B7-H3 targeting chimeric antigen receptor and application thereof in glioma treatment

Abstract

The invention discloses a B7-H3 targeting chimeric antigen receptor and application thereof in glioma treatment, and relates to the technical field of biological medicines, and the key point of the technical scheme is that the B7-H3 targeting chimeric antigen receptor is composed of a single-chain antibody region, a CD8alpha hinge region, a CD8alpha transmembrane region, a CD28 costimulatory factor region and a CD3zeta signal transduction region. The B7-H3 CAR constructed by the invention can more effectively kill glioma cells highly expressed by B7-H3, and can effectively control the progress of glioma in vivo.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, more specifically, it relates to a chimeric antigen receptor targeting B7-H3 and its application in glioma treatment. BACKGROUND

[0002] Glioma is a primary intracranial tumor caused by canceration of glial cells in the central nervous system, accounting for 40-50% of intracranial tumors. The annual incidence of glioma is 3-8 per 100,000 people, and it occurs in both adults and children. Glioma is divided into various types, and most types of glioma are malignant tumors, of which half of the glioblastoma is particularly serious, and the 5-year survival rate of patients is less than 5%. At present, the main treatment method for glioma is surgery combined with radiotherapy and chemotherapy. However, due to the location of the tumor in the brain, surgery cannot completely remove it, and there are not many chemotherapy drugs to choose from, so new treatment methods and means for glioma are urgently needed.

[0003] Chimeric antigen receptor-mediated T (CAR-T) cell therapy as a tumor immunotherapy has become a key method for cancer treatment, especially for hematological tumor treatment. It has shown excellent results in the clinical treatment of hematological tumors, making products targeting CD19, BCMA, etc. approved for listing and achieved good results in the real world. However, compared with the treatment of hematological tumors, CAR-T cell therapy has not yet achieved breakthrough results in the clinical treatment of solid tumors (brain tumors, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, etc.). The main problems it faces include the lack of effective CAR-T target points in solid tumors, the difficulty of CAR-T to reach the tumor site, and the immunosuppressive microenvironment of the tumor. Among them, the target point problem is the key to limiting the application of CAR-T in the treatment of solid tumors. The target points selected by many clinical studies of CAR-T treatment of solid tumors are different, and many target points have the problems of low expression level or frequency of tumor cells, high expression of healthy tissues, etc., resulting in poor treatment effect on tumors or serious side effects. Therefore, selecting a target point that is highly expressed in tumor cells and lowly expressed or not expressed in healthy tissues is particularly important for CAR-T in the treatment of solid tumors.

[0004] B7-H3 belongs to the B7 immunomodulator family as PD-L1 and PD-L2, and its ligand is not clear. TLT-2, IL20RA and PLA2R1 are reported in the literature as potential receptors of B7-H3. B7-H3 is a type I transmembrane protein, and its structure includes a long extracellular region, a transmembrane region and a short intracellular region. There are two subtypes of 2Ig-B7-H3 and 4Ig-B7-H3 in human cells, and 4Ig-B7-H3 is the main subtype. Early literature reports that B7-H3 can co-stimulate T cell proliferation and IFNgamma secretion, but currently it is mainly considered that B7-H3 is an immunosuppressive molecule that can inhibit the killing function of T cells and NK cells, and can inhibit graft-versus-host reaction and some autoimmune diseases in animal models. B7-H3 is highly expressed in various types of solid tumors (such as glioma, head and neck cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, etc.), and its expression in normal human tissues is very low, only weakly expressed in the basal membrane of the stomach, gallbladder, prostate, cervix and endometrium. At the same time, there are reports that B7-H3 is also highly expressed in tumor stem cells, tumor-related blood vessels and fibroblasts, and its level is closely related to the rapid progression of various cancers and poor clinical prognosis. Therefore, B7-H3 is a good target for the treatment of various solid tumors.

[0005] Currently, there are various drugs that target B7-H3 to treat solid tumors, including antibodies, ADCs, bi-specific antibodies, CAR-Ts, etc. Compared with other forms, CAR-T cells have the advantages of long drug efficacy duration, good efficacy, low immune rejection, etc., and can achieve better tumor treatment effect. Therefore, there are many CAR-T cells targeting B7-H3 in clinical research for the treatment of different types of solid tumors. However, the CAR-T targeting B7-H3 has not yet achieved a breakthrough effect in the treatment of most solid tumors, which is closely related to the antigen epitope targeted by B7-H3, the antibody affinity of the derived CAR, etc. Therefore, it is particularly important to develop new CAR-T targeting B7-H3 to improve the treatment effect. SUMMARY

[0006] In view of the deficiencies in the prior art, the purpose of the present application is to provide a chimeric antigen receptor targeting B7-H3 and its application in the treatment of glioma.

[0007] The above technical purpose of the present application is realized by the following technical scheme: a chimeric antigen receptor targeting B7-H3, which is composed of a single-chain antibody region, a CD8alpha hinge region, a CD8alpha transmembrane region, a CD28 co-stimulatory factor region and a CD3zeta signal transduction region. The amino acid sequence of the single-chain antibody region is shown in SEQ ID NO: 2. The amino acid sequence of the CD8 alpha hinge region is shown as SEQ ID NO: 11; The amino acid sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO: 12; The amino acid sequence of the CD28 co-stimulatory factor region is shown as SEQ ID NO: 13; The amino acid sequence of the CD3 zeta signal transduction region is shown as SEQ ID NO: 14.

[0008] Further, the single-chain antibody region comprises an antibody light chain, a GS linker, an antibody heavy chain, the amino acid sequence of the antibody light chain is shown as SEQ ID NO: 3; and the amino acid sequence of the antibody heavy chain is shown as SEQ ID NO: 7.

[0009] Further, the antibody light chain comprises three variable regions, which are a light chain CDR1, a light chain CDR2, and a light chain CDR3, respectively; the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO: 4; the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO: 5; and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO: 6.

[0010] Further, the antibody heavy chain comprises three variable regions, which are a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, respectively; the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO: 8; the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO: 9; and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO: 10.

[0011] A nucleic acid molecule capable of expressing the single-chain antibody region of the single-chain antibody region of the above-mentioned chimeric antigen receptor.

[0012] Further, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO: 1.

[0013] A retrovirus prepared from a retroviral vector and a packaging vector comprising the above-mentioned chimeric antigen receptor in 293T cells.

[0014] A T cell prepared from a healthy person's T cell after being stimulated by the above-mentioned retrovirus transduction.

[0015] A pharmaceutical composition for treating glioma, comprising the above-mentioned chimeric antigen receptor, nucleic acid molecule, carrier virus, and T cell.

[0016] In summary, the present application has the following advantages: The B7-H3 CAR constructed in the application can more effectively kill glioma cells with high expression of B7-H3, and can effectively control the progression of glioma in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The expression level of the antigen B7-H3 on the surface of two glioma cell lines; Figure 2 The expression level of the antigen B7-H3 in patient glioma tissues and normal tissues; Figure 3 The basic structure of the B7-H3 chimeric antigen receptor targeting; Figure 4 The expression of several B7-H3 chimeric antigen receptors on T cells; Figure 5 The co-culture of several B7-H3 chimeric antigen receptor-T cells and glioma cells LN229; Figure 6 The cytokine levels released by several B7-H3 chimeric antigen receptor-T cells when killing glioma cells LN229; Figure 7 The killing of glioma by the B7-H3 chimeric antigen receptor-T cells of the application in a glioma mouse model in vivo. DETAILED DESCRIPTION

[0018] The application will be described in detail below in conjunction with the drawings and examples.

[0019] Examples: The cell lines used in the application include 293T cells, which are cultured in IMDM medium containing 10% FBS, 1% GlutaMAX, and 1% double antibody. Glioma cell lines LN229 and U87 cells are cultured in RPMI-1640 or DMEM containing 10% FBS. The GFP-positive LN229 cells used in the CAR-T and tumor cell co-culture experiment were prepared by transducing wild-type LN229 cells with a retrovirus encoding GFP. The LN229 cell line expressing luciferase used in the mouse experiment was obtained by transducing wild-type LN229 with a lentivirus encoding luciferase. Primary T cells were cultured in X-VIVO 15 medium containing 2% FBS, and IL-7 at a final concentration of 10 ng / mL and IL-15 at a final concentration of 5 ng / mL were added during the culture process.

[0020] Immunohistochemical staining, paraffin sections containing patient glioma tissue were deparaffinized with xylene and rewetted with alcohol, then antigen was repaired with sodium citrate, and then blocked in serum for 1 hour. The sections were incubated with rabbit anti-human B7-H3 antibody overnight at 4°C, then incubated with horseradish peroxidase-conjugated secondary antibody for 30 minutes. After washing, develop color with diaminobenzidine (DAB), then stain with hematoxylin. After staining, observe under a microscope and take pictures.

[0021] Construction of chimeric antigen receptor, the chimeric antigen receptor is composed of a single-chain antibody region (scFv), a CD8 alpha hinge region, a CD8 alpha transmembrane region, a CD28 costimulatory factor region, and a CD3 zeta signal transduction region. The single-chain antibody region of the chimeric antigen receptor is derived from the antibody against B7-H3 produced by the screened monoclonal hybridoma cells. The full-length chimeric antigen receptor is produced by gene synthesis method, and then cloned into a retrovirus vector. After cloning, the correctness of the sequence is confirmed by sequencing. The scFv region of the chimeric antigen receptor in other patents is synthesized by gene synthesis, and then cloned into a retrovirus vector containing the CAR hinge region, transmembrane region, costimulatory factor region, and signal transduction region by enzyme digestion and ligation method. After cloning, the correctness is confirmed by sequencing.

[0022] Preparation of retrovirus of chimeric antigen receptor, murine leukemia retrovirus (MMLV) is prepared by transient transfection of 293T cells with SFG (containing CAR gene), PagPam-e, and RDF three-plasmid system. After 48 and 72 hours of plasmid transfection, the 293T cell supernatant is collected for transfection of T cells.

[0023] Transduction and expansion of T cells, peripheral blood donated by healthy people is used to isolate peripheral blood mononuclear cells. The mononuclear cells are isolated by lymphocyte density gradient separation method, and the cells are activated after being coated with anti-human CD3 and CD28 antibodies in a 24-well plate. The activated T cells are collected and added to a 24-well plate coated with retrovirus supernatant, and centrifuged for transduction. After 48 to 72 hours of retrovirus transduction, the successfully transduced chimeric antigen receptor-transfected T cells are collected and transferred to a T25 or T75 culture flask for expansion, which is used for subsequent detection of chimeric antigen receptor expression and co-culture killing experiment with tumor cells.

[0024] Detection of B7-H3 chimeric antigen receptor expression, centrifugal collection of part of the expanding chimeric antigen receptor-T (CAR-T) cells, after washing with PBS, incubated with B7-H3 and antibody Fc fragment fusion protein on ice for 30 minutes, and then washed with PBS to remove residual protein, and incubated with APC fluorescent dye-labeled antibody Fc binding protein for 30 minutes. After washing with PBS, the expression of chimeric antigen receptor on the surface of CAR-T cells is detected by flow cytometry.

[0025] Expression of antigen B7-H3 in tumor cell lines, collect part of the logarithmic growth of tumor cells, using PBS wash three times, and then use mouse anti-human B7-H3 antibody to stain on ice for 30 minutes, after PBS washing, add APC conjugated secondary antibody, incubate at room temperature for 20 minutes, after PBS washing, use flow cytometry to detect the expression level of B7-H3 on the surface of tumor cells.

[0026] CAR-T cell co-culture with tumor cells, when CAR-T cells are cultured for 8-14 days, 1-2x105 GFP-labeled LN229 tumor cells are inoculated in each well of a 24-well plate, 24 hours later, the corresponding number of T cells are added according to the ratio of T cells to tumor cells = 1:1. After 3-7 days, the cells in the wells are collected and detected by flow cytometry. Among them, T cells are detected by mouse anti-human CD3 antibody, and tumor cells are detected by GFP.

[0027] Enzyme-linked immunosorbent assay (ELISA), 24 hours after adding T cells in the CAR-T cell co-culture experiment, the cell supernatant is collected. Then the level of cytokines IFN-gamma and IL-2 in the supernatant is detected by ELISA kit.

[0028] Glioma xenograft mouse experiment, CAR-T cell targeting glioma in vivo experiment is completed in 6-8 week old nude mice. First, luciferase-labeled LN229 tumor cells are implanted intracranially in nude mice by stereotactic method, 2-3 weeks later, the successful transplantation of glioma is confirmed by in vivo imaging system, and then PBS solvent is injected into the mouse brain ventricle by stereotactic method, and untransduced T cells (NT) or B7-H3-targeted CAR-T cells are injected. After that, imaging is performed once a week to observe the killing of T cells on glioma.

[0029] Immunohistochemical staining, paraffin sections of patient glioma tissue and normal tissue, the expression level of B7-H3 in tumor and normal tissue is detected by immunohistochemical method. After dewaxing with xylene and rewetting with alcohol, the paraffin sections are subjected to antigen retrieval with sodium citrate. After blocking in serum for 1 hour, incubate with anti-B7-H3 antibody at 4°C overnight, then incubate with horseradish peroxidase-conjugated goat anti-mouse secondary antibody for 30 minutes. After washing, develop with DAB, then stain with hematoxylin. After staining, observe under a microscope and take pictures.

[0030] In order to determine the expression level of B7-H3 in glioma, two different glioma cell lines, U87-MG and LN-229, were tested. The tumor cells were stained with B7-H3 specific antibody, and then the expression of B7-H3 was detected by flow cytometry. As shown in Figure 1, the expression of B7-H3 in U87-MG and LN-229 cells was detected by flow cytometry. The results showed that the expression of B7-H3 in U87-MG and LN-229 cells was significantly higher than that in the control group. Figure 1As shown, both glioma cell lines highly express B7-H3 antigen compared to the negative control, thus can be used to test the killing effect of B7-H3 CAR-T on glioma.

[0031] To further confirm the expression of B7-H3 in patient glioma samples, we used the specific antibody of B7-H3 to perform immunohistochemical staining on the tumor tissue chip of glioma patients. As shown in the attached figure, B7-H3 has a high expression (darker color) on the glioma tissue of patients, while the expression level in normal tissue is very low. The above results prove that B7-H3 is an effective target for glioma treatment. Figure 2

[0032] The present application screened a brand new monoclonal antibody (clone number: 24A11A11) for B7-H3, and then integrated the scFv sequence into the second generation of chimeric antigen receptor, the structure of which is shown in the attached figure Figure 3 As shown, it includes the single-chain antibody sequence recognizing B7-H3 (consisting of the light chain of the antibody, the GS linker, and the heavy chain of the antibody), the hinge region and transmembrane region of CD8alpha, the costimulatory region of CD28, and the signal transduction region of CD3zeta.

[0033] To test whether the newly constructed B7-H3 CAR can be expressed on the surface of T cells, we used the retrovirus vector containing the CAR and the packaging vector to prepare retrovirus in 293T cells, and used the retrovirus to transduce the isolated T cells of healthy people after stimulation. Then we used B7-H3 and human IgG Fc fusion protein to stain the transduced T cells, and used flow cytometry to detect the expression of B7-H3 CAR on the surface of T cells. We also constructed the B7-H3 CAR vectors in other patents (WO2017044699A1, US10233226B2, CN113667021B, CN109609533B, CN113461818B, CN112390892A) at the same time to prepare CAR-T for subsequent synchronous comparison test. The results are shown in the attached figure Figure 4 As shown, the B7-H3 CAR we constructed can be effectively expressed on the surface of T cells as the CAR in other patents, and the expression levels of each CAR are basically equivalent.

[0034] ​To determine whether the B7-H3 CAR constructed in the present application can more effectively kill glioma cells with high expression of B7-H3, we co-cultured the B7-H3 CAR-T in the present application and the B7-H3 CAR-T in other patents with the glioma cell line LN229, respectively, and then observed the killing effect of several B7-H3 CAR-T on LN229. After the LN229 cells adhered to the wall for 24 hours, we added several B7-H3 CAR-T cells at a ratio of 1:1, and after co-culturing for 3 days, we detected the proportion of residual T cells and LN229 cells by flow cytometry. As shown in Fig. 1, compared with the control CD19 CAR-T, several single-chain antibody different B7-H3 CAR-T cells can effectively reduce the proportion of glioma cells LN229, and the proportion of residual LN229 cells in the B7-H3 CAR-T group of the present application is lower, indicating that the B7-H3 CAR-T of the present application can more effectively kill glioma cells than the CAR-T in other patents. Figure 5 As can be seen, compared with the control CD19 CAR-T, several single-chain antibody different B7-H3 CAR-T cells can effectively reduce the proportion of glioma cells LN229, and the proportion of residual LN229 cells in the B7-H3 CAR-T group of the present application is lower, indicating that the B7-H3 CAR-T of the present application can more effectively kill glioma cells than the CAR-T in other patents. Figure 6 As shown in Fig. 2, the cytokine release level during the killing of tumor by different CAR-T was detected by ELISA method, and the results are shown in the figure. The cytokine released by the control CD19 CAR-T cannot be detected, while the B7-H3 CAR-T of the present application releases more IFN-gamma and IL-2 than other CAR-T, indicating that it can more effectively activate the body's immune ability to tumor.

[0035] To determine whether the B7-H3 CAR-T of the present application can effectively control the progression of glioma in vivo, we used luciferase-labeled LN229 cell line to construct a mouse glioma model in the nude mouse brain. We used a stereotactic method to transplant LN229 cells into the nude mouse brain, and after imaging confirmed the formation of glioma, we used the same method to reinfuse solvent PBS, control untransduced T cells (NT) and B7-H3 CAR-T of the present application into the tumor-bearing mice, and then observed the progression of intracranial tumor in mice at different times using a real-time imaging instrument. As shown in Fig. 3, the tumor in the solvent PBS and control NT groups continued to progress, while the glioma in most mice in the B7-H3 CAR-T group regressed, proving that the B7-H3 CAR-T of the present application can effectively control the progression of glioma in vivo and can be used as a glioma treatment method. Figure 7 As shown in Fig. 3, the tumor in the solvent PBS and control NT groups continued to progress, while the glioma in most mice in the B7-H3 CAR-T group regressed, proving that the B7-H3 CAR-T of the present application can effectively control the progression of glioma in vivo and can be used as a glioma treatment method.

[0036] The above merely describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above-described embodiments. Any technical solution falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application shall also be considered as falling within the protection scope of the present application.

Claims

1. A chimeric antigen receptor targeting B7-H3, characterized in that: The chimeric antigen receptor is composed of a single-chain antibody region, a CD8 alpha hinge region, a CD8 alpha transmembrane region, a CD28 costimulatory factor region and a CD3 zeta signal transduction region. The amino acid sequence of the single-chain antibody region is shown as SEQ ID NO:

2. The amino acid sequence of the CD8 alpha hinge region is shown as SEQ ID NO:

11. The amino acid sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO:

12. The amino acid sequence of the CD28 costimulatory factor region is shown as SEQ ID NO:

13. The amino acid sequence of the CD3 zeta signal transduction region is shown as SEQ ID NO:

14.

2. The chimeric antigen receptor targeting B7-H3 according to claim 1, characterized in that: The single-chain antibody region comprises an antibody light chain and an antibody heavy chain, the amino acid sequence of the antibody light chain is shown as SEQ ID NO: 3, and the amino acid sequence of the antibody heavy chain is shown as SEQ ID NO:

7.

3. The chimeric antigen receptor targeting B7-H3 of claim 2, wherein: The antibody light chain comprises three variable regions, i.e., a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO: 4, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO: 5, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO:

6.

4. The chimeric antigen receptor targeting B7-H3 of claim 2, wherein: The antibody heavy chain comprises three variable regions, i.e., a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO: 8, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO: 9, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO:

10.

5. A nucleic acid molecule, characterized in that: The nucleic acid molecule is capable of expressing the single-chain antibody region as claimed in claim 1.

6. A nucleic acid molecule according to claim 5, wherein: The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:

1.

7. A retrovirus, characterized by: The retrovirus is prepared from a retrovirus vector and a packaging vector comprising the chimeric antigen receptor as claimed in claim 1 in 293T cells.

8. A T cell, characterized by: The T cell is prepared from a T cell of a healthy person after being separated, stimulated and transduced by the retrovirus as claimed in claim 7.

9. A pharmaceutical composition for use in the treatment of glioma, characterized in that: The chimeric antigen receptor as claimed in any one of claims 1-4, the nucleic acid molecule as claimed in any one of claims 5 and 6, the vector virus as claimed in claim 7, and the T cell as claimed in claim 8.

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