A car-gammadelta t cell targeting gp350 and preparation method and use thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a CAR-gammadelta T cell targeting GP350 and a preparation method and application thereof. The application provides an antibody or antigen binding fragment thereof targeting GP350. The GP350-targeting antibody screened is used as an antigen binding domain to construct a chimeric antigen receptor, and a gammadelta T cell isolated from peripheral blood of a healthy donor is used to prepare a CAR-gammadelta T cell targeting GP350. In-vivo and in-vitro experiments show that the CAR-gammadelta T cell targeting GP350 has obvious killing activity on an EBV-positive nasopharyngeal carcinoma cell (GP350 + ). The application can be applied to the diagnosis of EBV-positive diseases, and has a broad prospect in the preparation of products for detecting, preventing and / or treating GP350-positive tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a CAR-γδT cell targeting GP350, its preparation method, and its uses. Background Technology

[0002] In the field of tumor immunotherapy, chimeric antigen receptor (CAR) T-cell therapy has become a revolutionary technology. By genetically modifying a patient's T cells to specifically recognize and kill tumor cells, it enables precise treatment of tumors. Although CAR-T cells have achieved remarkable results in treating certain hematologic malignancies, their effectiveness in treating solid tumors is still limited by several factors, such as the inhibition of the tumor microenvironment, off-target effects, and potential serious side effects.

[0003] γδT cells are a unique subset of T cells with dual functions of innate and adaptive immunity. They can directly recognize stress cells and possess anti-tumor activity. Furthermore, γδT cells do not rely on the conventional major histocompatibility complex (MHC) to recognize antigens, which can reduce rejection reactions in allogeneic transplantation. Therefore, γδT cells show significant potential in tumor immunotherapy.

[0004] GP350 is a membrane glycoprotein encoded by Epstein-Barr virus (EBV) and is highly expressed in various EBV-related tumors, such as nasopharyngeal carcinoma, Burkitt lymphoma, and gastric cancer. This makes GP350 an ideal tumor-specific target. However, current research on CAR-T cell therapy targeting GP350 mainly focuses on αβT cells, with relatively little research on γδT cells, and the preparation methods for CAR-γδT cells targeting this target are not yet mature.

[0005] Therefore, constructing a CAR-γδT cell that targets GP350 and makes it highly effective in fighting tumors is a problem and challenge in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides CAR-γδT cells targeting GP350 and their preparation method, with the aim of effectively combating EBV-related tumors.

[0007] The present invention provides an antibody or antigen-binding fragment thereof targeting GP350, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises three complementarity-determining regions CDRL1, CDRL2, and CDRL3.

[0008] The amino acid sequence of CDRH1 is selected from any one of SEQ ID No. 1, SEQ ID No. 10, SEQ ID No. 19, and SEQ ID No. 28;

[0009] The amino acid sequence of CDRH2 is selected from any one of SEQ ID No. 2, SEQ ID No. 11, SEQ ID No. 20, and SEQ ID No. 29;

[0010] The amino acid sequence of CDRH3 is selected from any one of SEQ ID No. 3, SEQ ID No. 12, SEQ ID No. 21, and SEQ ID No. 30;

[0011] The amino acid sequence of CDLH1 is selected from any one of SEQ ID No. 4, SEQ ID No. 13, SEQ ID No. 22, and SEQ ID No. 31;

[0012] The amino acid sequence of CDLH2 is selected from any one of SEQ ID No. 5, SEQ ID No. 14, SEQ ID No. 23, and SEQ ID No. 32;

[0013] The amino acid sequence of CDLH3 is selected from any one of SEQ ID No. 6, SEQ ID No. 15, SEQ ID No. 24, and SEQ ID No. 33.

[0014] Preferably, the combination of the heavy chain variable region and the light chain variable region includes any one of the following combinations:

[0015] The amino acid sequences are CDRH1, CDRH2 and CDRH3 as shown in SEQ ID No. 1 to 3, and CDRL1, CDRL2 and CDRL3 as shown in SEQ ID No. 4 to 6, respectively.

[0016] Alternatively, the amino acid sequences are as shown in SEQ ID No. 10-12, CDRH1, CDRH2 and CDRH3, and the amino acid sequences are as shown in SEQ ID No. 13-15, CDRL1, CDRL2 and CDRL3.

[0017] Alternatively, the amino acid sequences are as shown in SEQ ID No. 19–21, CDRH1, CDRH2, and CDRH3, and the amino acid sequences are as shown in SEQ ID No. 22–24, CDRL1, CDRL2, and CDRL3, respectively.

[0018] Alternatively, the amino acid sequences are as shown in SEQ ID No. 28–30, CDRH1, CDRH2, and CDRH3, and the amino acid sequences are as shown in SEQ ID No. 31–33, CDRL1, CDRL2, and CDRL3.

[0019] Preferably, the heavy chain variable region and the light chain variable region further include a skeleton region, and the combination of the heavy chain variable region and the light chain variable region includes any one of the following combinations:

[0020] The amino acid sequences are VH and VL as shown in SEQ ID No. 7 and 8, respectively.

[0021] Alternatively, the amino acid sequences are VH and VL as shown in SEQ ID No. 16 and 17, respectively.

[0022] Alternatively, the amino acid sequences are VH and VL as shown in SEQ ID No. 25 and 26, respectively.

[0023] Alternatively, the amino acid sequences are VH and VL as shown in SEQ ID No. 34 and 35, respectively;

[0024] And / or, the heavy chain variable region and the light chain variable region are linked by three repeating GGGGS amino acid fragments.

[0025] Preferably, the antibody or its antigen-binding fragment further includes a constant region;

[0026] Preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

[0027] Preferably, the species source of the constant region is cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans;

[0028] Preferably, the antibody is selected from any one of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, chimeric antibodies, and full-length antibodies;

[0029] Preferably, the antigen-binding fragment is selected from any one of the antibody F(ab')2, Fab', Fab, Fv and scFv;

[0030] Preferably, when the antigen-binding fragment is scFv, the amino acid sequence of scFv is selected from any one of SEQ ID No. 9, SEQ ID No. 18, SEQ ID No. 27, and SEQ ID No. 36.

[0031] The present invention provides a nucleic acid characterized in that it encodes an antibody or an antigen-binding fragment thereof as described in any of the preceding claims.

[0032] Preferably, the nucleotide sequence of the nucleic acid is selected from any of the nucleotide sequences shown in SEQ ID No. 37-40.

[0033] The present invention provides a recombinant vector comprising the nucleic acid as described above.

[0034] Preferably, the recombinant vector is selected from at least one of pcDNA.

[0035] The present invention provides a host cell comprising the recombinant vector described above.

[0036] This invention provides a method for preparing the antibody targeting GP350 or its antigen-binding fragment as described in any one of the preceding claims, comprising:

[0037] The gene fragment encoding the antibody or its antigen-binding fragment targeting GP350 described above is inserted into a recombinant vector, transformed into host cells for expression, and then isolated and purified to obtain the final product.

[0038] The present invention provides a chimeric antigen receptor, wherein the antigen-binding domain of the chimeric antigen receptor includes an antibody or antigen-binding fragment thereof targeting GP350 as described in any of the preceding claims.

[0039] Preferably, the chimeric antigen receptor further includes a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.

[0040] Preferably, the signal transduction structural domain includes CD3ζ;

[0041] Preferably, the signal transduction domain further includes a 4-1BB intracellular region.

[0042] The present invention provides a CAR-γδT cell comprising the chimeric antigen receptor as described above.

[0043] This invention provides a method for preparing the above-mentioned CAR-γδT cells, which includes the following steps:

[0044] Step 1: Extract γδT cells from blood and expand and culture them;

[0045] Step 2: Culture host cells containing lentiviruses and collect viral fluid;

[0046] Step 3: Infect γδT cells with the viral fluid and RetroNectin protein to obtain the final product.

[0047] Preferably, the lentivirus packaging uses a three-plasmid packaging system, wherein the three plasmids include CAR-γδTvector.

[0048] The present invention provides the use of the antibody or antigen-binding fragment thereof targeting GP350 as described above, the nucleic acid as described above, the recombinant vector as described above, the host cell as described above, the chimeric antigen receptor as described above, and the CAR-γδT cell as described above in the preparation of products for detecting, preventing and / or treating GP350-positive tumors;

[0049] The product includes at least one of the following: testing reagents, testing kits, microfluidic chips, immune cells, and drugs.

[0050] The present invention provides a cell injection solution comprising any one of the following: an antibody targeting GP350 as described above or an antigen-binding fragment thereof, a nucleic acid as described above, a recombinant vector as described above, a host cell as described above, a chimeric antigen receptor as described above, or a CAR-γδT cell as described above.

[0051] In this invention, the "backbone region" or "FR" region refers to the region in the antibody heavy chain variable region or light chain variable region other than the CDRs. Taking the heavy chain variable region as an example, the heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4), wherein the heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs, including the FRH1, FRH2, FRH3, and FRH4 backbone regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs with FRs (from the amino terminus to the carboxyl terminus): FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4.

[0052] The "antigen-binding fragment" is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv (single-chain antibody). The "antigen-binding fragment" is a portion of the intact antibody that specifically binds to the antigen to which the intact antibody is bound. Those skilled in the art will readily understand from the description of this invention that the antigen-binding fragment can be prepared by methods known in the art, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds, or by recombinant genetics techniques or by an automated peptide synthesizer (such as an automated peptide synthesizer from AppliedBioSystems).

[0053] The "recombinant vector" is an expression vector or cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged as a virus) into host cells through transformation, transfection, or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can ligate the target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package the target DNA fragment into the viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0054] The term "host cell" includes at least one of prokaryotic host cells, eukaryotic host cells, and bacteriophages. The prokaryotic host cell can be *Escherichia coli*, *Streptomyces*, or *Bacillus subtilis*, etc. The eukaryotic host cell can be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6 cells, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Saccharomyces hansenii*, *Candida*, some insect cells, and plant cells. The 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to those skilled in the art.

[0055] The present invention does not specifically limit the culture conditions of the host cells. Based on conventional technical knowledge, culture conditions that enable the host cells to express and produce the antibody targeting GP350 can be obtained.

[0056] The term "GP350-positive tumor" refers to tumors whose cell surface expresses the Epstein-Barr Virus (EBV) envelope glycoprotein GP350, including EBV-positive nasopharyngeal carcinoma, Burkitt lymphoma, chronic B-cell lymphocytic leukemia, and EBV-positive gastric cancer.

[0057] This invention provides antibodies targeting GP350 or their antigen-binding fragments. Using the selected GP350-targeting antibodies as antigen-binding domains, a chimeric antigen receptor was constructed, and CAR-γδT cells targeting GP350 were prepared using γδT cells isolated from healthy donor peripheral blood. In vitro and in vivo experiments showed that CAR-γδT cells targeting GP350 effectively target EBV-positive nasopharyngeal carcinoma cells (GP350). + It exhibits significant cytotoxic activity. This invention can be applied to the diagnosis of EBV-positive diseases and has broad prospects in the preparation of products for the detection, prevention, and / or treatment of GP350-positive tumors.

[0058] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0059] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0060] Figure 1 This is a diagram showing the binding effect of immunofluorescence (IFA) on hybridoma supernatant and HeLa cells overexpressing GP350 in Example 1.

[0061] Figure 2 This is an SDS-PAGE result of the purified recombinant single-chain antibody from Example 2;

[0062] Figure 3 This is a graph showing the results of ELISA identification of the reactivity between the recombinant scFv single-chain antibody and the antigen in Example 2;

[0063] Figure 4 This is a graph showing the results of flow cytometry analysis of the binding of four recombinant scFv single-chain antibodies to GP350 positive tumor cells in Experiment Example 1.

[0064] Figure 5 The image shows the results of the label-free killer detection (RTCA) of allogeneic GP350-CAR-γδT on target cells in Experiment Example 3.

[0065] Figure 6 The figure shows the results of evaluating the in vivo antitumor activity of GP350-CAR-γδT cells using the NCG mouse xenograft model in Experiment Example 4. Detailed Implementation

[0066] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0067] Example 1: Antibody targeting GP350 and its preparation method

[0068] The antibody targeting GP350 in this embodiment was prepared by the following method:

[0069] 1. Preparation of GP350 recombinant protein

[0070] Using the GP350 gene synthesized by Qingke Biotechnology as a template, the dominant epitope region containing amino acid Met1-Pro490 of the GP350 gene was selected as the immunotargeting fragment and cloned into the pFastBacHT B expression vector carrying a His tag at the C-terminus. Then, it was expressed using an insect baculovirus expression system, and the recombinant GP350 protein was subsequently purified using the His tag.

[0071] 2. Animal immunization

[0072] Five- to six-week-old female Balb / c mice were used as immunization animals, with an immunization dose of 100 μg per mouse. For the initial immunization, 100 μl of Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant GP350 protein, thoroughly emulsified, and then injected subcutaneously at multiple sites. Two weeks later, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with recombinant GP350 protein, thoroughly emulsified, and then injected subcutaneously at multiple sites. Four booster immunizations were administered. On day 10 after the last booster immunization, blood was collected to detect antibody titers in the mice. Three days before cell fusion, 100 μg of recombinant protein was administered via intraperitoneal pulse.

[0073] 3. Cell fusion and hybridoma screening

[0074] Under aseptic conditions, mouse spleens were harvested, and a suspension rich in B cells was prepared. Cells were then fused with SP2 / 0 cells using the classic PEG (Sigma) method. The fused cells were resuspended in HAT medium and cultured. On days 5 and 10 post-fusion, the medium was partially replaced with fresh HAT medium. Immunofluorescence was performed on days 11-15 post-fusion to screen for positive clones.

[0075] Immunofluorescence screening was performed using 96-well plates. In short, HeLa cells overexpressing GP350 were seeded at a density of 20,000 cells / well in the plates. The next day, 50 μl of hybridoma supernatant was collected and added to the 96-well plates. After incubation, the cells were washed twice, fixed with paraformaldehyde for 5 minutes, washed twice with PBS, and then incubated with 594-fluorescently labeled goat anti-mouse IgG antibody. After 1 hour of incubation, the plates were washed three times, and the results were observed under a fluorescence microscope. Based on the immunofluorescence results, four optimal hybridoma clones were finally identified (named 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ... # 15 # ,twenty two # 36 # ()( Figure 1 (This information is used for subsequent experiments such as sequence cloning and affinity analysis.)

[0076] 4. Sequencing analysis of specific antibodies

[0077] Hybridoma antibody variable region sequence cloning: Optimal hybridoma clones in logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen) and reverse transcribed (PrimeScript™ Reverse Transcriptase, Takara). The cDNA obtained from reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen) and then sequenced to obtain the variable region sequences of the heavy and light chains. The CDR sequences of the variable regions of the heavy and light chains and the single-chain antibody sequences (scFv) are shown in Table 1.

[0078] Table 1. CDR sequences and single-chain antibody sequences contained in the heavy and light chain variable regions of mouse monoclonal antibodies.

[0079]

[0080]

[0081]

[0082]

[0083] Example 2: Expression, purification, and reactivity of single-chain antibody (scFv) with antigen

[0084] A single-chain antibody (scFv) expression platform was constructed based on a eukaryotic expression vector. VH and VL gene fragments from hybridoma clones 1#, 15#, 22#, and 36# were tandemly constructed into the pcDNA3.1 vector using a (G4S) 3-linker. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system. SDS-PAGE analysis showed that the size of the single-chain antibody (scFv) conformed to the theoretical molecular weight. Figure 2 Single-chain antibody 1 # scFv, 15 # scFv, 22 # scFv, 36 # The amino acid sequence of scFv is shown in Table 1. It encodes single-chain antibody 1. # scFv, 15 # scFv, 22 # scFv, 36 # The nucleotide sequence of scFv is shown in Table 2.

[0085] Table 2. Nucleotide sequences encoding single-chain antibodies

[0086]

[0087]

[0088]

[0089] Table Note: The nucleotide sequences marked with a single underscore "" are the nucleotide sequences encoding the heavy chain variable region in single-chain antibodies, while the wavy lines... The labeled nucleotide sequence is the nucleotide sequence encoding the light chain variable region in a single-chain antibody.

[0090] To assess the reactivity of single-chain antibodies with antigens, 200 ng / well of GP350 recombinant protein was pre-coated onto an ELISA plate, incubated overnight at 4°C, and then the plate was blocked. Different amounts of recombinant antibody (dilution: 10) were then added. 2 ~10 -5 μg / mL), added secondary antibody for washing, color development, and termination of the reaction, and the optical density (OD450) at 450 nm was measured using a microplate reader. The binding capacity was determined using a four-parameter nonlinear regression curve fitting. Results showed that GP350 binds to four single-chain antibodies (1 μg / mL). # scFv, 15 # scFv, 22 # scFv, 36 # Both scFv and GP350 recombinant protein exhibit high specificity binding. Figure 3 ).

[0091] Example 3: Allogeneic γδT cell expansion and CAR-γδT cell construction

[0092] 1. Extraction and expansion of γδT cells

[0093] Collect 10 mL of peripheral blood from a healthy adult donor into a sterile anticoagulant tube. First, dilute the blood 1:1 with an equal volume of PBS and mix by inverting the tube. Add 3 mL of lymphocyte separation medium to each separation tube and centrifuge at 1500 rpm for 1 min at room temperature. After centrifugation, add the diluted blood to the separation tube and centrifuge at 1500 rpm for 15 min at room temperature (both speed settings are 2). The centrifuged tube will separate into three layers: the top layer is plasma, the middle layer is a ring of milky white cells (lymphocytes), and the bottom layer is red blood cells. Insert the pipette tip into the white layer and slowly aspirate the lymphocytes into a new 15 mL centrifuge tube. Add an appropriate amount of PBS to the tube, mix well, and centrifuge at 1500 rpm for 5 min. Discard the supernatant, resuspend the cells in an appropriate amount of PBS, mix by inverting the tube, and centrifuge at 1500 rpm for 5 min. Repeat the washing process once. After discarding the supernatant, resuspend the cells in an appropriate amount of RPMI 1640 complete medium (serum inactivated) containing zoledronic acid at a concentration of 1.00 μg / ml to 4 μg / ml, and then count the lymphocytes using a cell counter. Adjust the count to 1 × 10⁻⁶ cells / ml after completion. 6 A concentration of 10 cells / mL was added to the cell culture system to stimulate the proliferation and activation of γδT cells.

[0094] 2. Construction of CAR-γδT cells

[0095] (1) Construction of antigen-binding receptor GP350-CAR

[0096] Select 4 candidate single-chain antibody sequences (1 # scFv, 15 # scFv, 22 # scFv, 36 # The antigen-binding receptor GP350-CAR was constructed using scFv, with the following structure: CD8αsignal peptide-GP350(scFv)-CD8αhinge-CD28αTm-4-1BB-CD3ζ-P2A-EGFP.

[0097] (2) Preparation of CAR-γδT cells

[0098] HEK293T cells were used as lentiviral packaging cells. Lentiviral packaging was performed using a three-plasmid packaging system (psPAX2, pMD2.G, CAR-γδT vector). After 48 hours, the supernatant viral solution was collected and concentrated by ultracentrifugation. RetroNectin protein was pre-coated and added to the culture medium containing γδT cells along with the concentrated viral solution for γδT cell infection. After 48 hours of infection, flow cytometry analysis showed that the transfection efficiency of the four GP350 CAR-γδT cell lines was above 80.00%. Four GP350-targeting CAR-γδT cell lines were obtained, namely GP350 1... # CAR-γδT cells, GP350 15 # CAR-γδT cells, GP350 22 # CAR-γδT cells, GP350 36 # CAR-γδT cells.

[0099] The technical solution of the present invention will be further explained through experiments below. The antibody 1 detected in the following experimental examples... # scFv, 15 # scFv, 22 # scFv, 36 # All scFv cells were prepared using the method described in Example 2; four types of CAR-γδT cells targeting GP350 were prepared using the method described in Example 3.

[0100] Experimental Example 1: Analysis of antibody binding to endogenously expressed GP350 cells

[0101] I. Experimental Methods

[0102] The C666-1 human nasopharyngeal carcinoma cell line, which expresses GP350 positively, was selected and compared with 1... # scFv, 15 # scFv, 22 # scFv, 36 # scFv antibodies were analyzed by flow cytometry.

[0103] II. Experimental Results

[0104] The results are as follows Figure 4 As shown, C666-1 cells expressing GP350 were able to bind to four GP350 single-chain antibodies (scFv), resulting in almost 100% positive staining. This indicates that the four GP350 single-chain antibodies (1 # scFv, 15 # scFv, 22 # scFv, 36 # Both scFv and scFv can specifically recognize GP350 molecules expressed endogenously in cells.

[0105] Experiment Example 2: Affinity of Four GP350 Antibodies

[0106] I. Experimental Methods

[0107] Surface plasmon resonance experiments were used to verify four single-chain antibodies (1 # scFv, 15 # scFv, 22 # scFv, 36 # The affinity of scFv for GP350 antigen.

[0108] II. Experimental Results

[0109] The results are shown in Table 2: Biacore data indicate that four single-chain antibodies against GP350 (1 # scFv, 15 # scFv, 22 # scFv, 36 # All scFvs can bind to the GP350 antigen. Among them, 22 has the highest binding capacity. # scFv, KD is 1.26×10 -10 Then 15 # scFv(2.17×10 -9 ), 36 # scFv(4.85×10 -8 ),1 # scFv(1.12×10 -8 Furthermore, the dissociation rates of the four antibodies were relatively slow. Therefore, among the four GP350 single-chain antibodies, 22... #scFv is the preferred antibody with the strongest affinity.

[0110] Example 3: Evaluation of the in vitro killing effect of allogeneic CAR-γδT cells on target cells.

[0111] I. Experimental Methods

[0112] GP350 + C666-1 cells were added at a rate of 5000 cells per well (100 μL / well, replicates) to both a label-free killer detection instrument (RTCA) 96-well plate and a regular 96-well cell culture plate. The RTCA 96-well plates were placed in the instrument and cultured until the Cell Index value was between 1.0 and 2.0. Then, the four types of GP350-targeting CAR-γδT cells prepared in Example 3 were mixed with GP350 at an effector-to-target ratio of 2:1. + C666-1 cells were co-incubated with CAR-γδT cells, and the RTCA monitoring time was 65 h to evaluate the killing activity of CAR-γδT cells.

[0113] II. Experimental Results

[0114] The results are as follows Figure 5 As shown, all four types of GP350 CAR-γδT cells exhibited good killing activity against C666-1 cells, among which 22... # CAR-γδT cells and 15 # CAR-γδT cells have strong cytotoxic activity, 22 # CAR-γδT cells exhibit the strongest cytotoxic activity.

[0115] Experiment Example 4: Anti-tumor experiment using a xenograft mouse model

[0116] This experiment uses a xenograft mouse model to evaluate the in vivo antitumor activity of GP350-targeting CAR-γδT cells (four types of GP350 CAR-γδT cells prepared in Example 3).

[0117] I. Experimental Methods

[0118] 1. EBV-positive nasopharyngeal carcinoma mouse model

[0119] A mouse model of EBV-positive nasopharyngeal carcinoma was established using the C666-1 cell line for evaluation. 1×10⁶ cells were subcutaneously injected into the right hind limb of NCG mice. 6 One C666-1-mCherry.ffLuc cell.

[0120] 2. Experimental Grouping

[0121] Five days after NCG mice were injected with C666-1 cells, the mice were randomly divided into 6 groups of 5 mice each. These groups were: control group, γδT group, and 1... # -CAR-γδT group, 15 # -CAR-γδT group, 22 # -CAR-γδT group, 36 # -CAR-γδT group. The control group received PBS, while the γδT group received 1×10 7 1 NT-γδT cell, 1 # The drug in the CAR-γδT group was 1×10 7 GP3501 # CAR-γδT cells, 15 # The drug in the CAR-γδT group was 1×10 7 GP350 15 # CAR-γδT cells, 22 # The drug in the CAR-γδT group was 1×10 7 GP350 22 # CAR-γδT cells, 36 # The drug in the CAR-γδT group was 1×10 7 GP35036 # CAR-γδT cells. The drug was administered via tail vein injection. The growth and survival of mice were observed daily, and the mice were weighed and tumor size was measured every 3 days.

[0122] When the tumor volume in mice reaches 1500 mm 3 If obvious ulceration occurs on the surface of the tumor, the mouse is euthanized, and the animal experiment is terminated.

[0123] 3. Calculation of tumor volume

[0124] The average volume of the transplanted tumor is calculated using the formula V = 1 / 2(L × W2), where L represents the length of the tumor and W represents the width of the tumor.

[0125] 4. Calculation of tumor inhibition rate

[0126] Calculate using the following formula:

[0127] Tumor inhibition rate (%) = [1 - (tumor volume in experimental group / tumor volume in control group)] × 100%.

[0128] 5. Data Processing

[0129] Data analysis was performed using one-way ANOVA. ns indicates no statistical difference, **** indicates P < 0.0001, and * indicates P < 0.05.

[0130] II. Experimental Results

[0131] The results are as follows Figure 6 As shown, compared with the control group, the tumor size of the four GP350 CAR-γδT groups was significantly reduced; the tumor inhibition rate was significantly higher than that of the control group and the γδT group. This confirms that GP350 CAR-γδT has a significant inhibitory effect on the growth of C666-1 cells, with GP350 15... # CAR-γδT cells and GP350 22 # CAR-γδT showed the best inhibitory effect.

[0132] As can be seen from the above embodiments and experimental examples, the present invention provides an antibody or antigen-binding fragment targeting GP350. Using the screened GP350-targeting antibody as the antigen-binding domain, a chimeric antigen receptor was constructed, and CAR-γδT cells targeting GP350 were prepared using γδT cells isolated from healthy donor peripheral blood. In vitro and in vivo experiments showed that CAR-γδT cells targeting GP350 effectively targeted EBV-positive nasopharyngeal carcinoma cells (GP350). + It exhibits significant cytotoxic activity. This invention can be applied to the diagnosis of EBV-positive diseases and has broad prospects in the preparation of products for the detection, prevention, and / or treatment of GP350-positive tumors.

Claims

1. An antibody or antigen-binding fragment thereof targeting GP350, characterized in that: It includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes three complementary determinant regions CDRH1, CDRH2, and CDRH3, and the light chain variable region includes three complementary determinant regions CDRL1, CDRL2, and CDRL3. The amino acid sequence of CDRH1 is selected from any one of SEQ ID No. 1, SEQ ID No. 10, SEQ ID No. 19, and SEQ ID No. 28; The amino acid sequence of CDRH2 is selected from any one of SEQ ID No. 2, SEQ ID No. 11, SEQ ID No. 20, and SEQ ID No. 29; The amino acid sequence of CDRH3 is selected from any one of SEQ ID No. 3, SEQ ID No. 12, SEQ ID No. 21, and SEQ ID No. 30; The amino acid sequence of CDLH1 is selected from any one of SEQ ID No. 4, SEQ ID No. 13, SEQ ID No. 22, and SEQ ID No. 31; The amino acid sequence of CDLH2 is selected from any one of SEQ ID No. 5, SEQ ID No. 14, SEQ ID No. 23, and SEQ ID No. 32; The amino acid sequence of the CDLH3 is selected from any one of SEQ ID No. 6, SEQ ID No. 15, SEQ ID No. 24, and SEQ ID No.

33.

2. The antibody targeting GP350 or its antigen-binding fragment according to claim 1, characterized in that: The combination of the heavy chain variable region and the light chain variable region includes any one of the following combinations: The amino acid sequences are CDRH1, CDRH2 and CDRH3 as shown in SEQ ID No. 1 to 3, and CDRL1, CDRL2 and CDRL3 as shown in SEQ ID No. 4 to 6, respectively. Alternatively, the amino acid sequences are as shown in SEQ ID No. 10-12, CDRH1, CDRH2 and CDRH3, and the amino acid sequences are as shown in SEQ ID No. 13-15, CDRL1, CDRL2 and CDRL3. Alternatively, the amino acid sequences are as shown in SEQ ID No. 19–21, CDRH1, CDRH2, and CDRH3, and the amino acid sequences are as shown in SEQ ID No. 22–24, CDRL1, CDRL2, and CDRL3, respectively. Alternatively, the amino acid sequences are as shown in SEQ ID No. 28–30, CDRH1, CDRH2, and CDRH3, and the amino acid sequences are as shown in SEQ ID No. 31–33, CDRL1, CDRL2, and CDRL3.

3. The antibody targeting GP350 or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region and the light chain variable region further include a skeleton region, and the combination of the heavy chain variable region and the light chain variable region includes any one of the following combinations: The amino acid sequences are VH and VL as shown in SEQ ID No. 7 and 8, respectively. Alternatively, the amino acid sequences are VH and VL as shown in SEQ ID No. 16 and 17, respectively. Alternatively, the amino acid sequences are VH and VL as shown in SEQ ID No. 25 and 26, respectively. Alternatively, the amino acid sequences are VH and VL as shown in SEQ ID No. 34 and 35, respectively; And / or, the heavy chain variable region and the light chain variable region are linked by three repeating GGGGS amino acid fragments.

4. A nucleic acid, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-3.

5. A recombinant vector, characterized in that: It contains the nucleic acid as described in claim 4.

6. A host cell, characterized in that, It contains the recombinant vector as described in claim 5.

7. The method for preparing the antibody targeting GP350 or its antigen-binding fragment according to any one of claims 1-3, characterized in that, It includes: The gene fragment encoding the antibody targeting GP350 or its antigen-binding fragment is inserted into a recombinant vector, transformed into host cells for expression, and then isolated and purified to obtain the final product.

8. A chimeric antigen receptor, characterized in that: The antigen-binding domain of the chimeric antigen receptor includes an antibody or antigen-binding fragment thereof targeting GP350 as described in any one of claims 1-3.

9. A CAR-γδT cell, characterized in that: It includes the chimeric antigen receptor as described in claim 8.

10. Use of the antibody targeting GP350 according to any one of claims 1-3 or its antigen-binding fragment, the nucleic acid according to claim 4, the recombinant vector according to claim 5, the host cell according to claim 6, the chimeric antigen receptor according to claim 8, and the CAR-γδT cell according to claim 9 in the preparation of products for detecting, preventing, and / or treating GP350-positive tumors; The product includes at least one of the following: testing reagents, testing kits, microfluidic chips, immune cells, and drugs.

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