CAR-T immune cell targeting PD-1 antigen as well as preparation method and application of CAR-T immune cell

Abstract

The invention discloses a PD-1 antigen targeting CAR-T immune cell as well as a preparation method and application thereof. A PD-L1 signal peptide, a PD-L1 extracellular terminal, a CD28 hinge, a transmembrane region, a CD28 intracellular costimulatory domain and a CD 3 zeta intracellular costimulatory domain are sequentially connected to obtain a coding sequence of the chimeric antigen receptor, and the coding sequence is transferred into an immune cell for expression to obtain the CAR-T immune cell. The CAR-T immune cell effectively kills PD-1 < + > cells in vivo and in vitro, can effectively eliminate liver CD8 < + > Trm and relieve inflammation of a liver converging region when being transferred into a PBC model mouse, can also effectively relieve secondary sicca syndrome, and can be used for preparing a medicine for treating primary biliary cholangitis and a medicine for treating sicca syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and specifically relates to a CAR-T immune cell targeting PD-1 antigen and a preparation method and application thereof. Background Art

[0002] Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis, is an autoimmune liver disease caused by the immune system attacking the small bile ducts in the liver, resulting in chronic cholestasis and liver damage. It is more common in middle-aged women. High titers of anti-mitochondrial antibodies AMA against PDC-E2 can be detected in the serum of PBC patients, accompanied by elevated IgM and various inflammatory cytokines. Its pathological process can be divided into four stages: stage I bile duct inflammation; stage II portal inflammation and bile duct hyperplasia, including secondary destruction of small and medium bile ducts in the liver; stage III progressive fibrosis; and stage IV cirrhosis. In the absence of effective treatment, PBC disease will gradually evolve from chronic inflammation at the beginning to fibrosis, and eventually lead to cirrhosis and liver failure, and even develop into liver cancer.

[0003] It is estimated that globally, one in every 1,000 women over the age of 40 suffers from PBC and is at risk of developing cirrhosis and liver failure. There are no accurate statistics on the incidence and prevalence of PBC in China, but with the development of the economy and the improvement of medical standards, especially the diagnosis of PBC, the number of PBC patients in my country is on the rise every year. Therefore, promoting basic and clinical research on PBC is of great significance to promoting the improvement of PBC diagnosis and treatment and improving people's health.

[0004] High titers of autoantibodies and a large number of CD4 + T and CD8 + The infiltration of T cells indicates that the activation of the adaptive immune system plays a very important role. PDC-E2 is the main antigen of AMA autoantibodies in PBC patients. In PBC patients, there are indeed CD4 + and CD8 +Autoreactive T cells indicate that AMA is not only a serological indicator, but also that the recognition of autoantigens such as PDC-E2 by autoimmune lymphocytes may be the driving force in the immunological pathogenesis of PBC. Although autoimmune response is an important cause of PBC, the efficacy of immunosuppressants has not been confirmed. The possible reason is that severe liver symptoms usually exist when PBC is diagnosed, and the opportunity to intervene in early immune responses is missed. Before 2016, ursodeoxycholic acid (UDCA) was the only drug approved for the treatment of PBC, but about 40% of PBC patients did not respond well to its treatment. In 2016, obeticholic acid (OCA), as a farnesoid X receptor (FXR) agonist, was approved by the FDA for use in combination with UDCA for PBC patients who did not respond well to UDCA monotherapy or for PBC patients who could not tolerate it, becoming the first drug approved for the treatment of PBC in nearly 20 years. The principles of the two drugs for treating PBC are, respectively, competitive inhibition of toxic endogenous bile acid absorption and inhibition of bile acid synthesis, thereby inhibiting liver toxicity caused by cholestasis. However, UDCA treatment can only maintain the stability of the disease in patients with stage I and II but cannot reverse the disease, and patients with stage III and IV do not respond well to it. Other nuclear receptor agonists, therapeutic bile acids, macromolecular drugs, etc. need to be further evaluated as second-line or third-line treatments for PBC (combined with UDCA) in the next few years. Finally, liver transplantation has been the standard for terminal treatment of PBC for nearly four decades, but PBC patients who undergo liver transplantation still have a 10.9-42.3% chance of recurrence. Therefore, new treatment options urgently need to be further developed. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the deficiencies and defects of the prior art and to provide a chimeric antigen receptor and its encoding nucleic acid.

[0006] Another object of the present invention is to provide a vector for expressing the chimeric antigen receptor.

[0007] Another object of the present invention is to provide a CAR-T immune cell that can express the above-mentioned chimeric antigen receptor.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned CAR-T immune cells and applications thereof.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A chimeric antigen receptor comprises a PD-L1 signal peptide, a PD-L1 extracellular end, a CD28 hinge and transmembrane region, a CD28 intracellular co-stimulatory domain, and a CD 3ζ intracellular co-stimulatory domain, which are connected in sequence.

[0011] The amino acid sequence of the PD-L1 signal peptide is shown in SEQ ID NO.6.

[0012] The amino acid sequence of the extracellular end of PD-L1 is shown in SEQ ID NO.7.

[0013] The amino acid sequences of the CD28 hinge and transmembrane region are shown in SEQ ID NO.8.

[0014] The amino acid sequence of the intracellular co-stimulatory domain of CD28 is shown in SEQ ID NO.9.

[0015] The amino acid sequence of the CD 3ζ intracellular co-stimulatory domain is shown in SEQ ID NO.10.

[0016] A nucleic acid molecule is a nucleic acid encoding the chimeric antigen receptor.

[0017] The nucleic acid sequence encoding the PD-L1 signal peptide is preferably as shown in SEQ ID NO.1.

[0018] The encoding nucleic acid sequence of the extracellular end of PD-L1 is preferably as shown in SEQ ID NO.2.

[0019] The nucleic acid sequence encoding the CD28 hinge and transmembrane region is preferably as shown in SEQ ID NO.3.

[0020] The nucleic acid sequence encoding the intracellular co-stimulatory domain of CD28 is preferably as shown in SEQ ID NO.4.

[0021] The nucleic acid sequence encoding the CD 3ζ intracellular co-stimulatory domain is preferably as shown in SEQ ID NO.5.

[0022] A vector for expressing the chimeric antigen receptor, comprising the nucleic acid molecule, namely, the coding sequence of the chimeric antigen receptor.

[0023] The backbone of the vector is preferably a retroviral vector; more preferably a pMSCV-IRES-mCherry FP vector.

[0024] A CAR-T immune cell contains the above-mentioned vector expressing the above-mentioned chimeric antigen receptor, which can express the above-mentioned chimeric antigen receptor.

[0025] The starting cells of the CAR-T immune cells are CD8 + T cells.

[0026] The method for preparing the above-mentioned CAR-T immune cells comprises the following steps: transferring the above-mentioned vector expressing the above-mentioned chimeric antigen receptor into the starting immune cells to obtain CAR-T immune cells; more preferably comprises the following steps: firstly packaging the above-mentioned vector expressing the above-mentioned chimeric antigen receptor through packaging cells, and the obtained virus infects the starting immune cells to obtain CAR-T immune cells.

[0027] The starting immune cells are CD8 + T lymphocytes.

[0028] The above CAR-T immune cells can target and eliminate PD-1 + T cells, which can be used to prepare and eliminate PD-1 + Cell preparation; preferably used for preparing drugs for treating primary biliary cholangitis.

[0029] During the experiment, the inventors of the present invention also found that the above-mentioned CAR-T immune cells can effectively alleviate secondary Sjögren's syndrome, so the above-mentioned CAR-T immune cells can also be used to prepare drugs for treating Sjögren's syndrome.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] The current treatment options for PBC are limited, and the mainstream treatment options are mainly to inhibit the hepatotoxicity caused by cholestasis. In previous studies, the research team of this invention used PBC model mice and found that the main pathogenic cells of PBC are tissue-resident memory CD8 + T cells (CD8 + Trm), which specifically overexpresses PD-1. Therefore, in further studies, it was found that targeted clearance of PD-1 + The cells have a therapeutic effect on PBC. The present invention provides a CAR-T-based PD-1+ cell-specific elimination method for the first time, and finds that the anti-PD-1CAR-T immune cells can effectively eliminate CD8 + Trm can relieve inflammation in the portal area of ​​the liver and achieve the purpose of treating PBC; in addition, it can also effectively relieve secondary Sjögren's syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the anti-PD-1-CAR vector element and the result of enzyme digestion identification; among them, (A) is a schematic diagram of anti-PD-1CAR; (B) is a plasmid enzyme digestion electrophoresis diagram, lanes 1 and 2 are CAR plasmids, lanes 3 and 4 are empty plasmids, and lanes 2 and 4 are the products obtained by digesting the plasmid with EcoRI and ClaI; (C) is a second-generation sequencing comparison diagram, and the red arrows represent consistent sequence alignment.

[0033] Figure 2 Is anti-PD-1-CAR in primary CD8 + T cell expression efficiency results.

[0034] Figure 3 It is a result diagram of the reactivity of anti-PD-1-CAR-T cells to PD-1 recombinant protein; wherein, Ctrl-T is a control T cell transfected with an empty vector, CAR-T is the CAR-T cell prepared in Example 2, the average fluorescence intensity of CD69 of CAR-T cells and control T cells under different conditions, PD-1-Fc recombinant protein and α-PD-L1 blocking antibody are added according to the groups in the figure.

[0035] Figure 4 The results of the in vitro reactivity of anti-PD-1-CAR-T cells to PD-1+T cells are shown in Figure 2. (A) is the reactivity of anti-PD-1-CAR-T cells to PD-1+T cells. + The mean fluorescence intensity of CD69 in CAR-T cells and Ctr-T cells co-cultured with T cells. α-PD-L1 blocking antibody was added to some groups; (B) CFSE indicates the interaction with PD-1 + Proliferation levels of CAR-T cells and control T cells co-cultured with T cells, and α-PD-L1 blocking antibodies were added in some groups.

[0036] Figure 5 The results of the killing effect of anti-PD-1-CAR-T cells on PD-1+T cells in vitro; (A) is PD-1 + The activity of PD-1+T cells after T cells were co-cultured with CAR-T or control T cells at the corresponding effector-target ratio; (B) The killing efficiency of CAR-T and control T cells at an effector-target ratio of 0.5:1, and α-PD-L1 blocking antibodies were added to some groups as shown in the figure.

[0037] Figure 6 This is a diagram showing the results of the clearance of PD-1+T cells by anti-PD-1-CAR-T cells in vivo; (A) is PD-1 + CD4 + T cells; (B) PD-1 + CD8 + T cells; (C) is the dynamic change curve of the ratio of transferred cells at different times after transfer.

[0038] Figure 7 This is the result of the clearance of Trm in the liver by anti-PD-1-CAR-T cells in vivo; (A) is the number of liver mononuclear cells, (B) is the number of PD-1 + CD8 + The proportion of T cells, (C) is CD8 +The ratio of Trm, (D) is CD8 + The number of Trm, (E) is CD8 + The number of Tcm, (F) is CD8 + The number of Tem.

[0039] Figure 8 The diagram shows the results of the alleviating effect of anti-PD-1-CAR-T cells on liver inflammation in vivo; (A) is the histopathological diagram of the liver portal area, and (B) is the corresponding pathological score.

[0040] Fig. 9 This is a diagram showing the therapeutic effect of anti-PD-1-CAR-T cells on Sjögren's syndrome in vivo; (A) is the number of salivary gland immune cells, and (B) is the number of salivary gland PD-1 + (C) is the number of T cells, (C) is the salivary flow rate, and (D) is the histopathological image of the area around the salivary duct. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0042] Example 1 Construction of anti-PD-1-CAR vector

[0043] The commissioned company artificially synthesized the full-length sequence of anti-PD-1CAR, which is obtained by connecting the coding sequence of the PD-L1 signal peptide as shown in SEQ ID NO.1, the coding sequence of the PD-L1 extracellular segment as shown in SEQ ID NO.2, the CD28 hinge domain and transmembrane region as shown in SEQ ID NO.3, the CD28 intracellular co-stimulatory domain as shown in SEQ ID NO.4, and the CD3ζ intracellular co-stimulatory domain as shown in SEQ ID NO.5 in sequence.

[0044] The synthetic CAR full-length sequence was inserted into the multiple cloning site EcoRI of the retroviral vector pMSCV-IRES-mCherry FP (Addgene) to obtain the anti-PD-1-CAR vector. After double digestion with EcoRI and ClaI, two bands of 2720 and 5057 bp were obtained, which were consistent with expectations. Sanger sequencing was further used to confirm that all vector sequences were correct. Figure 1 shown.

[0045] Example 2 Preparation of PD-1-targeted CAR-T cells and detection of CAR expression efficiency

[0046] Using the retroviral packaging system, the anti-PD-1-CAR vector constructed in Example 1 and the pcl-eco plasmid were transiently transfected into 293-T cells at a mass ratio of 3:2 to package the retrovirus. CD8 + T cells. T cells (1×10 6 The cells were activated by centrifugation (2000 rpm for 2 hours) and then infected with retrovirus 48 hours later to obtain CAR-T cells. Thy1.1 was used as a screening marker to detect the expression efficiency of CAR. CAR-T cells labeled with anti-Thy1.1 PE antibody (Cat#202523, BioLegend) were detected by flow cytometry. The results are shown in Figure 2. Figure 2 As shown, it can be seen that the CAR expression efficiency is 50-60%.

[0047] Example 3. Reactivity of anti-PD-1-CAR-T cells to PD-1

[0048] The CAR-T cells (1×10 6 cells / mL) and control T cells transfected with empty vector (1×10 6 cells / mL). The results are as follows Figure 3 As shown, it can be seen that PD-1 recombinant protein can specifically upregulate the expression of CAR-T activation molecule CD69. Adding PD-L1 blocking antibody (Cat#124301, BioLegend) can inhibit CAR-T cell activation, proving that this activation is PD-L1 dependent and verifying the specificity of anti-PD-1-CAR-T cell response. We used 4μg / mL coated CD3 antibody and 2μg / mL CD28 antibody to induce CD8 + T cells express PD-1, and the PD-1 thus obtained is further + T cells were used as target cells and co-cultured with CAR-T cells. CAR-T cells could upregulate the expression and proliferation of the activation molecule CD69, and this was PD-L1 dependent, further proving that anti-PD-1-CAR-T cells could inhibit PD-1. + Cellular responses, such as Figure 4 shown.

[0049] Example 4 Killing effect of anti-PD-1-CAR-T cells on PD-1+ T cells in vitro

[0050] The CAR-T cells prepared in Example 2 and the control T cells were respectively incubated with CD3 / CD28-induced PD-1 + T cells were co-cultured for 24 h at the corresponding effector-target ratio (E:Tratio), and their killing efficiency was detected using Annexin V. Figure 5 As shown. Combining CAR-T with PD-1 + The killing efficiency of T cells incubated at a ratio of 1:1 was 70%, and the killing efficiency at a ratio of 1:2 was 50%. Compared with control T cells, CAR-T cells significantly killed all target targets, and this activation was PD-L1 dependent, such as Figure 5 shown.

[0051] Example 5 Anti-PD-1-CAR-T cells kill PD-1+T cells in vivo and treat liver inflammation

[0052] In PBC model mice (p40 - / - IL-2Rα - / - ) (According to the literature "Yao Y, et al. Distinct from its canonical effects, deletion of IL-12p40 induces cholangitis and fibrosis in interleukin-2Rα- / -mice. Journal of Autoimmunity, Volume 51, June 2014, Pages99-108. http: / / dx.doi.org / 10.1016 / j.jaut.2014.02.009 ” operation) 8 weeks and 10 weeks old were injected into the tail vein with 1×10 6 CAR-T cells / mouse, blood was collected from the tail vein every week after transfusion. From 1 week after transfusion, blood was collected from the microvenous vein of mice, and the cell ratio and PD-1 in peripheral blood were detected by flow cytometry. + The cells will be cleared away, accompanied by the expansion of CAR-T cells, such as Figure 6 In addition, liver cytology was performed 4 weeks after treatment. + The cells were completely cleared, and the number of infiltrating mononuclear cells was significantly reduced. + In the case of T cell subsets, pathogenic CD8 + Trm is significantly eliminated, such as Figure 7 From the perspective of histopathology, the inflammatory infiltration in the portal area of ​​the liver was also significantly alleviated. After scoring by pathologists (scoring criteria according to the aforementioned literature), the inflammatory infiltration score in the portal area was reduced, as shown in Figure 2. Figure 8 shown.

[0053] Example 6 The therapeutic effect of anti-PD-1-CAR-T cells on Sjögren's syndrome in vivo

[0054] In previous studies, p40 - / - IL-2Rα - / - Secondary Sjögren's syndrome (GAO CY, et al. Tissue-Resident Memory CD8 + T Cells Acting as Mediators of Salivary Gland Damage ina Murine Model of Syndrome.Journal of Autoimmunity,Volume 71,Jan2019,Pages 121-132.https: / / doi.org / 10.1002 / art.4067”). After CAR-T transfer therapy as in Example 5, flow cytometry was performed on the salivary glands, and the results showed that the number of salivary gland immune cells and PD-1+T cells was significantly reduced (see Fig. 9 In addition, there was a significant recovery in salivary flow rate (see Fig. 9 (C)); from the histopathological point of view, the inflammatory infiltration of salivary glands was significantly alleviated (see Fig. 9 (D) in the.

[0055] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. It should be pointed out that those skilled in the art may make certain improvements and modifications without departing from the spirit and principles of the present invention, but these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that: It contains the PD-L1 signal peptide, the extracellular end of PD-L1, the CD28 hinge and transmembrane region, the CD28 intracellular co-stimulatory domain, and the CD 3ζ intracellular co-stimulatory domain, which are connected in sequence.

2. The chimeric antigen receptor according to claim 1, characterized in that: The amino acid sequence of the PD-L1 signal peptide is shown in SEQ ID NO.6; The amino acid sequence of the extracellular end of PD-L1 is shown in SEQ ID NO.7; The amino acid sequence of the CD28 hinge and transmembrane region is shown in SEQ ID NO.8; The amino acid sequence of the intracellular co-stimulatory domain of CD28 is shown in SEQ ID NO.9; The amino acid sequence of the CD 3ζ intracellular co-stimulatory domain is shown in SEQ ID NO.

10.

3. A nucleic acid molecule, characterized in that: The chimeric antigen receptor encoding nucleic acid according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleic acid sequence encoding the PD-L1 signal peptide is shown in SEQ ID NO.1; The coding nucleic acid sequence of the extracellular end of PD-L1 is shown in SEQ ID NO.2; The nucleic acid sequence encoding the hinge and transmembrane region of CD28 is shown in SEQ ID NO.3; The nucleic acid sequence encoding the intracellular co-stimulatory domain of CD28 is shown in SEQ ID NO.4; The nucleic acid sequence encoding the CD 3ζ intracellular co-stimulatory domain is shown in SEQ ID NO.

5.

5. A vector for expressing the chimeric antigen receptor, characterized in that: Containing the nucleic acid molecule according to claim 3 or 4.

6. A CAR-T immune cell, characterized in that: Containing the chimeric antigen receptor expressed as described in claim 2.

7. The method for preparing CAR-T immune cells according to claim 6, characterized in that The method comprises the following steps: transferring the vector expressing the chimeric antigen receptor as described in claim 5 into starting immune cells to obtain CAR-T immune cells.

8. The CAR-T immune cell of claim 7 is used in the preparation of a method for clearing PD-1 + Application in cell preparations.

9. Use of the CAR-T immune cells according to claim 7 in the preparation of a drug for treating primary biliary cholangitis.

10. Use of the CAR-T immune cells according to claim 7 in the preparation of a drug for treating Sjögren's syndrome.

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