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

Abstract

The invention relates to application of anti-PTPRZ1 chimeric antigen receptor macrophages in lung cancer treatment, and belongs to the technical field of tumor targeted therapy. The invention constructs a novel chimeric antigen receptor (CAR) which comprises a coding sequence of a single-chain variable region fragment for specifically recognizing PTPRZ1, and a coding sequence of a CD8TM transmembrane region and a coding sequence of a CD3zeta intracellular signal transduction structural domain. The design ingeniously endows the chimeric antigen receptor (CAR) with high specificity, so that the chimeric antigen receptor (CAR) can accurately recognize and firmly bind to the surface of a tumor cell expressing PTPRZ1 protein. According to the present invention, the chimeric antigen receptor targeting PTPRZ1 is successfully transfected into the human macrophage to form the CAR-M. A flow cytometry verifies that compared with non-transfected macrophages (Mphi) or transfected macrophages lacking a specific target binding domain (CAR-M-delta), the CAR-M has the advantage that the efficiency of swallowing PTPRZ1 positive tumor cells is remarkably improved in the aspect of the efficiency of swallowing PTPRZ1 positive tumor cells. Therefore, the CAR-M constructed by the invention can be used as a potential treatment means for targeting PTPRZ1 expression tumors.

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 brain metastasis of lung cancer. Background Art

[0002] Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the leading 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 deepening of understanding of the tumor microenvironment, targeted therapy has gradually become a research hotspot, which specifically acts on molecules in tumor cells or tumor matrix in order to achieve the purpose of inhibiting tumor growth and metastasis.

[0003] PTPRZ1, or protein tyrosine phosphatase receptor Z1, is a transmembrane receptor protein tyrosine phosphatase that is abnormally expressed in a variety of tumors. In lung cancer, high expression of PTPRZ1 is closely associated with tumor invasiveness, metastasis, and poor prognosis. Therefore, PTPRZ1 is considered a potential target for tumor therapy.

[0004] Chimeric Antigen Receptor (CAR) technology is an advanced bioengineering technology that combines single-chain variable region antibody fragments that can specifically recognize tumor-associated antigens with T cells or macrophages, thereby giving these immune cells the ability to highly specifically recognize and kill tumor cells. This single-chain variable region antibody fragment is formed by connecting the heavy chain variable region (VH) and light chain variable region (VL) of a tumor antigen-reactive antibody through a flexible joint. This design enables CAR-T cells or CAR-M cells to accurately 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 blood tumors, but it 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 properties of the tumor microenvironment, endogenous macrophages are often induced to become M2 phenotypes that promote 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 this, the purpose of the present invention is to construct a chimeric antigen receptor macrophage (CAR-M) with targeted recognition of PTPRZ1 to improve the phagocytosis of tumor cells by macrophages. The present invention aims to provide a new lung cancer treatment method, by constructing a CAR-M targeting PTPRZ1 to enhance the immune response to lung cancer, especially tumor cells resistant to conventional treatment. The present invention introduces CAR targeting PTPRZ1 into human macrophages through transfection technology, so that it can specifically recognize and phagocytize tumor cells expressing PTPRZ1, thereby improving the therapeutic effect without increasing normal tissue damage. Through the implementation of the present invention, the targeting and effect of lung cancer treatment can be significantly improved, providing new options for clinical treatment, especially for patients who are ineffective or resistant to conventional treatment, which may bring new hope.

[0007] In order 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 PTPRZ1 protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region comprises:

[0010] CDR1:GFSLTDYG;

[0011] CDR2:MWGGGNT;

[0012] CDR3:AKQTSYRYDAYAMDY;

[0013] The light chain variable region comprises:

[0014] CDR1:QNVNTN;

[0015] CDR2:SAS;

[0016] CDR3:QQYNNYPLT.

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

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

[0019] 4. The present invention provides a host cell comprising 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 comprises a single-chain variable region fragment (Anti-PTPRZ1 ScFv) that can specifically bind to the PTPRZ1 protein. The single-chain variable region fragment is an antigen binding fragment that has been screened and optimized to ensure that it has 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, and its amino acid sequence is shown in SEQ ID NO: 20.

[0021] The above-mentioned chimeric antigen receptor also includes a signal peptide (CD8αLeader) that guides the expression of CAR protein to the cell membrane, a tag protein (MYC), a linker (CD8αHinge), a transmembrane domain (CD28-TM), an intracellular signal transduction domain (CD3zeta), and 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 technology. These CAR-M cells show targeted recognition and phagocytic ability of tumor cells in in vitro experiments, showing 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 to the function of CAR-M cells because 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 CAR confirmed the successful construction of the chimeric antigen receptor vector plasmid targeting PTPRZ1, and provided a reliable vector structure for subsequent research; by utilizing adenovirus-mediated gene transfection technology, we successfully achieved the expression of a chimeric antigen receptor (CAR) targeting PTPRZ1 on the surface of macrophages. The experimental results showed that these modified macrophages (CAR-M) showed a significantly increased phagocytic ratio when co-cultured with tumor cells expressing PTPRZ1 compared with the control group macrophages that had not undergone targeted modification. Therefore, the humanized anti-PTPRZ1 chimeric antigen receptor macrophages of the present invention can treat patients who are ineffective or resistant to conventional treatment for PTPRZ1 expression.

[0025] 7. Therefore, the present invention provides the use of the above-mentioned antibodies or antigen-binding fragments that specifically bind to PTPRZ1 protein, nucleic acid molecules, expression vectors, host cells, chimeric antigen receptors, and engineered macrophages expressing the chimeric antigen receptors in the preparation of drugs for treating lung cancer or brain metastasis of lung cancer.

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

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

[0028] 2. The present invention achieves transfection of humanized anti-PTPRZ1 chimeric antigen receptors in human monocyte-derived macrophages, ensuring that CAR-M cells can express CAR in vivo for a long time, thereby continuously exerting anti-tumor effects.

[0029] 3. Through co-culture phagocytosis experiments, the present invention found that macrophages transfected with anti-PTPRZ1 (CAR-M) were more potent than untransfected macrophages. The phagocytic ratio of PTPRZ1-positive tumor cells by CAR-M-Δ and macrophages without specific targets (CAR-M-Δ) was significantly increased. This result shows that CAR-M cells have stronger targeted phagocytic ability and can effectively identify and eliminate tumor cells.

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

[0031] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 For monoclonal antibody affinity testing;

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

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

[0036] Figure 4 Human macrophages were transfected with anti-PTPRZ1 chimeric antigen receptors to detect their stable surface expression;

[0037] Figure 5 Transfection of human macrophages with anti-PTPRZ1 chimeric antigen receptors increased the phagocytic ability of PTPRZ1-positive tumor cells. DETAILED DESCRIPTION

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

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

[0040] Depository: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, deposit date: June 28, 2023, deposit number: CCTCC NO: C2023175, culture name and indicated 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) was selected (the selected fragment was the gene encoding the amino acid 26-300 segment of human PTPRZ1) and cloned into the eukaryotic expression vector pSIN-EF-1a-puro (Shanghai Shengbo Biopharmaceutical Co., Ltd.) to obtain a eukaryotic expression plasmid. The expression plasmid was transfected into HEK293 cells and then cultured in suspension for 5 days. The cell supernatant was collected by centrifugation, and the target protein (recombinant PTPRZ1 protein) was obtained by nickel column purification and anion exchange purification.

[0044] 2. Mouse Immunization and Antibody Detection

[0045] Freund's complete adjuvant and antigen protein (recombinant PTPRZ1 protein) at a concentration of 2 mg / mL were mixed and emulsified in equal volumes, and the emulsified antigen was used to immunize 6-8 week old SPF female Balb / c mice, and 100 μg of antigen protein was injected into each mouse by foot injection. Two weeks after the initial immunization, the antigen protein was mixed and emulsified with Freund's incomplete adjuvant, and 100 μg of antigen protein was injected into each mouse by foot injection again. Two weeks later, blood was collected through the tail vein, the supernatant was collected by centrifugation, and the serum titer was detected by ELISA, and mice with serum titer>80000 were selected for booster immunization before fusion.

[0046] 3. B cell fusion and screening and subcloning of positive hybridoma cells

[0047] 1) Preparation of splenic B lymphocyte and SP2 / 0 cell suspension

[0048] Female Balb / c mice immunized with recombinant PTPRZ1 protein were taken, the eyeballs were removed and bled to death, the eye blood was centrifuged and the serum was collected as a positive control for ELISA, the inguinal lymph nodes of the mice were removed aseptically, placed in a glass dish containing 10 mL of incomplete culture medium, washed, and the surrounding connective tissue and adipose tissue were carefully peeled off, and a glass dish was changed to take out the spleen, placed in a 200-mesh stainless steel mesh, and ground with the inner core of a syringe, and rinsed with incomplete culture medium from time to time to allow spleen cells to pass through the mesh and enter the solution, and the spleen cells were transferred to a 10 mL glass centrifuge tube, centrifuged horizontally at 1500 rpm for 10 minutes, and the supernatant was removed. In the same way, the cells were washed once with 10 mL of incomplete culture medium, the precipitated cells were collected by centrifugation, and the cells were resuspended and mixed with 10 mL of incomplete culture medium. The cell count was about 1×10 8 cells.

[0049] Take out the SP2 / 0 cells from liquid nitrogen, quickly put them in a 37°C water bath, and shake them continuously until the cell solution is completely dissolved. Transfer the cells to a 10mL centrifuge tube, centrifuge horizontally at 1500rpm for 10min, discard the supernatant, resuspend the precipitate with 10mL complete culture medium, transfer the cell suspension to a 50mL culture flask, and culture it in a 37°C, 5% CO2 incubator. After the cells grow well, screen the cells for one week with a selection medium containing 8-AG; 2 days before fusion, transfer 1 bottle of cells to 4 bottles. On the day of fusion, the cells are in the logarithmic growth phase, with just the right vitality, and the cells are uniform in size, round and translucent. On the day of fusion, use an elbow dropper to gently blow the SP2 / 0 cells off the tube wall, collect them in a centrifuge tube, centrifuge, discard the supernatant, wash the precipitate with incomplete culture medium, resuspend it with 10mL incomplete culture medium, and count the cells, which is about 5×10 7 indivual.

[0050] 2) Preparation of feeder cells

[0051] Take a non-immunized female rat, remove the eyeball and bleed to death, soak in 70% ethanol for disinfection for 5 minutes, cut the rat skin, lift the peritoneum with forceps, cut a small hole with scissors, and use an elbow dropper to draw pre-cooled incomplete culture medium to flush the abdominal cavity, and draw the washing liquid into a 50mL centrifuge tube. In the same way, flush the abdominal cavity with incomplete culture medium 3 times, collect the washing liquid, centrifuge horizontally at room temperature for 10 minutes at 1000rpm, remove the supernatant, resuspend the cells in 10mL incomplete culture medium and count them.

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

[0053] Before fusion, PEG 1450 was placed in a 37°C incubator for pre-warming and 1×10 7 Myeloma cell suspension and 1×10 8 Add a splenic B lymphocyte suspension to a 50mL sterile centrifuge tube, add 30mL RPMI-1640 medium, mix thoroughly, centrifuge at 1500rpm for 10min, discard the supernatant, flick the bottom of the tube to loosen the cell clusters into a paste, place the centrifuge tube in a 37℃ water bath, use a dropper to absorb 0.8mL of pre-warmed 50% PEG1450 solution, and slowly add it to the cells along the tube wall about 2cm from the bottom of the tube, turning the centrifuge tube while adding, add it in about 1min, then let it stand for 90s, add 30mL of 37℃ pre-warmed RPMI-1640 medium dropwise to terminate fusion, add it within 3min, slow first and then fast, and be gentle, let the centrifuge tube stand in a 37℃ incubator for 5min, take out the centrifuge tube, centrifuge at 1500rpm for 5min, discard the supernatant, add 10mL Resuspend the cells in HAT medium, gently blow and mix, and inoculate the fused cells into a 96-well cell culture plate that has been plated with feeder cells at 100 μL / well. Leave 6 wells of each culture plate to inoculate SP2 / 0 cells as a negative control for HAT selection, and culture them in a 37°C, 5% CO2 incubator.

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

[0055] 4) Screening of specific hybridoma cells

[0056] Preliminary screening: 12-15 days after fusion, when the cells grow to 1 / 4-1 / 2 of the bottom of the culture well, use the free ELISA method to detect the culture supernatant and screen the positive clones; coat the ELISA plate with goat anti-mouse polyclonal antibody (0.5μg / well), overnight at 4°C, wash 5 times with washing buffer, 5 minutes each time, pat dry the liquid, add 50μL of cell culture supernatant and 50μL of 0.2% PBST, select mouse immune serum as the positive control, select SP2 / 0 culture supernatant as the negative control, and use washing buffer for blank control, incubate at 37°C for 1h; use biotin to label the recombinant PTPRZ1 protein to obtain PTPRZ1-biotin, wash the ELISA plate, add 100μLPTPRZ1-biotin diluent (about 0.5μg / mL) and incubate at 37°C for 1h. After washing the ELISA plate, add 1:5000 diluted streptavidin-HRP and incubate at 37°C for 30min. Wash, pat dry the liquid, add 100 μL / well of freshly prepared TMB solution, react in the dark at room temperature for 3-5 minutes, add 50 μL of stop solution to each well to stop the reaction, and detect the absorbance value at 450 nm with an enzyme-labeled instrument.

[0057] 5) Review and screening

[0058] The cell ELISA method was used for verification and screening. Select the color value OD in the previous step 450 The cell wells with >0.5 were used for re-examination and screening, and the rest of the cell wells were eliminated. Glioma sphere cells were cultured in 96-well cell culture plates, with approximately 4×10 4 cells / well, adhere to the wall and culture overnight. On the second day, aspirate the culture medium, wash twice with PBS, and fix with 4% PFA for 10 minutes. Aspirate the fixative and wash twice with PBS. Take 50μL of the supernatant of the cells to be tested and add it to the cell ELISA well, incubate at room temperature at 300rpm for 1h. Aspirate the supernatant, wash twice with washing buffer, pat dry the liquid, add 1:5000 diluted HRP-labeled goat anti-mouse polyclonal antibody, and incubate at room temperature at 300rpm for 1 hour. Wash, pat dry the liquid, add 100μL / well of freshly prepared TMB solution, react in the dark at room temperature for 3-5min, add 50μL of stop solution per well to stop the reaction, and detect the absorbance value at 450nm with an enzyme marker. Select cells with a color value >0.3 for subsequent cloning culture.

[0059] The results showed that six female Balb / c mice were immunized with recombinant PTPRZ1 protein as antigen and fused twice. Five hybridoma cell lines that can secrete high-affinity PTPRZ1 antibodies were screened. After three cloning and ELISA screening, hybridoma cell lines secreting PTPRZ1 monoclonal antibodies were obtained. These hybridoma cells were frozen several times and could stably secrete PTPRZ1 monoclonal antibodies after in vitro subculture for more than three months, and were finally frozen in liquid nitrogen tanks.

[0060] 4. Antibody Purification and Preparation

[0061] The hybridoma cells obtained after screening and cloning were expanded and inoculated into shake flasks for serum-free shake flask culture and fermentation. 6 The shake flask was inoculated with a concentration of 1000 μg / mL and cultured continuously for 4 days at 37°C and 65 rpm under sterile conditions. After the fermentation was completed, the culture solution was collected and centrifuged at 15°C and 4000 g for 30 minutes. The supernatant was collected and filtered with a 0.45 μm filter membrane to collect the filtrate. Affinity purification was performed using a protein G purification column to obtain the target antibody.

[0062] 5. Antibody subtype identification and gene sequence cloning

[0063] The heavy chain and light chain subtypes of the monoclonal antibody were identified using the SBA Clonotyping System-HRP kit from Southern Biothech according to the instructions.

[0064] The specific operation is as follows: dilute the capture antibody to 1 μg / mL with coating solution (0.05M carbonate and bicarbonate buffer at pH = 9.5), add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer (washing solution) containing 0.05% Tween-20. Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-RP, kappa-HRP, lambda-HRP) with diluent at a ratio of 1:3000. After washing the plate three times with washing solution, add 100 μL of the diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate 3 times again, add the color developing solution, add 2M sulfuric acid to terminate the reaction after about 5 minutes (depending on the strength of the reaction), and read the absorbance at 450nm. 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, the antibody gene sequence is cloned using a mature technical route. Specifically, hybridoma cells with good growth status are collected, and the total RNA of the hybridoma cells is extracted using Trizol from Thermo Company. According to the operating method of the PrimerScript II ReverseTranscriptase instruction manual of Takara Company, the mRNA is reversely transcribed into cDNA, and frozen at -20°C for use.

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

[0066] First, prepare a 10 μL total volume of template RNA / primer DNA premix, including 1 μL 10 μM reverse transcription primer (Oligo d(T)18 Primer), 4 μL 2.5 mM dNTP mixture, and 5 μL RNA (total amount less than 5 μg). After mixing, treat at 65°C for 5 minutes and immediately put on ice. Add 4 μL 5× PrimeScript II buffer, 20 units of RNase inhibitor, and 1 μL Primer Script II RTase (200 units) to the premix, mix, react at 42°C for 60 minutes, and then treat at 70°C for 15 minutes, then cool on ice for later use.

[0067] Then, using cDNA as a template, 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.) were used to perform amplification attempts independently, and primers that can efficiently amplify antibody genes were screened. PCR was performed according to the operating protocol of the Phanta Max Super-Fidelity DNA Polymerase instruction manual of Nanjing Novozymes.

[0068] The PCR reaction system was: 25 μL 2×Phanta, 1 μL dNTP, 4 μL 10uM primer pair, 4 μL hybridoma cell cDNA, 1 μL DNA polymerase, 15 μL dd H2O, and the total reaction volume was 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 product was recovered by gel according to the instructions of OMEGA GelExtraction Kit of OMEGA Company, and the antibody gene sequence was obtained by plasmid sequencing after the amplified product was linked to the T vector.

[0069] 6. Flow cytometry to detect antibody binding specificity

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

[0071] 2) Wash once with PBS;

[0072] 3) The secondary antibody was diluted 1:1000 and incubated on ice for 20 min;

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

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

[0075] 7. Capture molecule coupling and affinity determination

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

[0077] Use HEPES running buffer to dilute the ligand and analyte to the target concentration. Load the ligand and analyte into the EP tube and place it on the reagent rack; load the regeneration solution 10mM glycine (pH=1.7) into the EP tube and place it on the reagent rack. Place the reagent rack into the Biacore T200 sample compartment, open the affinity determination program, select the "2-1, 3-1, 4-1" channel for the chip flow path, and use the multi-cycle kinetic method (set parameters: capture channel 2 / 3 / 4, flow rate 10μL / min, capture 45s; analyte 1, 2, 3, 4 channels, flow rate 30μL / min, injection 120s, dissociation 900s; regeneration solution flow rate 30μL / min, regeneration time 90s. The temperature of the chip compartment and sample compartment is set to 25℃, and the affinity of the analyte and the ligand is determined. The kinetic data are analyzed using a 1:1 binding model.

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

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

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

[0081] The nucleotide sequence of the light chain of the mouse 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 target gene in anti-PTPRZ1 chimeric antigen receptor vector plasmid

[0101] 1.293T cells were transfected with plasmids with PTPRZ1 recognition target (pCAR-M) and plasmids without PTPRZ1 recognition target (pCAR-M-Δ)

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

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

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

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

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

[0107] The DNA encoding CD8αHinge (linker) is shown as SEQ ID NO:22.

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

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

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

[0111] The DNA encoding CopGFP is shown in SEQ ID NO:26.

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

[0113] 2). After 16 hours, the cell density of 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, mix gently and let stand at room temperature for 5 minutes to allow the formation of transfection complexes;

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

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

[0117] 6). Before transfection, replace the culture medium in the six-well plate with fresh antibiotic-free culture medium and add 2 mL to each well;

[0118] 7). Add all the transfection complexes formed in step 5 dropwise to the cell culture in each well;

[0119] 8) 4 hours after transfection, the culture medium was replaced with complete culture medium containing serum, and the culture plate was returned to a constant temperature incubator containing 5% CO2 for further culture;

[0120] 9). 48 hours after transfection, the transfection efficiency of cells was observed by fluorescence microscopy, the expression of plasmid RNA was detected by quantitative polymerase chain reaction (RT-qPCR), and the expression level of CAR protein was evaluated by flow cytometry.

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

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

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

[0124] (2) Add 500 μL RA2 lysis buffer, invert to mix and let 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 discard the supernatant;

[0126] (4) Add 500 μL of washing solution and repeat the centrifugation and discarding steps to wash thoroughly;

[0127] (5) Centrifuge again to remove the remaining washing solution;

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

[0129] (7) Use a spectrophotometer to measure the concentration and purity of RNA.

[0130] 2) Reverse transcription of RNA into cDNA

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

[0132] The reaction system is as follows:

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

[0134] The reaction procedure is as follows:

[0135] 37℃ 15-30min 85℃ 5s 4℃ Forever

[0136] 3) qPCR amplification

[0137] SYBR Premix Ex Taq II fluorescent quantitative kit was used to perform quantitative PCR on cDNA, and specific primers were designed to amplify the target gene. The primer sequences are as follows:

[0138]

[0139] Reaction system:

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

[0141] Three replicate wells were set up in 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, the cells are collected by centrifugation, the supernatant is removed, and the cell pellet is retained;

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

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

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

[0147] 5). Fixation: Use 1% PFA (Paraformaldehyde) to fix the cells for 15 minutes at room temperature. Then, add 1 mL FACS buffer for washing, centrifuge at 300g for 5 minutes, and remove the supernatant. Resuspend the cells with 100 μL FACS buffer, store at 4°C, and detect by flow cytometry.

[0148] The results are as follows Figure 3 As 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 cells in the transfected cells successfully expressed both marker proteins at the same time. These data provide strong evidence for the successful expression of the constructed anti-PTPRZ1 chimeric antigen receptor (CAR) at the RNA and protein levels. Specifically, the expression of GFP is used to mark the transfection efficiency, while the expression of MYC indicates the integration of CAR. Therefore, these results not only confirm the functionality of the transfected plasmid, but also verify the expression and functionality of the 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) Separate 7.5 mL of buffy coat from peripheral blood, add 22.5 mL of PBS to dilute to form a 30 mL mixture, and gently pipette to ensure uniform mixing;

[0153] (2) Gently add 15 mL of Ficoll along the wall of a new 50 mL centrifuge tube, and then slowly add the diluted buffy coat blood to the top layer of 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, use a pipette to carefully aspirate the mononuclear cells and platelets in the middle cloud layer, avoiding the red blood cell layer;

[0156] (5) The collected mononuclear cells were washed with PBS, centrifuged at 800 rcf for 5 min, and washed twice to remove residual red blood cells;

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

[0158] 2).CD14 + Isolation of monocytes

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

[0160] (2) Magnetic separation was performed using an LS column to collect target CD14+ monocytes.

[0161] 3). Induction of macrophages

[0162] The sorted monocytes were placed in ultra-low adhesion 6-well plates and induced for 5 days using 1640 medium + 10% FBS + 20ng / mL m-CSF to form macrophages;

[0163] 2. CAR adenovirus production

[0164] The AdMax adenovirus packaging system was established by Professor Frank L. Graham and is distributed by Microbix, a Canadian company. Its working principle is to co-transfect HEK293 cells with an adenovirus shuttle plasmid carrying an exogenous gene and an auxiliary packaging plasmid carrying most of the adenovirus genome, and use the Cre / loxP (or FLP / frt) recombinase system to achieve recombination and 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 with anti-PTPRZ1 chimeric antigen receptor adenovirus

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

[0167] 2). Add recombinant adenovirus at an MOI of 200, and collect cells for flow cytometry after 48 hours.

[0168] 4. Flow cytometry

[0169] Flow cytometry was used to analyze the expression of MYC in transfected human macrophages. The experimental steps were the same as those in Example 2. In the experiment, macrophages transfected with anti-PTPRZ1 chimeric antigen receptor (CAR-M) were compared with untransfected Or control macrophages transfected with a cell line without the target binding domain (CAR-M-Δ) were compared.

[0170] like Figure 4 As shown, compared with untransfected Compared with the control group macrophages (CAR-M-Δ), the macrophages transfected with the anti-PTPRZ1 chimeric antigen receptor (CAR-M) showed a positive ratio of about 90%, indicating that the anti-PTPRZ1 Scfv was effectively expressed in the transfected macrophages. In addition, GFP, as a reporter gene, also showed strong expression in the transfected macrophages, which further verified the transfection efficiency and the expression of the chimeric antigen receptor. The experimental results showed that the anti-PTPRZ1 chimeric antigen receptor can be expressed in human macrophages. These findings demonstrate that the constructed anti-PTPRZ1 chimeric antigen receptor has the ability to be specifically expressed in human macrophages.

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

[0172] 1. Anti-PTPRZ1 chimeric antigen receptor adenovirus infection of human macrophages

[0173] According to the experimental steps of Example 3, human macrophages were extracted and infected with adenovirus.

[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, centrifuge at 800g for 5 minutes, and discard the supernatant;

[0176] 2). Add culture medium containing 20 ng / mL m-CSF, count the cells and place them at 10 per well. 5 (1×10 5 ) were cultured in ultra-low adhesion 96-well plates, 100 μL per well, overnight;

[0177] 3). Digestion of PTPRZ1-overexpressing tumor cells and staining: Add 2.5 μL PKH26 dye to 500 μL starting solvent of PTPRZ1-overexpressing cells and incubate at 37°C for 15 min;

[0178] 4). Add 5 mL of serum to stop staining, mix well and centrifuge, discard the supernatant, resuspend the cells in serum-containing medium, count, wash and centrifuge at 300g for 5 minutes;

[0179] 5). Resuspend the cells to 2×10 in serum-free 1640 medium. 5 cells / 100 μL, added to a 96-well plate and incubated at 37°C for 4 h.

[0180] 3. Flow cytometry to detect the phagocytic ability of transfected anti-PTPRZ1 chimeric antigen receptor

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

[0182] Flow cytometry results Figure 5 As shown in the figure, the proportion of tumor cells with high PTPRZ1 expression in the CAR-M group transfected with the target increased significantly by 8 times compared with the CAR-M-Δ group without the target. This result confirms that the anti-PTPRZ1 chimeric antigen receptor we constructed can significantly increase the phagocytic 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 solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment that specifically binds to PTPRZ1 protein, characterized in that: The antibody or antigen-binding fragment 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 that specifically binds to the PTPRZ1 protein according to claim 1.

3. An expression vector comprising the nucleic acid molecule of claim 2.

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

5. An anti-PTPRZ1 chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the antigen binding fragment according to claim 1, wherein the antigen binding fragment is a single-chain variable region fragment that specifically binds to the PTPRZ1 protein.

6. An anti-PTPRZ1 chimeric antigen receptor according to claim 5, characterized in that: The amino acid sequence of the single-chain variable region fragment is shown in SEQ ID NO:

20.

7. The anti-PTPRZ1 chimeric antigen receptor according to claim 5, characterized in that: The chimeric antigen receptor comprises a promoter, a signal peptide for directing the expression of the chimeric antigen receptor protein to the cell membrane, a single-chain variable region fragment specifically binding to the PTPRZ1 protein, a tag protein, a linker, a transmembrane domain, and an intracellular signal transduction domain.

8. An engineered macrophage, characterized in that: The macrophages express the anti-PTPRZ1 chimeric antigen receptor according to any one of claims 5 to 7 through genetic engineering technology.

9. Use of the antibody or antigen-binding fragment that specifically binds to the PTPRZ1 protein according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3, the host cell according to claim 4, the anti-PTPRZ1 chimeric antigen receptor according to any one of claims 5 to 7, and the engineered macrophage according to claim 8 in the preparation of a drug for treating lung cancer or brain metastasis of lung cancer.

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