CD147 chimeric antigen receptors and methods of use

Abstract

A modified single-chain variable fragment (scFv) specifically binding to CD147 is provided. A chimeric antigen receptor (CAR) comprising the modified CD147scFv, a nucleic acid encoding the CAR, a vector comprising the nucleic acid encoding the CAR, and an immune cell expressing the CAR are also provided. A method for treating a subject with cancer is also provided, the method comprising administering to the subject an immune cell expressing the disclosed CD147-CAR.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 819,403, filed on March 15, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to immunotherapy, in particular to chimeric antigen receptors targeting CD147 and their use for treating cancer.

[0004] Government Support Statement

[0005] This invention was made with government support under Grant Nos. AI130197, HL125018, AI124769-01, and AI129594 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0006] Liver cancer is the second most common cause of cancer-related death worldwide. The burden of liver cancer is expected to exceed 1 million cases by 2030. Liver cancer ranks fifth in cases worldwide and second in deaths among men. More than 500,000 patients die from hepatocellular carcinoma (HCC) each year.

[0007] Primary liver cancer includes hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (iCCA), fibrocarcinoma and hepatoblastoma. HCC and iCCA are the most common primary liver cancers, accounting for more than 99% of primary liver cancer cases. HCC alone (nearly 800,000 new cases per year) accounts for 90% of all primary liver cancer cases. Currently, there is no effective therapy available for the treatment of HCC. Sorafenib (CheckMate-040, a multikinase inhibitor with low efficacy and severe side effects widely used in patients with advanced HCC) is a first-line standard systemic agent for HCC. Currently, PD-1 blockade Opdivo (Nivolumab) has been approved by the U.S. Food and Drug Administration (FDA) as a second-line treatment strategy for HCC patients previously treated with sorafenib. Clinical trials testing PD-1 blockade as a first-line treatment for HCC are underway. At the same time, various clinical trials using PD-1 or PD-L1 blockade in combination with other interventions are also underway. For example, a study evaluating the combination of anti-PD-1 antibody and anti-CTLA-4 antibody in patients with resectable and potentially resectable HCC is being tested in a clinical trial (NCT03222076).

[0008] Chimeric antigen receptor (CAR)-modified T cell therapy has become a promising immunotherapeutic strategy for the treatment of various blood cancers. Although CAR-modified T cell immunotherapy has recently made progress in blood cancers, its widespread use is hindered by high costs and severe toxicity. At the same time, CAR-T cells face additional challenges such as maintaining persistent proliferation and persistence in the tumor microenvironment during targeting of solid tumors. Another challenge for CAR-mediated liver cancer immunotherapy is to find effective targets. Summary of the invention

[0009] CD147 is expressed at different levels on different cell types (e.g., hematopoietic cells, epithelial cells, and endothelial cells). However, CD147 is significantly upregulated in disease states such as HCC, breast cancer, bladder cancer, colorectal cancer, ovarian cancer, melanoma, and osteosarcoma. CARs for cells specifically targeting expression of CD147 are provided. These CARs can be used for immunotherapy of cancers expressing or overexpressing CD147.

[0010] Disclosed herein are modified single-chain variable fragments (scFv) that specifically bind to CD147. In some embodiments, the scFv has an amino acid sequence comprising the variable heavy chain (VH) domain complementary determining region 1 (CDR1), CDR2 and CDR3 amino acid sequences of SEQ ID NO:8 and the variable light chain (VL) domain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:9. In some examples, the scFv has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2, or comprises the amino acid sequence of SEQ ID NO:2 or consists of the amino acid sequence of SEQ ID NO:2. Also provided are nucleic acids encoding the modified CD147 scFv, such as nucleic acids having at least 90% sequence identity with the nucleic acid molecule of SEQ ID NO:1, or nucleic acids comprising the nucleic acid sequence of SEQ ID NO:1 or consisting of the nucleic acid sequence of SEQ ID NO:1; and vectors comprising the nucleic acid sequence. In another embodiment, a vector encoding the modified CD147 scFv (such as SEQ ID NO: 1) is provided, the vector further comprising an inducible promoter or enhancer nucleic acid molecule operably connected to the CD147 scFv nucleic acid molecule. In some examples, the enhancer nucleic acid is a Gal4 upstream activation sequence (UAS) operably connected to the nucleic acid encoding the CD147 scFv. In another example, the vector is a synNotch construct, such as a vector comprising a nucleic acid sequence of a modified CD147 scFv nucleic acid molecule (such as SEQ ID NO: 1) connected to a synNotch and Gal4-VP64 coding sequence (such as SEQ ID NO: 17).

[0011] Also provided is a CAR, comprising a modified CD147 scFv, a hinge domain, a transmembrane domain, an intracellular domain comprising one or more co-stimulatory molecule intracellular domains and an intracellular signaling domain provided herein. In one embodiment, the CD147-CAR comprises a modified CD147 scFv, an IgG1 hinge domain, a CD28 transmembrane domain, a CD28 and 4-1BB co-stimulatory domain, and a CD3ζ signaling domain provided herein. In some examples, the CD147-CAR comprises an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO: 5, or comprises an amino acid sequence of SEQ ID NO: 5 or consists of an amino acid sequence of SEQ ID NO: 5.

[0012] In some embodiments, the CD147-CAR further includes inducible suicide molecules such as caspase 9. In some examples, the expression of the suicide molecule is induced by tetracycline, doxycycline or rapamycin. In one example, the CD147-CAR with an inducible suicide gene includes an amino acid sequence with at least 90% homology to the amino acid sequence of SEQ ID NO:7 or includes an amino acid sequence of SEQ ID NO:7 or is composed of an amino acid sequence of SEQ ID NO:7. The CD147-CAR may further include a cytokine receptor intracellular domain, such as an interleukin-15 receptor intracellular domain (e.g., SEQ ID NO:12), an interleukin-12 receptor intracellular domain, or an interleukin-18 receptor intracellular domain.

[0013] Also provided are nucleic acids encoding CD147-CAR disclosed herein and vectors (such as viral vectors) including the nucleic acids. In some instances, the CD147-CAR is encoded by a nucleic acid sequence having at least 90% homology to a nucleic acid sequence of SEQ ID NO:4 or SEQ ID NO:6. In other instances, the CD147-CAR is encoded by a nucleic acid sequence including SEQ ID NO:4 or SEQ ID NO:6 or by a nucleic acid sequence consisting of SEQ ID NO:4 or SEQ ID NO:6.

[0014] In another embodiment, there is provided a vector encoding CD147-CAR (such as SEQ ID NO:4 or SEQ ID NO:6), the vector further comprising an inducible promoter or enhancer nucleic acid molecule operably connected to CD147-CAR nucleic acid molecules. In some examples, the enhancer nucleic acid is a Gal4 upstream activation sequence (UAS) (eg, SEQ ID NO:14) operably connected to the nucleic acid encoding CD147-CAR. In one example, the vector includes the CD147-CAR in the synNotch construct, for example, including a vector of a nucleic acid sequence of SEQ ID NO:15. In other examples, the CD147-CAR nucleic acid molecule (eg, SEQ ID NO:1) is connected to synNotch and Gal4-VP64 coding sequences (eg, SEQ ID NO:17).

[0015] Also provided are T cells, natural killer (NK) cells, natural killer T (NKT) cells, double negative T (DNT) cells (CD3 + CD4 - CD8 -), neutrophils or macrophages, such as T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages containing nucleic acids encoding the disclosed CD147 scFv or CD147-CAR or vectors encoding the disclosed CD147 scFv or CD147-CAR. In some examples, the NK cells are NK-92 or NK-92MI cells. Methods for producing cells expressing the CAR are provided, including but not limited to CD147-CAR-NK cells, CD147-CAR-T cells or CD147-CAR-macrophages. These methods include transducing or transfecting T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages with vectors encoding the disclosed CAR.

[0016] In another embodiment, a T cell, NK cell, NKT cell, DNT cell, neutrophil or macrophage expressing a CD147-CAR operably connected to an activator of an inducible promoter or an enhancer element is provided. In some examples, the T cell, NK cell, NKT cell, DNT cell, neutrophil or macrophage further expresses an anti-GPC3 specific binding agent (such as anti-GPC3 scFv) encoding an activator of an inducible promoter or an enhancer element. In other embodiments, a T cell, NK cell, NKT cell, DNT cell, neutrophil or macrophage is provided, and the T cell, NK cell, NKT cell, DNT cell, neutrophil or macrophage expresses a CD147 scFv operably connected to an inducible promoter or an enhancer element and further comprises a nucleic acid molecule encoding an anti-GPC3 chimeric antigen receptor operably connected to an inducible promoter or an enhancer.

[0017] Disclosed herein is a method for treating a subject with cancer, for example, by administering to the subject NK cells, T cells, NKT cells, DNT cells, neutrophils or macrophages (eg, CD147-CAR NK cells, CD147-CAR-T cells or CD147-CAR macrophages) expressing CAR disclosed herein. In some instances, the subject suffers from cancer expressing CD147. In a specific non-limiting example, the subject suffers from hepatocellular carcinoma, neuroblastoma, breast cancer, pancreatic cancer, leukemia, lymphoma, multiple myeloma, colorectal cancer, lung cancer, melanoma, renal cell carcinoma, sarcoma or nasopharyngeal carcinoma.

[0018] The foregoing and other features of the present disclosure will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figures 1A-1E Overexpression of CD147 in hepatocellular carcinoma cells is shown. Figure 1A The prognostic value of the total survival rate of human cancer patients for CD147 up-regulated expression from TCGA data set is shown. The survival rate curves (upper figure) of different patient populations based on relative CD147 high expression and low expression (lower figure) are collected for analysis. The data of LUAD (lung adenocarcinoma), SKCM (skin melanoma), LIHC (hepatocellular carcinoma) and LGG (lower grade glioma of the brain) are analyzed. Figure 1B The comparison of CD147 expression between normal tissue (NT) and tumor sample (TP) in various cancer types from TCGA data set is shown. Data represent the mean ± SEM of three separate experiments. Unpaired Student's t test is used. *p<0.05, **p<0.01, ***p<0.001 and ns (no significant difference). According to TCGA database, the full name of each cancer type is: BRCA (invasive breast cancer), CHOL (cholangiocarcinoma), GBM (glioblastoma multiforme), LGG (low-grade glioma of brain), HNSC (head and neck squamous cell carcinoma), KICH (chromophobe renal cell carcinoma), KIPAN pan-renal cohort (KICH+KIRC+KIRP), KIRP (renal papillary cell carcinoma), LIHC (hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PRAD (prostate cancer), UCEC (endometrial cancer). Figure 1C Western blot analysis of CD147 in HCC cell lines is shown. 1×10 6 Each cell was lysed in 200 μl RIPA buffer and mixed with 50 μl 5X SDS loading buffer before being loaded onto SDS-PAGE independently. Mouse anti-human CD147 (HIM6, mouse IgG1) was used for Western blot analysis. Anti-GAPDH was used as a loading control. Figure 1D CD147 in HCC cell lines (SK-Hep1 and HepG2) is shown. SK-Hep1 and HepG2 (1×10 6 Cells) were stained. After incubation and washing, samples were analyzed by flow cytometry. Numbers represent the mean fluorescence intensity (MFI) of each sample. Figure 1EIs a series of small figures showing the histopathological analysis of CD147 antigen expression on human HCC tumors isolated from PDX mouse models. Representative H&E (upper row) and CD147 IHC staining (middle row) of tumor samples from different patient-derived xenograft (PDX) mice treated with PBS, NK-92MI and CD147-CAR NK-92MI, respectively. The bottom row shows IHC staining without primary antibodies. The scale bar represents 50 μm. The data represent three independent experiments.

[0021] Figures 2A-2F The design of CD147-CAR and phenotyping of CAR-modified NK-92MI cells are shown. Figure 2A The schematic design of the CD147-specific CAR based on the SFG retroviral vector is shown. The construct includes a CD147-specific single-chain antibody fragment (modified scFv from clone 5F6, mIgG1), a human IgG1 CH2CH3 hinge region and a CD28 transmembrane region, followed by the intracellular domain of the co-stimulatory CD28, 4-1BB and the intracellular domain of CD3ζ. Figure 2B Flow cytometric analysis of CAR expression and CD56 on the surface of parental NK-92MI and CD147-CAR-NK-92MI is shown. Data are representative of at least three experiments. Figure 2C Shown is a Western blot analysis of CAR expression in parental NK-92MI and CD147-CAR-NK-92MI cells detecting endogenous CD147 and CD147-CAR fusion protein by anti-human CD3ζ specific antibody. Figure 2D NK activation and inhibition markers in parental NK-92MI, CAR-CD19 (4-1BB)-NK-92MI and CD147-CAR-NK-92MI cells are shown. Each data represents at least three or four experiments. The numbers in the flow chart represent the mean fluorescence intensity (MFI) of each sample. Figure 2E Flow cytometric analysis of CAR expression on CD19-CAR-NK-92MI and CD147-CAR-NK-92MI using goat anti-human IgG (H+L) is shown. Wild-type NK-92MI cells were used as a control. Figure 2F Flow cytometric analysis of CD147 expression on NK-92MI, CD19-CAR-NK-92MI, and CD147-CAR-NK-92MI is shown. Figure 2G Overlay flow cytometry profiles of CD147 expression levels on NK-92MI, CD19-CAR-NK-92MI, and CD147-CAR-NK-92MI are shown. Data represent two independent experiments.

[0022] Figures 3A-3D Shown are CD107a degranulation and cytokine production in CD147-CAR-NK-92MI cells stimulated with their sensitive target cells. Figure 3A Representative flow cytometry data are shown, illustrating CD107a degranulation on NK-92MI, CD19-4-1BB-CAR, CD19-CD28-CAR and CD147-CAR after 10 hours with culture medium (control), SK-Hep1 and HepG2. The ratio of effector to target cells was 1:1.2. Cells were gated according to CD56 positive subpopulations to quantify surface CD107a expression. Figure 3B Quantitative data of the percentage of surface CD107a expression on CD147-CAR-NK-92MI cells under different stimulations are shown, as shown. Cytokine TNF-α (TNF-α) was produced by CD147-CAR-NK-92MI, CD19-4-1BB-CAR-NK-92MI, CD19-CD28-CAR-NK-92MI and wild-type NK-92MI stimulated by different conditions. Figure 3C ) and IFN-γ( Figure 3D NK-92MI cells were co-cultured with SK-Hep1 cells at an effector / target ratio of 1:1 or in culture medium for 12 h. Phorbol-12-myristate-13-acetate (PMA) / ionomycin (IONO) was used for positive control. Cytokine release ratios were calculated by the following equation: sample value / mean value of positive control value × 100 (%). Data were pooled from at least three or four experiments.

[0023] Figures 4A-4C CD147-CAR-NK-92MI killing of two HCC cell lines is shown. Figure 4A Representative flow cytometric staining of surface CD147 molecules on Huh7 (left) and HCO2 (right) cell lines. Figure 4B It is shown that the standard of passing is 4 hours 51 Graph of the cytotoxicity of CD147-CAR-NK-92MI measured by Cr release assay. CD147-positive Huh7 cells were used as CD147-CAR-NK-92MI susceptible target cells. Wild-type NK-92MI was used as a control. Figure 4C It is shown that the standard of passing is 4 hours 51 Figure 3 shows the cytotoxicity of CD147-CAR-NK-92MI measured by Cr release assay. CD147 positive HCO2 cells were used as CD147-CAR-NK-92MI susceptible target cells. Wild-type NK-92MI was used as a control. Data represent mean ± SEM from three independent experiments. *p<0.05, **p<0.01 and ***p<0.001.

[0024] Figure 5A-5G Activation of CD147-CAR-NK-92MI cells upon stimulation with CD147-positive target cells is shown. Figure 5A Representative data are shown, showing the percentage of surface CD107a expression on CD147-CAR-NK-92MI cells at different stimulations, as shown. CD147-CAR-NK-92MI cells were stimulated with SK-Hep1 or HepG2 cells for 4 hours. In order to block the interaction between CD147-CAR and CD147 molecules, 5 μg of mouse anti-human CD147 (HIM6) was added to the mixture of effector and target cells. As a control, 5 μg of isotype mouse IgG (IgG) or PBS (vehicle control group) was used, as shown. Figure 5B and 5C Figure 2 shows the expression of CD147 positive SK-Hep1 ( Figure 5B ) and CD147 positive HepG2 ( Figure 5C ) Graph of quantitative data of the percentage of surface CD107a staining on CD147-CAR-NK-92MI cells stimulated by cell lines. Wild-type NK-92MI cells alone and CD147-CAR-NK-92MI cells alone were used as controls, as indicated. Figure 5D Representative data are shown, showing the expression of CD147-positive wild-type (WT) SK-Hep1 cell line (upper panel) and CD147-knockout (CD147 - / - ) The percentage of surface CD107a expression on CD147-CAR-NK-92MI cells stimulated by SK-Hep1 cell line (middle figure). The medium group alone was used as a control. Naive NK-92MI and 4-1BB-CD19-CAR (CD19-CAR) were used as control effector cells. Figure 5E The results show that CD147 positive SK-Hep1 (WT) and CD147-knockout SK-Hep1 (CD147 - / - ) Quantitative data of the percentage of surface CD107a staining on CD147-CAR-NK-92MI cells stimulated with the cell line. Fig. 5F Representative data are shown, showing the percentage of surface CD107a expression on CD147-CAR-NK-92MI cells stimulated with CD147-positive wild-type (WT) HepG2 cell lines (upper graph) and CD147-knockout HepG2 cell lines (middle graph). Only the culture medium group was used as a control. Naive and CD19-4-1BB-CAR-NK-92MI (CD19-CAR) were used as control effector cells. Figure 5G The results show that CD147 positive HepG2 (WT) and CD147-knockout HepG2 (CD147- / - ) Quantitative data of the percentage of surface CD107a staining on CD147-CAR-NK-92MI cells stimulated by cell lines. The ratio of effector to target cells was 1:1.2. CD147-CAR-NK-92MI cells were gated by CD56 antibody surface staining. NK degranulation was measured by flow cytometry with CD107a surface staining. Data represent the mean ± SEM of three separate experiments. *p<0.05, **p<0.01 and ***p<0.001.

[0025] Figure 6 Mouse anti-human CD147 (HIM6) showed that it did not affect the cytotoxicity of CD19-CAR-NK cells. The cytotoxicity of CD19-CAR-NK-92MI was measured using the FFLuc reporter system. Briefly, Daudi-FFLuc cells (1×10 4 The 96-well optical bottom microplates were pre-seeded with Matrigel (BD) overnight. The effector cells (CD19-CAR-NK-92MI) under two different effector / target ratios (5: 1 and 1: 1, as shown) were co-cultured for 6 hours. The luminescent signal was quantified by an ELISA instrument, and the percentage of specific lysis was calculated. Data were collected from three independent experiments. Error bars show ± SEM (standard error of the mean). *p<0.05, **p<0.01 and ***p<0.001.

[0026] Fig. 7A and 7B Representative images of CD147-CAR-NK-92MI cytotoxicity are shown. Effector cells (1×10 4 CD147-CAR-NK-92MI and NK-92MI and target cells FFLuc-GFP-SK-Hep1 ( Fig. 7A , top)(1×10 4 ) and FFLuc-GFP-HepG2 ( Fig. 7A , bottom) were co-cultured in 96-well optical bottom microplates for 12 hours. Conventional fluorescence microscopy detected GFP fluorescence (upper lane), and bright field (lower lane) was used to visualize CD147-CAR-NK-92MI killing activity under the same settings. GFP fluorescence intensity was quantified by ImageJ software (NIH) ( Figure 7B ). The quantitative mean fluorescence intensity (MFI) of GFP was plotted using Graph Prism 5 software (GraphPad Software, San Diego, CA, USA).

[0027] Figures 8A-8NIt was shown that CD147-CAR-T cells and CD147-CAR-NK cells specifically killed CD147-positive tumor cells in vitro. Fig. 8A The cytotoxicity of primary CD147-CAR-T cells measured by FFLuc reporter gene assay is shown. CD147 positive FFLuc-GFP-SK-Hep1 was used as CD147-CAR-T susceptible target cells. κ-CAR T cells were used as the control group for each experiment. Figure 8B and 8C The cytotoxicity of CD147-CAR-T cells using knockout-CD147 FFLuc-GFP-SK-Hep1 cell line and HepG2 cell line was shown to be significantly reduced by FFLuc reporter gene assay. The data represent the mean ± SEM from three independent experiments. Fig.8D Shows passing the 4-hour standard 51 Cytotoxicity of primary CD147-CAR-NK cells measured by Cr release assay. CD147-positive FFLuc-GFP-SK-Hep1 were used as CD147-CAR-T susceptible target cells. κ-CAR T cells were used as control group for each experiment. Fig. 8E and 8F Indicates passing the 4-hour standard 51 Cr release assay, cytotoxicity of primary CD147-CAR-NK cells using knockout-CD147FFLuc-GFP-SK-Hep1 cell line and HepG2 cell line was significantly reduced. Data represent mean ± SEM from three independent experiments. Figure 8G Anti-NKG2D antibody blocks natural killing of FFLuc-GFP-SK-Hep1 by primary CD147-CAR-NK. Different ratios of primary CD147-CAR-NK cells were co-cultured with FFLuc-GFP-SK-Hep1, knockout-CD147 FFLuc-GFP-SK-Hep1 cell lines or knockout-CD147 FFLuc-GFP-SK-Hep1 cell lines and 5 μg anti-NKG2D for 4 hours. FFLuc reporter gene assay was used. Data represent mean ± SEM from three independent experiments. Figure 8H Shows passing standard 4 hours 51 Cr release assay was used to measure the cytotoxicity of CD147-CAR-NK-92MI against SK-Hep1 cells. Effector cells (CD147-CAR-NK-92MI and NK-92MI) were incubated with 1×10 4 Four hours later, the supernatant was collected and the released 51 Cr. Fig.8I and 8JIt is an FFLuc reporter gene system assay for CD147-CAR-NK-92MI to specifically kill FFLuc-GFP-SK-Hep1 and FFLuc-GFP-HepG2 cell lines. Effector cells (CD147-CAR-NK-92MI and NK-92MI) were incubated with 1×10 4 FFLuc-GFP-SK-Hep1( Fig.8I ) or FFLuc-GFP-HepG2 ( Figure 8J ) Target cells were co-cultured in 96-well optical bottom microplates for 6 hours. After incubation with D-luciferin for 5 minutes, the luminescent signal was measured by a microplate reader to calculate the cytotoxicity of NK cells. The control group used was wild-type NK-92MI incubated with CD147 positive FFLuc-GFP-SK-Hep1 or CD147 positive FFLuc-GFP-HepG2. Figure 8K and 8L The FFLuc reporter gene system was used to measure the expression of knockout-CD147 FFLuc-GFP-SK-Hep1 ( Figure 8K ) and knockout-CD147-FFLuc-GFP-HepG2( Figure 8L ) showed reduced cytotoxicity of the CD147-CAR-NK-92MI cell line. Effector cells (CD147-CAR-NK-92MI and NK-92MI) were co-cultured with 1×10 4 Wild-type or CD147 knockout target cells were co-cultured in 96-well optical bottom microplates for 6 hours. After incubation with D-luciferin for 5 minutes, the luminescent signal was measured by a microplate reader to calculate the cytotoxicity of NK cells. Figure 8M and 8N Figure 2 shows the inhibition of anti-CD147 (clone, HIM6) against FFLuc-GFP-SK-Hep1 ( Figure 8M ) and FFLuc-GFP-HepG2( Figure 8N ) were added to the effector cells (CD147-CAR-NK-92MI and NK-92MI) and 1×10 4 FFLuc-GFP-SK-Hep1 or FFLuc-GFP-HepG2 target cells were co-cultured in 96-well optical bottom microplates for 6 hours. After incubation with D-luciferin for 5 minutes, the luminescence signal was measured by a microplate reader to calculate the cytotoxicity of NK cells. The data represent the mean ± SEM from three independent experiments. *p<0.05, **p<0.01 and ***p<0.001.

[0028] Fig.9A and 9BValidation of knockout-CD147 SK-Hep1 and HepG2 cell lines by flow cytometry and western blotting is shown. Fig.9A Wild-type (wt) SK-Hep1 and CD147 are shown - / - -SK-Hep1 cell line (top) as well as wild-type (wt) HepG2 and CD147 - / - - Staining of surface CD147 molecules on HepG2 cell line (bottom). Fig. 9B The expression of wild-type (wt) SK-Hep1 and CD147 - / - -SK-Hep1 cell line as well as wild-type (wt) HepG2 and CD147 - / - - Western blot analysis of CD147 molecules on HepG2 cell line. GAPDH was used as loading control (bottom).

[0029] Figures 10A-10C CD147-CAR-NK-T cells specifically kill CD147-positive tumor cells. The cytotoxicity of CD147-CAR-T cells was measured by the FFLuc reporter system. CD147-positive FFLuc-EGFP-Hep-G2 ( Fig. 10A ) and CD147 positive FFLuc-EGFP-SK-Hep1 ( Fig. 10B ) were used as CD147-CAR-T susceptible target cells. κ-CAR T cells were used as the control group in each experiment. Fig. 10C The cytotoxicity of CD147-CAR-T cells using the knockout-CD147 FFLuc-GFP-SK-Hep1 cell line was significantly reduced as measured by the FFLuc reporter system. Briefly, effector cells (CD147-CAR-T cells) were incubated with target cells FFLuc-EGFP-SK-Hep1 or CD147 knockout FFLuc-EGFP-SK-Hep1 (1×10 4 The cells were co-cultured in 96-well optical bottom microplates for 6 hours. The cytotoxicity of CD147-CAR-T cells was measured by luminescent signals read by a microplate reader. The data represent the mean ± SEM from three independent experiments. *p<0.05, **p<0.01 and ***p<0.001.

[0030] Figures 11A-11D Shown is the progression of CD147-CAR-T-92MI cells controlling HCC in a xenograft mouse model. Fig.11A Figure 2 is the experimental design of the HCC xenograft model. Briefly, NSG mice were subcutaneously injected with 4×10 6SK-Hep1 cells. The mice were monitored for tumor burden (reaching nearly 50 mm 2 ) and were randomly divided into groups on day 4. On day 5 (D5), mice were injected (intravenously) with a dose of 1×10 7 Effector CD147-CAR-T cells (Group 1) and 2×10 4 IU IL-2. The control group was injected with vehicle (PBS) control only (Group 2). On days 7, 9 and 16, the same treatment was administered to each group. Fig. 11B Quantification of tumor burden of SK-Hep1 xenografts treated with CD147-CAR-T and PBS (vehicle control group) is shown. All results are mean ± SEM. The differences in each group were analyzed by two-way ANOVA analysis. Fig. 11C Quantitative body weight was assessed for each group at the indicated time points. Fig.11D Shown are Kaplan-Meier survival curves of tumor-bearing mice after treatment with CD147-CAR-T cells and PBS (vehicle control group). P values ​​were analyzed by log-rank (Mantel-Cox) test.

[0031] Figures 12A-12D The antitumor efficacy of CD147-CAR-NK-92-MI cells against HCC in a mouse xenograft model is shown. Fig. 12A Figure 1 is an experimental design of the antitumor efficacy of primary CD147-CAR-NK in HCC xenograft model. Five days after tumor implantation (day 5), mice were injected (intravenously) with a dose of 1×10 7 Effector primary CD147-CAR-NK cells and 2×10 4 The control group was injected with the same number of untransduced primary NK cells and 2×10 4 IU IL-2 (Group 2) or PBS alone (Group 3). On days 5, 7, 9, 16 and 18, the same treatments were administered to each group as indicated. Fig. 12B The quantitative tumor burden of HCC xenograft mice treated with primary CD147-CAR-NK, untransduced primary NK and PBS (vehicle control group) is shown. All results are mean ± SEM. The difference of each group is analyzed by two-way ANOVA analysis. Fig. 12C Quantitative body weights of each group assessed at the indicated time points are shown. Fig.12DIt is the Kaplan-Meyer survival curve of tumor-bearing mice after treatment with primary CD147-CAR-NK, parental primary NK group and PBS (vehicle control group). The p value analysis by log-rank (Mantel-Cox) test. The difference of each group is analyzed by two-way ANOVA analysis. *p<0.05, **p<0.01 and ***p<0.001 indicate the comparison of CD147-CAR modified cell therapy group with control group.

[0032] Fig.13 The results showed that the irradiated CD147-CAR-NK-92MI cells had comparable anti-HCC tumor activity to the non-irradiated CD147-CAR-NK-92MI cells in killing HCC cell lines in vitro. 51 Cr release assay measures cytotoxicity of irradiated and unirradiated CD147-CAR-NK-92MI. CD147-positive wild-type HepG2 tumor cells (experimental group, left figure) or CD147-knockout (CD147KO, right figure) HepG2 tumor cell lines were used as CD147-CAR-NK-92MI susceptible target cells. Irradiated and unirradiated wild-type NK-92MI cells were used as effector cell control groups. Data represent three independent experiments. All data are presented as mean ± SEM.

[0033] Figures 14A-14D It was shown that in a xenograft mouse model, irradiated CD147-CAR-NK-92MI cells had comparable anti-HCC tumor activity to non-irradiated CD147-CAR-NK-92MI cells in controlling HCC progression. Fig.14A Figure 1 is the experimental design of the HCC xenograft model. NSG mice were injected (subcutaneously) with 2×10 6 SK-Hep1 cells. One day before treatment (day 4), tumor burden (reaching nearly 50 mm 2 ) and the mice were randomly divided into groups. On day 5 (D5), the mice were injected (intravenously) with a dose of 1×10 7 effector CD147-CAR-NK-92MI (group 1) cells and 2×10 4 The control group was injected with the same number of irradiated CD147-CAR-NK-92MI in PBS and 2×10 4 IU IL-2 (Group 2) or vehicle control alone (Group 3). On days 7, 9, 16, and 18, the same treatment was administered to each group. Fig. 14BQuantification of tumor burden of SK-Hep1 xenografts treated with CD147-CAR-NK-92MI and PBS (vehicle control group) is shown. All results are mean ± SEM. Differences in each group were analyzed by two-way ANOVA analysis. Fig. 14C are graphs of quantitative body weight assessed for each group at the indicated time points. Fig.14D Kaplan-Meyer survival curves of tumor-bearing mice after treatment with CD147-CAR-NK-92MI cells and PBS (vehicle control group) are shown. P values ​​were analyzed by log-rank (Mantel-Cox) test. Data represent two independent experiments. All data are presented as mean ± SEM.

[0034] Figures 15A-15D It was shown that CD147-CAR-NK-92MI cells controlled the progression of HCC in a xenograft mouse model. Fig.15A Figure 2 is the experimental design of the HCC xenograft model. Briefly, NSG mice were subcutaneously injected with 4×10 6 On day 0 (the day before day 1), tumor burden (approximately 50 mm 2 ) and the mice were randomly divided into groups. On day 1 (D1), mice were injected (intravenously) with a dose of 1×10 7 The control group was injected with the same number of NK-92MI and 2×104 IU IL-2 in PBS. 4 IU IL-2 (Group 2) or vehicle control alone (Group 3). On days 3 and 5, the same treatment was administered to each group. Fig. 15B Quantification of tumor burden of SK-Hep1 xenografts treated with CD147-CAR-NK-92MI, parental NK-92MI cells (control group) and PBS (vehicle control group) is shown. All results are mean ± SEM. The differences in each group were analyzed by two-way ANOVA analysis. *p<0.05, **p<0.01 and ***p<0.001 indicate comparison of CD147-CAR treatment group with NK-92MI treatment group. +p<0.05, ++p<0.01 and +++p<0.001 indicate comparison of CD147-CAR treatment group with vehicle control treatment group. Fig. 15C Quantitative body weights of each group assessed at the indicated time points are shown, and Fig.15DShown are Kaplan-Meier survival curves of tumor-bearing mice after treatment with CD147-CAR-NK-92MI cells, parental NK-92MI group, and PBS (vehicle control group). P values ​​were analyzed by log-rank (Mantel-Cox) test.

[0035] Figures 16A-16D It was shown that CD147-CAR-NK-92MI cells controlled the progression of HCC in the PDX mouse model. Fig.16A Figure 1 shows the experimental design of the anti-tumor efficacy of CD147-CAR-NK-92MI in a liver PDX model generated by The Jackson Laboratory. Patient-derived xenograft hepatocellular carcinoma mice were purchased from The Jackson Laboratory. Tumor burden (approximately 50 mm 2 ) and the mice were randomly divided into groups. The indicated mice were then injected (intravenously) with a dose of 5×106 effector CD147-CAR-NK-92MI cells and 2×10 4 The control group was injected with the same number of NK-92MI cells in PBS and 2×10 4 IU IL-2 (Group 2) or PBS alone (Group 3). On days 6, 8, 11, 15, 18, 22, and 26, the same treatments were administered to each group as indicated. Fig. 16B It is the quantitative tumor burden of PDX mice treated with CD147-CAR-NK-92MI cells, parental NK-92MI cells (control group) and PBS (vehicle control group) respectively. All results are mean ± SEM. The difference of each group was analyzed by two-way ANOVA analysis. *p<0.05, **p<0.01 and ***p<0.001 are indicated as the comparison between the CD147-CAR treatment group and the NK-92MI treatment group. +p<0.05, ++p<0.01 and +++p<0.001 are indicated as the comparison between the CD147-CAR treatment group and the vehicle control treatment group. Fig. 16C Quantitative body weights of each group assessed at the indicated time points are shown. Fig.16D Figure 1 is the Kaplan-Meier survival curve of tumor-bearing mice after treatment with CD147-CAR-NK-92MI cells, parental NK-92MI group and PBS (vehicle control group). p-value analysis by log-rank (Mantel-Cox) test.

[0036] Figures 17A-17DThe killing of CD147-positive HCC cells by CD147-CAR-T cells is shown. FFLuc reporter gene system assay for CD147-CAR-T cell-specific killing of FFLuc-EGFP-HepG2 ( Fig.17A The control group used wild-type κ-CAR-T cells incubated with CD147-positive FFLuc-EGFP-HepG2. Fig. 17B Shown is the reduced cytotoxicity of CD147-CAR-T cells using knockout-CD147 FFLuc-GFP-HepG1 as measured by the FFLuc reporter system. Fig. 17C Shows passing standard 4 hours 51 Cytotoxicity of CD147-CAR-T cells measured by Cr release assay. Fig.17D Shows passing standard 4 hours 51 The Cr release assay showed that the cytotoxicity of CD147-CAR-T cells using knockout-CD147 FFLuc-GFP-HepG1 was significantly reduced.

[0037] Fig.18A and 18B CD107a degranulation of CD147 CAR-T or CD147 CAR-NK cells is shown. Fig.18A Representative flow cytometry data are shown, indicating that CD107a degranulation on CD147-CAR-T cells after 10 hours with culture medium (control), SK-N-SH tumor cells. Cells were gated according to CD56 positive subpopulations to quantify surface CD107a expression. Fig.18B Quantitative data of the percentage of surface CD107a expression on CD147-CAR-NK-92MI cells under different stimulations are shown, as indicated. Data were pooled from at least three or four experiments.

[0038] Fig.19 The cytotoxicity of CD147-CAR-NK-92MI cells against DaoY cells in vitro is shown.

[0039] Fig. 20 is an alignment showing the optimized CD147 scFv nucleic acid sequence (SEQ ID NO: 1) compared to the original scFv sequence (SEQ ID NO: 3) and the consensus sequence (SEQ ID NO: 13).

[0040] Figures 21A-21E Patient-derived primary CD147-CAR-NK cells were shown to specifically kill CD147-positive tumor cells in vitro. Fig.21ARepresentative H&E (top) and IHC (bottom) staining of liver samples from HCC patients at different stages. Fig. 21C The experimental design diagram of collecting HCC samples from different areas of liver cancer tissue. In brief, three areas of interest (tumor area, adjacent area and non-tumor area) were obtained. Primary NK cells were isolated from these areas ( Fig.21B shown). Fig.21D Figure 3 Flow cytometric analysis of CD147-CAR positive primary NK cells from different regions of liver tissue. Fig.21E Shows passing the 4-hour standard 51 Cytotoxicity of primary CD147-CAR-NK cells measured by Cr release assay.

[0041] Fig.22A and 22B is a representative flow cytometric analysis of CD147 expression on different cell types ( Fig.22A ) and pass standard 4 hours 51 Cr release assay to measure the cytotoxicity of CD147-CAR-NK-92MI against target cells with different CD147 expression levels ( Fig. 22B ). Data represent two independent experiments. All data are presented as mean ± SEM.

[0042] Figures 23A-23H These results suggest that SynNotch GPC3-inducible CD147-CAR T cells selectively target GPC3+CD147+HepG2 cells, but not GPC3+CD147- or GPC3-CD147+HepG2 cells. Fig.23A It is a schematic design of GPC3-Gal4VP64-synNotch receptor and CD147-CAR based on pHR lentiviral vector in SFG retroviral vector.SFG retroviral vector contains eGFP, which can be used as a marker for selecting GPC3-Gal4VP64-synNotch positive cells.pHR construct includes CD147 specific single-chain antibody fragment (clone, 5F6), human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domain of costimulation CD28, 4-1BB and the intracellular domain of CD3ζ.pHR lentiviral vector contains mCherry, which can be used as a marker for selecting CD147-CAR positive cells. Fig. 23B is a schematic design of “logic-gated” GPC3-synNotch and CD147-CAR, which showed induced cytotoxicity when the two antigens were co-expressed, but not when the two antigens were expressed alone on adjacent or healthy cells. Fig.23C and 23DSchematic experimental design of T cells co-transduced with GPC3-synNotch-GFP and CD147-CAR-mCherry vectors ( Fig.23C ) and representative flow cytometry analysis of GPC3-synNotch-GFP and CD147-CAR-mCherry expression ( Fig.23D ). Fig.23E GPC3-synNotch-GFP and CD147-CAR-mCherry vectors are expressed through GPC3 高 CD147 低 Schematic experimental design of sensitized co-transduction of HepG2 cell line into T cells and subsequent analysis of CD147-CAR expression in different transduced T cell subsets, including mCherry-positive only, GFP-positive only, GFP and mCherry double-positive, and GFP and mCherry double-negative. Fig.23F Representative flow cytometry analysis of CD147-CAR expression on the surface of different transduced T cell subsets is shown. Both mean fluorescence intensity (MFI) and CD147-CAR percentage are shown in each representative flow cytometry graph. Figure 23G Representative flow cytometric analysis of CD147 and GPC3 expression on HepG2 tumor cell line. Fig.23H Shown is the quantitative analysis of surface CD107a expression on different transduced T cell subsets 2 hours after a "priming and triggering" protocol by different HepG2 tumor cell lines. Data are representative of two independent experiments.

[0043] Fig.24Gamma secretase inhibitors (MK-0752, Notch signaling inhibitors) are shown to specifically block GPC3-SynNotch-eGFP+ and CD147-CAR-mCherry+ induction CD147-CAR expression in primary T cell subsets, but do not block expression in other subsets of primary T cells. Representative flow cytometry analysis (middle figure) of CD147-CAR expression in different primary T cell subsets. Transduced T cells were treated with a combination of DMSO (0.3%; control), MK-0752 (10 μM), TAPI-1 (10 μM), GI254023X (10 μM) and MK-0752+TAPI-1+GI254023X, respectively. At the same time, these cells were sensitized in the presence of CD147KO GPC3high HepG2 cells. CD147-CAR expression on the surface of different transduced T cell subsets was analyzed by flow cytometry. Both mean fluorescence intensity (MFI) and CD147-CAR percentage are shown in each representative flow cytometry figure. Data represent two independent experiments. All data are presented as mean ± SEM.

[0044] Figures 25A-25H It is shown that SynNotch CD147-inducible GPC3-CAR T cells selectively target GPC3+CD147+HepG2 cells, but not GPC3+CD147- or GPC3-CD147+HepG2 cells. Fig.25A It is a schematic design of Myc-CD147-Gal4VP64-SynNotch receptor in SFG retroviral vector and GPC3-CAR based on pHR lentiviral vector.SFG retroviral vector contains Myc tag, which can be used as a marker for selecting CD147-Gal4VP64-SynNotch positive cells.pHR construct consists of GPC3 specific single-chain antibody fragment (scFv, clone 5F6, mIgG1), human IgG1CH2CH3 hinge region and CD28 transmembrane region, followed by costimulation CD28, 4-1BB intracellular domain and CD3ζ intracellular domain composition.pHR lentiviral vector contains mCherry, which can be used as a marker for selecting cells with GPC3-CAR positive cells. Fig.25B is a schematic design of “logic-gated” CD147-SynNotch and GPC3-CAR, which showed induced cytotoxicity when the two antigens were co-expressed but were not activated when the two antigens were expressed alone on adjacent or healthy cells. Fig.25C and 25DSchematic experimental design of T cells co-transduced with Myc-CD147-SynNotch and GPC3-CAR-mCherry vectors ( Fig.25C ) and representative flow cytometry analysis of Myc-CD147-SynNotch and GPC3-CAR-mCherry expression ( Fig.25D ). Fig.25E Myc-CD147-SynNotch and GPC3-CAR-mCherry vectors are expressed through GPC3 高 CD147 低 Schematic experimental design of HepG2 cell line sensitization co-transduction in T cells and subsequent GPC3-CAR expression analysis in different transduced T cell subsets, including mCherry-positive only, GFP-positive only, GFP and mCherry double-positive, and GFP and mCherry double-negative. Fig.25F It is a representative flow cytometry analysis of GPC3-CAR expression on the surface of different transduced T cell subsets. Both mean fluorescence intensity (MFI) and GPC3-CAR percentage are shown in each representative flow cytometry graph. Figure 25G Representative flow cytometric analysis of surface CD107a expression on different transduced T cell subsets after a “priming and triggering” protocol with different HepG2 tumor cell lines. Fig.25H Shown is a quantitative analysis of the surface CD107a expression on different transduced T cell subsets after "sensitization and triggering" protocols by different HepG2 tumor cell lines. The fold change of CD107a MFI is calculated as follows: [(sample MFI-sensitization MFI only) / sensitization MFI only]. Data represent two independent experiments. All data are presented as mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001.

[0045] Figures 26A-26D SynNotch GPC3-inducible CD147-CAR T cells selectively kill GPC3 高 CD147 高 HepG2 cells, but not CD147 敲除 GPC3 高 HepG2 cells. Fig.26A and 26BRepresentative flow cytometric analysis of CD3, CD56, GPC3-synNotch-GFP, and CD147-CAR-mCherry expression. Primary PBMCs were transduced with CD147-CAR-mCherry lentivirus. These mCherry-positive T cells were sorted by flow cytometry and then transduced with GPC3-synNotch-GFP retrovirus. Expression of CD3 and CD56 ( Fig.26A ) and GPC3-synNotch-GFP and CD147-CAR-mCherry expression ( Fig.26B ) Representative flow cytometry analysis. Fig.26C Primary GPC3-synNotch-GFP-CD147-CAR-mCherry T cells were measured by 7-hour FFluc reporter assay to detect the effects of primary GPC3-synNotch-GFP-CD147-CAR-mCherry T cells on HepG2-CD147 高 -GPC3 高 and HepG2-CD147 敲除 -GPC3 高 Figure 3 shows the cytotoxicity of . Fig.26D Figure 3 shows the expression of primary GPC 3-synNotch-GFP-CD147-CAR-mCherry T cells on HepG2-CD147 measured by a 7-hour Cr-51 release assay. 高 -GPC3 高 and HepG2-CD147 敲除 -GPC3 高 Data are representative of two independent experiments.

[0046] Sequence Listing

[0047] Any nucleic acid and amino acid sequence listed herein or in the accompanying sequence listing is shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 CFR § 1.822. In at least some cases, only one strand is shown for each nucleic acid sequence, but any reference to the displayed strand is understood to include the complementary strand.

[0048] SEQ ID NO: 1 is a nucleic acid sequence encoding a modified CD147 scFv. Nucleotides 349-409 are linker sequences.

[0049] SEQ ID NO: 2 is the amino acid sequence of the modified CD147 scFv.

[0050] SEQ ID NO: 3 is the nucleic acid sequence encoding the starting CD147 scFv.

[0051] SEQ ID NO: 4 is a nucleic acid sequence encoding CD147-CAR. Signal peptide: nucleotides 1-57; VH domain: nucleotides 58-411; Linker sequence: nucleotides 412-468; VL domain: nucleotides 469-792; CD28 TM domain-41BB intracellular domain-CD3 ζ domain: nucleotides 793-2202.

[0052] SEQ ID NO: 5 is the amino acid sequence of CD147-CAR. Signal peptide: amino acids 1-19; VH domain: amino acids 20-137; Linker sequence: amino acids 138-156; VL domain: amino acids 157-264; CD28 TM domain-41BB intracellular domain-CD3 ζ domain: amino acids 269-734.

[0053] SEQ ID NO: 6 is a nucleic acid sequence encoding a CD14-CAR with inducible caspase 9. The iCaspase9 sequence is nucleotides 355-1200.

[0054] SEQ ID NO: 7 is the amino acid sequence of CD147-iCaspase 9-CAR. Amino acids 119-400 are iCaspase9.

[0055] SEQ ID NO: 8 is the amino acid sequence of the anti-CD147 VH CDR domain.

[0056] SEQ ID NO: 9 is the amino acid sequence of the anti-CD147 VL CDR domain.

[0057] SEQ ID NOs: 10 and 11 are guide RNAs targeting CD147 for generating CD147 knockout cell lines.

[0058] SEQ ID NO: 12 is an exemplary IL-15 receptor intracellular domain.

[0059] SEQ ID NO: 13 is the consensus nucleic acid sequence of the optimized CD147 scFv nucleic acid sequence (SEQ ID NO: 1) compared to the original scFv sequence (SEQ ID NO: 3).

[0060] SEQ ID NO: 14 is the nucleic acid sequence of the Gal4UAS CD147-CAR construct.

[0061] SEQ ID NO: 15 is the nucleic acid sequence of pHR_Gal4UAS-CD147-CAR-pGK_mCherry vector.

[0062] SEQ ID NO: 16 is the nucleic acid sequence of GPC3-CAR.

[0063] SEQ ID NO: 17 is a nucleic acid encoding a GAL4-VP64 activator.

[0064] SEQ ID NOs: 18-35 are the nucleic acid sequences of primers used for plasmid construction. DETAILED DESCRIPTION

[0065] Disclosed herein are immune cells (including T cells and NK cells) expressing new CD147 targeting CARs. The biological properties of the CD147 antigen allow CD147-CAR-NK cells and CD147-CAR-T cells to produce potent anti-tumor activity against hepatocellular carcinoma in vitro and in vivo. In addition, CD147-CAR-NK cells are also able to kill human neuroblastoma cells in vitro.

[0066] Since CD147 is also expressed at different expression levels on several organs, CD147-CAR modified immune cells may potentially exhibit "on-target, off-tumor" toxicity. Constructs and methods for addressing this potential toxicity are disclosed herein, including "suicide genes" (such as inducible caspase 9) and combination therapies such as cells expressing CAR that are activated only when combined with two antigens such as CD147 and GPC3.

[0067] I. Terminology

[0068] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in: Lewin's Genes X, Krebs et al., ed., Jones & Bartlett Publishers, 2009 (ISBN 0763766321); Kendrew et al. (ed.), The Encyclopedia of Molecular Biology, Blackwell Publishers, 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and George P. Rédei, Encyclopedic Dictionary of Genetics, Genomics, Proteomics and Informatics, 3rd edition, Springer, 2008 (ISBN: 1402067534) and other similar references.

[0069] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Unless the context clearly indicates otherwise, the singular terms "a", "an", and "the" include plural references. "Comprising A or B" means including A or B or A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for description.

[0070] Although methods and materials similar to or equivalent to methods and materials described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety, as are GenBank accession numbers. In the event of a conflict, this specification sheet (comprising the explanation of terms) shall prevail. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive.

[0071] To facilitate review of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0072] Antibody: A polypeptide ligand that contains at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an antigenic epitope. Mammalian immunoglobulin molecules are composed of heavy (H) chains and light (L) chains, each of which has a variable region, referred to as a variable heavy chain (V H ) region and variable light chain (V L ) area. H Area and V L The regions are together responsible for binding the antigen recognized by the antibody. There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE.

[0073] The variable region of an antibody contains a "framework" region and a hypervariable region called a "complementarity determining region" or "CDR." The CDR is primarily responsible for binding to the epitope of an antigen. The framework region of an antibody is used to position and align the CDR in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; "Kabat" numbering scheme); Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273, 927-948, 1997; "Chothia" numbering scheme), and the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; "IMGT" numbering scheme). The Kabat and IMGT databases are maintained online.

[0074] Single-chain antibodies (scFv) are genetically engineered molecules that contain one or more V fragments of an antibody connected by a suitable polypeptide linker. H and V LThe V domains are genetically fused to the single-chain molecules (see, for example, Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, ID Rugs, 13:543-549, 2010). H -Domain and V L The intramolecular orientation of the -domain is usually not critical for scFv. Therefore, two possible arrangements (V H Domain-Linker Domain-V L Domain; V L Domain-Linker Domain-V H In dsFv, V H and V L Mutations have been made to introduce disulfide bonds to stabilize the chain association. Also included are diabodies, which are bivalent, bispecific antibodies in which V H and V L The domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0075] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Edition, WH Freeman & Co., New York, 1997.

[0076] Cancer: A malignant tumor characterized by abnormal or uncontrolled cell growth. Other characteristics commonly associated with cancer include metastasis, interference with the normal function of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or exacerbation of inflammation or immune responses, invasion of surrounding or distant tissues or organs such as lymph nodes. "Metastatic disease" means that cancer cells have left the original tumor site and migrated to other parts of the body, for example, through the bloodstream or lymphatic system.

[0077] CD147: Also known as basigin (BSG), extracellular matrix metalloproteinase inhibitor (EMMPRIN or EMPRIN). A transmembrane glycoprotein with multiple functions in normal cell function and disease (Hahn et al., J. Leukocyte Biol. 98: 33-48, 2015). CD147 is important for T cell activation and proliferation as well as cell migration, adhesion and invasion in immune cells (Hahn et al., J. Leukocyte Biol. 98: 33-48, 2015). CD147 is expressed at different levels on different cell types (e.g., hematopoietic cells, epithelial cells and endothelial cells) (Liao et al., Mol. Cell Biol. 31: 2591-2604, 2011) and can be significantly upregulated in disease states such as HCC.

[0078] CD147 sequences are publicly available. For example, GenBank accession numbers: NM_198590, NM_198591, NM_001728, NM_198589 and NM_001322243 disclose human CD147 nucleic acid sequences, and GenBank accession numbers: NP_940992, NP_940993, NP_001719, NP_940991 and NP_001309172 disclose human CD147 amino acid sequences. Similarly, NM_009768 and NM_001077184 disclose mouse CD147 nucleic acid sequences and GenBank accession numbers NP_033898 and NP_001070652 disclose mouse CD147 amino acid sequences. All of these sequences are incorporated by reference in the form present in GenBank on March 15, 2019.

[0079] Chimeric antigen receptor (CAR): a chimeric molecule including an antigen binding portion (such as a single domain antibody or scFv) and a signaling domain such as a signaling domain from a T cell receptor (e.g., CD3ζ). Typically, CAR includes an antigen binding portion, a transmembrane domain, and an intracellular domain. The intracellular domain typically includes a signaling chain with an immunoreceptor tyrosine-based activation motif (ITAM) such as CD3ζ or FcεRIγ. In some cases, the intracellular domain also includes an intracellular portion of at least one additional costimulatory domain such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10.

[0080] Complementarity Determining Region (CDR): A region of highly variable amino acid sequence that defines the binding affinity and specificity of an antibody. The light chain and heavy chain of a mammalian immunoglobulin each have three CDRs, named VL-CDR1, VL-CDR2, VL-CDR3 and VH-CDR1, VH-CDR2, VH-CDR3.

[0081] Glypican-3 (GPC3): A cell surface heparan sulfate proteoglycan that binds to CD26 and inhibits CD26 activity. GPC3 can induce apoptosis in some cell types. GPC3 is expressed by some tumors, including hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma, hepatoblastoma, and Wilms tumor.

[0082] GPC3 sequences are publicly available. For example, GenBank Accession Numbers: NM_001164619, NM_001164618, NM_004484, and NM_001164617 disclose exemplary human GPC3 nucleic acid sequences, and GenBank Accession Numbers: NP_001158091, NP_001158090, NP_004475, and NP_001158089 disclose exemplary human GPC3 amino acid sequences. Similarly, GenBank Accession Number: NM_016697 discloses an exemplary mouse GPC3 nucleic acid sequence, and GenBank Accession Number: NP_057906 discloses an exemplary mouse GPC3 amino acid sequence. Each of these sequences is incorporated by reference as it exists in GenBank on February 27, 2020.

[0083] Isolated: An "isolated" biological component such as a nucleic acid, protein (including antibodies) or organelle has been substantially separated or purified from other biological components in the environment (e.g., a cell) in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins and organelles. "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0084] Liver cancer: Hepatocellular carcinoma (HCC) is the most common type of primary malignant liver tumor and often occurs in patients with viral hepatitis (such as hepatitis B or hepatitis C), toxin exposure, or cirrhosis (sometimes caused by alcoholism). Other types of liver cancer include intrahepatic cholangiocarcinoma (iCCA), fibrocarcinoma, and hepatoblastoma.

[0085] Natural Killer (NK) Cell: An immune system cell that kills target cells in the absence of specific antigenic stimulation and without restriction, depending on the MHC class. The target cell can be a tumor cell or a cell carrying a virus. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers. NK cells typically comprise approximately 10% to 15% of the mononuclear cell fraction in normal peripheral blood. Historically, NK cells were first identified by their ability to lyse certain tumor cells without prior immunization or activation. NK cells are thought to provide a "backup" protection mechanism against viruses and tumors that can evade CTL responses by downregulating MHC class I presentation. In addition to participating in direct cytotoxic killing, NK cells also play a role in cytokine production, which may be important in controlling cancer and infection.

[0086] In some examples, "modified NK cells" are NK cells transduced or transfected with heterologous nucleic acids (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins. The terms "modified NK cells" and "transduced NK cells" are used interchangeably in some examples herein.

[0087] Purified: The term purified does not require absolute purity; on the contrary, it is intended as a relative term. Thus, for example, a purified protein or nucleic acid preparation is one in which the protein or nucleic acid is more enriched than the protein or nucleic acid in its natural environment (e.g., within a cell). In one embodiment, the preparation is purified so that the protein or nucleic acid accounts for at least 50% of the total protein or nucleic acid content of the preparation. Substantially purified refers to purification from other proteins or cellular components. Substantially purified proteins or nucleic acids are at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in a specific non-limiting example, a substantially purified protein or nucleic acid is 90% free of other components.

[0088] Recombinant: A nucleic acid or protein that has a sequence that is not naturally occurring or that is made by the artificial combination of two otherwise isolated sequence fragments (e.g., a "chimeric" sequence). This artificial combination can be accomplished by chemical synthesis or artificial manipulation of isolated nucleic acid fragments, such as through genetic engineering techniques.

[0089] Subject: A living multicellular vertebrate organism, a category that includes both human and veterinary subjects, including human and non-human mammals.

[0090] T cell: A white blood cell (lymphocyte) that is an important mediator of the immune response. T cells include but are not limited to CD4 + T cells and CD8 + T cells. CD4 +T lymphocytes are immune cells that carry a marker on their surface called cluster of differentiation 4 (CD4). These cells, also known as helper T cells, help orchestrate immune responses, including antibody responses as well as killer T cell responses. + T cells carry the "cluster of differentiation 8 (CD8)" marker. In one embodiment, CD8 + The T cells are cytotoxic T lymphocytes (CTL). In another embodiment, CD8 + The cells are suppressor T cells.

[0091] Activated T cells can be detected by increased cell proliferation and / or expression or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFNγ or TNFα). + Activation of T cells can also be detected by an increase in cytolytic activity in response to antigen.

[0092] In some examples, "modified T cells" are T cells that are transduced or transfected with heterologous nucleic acids (such as one or more of the nucleic acids or vectors disclosed herein) or express one or more heterologous proteins. The terms "modified T cells" and "transduced T cells" are used interchangeably in some examples herein.

[0093] Transduced or transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the terms transduction and transformation encompass all techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, use of plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0094] Treat or ameliorate a disease: "Treatment" refers to therapeutic intervention that reduces or inhibits the signs or symptoms of a disease or pathological condition after it has begun to develop, such as reducing tumor size or tumor burden. "Amelioration" refers to a decrease in the number or severity of signs or symptoms of a disease, such as cancer.

[0095] Vector: A nucleic acid molecule that can be introduced into a host cell (e.g., by transfection or transduction) to produce a transformed host cell. A recombinant DNA vector is a vector with recombinant DNA. The vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements known in the art. A viral vector is a recombinant nucleic acid vector with at least some nucleic acid sequences derived from one or more viruses. A replication-defective viral vector is a vector that needs to complement one or more regions in the viral genome required for replication due to a defect in at least one replication-essential gene function.

[0096] II. Overview of Several Implementation Schemes

[0097] Disclosed herein are CD147 specific binding agents, including modified CD147 scFv. Also disclosed are chimeric antigen receptors (CARs), which encode CD147 specific binding agents fused to hinge regions, transmembrane domains, and intracellular domains including costimulatory domains and intracellular signaling domains. In some instances, the costimulatory domains are from CD28 and / or 4-1BB, and the signaling domains are from CD3ζ. Also provided are nucleic acids encoding CD147 specific binding agents and CD147-CARs and vectors comprising the nucleic acids.

[0098] Also provided herein are modified immune cells (such as T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages) expressing CD147-CAR. In some embodiments, modified immune cells express one or more additional CARs such as CARs targeting hepatitis viruses (such as hepatitis B or hepatitis C). In other embodiments, modified immune cells express inducible CD147-CAR and constructs that induce CD147-CAR expression, such as anti-GPC3 SynNotch constructs that drive CD147-CAR inducing agents to express. In a further embodiment, modified immune cells express inducible GPC3-CAR and constructs that induce GPC3-CAR expression, such as anti-CD147SynNotch constructs that drive GPC3-CAR inducing agents to express. In a particular example, CD147-CAR and / or anti-CD147 SynNotch constructs include modified CD147 scFv disclosed herein.

[0099] Also provided is a method for treating cancer expressing CD147 in a subject. In some embodiments, the method includes administering to the subject an effective amount of modified immune cells (such as T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages) comprising nucleic acids encoding CD147-CAR. In other embodiments, the method includes administering to the subject an effective amount of modified immune cells (such as T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages), the modified immune cells include nucleic acids encoding inducible CD147-CAR and anti-GPC3 SynNotch nucleic acids expressing the inducer of CD147-CAR expression. In other embodiments, the method includes administering to the subject an effective amount of modified immune cells (such as T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages), the modified immune cells include nucleic acids encoding GPC3-CAR and anti-CD147SynNotch constructs expressing the inducer of GPC3-CAR expression. In some instances, the modified immune cells are autologous. In other instances, the immune cells are allogeneic. In some embodiments, the subject has hepatocellular carcinoma or neuroblastoma.

[0100] III. CD147-specific binding agents

[0101] Disclosed herein are CD147 binding agents that are used in some examples as targeting moieties of chimeric antigen receptors. In some embodiments, the CD147 binding agent is a CD147 scFv, which is a modified fragment encoding a CD147-specific scFv from the 5F6 clone (U.S. Pat. No. 8,618,264). Fig. 20 An alignment of the starting CD147 scFv nucleic acid sequence (SEQ ID NO: 3) and the modified scFv nucleic acid sequence (SEQ ID NO: 1) is shown. In some examples, the modified CD147-specific scFv binds to cells that highly express CD147.

[0102] In some embodiments, the CD147-specific binding agent is a single domain antibody (such as scFv) that specifically binds to CD147 and includes the CDR sequences provided in Table 1. In some examples, the scFv specifically binds to CD147 and includes an amino acid sequence comprising the variable heavy chain (VH) domain complementary determining region 1 (CDR1), CDR2 and CDR3 amino acid sequences of SEQ ID NO: 8 and the variable light chain (VL) domain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 9. In some embodiments, the scFv includes the CDR amino acid sequences provided in Table 1 and has at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity) with the amino acid sequence of SEQ ID NO: 1. In other embodiments, the scFv includes the amino acid sequence of SEQ ID NO: 1 or consists of the amino acid sequence of SEQ ID NO: 1.

[0103] Table 1: Position of CDRs in the CD147 scFv sequence (determined using the Kabat numbering scheme)

[0104]

[0105]

[0106] In another example, the scFv is encoded by a nucleic acid comprising the CDR sequences provided in Table 1 and having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to the nucleotide sequence of SEQ ID NO: 2. In other embodiments, the scFv is encoded by a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO: 2.

[0107] Also provided is a vector comprising a nucleic acid encoding the above-mentioned CD147-specific binding agent. In some examples, the vector includes a nucleic acid having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with SEQ ID NO: 1, for example, encoding a nucleic acid having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with SEQ ID NO: 2.

[0108] In another example, provided herein is a nucleic acid encoding a CD147-specific binding agent (e.g., a nucleic acid having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to SEQ ID NO: 1) operably linked to a nucleic acid encoding Gal4-VP64 (e.g., SEQ ID NO: 17). In some examples, the nucleic acid is part of a SynNotch construct. When CD147 binds to a protein encoded by this construct, Gal4-VP64 is activated and expression of the construct can be induced using a Gal4 response element (e.g., Gal4UAS). Fig.25A An exemplary vector encoding an anti-CD147 scFv SynNotch inducer construct is shown in FIG.

[0109] IV. CD147 Chimeric Antigen Receptor

[0110] Provided herein is a CD147-CAR comprising a CD147-specific binding agent described in Section III above. In some embodiments, CAR includes an antigen binding domain comprising a CD147-specific scFv (such as SEQ ID NO: 2), a hinge domain, a transmembrane domain, and an intracellular domain comprising at least one co-stimulatory domain and an intracellular signaling domain.

[0111] In some embodiments, the antigen binding domain is a CD147-specific scFv, which has, for example, an amino acid sequence that has at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO:2, or comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0112] In some embodiments, the hinge domain is an IgG hinge region. In one example, the hinge domain is an IgG1 hinge. Other hinge domains can be used, such as hinge regions from other immunoglobulins (e.g., IgG4 or IgD) or hinge regions from CD8, CD28, or CD40.

[0113] In another embodiment, the transmembrane domain is a CD28 transmembrane domain. In one example, the transmembrane domain is from CD28. The transmembrane domain can also be from other T cell proteins, such as CD8, CD4, CD3ζ, CD40, OX40L, 41BBL, ICOS, ICOS-L, CD80, CD86, ICAM-1, LFA-1, ICAM-1, CD56, CTLA-4, PD-1, TIM-3, NKP30, NKP44, NKP40, NKP46, B7-H3, PD-L1, PD-2, and CD70.

[0114] In another embodiment, the intracellular domain includes one or more intracellular regions or parts thereof from costimulatory molecules.Exemplary costimulatory molecules include CD28, 4-1BB, CD8, CD40, OX-40, ICOS, CD27 and DAP10, OX40-L, 4-1BBL, ICOS-L, CD80, CD86, ICAM-1, LFA-1, CD56, CTLA-4, PD-1, TIM-3, NKP30, NKP44, NKP40, NKP46, B7-H3, PD-L1, PD-2 and CD70.In a particular example, the costimulatory domain is from CD28 and / or 4-1BB.In one example, the costimulatory domain includes domains from both CD28 and 4-1BB.The intracellular domain also includes an intracellular signaling domain from CD3ζ. In other examples, the intracellular signaling domain is from DAP10, DAP12, PDK or FcεRIγ. In one example, the intracellular signaling domain is from CD3ζ.

[0115] In some embodiments, CD147-CAR also includes a signal sequence located at the N-terminus of the scFv domain. In some instances, the signal sequence is an IgG signal sequence or a GM-CSF signal sequence. In one example, the signal sequence is SEQ ID NO:5 amino acids 1-19.

[0116] In certain embodiments, CD147-CAR includes an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO: 5. In some examples, CD147-CAR includes or consists of an amino acid sequence of SEQ ID NO: 5.

[0117] In another embodiment, CD147-CAR further includes an inducible gene (eg, "suicide" gene) that can be used to eliminate cells expressing CD147-CAR. Inducible genes can be activated when there are off-target side effects (or on-target / off-tumor effects) such as cytokine release syndrome ("cytokine storm"), etc. In some examples, the expression of suicide genes can be induced by small molecules such as tetracycline or doxycycline ("TET ON" system) or rapamycin. See, for example, Gargett et al., Front.Pharmacol.5:235,2014; Stavrou et al., Mol.Ther.6:1266-1276,2018. In other examples, suicide genes can be induced by Fas domain inducible systems. In some examples, the inducible suicide domain is located at the N-terminus or C-terminus of the antigen binding domain of CAR, and in other examples, the inducible suicide domain is located at the C-terminus of the CD3ζ domain of CAR. The inducible suicide domain is separated from CAR by a self-cleaving peptide (such as a P2A peptide or a T2A peptide). In some embodiments, the inducible suicide domain includes caspase 9, such as SEQ ID NO:7 amino acids 119-400. In some examples, the CD147-CAR including tetracycline-inducible caspase 9 includes an amino acid sequence with SEQ ID NO:7 having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity). In other examples, the CD147-CAR including tetracycline-inducible caspase 9 includes SEQ ID NO:7 amino acid sequence or is composed of SEQ ID NO:7 amino acid sequence.

[0118] In other embodiments, CD147-CAR further includes a domain that increases the survival or persistence of modified immune cells expressing CAR. In some instances, the domain is an intracellular domain from a cytokine receptor, for example, from an intracellular domain of interleukin (IL) receptor 15 (eg, SEQ ID NO: 12), IL-12 receptor or IL-18 receptor. In other examples, the domain is an intracellular domain of a growth factor receptor, such as an intracellular domain of CD40, NKG2D, NKP40 or NKP46. In some instances, the domain is located at the C-terminus of the CD3 ζ domain of CAR.

[0119] In some examples, CD147-CAR further includes one or more additional antigen binding domains, and the one or more additional antigen binding domains specifically bind to antigens co-expressed with CD147 on tumor cells. In some non-limiting examples, CD147-CAR includes at least one additional antigen binding domain that specifically binds to one or more of liver cancer antigens such as glypican-3, alpha-fetoprotein or mucin-1. Additional tumor antigens can be selected based on the type of cancer being treated.

[0120] Also provided is a nucleic acid encoding CD147-CAR disclosed herein. In some embodiments, the nucleic acid encoding includes CD147 specific scFv CAR, such as with SEQ ID NO: 1 having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity) nucleic acid sequence or including SEQ ID NO: 1 nucleic acid sequence or consisting of SEQ ID NO: 1 nucleic acid sequence. In some examples, CD147 specific CAR nucleic acid also encodes IgG hinge domain, CD28 transmembrane domain, CD28 and 4-1BB costimulatory domain and CD3 ζ domain. In one example, the CD147-specific CAR is encoded by a nucleic acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity) with SEQ ID NO: 4 or includes a nucleic acid sequence of SEQ ID NO: 4 or consists of a nucleic acid sequence of SEQ ID NO: 4. In some examples, the CD147-specific CAR nucleic acid also encodes an inducible caspase 9 domain, such as a nucleic acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity) with SEQ ID NO: 6 or includes a nucleic acid sequence of SEQ ID NO: 6 or consists of a nucleic acid sequence of SEQ ID NO: 6.

[0121] Also provided are functional variants of CAR or its domain as described herein, which retain the biological activity of the CAR as a variant or retain the biological activity of a specific domain. Functional variants can have at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homogeneity with the parent CAR or domain in amino acid sequence. It can be substituted, for example, in one or more of an extracellular targeting domain, a hinge domain, a transmembrane domain, and an intracellular domain.

[0122] In some examples, the functional variant includes an amino acid sequence of a parent CAR or domain having at least one conservative amino acid substitution (such as up to 10 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions). In other examples, the functional variant includes an amino acid sequence of a parent CAR or domain having at least one non-conservative amino acid substitution (such as up to 10 non-conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-conservative substitutions). In this case, the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent CAR or domain.

[0123] In some examples, CAR or its domain may include one or more synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole-2-carboxylic acid , 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N', N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, oc-aminocycloheptanecarboxylic acid, -(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, α, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. CAR can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized by, for example, a disulfide bond, or converted into an acid addition salt and / or optionally dimerized or polymerized or conjugated.

[0124] In some embodiments, the nucleic acid molecules encoding the disclosed CAR are included in an expression vector (such as a viral vector) for expression in host cells such as T cells or NK cells. In some examples, the expression vector includes a promoter operably connected to the nucleic acid molecules encoding CAR. Other expression control sequences, such as one or more enhancers, transcription and / or translation terminators and initial sequences, may also be included in the expression vector. In some embodiments, the nucleic acid encoding the CD147-CAR provided herein is included in a viral vector. Examples of suitable viral vectors include retroviruses (such as MoMLV viruses or slow viruses), adenoviruses, adeno-associated viruses, vaccinia viruses and fowl pox vectors. In a specific example, the CD147-CAR encoding nucleic acid is included in a MoMLV vector such as a SFG retroviral vector or a pHAGE-CPPT slow virus vector. In other examples, the vector may be a DNA vector.

[0125] In some embodiments, the vector further includes an upstream activation sequence (UAS) that allows CD147-CAR inducible expression. In a non-limiting example, UAS is Gal4 UAS activated by Gal4. However, those skilled in the art can recognize other transactivation systems that can be used. In one example, the Gal4UAS CD147-CAR nucleic acid includes a nucleic acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with SEQ ID NO: 14. An exemplary vector encoding a Gal4UAS CD147-CAR construct is in Fig.23A As shown in pHR_Gal4UAS-CD147-CAR-pGK_mCherry. In some examples, the vector includes a nucleic acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with SEQ ID NO: 15. In some examples, the vector includes a selectable marker (e.g., mCherry in SEQ ID NO: 15), but in other examples, the selectable marker is not included in the vector.

[0126] In some examples, the carrier further includes a nucleic acid sequence encoding at least one additional CAR. In some examples, the additional CAR is specific to additional tumor antigens, such as to increase the specificity of CD147-CAR targeted expression or overexpression of CD147 tumor cells. In some examples, the carrier includes nucleic acids encoding one or more CARs, and the one or more CARs include antigen binding domains that specifically bind to liver cancer antigens such as glycan-3, alpha-fetoprotein or mucin-1. Additional tumor antigens / CARs can be selected based on the type of cancer treated. In other examples, the carrier includes nucleic acids encoding CAR, and the CAR includes antigen binding domains that are specific to hepatitis B or hepatitis C. In some examples, the additional CAR binds to HBV envelope protein or hepatitis B surface antigen (HBsAg). In other examples, the additional CAR binds to HCV E2 glycoprotein. In some examples, the one or more additional CARs are included in a carrier with CD147-CAR, for example, separated by self-cleaving peptides such as P2A peptide sequences.

[0127] V. Cells expressing CD147 CAR or CD147-specific binding agents

[0128] Also provided herein is a cell (eg, immune cell) expressing a disclosed CD147-CAR or CD147 specific binding agent. In a specific embodiment, the cell includes a T cell, a NK cell, a NKT cell, a DNT cell, a neutrophil or a macrophage. In some embodiments, the cell is a T cell, a NK cell or a macrophage expressing CD147-CAR.

[0129] In some examples, the cells further express a GPC3 specific binding agent such as an anti-GPC3 scFV. In a specific example, the cells express an anti-GPC3 scFV operably linked to a nucleic acid encoding Gal4-VP64 (e.g., a SynNotch construct). When GPC3 binds to the protein encoded by this construct, Gal4-VP64 is activated and can induce expression of the construct using a Gal4 response element (e.g., Gal4UAS). Fig.23A An exemplary vector encoding an anti-GPC3scFv SynNotch inducer construct is shown in . Thus, in some examples, the T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages express inducible CD147-CAR (eg, SEQ ID NO: 14) and an anti-GPC3 binder construct that induces inducible CD147-CAR expression.

[0130] In other embodiments, the cell is a T cell, NK cell, NKT cell, DNT cell, neutrophil or macrophage expressing CD147 specific binding agents such as CD147 scFv (e.g., SEQ ID NO: 1) and GPC3-CAR. In some instances, GPC3-CAR includes a nucleic acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with SEQ ID NO: 16. In a particular example, the cell expresses an anti-CD147 scFV operably connected to a nucleic acid encoding Gal4-VP64 (e.g., SynNotch construct). When CD147 is bound to a protein encoded by this construct, Gal4-VP64 is activated and the expression of the construct can be induced using a Gal4 response element (e.g., Gal4UAS). Fig.25A An exemplary vector encoding an anti-CD147 scFv SynNotch inducer construct and an anti-GPC3-CAR is shown in . Therefore, in some examples, the T cells, NK cells, NKT cells, DNT cells, neutrophils, macrophages express inducible GPC3-CAR (e.g., SEQ ID NO: 16) and an anti-CD147 binder construct that induces inducible GPC3-CAR expression.

[0131] In some examples, the immune cells are transduced or transfected with one or more expression vectors, and the one or more expression vectors include one or more nucleic acids, including encoding CD147-CAR, inducible GPC3-CAR, inducible CD147-CAR, CD147 specific binding agents operably connected to an inducer, GPC3 specific binding agents operably connected to an inducer, or nucleic acids of any combination thereof of two or more. In other examples, a vector (or DNA encoding a construct) can be introduced by contacting a cell with a nanoparticle including a vector or DNA. In some examples, the cells are irradiated (e.g., treated with gamma-irradiation, such as at a dose of at least 1,000 rads, at least 2,000 rads, at least 3,000 rads, at least 5,000 rads, at least 7,000 rads, at least 8,000 rads, at least 9,000 rads, at least 10,000 rads, at least 11,000 rads, at least 12,000 rads, or at least 15,000 rads, or about 1,000-15,000 rads, 2,000-12,000 rads, 1,000-5,000 rads, 5,000-10,000 rads, or 8,000-12,000 rads, or about 10,000 rads), e.g., prior to administration to a subject, following transduction or transfection.

[0132] In some examples, the transduced or transfected cells are separated T cells (such as primary T cells or T cells obtained from a subject), separated NK cells (such as primary NK cells or NK cells obtained from a subject), separated NKT cells, separated DNT cells, separated neutrophils or separated macrophages (such as primary macrophages or macrophages obtained from a subject). In some examples, the T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages are obtained from peripheral blood, cord blood, lymph node tissue, bone marrow or tumor tissue. In some examples, for example, before and / or after transduction with one or more of the disclosed expression vectors, T cells, NK cells, NKT cells or DNT cells are also enriched, purified and / or amplified from samples from a subject.

[0133] In one non-limiting embodiment, the cell is a NK-92 cell. NK-92 cells are a NK cell line derived from a patient with non-Hodgkin's lymphoma (e.g. CRL-2407 TM ). This cell line has the properties of activated NK cells (see, e.g., Gong et al., Leukemia 8:652-658, 1994). In another embodiment, the cell is a NK-92MI cell (e.g. CRL-2408 TM ). The NK-92MI cell line is an interleukin-2 (IL-2)-independent NK cell line derived from NK-92, which stably expresses human IL-2 (see, e.g., Tam et al., Hum. Gene Ther. 10: 1359-1373, 1999). NK-92 or NK-92MI cells expressing CAR (such as CD147-CAR and / or other nucleic acids disclosed herein) can be used as "off-the-shelf" immunotherapy herein because autologous NK cells do not have to be produced for each subject. Other NK cell lines that can be used with the CD147-CAR (or other nucleic acids) described herein include NKL, KHYG-1, and YTS cells.

[0134] NK-92-mediated immunotherapy is currently undergoing phase I / II clinical trials (Arai et al., 2008; Tonn et al., 2013). Typically, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment. The required irradiation dose is approximately 10 Gy. The infusion dose of irradiated NK-92 can be up to 10 10 NK92 cells / m 2Importantly, irradiated NK-92 cells have been shown to be safe for infusion in patients, as demonstrated in several NK-92 clinical trials (NCT00900809, NCT00990717, NCT00995137, and NCT01974479).

[0135] In some non-limiting embodiments, immune cells are transduced with vectors encoding CD147-CAR. After transduction, cells expressing CD147-CAR can be detected and / or enriched, for example, by flow cytometry using labeled antibodies in combination with CD147. In some examples, transduced cells (such as NK cells or T cells) are amplified, for example, by cell culture for a period of time after transduction. In some examples, some or all of the modified cells are cryopreserved for later use.

[0136] VI. Immunotherapeutic Methods

[0137] A method for treating a subject's cancer (such as cancer expressing or overexpressing CD147) with a CD147-CAR disclosed herein is provided. In some embodiments, the method includes administering a composition to the subject, the composition including modified T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages (e.g., transduced with a vector encoding CAR) and a pharmaceutically acceptable carrier expressing CD147-CAR. In other examples, the method includes administering a pharmaceutical composition to the subject, the pharmaceutical composition including an expression vector encoding CD147-CAR and a pharmaceutically acceptable carrier.

[0138] Also provided is a method for treating a subject's cancer (such as cancer expressing or overexpressing CD147) with an inducible CD147-CAR and a GPC3 specific binding agent connected to the expression of an inducible CD147-CAR disclosed herein. In some embodiments, the method includes administering a composition to a subject, the composition including modified T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages expressing inducible CD147-CAR and a GPC3 specific binding agent connected to an inducing agent (e.g., transduced with one or more vectors encoding CD147-CAR and GPC3 specific binding agents) and a pharmaceutically acceptable carrier. In other examples, the method includes administering to a subject one or more expression vectors encoding inducible CD147-CAR (e.g., SEQ ID NO: 15) and a GPC3 specific binding agent connected to an inducing agent and a pharmaceutically acceptable carrier.

[0139] Also provided is a method for treating a subject's cancer (such as cancer expressing or overexpressing CD147) with an inducible GPC3-CAR and a CD147 specific binding agent connected to an inducible GPC3-CAR disclosed herein. In some embodiments, the method includes administering a composition to a subject, the composition including modified T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages expressing inducible GPC3-CAR and CD147 specific binding agents connected to an inducing agent (e.g., transduced with one or more vectors encoding GPC3-CAR (e.g., SEQ ID NO: 16) and CD147 specific binding agents) and a pharmaceutically acceptable carrier. In other examples, the method includes administering one or more expression vectors to a subject, the one or more expression vectors encoding inducible GPC3-CAR and CD147 specific binding agents connected to an inducing agent and a pharmaceutically acceptable carrier.

[0140] Modified cells (such as T cells, NK cells, NKT cells, DNT cells, neutrophils or macrophages) expressing CD147-CAR or CD147 specific binding agents as described herein can be incorporated into pharmaceutical compositions. Such compositions generally include cell populations (such as CD147-CAR-NK cells or CD147-CAR-T cells) and pharmaceutically acceptable carriers. "Pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents compatible with drug administration (see, for example, Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st edition, 2005). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, balanced salt solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds may also be incorporated into the composition. Practical methods for preparing administrable compositions include those provided in Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st edition, (2005).

[0141] In some examples, the composition includes about 10 4 to 10 12 Modified NK cells or T cells (e.g., about 10 4 -10 8 cells, about 10 6 -10 8 cells or about 10 6 -10 12 For example, the composition can be prepared so that about 10 4 to 10 10 Modified NK cells or modified T cells / kg (e.g., about 10 4 , 10 5 , 10 6 , 10 7 or 10 8 In a specific example, the composition comprises at least 10 4 , 10 5 , 10 6 or 10 7 Individual CD147-CAR-NK cells or CD147-CAR-T cells. Modified NK cell populations or modified T cell populations are usually administered parenterally, such as intravenously; however, injection or infusion (local administration) to a tumor or near a tumor or administration to the peritoneal cavity can also be used. Appropriate administration routes can be determined based on factors such as the subject, the condition being treated, and other factors.

[0142] Multiple doses of modified NK cell populations or modified T cell populations can be administered to a subject. For example, CD147-CAR-NK cells or CD147-CAR-T cells can be administered daily, every other day, twice a week, weekly, every other week, every three weeks, monthly or at a lower frequency. A skilled clinician can select an administration regimen based on the subject, the condition being treated, previous treatment history, and other factors.

[0143] In another example, at least one, at least one, at least two, at least three or at least four cytokines (such as IL-2, IL-15, IL-21 and / or IL-12) are also administered to the subject to support the survival and / or growth of modified NK cells or modified T cells. In a specific non-limiting example, at least one cytokine includes IL-2 and IL-15. The cytokine is administered before, after or substantially simultaneously with modified NK cells or modified T cells. In a specific example, at least one cytokine (such as IL-2) is administered, for example, simultaneously with CD147-CAR-NK cells.

[0144] In some examples, the treated subject has a solid tumor, such as a solid tumor expressing CD147. Examples of solid tumors include sarcomas (such as fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, soft tissue sarcomas and other sarcomas), synovial tumors, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, peritoneal cancer, esophageal cancer (such as esophageal squamous cell carcinoma), pancreatic cancer, breast cancer (including basal breast cancer, ductal carcinoma and lobular breast cancer), endometrial cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, prostate cancer, liver cancer (including hepatocellular carcinoma), gastric cancer, squamous cell carcinoma (including head and neck squamous cell carcinoma), basal cell carcinoma, Adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, fallopian tube cancer, testicular tumors, seminoma, bladder cancer (such as renal cell carcinoma), melanoma and CNS tumors (such as glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma). Solid tumors also include tumor metastasis (e.g., metastasis to the lung, liver, brain or bone). In some instances, the subject is suffering from hepatocellular carcinoma, neuroblastoma, breast cancer, gastric cancer, endometrial cancer, bladder cancer (such as renal cell carcinoma), lung cancer (such as non-small cell lung cancer), cervical cancer, medulloblastoma, esophageal cancer (such as esophageal squamous cell carcinoma), prostate cancer, seminoma, glioblastoma, osteosarcoma, astrocytoma or soft tissue sarcoma. In a specific instance, the subject is suffering from hepatocellular carcinoma or neuroblastoma.

[0145] In other examples, the subject has a hematological malignancy, such as a hematological malignancy that expresses CD147. Examples of hematological malignancies include leukemias, including acute leukemias (e.g., 11q23-positive acute leukemia, acute lymphocytic leukemia (ALL), T-cell ALL, acute myeloid leukemia, acute myeloid leukemia (AML), and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), lymphoblastic leukemia, polycythemia vera, lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. In certain examples, the subject has acute lymphoblastic leukemia (ALL), T-cell ALL, acute myeloid leukemia, or acute myeloid leukemia (AML).

[0146] In some instances, the subject is also treated with one or more of surgery, radiotherapy, and chemotherapeutic agents. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil nitrogen mustard, or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozotocin, or dacarbazine), etc.; antimetabolites such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU or cytarabine), and purine analogs such as mercaptopurine or thioguanine; or natural products such as vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (e.g., L-asparaginase). Additional agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocortical suppressants (such as mitotane and aminoglutethimide); hormones and antagonists, such as adrenocortical steroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinylestradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include doxorubicin, melphalan, and phenoxyprogesterone acetate. Ara-C (cytarabine), carmustine, busulfan, lomustine, carboplatin, cisplatin, cyclophosphamide Daunorubicin, dacarbazine, 5-fluorouracil, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes such as docetaxel), vinblastine, vincristine, VP-16, and newer drugs including gemcitabine Trastuzumab Irinotecan (CPT-11), clalipid, navelbine, rituximab Imatinib (STI-571), topotecan Capecitabine, ibritumomab A skilled clinician can select an appropriate additional therapy for a subject (from among those listed herein or other current therapies) based on factors such as the subject, the cancer being treated, treatment history, and other factors.

[0147] Example

[0148] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

[0149] Example 1

[0150] Materials and methods

[0151] Antibodies and reagents: purified anti-CD247 (also known as T cell surface glycoprotein CD3 ζ chain, CD3) antibody (clone 6B10.2, BioLegend), purified anti-human CD147, FITC-conjugated anti-human CD147 (clone HIM6, BioLegend), PE- or APC-conjugated anti-human CD3 antibody (clone OKT3, BioLegend), FITC- or BV 510-conjugated anti-human CD56 antibody (clone HCD56, BioLegend), PE-conjugated anti-human CD69 antibody (clone FN50, BioLegend), APC / Fire 750-conjugated anti-human CD226 antibody (also known as DNAM-1, clone 11A8, BioLegend), APC / Fire 750-conjugated anti-human KLRG1 (MAFA) antibody (clone SA231A2, BioLegend), BV421-conjugated anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend), PE / Cy7-conjugated anti-human CD158b (KIR2DL2 / L3, BioLegend) antibody (clone DX27, BioLegend), PE / Cy7-conjugated anti-human CD244 (2B4) antibody (clone C1.7, BioLegend), PE-conjugated anti-human CD152 (CTLA-4) antibody (clone BNI3), APC-conjugated anti-human CD366 (Tim-3) antibody (clone F38-2E2), PerCP / Cy5.5 anti-human TIGIT (VSTM3) antibody (clone A15153G), FITC-conjugated anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend), and PerCP / Cy5.5-conjugated anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA).

[0152] APC-conjugated anti-human CD16 antibody (clone B73.1, BD Biosciences), FITC-conjugated anti-human CD3 antibody (clone UCHT1, BD Biosciences), BV480-conjugated anti-human CD85j antibody (LIR-1) antibody (clone GHI / 75, BD Biosciences), BV711-conjugated anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and PE- or FITC-conjugated anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA).

[0153] FITC-conjugated anti-human KIR / CD158 antibody (clone 180704, R&D Systems), PE-conjugated anti-human KIR2DL1 / KIR2DS5 antibody (clone 143211, R&D Systems), APC-conjugated anti-human KIR3DL1 antibody (clone DX9, R&D Systems), AF405-conjugated anti-human KIR3DL2 / CD158k antibody (clone 539304, R&D Systems), APC-conjugated anti-human NKG2A / CD159a antibody (clone 131411, R&D Systems), and PE-conjugated anti-human NKG2C / CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems. AF647 goat anti-human IgG F(ab') 2 Fragment antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

[0154] Bioinformatics analysis from public cancer patient databases: Patient survival data and RSEM (RNA-Seq by expectation maximization) normalized expression data sets for CD147 were generated from The Cancer Genome Atlas (TCGA) and downloaded from OncoLnc (oncolnc.org). Data were plotted using GraphPad Prism 5.0 (GraphPad) Kaplan-Meier curves. RSEM normalized expression data sets derived from TCGA come from FireHose Broad GDAC developed by The Broad Institute (gdac.broadinstitute.org). Figures are generated using GraphPad Prism 5.

[0155] Cell lines: 293T, K562, Daudi cells, SK-Hep1, and HepG2 cell lines were purchased from the American Type Culture Collection (ATCC). To establish Daudi-FFluc cells, CD147-positive HepG2 and SK-Hep1 cells were transduced with a lentiviral vector encoding FFLuc-GFP as previously described. The protocol for collecting peripheral blood from healthy donors was approved by the Institutional Review Board (IRB) and the Ethics Review Committee of Rutgers-New Jersey Medical School (Newark, NJ).

[0156] NK-92MI cell culture and generation of CAR-modified NK-92MI cells: NK-92MI cell line was purchased from (CRL-2408 TM NK-92MI, an interleukin-2 (IL-2)-independent NK cell line, is derived from NK-92 ( CRL-2407 TM ) cell line (Gong et al., Leukemia 8:652-658, 1994) (Tam et al., Hum Gene Ther 10:1359-1373, 1999; Tam et al., J. Hematother. 8:281-290, 1999). The NK-92MI cell line was maintained in a specific NK-92MI medium (α-minimal essential medium, α-MEM) without ribonucleosides and deoxyribonucleosides but containing 2 mM L-glutamine and 1.5 g / L sodium bicarbonate. To prepare a complete growth medium, the following components were added to the basal medium: 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, horse serum at a final concentration of 12.5%, and fetal bovine serum at a final concentration of 12.5%. On day 3, NK-92MI cells were transduced with retroviral supernatant in plates coated with recombinant fibronectin fragments (FN CH-296; Retronectin; Takara, Japan). After transduction, NK cells were expanded using IL-2. To examine the percentage of CD147-CAR expression on NK-92MI cells, these cells were stained for CD3 and CD56 to stain NK cells, and then flow cytometry analysis was performed.

[0157] Generation of CD147-knockout cell lines: To generate CD147 knockout hepatocyte cell lines, a lentiviral delivery system was used, with guide RNAs targeting CD147 sequences: #1 (5-TTGACATCGTTGGCCACCGC-3; SEQ ID NO: 10), #3 (5-GTGGACGCAGATGACCGCTC-3; SEQ ID NO: 11). Lentivirus was produced in HEK 293T by transfecting lenti-CRISPR v2 with packaging plasmids pSPAX2 and pMD2G. After 3 days, the supernatant was filtered (0.45 μm) and incubated with hepatocytes and 8 μg / mL polybrene (Sigma). After 48 hours of incubation, the transduced cells were replaced with fresh medium and selected with 8.0 μg / ml puromycin for 5 days. Western blot and flow cytometry analysis were performed to confirm the efficacy of the knockout cell lines.

[0158] Plasmid construction and retrovirus production: The codon-optimized DNA fragment encoding the CD147-specific scFv from 5F6 clones was synthesized by GENEWIZ, and the frame was subcloned into the SFG retroviral vector retroviral backbone, and the hinge components of human IgG1, CD28 transmembrane domain, CD28 plus 4-1BB intracellular domain and human TCR / CD3 complex ζ chain conformed to the reading frame. To produce CD147-CAR retrovirus, 293T cells were transfected with a plasmid combination containing CD147-specific scFv, RDF and PegPam3, as previously described (Loskog et al., Leukemia 20: 1819-1828, 2006). The constructs of CD19-CD28-CAR and CD19-4-1BB-CAR have been previously described (Xiong et al., Mol. Ther. 26: 963-975, 2018).

[0159] Transduction of NK-92MI cells with CD147-CAR: NK cells were harvested on day 7 of expansion and retrovirally transduced with CD147-CAR in plates coated with Retronectin (FN CH-296, Takara, Japan). Two days later, cells were transferred to G-Rex 6 multiwell cell culture plates and maintained in 35 ml complete RPMI-1640 medium containing 200 U / ml IL-2 (PeproTech). The medium was changed every 3-4 days, and 2×10 7 Cells were kept in each well for further culture. The total number of cells was counted using trypan blue exclusion. To examine the percentage of NK cells and the expression of CAR, cells were stained for CD3, CD56, and IgG F(ab')2 and analyzed by flow cytometry.

[0160] Flow cytometry analysis and sorting: CAR-NK cells were stained with fluorescently conjugated antibodies on ice for 30 minutes in FACS staining buffer containing 1% fetal bovine serum (FBS), washed with PBS and analyzed on a FACS LSRII or LSR Fortessa flow cytometer (BD). PMT voltages were adjusted and compensation values ​​were calculated before data collection. Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (Tree Star).

[0161] For flow cytometry single live cell sorting, all sample cells were stained with fluorescent conjugated antibodies (with RPMI1640 containing 1% FBS) on ice for 30 minutes, washed twice with PBS, resuspended in complete medium and sorted by SORP BDFACS Aria III. After sorting, the collected samples were washed once with pre-warmed medium and cultured for use.

[0162] CAR-NK degranulation assay (CD107a): CAR-NK cells (1×10 5 ) were incubated with target cells in a U-bottom 96-well plate in complete NK-92MI medium at 37° C. for 4 hours or overnight. Afterwards, cells were harvested, washed, stained for CD3, CD56, and CD107a with GolgiStop (BD) on ice for 30 minutes, and analyzed by flow cytometry.

[0163] Cytokine release assay: IFNγ and TNF-α cytokines secreted by CAR-NK were measured by commercial ELISA kits (Invitrogen-Thermo Fisher Scientific) according to the manufacturer's protocol.

[0164] 51 Cr release assay: To evaluate the cytotoxic activity of CAR-NK cells, a standard 4-hour 51 Cr release assay. Briefly, the 51 Cr was labeled for 2 h, and then 2 × 10 5 / mL was resuspended in NK-92MI medium containing 10% FBS and without IL2. Then, 2×10 4 Target cells were incubated with serially diluted CAR-NK cells at 37°C for 4 h. After centrifugation, the supernatant was collected and the released NK was measured using a gamma counter (Wallac, Turku, Finland). 51 Cr. Cytotoxicity (in percentage) was calculated as follows: [(sample-spontaneous release) / (maximum release-spontaneous release)]×100.

[0165] FFLuc reporter gene system assay: To quantify the cytotoxicity of CAR-modified immune cells, a FFLuc reporter gene system assay was developed. Briefly, on day 1, target cells were plated at 2 × 10 4 or 3×10 4 Target cells / well (FFluc-GFP stably transduced cells) were pre-seeded into an optical 96-well plate (Greiner Bio-One TM No.: 655098)) in 100 μl / well of target cell complete nutrient medium and incubated overnight at 37°C with 5% CO2. The next day, serial dilutions of effector cells were prepared according to the effector / target ratio, and the indicated effector cells were added to each well (100 μl / well). The reaction was incubated at 37°C with 5% CO2 for 4 hours, and the supernatant was gently discarded. 100 μl of working D-luciferin was added to each well and incubated at 37°C with 5% CO2 with the lights off for 5 minutes. The data was quantified using a microplate reader (PerkinElmer, USA). The data were quantified by converting the obtained values ​​into specific lysis percentages by the following formula: specific lysis percentage (%) = [1-(SE) / (TM)]*100, where S is the luminescence value of the sample well, E is the luminescence value of the "effect cell only" well compared to the sample well, T is the average luminescence value of the "target cell only" well, and M is the average luminescence value of the "blank medium only" well.

[0166] Animal studies: All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC). NOD.Cg-Prkdc was obtained from The Jackson Laboratory (Bar Harbor, ME). scid Iq tm1Wjl / SzJ (NSG) mice were used for all in vivo experiments. To establish the hepatocellular carcinoma cell line xenograft model, both male and female NSG mice (8 weeks old) were subcutaneously injected with 100 μL PBS in the right flank. 4×10 in Matrix 6 SK-Hep1 cells. When the tumor burden reaches about 40-50 mm 2 The mice were randomly divided into three groups. Treatment started on day 1 by injecting (iv) 5×10 6CD147-CAR-NK-92MI cells. The control group was infused with parental NK-92MI cells or vehicle (PBS). The next day (day 2), all animals were injected with (iv) IL-2 (20,000 units / mouse). Animal weight and tumor burden were collected twice a week. Tumor size was measured with a caliper and the maximum longitudinal diameter (length) and maximum transverse diameter (width) were recorded. The tumor size based on the caliper measurement results was calculated by a modified ellipsoid formula. Tumor size was calculated as follows: Tumor size (mm 2 )=1 / 2(length×width 2 ). When the tumor burden exceeds 2000mm 2 Or when the body weight of the animal decreased by >20%, the mice were euthanized according to IACUC guidelines. The animal survival data were also recorded.

[0167] For patient-derived xenograft (PDX) models, patient hepatocellular carcinoma animal models were developed by The Jackson Laboratory. Briefly, fresh PDX samples were subcutaneously implanted into the flank of 6-8 week old NOD SCIDγ (NSG) mice. When the tumor burden reached approximately 40-50 mm 2 After that, the mice were randomly divided into three groups for further analysis. The main treatment procedures used were as described above. Xenograft samples were fixed with 10% formalin and embedded in paraffin for cutting and IHC staining or directly frozen into liquid nitrogen for further analysis.

[0168] Statistical analysis: Tumor size statistical analysis was performed by two-way ANOVA with Bonferroni post-test. Overall survival statistics were calculated using the log-rank test. Other statistical significance was determined using two-tailed unpaired Student's t-test and two-tailed paired Student's t-test. All statistical calculations were generated using GraphPad Prism 5.0. P < 0.05 (*), P < 0.01 (**), and P < 0.001 were considered statistically significant.

[0169] Example 2

[0170] CD147 is expressed in hepatocellular carcinoma cell lines and patient-derived liver cancer samples

[0171] To determine whether CD147 is an effective and rational target for HCC and other types of cancer, the correlation between patient survival and the expression level of CD147 from the TCGA (The Cancer Genome Atlas, cancergenome.nih.gov) dataset was analyzed. 高 and CD147 低) showed a strong negative correlation between CD147 and survival percentage ( Figure 1A ). Specifically, CD147 in multiple tumor tissues 高 The survival rate was low ( Figure 1A In addition, the comparison of CD147 expression between normal tissue (NT) and tumor samples (TP) in various cancer types showed that CD147 expression was significantly upregulated in different types of tumor tissues ( Figure 1B ).

[0172] To validate the data from the bioinformatics analysis, the expression of CD147 in different tumor cell lines and other tissues was analyzed by Western blotting, including wild-type NK-92 (human NK cell line), T2 (mutant T×B cell hybrid), 721.221 (human cell line without HLA-A, HLA-B, HLA-C), MDA-MB-231 (human breast cancer cell line), K562 (human myeloid leukemia cell line), HepG2 (human hepatocellular carcinoma cell line), SK-Hep1 (human hepatoadenocarcinoma cell line), Raji (human B lymphocyte Burkitt's lymphoma cell line), Daudi (human B lymphoblastoid cell line), NK-92-MI (interleukin-2-independent natural killer cell line) and human peripheral blood mononuclear cells (PBMCs). Compared with PBMCs, the CD147 molecule was highly upregulated in HepG2 and SK-Hep1 cell lines (two hepatocellular carcinoma cell lines) ( Figure 1C ). Compared with tumor cell lines, CD147 expression on PBMCs is relatively low ( Figure 1C Similar results were obtained by flow cytometry analysis ( Figure 1D In addition, the results of immunohistochemistry (IHC) assay confirmed that CD147 was significantly upregulated in HCC tissues isolated from PDX mouse models ( Figure 1E ).

[0173] Example 3

[0174] Generation and characterization of CD147-CAR-NK cells

[0175] A CD147-CAR using a SFG vector was constructed (Loskog et al., Leukemia 20:1819-1828, 2006; Xiong et al., Mol. Ther. 26:963-975, 2018). The CD147-CAR contains a modified single-chain variable fragment (scFv) of an anti-CD147 antibody (as described in Example 1, derived from clone 5F6), an IgG-CH2CH3 spacer, a transmembrane domain of CD28, an intracellular domain of CD28-4-1BB, and an intracellular signaling domain of a TCR-ζ chain ( Figure 2A).

[0176] First, this CAR construct was tested in the NK-92MI cell line. After transduction, NK-92MI expressed the CD147-CAR molecule ( Figure 2B After flow cytometry sorting, the percentage of CD147 positive NK-92MI cells was above 96% ( Figure 2B ). The expression of CD147-CAR molecules in NK-92MI cells was further confirmed by Western blotting. Compared with the parental NK-92MI cell line, CD147-CAR-NK-92MI expressed chimeric scFv-CD147-CAR. The approximate molecular weight is about 80-85kD ( Figure 2C The CD147-CAR-NK-92MI cell line was further characterized by flow cytometry. Comparable expression of activating receptors (e.g., CD56, NKG2D, NKP46, NKG2A, CD16, CD94 / NKG2C, CD226 (also known as DNAM-1), and CD244 (also known as 2B4)) and inhibitory receptors (e.g., KLRG1, LAG-3, CTLA-4, TIM-3, PD-1, and TIGIT) was observed ( Figure 2D Given the low level of CD147 expression on NK92-MI cells, CD147 expression on the CD147-CAR-NK-92MI cell line was also analyzed by flow cytometry. After transduction, CD147-CAR expression on NK-92MI was stable for more than 30 days. However, CD147-CAR expression was associated with loss of CD147 on the NK-92MI cell line, indicating limited autokilling in CD147-CAR-NK-92MI cells ( Figure 2E-2G ). It is noteworthy that the loss of CD147 molecule expression on CD147-CAR-NK-92MI cells did not affect their in vitro functionality and expression.

[0177] Example 4

[0178] CD147-CAR NK cells specifically kill hepatocellular carcinoma (HCC) in vitro

[0179] After the successful establishment of CD147-CAR-NK cells, CD147-CAR-NK-92MI cells were tested for their ability to eliminate CD147 + Compared with the control CD19-CD28-CAR-NK-92MI and CD19-4-1BB-CAR-NK-92MI cells, CD147-CAR-NK-92MI cells showed a significant improvement in the ability of the two HCC cell lines, SK-Hep1 and HepG2 ( Figure 3Aand 3B ) and Huh5 and HCO2 cell lines ( Figures 4A-4C ) showed significant cytotoxicity. In addition, the production of TNF-α and IFN-γ by CD147-CAR-NK-92MI cells was significantly higher than that of CD19-CD28-CAR-NK-92MI and CD19-4-1BB-CAR-NK-92MI cells stimulated by SK-Hep1 and HepG2 cells ( Figure 3C and 3D Interestingly, activation of CD147-CAR-NK-92MI cells by their susceptible target cells could be blocked by anti-CD147 antibody (clone HIM6) but not by control IgG1 ( Figures 5A-5C ). The specificity of this anti-CD147 antibody was further verified by testing its effect on the cytotoxicity of CD19-4-1BB-CAR-NK-92MI cells. This anti-CD147 antibody was unable to block the cytotoxicity of CD19-4-1BB-CAR-NK-92MI cells to the CD19-positive Daudi cell line ( Figure 6 ), indicating the selectivity of CD19-4-1BB-CAR-NK-92MI cells and anti-CD147 antibodies.

[0180] To further confirm the specificity of CD147-CAR-NK-92MI cells, CD147-knockout (CD147 - / - )SK-Hep1 cell line (CD147 - / - -SK-Hep1) and CD147-knockout (CD147 - / - )HepG2 cell line (CD147 - / - -HepG2). CD147 - / - -HepG2 and CD147 - / - -SK-Hep1 cells were not recognized by CD147-CAR-NK-92MI cells ( Figure 5D-5G ), the CD147-CAR-NK-92MI cells were quantified by CD107a surface expression when co-cultured with the CD147-KO cell line.

[0181] Both CD107a assay and cytokine production assay can be used to assess the activation of CD147-CAR-NK-92MI cells by susceptible target cells. To directly test whether CD147-CAR-NK-92MI cells can kill CD147-positive HCC cells, a 4-hour standard chromium-51 ( 51Cr) release assay (the gold standard assay for evaluating the cytotoxicity of CTL and NK cells in the field of immunology). CD147-CAR-NK-92MI cells kill SK-Hep1 and Daudi cells. CD147-CAR-NK-92MI cells have similar killing activity against other HCC cell lines such as Huh7 cell line (Kasai et al., Hum Cell 31:261-267, 2018) and HCO2 cell line (Trinh et al., PLoS One 10:e0136673, 2015).

[0182] To further verify the killing activity of CD147-CAR-NK-92MI cells, a novel, easy-to-use and non-radioactive method for evaluating the cytotoxicity of CD147-CAR-NK-92MI cells using luciferase bioluminescent signals was developed. First, FFLuc-EGFP-SK-Hep1 and FFLuc-EGFP-HepG2 cell lines were generated. To evaluate the direct killing of target cells, CD147-CAR-NK-92MI cells were co-cultured with FFLuc-EGFP-SK-Hep1 and FFLuc-EGFP-HepG2 cell lines, respectively. After CD147-CAR-NK-92MI cells were incubated for 4 hours in a 96-well optical bottom microplate pre-seeded with target cells stably expressing the EGFP-firefly luciferase fusion gene (EGFP-FFluc), the chemiluminescent signal of EGFP-FFluc was quantified by a fluorescent microplate reader. As described in Example 1, similar to 51 Cr release assay, converting FFLuc signal into percentage of specific lysis ( Figure 8H and 8I ).

[0183] First, CD147-CAR-modified primary T cells and NK cells isolated from human peripheral blood mononuclear cells (PBMCs) can eliminate multiple HCC cell lines (including SK-Hep1, Huh7, and HepG2, etc.), but κ-CAR-modified T cells cannot ( Figures 8A-8C ). We pass 51 Cr release assay also demonstrated that CD147-CAR modified human primary NK cells effectively killed HCC cell lines in vitro ( Figures 8D-8F To further demonstrate the natural killing ability of primary NK cells through NKGD / NKG2DL interaction in addition to CD147-CAR primary NK cytotoxicity, we found that anti-NKG2D further blocked CD147-CAR-NK cytotoxicity to CD147 - / - - Killing of SK-Hep1 cells ( Figure 8G ).

[0184] Dose-dependent specific lysis and 51 The cytotoxic activity measured by this method was further quantified under a common inverted fluorescence microscope to assess the morphology and dynamics of the EGFP signal in target cells ( Fig. 7A and 7B ). Thus, two complementary approaches demonstrated that CD147-CAR NK cells specifically kill hepatocellular carcinoma (HCC) in vitro.

[0185] Compared with parental SK-Hep1 and HepG2 cells, CD147-CAR-NK-92MI cells were unable to kill CD147 - / - -SK-Hep1 and CD147 - / - -HepG2( Figure 8K and 8L The specificity of CD147-CAR-NK-92MI cell cytotoxicity was further verified by adding anti-CD147 antibody into the co-culture system of effector cells and target cells ( Figure 8M and 8N To further validate CD147 as an effective and rational target for HCC, we tested CD147-CAR-T cells against two different HCC cell lines—HepG2 ( Fig. 10A ) and SK-Hep1( Fig. 10B ) cytotoxicity. When the CD147 molecule was deleted in the SK-Hep1 cell line (CD147-knockout SK-Hep1 cell line), the specific lysis of CD147-CAR-T cells was significantly reduced ( Fig. 10C ), further verifying the specific cytotoxicity of CD147-CAR-T cells against CD147-positive HCC cell lines.

[0186] Example 5

[0187] CD147-CAR-NK cells control HCC progression in vivo

[0188] To evaluate whether CD147-CAR can kill HCC in vivo, two different xenograft models were used. First, CD147-CAR-modified primary T cells and NK cells derived from PBMC were evaluated in the SK-Hep1 xenograft mouse model. CD147-CAR-modified primary T cells significantly inhibited tumor size and prolonged survival ( Figures 11A-11D To further evaluate the efficacy of CD147-CAR modified primary NK cells, we included a non-transduced (NT) primary NK group as an additional control ( Figures 12A-12D). Mice receiving the parental NT-NK control group and the PBS vehicle control group developed rapid disease progression. In contrast, mice receiving CD147-CAR primary T cells and NK cells were significantly protected from rapid progression and had prolonged median survival (P<0.05), with comparable body weights in the different groups ( Fig. 11C and 12C ), indicating that the in vivo toxicity of primary T cells and NK cells modified with CD147-CAR is tolerable.

[0189] In addition, to further develop additional “off-the-shelf” cell therapy strategies, we evaluated the efficacy of CD147-CAR-modified NK-92MI cells. Due to the malignant nature of NK-92MI, CAR-modified NK-92MI cells need to be irradiated before administration to patients46,61. 51 The Cr release assay compared the cytotoxicity of non-irradiated and irradiated CD147-CAR-NK-92MI cells ( Fig.13 Comparable cytotoxicity was observed between non-irradiated and irradiated CD147-CAR-NK-92MI cells in vitro ( Fig.13 ).

[0190] We further compared the efficacy of non-irradiated and irradiated CD147-CAR-NK-92MI cells in a xenograft NSG mouse model ( Figures 14A-14D Comparable in vivo efficacy measured by median survival was observed between non-irradiated and irradiated CD147-CAR-NK-92MI-infused mice ( Figures 14A-14D ).

[0191] To further evaluate the efficacy of CD147-CAR-NK-92MI cells (injected on days 1, 3, and 5 after tumor implantation) in controlling tumor growth, disease progression was measured by tumor size ( Fig.15A Mice receiving parental NK-92MI and PBS vehicle control groups developed rapid disease progression ( Fig. 15B In contrast, mice receiving CD147-CAR-NK-92MI cells were significantly protected from rapid progression and their median survival was prolonged (P<0.01), with comparable body weights in the different groups ( Fig. 15C and 15D ), indicating that the in vivo toxicity of CD147-CAR-NK-92MI cells is tolerable.

[0192] Although cancer cell lines may have significant limitations in their ability to accurately model biological and therapeutic effects, patient-derived xenograft (PDX) models are biologically stable and can simulate human clinical conditions regarding mutation status, gene expression patterns, and tumor heterogeneity. Therefore, a second xenograft mouse model was used using metastatic liver cancer tissue from patients. The effects of CD147-CAR-NK-92MI cells administered on days 0, 4, 8, 11, 15, 22, 25, and 35 after transplantation were tested. The median survival of mice treated with CD147-CAR-NK-92MI cells was 63 days, which was significantly higher than that of control mice (median survival was about 42 days). Reduced tumor burden and reduced disease progression were observed in mice treated with CD147-CAR-NK-92MI cells ( Figures 16A-16D ), indicating the effectiveness of CD147-CAR-NK-92MI cells in inhibiting HCC progression in PDX mouse models.

[0193] Example 6

[0194] CD147-CAR-T cells specifically kill CD147-positive tumor cells

[0195] The ability of CD147-CAR-T cells to kill HCC cells was tested against 8 HCC cell lines (Huh7, Huh7.5, HepG2, SK-Hep1, Hep3B, Hu1545, HCO2, and LH86). Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors. To transduce PBMCs, cells were activated in 10% FBS RPMI-1640 medium with 1 μg / ml anti-CD3 (clone, OKT3, OrthoBiotech, Bridgewater, NJ, USA) and 1 μg / ml anti-CD28 and 100 U / ml recombinant human IL-2 (aldesleukin; Chiron, Emeryville, CA, USA). To generate CD147-CAR-T cells, activated T cells were transduced with retroviral supernatant in plates coated with recombinant fibronectin fragments (FN CH-296; Retronectin; Takara Shuzo, Otsu, Japan) on day 3. After transduction, T cells were expanded with IL-2 and then used in the assay.

[0196] The specific killing of FFLuc-EGFP-HepG2 by CD147-CAR-T cells was tested using the FFLuc reporter gene system as described in Example 1 ( Fig.17A Effector cells (CD147-CAR-T cells) were added to each well at 1×10 4Target cells of 10 cells were co-cultured in 96-well optical bottom microplates for 6 hours. The control group used wild-type κ-CAR-T cells incubated with CD147 positive FFLuc-EGFP-HepG2. The FFLuc reporter system assay showed that the cytotoxicity of CD147-CAR-T cells using knockout-CD147 FFLuc-GFP-HepG1 was reduced ( Fig. 17B Effector cells (CD147-CAR-T and κ-CAR-T cells) were mixed with target cells CD147 positive FFLuc-EGFP-HepG2 (1×10 4 ) were co-cultured in a 96-well optical bottom microplate for 6 hours. The cytotoxicity of CD147-CAR-T cells was measured by luminescence signal read by a microplate reader. 51 Cr release assay to measure the cytotoxicity of CD147-CAR-T cells ( Fig. 17C ). κ-CAR-T cells were used as a negative control group.

[0197] Example 7

[0198] Effects of CD147-CAR-T cells on neuroblastoma cells

[0199] In the presence of culture medium (control) or SK-N-SH tumor cells ( Fig.18A ) and DaoY and D283 cells for 10 hours, CD107a degranulation was observed on CD147-CAR-T cells. The effector and target ratio was 1:1.2. Cells were gated according to the CD56 positive subpopulation to quantify surface CD107a expression. Quantitative data of the percentage of surface CD107a expression on CD147-CAR-NK-92MI cells under different stimulations are shown in ( Fig.18B CD147-CAR-NK-92MI cells were also cytotoxic to DaoY cells after 3 hours ( Fig.19 ).

[0200] Example 8

[0201] Primary CD147-CAR-NK cells derived from HCC patients specifically kill CD147-positive tumor cells in vitro

[0202] Due to the broad expression pattern of CD147 across multiple solid tumor types, CD147 is an attractive target for CD147-CAR-based cancer immunotherapy. Here we examined whether CD147 is upregulated in human HCC tissue samples. Compared with healthy liver tissue, HCC tumor tissues at different stages showed strong positive staining for CD147 ( Fig.21A ).

[0203] To evaluate whether CD147-CAR modified primary NK cells directly isolated from HCC livers can kill HCC in vitro, NK cells were isolated from different regions of liver tissue ( Fig.21B ), wherein the different regions include tumor region, tumor adjacent region and non-tumor region in the liver of a person with HCC. Then, these NK cells ( Fig. 21C ). CD147-CAR was generated using these expanded NK cells from human HCC liver tissue. The transduction efficiency of activated NK cells was generally greater than 70% ( Fig.21D ). CD147-CAR-NK cells specifically recognize tumor cells expressing CD147. The anti-tumor activity of CD147-CAR-NK was evaluated against HCC cell lines ( Fig.21E ). Taken together, this indicates that primary human liver NK cells redirected by CD147-CAR specifically and selectively kill CD147-positive target cells.

[0204] Example 9

[0205] SynNotch GPC3-inducible CD147-CAR T cells selectively target GPC3+CD147+ HCC cells, but not GPC3+CD147- or GCP3-CD147+ HCC cells

[0206] To generate an anti-GPC3 synNotch-induced receptor vector, an anti-GPC3 (mouse GC33 clone) scFv that specifically binds to the human GPC3 antigen was synthesized by GENEWIZ. The sequence encoding the signal peptide and myc tag at the N-terminus was fused to the synNotch-Gal4VP64 inducible element from (Addgene plasmid number 79125) by overlapping PCR. The fragment was inserted into the SFGγ retroviral vector digested with restriction endonucleases NcoI and XhoI.

[0207] To construct the anti-CD147-CAR-mCherry vector, the entire CD147-CAR element was inserted into pHR_Gal4UAS_pGK_mCherry (Addgene plasmid number 79124) digested with restriction endonucleases MluI and NdeI. The expression of the mCherry gene is controlled by the pGK promoter. In this strategy, eGFP and mCherry double-positive cells were gated as cells modified with synNotchCAR for further analysis and functional evaluation.

[0208] To generate the anti-CD147 synNotch inducible receptor vector, the anti-CD147 scFv was fused to the synNotch-Gal4VP64 inducible element derived from pHR_PGK_antiCD19_synNotch_Gal4VP64 (Addgene plasmid number 79125) by overlapping PCR. A myc tag was added to the N-terminus. The fragment was inserted into the SFGγ retroviral vector digested with restriction endonucleases SalI and MluI after the signal peptide.

[0209] To construct the anti-GPC3-CAR-mCherry vector, the complete GPC3-CAR element containing the anti-GPC3 (mouse GC33 clone) scFv was inserted into pHR_Gal4UAS_pGK_mCherry (Addgene plasmid number 79124) digested with restriction endonucleases MluI and NdeI. The expression of the mCherry gene is controlled by the pGK promoter. In this strategy, myc tag and mCherry double-positive cells were gated as cells modified with synNotch CAR for further analysis and functional evaluation.

[0210] Table 2: Primers used for plasmid construction

[0211]

[0212]

[0213] To mitigate the off-tumor toxicity to normal tissues, the effect of CD147 expression density on the anti-tumor activity of CD147-CAR in different cell types (focusing on hematopoietic cells) was tested. CD147 expression in HepG2, Raji, Daudi, and PBMC was evaluated. Different expression levels of CD147 ( Fig.22A Notably, cells expressing low levels of CD147 (e.g., PBMCs) did not trigger the cytotoxic activity of CD147-CAR-NK-92MI cells, even when CD147-CAR cells were cultured with target cells at a high effector to target ratio (E:T ratio) of 10 to 1 ( Fig. 22B ). These findings suggest that the optimized scFv sequence of anti-CD147 allows only the specific scFv domain to bind to cells with high expression of CD147 molecules, which can alleviate the off-tumor toxicity to normal tissues expressing low levels of CD147 molecules.

[0214] To further reduce the off-tumor toxicity of CD147-CAR, a synNotch receptor that can release transcription factors was used, which in turn drove the expression of CARs targeting different tumor antigens. This "logic gating" synNotch CAR can only kill dual-antigen-positive tumor cells, but not single tumor antigen-positive tumor cells. A combination approach consisting of GPC-3 and CD147 was designed to reduce off-tumor toxicity. In brief, an SFG retroviral vector was constructed, which encodes an anti-GPC3-specific synNotch receptor connected to the Gal4-VP64 intracellular transcriptional activation domain. The constitutively expressed enhanced GFP (eGFP) was placed downstream of GPC3-synNotch to identify transduced cells ( Fig.23A ).

[0215] A lentiviral vector was constructed in which the anti-CD147-CAR was placed under the control of an upstream activation sequence (UAS) promoter, which can be activated by the Gal4-VP64 transcription factor released after synNotch receptor engagement. The constitutively expressed monomeric red fluorescent protein Cherry (mCherry) was placed downstream of the inducible CD147-CAR to identify transduced cells ( Fig.23A ).

[0216] Human PBMCs were co-transduced with both lentiviral and retroviral vectors ( Fig.23C Flow cytometry analysis was used to verify double positive cells by eGFP (a marker for anti-GPC3-synNotch) and mCherry (a marker for CD147-CAR). Fig.23D Four subpopulations of transduced T cells (including mCherry-only positive, GFP-only positive, GFP and mCherry double positive, and GFP and mCherry double negative subpopulations) were analyzed ( Fig.23E These transduced T cells are composed of GPC3 高 CD147 低 HepG2 cell line was sensitized to induce CD147-CAR expression on the surface ( Fig.23F ). In the absence of synNotch engagement, no CD147-CAR expression was observed. No leakage of CAR expression was observed in transduced primary T cells. However, approximately 10% leakage of CAR expression was observed in transduced NK-92MI cells (data not shown). This observation was further validated by treatment with a γ-secretase inhibitor (MK-0752, a Notch signaling inhibitor) ( Fig.24). MK-0752 treatment significantly inhibited CD147-CAR expression on GPC3-synNotch-GFP and CD147-CAR-mCherry double-positive T cells ( Fig.24 ).

[0217] In T cells co-transduced with GPC3-synNotch-GFP and CD147-CAR-mCherry and expressed by GPC3 高 CD147 低 After sensitization of HepG2 cell line, the activity of transduced T cells was triggered by different HCC cell line subsets for 2 hours to evaluate the killing efficacy. The different subsets of HCC cell lines are: CD147+GPC3 高 HepG2 cell line, CD147 ko GPC3 高 HepG2 cell line, CD147+GPC3 低 HepG2 cell line, CD147 ko GPC3 低 HepG2 cell line ( Figure 23G ). Phorbol-12-myristate-13-acetate (PMA) / ionomycin (IONO) was used as a positive control.

[0218] With CD147 ko GPC3 高 HepG2 cell-sensitized GPC3-synNotch-GFP and CD147-CAR-mCherry double positive T cells can be 高 HepG2 cell-specific activation ( Fig.23H ), quantified by CD107a surface expression when co-cultured with different target cell lines. Similar results were obtained when myc-tagged CD147-specific synNotch-GFP and inducible GPC3-CAR-mCherry were co-transduced into T cells ( Figures 25A-25H ).

[0219] In summary, the data show that only synNotch GPC3-inducible CD147-CAR T cells can be specifically activated by GPC3+CD147+HepG2 cells, but not by GPC3+CD147- or GPC3-CD147+HepG2 cells. These activated CD147-CAR T cells can kill CD147+GPC3+HepG2 cells, but not CD147-GPC3+HepG2 cells ( Figures 26A-26D ).

[0220] In view of the many possible embodiments to which the principles of the present disclosure may be applied, it should be appreciated that the illustrated embodiments are merely examples and should not be considered to limit the scope of the present invention. Instead, the scope of the present invention is defined by the following claims. Therefore, we claim protection for all that falls within the scope and spirit of these claims as the present invention. Sequence Listing <110> Rutgers, The State University of New Jersey <120> CD147 chimeric antigen receptors and methods of use <130> 7213-102200-02 <150> US 62 / 819,403 <151> 2019-03-15 <160> 35 <170> PatentIn Version 3.5 <210> 1 <211> 729 <212> DNA <213> Artificial sequence <220> <223> Modified CD147 ScFv <400> 1 gagatgaagc tggaagagag cggcggcgga ctggtgcagc ccggcggcag catgaagctg 60 agctgcgtgg cctccggctt caccttcagc aactactgga tgaactgggt gagacagtcc 120 cccgaaaagg gcctggagtg ggtggcccag atcagactga agtcctacaa ctacgccacc 180 cactacgccg agagcgtgaa gggcagattc accatctcca gggacgactc caagtcctcc 240 gtgtacctgc agatgaacaa cctgagagcc gaggacaccg gcatctacta ctgcaccccc 300 gatggcagcg actattgggg ccagggcaca acactcaccg tgagctccgg cggcggaggc 360 agcggcggcg gcggcagcgg cggcggcggc tccggcggag gcggcagcag catcgtgatg 420 acccagaccc ccaagttcct cctggtgagc gccggcgata gggtgaccat tacctgcaag 480 gcctcccagt ccgtgagcaa cgatgtggcc tggtaccagc agaagcccgg ccagagccct 540 aagctgctga tctactacgc cagcaacagg tacacaggcg tgcccgacag gtttaccggc 600 tccggctacg gcaccgactt caccttcacc atcagcacag tgcaggccga ggacctggcc 660 gtgtacttct gccagcagga ctactccagc ccctacacct tcggaggcgg caccaagctg 720 gagatcaag 729 <210> 2 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Modified CD147 ScFv <400> 2 Glu Met Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Tyr Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys Thr Pro Asp Gly Ser Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val 130 135 140 Ser Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val 145 150 155 160 Ser Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 165 170 175 Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg 180 185 190 Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr 195 200 205 Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser 210 215 220 Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 225 230 235 <210> 3 <211> 729 <212> DNA <213> Artificial sequence <220> <223> CD147 scFv <400> 3 gagatgaagc ttgaggagag tggtggtggt cttgttcagc ctggtggtag tatgaagctt 60 agttgcgttg ctagtggttt cactttcagt aactactgga tgaactgggt tcgtcagagt 120 cctgagaagg gtcttgagtg ggttgctcag atccgtctta agagttacaa ctacgctact 180 cactacgctg agagtgttaa gggtcgtttc actatcagtc gtgacgacag taagagtagt 240 gtttaccttc agatgaacaa ccttcgtgct gaggacactg gtatctacta ctgcactcct 300 gacggtagtg actactgggg tcagggtact actcttactg ttagtagtgg tggtggtggt 360 tctggtggtg gtggttctgg cggcggcggc tccggtggtg gtggctccag tatcgttatg 420 actcagactc ctaagttcct tcttgttagt gctggtgacc gtgttactat cacttgcaag 480 gctagtcaga gtgttagtaa cgacgttgct tggtaccagc agaagcctgg tcagagtcct 540 aagcttctta tctactacgc tagtaaccgt tacactggtg ttcctgaccg tttcactggt 600 agtggttacg gtactgactt cactttcact atcagtactg ttcaggctga ggaccttgct 660 gtttacttct gccagcagga ctacagtagt ccttacactt tcggtggtgg tactaagctt 720 gagatcaag 729 <210> 4 <211> 2202 <212> DNA <213> Artificial sequence <220> <223> CD147 CAR <400> 4 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgcgtc 60 gacgagatga agctggaaga gagcggcggc ggactggtgc agcccggcgg cagcatgaag 120 ctgagctgcg tggcctccgg cttcaccttc agcaactact ggatgaactg ggtgagacag 180 tcccccgaaa agggcctgga gtgggtggcc cagatcagac tgaagtccta caactacgcc 240 acccactacg ccgagagcgt gaagggcaga ttcaccatct ccagggacga ctccaagtcc 300 tccgtgtacc tgcagatgaa caacctgaga gccgaggaca ccggcatcta ctactgcacc 360 cccgatggca gcgactattg gggccagggc acaacactca ccgtgagctc cggcggcgga 420 ggcagcggcg gcggcggcag cggcggcggc ggctccggcg gaggcggcag cagcatcgtg 480 atgacccaga cccccaagtt cctcctggtg agcgccggcg atagggtgac cattacctgc 540 aaggcctccc agtccgtgag caacgatgtg gcctggtacc agcagaagcc cggccagagc 600 cctaagctgc tgatctacta cgccagcaac aggtacacag gcgtgcccga caggtttacc 660 ggctccggct acggcaccga cttcacctt accatcagca cagtgcaggc cgaggacctg 720 gccgtgtact tctgccagca ggactactcc agcccctaca ccttcggagg cggcaccaag 780 ctggagatca agacgtacgt caccgtctct tcacaggatc ccgccgagcc caaatctcct 840 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 900 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 960 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 1020 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 1080 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggatcaag 1140 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1200 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1260 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1320 tgggagagca atgggcaacc gggaacaac tacaagacca cgcctcccgt gctggactcc 1380 gacggctcct tcttctctca cagcaagctc accgtggaca agagcaggtg gcagcagggg 1440 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1500 ctctccctgt ctccgggtaa aaaagatccc aaattttggg tgctggtggt ggttggtgga 1560 gtcctggctt gctatagctt gctagtaaca gtggccttta ttatttttg ggtgaggagt 1620 aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1680 acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaaa 1740 cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 1800 actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 1860 ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1920 ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1980 ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 2040 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 2100 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 2160 acctacgacg cccttcacat gcaggccctg ccccctcgct aa 2202 <210> 5 <211> 733 <212> PRT <213> Artificial Sequence <220> <223> CD147 CAR <400> 5 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Val Asp Glu Met Lys Leu Glu Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe 35 40 45 Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys 50 55 60 Gly Leu Glu Trp Val Ala Gln Ile Arg Leu Lys Ser Tyr Asn Tyr Ala 65 70 75 80 Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 85 90 95 Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu 100 105 110 Asp Thr Gly Ile Tyr Tyr Cys Thr Pro Asp Gly Ser Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val 145 150 155 160 Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val 165 170 175 Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp 180 185 190 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Tyr Ala 195 200 205 Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Tyr 210 215 220 Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala Glu Asp Leu 225 230 235 240 Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr Thr Phe Gly 245 250 255 Gly Gly Thr Lys Leu Glu Ile Lys Thr Tyr Val Thr Val Ser Ser Gln 260 265 270 Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro 275 280 285 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 290 295 300 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 305 310 315 320 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 325 330 335 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 340 345 350 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 355 360 365 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 370 375 380 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 385 390 395 400 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 405 410 415 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 420 425 430 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 435 440 445 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 450 455 460 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 465 470 475 480 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 485 490 495 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Pro Lys Phe 500 505 510 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 515 520 525 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 530 535 540 Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 545 550 555 560 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 565 570 575 Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 580 585 590 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 595 600 605 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 610 615 620 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 625 630 635 640 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 645 650 655 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 660 665 670 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 675 680 685 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 690 695 700 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 705 710 715 720 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 725 730 <210> 6 <211> 3402 <212> DNA <213> Artificial Sequence <220> <223> CD14 CAR with Inducible Caspase 9 <400> 6 atgctcgagg gagtgcaggt ggagactatc tccccaggag acgggcgcac cttccccaag 60 cgcggccaga cctgcgtggt gcactacacc gggatgcttg aagatggaaa gaaagttgat 120 tcctcccggg acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga 180 ggctgggaag aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct 240 ccagattatg cctatggtgc cactgggcac ccaggcatca tcccaccaca tgccactctc 300 gtcttcgatg tggagcttct aaaactggaa tctggcggtg gatccggagt cgacggattt 360 ggtgatgtcg gtgctcttga gagtttgagg ggaaatgcag atttggctta catcctgagc 420 atggagccct gtggccactg cctcattatc aacaatgtga acttctgccg tgagtccggg 480 ctccgcaccc gcactggctc caacatcgac tgtgagaagt tgcggcgtcg cttctcctcg 540 ctgcatttca tggtggaggt gaagggcgac ctgactgcca agaaaatggt gctggctttg 600 ctggagctgg cgcagcagga ccacggtgct ctggactgct gcgtggtggt cattctctct 660 cacggctgtc aggccagcca cctgcagttc ccaggggctg tctacggcac agatggatgc 720 cctgtgtcgg tcgagaagat tgtgaacatc ttcaatggga ccagctgccc cagcctggga 780 gggaagccca agctcttttt catccaggcc tgtggtgggg agcagaaaga ccatgggttt 840 gaggtggcct ccacttcccc tgaagacgag tcccctggca gtaaccccga gccagatgcc 900 accccgttcc aggaaggttt gaggaccttc gaccagctgg acgccatatc tagtttgccc 960 acacccagtg acatctttgt gtcctactct actttcccag gttttgtttc ctggagggac 1020 cccaagagtg gctcctggta cgttgagacc ctggacgaca tctttgagca gtgggctcac 1080 tctgaagacc tgcagtccct cctgcttagg gtcgctaatg ctgtttcggt gaaagggatt 1140 tataaacaga tgcctggttg ctttaatttc ctccggaaaa aacttttctt taaaacatca 1200 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagcggcc 1260 gctgagatga agctggaaga gagcggcggc ggactggtgc agcccggcgg cagcatgaag 1320 ctgagctgcg tggcctccgg cttcaccttc agcaactact ggatgaactg ggtgagacag 1380 tcccccgaaa agggcctgga gtgggtggcc cagatcagac tgaagtccta caactacgcc 1440 acccactacg ccgagagcgt gaagggcaga ttcaccatct ccagggacga ctccaagtcc 1500 tccgtgtacc tgcagatgaa caacctgaga gccgaggaca ccggcatcta ctactgcacc 1560 cccgatggca gcgactattg gggccagggc acaacactca ccgtgagctc cggcggcgga 1620 ggcagcggcg gcggcggcag cggcggcggc ggctccggcg gaggcggcag cagcatcgtg 1680 atgacccaga cccccaagtt cctcctggtg agcgccggcg atagggtgac cattacctgc 1740 aaggcctccc agtccgtgag caacgatgtg gcctggtacc agcagaagcc cggccagagc 1800 cctaagctgc tgatctacta cgccagcaac aggtacacag gcgtgcccga caggtttacc 1860 ggctccggct acggcaccga cttcacctt accatcagca cagtgcaggc cgaggacctg 1920 gccgtgtact tctgccagca ggactactcc agcccctaca ccttcggagg cggcaccaag 1980 ctggagatca agtcgtacgt caccgtctct tcacaggatc ccgccgagcc caaatctcct 2040 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2100 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2160 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 2220 ggcgtggagg tgcataatgc caacaaag ccgcgggagg agcagtacaa cagcacgtac 2280 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggatcaag 2340 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2400 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2460 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2520 tgggagagca atgggcaacc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2580 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 2640 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2700 ctctccctgt ctccgggtaa aaaagatccc aaatttggg tgctggtggt ggttggtgga 2760 gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttttg ggtgaggagt 2820 aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 2880 acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaaa 2940 cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 3000 actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg agatgtgaa 3060 ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 3120 ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 3180 ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 3240 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 3300 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 3360 acctacgacg cccttcacat gcaggccctg ccccctcgct aa 3402 <210> 7 <211> 1133 <212> PRT <213> Artificial Sequence <220> <223> CD14 CAR with Inducible Caspase 9 <400> 7 Met Leu Glu Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg 1 5 10 15 Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met 20 25 30 Leu Glu Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro 35 40 45 Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu 50 55 60 Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser 65 70 75 80 Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro 85 90 95 His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly 100 105 110 Gly Gly Ser Gly Val Asp Gly Phe Gly Asp Val Gly Ala Leu Glu Ser 115 120 125 Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys 130 135 140 Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly 145 150 155 160 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg 165 170 175 Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr 180 185 190 Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His 195 200 205 Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln 210 215 220 Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys 225 230 235 240 Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys 245 250 255 Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly 260 265 270 Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu 275 280 285 Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln 290 295 300 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro 305 310 315 320 Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val 325 330 335 Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp 340 345 350 Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu 355 360 365 Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 370 375 380 Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser 385 390 395 400 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 405 410 415 Gly Pro Ala Ala Ala Glu Met Lys Leu Glu Glu Ser Gly Gly Gly Leu 420 425 430 Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe 435 440 445 Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys 450 455 460 Gly Leu Glu Trp Val Ala Gln Ile Arg Leu Lys Ser Tyr Asn Tyr Ala 465 470 475 480 Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 485 490 495 Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu 500 505 510 Asp Thr Gly Ile Tyr Tyr Cys Thr Pro Asp Gly Ser Asp Tyr Trp Gly 515 520 525 Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 530 535 540 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val 545 550 555 560 Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val 565 570 575 Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp 580 585 590 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Tyr Ala 595 600 605 Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Tyr 610 615 620 Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala Glu Asp Leu 625 630 635 640 Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr Thr Phe Gly 645 650 655 Gly Gly Thr Lys Leu Glu Ile Lys Ser Tyr Val Thr Val Ser Ser Gln 660 665 670 Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro 675 680 685 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 690 695 700 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 705 710 715 720 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 725 730 735 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 740 745 750 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 755 760 765 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 770 775 780 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 785 790 795 800 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 805 810 815 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 820 825 830 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 835 840 845 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 850 855 860 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 865 870 875 880 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 885 890 895 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Pro Lys Phe 900 905 910 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 915 920 925 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 930 935 940 Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 945 950 955 960 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 965 970 975 Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 980 985 990 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 995 1000 1005 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 1010 1015 1020 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 1025 1030 1035 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 1040 1045 1050 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 1055 1060 1065 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 1070 1075 1080 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 1085 1090 1095 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 1100 1105 1110 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 1115 1120 1125 Ala Leu Pro Pro Arg 1130 <210> 8 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Anti - CD147 VH CDR Domain <400> 8 Glu Met Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Tyr Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys Thr Pro Asp Gly Ser Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 9 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Anti-CD147 VL CDR domain <400> 9 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Guide RNA targeting CD147 <400> 10 ttgacatcgt tggccaccgc 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Guide RNA targeting CD147 <400> 11 gtggacgcag atgaccgctc 20 <210> 12 <211> 489 <212> DNA <213> Artificial sequence <220> <223> IL-15 receptor intracellular domain <400> 12 atgcggatca gcaagcccca cctgcggagc atcagcatcc agtgctacct gtgcctgctg 60 ctgaacagcc acttcctgac cgaggccggc atccacgtgt tcatcctggg ctgcttcagc 120 gccggactgc ccaagaccga ggccaactgg gtgaacgtga tcagcgacct gaagaagatc 180 gaggacctga tccagagcat gcacatcgac gccaccctgt acaccgagag cgacgtgcac 240 cccagctgca aggtgaccgc catgaagtgc tttctgctgg aactgcaggt gatcagcctg 300 gaaagcggcg acgccagcat ccacgacacc gtggagaacc tgatcatcct ggccaacaac 360 agcctgagca gcaacggcaa cgtgaccgag agcggctgca aagagtgcga ggaactggaa 420 gagaagaaca tcaaagagtt tctgcagagc ttcgtgcaca tcgtgcagat gttcatcaac 480 accagctga 489 <210> 13 <211> 729 <212> DNA <213> Artificial sequence <220> <223> Consensus scFv sequence <400> 13 gagatgaagc tggaagagag cggcggcgga ctggtgcagc ccggcggcag catgaagctg 60 agctgcgtgg ccaccggctt caccttcagc aactactgga tgaactgggt gagacagacc 120 cccgaaaagg gcctggagtg ggtggcccag atcagactga agacctacaa ctacgccacc 180 cactacgccg agagcgtgaa gggcagattc accatcacca gggacgacac caagaccacc 240 gtgtacctgc agatgaacaa cctgagagcc gaggacaccg gcatctacta ctgcaccccc 300 gacggcagcg actactgggg ccagggcaca acactcaccg tgagcaccgg cggcggaggc 360 accggcggcg gcggcaccgg cggcggcggc tccggcggag gcggcaccag catcgtgatg 420 acccagaccc ccaagttcct cctggtgagc gccggcgaca gggtgaccat cacctgcaag 480 gccacccaga ccgtgagcaa cgacgtggcc tggtaccagc agaagcccgg ccagagccct 540 aagctgctga tctactacgc cagcaacagg tacacaggcg tgcccgacag gttcaccggc 600 accggctacg gcaccgactt caccttcacc atcagcacag tgcaggccga ggacctggcc 660 gtgtacttct gccagcagga ctacaccagc ccctacacct tcggaggcgg caccaagctg 720 gagatcaag 729 <210> 14 <211> 3833 <212> DNA <213> Artificial Sequence <220> <223> Gal4UAS CD147-CAR <400> 14 ggagcactgt cctccgaacg tcggagcact gtcctccgaa cgtcggagca ctgtcctccg 60 aacgtcggag cactgtcctc cgaacggagc atgtcctccg aacgtcggag cactgtcctc 120 cgaacgacta gttaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa 180 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga 240 ccgatccagc ctctcgacat tcgttggatc cacgcgtatg gagtttgggc tgagctggct 300 ttttcttgtg gctattttaa aaggtgtcca gtgcgtcgac gagatgaagc tggaagagag 360 cggcggcgga ctggtgcagc ccggcggcag catgaagctg agctgcgtgg cctccggctt 420 caccttcagc aactactgga tgaactgggt gagacagtcc cccgaaaagg gcctggagtg 480 ggtggcccag atcagactga agtcctacaa ctacgccacc cactacgccg agagcgtgaa 540 gggcagattc accatctcca gggacgactc caagtcctcc gtgtacctgc agatgaacaa 600 cctgagagcc gaggacaccg gcatctacta ctgcaccccc gatggcagcg actattgggg 660 ccagggcaca acactcaccg tgagctccgg cggcggaggc agcggcggcg gcggcagcgg 720 cggcggcggc tccggcggag gcggcagcag catcgtgatg acccagaccc ccaagttcct 780 cctggtgagc gccggcgata gggtgaccat tacctgcaag gcctcccagt ccgtgagcaa 840 cgatgtggcc tggtaccagc agaagcccgg ccagagccct aagctgctga tctactacgc 900 cagcaacagg tacacaggcg tgcccgacag gtttaccggc tccggctacg gcaccgactt 960 caccttcacc atcagcacag tgcaggccga ggacctggcc gtgtacttct gccagcagga 1020 ctactccagc ccctacacct tcggaggcgg caccaagctg gagatcaaga cgtacgtcac 1080 cgtctcttca caggatcccg ccgagcccaa atctcctgac aaaactcaca catgcccacc 1140 gtgcccagca cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa 1200 ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca 1260 cgaagaccct gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa 1320 gacaaagccg cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt 1380 cctgcaccag gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct 1440 cccagcccc atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt 1500 gtacaccctg cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct 1560 ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcaaccgga 1620 gaacactac aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag 1680 caagctcacc gtgggaaga gcaggtggca gcagggggaac gtcttctcat gctccgtgat 1740 gcatgaggct ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggataaaaa 1800 agatcccaaa ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct 1860 agtaacagtg gcctttatta ttttctgggt gaggatag aggagcaggc tcctgcacag 1920 tgactacatg aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta 1980 tgccccacca cgcgacttcg cagcctatcg ctccaaacgg ggcaagaaga aactcctgta 2040 tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag atggctgtag 2100 ctgccgattt ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag 2160 cgcagacgcc cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg 2220 acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg 2280 aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat 2340 ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga 2400 tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca 2460 ggccctgccc cctcgctaac atatgttgac ttgcggccgc aactcccacc tgcaacatgc 2520 gtgactgact gaggccgga ctctagagtc gacctgcatc taggcgccgg aattagatct 2580 ctcgaggtta acgaattcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca 2640 tgcgctttag cagccccgct gggcacttg cgctacacaa gtggcctctg gcctcgcaca 2700 cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc cttcgcgcca 2760 ccttctactc ctcccctagt caggaagttc cccccgccc cgcagctcgc gtcgtgcagg 2820 acgtgaaaa tggagtagc acgtctcact agtctcgtgc agatggacag caccgctgag 2880 caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc 2940 tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc 3000 ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg 3060 tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcag cccaagctta 3120 ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca 3180 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg 3240 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc 3300 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc 3360 accccgccga catccccgac tacttgaagc tgtccttccc cgaggcttc aagtgggagc 3420 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg 3480 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttccccctcc gacggccccg 3540 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg 3600 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg 3660 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg 3720 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 3780 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taa 3833 <210> 15 <211> 12221 <212> DNA <213> Artificial sequence <220> <223> pHR_Gal4UAS-CD147-CAR-pGK_mCherry vector <400> 15 cgataccgtc gaccaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac 60 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc 120 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 180 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 240 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatct 300 agaatttaatt ccgtgtattc father cctaaatcgt atgtgtatga tacataaggt 420. ttgtattaa ttgtagccgc gttctacga caatatgtac aagcctaatt gtgtagcatc tggcttactg aagcagaccc tatcatctct ctcgtaaact gccgtcagag tcggtttggt 480 tggacgaacc ttctgagttt ctggtaacgc cgtcccgcac ccggaaatgg tcagcgaacc 540 aatcagcagg gtcatcgcta gccagatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg 660 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt 720 ggccgggggga ctgttggggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct 780 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg 840. tcgaatggtg cactctcagt acaatctagc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 960 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgctatttt tataggttaa tgtcatgata 1080 ataatggttt cttagacgtc aggtggcact tttcgggggaa atgtgcgcgg aacccctatt 1140 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 1200 atgcttcaat aatattgaaa aaagaagagt atgagtattc aacatttccg tgtcgccctt 1260 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 1320 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 1380 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 1440 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 1500 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 1560 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 1620 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 1680 caacacatgg gggatcatgt aactcgcctt gatcgttgg aaccggagct gaatgaagcc 1740 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 1800 ctattaactg gcgaactact tactctagct tcccggcaac attaataga ctggatggag 1860 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gttattgct 1920 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 1980 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 2040 cgaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 2100 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 2160 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 2220 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc ttttttctg 2280 cgcgtaatct gctgcttgca aaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 2340 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 2400 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 2460 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 2520 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 2580 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 2640 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 2700 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 2760 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 2820 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 2880 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 2940 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3000 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3060 gcgcgttggc cgattcatta atgcagctgt ggaatgtgtg tcagttaggg tgtggaaagt 3120 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 3180 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 3240 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 3300 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 3360 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 3420 gcaaaaagct tggacacaag acaggcttgc gagatatgtt tgagaatacc actttatccc 3480 gcgtcaggga gaggcagtgc gtaaaaagac gcggactcat gtgaaatact ggtttttagt 3540 gcgccagatc tctataatct cgcgcaacct attttcccct cgaacacttt ttaagccgta 3600 gataaacagg ctgggacact tcacatgagc gaaaaataca tcgtcacctg ggacatgttg 3660 cagatccatg cacgtaaact cgcaagccga ctgatgcctt ctgaacaatg gaaaggcatt 3720 attgccgtaa gccgtggcgg tctgtaccgg gtgcgttact ggcgcgtgaa ctgggtattc 3780 gtcatgtcga taccgtttgt atttccagct acgatcacga caaccagcgc gagcttaaag 3840 tgctgaaacg cgcagaaggc gatggcgaag gcttcatcgt tattgatgac ctggtggata 3900 ccggtggtac tgcggttgcg attcgtgaaa tgtatccaaa agcgcacttt gtcaccatct 3960 tcgcaaacc ggctggttcgt ccgctggttg atgactatgt tgttgatatc ccgcaagata 4020 cctggattga acagccgtgg gatatgggcg tcgtattcgt cccgccaatc tccggtcgct 4080 aatcttttca acgcctggca ctgccgggcg ttgttcttt taacttcagg cgggttacaa 4140 tagtttccag tagtattct ggaggctgca tccatgacac aggcaaacct gagcgaaacc 4200 ctgttcaaac cccgctttaa acatcctgaa acctcgacgc tagtccgccg ctttaatcac 4260 ggcgcacaac cgcctgtgca gtcggccctt gatggtaaaa ccatccctca ctggtatcgc 4320 atgattaacc gtctgatgtg gatctggcgc ggcattgacc cacgcgaaat cctcgacgtc 4380 caggcacgta ttgtgatgag cgatgccgaa cgtaccgacg atgatttata cgatacggtg 4440 attggctacc gtggcggcaa ctggatttat gagtgggccc cggatctttg tgaaggaacc 4500 ttacttctgt ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt 4560 aaaataaaaa tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt 4620 ttagattcca acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga 4680 aaacctgttt tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca 4740 acattctact cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga 4800 attgctaagt ttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat 4860 ttacaccaca aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatattctgt 4920 aacctttata agtaggcata acagttataa tcataacata ctgttttttc ttactccaca 4980 caggcataga gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt 5040 aatttgtaaa ggggttaata aggaatattt gatgtatagt gccttgacta gagatcataa 5100 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 5160 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 5220 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 5280 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga 5340 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca 5400 attacctagt ggtttcattt actctaaacc tgtgattcct ctgaattatt ttcattttaa 5460 agaaattgta tttgttaaat atgtactaca aacttagtag ttggaagggc taattcactc 5520 ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga 5580 ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta 5640 caagctagta ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag 5700 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg 5760 gaggttgac agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt 5820 caagaactgc tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag 5880 gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc 5940 ttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct 6000 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt 6060 gtgcccgtct gttgtgtgac tctgtaact agagatccct cagaccctt tagtcagtgt 6120 ggaaaatctc tagcagtggc gcccgaacag ggacttgaa gcgaaaggga aaccagagga 6180 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg agggcggcg 6240 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga 6300 gagcgtcagt attaagcgggg ggagaattag atcgcgatgg gaaaaaatttc ggttaaggcc 6360 aggggaaag aaaaaata aattaaaaca tatagtatgg gcaagcaggg agctagaacg 6420 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca 6480 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc 6540 aaccctt tgtgtgcatc aaggataga gataaaagac accaggaag ctttagacaca 6600 gatgaggaa gaggaaaca aaagtaagac caccgcacag caagcggccg gtgatcttca 6660 gacctggacg atatatga gggacaattg gagaagtgaa tatataat attaagtagt 6720 aaaaattgaa ccattaggag tagcaccac ccaggchaag agaagtgg tgxagaga 6780 aaaaagagca gtgggaatag gagctttgtt ccttggttc tgggagcag caggaagcac 6840 tatgggcgca gcgtcaatga cgctgacggt acaggccaga cattattgt ctggtatagt 6900 gcagcagcag aacaatttgc tgaggctat tgaggcgca cagcatctgt tgcactcac 6960 agtctggggc atcagcagc tccaggcag aatcctggct gtggaagat acctaagga 7020 tcacagctc ctggggattt ggggttgctc tggaaactc atttgcacca ctgctgtgcc 7080 ttggaatgct agttggagta ataatctct ggaacagatt tggaatcaca cgacctggat 7140 ggagtgggac agagaatta acattacac aagcttaata cactccttaa ttgagaatc 7200 gcaaaccag caaaaaga attattgaa ttagataat gggcaagtttt 7260 gtggaattgg tttaacataa caattggct gtgtatata aattattca taatgatagt 7320 aggaggcttg gtaggtttaa gatagtttt tgctgtactt tctatagtga atagagttag 7380 gcagggatat tcaccattat cgtttcagac ccaccccca accccgaggg gacccgacag 7440 gcccgaagga atagagaag aggtggaga gagagacaga gatacca ttcgattagt 7500 gaacggatct cgacggtcgc caaatggcag tattcatcca caattttaaa agaaaagggg 7560 ggattggggg gtacagtgca gggaaaagaa tagtagacat aatagcaaca gacatacaaa 7620 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca 7680 gcagagatcc agtttggatc gataagcttg atatcgaatt cggagcactg tcctccgaac 7740 gtcggagcac tgtcctccga acgtcggagc actgtcctcc gaacgtcgga gcactgtcct 7800 ccgaacggag catgtcctcc gaacgtcgga gcactgtcct ccgaacgact agttaggcgt 7860 gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 7920 cgccatccac gctcttttga cctccataga agacaccggg accgatccag cctctcgaca 7980 ttcgttggat ccacgcgtat ggagtttggg ctgagctggc tttttcttgt ggctatttta 8040 aaaggtgtcc agtgcgtcga cgagatgaag ctggaagaga gcggcgggggg actggtgcag 8100 cccggcggca gcatgaagct gagctgcgtg gcctccggct tcaccttcag caactactgg 8160 atgaactggg tgagacagtc ccccgaaaag ggcctggagt gggtggccca gatcagactg 8220 aagtcctaca actacgccac ccactacgcc gagagcgtga agggcagatt caccatctcc 8280 agggacgact ccaagtcctc cgtgtacctg cagatgaaca acctgagagc cgaggacacc 8340 ggcatctact actgcacccc cgatggcagc gactattggg gccagggcac aacactcacc 8400 gtgagctccg gcggcggagg cagcggcggc ggcggcagcg gcggcggcgg ctccggcgga 8460 ggcggcagca gcatcgtgat gacccagacc cccaagttcc tcctggtgag cgccggcgat 8520 agggtgacca ttacctgcaa ggcctcccag tccgtgagca acgatgtggc ctggtaccag 8580 cagaagcccg gccagagccc taagctgctg atctactacg ccagcaacag gtacacaggc 8640 gtgcccgaca ggtttaccgg ctccggctac ggcaccgact tcaccttcac catcagcaca 8700 gtgcaggccg aggacctggc cgtgtacttc tgccagcagg actactccag cccctacacc 8760 ttcggaggcg gcaccaagct ggagatcaag acgtacgtca ccgtctcttc acaggatccc 8820 gccgagccca aatctcctga caaaactcac acatgcccac cgtgcccagc acctgaactc 8880 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 8940 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 9000 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 9060 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 9120 aatggcaagg agtacaagtg caaggtctcc aaaaagccc tcccagcccc catcgagaaa 9180 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 9240 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 9300 agcgacatcg ccgtggagtg ggagagcaat gggcaaccgg agaacaacta caagaccacg 9360 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 9420 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 9480 cactacacgc agaagagcct ctccctgtct ccgggtaaa aagatcccaa attttgggtg 9540 ctggtggtgg ttggtggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt 9600 atttctggg tgaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact 9660 ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc 9720 gcagcctatc gctccaaacg gggcagaaag aaactcctgt atatattcaa aaaccattt 9780 atgagaccag tacaaactac tcaaggaa gatggctgta gctgccgatt tccagaagaa 9840 gaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 9900 9960 gttttggaca agagacgtgg ccgggaccct gagatggggg gaagccgag agaagaac 10020 cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 10080 attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 10140 agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctaa 10200 catatgttga cttgcggccg caactcccac ctgcaacatg cgtgactgac tgaggccgcg 10260 actctagagt cgacctgcat ctaggcgccg gaattagatc tctcgaggtt aacgaattct 10320 accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc 10380 tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta 10440 ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag 10500 tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag 10560 cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc 10620 tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa 10680 ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt 10740 cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct 10800 cttcctcatc tccgggcctt tcgacctgca gcccaagctt accatggtga gcaagggcga 10860 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc 10920 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac 10980 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct 11040 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga 11100 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga 11160 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa 11220 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat 11280 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat 11340 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta 11400 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat 11460 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca 11520 ctccaccggc ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gagtcgacct 11580 gcaggcatgc aagcttgcag gcatgcaagc ttgatatcaa gcttatcgat aatcaacctc 11640 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc 11700 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca 11760 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg 11820 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca 11880 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg 11940 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg 12000 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg 12060 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg 12120 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc 12180 ctcagacgag tcggatctcc ctttgggccg cctccccgca t 12221 <210> 16 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> GPC3-CAR <400> 16 atggagtttg gtttaagctg gctgttttta gtggccattt taaagggcgt gcagtgcgag 60 caaaaactta tctctgaaga ggacctcgtc gaccaagttc agctgcagca gagcggagcc 120 gaactggtga gacccggcgc tagcgtgaag ctgtcttgta aggctagcgg ctacaccttc 180 accgactacg agatgcattg ggtcaagcag acccccgttc acggtttaaa gtggatcggc 240 gctctcgatc ctaagactgg tgacaccgcc tacagccaga agttcaaagg caaggccact 300 ttaaccgccg acaagtccag cagcaccgct tacatggagc tgaggagcct cacctccgag 360 gacagcgccg tgtattactg cacaaggttc tacagctaca catactgggg ccaaggtaca 420 ctggtgaccg tgagcgctgg cggcggaggc tccggtggcg gcggcagcgg aggcggcggt 480 tctgacgtgg tgatgaccca gacaccttta agcctccccg tttctttagg cgatcaagct 540 agcatctctt gtcgttcctc ccagtcttta gtgcactcca acggcaacac ctatttacac 600 tggtatctgc agaagcccgg ccagagcccc aagctgctga tctacaaggt cagcaatagg 660 ttcagcggcg tgcccgatcg ttttagcgga agcggcagcg gcaccgactt cactttaaaa 720 atctctcgtg tggaggccga agatttaggc gtgtactttt gttcccagaa cacccacgtg 780 ccccccacat tcggctccgg aaccaagctg gagattaag 819 <210> 17 <211> 87 <212> DNA <213> Artificial sequence <220> <223> GAL4-VP64 <400> 17 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc 60 ctctcgacat tcgttggatc cacgcgt 87 <210> 18 <211> 65 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 18 tgcgtcgacg agcagaaact catctctgaa gaggatctgg agatgaagct ggaagagagc 60 ggcgg 65 <210> 19 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 19 ggcaccaagc tggagatcaa gatcctggac tacagcttca cagg 44 <210> 20 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 20 cctgtgaagc tgtagtccag gatcttgatc tccagcttgg tgcc 44 <210> twenty one <211> 37 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> twenty one ctaacgcgtt catgatccga gcatgtccag gtcaaag 37 <210> twenty two <211> 42 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> twenty two tcgacattcg ttggatccgc cagcatggag tttggtttaa gc 42 <210> twenty three <211> 55 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> twenty three cggctccgga accaagctgg agattaagga gcccaaatct cctgacaaaa ctcac 55 <210> twenty four <211> 55 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> twenty four gtgagttttg tcaggagatt tgggctcctt aatctccagc ttggttccgg agccg 55 <210> 25 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 25 tagaattcgt taacctcgag ttagcgaggg ggcagggcct gc 42 <210> 26 <211> 64 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 26 tgccaccatg gcaatggagt ttggtttaag ctggctgttt ttagtggcca ttttaaaggg 60 cgtg 64 <210> 27 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 27 caggatacgc gtcttaatct ccagcttggt tccgg 35 <210> 28 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 28 ttaagacgcg tatcctggac tacagcttca caggtg 36 <210> 29 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 29 tcccgctcga gtcatgatcc gagcatgtcc agg 33 <210> 30 <211> 61 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 30 tcgttggatc cacgcgtcgt acgttaatta acccgggcat atgttgactt gcggccgcaa 60 c 61 <210> 31 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 31 ccattgctca gcggtgctg 19 <210> 32 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 32 gatccacgcg tatggagttt gggctgagct ggc 33 <210> 33 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 33 gtcaacatat gttagcgagg gggcagggcc tgcatg 36 <210> 34 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 34 ctagactgcc atggagtttg ggctgagctg 30 <210> 35 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide primers <400> 35 gacggtgacg tacgtcttga tctccagctt ggtg 34

Claims

1. A chimeric antigen receptor comprising: (a) an antigen-binding domain that specifically binds to CD147, wherein the antigen-binding domain is encoded by a nucleic acid comprising a nucleic acid sequence of SEQ ID NO: 1; (b) hinge domain; (c) a transmembrane domain; and (d) an intracellular domain, which comprises one or more intracellular domains of co-stimulatory molecules and an intracellular signaling domain.

2. The chimeric antigen receptor of claim 1, wherein the antigen binding domain binds to cells expressing high levels of surface CD147.

3. The chimeric antigen receptor of claim 1 or 2, wherein the one or more co-stimulatory molecule intracellular domains comprise the intracellular domains of CD28 and 4-1BB.

4. The chimeric antigen receptor of claim 1 or 2, wherein the intracellular signaling domain comprises the signaling domain of CD3ζ.

5. The chimeric antigen receptor of claim 1 or 2, wherein the hinge domain comprises an IgG1 hinge domain.

6. The chimeric antigen receptor according to claim 1 or 2, wherein the transmembrane domain is a CD28 transmembrane domain or a CD8a transmembrane domain.

7. The chimeric antigen receptor according to claim 1 or 2, comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

5. The chimeric antigen receptor according to claim 7, comprising the amino acid sequence of SEQ ID NO:

5.

9. The chimeric antigen receptor of claim 1 or 2, further comprising one or more additional antigen binding domains.

10. The chimeric antigen receptor of claim 9, wherein the one or more additional antigen binding domains specifically bind to Glypican 3, AFP, or Mucin-1. The chimeric antigen receptor according to claim 1 or 2, further comprising an inducible suicide molecule.

12. The chimeric antigen receptor of claim 11, wherein the suicide molecule comprises caspase 9.

13. The chimeric antigen receptor of claim 11, wherein expression of the suicide molecule is inducible by tetracycline, doxycycline or rapamycin.

14. The chimeric antigen receptor of claim 11, comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

7. The chimeric antigen receptor according to claim 14 , comprising the amino acid sequence of SEQ ID NO:

7.

16. The chimeric antigen receptor of claim 1 or 2, further comprising a cytokine receptor intracellular domain.

17. The chimeric antigen receptor of claim 16, wherein the cytokine receptor intracellular domain is an interleukin-15 receptor intracellular domain, an interleukin-12 receptor intracellular domain, or an interleukin-18 receptor intracellular domain.

18. The chimeric antigen receptor of claim 17, wherein the cytokine receptor intracellular domain is an interleukin-15 receptor intracellular domain comprising the amino acid sequence of SEQ ID NO:

12.

19. A nucleic acid molecule encoding the chimeric antigen receptor according to claim 1 or 2.

20. The nucleic acid molecule according to claim 19, comprising the nucleic acid sequence of SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:

14. A vector comprising the nucleic acid molecule according to claim 20 .

22. The vector of claim 21, further comprising a nucleic acid molecule encoding a second chimeric antigen receptor targeting hepatitis B virus or hepatitis C virus.

23. The vector of claim 21, further comprising an inducible promoter or enhancer nucleic acid molecule operably linked to the chimeric antigen receptor nucleic acid molecule.

24. The vector of claim 23, wherein the enhancer nucleic acid molecule is a Gal4 upstream activating sequence.

25. The vector according to claim 24, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:

15.

26. The vector of claim 22, wherein the vector is a viral vector.

27. The vector of claim 26, wherein the viral vector is a lentiviral vector or a retroviral vector.

28. A cell expressing the chimeric antigen receptor according to claim 1 or 2, wherein the cell is a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a double negative T (DNT) cell, a neutrophil or a macrophage.

29. A cell comprising the nucleic acid of claim 19, wherein the cell is a T cell, a NK cell, a natural killer T (NKT) cell, a double negative T (DNT) cell, a neutrophil or a macrophage.

30. The cell of claim 29, wherein the cell comprises the vector of claim 21.

31. The cell of claim 29, further comprising a nucleic acid molecule encoding an activator of an inducible promoter or enhancer.

32. The cell of claim 31, wherein the nucleic acid encoding the activator further encodes an anti-GPC3 specific binding agent.

33. A cell comprising the vector of claim 23, further comprising a nucleic acid molecule encoding an activator of an inducible promoter or enhancer and a vector comprising an anti-GPC3 chimeric antigen receptor operably linked to the inducible promoter or enhancer, wherein the cell is a T cell, a NK cell, a natural killer T (NKT) cell, a double negative T (DNT) cell, a neutrophil or a macrophage.

34. The cell of claim 33, wherein the vector comprising the anti-GPC3 chimeric antigen receptor comprises the nucleic acid sequence of SEQ ID NO:

16.

35. The cell of claim 28, wherein the cell is obtained from peripheral blood, umbilical cord blood, lymph nodes, bone marrow, tumor tissue, or a cell line.

36. The cell of claim 28, wherein the cell is a NK-92 cell or a NK-92MI cell.

37. A method for producing CD147-CAR-T cells or CD147-CAR-NK cells, the method comprising transducing or transfecting T cells or NK cells with the vector according to claim 21.

38. Use of the cell of claim 28 in the preparation of a medicament for treating a subject suffering from cancer.

39. The use according to claim 38, wherein the subject suffers from a cancer expressing CD147.

40. The use of claim 38, wherein the subject has hepatocellular carcinoma, neuroblastoma, breast cancer, pancreatic cancer, leukemia, lymphoma, multiple myeloma, colorectal cancer, lung cancer, melanoma, renal cell carcinoma, sarcoma, or nasopharyngeal carcinoma.

41. The use of claim 38, further comprising treating the subject with at least one of: surgery, radiation, chemotherapy, additional immunotherapy, or a combination of two or more thereof.

42. The use according to claim 38, wherein the subject has hepatocellular carcinoma.

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