A CART targeting PD-1 and VEGF and its use in preparing a drug for treating tumors

Abstract

The present invention provides a CART targeting PD-1 and VEGF and its use in preparing a drug for treating tumors. The chimeric antigen receptor comprises a signal peptide, antiPD-1scFv, antiVEGF scFv, a hinge region, a transmembrane region, a co-stimulatory factor and a CD3ζ signaling domain; the chimeric antigen receptor can efficiently bind to the target antigen; the CAR-T cell can inhibit the proliferation of tumor cells in vivo and in vitro, and can also promote the expression of immune factors to exert an anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a CART targeting PD-1 and VEGF and its use in preparing a drug for treating tumors. Background Art

[0002] In recent years, malignant tumors have become more common, among which cervical cancer is the most common malignant tumor of the female reproductive system, and is more common in women aged 40 to 60. At present, research on cervical cancer cannot fully explain the pathogenesis of cervical cancer, but there is evidence that the incidence of cervical cancer may be related to a series of factors such as early marriage, multiple births, cervical lacerations, and poor local hygiene. Since the 1990s, studies have further confirmed that human papillomavirus (HPV) plays an important role in the pathogenesis of cervical cancer (see Olusola P, Banerjee HN, Philley JV, et al., Human papilloma virus-associated cervical cancer and health disparities, Cells (2019), 8(6): 622).

[0003] According to the different stages and specific circumstances of cervical cancer progression, five treatments can be used clinically, including surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. In traditional cervical cancer treatment, surgery can directly eradicate the cancer, but there is a risk of recurrence, and when the cancer metastasizes, the surgical effect is not good. Radiotherapy and chemotherapy use the different sensitivities of cancer cells and normal cells to radiation and drugs to treat cancer. However, the similarities between cancer cells and normal cells make these two therapies have a strong negative impact on normal tissues. In order to prevent excessive damage, their application is greatly limited and they cannot eradicate cancer cells. Targeted drugs can target specific types of cancer cells to kill cancer cells without killing normal cells. Targeted drugs have played a revolutionary role in the treatment of specific cancers. However, only a small number of known cancers can be treated with targeted drugs. At the same time, the discovery of new anti-cancer drugs is slow and costly, which limits the application of targeted drugs.

[0004] As a new treatment, immunotherapy aims to make the human immune system respond more effectively to cancer cells. Immunotherapy includes immune checkpoint inhibitors, monoclonal antibodies, vaccines, immunomodulators and T cell transfer therapy. Through in vitro editing, T cells with stronger response to cancer cells are cultivated and introduced into the body to treat cancer. Adoptive T cell therapy (ACT) is a cancer immunotherapy that uses the patient's immune cells to find and eliminate tumor cells. In some cases, donor immune cells can also be used. Chimeric antigen receptor T cells (CAR-T cells) are the most popular in ACT therapy.

[0005] Chimeric antigen receptor (CAR) is an artificial hybrid receptor. Through genetic engineering, scFv containing monoclonal antibodies is used as the antigen binding extracellular domain, and the intracellular CD3 z chain is used as the TCR signaling domain and additional auxiliary signaling domains (mainly CD28 and 4-1BB (CD137)) to provide co-stimulation. The CAR gene is transferred into T cells to form CAR-T cells, which are then returned to the patient to exert anti-tumor effects. After nearly 30 years of development, CAR-T cells can be divided into four development stages according to different CARs: non-co-stimulatory signal CAR-T cells (first generation), single co-stimulatory signal CAR-T cells (second generation), dual / multiple co-stimulatory signal CAR-T cells (third generation) and precision CAR-T cells (fourth generation). The extracellular recognition regions and transmembrane regions of these four generations of CAR-T cells are not much different, and their stages are mainly divided according to the structure of the intracellular fragment of CAR. In the first generation of CAR-T, the structure of the intracellular fragment of CAR is relatively simple, mainly composed of ITAM of the ζ chain of the CD3 molecule. Based on the first generation of CAR-T cells, the second generation of CAR-T cells added ITAM regions from the co-stimulatory molecules CD28 or CD137 (4-1BB) to the intracellular fragments. After the extracellular antigen recognition region binds to the target antigen, the T cells can simultaneously obtain antibody stimulation signals and co-stimulatory signals (see Maher J, Brentjens RJ, Gunset G, et al., Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta / CD28 receptor, Nat Biotechnol (2002), 20 (1): 70–5). The main structure of the third-generation CAR-T cells is similar to that of the second-generation CAR-T cells, but the third-generation CAR-T cells use lentivirus as a transfection vector, which can carry larger gene fragments into T lymphocytes. In theory, the third-generation CAR-T cells should have stronger activation and killing capabilities than the second-generation CAR-T cells (see Morgan RA, Yang JC, Kitano M, et al., Case report of a serious adverse event following the administration of T cells transduced with achimeric antigen receptor recognizing ERBB2, Mol Ther (2010), 18(4): 843–51).The fourth generation of CAR-T adds suicide genes or controllable suicide genes (such as some drug-sensitive genes) to the CAR structure to control the survival time of CAR-T cells in the body (see Chmielewski M, Abken H. TRUCKs: the fourth generation of CARs, Expert Opin Biol Ther (2015), 15(8): 1145–54).

[0006] Mesothelin, CA125, VEGF, and folate receptors are the most widely studied antigens in CAR-T cell therapy trials. At present, there are limited clinical trials related to the application of CAR-T in cervical cancer, and NCT01583686, NCT04556669, NCT03356795, NCT04556669, etc. are among the few studies. In addition, some studies have shown that CAR-T has weak sensitivity and affinity for cancer cells, and CAR-T cells need to be triggered by multiple targets at the same time to work (see Yu LF, Gong LQ, Huang ZY, et al. T cell immunotherapy for cervical cancer: challenges and opportunities, Front Immunol. (2023), 14: 1105265).

[0007] To this end, the present invention uses PD-1 and VEGF as targets to construct bispecific CAR-T cells for the treatment of cervical cancer, which can effectively inhibit tumor growth in vitro and in vivo, improve the tumor immune environment, and provide a research basis for the development of new tumor immunotherapy. Summary of the invention

[0008] The first aspect of the present invention provides a chimeric antigen receptor, which comprises a signal peptide, an antiPD-1scFv, an antiVEGF scFv, a hinge region, a transmembrane region, a co-stimulatory factor and a CD3ζ signaling domain, wherein the antiPD-1scFv comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:2; the antiVEGF scFv comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:3 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:4.

[0009] Immune checkpoints are molecules expressed on immune cells that negatively regulate immune responses. The presence and high expression of these molecules are important indicators of immune suppression. For cervical cancer, PD-1 / PD-L1 expression is most relevant to HPV-related cancers, especially HPV16+ and HPV18+ cases. Studies have shown that when HPV vaccines are combined with anti-PD-1 antibodies, they are beneficial to the mechanism of tumor clearance (see Rice AE, Latchman YE, Balint JP, et al. An HPV-E6 / E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression, Cancer Gene Ther (2015), 22(9): 454–62).

[0010] VEGF is also an important anti-tumor target and is highly correlated with the occurrence and development of various tumors, including cervical cancer. A study showed that the use of PM8002, a bispecific antibody targeting both PD-L1 and VEGF-A, can effectively inhibit the growth of cervical cancer cells, with an ORR of 28% (7 / 25) in 25 patients with cervical cancer (see Guo Y, Guo J, Cheng Y, et al., Phase Ib / IIa safety and efficacy of PM8002, a bispecific antibody targeting PD-L1 and VEGF-A, as a monotherapy in patients with advanced solid tumors, Journal of Clinical, Oncology (2023), 41(16): 2536).

[0011] Therefore, in the present invention, PD-1 and VEGF are selected as dual targets to construct CAR-T cells, and their antigen binding domains are optimized so that the chimeric antigen receptor can maintain a high affinity for the target, thereby effectively exerting an anti-tumor effect.

[0012] Furthermore, the co-stimulatory factor is 4-1BB.

[0013] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:5.

[0014] Furthermore, the chimeric antigen receptor nucleotide sequence is shown in SEQ ID NO:6.

[0015] .The second aspect of the present invention provides a vector carrying the nucleotide described in claim 4.

[0016] The third aspect of the present invention provides a chimeric antigen receptor immune cell, wherein the immune cell expresses the chimeric antigen receptor described in the present invention.

[0017] Furthermore, the immune cell is at least one of a T cell, a NK cell, a CIK cell and a macrophage.

[0018] Furthermore, the immune cells are T cells.

[0019] The fourth aspect of the present invention provides a use of the chimeric antigen receptor and / or the chimeric antigen receptor immune cell in the preparation of a drug for treating tumors.

[0020] Furthermore, the tumor includes a liquid tumor or a solid tumor.

[0021] Furthermore, the tumor includes but is not limited to any one of leukemia, lymphoma, myeloma, lung cancer, prostate cancer, pancreatic cancer, colon cancer, breast cancer, liver cancer, cervical cancer or gastric cancer.

[0022] The tumor is cervical cancer.

[0023] Beneficial Effects

[0024] The present invention provides a CART targeting PD-1 and VEGF and its use in preparing a drug for treating tumors, with the following specific beneficial effects:

[0025] (1) Provides a chimeric antigen receptor targeting PD-1 and VEGF that can efficiently bind to the target antigen;

[0026] (2) The CAR-T cells are able to inhibit tumor cell proliferation in vivo and in vitro;

[0027] (3) The CAR-T cells can promote the expression of immune factors and exert anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Schematic diagram of CAR structure;

[0029] Figure 2 :CAR-T expresses green fluorescent protein;

[0030] Figure 3 : CAR-T cell killing effect in vitro;

[0031] Figure 4 : Diagram of cell volume development in animal models;

[0032] Figure 5 : Expression levels of inflammatory factors in serum. DETAILED DESCRIPTION

[0033] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0034] Example 1 Structural design of CAR

[0035] Currently, CAR technology has developed to the fourth generation, but the second-generation CAR with a co-stimulatory factor domain is the most classic. It can not only effectively identify and kill tumor cells, but also has a relatively simple structure and is easy to prepare and use. Most of the CAR-T therapies currently being tested in clinical trials use the second-generation CAR structure. Therefore, this classic structure is also used in the present invention to construct CAR-T cells.

[0036] The present invention first screened and obtained an antigen binding domain (antiPD-1scFv) targeting PD-1 based on phage display library technology, which includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2. With reference to the well-known anti-VEGF antibody, bevacizumab, an antigen binding domain (antiVEGF scFv) targeting VEGF was designed, and while retaining its heavy chain variable region, the structure of the light chain variable region was adjusted, which includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 4.

[0037] The structure of the CAR is as follows Figure 1 The scFv comprises a signal peptide, an antiPD-1 scFv, an antiVEGF scFv, a hinge region, a transmembrane region, a 4-1BB co-stimulatory factor and a CD3ζ signal domain, and its amino acid sequence is shown in SEQ ID NO: 5. After the CAR is constructed, its nucleotide sequence (as shown in SEQ ID NO: 6) is introduced into a saccharomyces cerevisiae expression system, and after expression and purification, a biomolecular interaction analyzer (purchased from Pall, USA) is used to detect the affinity of the CAR for the target antigen. The results are shown in Table 1. After the scFv targeting PD-1 and VEGF is constructed into CAR, although the affinity with the target antigen decreases to a certain extent, this may be related to the formation of spatial barriers due to the increase in the molecular structure, but it still maintains a high level of affinity, which can meet the requirements of specific recognition of tumor cells.

[0038] Table 1 Affinity of antigen binding domain and target antigen

[0039]

[0040] Example 2 CAR-T cell preparation

[0041] 2.1 Preparation of lentiviral vector carrying CAR gene

[0042] The second-generation packaging system was used to construct a lentiviral vector. The CAR nucleotide sequence was inserted into the pLVX plasmid (carrying the green fluorescent protein gene) through enzyme digestion and ligation reaction to form the pLVX-CAR plasmid; the transfection reagent Lipofectamine TM 2000 (purchased from Invitrogen) and Opti-MEM culture medium (purchased from Gibco) were mixed and allowed to stand for 5 minutes, and then pLVX-CAR plasmid, auxiliary plasmid psPAX2, and pMD2.G were added to Opti-MEM culture medium at a ratio of 4:3:1 and mixed. HEK-293T cells were revived and cultured, and transfection was performed when the confluence of HEK-293T cells reached 80%. After continuing to culture in a cell culture incubator for 6 hours, fresh culture medium was added; viruses at 48 hours and 72 hours were collected, inverted and mixed evenly, and the viruses were collected by centrifugation at 500g for 10 minutes. After resuspension, cell debris was removed through a 0.22μm filter to obtain purified virus fluid, which was stored in a -80℃ refrigerator.

[0043] 2.2 Isolation of mononuclear cells

[0044] Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy volunteers and cultured in RPMI1640 medium containing 100 mL / L FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. All cell lines were kept at 37°C and 5% CO. 2 The cells were cultured in an incubator until the logarithmic growth phase, and CD3+CD28+ cells were screened using flow cytometry to obtain target T cells.

[0045] 2.3 Preparation of CAR-T cells

[0046] Adjust the T cell density to 1×10 6 / mL, 300μL of cell suspension was inoculated into a 6-well plate, and lentiviral vector was added at a multiplicity of infection (MOI) of 5. After culturing at 37°C and 5% CO2 for 24h, the virus solution was removed and replaced with X-VIVO 15 culture medium containing 100IU / mL human IL-2 and 10% FBS, and culture and amplification were continued. Observe under a fluorescence microscope, such as Figure 2 As shown, the T cells can express green fluorescence, namely, CAR-T cells.

[0047] Example 3 CAR-T cells inhibit the growth of cervical cancer cells in vitro

[0048] Cervical cancer cell line HeLa 229 was used as target cells, CAR-T cells were used as effector cells, and the effector cells and target cells were mixed at a ratio of 5:1 and added to a 96-well plate; an equal volume of saline was added to the target cells as a control well, and effector cell wells and target cell wells were set at the same time. Each group had 3 replicate wells, and was cultured at 37°C and 5% CO2 for 24 hours. 20 μL of CCK8 solution was added to each well, and the OD value at 450 nm was detected using an ELISA reader after incubation at 37°C for 4 hours. The cell killing rate was calculated according to the following formula: Cell killing rate (%) = (1-(OD 实验孔 -OD 效应孔 ) / OD 靶标孔 )×100%. The result is Figure 3 As shown, the CAR-T cells provided by the present invention can effectively kill tumor cells and significantly inhibit their growth.

[0049] Example 4 Anti-tumor effect of CAR-T cells in vivo

[0050] 4.1 Preparation of mouse animal model

[0051] Select 6-8 week old SPF female BALB / c mice and place them in an environment with relative humidity (95±5)% and room temperature (32±2)℃ every day. Drink water freely every day and feed them for 1 week. Cultivate HeLa 229 cells to the logarithmic growth phase and adjust the cell density to 6×10 6 Each mouse was injected subcutaneously with 0.5 mL of cell suspension. One week after injection, obvious tumor tissue was visible, indicating that the model was successfully established.

[0052] 4.2 Grouping and Dosing

[0053] The model mice were randomly divided into two groups, 10 mice in each group: CAR-T group: intravenous injection of 5×10 6 CAR-T cells; control group: intravenous injection of an equal volume of normal saline. The health status of the mice was observed and recorded daily.

[0054] 4.3 Tumor volume measurement

[0055] The tumor volume of mice was measured weekly for 4 weeks. The long diameter (L) and short diameter (W) of the tumor tissue were measured using a vernier caliper, and the formula: L×W 2 / 2 Calculate tumor volume. The results are shown in 4. CAR-T cell therapy can significantly slow down the growth rate of tumor tissue in animals, especially after the third week, when the tumor volume is significantly lower than that of the control group.

[0056] 4.4 Detection of inflammatory factors

[0057] After 4 weeks of administration, the experimental animals were killed, peripheral blood was centrifuged to collect serum, and ELISA kits (purchased from Shanghai ELISA Biologicals Co., Ltd.) were used to detect the secretion of IFN-γ and IL-2 in the serum. Figure 5 As shown in the figure, the secretion levels of inflammatory factors such as IFN-γ and IL-2 increased significantly during CAR-T cell therapy. This shows that the CAR-T cells provided by the present invention can effectively activate the humoral immune response in the body and exert anti-tumor effects; on the other hand, it also suggests that the release of inflammatory factors should be closely monitored in subsequent animal and clinical trials, and drug treatment measures should be taken when necessary to prevent the occurrence of inflammatory factor storms.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that The chimeric antigen receptor includes a signal peptide, an antiPD-1 scFv, an antiVEGF scFv, a hinge region, a transmembrane region, a co-stimulatory factor and a CD3ζ signal domain, wherein the antiPD-1 scFv includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; the antiVEGF scFv includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

4.

2. The chimeric antigen receptor according to claim 1, characterized in that The co-stimulatory factor is 4-1BB.

3. The chimeric antigen receptor according to claim 1, characterized in that The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:

5.

4. The chimeric antigen receptor according to claim 3, characterized in that The chimeric antigen receptor nucleotide sequence is shown in SEQ ID NO:

6.

5. A carrier, characterized in that The vector carries the nucleotide described in claim 4.

6. A chimeric antigen receptor immune cell, characterized in that The immune cell expresses the chimeric antigen receptor according to any one of claims 1 to 4.

7. The chimeric antigen receptor immune cell according to claim 6, wherein the immune cell is at least one of a T cell, a NK cell, a CIK cell and a macrophage. The chimeric antigen receptor immune cell according to claim 7 , wherein the immune cell is a T cell.

9. Use of the chimeric antigen receptor according to any one of claims 1 to 4 and / or the chimeric antigen receptor immune cell according to any one of claims 6 to 7 in the preparation of a drug for treating tumors, wherein the tumor is cervical cancer.

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