Preparation method and application of CAR-γδT cells targeting B7-H3

Abstract

The present invention discloses a preparation method and application of CAR-γδT cells targeting B7-H3, and relates to the field of biomedicine. The present invention provides a monoclonal antibody or a functional fragment thereof that specifically binds to human B7-H3, which can specifically bind to human B7-H3 antigen with good affinity. After humanization, the antibody affinity can be measured to show that the antibody affinity is within 10 ‑9 ~10 ‑8 In addition, the antibodies obtained by screening are used as antigen binding domains to construct chimeric antigen receptors, and γδT cells isolated from the peripheral blood of healthy donors are used to prepare allogeneic CAR-γδT cells, which have obvious killing activity against B7-H3 positive acute myeloid leukemia cell lines. The present invention can be used to prepare drugs for diagnosis, prevention and treatment of human tumors.

Description

Technical Field

[0001] The present invention relates to biomedicine, and in particular to a preparation method and application of CAR-γδT cells targeting B7-H3. Background Art

[0002] B7-H3 (also known as CD276) is a human cell surface molecule and one of the new members of the B7 family. It plays an important role in immunoregulation and anti-tumor immunity. It is a member of the B7 family, including B7-1 (CD80), B7-2 (CD86), PD-L1 (CD274), PD-L2 (CD273), etc. The role of B7-H3 is similar to that of other immune checkpoint molecules, and it can play a key role in T cell activation and immune response. B7-H3 is usually highly expressed in tumors, inflammation, and autoimmune diseases. It can regulate the activity and proliferation of T cells by binding to T cell ligands (such as CD28 or CTLA-4). At the same time, B7-H3 can also directly participate in regulating the growth and metastasis of tumor cells, affecting the immune response and escape mechanism of the tumor microenvironment. Therefore, B7-H3 has become a hot target in the field of tumor treatment. At present, a variety of B7-H3 antibodies, CAR-T and other biological agents are under development to enhance the effect of tumor immunotherapy.

[0003] γδT cells are a subset of T cells in the immune system that express a T cell receptor (TCR) composed of γ and δ chains, unlike the more common αβT cells, which express a TCR composed of α and β chains. γδT cells are distributed in many tissues of the body, including the skin, intestines, and lungs, and participate in various immune responses, such as fighting infection and regulating inflammation. They are also thought to play a role in recognizing and destroying cancer cells. Compared with αβT cells, γδT cells have more limited TCR diversity and their functions are more similar to innate immunity. They can recognize antigens in a way that does not require the mediation of major histocompatibility complex (MHC) molecules, which is the main way αβT cells recognize antigens. In contrast, γδT cells can recognize a variety of antigens, including lipids and stress-induced molecules. Research on γδT cells is ongoing and has potential implications for the development of new immunotherapies for cancer and infectious diseases.

[0004] With the development of adoptive cellular therapy (ACT), autologous chimeric antigen receptor T (CAR-T) cell therapy has shown good anti-tumor effects, improved the remission rate and survival rate of patients with various hematological tumors, and promoted the development of other immune cell therapies. To date, the most successful CAR-T cell therapy is the treatment of patients with relapsed or refractory CD19-positive acute lymphoblastic leukemia with autologous CD19-CAR-T cells. Multiple research institutions have developed a variety of different CD19-CAR-T structures and preparation methods, and their efficacy is significant. CAR-γδT is a genetically modified γδT cell with the ability to target cancer cells. CAR stands for chimeric antigen receptor, which is constructed by combining the domain of an antibody with the signal transduction domain of a T cell receptor (TCR). CAR-γδT cells can recognize and bind to antigens on the surface of cancer cells through the domain of CAR, thereby killing these cancer cells. Compared with αβT cells, γδT cells have a wider range of ligand specificity and higher killing activity, and are therefore considered to be a better cell therapy carrier. At the same time, given that they can recognize antigens in a manner that does not require the mediation of major histocompatibility complex (MHC) molecules, more research is also devoted to developing them as an allogeneic cell therapy product. In addition, γδT cells have relatively low sensitivity to immunosuppressive factors in the tumor microenvironment, so they have great potential in treating tumors.

[0005] Currently, CAR-γδT cells have shown good anti-tumor effects in in vitro and in vivo experiments and have been preliminarily verified in clinical trials. Although the research on CAR-γδT cells is still in its early stages, it has been considered a potential tumor immunotherapy strategy that can provide better treatment options for cancer patients.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a preparation method and application of CAR-γδT cells targeting B7-H3.

[0008] The present invention is achieved in that:

[0009] In the first aspect, an embodiment of the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region include the CDRs shown in any one of (1) to (2); (1) the amino acid sequences of HCDR1 to 3 are sequentially shown in SEQ ID Nos. 1 to 3, and the amino acid sequences of LCDR4 to 6 are sequentially shown in SEQ ID Nos. 4 to 6; (2) the amino acid sequences of HCDR10 to 12 are sequentially shown in SEQ ID Nos. 10 to 12, and the amino acid sequences of LCDR13 to 15 are sequentially shown in SEQ ID Nos. 13 to 15.

[0010] In a second aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the above embodiments.

[0011] In a third aspect, an embodiment of the present invention provides a recombinant vector comprising the isolated nucleic acid as described in the preceding embodiments.

[0012] In a fourth aspect, an embodiment of the present invention provides a host cell comprising the recombinant vector described in the preceding embodiment.

[0013] In a fifth aspect, an embodiment of the present invention provides a method for preparing an anti-B7-H3 antibody or an antigen-binding fragment thereof, which comprises: culturing the host cell as described in the above embodiment to obtain the anti-B7-H3 antibody or an antigen-binding fragment thereof.

[0014] In a sixth aspect, an embodiment of the present invention provides a chimeric antigen receptor, wherein the antigen binding domain of the chimeric antigen receptor comprises the anti-B7-H3 antibody or antigen binding fragment thereof as described in the above embodiments.

[0015] In a seventh aspect, an embodiment of the present invention provides a CAR-γδT cell, comprising: a chimeric antigen receptor as described in the above embodiment.

[0016] In an eighth aspect, embodiments of the present invention provide the use of an anti-B7-H3 antibody or antigen-binding fragment thereof as described in the preceding embodiments, or an isolated nucleic acid as described in the preceding embodiments, or a recombinant vector as described in the preceding embodiments, or a host cell as described in the preceding embodiments, or a chimeric antigen receptor as described in the preceding embodiments, or a CAR-γδT cell as described in the preceding embodiments in detecting B7-H3 antigen for non-disease diagnosis purposes and / or treatment purposes, or in preparing a product for detecting B7-H3 antigen, or in preparing a product for preventing or treating tumors.

[0017] In the ninth aspect, an embodiment of the present invention provides a cell injection solution, which includes: an anti-B7-H3 antibody or an antigen-binding fragment thereof as described in the preceding embodiment, or an isolated nucleic acid as described in the preceding embodiment, or a recombinant vector as described in the preceding embodiment, or a host cell as described in the preceding embodiment, or a chimeric antigen receptor as described in the preceding embodiment, or a CAR-γδT cell as described in the preceding embodiment.

[0018] The present invention has the following beneficial effects:

[0019] The anti-B7-H3 antibody screened by the hybridoma technology platform of the present invention can bind to the B7-H3 antigen with high specificity and good affinity, with KD≤10 -9 ~10 -8 M, belongs to high affinity antibody.

[0020] In addition, the anti-B7-H3 antibody or its antigen-binding fragment provided by the present invention can be used as an antigen-binding domain to construct a chimeric antigen receptor, and the prepared CAR-γδT cells have obvious killing activity against B7-H3-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 To identify positive reaction clones using ELISA;

[0023] Figure 2 The purified recombinant chimeric antibody was identified by SDS-PAGE;

[0024] Figure 3 To identify the reactivity of recombinant antibodies with antigens by ELISA;

[0025] Figure 4 Identification of the specific binding of B7-H3 antibody to AML natural target cells;

[0026] Figure 5 SDS-PAGE was used to identify the antibody-hFc fusion protein expressed and purified after humanization;

[0027] Figure 6 In vitro co-culture of B7-H3 CAR-γδT cells and AML natural cells;

[0028] Figure 7The anti-tumor activity of CAR-γδT cells was verified in the MV411 xenograft model. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0030] In one aspect, an embodiment of the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the CDRs shown in any one of (1) to (2);

[0031] (1) HCDR1 to 3 with amino acid sequences as shown in SEQ ID Nos. 1 to 3, and LCDR4 to 6 with amino acid sequences as shown in SEQ ID Nos. 4 to 6;

[0032] (2) The amino acid sequences of HCDR10 to 12 are shown in SEQ ID Nos. 10 to 12, and the amino acid sequences of LCDR13 to 15 are shown in SEQ ID Nos. 13 to 15.

[0033] It has been verified that the antibody or antigen-binding fragment thereof provided by the present invention can be used for -9 ~10 -8 M binds to the B7-H3 antigen with high affinity.

[0034] In some embodiments, the heavy chain variable region and light chain variable region further comprise a framework region.

[0035] In the present invention, "framework region" or "FR" region refers to the region other than CDR in the heavy chain variable region and light chain variable region of an antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by LCDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (arranged from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (arranged from the amino terminus to the carboxyl terminus): LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0036] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 7 to 8, respectively.

[0037] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 16-17, respectively.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0039] In some embodiments, the constant region includes a heavy chain constant region and / or a light chain constant region. The light chain of a full-length antibody includes a light chain variable region domain VL and a constant region domain CL, VL is at the amino terminus of the light chain, the CL domain is at the carboxyl terminus, and the light chain includes a κ chain or a λ chain; the heavy chain of a full-length antibody includes a heavy variable region domain VH and a constant region CH, VH is at the amino terminus of the heavy chain, and the CH domain is at the carboxyl terminus.

[0040] In some embodiments, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0041] In some embodiments, the species of origin of the constant region is cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0042] In some embodiments, the antibody is selected from any one of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a murine antibody, a chimeric antibody, and a full-length antibody.

[0043] In some embodiments, the antigen-binding fragment is selected from the F(ab') 2 , Fab', Fab, Fv and scFv.

[0044] In some embodiments, when the antigen-binding fragment is scFv, the amino acid sequence of scFv is shown in any one of SEQ ID NO: 9, 18, 21, and 24.

[0045] "Antigen binding fragment" is a portion of an intact antibody that specifically binds to an antigen to which the intact antibody binds. It is readily understood by those skilled in the art based on the contents described in the present invention that antigen binding fragments can be prepared by methods known in the art, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds, or by recombinant genetics techniques or by automatic peptide synthesizers (such as Applied BioSystems' automatic peptide synthesizers).

[0046] On the other hand, an embodiment of the present invention further provides an isolated nucleic acid, characterized in that it encodes the antibody or antigen-binding fragment thereof as described in any of the above embodiments.

[0047] On the other hand, an embodiment of the present invention further provides a recombinant vector comprising the isolated nucleic acid as described in any of the above embodiments.

[0048] In some embodiments, the recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which may refer to any recombinant polynucleotide construct, which may directly or indirectly (such as packaged into a virus) introduce the target DNA fragment into a host cell by means of transformation, transfection or transduction, and express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can connect the target DNA fragment to the plasmid ring. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, slow virus, oncolytic virus). After these vectors enter the host cell, the target gene can be expressed.

[0049] On the other hand, an embodiment of the present invention further provides a host cell, which contains the recombinant vector as described in any of the above embodiments.

[0050] Specifically, the host cell includes at least one of a prokaryotic host cell, a eukaryotic host cell and a bacteriophage. The prokaryotic host cell may be Escherichia coli, Streptomyces or Bacillus subtilis, etc. The eukaryotic host cell may be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6, Saccharomyces cerevisiae, Pichia pastoris, Hansen yeast, Candida, some insect cells and plant cells. 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 of ordinary skill in the art.

[0051] On the other hand, an embodiment of the present invention further provides a method for preparing an anti-B7-H3 antibody or an antigen-binding fragment thereof, comprising: culturing the host cell as described in any of the preceding embodiments to obtain the anti-B7-H3 antibody or an antigen-binding fragment thereof.

[0052] Specifically, the present invention does not specifically limit the culture conditions of the host cells, and the culture conditions that enable the host cells to express and produce the anti-B7-H3 antibody can be obtained based on conventional technical knowledge.

[0053] On the other hand, an embodiment of the present invention further provides a chimeric antigen receptor, wherein the antigen binding domain of the chimeric antigen receptor comprises the anti-B7-H3 antibody or antigen binding fragment thereof as described in any of the aforementioned embodiments.

[0054] In some embodiments, the chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.

[0055] In some embodiments, the signaling domain comprises CD3ζ.

[0056] In some embodiments, the signal transduction domain further comprises a 4-1BB intracellular region.

[0057] On the other hand, an embodiment of the present invention further provides a CAR-γδT cell, which includes: a chimeric antigen receptor as described in any of the aforementioned embodiments.

[0058] The CAR-γδT cells prepared based on the antibodies or antigen-binding fragments thereof provided in the embodiments of the present invention show highly effective anti-tumor activity against B7-H3 positive tumors.

[0059] Specifically, the embodiments of the present invention also provide the use of an anti-B7-H3 antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, or an isolated nucleic acid as described in any of the preceding embodiments, or a recombinant vector as described in any of the preceding embodiments, or a host cell as described in any of the preceding embodiments, or a chimeric antigen receptor as described in any of the preceding embodiments, or a CAR-γδT cell as described in any of the preceding embodiments in detecting B7-H3 antigen for non-disease diagnosis purposes and / or treatment purposes, or in preparing a product for detecting B7-H3 antigen, or in preparing a product for preventing or treating tumors.

[0060] In some embodiments, the product includes at least one of: an immune cell, a reagent, a kit, a drug, and a pharmaceutical composition.

[0061] In some embodiments, the tumor comprises a B7-H3 positive tumor.

[0062] In some embodiments, the tumor comprises at least one of acute myeloid leukemia, chronic lymphocytic leukemia, glioma, lung cancer, breast cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, colorectal cancer and pancreatic cancer.

[0063] As used herein, "treating" includes preventing or alleviating a condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0064] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0065] In addition, an embodiment of the present invention further provides a cell injection solution, which includes: an anti-B7-H3 antibody or an antigen-binding fragment thereof as described in any of the preceding embodiments, or an isolated nucleic acid as described in any of the preceding embodiments, or a recombinant vector as described in any of the preceding embodiments, or a host cell as described in any of the preceding embodiments, or a chimeric antigen receptor as described in any of the preceding embodiments, or a CAR-γδT cell as described in any of the preceding embodiments.

[0066] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0067] Example 1 Preparation of B7-H3 recombinant protein

[0068] The purchased plasmid encoding human pMD18-B7-H3 was used as a template for PCR amplification. The extracellular domain (ECD) fragment was obtained by agarose gel electrophoresis. The pcDNA3.1 vector was used as a backbone to clone the extracellular domain of B7-H3 into a pcDNA3.1 expression vector with an Fc or His tag at the C-terminus. The Fc tag includes human Fc (hFc) and mouse Fc (mFc). Then, 293FT was transiently transfected and FreeStyle TM The cells were cultured in shake flasks in serum-free medium (Life Technologies) for 5-7 days, and the supernatant was collected and purified by Protein A / G or nickel column affinity chromatography and molecular sieve chromatography column to purify the recombinant B7-H3 protein carrying Fc or His tag.

[0069] Example 2 Cell fusion and hybridoma screening

[0070] (1) Animal immunization

[0071] 5-6 week old Balb / c female mice were used as immunized animals, and the immunization dose was 100 μg / mouse. For the first immunization, 100 μl of Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant B7-H3 protein, and multiple subcutaneous injections were performed after sufficient emulsification. After an interval of 2 weeks, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with the recombinant protein, and multiple subcutaneous injections were performed after sufficient emulsification. The booster immunization was performed 4 times in total. On the 10th day after the last booster immunization, blood was collected to detect the antibody titer of the mice. Three days before cell fusion, 100 μg of recombinant protein was intraperitoneally impacted once.

[0072] (2) Cell fusion and hybridoma screening

[0073] Under sterile conditions, mouse spleens were taken to prepare a suspension rich in B cells, and cells were fused with SP2 / 0 according to the classic PEG (Sigma) method. The fused cells were resuspended in HAT medium for culture. Fresh HAT medium was used for half-medium replacement culture on the 5th and 10th days after fusion. ELISA, immunofluorescence and flow cytometry were performed on the 11th to 15th day after fusion to screen positive clones.

[0074] ELISA screening was performed using a 96-well plate. Briefly, the B7-H3 recombinant protein was coated onto the bottom of the plate at 100 ng / well overnight at 4°C, and HRP-conjugated anti-mouse IgG antibody and chemiluminescent reagent (Beyotime Biotech) were used for color development, and the values ​​were read on a microplate reader at a wavelength of 450 nm.

[0075] According to the above ELISA analysis results, two optimal hybridoma clones (named B7-1 and B7-2, respectively) were finally determined ( Figure 1 ) for subsequent experiments such as sequence cloning and affinity analysis.

[0076] (3) Sequencing analysis of specific antibodies

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

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

[0079]

[0080]

[0081] Example 3 Expression, purification and reactivity of recombinant chimeric antibodies with antigens

[0082] A recombinant chimeric antibody expression platform was constructed based on a eukaryotic expression vector, and the VH and VL gene fragments of the hybridoma clones B7-1 and B7-2 were constructed on the pvax vector. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system. SDS-PAGE identification showed that the size of the recombinant chimeric antibody was consistent with the theoretical molecular weight of the light chain and heavy chain ( Figure 2 ).

[0083] To identify the reactivity of the recombinant chimeric antibody with the antigen, 200 ng / well of B7-H3 recombinant protein was coated on the ELISA plate in advance, the plate was blocked after overnight at 4°C, and different amounts of recombinant antibodies (dilution: 10 2 ~10 -5 μg / mL), added secondary antibody for washing, color development, terminated reaction, and measured the optical density (OD450) at 450nm using a microplate reader. The binding capacity was determined using a four-parameter nonlinear regression curve fitting. The results showed that the two recombinant chimeric antibodies of B7-H3 (B7-1 and B7-2) had high specificity binding to the B7-H3 recombinant protein ( Figure 3 ).

[0084] Example 4 Analysis of antibody binding to endogenously expressed B7-H3

[0085] Human acute myeloid leukemia cell lines MV411 and THP-1 with positive B7-H3 expression, as well as human lymphoma cells and human multiple myeloma cell lines with negative B7-H3 expression were selected for flow cytometry analysis. The results showed that MV411 and THP-1 cells with positive B7-H3 expression could be stained almost 100% positive with two B7-H3 recombinant chimeric antibodies (B7-1, B7-2), while human lymphoma cells and human multiple myeloma cell lines with negative B7-H3 expression were stained negative. This indicates that both B7-H3 recombinant chimeric antibodies can specifically recognize the B7-H3 molecules endogenously expressed in cells ( Figure 4 ).

[0086] Example 5 Humanization of B7-H3 antibody and its affinity determination

[0087] The B7-H3 monoclonal antibody obtained by screening in Example 2 was homologously compared and analyzed for its VH and VL genes. The antibody was automatically humanized using the BioPhi antibody humanization platform, and the CDR region of the original antibody was kept unchanged. The framework region (FR) of the humanized antibody was replaced in sequence to obtain the humanized antibody amino acid sequence. The designed humanized chimeric antibody was synthesized on the pcDNA3.1 vector, expressed and purified using the HEK293T eukaryotic protein expression system. After the expressed antibody was purified and tested by SDS-PAGE, it was found that the molecular weight of the humanized antibody was consistent with the theoretical value, and the ratio of VH and VL was moderate ( Figure 5 ).

[0088] Table 2 CDR sequences contained in the heavy and light chain variable regions of humanized monoclonal antibodies

[0089]

[0090]

[0091] The affinity of humanized monoclonal antibodies was verified by surface plasmon resonance, and it was determined that the highest binding was HuB7-1 (3.43×10 -9 ), followed by HuB7-2 (7.25×10 -8 ), and the dissociation rates of the two antibodies were also slow. In summary, the Biacore data showed that the two humanized monoclonal antibodies were successfully transformed, and the specific data of the detection are shown in Table 3.

[0092] Table 3 Affinity data summary of humanized antibodies

[0093] Humanized monoclonal antibodies Ka(1 / Ms) kd(1 / s) KD(M) HuB7-1 <![CDATA[2.15×10 5 ]]> <![CDATA[7.36×10 -4 ]]> <![CDATA[3.43×10 -9 ]]> HuB7-2 <![CDATA[4.77×10 5 ]]> <![CDATA[3.46×10 -4 ]]> <![CDATA[7.25×10 -8 ]]>

[0094] Example 6 Allogeneic γδT cell expansion and CAR-γδT cell construction

[0095] 10 mL of peripheral blood from healthy adult donors was collected in a sterile anticoagulant tube. First, the blood was diluted 1:1 with an equal volume of PBS and mixed by inverting. 3 mL of lymphocyte separation solution was added to each separation tube, centrifuged at room temperature at 1500 rpm for 1 min, and the diluted blood was added to the separation tube after removal, and centrifuged at room temperature at 1500 rpm for 15 min (the speed increase gear was adjusted to 2, and the speed decrease gear was adjusted to 2). The centrifuged separation tube was divided into three layers, the top layer was plasma, the milky white circle in the middle layer was lymphocytes, and the bottom layer was red blood cells; insert the gun tip into the white layer, slowly draw 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, add an appropriate amount of PBS to resuspend, mix well by inverting, and centrifuge at 1500 rpm for 5 min. Repeat the wash once. After discarding the supernatant, the cells were resuspended in an appropriate amount of RPMI1640 complete medium (serum inactivated) containing 1.00 μg / ml to 4 μg / ml of zoledronic acid, and the lymphocytes were counted using a cell counter. After counting, the cells were adjusted to an appropriate density and added to the cell culture system to stimulate the proliferation and activation of γδT cells.

[0096] According to Example 5, the humanized antibody sequence HuB7-1 with higher affinity is preferred to construct B7-H3-CAR, and the structure is as follows: CD8αsignal peptide-HuB7-1-CD8αhinge-CD28αTm-4-1BB-CD3ζ-P2A-EGFP. HEK293T cells were used as cells for lentiviral packaging, and lentiviral packaging was performed using a three-plasmid packaging system (psPAX2, pMD2.G, CAR-γδT vector). After 48 hours, the supernatant virus solution was collected, concentrated by ultracentrifugation, and then coated with RetroNectin protein in advance and concentrated virus was added to infect γδT cells. After 48 hours of infection, the transfection efficiency of CAR-γδT cells was evaluated by flow cytometry to be above 80.00%.

[0097] Example 7 Evaluation of the killing effect of allogeneic CAR-γδT cells on target cells in vitro

[0098] To evaluate the function of CAR-γδT cells (Example 6), B7-H3 CAR-γδT cells were co-cultured with MV411 cells, an AML antigen-positive cell line expressing luciferase, to determine the ability of CAR-γδT cells to lyse AML cells in an antigen-specific manner. Untreated γδT cells were used as controls (MockγδT cells), and cells were inoculated in 96-well plates at effect-target ratios of 1.25:1, 2.5:1, 5:1, and 10:1, respectively. IL-2 was not added to the culture system. After 24 hours of co-culture, the luciferase value was detected using a multifunctional enzyme labeling system to evaluate the killing effect of CAR-γδT cells on AML natural cells. The results showed that CAR-γδT cells killed target cells expressing B7-H3 antigen-positive in a dose-dependent manner, indicating that B7-H3 CAR-γδT has good killing activity ( Figure 6 ).

[0099] Example 8 Anti-tumor experiment in xenograft mouse model

[0100] A xenograft mouse model was used to evaluate the in vivo antitumor activity of CAR-γδT targeting B7-H3 (B7-H3 CAR-γδT in Example 6). The NCG mouse acute myeloid leukemia model was established using the MV411 cell line for evaluation. 5×10 6 The acute myeloid leukemia (AML) mouse tumor model was established with MV411-mCherry.ffLuc cells to verify the in vivo effect of B7-H3 CAR-γδT. On the 5th day after inoculation, NCG mice were randomly divided into 4 groups, 5 mice in each group, and injected with 1×10 7 Mock T cells, 1×10 7NT-γδT cells, 1×10 7 The B7-H3 CAR-γδT treatment group was treated with B7-H3 CAR-γδT, and a control group injected with PBS was set up. The growth status and survival of the mice were observed every day, and the survival period was recorded. The results showed that the B7-H3 CAR-γδT treatment group observed a significant delay in tumor progression, and the survival time of tumor-bearing mice was significantly prolonged ( Figure 7 ).

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-B7-H3 antibody or an antigen-binding fragment thereof, characterized in that: It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the CDRs shown in any one of (1) to (2); (1) HCDR1 to 3 with amino acid sequences as shown in SEQ ID Nos. 1 to 3, and LCDR4 to 6 with amino acid sequences as shown in SEQ ID Nos. 4 to 6; (2) The amino acid sequences of HCDR10 to 12 are shown in SEQ ID Nos. 10 to 12, and the amino acid sequences of LCDR13 to 15 are shown in SEQ ID Nos. 13 to 15.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The light chain variable region and the heavy chain variable region also include a framework region; The heavy chain variable region and light chain variable region are as shown in any one of (a) to (b); (a) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID No. 7-8 respectively; (b) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID No. 16-17 respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof also includes a constant region.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

5. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody is selected from any one of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a mouse antibody, a chimeric antibody and a full-length antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of an antibody.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: When the antigen-binding fragment is scFv, the amino acid sequence of scFv is shown in any one of SEQ ID NOs: 9, 18, 21, and 24.

9. An isolated nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A recombinant vector, characterized in that: It contains the isolated nucleic acid as claimed in claim 9.

11. A host cell, characterized in that It contains the recombinant vector as claimed in claim 10, and the host cell is a non-plant cell.

12. A method for preparing an anti-B7-H3 antibody or an antigen-binding fragment thereof, characterized in that: It includes: Cultivate the host cell as claimed in claim 11 to obtain an anti-B7-H3 antibody or an antigen-binding fragment thereof.

13. A chimeric antigen receptor, characterized in that The antigen binding domain of the chimeric antigen receptor comprises the scFv as described in claim 7 or 8; The chimeric antigen receptor also includes a signal transduction domain, which includes CD3ζ.

14. The chimeric antigen receptor according to claim 13, characterized in that The signal transduction domain also includes the 4-1BB intracellular region.

15. A CAR-γδT cell, characterized in that: It comprises the chimeric antigen receptor according to claim 13 or 14.

16. Use of the chimeric antigen receptor according to claim 13 or 14 or the CAR-γδT cell according to claim 15 in the preparation of a product for treating tumors; The tumor is selected from at least one of acute myeloid leukemia, glioma, lung cancer, gastric cancer, and ovarian cancer.

17. The use according to claim 16, characterized in that The product includes at least one of: immune cells, reagents, kits, drugs and pharmaceutical compositions.

18. A cell injection solution, characterized in that: It includes the CAR-γδT cell as described in claim 15.

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