 5 cells / 100 μL, add them to a 96-well plate, and incubate at 37 °C for 4 h.

[0180] 3. Detection of the phagocytic ability of macrophages transfected with anti-PTPRZ1 chimeric antigen receptor by flow cytometry

[0181] According to the experimental procedures of Example 2, flow cytometry was used to detect the phagocytic ability of macrophages transfected with anti-PTPRZ1 chimeric antigen receptor against tumor cells. The detection indicators were L / D - live / dead, tumor cells - DIL, and macrophages - CD11b.

[0182] The results of flow cytometry detection are as Figure 5 shown. Compared with the CAR-M-Δ group without a target, the proportion of tumor cells with high expression of PTPRZ1 phagocytosed by the CAR-M group with a target increased significantly by 8-fold. This result confirmed that the anti-PTPRZ1 chimeric antigen receptor we constructed could significantly increase the phagocytosis proportion of human macrophages by binding to PTPRZ1 on tumor cells.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the present technical solution, and they should all be covered within the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment that specifically binds to the PTPRZ1 protein, characterized in that, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises: CDR1: GFSLTDYG; CDR2: MWGGGNT; CDR3: AKQTSYRYDAYAMDY; The light chain variable region comprises: CDR1: QNVNTN; CDR2: SAS; CDR3: QQYNNYPLT.

2. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof that specifically binds to the PTPRZ1 protein as claimed in claim 1.

3. An expression vector comprising the nucleic acid molecule as claimed in claim 2.

4. A host cell comprising the nucleic acid molecule as claimed in claim 2 or the expression vector as claimed in claim 3.

5. A chimeric antigen receptor against PTPRZ1, characterized in that, The chimeric antigen receptor comprises a signal peptide that guides the expression of the chimeric antigen receptor protein to the cell membrane, a single-chain variable region fragment that specifically binds to the PTPRZ1 protein, a tag protein, a linker, a transmembrane domain, and an intracellular signaling domain, and the amino acid sequence of the single-chain variable region fragment is as shown in SEQ ID NO:

20.

6. An engineered macrophage, characterized in that, The macrophage expresses the anti-PTPRZ1 chimeric antigen receptor as claimed in claim 5 by genetic engineering techniques.

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