Preparation and use of GPR87-targeting allogeneic car-γδt cells

Abstract

The present invention relates to the field of biomedicine technology, and in particular, to preparation and use of GPR87-targeting allogeneic CAR-γδT cells. The antibody or an antigen-binding fragment thereof comprises CDRs in a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 6 or 14, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 7 or 15. The antibody or the antigen-binding fragment thereof is capable of specifically binding to the GPR87 antigen with good affinity. The antibody or the antigen-binding fragment thereof is used as an antigen-binding domain to construct a chimeric antigen receptor. The prepared CAR-γδT cells exhibit significant killing activity against GPR87-positive tumor cell lines, such as non-small cell lung cancer cells, and can be used in the preparation of a medicament for diagnosing, preventing, and treating GPR87-positive malignant tumors.

Description

Preparation and application of GPR87-targeted allogeneic CAR-γδT cells Technical Field

[0001] This disclosure relates to the biomedical field, and in particular to the preparation and application of GPR87-targeted allogeneic CAR-γδT cells. Background Technology

[0002] G protein-coupled receptor 87 (GPR87) is a membrane protein belonging to the G protein-coupled receptor (GPCR) family, primarily involved in cell signal transduction and regulation. Studies have shown that GPR87 is highly expressed in various malignant tumors (such as lung cancer, pancreatic cancer, and cervical cancer), and its activation is closely related to tumor proliferation, invasion, and drug resistance. Therefore, GPR87 has become an important tumor target, and its specific expression provides a potential precision targeting strategy for cancer therapy.

[0003] Currently, research on GPR87 mainly focuses on its functional mechanisms and the development of inhibitors. However, unlike traditional targets, the therapeutic potential of GPR87 has not been fully explored, especially in the field of immunocellular therapy. Therefore, designing an immunocellular therapy strategy that can target GPR87 will greatly promote the development of this field and provide cancer patients with more efficient and specific treatment options.

[0004] In view of this, this disclosure is made. Summary of the Invention

[0005] To address the aforementioned issues, the objectives of this disclosure include, for example, providing the preparation and application of GPR87-targeting allogeneic CAR-γδT cells.

[0006] To achieve the above objectives, this disclosure provides the following technical solution:

[0007] Embodiments of this disclosure provide an antibody against GPR87 or an antigen-binding fragment thereof, comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region and / or LCDR1, LCDR2 and LCDR3 in the light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6 or 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7 or 15.

[0008] Embodiments of this disclosure provide an isolated nucleic acid that encodes the anti-GPR87 antibody or its antigen-binding fragment as described in the foregoing embodiments.

[0009] Embodiments of this disclosure provide a recombinant vector containing the isolated nucleic acid as described in the foregoing embodiments.

[0010] Embodiments of this disclosure provide a host cell containing the recombinant vector as described in the foregoing embodiments.

[0011] The embodiments of this disclosure provide a method for preparing an antibody or an antigen-binding fragment thereof, which includes: culturing host cells as described in the foregoing embodiments.

[0012] Embodiments of this disclosure provide a chimeric antigen receptor, wherein the antigen-binding domain of the chimeric antigen receptor includes the antibody or its antigen-binding fragment described in the foregoing embodiments.

[0013] Embodiments of this disclosure provide a CAR-γδT cell that includes the chimeric antigen receptor as described in the foregoing embodiments.

[0014] The embodiments of this disclosure provide the use of antibodies or antigen-binding fragments thereof as described in the foregoing embodiments, or isolated nucleic acids as described in the foregoing embodiments, or recombinant vectors as described in the foregoing embodiments, or host cells as described in the foregoing embodiments, or chimeric antigen receptors as described in the foregoing embodiments, or CAR-γδT cells as described in the foregoing embodiments, in the preparation of products for the prevention or treatment of tumors and / or autoimmune diseases.

[0015] The embodiments of this disclosure provide a cell injection solution whose active ingredients include: an antibody or its antigen-binding fragment as described in the foregoing embodiments, or an isolated nucleic acid as described in the foregoing embodiments, or a recombinant vector as described in the foregoing embodiments, or a host cell as described in the foregoing embodiments, or a chimeric antigen receptor as described in the foregoing embodiments, or a CAR-γδT cell as described in the foregoing embodiments. Beneficial effects:

[0016] The embodiments of this disclosure provide an antibody against GPR87 or an antigen-binding fragment thereof, which can specifically bind to the GPR87 antigen and has good affinity.

[0017] The anti-GPR87 antibody or its antigen-binding fragment was used as the antigen-binding domain to construct a chimeric antigen receptor. The resulting CAR-γδT cells showed significant killing activity against GPR87-positive tumor cell lines, such as non-small cell lung cancer cells. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 shows the binding effect of immunofluorescence (IFA) on hybridoma supernatant and HeLa cells overexpressing GPR87 in an embodiment of this disclosure.

[0020] Figure 2 shows the SDS-PAGE identification of the purified recombinant chimeric antibody in an embodiment of this disclosure.

[0021] Figure 3 illustrates the use of ELISA to identify the reactivity of recombinant scFv single-chain antibody with antigen in an embodiment of this disclosure.

[0022] Figure 4 shows the flow cytometry analysis of the binding of recombinant scFv single-chain antibody to GPR87-positive tumor cells PC-9 in an embodiment of this disclosure.

[0023] Figure 5 shows the RTCA detection of the killing effect of allogeneic GPR87-CAR-γδT on target cells in an embodiment of this disclosure.

[0024] Figure 6 shows the in vivo antitumor activity of GPR87-CAR-γδT cells evaluated using an NCG mouse xenograft model. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of this disclosure, but not all embodiments.

[0026] Therefore, the following detailed description of the embodiments of this disclosure provided in the accompanying drawings is not intended to limit the scope of the claimed disclosure, but merely to illustrate selected embodiments of the disclosure. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure.

[0027] The embodiments of this disclosure are described in detail below with reference to the accompanying drawings; however, this disclosure may be implemented in a variety of different ways as defined and covered by the claims.

[0028] Embodiments of this disclosure provide an antibody or an antigen-binding fragment thereof, comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region and / or LCDR1, LCDR2 and LCDR3 in the light chain variable region;

[0029] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6 or 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7 or 15.

[0030] In some embodiments, the antibody or its antigen-binding fragment is an anti-GPR87 antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment exhibits high affinity and specificity for the GPR87 protein, and can avoid or reduce cross-reactions with other GPCRs in normal tissues. A chimeric antigen receptor is constructed using this GPR87-targeting antibody or its antigen-binding fragment as the antigen-binding domain, and CAR-γδT cells are prepared using γδT cells isolated from healthy donor peripheral blood. In vitro and in vivo experiments show that these CAR-γδT cells have significant killing activity against GPR87-positive cell lines such as non-small cell lung cancer cells (GPR87+). The antibody or the CAR-γδT cells constructed therefrom provided can be used to prepare drugs for the diagnosis, prevention, and treatment of GPR87-positive malignant tumors.

[0031] In some embodiments, HCDR1, HCDR2 and HCDR3 and / or LCDR1, LCDR2 and LCDR3 are defined by any one of the schemes Kabat, Chothia, AbM, Contact and IMGT or by a combination of multiple schemes.

[0032] In some embodiments, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:1-4, KVS, SEQ ID NO:5 or SEQ ID NO:9-12, KVS, SEQ ID NO:13, respectively.

[0033] In some embodiments, the antibody or its antigen-binding fragment further includes a backbone region, i.e., the antibody or its antigen-binding fragment includes the heavy chain variable region and / or the light chain variable region.

[0034] In this disclosure, the term "backbone region" is synonymous with "FR region," referring to the region of the antibody's heavy chain variable region excluding the CDR region. The heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4), which include the HFR1, HFR2, HFR3, and HFR4 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): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0035] In some embodiments, the antibody or its antigen-binding fragment further includes a constant region.

[0036] In some embodiments, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0037] In some embodiments, 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.

[0038] In some embodiments, the antibody is selected from any one of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, chimeric antibodies, and full-length antibodies.

[0039] In this document, "antigen-binding fragment" refers to a portion of a complete antibody that specifically binds to the antigen to which the complete antibody is bound. Those skilled in the art will readily understand from the contents of this disclosure that antigen-binding fragments can be prepared by methods known in the art, such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds, or by recombinant genetics techniques or by automated peptide synthesizers (such as Applied BioSystems' automated peptide synthesizers).

[0040] In some embodiments, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv.

[0041] In some embodiments, the antigen-binding fragment is scFv, and the amino acid sequence of the scFv is as shown in SEQ ID NO:8 or 16.

[0042] Embodiments of this disclosure provide an isolated nucleic acid that encodes the antibody or antigen-binding fragment thereof described in any of the foregoing embodiments.

[0043] Embodiments of this disclosure provide a recombinant vector containing isolated nucleic acids as described in any of the foregoing embodiments.

[0044] 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.

[0045] Embodiments of this disclosure provide a host cell containing the recombinant vector as described in any of the foregoing embodiments.

[0046] Specifically, the 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.

[0047] The embodiments of this disclosure provide a method for preparing an antibody or an antigen-binding fragment thereof, which includes: culturing host cells as described in any of the foregoing embodiments.

[0048] Specifically, this disclosure does not specifically limit the culture conditions of the host cells; culture conditions that enable the host cells to express and produce antibodies or their antigen-binding fragments can be obtained based on conventional technical knowledge.

[0049] Embodiments of this disclosure provide a chimeric antigen receptor, wherein the antigen-binding domain of the chimeric antigen receptor includes the antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0050] In some embodiments, the chimeric antigen receptor further includes any one or more of a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.

[0051] In some embodiments, the signal transduction structural domain includes CD3ζ.

[0052] In some embodiments, the signal transduction domain further includes a 4-1BB intracellular region.

[0053] Embodiments of this disclosure provide a CAR-γδT cell that includes a chimeric antigen receptor as described in any of the foregoing embodiments.

[0054] In some embodiments, the CAR-γδT cells include universal allogeneic CAR-γδT cells.

[0055] γδT cells are a special subset of T cells that lies between innate and adaptive immunity, attracting significant attention due to their unique antigen recognition mechanisms and tumor-killing capabilities. Unlike traditional αβT cells, γδT cells can directly recognize tumor-associated antigens without relying on the major histocompatibility complex (MHC), giving them a significant advantage in tumor immunotherapy.

[0056] The introduction of chimeric antigen receptors (CARs) has further enhanced the tumor-targeting ability of γδT cells. Traditional autologous CAR-T therapy faces significant challenges due to individual variability and high manufacturing costs. The development of allogeneic CAR-γδT cells offers a new approach to addressing this issue. By selecting γδT cells from healthy donors and combining them with GPR87-targeting CAR technology, highly homogeneous and ready-to-use immune cell products can be prepared.

[0057] The advantages of allogeneic CAR-γδT cells are: (1) overcoming the difficulties in collection and the delay in treatment time in autologous cell therapy; (2) reducing the risk of graft-versus-host disease (GvHD) through gene editing and other technologies; and (3) improving the accessibility and cost-effectiveness of treatment through mass production and standardized operation.

[0058] The embodiments of this disclosure provide the use of antibodies or antigen-binding fragments thereof as described in any of the foregoing embodiments, or isolated nucleic acids as described in any of the foregoing embodiments, or recombinant vectors as described in any of the foregoing embodiments, or host cells as described in any of the foregoing embodiments, or chimeric antigen receptors as described in any of the foregoing embodiments, or CAR-γδT cells as described in any of the foregoing embodiments, in the preparation of products for the prevention or treatment of tumors and / or autoimmune diseases.

[0059] In some embodiments, the tumor includes a GPR87-positive tumor.

[0060] In some embodiments, the tumor includes at least one of the following: glioma, neuroblastoma, medulloblastoma, meningioma, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, ureteral cancer, prostate cancer, skin cancer, melanoma, ovarian cancer, endometrial cancer, cervical cancer, soft tissue sarcoma, acute and chronic leukemia, Hodgkin and non-Hodgkin lymphoma, gastric cancer, or head and neck tumors.

[0061] In some embodiments, the lung cancer includes non-small cell lung cancer.

[0062] In some embodiments, the autoimmune disease includes at least one of lupus erythematosus, ankylosing spondylitis, multiple sclerosis, psoriasis, antiphospholipid syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune hepatitis, arthritis, rheumatoid arthritis, pemphigus, Guillain-Barré syndrome, Crohn's disease, vasculitis, or autoimmune diabetes.

[0063] In some embodiments, the product includes at least one of immune cells, reagents, kits, drugs, and drug compositions.

[0064] The embodiments of this disclosure provide a cell injection solution whose active ingredients include: the antibody or its antigen-binding fragment as described in any of the foregoing embodiments, or the isolated nucleic acid as described in any of the foregoing embodiments, or the recombinant vector as described in any of the foregoing embodiments, or the host cell as described in any of the foregoing embodiments, or the chimeric antigen receptor as described in any of the foregoing embodiments, or the CAR-γδT cell as described in any of the foregoing embodiments.

[0065] "Treatment" in this disclosure includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0066] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0067] To further illustrate this disclosure, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an anti-GPR87 antibody or its antigen-binding fragment, as well as CAR-γδT cells and related applications, but these should not be construed as limiting the scope of protection of this disclosure.

[0068] Example 1: Preparation of GPR87 recombinant protein

[0069] Using the GPR87 gene (NM_023915.3) synthesized by Qingke Biotechnology as a template, the full-length GPR87 gene was selected as the immune targeting fragment and cloned into the pFastBac gene carrying a His tag at the C-terminus. TM The protein was expressed in an expression vector. Then, it was expressed using an insect baculovirus expression system, and subsequently purified using a His tag.

[0070] Example 2: Cell Fusion and Hybridoma Screening

[0071] (1) 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 GPR87 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 the recombinant protein, thoroughly emulsified, and then injected subcutaneously at multiple sites. Four booster immunizations were administered. On day 10 after the last booster immunization, blood samples were collected to measure antibody titers. Three days before cell fusion, mice were administered a 100 μg intraperitoneal pulse of recombinant protein.

[0073] (2) 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. Positive clones were screened for immunofluorescence on days 11-15 post-fusion.

[0075] Immunofluorescence screening was performed using 96-well plates. In short, HeLa cells overexpressing GPR87 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, two optimal hybridoma clones were finally identified (named 1 and 2 respectively). # and 2 # (Figure 1), used for subsequent experiments such as sequence cloning and affinity analysis.

[0076] (3) 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 (scFv) contained in the heavy and light chain variable regions of mouse monoclonal antibodies.

[0079] Example 3: Expression, purification, and reactivity with antigens of recombinant chimeric antibodies and single-chain antibodies (scFv)

[0080] A recombinant chimeric antibody expression platform was constructed based on a eukaryotic expression vector, and hybridoma clone 1 was expressed separately. # and 2 # The VH and VL gene fragments were constructed into the pvax vector. The constructed expression vectors were expressed and purified using the HEK293T eukaryotic protein expression system. SDS-PAGE analysis showed that the size of the recombinant chimeric antibody conformed to the theoretical molecular weight of the light and heavy chains (Figure 2).

[0081] A recombinant single-chain antibody (scFv) expression platform was constructed based on a eukaryotic expression vector, and hybridoma clone 1 was expressed separately. # and 2 # The VH and VL gene fragments 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, and the sequence of the single-chain antibody is shown in Table 1.

[0082] To identify the reactivity of the single-chain antibody (scFv) with the antigen, 200 ng / well of GPR87 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 Add secondary antibody (μg / mL), wash, develop color, terminate the reaction, and measure the optical density (OD) at 450 nm using a microplate reader. 450 The binding affinity was determined by measuring the binding affinity using a four-parameter nonlinear regression curve. The results showed that both single-chain antibodies (scFv) of GPR87 had high specificity binding to the recombinant GPR87 protein (Figure 3).

[0083] Example 4: Analysis of antibody binding to endogenously expressed GPR87 in cells

[0084] GPR87-positive PC-9 human lung cancer cell lines were selected for flow cytometry analysis. The results showed that GPR87-positive PC-9 cells could specifically bind to two GPR87 single-chain antibodies (scFv). This indicates that both GPR87 single-chain antibodies (scFv) can specifically recognize endogenously expressed GPR87 molecules (Figure 4).

[0085] Example 5: Allogeneic γδT cell expansion and CAR-γδT cell construction

[0086] 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, the cells were resuspended 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 lymphocytes were counted using a cell counter. After counting, the cells were adjusted to a suitable density and added to the cell culture system to stimulate the proliferation and activation of γδT cells.

[0087] Two candidate single-chain antibody sequences (scFv) were selected to construct GPR87-CAR, with the following structure: CD8αsignal peptide-GPR87(scFv)-CD8αhinge-CD28αTm-4-1BB-CD3ζ-P2A-EGFP. HEK293T cells were used as the cells for lentiviral packaging. 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, concentrated by ultracentrifugation, pre-coated with RetroNectin protein, and then added to infect γδT cells. After 48 hours of infection, the transfection efficiency of GPR87 CAR-γδT cells was assessed by flow cytometry and found to be above 80.00%.

[0088] Example 6: Evaluation of the in vitro killing effect of allogeneic CAR-γδT cells on target cells

[0089] GPR87 + 5000 PC-9 cells per well, 100 μL / well (replicas), were added to both a label-free killer detection instrument (RTCA) 96-well plate and a regular 96-well cell culture plate. The RTCA 96-well plate was placed in the instrument and cultured until the Cell Index value was between 1.0 and 2.0. Then, the prepared GPR87 CAR-γδT cells were co-incubated at an effector-to-target ratio of 2:1. RTCA monitoring was performed for 65 h to evaluate their killing activity. The results are shown in Figure 5. Both GPR87 CAR-γδT cells showed strong killing activity against PC-9 cells, and 2# It has a stronger killing effect.

[0090] Example 7: Antitumor Experiment with Xenograft Mouse Model

[0091] The in vivo antitumor activity of GPR87-targeting CAR-γδT cells (GPR87 CAR-γδT cells in Example 6) was evaluated using a xenograft mouse model. A GPR87-positive non-small cell lung cancer cell model was established using the PC-9 cell line for evaluation.

[0092] 1×10⁻⁶ mice were subcutaneously injected into the right hind limb of NCG mice. 6 A mouse xenograft lung cancer model was established using PC-9-mCherry.ffLuc cells to verify the in vivo efficacy of GPR87 CAR-γδT. On day 5 post-inoculation, NCG mice were randomly divided into three groups of five mice each, and were injected via tail vein with 1×10⁷ NTγδT cells, 1×10⁷ CAR-γδT cells, and 1×10⁷ CAR-γδT cells, respectively. 7 GPR87 CAR-γδT(1 # 2 # Mice were divided into groups and a control group was set up that received PBS injections. The growth and survival of mice were observed daily, and the mice were weighed and tumor size measured every 3 days. The average tumor volume was calculated using the formula V = 1 / 2(L × W). 2 The calculation is performed, where L represents the length of the tumor and W represents the width of the tumor. When the mouse tumor volume reaches 1500 mm²... 3 If obvious ulceration appeared on the tumor surface, the mice were euthanized, and the animal experiment was terminated. The tumor inhibition rate of GPR87 CAR-γδT was calculated based on the tumor volume of the experimental and control groups. The calculation formula was: Tumor inhibition rate (%) = [1 - (tumor volume of experimental group / tumor volume of control group)] × 100%. The experimental results are shown in Figures A and B of Figure 6. Compared with the control group, the tumor volume of the two GPR87 CAR-γδT groups was significantly smaller than that of the control group (PBS and NTγδT), confirming that GPR87 CAR-γδT has a significant inhibitory effect on the growth of PC-9 cells. The data analysis was performed using one-way ANOVA. ns indicates no statistical difference, and **** indicates P < 0.0001 (Figure 6).

[0093] Although the above embodiments have provided a detailed description of this disclosure, they are only some embodiments of this disclosure, not all embodiments. People can obtain other embodiments based on this disclosure without creative effort, and these embodiments all fall within the protection scope of this disclosure. Industrial applicability

[0094] In summary, the embodiments of this disclosure provide an antibody or its antigen-binding fragment that specifically binds to the GPR87 antigen with good affinity. A chimeric antigen receptor was constructed using this antibody or its antigen-binding fragment as the antigen-binding domain, and the resulting CAR-γδT cells exhibited significant killing activity against GPR87-positive tumor cell lines, such as non-small cell lung cancer cells.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, It includes: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region and / or LCDR1, LCDR2 and LCDR3 in the light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6 or 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7 or 15.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The HCDR1, HCDR2, and HCDR3 and / or the LCDR1, LCDR2, and LCDR3 are defined by any one of the following schemes or a combination of multiple schemes: Kabat, Chothia, AbM, Contact, and IMGT. Optionally, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:1-4, KVS, SEQ ID NO:5 or SEQ ID NO:9-12, KVS, SEQ ID NO:13, respectively.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a backbone region; Optionally, the antibody or its antigen-binding fragment further includes a constant region; Optionally, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; Optionally, 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; Optionally, the antibody is selected from any one of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, chimeric antibodies, and full-length antibodies; Optionally, the antibody or its antigen-binding fragment is an antibody against GPR87 or its antigen-binding fragment.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv. Optionally, the antigen-binding fragment is scFv, and the amino acid sequence of the scFv is as shown in SEQ ID NO:8 or 16.

5. An isolated nucleic acid, characterized in that, Its encoding is the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4.

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

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

8. A method for preparing an antibody or its antigen-binding fragment, characterized in that, It includes: Culture the host cells as described in claim 7.

9. A chimeric antigen receptor, characterized in that, The antigen-binding domain of the chimeric antigen receptor includes the antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

10. The chimeric antigen receptor according to claim 9, characterized in that, The chimeric antigen receptor further includes a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain; Optionally, the signal transduction structure domain includes CD3ζ; Optionally, the signal transduction domain further includes a 4-1BB intracellular region.

11. A CAR-γδT cell, characterized in that, It includes the chimeric antigen receptor as described in claim 9 or 10.

12. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, or the isolated nucleic acid as described in claim 5, or the recombinant vector as described in claim 6, or the host cell as described in claim 7, or the chimeric antigen receptor as described in claim 9 or 10, or the CAR-γδT cell as described in claim 11, in the preparation of products for the prevention or treatment of tumors and / or autoimmune diseases.

13. The application according to claim 12, characterized in that, The tumors include GPR87-positive tumors; Optionally, the tumor includes at least one of the following: glioma, neuroblastoma, medulloblastoma, meningioma, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, ureteral cancer, prostate cancer, skin cancer, melanoma, ovarian cancer, endometrial cancer, cervical cancer, soft tissue sarcoma, acute and chronic leukemia, Hodgkin and non-Hodgkin lymphoma, gastric cancer, or head and neck tumors; Optionally, the lung cancer includes non-small cell lung cancer.

14. The application according to claim 12, characterized in that, The autoimmune diseases mentioned include at least one of the following: lupus erythematosus, ankylosing spondylitis, multiple sclerosis, psoriasis, antiphospholipid syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune hepatitis, arthritis, rheumatoid arthritis, pemphigus, Guillain-Barré syndrome, Crohn's disease, vasculitis, or autoimmune diabetes.

15. The application according to any one of claims 12 to 14, characterized in that, The products include at least one of the following: immune cells, reagents, kits, drugs, and drug compositions.

16. A cell injection solution, characterized in that, This includes the antibody or its antigen-binding fragment as described in any one of claims 1 to 4, the isolated nucleic acid as described in claim 5, the recombinant vector as described in claim 6, the host cell as described in claim 7, the chimeric antigen receptor as described in claim 9 or 10, or the CAR-γδT cell as described in claim 11.

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