Lung exempt MSLNCAR-T cell, application and anti-tumor preparation

Abstract

The invention relates to the technical field of biological genetic engineering, in particular to a lung exempt type MSLN CAR-T cell, application and an anti-tumor preparation. The CAR-T cell is constructed by adopting a recombinant vector, the recombinant vector comprises an MSLN binding structural domain nucleotide sequence, and further comprises an LIR-1 inhibitory nucleotide sequence and a nucleotide sequence used for being specifically bound with a lung high-expression gene, and a lung high-expression molecule is an AQP3 gene. When the CAR-T cell enters lung tissue, the LIR-1 inhibitory sequence inhibits the killing function of the CAR-T by being combined with lung high-expression molecules, so that lung toxicity is avoided; when the CAR-T cells infiltrate tumor tissues, the inhibitory sequence is inactivated, and the CAR-T is activated and normally plays a role, so that the tumor cells are effectively killed; the medicine has the advantages of accurate treatment, low side effect and safety.

Description

Technical Field

[0001] The present invention relates to the field of biogenetic engineering technology, and in particular to a lung-exempt MSLN CAR-T cell, application and anti-tumor preparation. Background Art

[0002] Mesothelin (MSLN) is a protein that is only expressed on distributable tissues (limited to mesothelial cells of the pleura, peritoneum and pericardium) and has very low nonspecific toxicity. Therefore, anti-tumor agents developed with it as a target have low side effects and have received increasing attention.

[0003] For most cancers, MSLN is evenly expressed on the cell surface with low cytoplasmic expression, but is highly overexpressed in mesothelioma, epithelial ovarian cancer, and pancreatic cancer.

[0004] The above cancers are generally highly malignant and have a high mortality rate. In addition, they lack typical symptoms in the early stages. Most patients are diagnosed in the late stages and have a poor prognosis. Surgery and chemotherapy are still the main means of treating these cancers targeting MSLN, but recurrence and chemotherapy resistance still result in unsatisfactory treatment effects and low survival rates. In recent years, immunotherapy, especially chimeric antigen receptor T cell (CAR-T) therapy, has shown significant efficacy in the treatment of a variety of solid tumors. However, for cancers targeting MSLN, CAR-T therapy still faces a series of challenges, and off-target toxicity is an important limiting factor in the use and promotion of CAR-T therapy.

[0005] Although mesothelin (MSLN) is a highly specific and highly expressed target in cancers targeting MSLN, MSLN is not only highly expressed on cancer cells, but also has a certain level of expression in normal lung tissue. Clinical trials have shown that traditional MSLN-CAR-T cells may cause dose-dependent pulmonary toxicity and lead to severe lung damage or even death in patients. This phenomenon significantly limits the clinical application of MSLN-CAR-T. Therefore, how to reduce the pulmonary toxicity of MSLN-CAR-T while maintaining its anti-cancer efficacy has become a key issue that needs to be solved urgently.

[0006] In recent years, researchers have worked hard to reduce off-target toxicity by improving CAR structural design, developing dual-target or logic gate CAR-T, and regulating CAR-T cell activity. However, these methods are complex, costly, and have limited effectiveness. In addition, there are currently few studies on CAR-T optimization for cancers targeting MSLN, especially in basic research on how to avoid lung toxicity, and there has been no breakthrough progress. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a lung-exempt MSLN CAR-T cell, application and anti-tumor preparation.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] The present invention provides a recombinant vector, which comprises a MSLN binding domain nucleotide sequence, a LIR-1 inhibitory nucleotide sequence and a nucleotide sequence for specifically binding to a lung highly expressed gene.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, the lung highly expressed molecule is the AQP3 gene.

[0012] Furthermore, the LIR-1 inhibitory nucleotide sequence is as shown in SEQ ID NO.1, and the nucleotide sequence for specifically binding to the lung highly expressed gene is as shown in SEQ ID NO.2, or as shown in SEQ ID NO.3.

[0013] Furthermore, the N-terminus of the LIR-1 inhibitory nucleotide sequence is sequentially connected to a HINGE sequence and the nucleotide sequence for specifically binding to the lung highly expressed gene, and the C-terminus of the LIR-1 inhibitory nucleotide sequence is sequentially connected to a T2A linker sequence, the MSLN binding domain nucleotide sequence and an activating sequence.

[0014] Furthermore, the T2A linker sequence is shown as SEQ ID NO.4, the MSLN binding domain nucleotide sequence is shown as SEQ ID NO.5, the activation sequence is shown as SEQ ID NO.6, and the HINGE sequence is shown as SEQ ID NO.7.

[0015] The present invention also provides a lentivirus, which is constructed using the above-mentioned recombinant vector.

[0016] The present invention also provides a lung-immune MSLN CAR-T cell, which is constructed using the above-mentioned lentivirus.

[0017] The present invention also provides a use of the lung-exempt MSLN CAR-T cells as described above, which can be used to prepare anti-tumor preparations, wherein the target of the tumor is MSLN.

[0018] The present invention also provides an anti-tumor preparation, comprising the lung-exempt MSLN CAR-T cells as described above.

[0019] Furthermore, the preparation is selected from drugs, pharmaceutical raw materials, vaccines, targeted inhibitors, gene therapy vectors or biological materials, and the preparation also includes pharmaceutically acceptable excipients.

[0020] The beneficial effects of the present invention are:

[0021] (1) The lung-exempt MSLN CAR-T cells of the present invention introduce the LIR-1 inhibitory receptor and connect it to the lung highly expressed gene. When the CAR-T cells enter the lung tissue, the LIR-1 inhibitory sequence will inhibit the killing function of CAR-T by binding to the lung highly expressed molecules, thereby avoiding lung toxicity; when the CAR-T cells infiltrate cancer tissue, the inhibitory sequence is inactivated, CAR-T is activated, and it functions normally, effectively killing cancer cells;

[0022] (2) The lung-exempt MSLN CAR-T cells of the present invention can achieve precise treatment with low side effects, can effectively reduce CAR-T off-target toxicity, and provide a safer treatment strategy for the clinical application of MSLN-CAR-T;

[0023] (3) In the lung-exempt MSLN CAR-T cells of the present invention, the AQP3 gene is a highly expressed molecule in the lung; this gene is highly expressed in lung tissue and is lowly expressed in most cancer tissues such as ovarian cancer, and is suitable as a target. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison chart of the sequencing data of the lung-exempt MSLN CAR-T cells of the present invention, and the sequencing data of the normal lung tissue single cell and the ovarian cancer single cell in Example 1;

[0025] Figure 2 This is the lung-exempt MSLN CAR-T cell of the present invention, in Example 1, the immunohistochemical staining diagram; Figure 2 A in the middle is the staining image of ZIP8 in ovarian cancer. Figure 2 B is the staining image of ZIP8 in normal lung tissue. Figure 2 Middle C is the staining image of AQP3 in ovarian cancer. Figure 2 Middle D is the staining image of AQP3 in normal lung tissue;

[0026] Figure 3 The lung-exempt MSLN CAR-T cells of the present invention are fluorescence images of the cell lines constructed in Example 3. Figure 3 A in the middle is the fluorescence image of Hela-AQP3-GFP. Figure 3 B in the middle is the fluorescence image of Hela-mCherry;

[0027] Figure 4The lung-exempt MSLN CAR-T cells of the present invention are Western Blot electrophoresis images of cells collected after lentivirus transfection of Hela cell line in Example 3. Figure 4 A in the middle is CD19, Figure 4 B in the middle is AQP3;

[0028] Figure 5 This is the lung-exempt MSLN CAR-T cell of the present invention. In Example 3, the fluorescence comparison diagram of the killing effect of CAR-T on the Hela-AQP3-GFP cell line and the Hela-mCherry cell line is shown. Figure 5 A in the middle is a Hela-mCherry cell line that is not infected with CAR-T. Figure 5 B in the middle is the Hela-mCherry cell line infected with CAR-T. Figure 5 Middle C is a Hela-AQP3-GFP cell line that is not infected with CAR-T. Figure 5 Middle D is the Hela-AQP3-GFP cell line infected with CAR-T;

[0029] Figure 6 This is a comparison chart of the lung-exempt MSLN CAR-T cells of the present invention, and the effects of CAR-T on the Hela-AQP3-GFP cell line and the Hela-mCherry cell line in Example 3, Figure 6 A in the middle is a comparison chart of relative cell activity. Figure 6 B in the middle is the fluorescence intensity value of Hela-mCherry after adding CAR-T cells. Figure 6 Middle C is the fluorescence intensity value of Hela-AQP3-GFP after adding CAR-T cells;

[0030] Figure 7 For the lung-exempt MSLN CAR-T cells of the present invention, in Example 3, the flow cytometry analysis results of T cells are: Figure 7 A in the middle is the analysis result diagram of CAR-T. Figure 7 Figure B shows the analysis results of the Hela-AQP3-GFP cell line infected with CAR-T. Figure 7 Middle C is the analysis result of Hela-mCherry cell line infected with CAR-T;

[0031] Figure 8 This is the lung-exempt MSLN CAR-T cell of the present invention. In Example 3, the comparison chart of different T cell contents in each experimental group is as follows: Figure 8 A in the middle is CD4 + / CD45 + T cells, Figure 8 B is CD8 + / CD45 +T cells, Figure 8 C in the middle is CD25 + / CD45 + T cells, Figure 8 D in the middle is CD69 + / CD45 + T cells;

[0032] Fig. 9 Schematic diagram of the action of the lung-immune MSLN CAR-T cells of the present invention. DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0034] The lung-exempt MSLN CAR-T cell of the present invention is a novel dual-target CAR-T cell, which is constructed by a recombinant vector and a lentivirus. The CAR-T cell has a nucleotide sequence of the MSLN binding domain, and also includes a LIR-1 inhibitory nucleotide sequence and a nucleotide sequence for specific binding to a high-expression gene in the lung.

[0035] The introduction of inhibitory receptors provides a new idea for optimizing the function of CAR-T. Inhibitory receptors can inhibit the activation of T cells by binding to specific molecules, thereby selectively reducing the activity of CAR-T and achieving precision treatment.

[0036] like Fig. 9 As shown, the lung-exempt MSLN CAR-T cells of the present invention introduce the LIR-1 inhibitory sequence and connect it to the lung highly expressed gene. In this way, when the CAR-T cells enter the lung tissue, the LIR-1 inhibitory sequence will inhibit the killing function of CAR-T by binding to the lung highly expressed molecules, thereby avoiding lung toxicity; when the CAR-T cells infiltrate cancer tissue, the inhibitory sequence is inactivated, CAR-T is activated, and it functions normally, effectively killing cancer cells.

[0037] The lung-exempt MSLN CAR-T cells of the present invention can effectively reduce the off-target toxicity of CAR-T, providing a safer treatment strategy for the clinical application of MSLN-CAR-T.

[0038] Preferably, the highly expressed molecule in the lung of the present invention is the AQP3 gene; this gene has the characteristics of being highly expressed in lung tissue and lowly expressed in cancer tissue, and is suitable as a target.

[0039] Preferably, the LIR-1 inhibitory nucleotide sequence is as shown in SEQ ID NO.1, and the nucleotide sequence for specifically binding to highly expressed genes in the lung is as shown in SEQ ID NO.2, or as shown in SEQ ID NO.3; the above two nucleotide sequences that specifically bind to highly expressed genes in the lung have the best affinity with the AQP3 peptide segment and the best antigen-antibody binding efficiency.

[0040] Preferably, the nucleotide sequence for specifically binding to highly expressed genes in the lung is located on the N-terminal side of the LIR-1 inhibitory nucleotide sequence, and the C-terminal of the LIR-1 inhibitory nucleotide sequence is sequentially connected to the T2A linker sequence, the MSLN binding domain nucleotide sequence and the activation sequence.

[0041] The LIR-1 inhibitory sequence can transmit the stimulation of the AQP3-ScFv extracellular segment into cells, generating inhibitory signals, thereby inhibiting the function of CAR-T cells. T2A is a connecting sequence that connects two CAR molecules. The activating sequence is CD8a-CD28-CD3z, which can transmit the stimulation signal of MSLN-ScFv into cells, generate activating signals, and activate the killing function of CAR-T.

[0042] Preferably, the T2A linker sequence is shown as SEQ ID NO.4, the MSLN binding domain nucleotide sequence is shown as SEQ ID NO.5, and the activation sequence is shown as SEQ ID NO.6.

[0043] Preferably, the recombinant vector also includes a HINGE sequence as shown in SEQ ID NO.7, which is located between the nucleotide sequence for specifically binding to highly expressed genes in the lung and the LIR-1 inhibitory nucleotide sequence. The function of the HINGE sequence is to connect LIR-1 and the nucleotide sequence for specifically binding to highly expressed genes in the lung.

[0044] The lung-exempt MSLN CAR-T cells of the present invention can be used to prepare anti-tumor preparations, and the tumor target is MSLN.

[0045] The anti-tumor preparation of the present invention includes the lung-exempt MSLN CAR-T cells as described above.

[0046] Preferably, the tumors targeted by the above-mentioned applications and preparations of the present invention are specifically tumors targeting MSLN, and their types include mesothelioma, ovarian cancer tumors, pancreatic cancer tumors, liver cancer tumors, esophageal cancer tumors, gastric cancer tumors, peritoneal cancer tumors, and breast cancer tumors.

[0047] Furthermore, the preparation is selected from drugs, pharmaceutical raw materials, vaccines, targeted inhibitors, gene therapy vectors or biological materials, and the preparation also includes pharmaceutically acceptable excipients.

[0048] The present invention is described below by means of specific examples.

[0049] Example 1 Single Cell Data Screening

[0050] The rapid development of single-cell sequencing technology has provided an important tool for discovering differentially expressed genes between tumors and normal tissues. By analyzing the transcriptome data of different tissues, molecules that are highly expressed in specific tissues but lowly expressed in tumors can be screened out, thus providing a basis for CAR-T target optimization.

[0051] This example uses normal lung tissue single-cell sequencing data and ovarian cancer single-cell sequencing data for analysis to find genes that are highly expressed in lung tissue and lowly expressed in ovarian cancer tissue.

[0052] like Figure 1 As shown in the figure, 21 candidate genes were obtained after screening. The Human Protein Atlas website was used to query the cellular expression location information of each gene, as well as the protein expression levels of ovarian cancer and lung tissues, and 3 genes were screened, namely SLC39A8, LAMP3 and AQP3. Immunohistochemistry experiments were used to verify the expression of these genes in normal lung tissue and ovarian cancer. Figure 2 It can be seen that the expression levels of LAMP3 and SLC39A8 in ovarian cancer are significantly higher than AQP3. Therefore, LAMP3 and SLC39A8 are excluded, and AQP3 (Aquaporin-3, Ensembl: ENSG00000165272) is determined to be the most suitable candidate gene.

[0053] The normal lung tissue single-cell sequencing data used in this example comes from https: / / www.synapse.org / Synapse:syn21041850 / wiki / 600865 / , and the ovarian cancer single-cell sequencing data comes from https: / / www.synapse.org / Synapse:syn25569736 / files / .

[0054] Example 2 Lentivirus Synthesis

[0055] In this example, a monoclonal antibody that specifically binds to the extracellular segment of AQP3 and the MSLN binding domain were used to construct plasmids AQP3-CscFV-LIR1-T2A-MSLN scFv-CD8a-CD28-CD3z and AQP3-J scFV-LIR1-T2A-MSLN scFv-CD8a-CD28-CD3z to package lentivirus and infect primary human T cells.

[0056] The above sequences were synthesized into plasmids using vector pXL0091, which was obtained from Nanjing Reindeer Company and synthesized by Genetron. Lentivirus packaging was completed by Nanjing GenScript.

[0057] The specific steps of using the obtained lentivirus to infect primary human T cells are:

[0058] (1) Before transfection, adjust the T cell density to 10 6 / mL, replace with 2mL of fresh T cell complete medium and add it to the six-well plate. Add 200μL of virus stock solution and polybrene with a final concentration of 5μg / mL to the six-well plate and mix gently. Incubate at 37℃ and 5% CO 2 incubator for 24 hours.

[0059] (2) After the culture is completed, replace the infection medium with fresh complete culture medium and continue to culture in the incubator for 24-48 hours to complete the infection and perform in vitro killing experiments.

[0060] In this example, the sequences of monoclonal antibodies specifically binding to the extracellular segment of AQP3 are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and the amino acid sequences encoded by the two are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively. In this example, lentiviruses containing the above two monoclonal antibodies were obtained.

[0061] The nucleotide sequence of the MSLN binding domain is shown in SEQ ID NO. 5. The primary human T cells used in this example were commercially available.

[0062] Example 3 Efficacy Test

[0063] In this example, the lentivirus constructed by plasmid AQP3-C scFV-LIR1-T2A-MSLN scFv-CD8a-CD28-CD3z obtained in Example 2 was used to construct a stable cell line Hela-AQP3-GFP co-expressing MSLN and AQP3. At the same time, a Hela-mCherry cell line for control was also constructed, and the core sequences and vector types of the two are shown in Table 1.

[0064] Table 1

[0065]

[0066] The specific steps for constructing the Hela-AQP3-GFP cell line are as follows:

[0067] (1) After digesting the cells in the logarithmic growth phase, add 5*10 4For each cell, use 0.5 mL of complete medium and incubate at 37°C, 5% CO 2 Incubate overnight under 4% CO 2 conditions.

[0068] (2) Add 25 μL of the original virus solution and polybrene with a final concentration of 5 μg / mL to 400 μL of diluent (complete culture medium of target cells) to obtain the virus solution. Aspirate the cell culture medium and add the diluted virus solution. At the same time, establish a control, that is, target cells without virus solution, and incubate at 37°C and 5% CO. 2 incubator for 24 hours.

[0069] (3) After the culture is completed, remove the virus solution, add 500 μL of complete culture medium, and continue to culture for 24 hours. Add complete culture medium containing puromycin at a final concentration of 10 ug / mL and continue to culture for 48 hours. Observe the fluorescence under the microscope. Figure 3 As shown, cells were collected for Western Blot verification, and the obtained electrophoresis pattern was as shown Figure 4 shown.

[0070] Human primary T cells infected with CAR lentivirus were co-cultured with Hela cells transfected / not transfected with AQP3 to detect the killing of Hela cells by CAR-T and the protection of Hela-AQP3 cells. The results are as follows Figure 5 and 6 As shown. Figure 6 Medium B and Figure 6 As can be seen in Figure C, after the addition of CAR-T, the fluorescence intensity of Hela-mCherry cells decreased, proving that CAR-T killed the target cells, while the fluorescence intensity of Hela-AQP3-GFP cells after the addition of CAR-T had no statistical difference compared with that before the addition of CAR-T, proving that CAR-T can effectively protect target cells from being killed.

[0071] The supernatant T cells were collected for flow cytometry analysis, and the results were as follows: Figure 7 and 8 As shown. Figure 7 and Figure 8 It can be seen that T cells are activated, indicating that CAR-T can effectively kill Hela cells, but has no killing effect on Hela-AQP3 cells.

[0072] It can be seen from the above examples that the lung-exempt MSLN CAR-T cells of the present invention can effectively kill cancer cells while significantly avoiding lung toxicity. For cancers targeting MSLN other than ovarian cancer, the lung-exempt MSLN CAR-T cells have a similar mechanism of action. Therefore, for other cancers targeting MSLN that low-express or do not express AQP3, the lung-exempt MSLN CAR-T cells of the present invention also have similar effects.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant vector, characterized in that: The recombinant vector comprises a nucleotide sequence of the MSLN binding domain, a LIR-1 inhibitory nucleotide sequence and a nucleotide sequence for specifically binding to a high-expression gene in the lung.

2. The recombinant vector according to claim 1, characterized in that The lung highly expressed molecule is the AQP3 gene.

3. The recombinant vector according to claim 2, characterized in that The LIR-1 inhibitory nucleotide sequence is shown as SEQ ID NO.1, and the nucleotide sequence for specifically binding to the lung highly expressed gene is shown as SEQ ID NO.2 or as SEQ ID NO.

3.

4. The recombinant vector according to any one of claims 1 to 3, characterized in that The N-terminus of the LIR-1 inhibitory nucleotide sequence is sequentially connected to a HINGE sequence and the nucleotide sequence for specifically binding to the lung highly expressed gene, and the C-terminus of the LIR-1 inhibitory nucleotide sequence is sequentially connected to a T2A linker sequence, the MSLN binding domain nucleotide sequence and an activation sequence.

5. The recombinant vector according to claim 4, characterized in that The T2A linker sequence is shown in SEQ ID NO.4, the MSLN binding domain nucleotide sequence is shown in SEQ ID NO.5, the activation sequence is shown in SEQ ID NO.6, and the HINGE sequence is shown in SEQ ID NO.

7.

6. A lentivirus, characterized in that The recombinant vector is constructed using any one of claims 1 to 5.

7. A lung-exempt MSLN CAR-T cell, characterized in that: It is obtained by using the recombinant vector according to any one of claims 1 to 5 or by using the lentivirus according to claim 6.

8. A use of lung-exempt MSLN CAR-T cells as claimed in claim 7, characterized in that: An anti-tumor preparation can be prepared, wherein the target of the tumor is MSLN.

9. An anti-tumor preparation, characterized in that: Including the lung-exempt MSLN CAR-T cells as described in claim 7.

10. An anti-tumor preparation according to claim 9, characterized in that: The preparation is selected from drugs, pharmaceutical raw materials, vaccines, targeted inhibitors, gene therapy vectors or biological materials, and the preparation also includes pharmaceutically acceptable excipients.

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