Artificial-natural antigen logic gating CAR-T and application thereof in solid tumor treatment

Abstract

The invention discloses a chimeric antigen receptor T cell (CAR-T) of modular artificial antigen-natural antigen logic gating (MANAGE) and application of the chimeric antigen receptor T cell in treatment of solid tumors. At present, solid tumors still lack efficient targets capable of covering tumor cells of the same focus and crossing tumor species, and most solid tumor targets are also expressed on normal tissue cells. Therefore, the application of single-target CAR-T therapy in solid tumors is limited by tumor heterogeneity and non-tumor targeted toxicity (OTOT). According to the invention, proteins and antibodies of targeted tumor cells modified by the artificial antigen FITC are constructed, and CAR-T double-gated by the FITC and the natural tumor antigen is researched and developed. When the two compounds are combined for use, the toxicity of OTOT can be reduced, the range of applicable cells and tumor species can be expanded, the injection time of FITC can be regulated and controlled, the effect of CAR-T can be'switched ', cytokine syndromes can be reduced, and a new method is provided for solid tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an artificial-natural antigen logic-gated CAR-T and its application in the treatment of solid tumors. Background Technology

[0002] The application of chimeric antigen receptor T-cell (CAR-T) therapy in solid tumors is limited by tumor heterogeneity, on-target off-tumor toxicity (OTOT), and cytokine syndrome.

[0003] Currently, there is a lack of specific targets for solid tumors that can cover all cells within the same tumor type, and there is also a lack of targets applicable to a variety of different solid tumors. CAR-T therapy targeting a single target cannot kill tumor cells that do not express the target antigen, resulting in immune escape. When CAR-T therapy efficiently kills tumor cells that express the target antigen, it also causes a large amount of pro-inflammatory cytokines to be secreted in a short period of time, which can lead to toxic side effects such as cytokine syndrome (CRS).

[0004] Furthermore, most target antigens highly expressed in solid tumors are also expressed on normal tissue cells, causing CAR-T cells targeting these antigens to mistakenly damage normal tissue cells, leading to OTOT toxicity. Unlike hematologic malignancies, which are distributed throughout the body, solid tumors are located only in specific locations. CAR-T cells have a higher probability of binding to tumor targets on normal cells in patients with solid tumors, thus making the OTOT toxicity of CAR-T cells more significant when treating solid tumors. SynNotch CAR-T cells, after recognizing one tumor antigen, promote CAR expression; the CAR then recognizes another tumor antigen and kills tumor cells. Although synNotch CAR-T cells require recognition of dual tumor antigens to kill tumor cells, reducing OTOT toxicity, in the treatment of solid tumors lacking highly effective targets, it increases the difficulty of target selection and exacerbates the immune escape of solid tumors.

[0005] Therefore, there is an urgent need to develop CAR-T therapies that are widely applicable, safe, and modulotropic in vivo for the efficient and precise treatment of solid tumors. Summary of the Invention

[0006] To address the aforementioned technical challenges, this invention constructs FITC-modified serum albumin (BSA-FITC) or PD-L1 antibody (aPD-L1-FITC) as tumor-targeting artificial antigens and develops a modular, dual-gated (MANAGE: Modular Artificial and Natural Antigen-GatEd) logic-gated CAR-T. The combined use of these two methods not only treats various tumors and reduces the OTOT toxicity of CAR-T, but also regulates the efficacy of CAR-T in vivo, reduces CRS, and improves safety, providing a new approach for the efficient and precise treatment of solid tumors.

[0007] The first objective of this invention is to provide a medical product with dual gating of artificial antigens and natural tumor antigens, said medical product being used in tumor treatment to increase the recognition range of tumor cells, reduce non-tumor-targeted toxicity and CRS, comprising:

[0008] A first formulation contains a tumor-targeting artificial antigen, wherein the tumor-targeting artificial antigen comprises a protein that modifies the artificial antigen or an antibody that modifies the artificial antigen (preferably a protein that modifies the artificial antigen); wherein the artificial antigen is a marker not expressed on the surface of cells (including normal cells and tumor cells), and the protein or antibody can target tumor cells;

[0009] The second formulation contains CAR-T cells, which contain a first gene expression cassette and a second gene expression cassette. The first gene expression cassette contains a coding sequence for an extracellular recognition region, a coding sequence for a transmembrane core region, and a coding sequence for an intracellular effector region. The extracellular recognition region contains a single-chain antibody sequence (scFv) of the artificial antigen, the transmembrane core region contains a Notch core sequence, and the intracellular effector region contains a transcription factor sequence. The second gene expression cassette contains an upstream activation sequence that specifically binds to the transcription factor, and a chimeric antigen receptor (CAR) located after the upstream activation sequence. The chimeric antigen receptor targets a natural antigen, which is a marker highly expressed on the surface of tumor cells.

[0010] Furthermore, the ratio of the artificial antigen to the antibody or protein connected is (0.1-10):1, such as (0.1-5):1.

[0011] Furthermore, the artificial antigen includes one or more of fluorescent molecules, biotin, and artificial peptide chains; the fluorescent molecules include one or more of FITC, Cy3, Cy5, and Cy7.

[0012] Furthermore, in the modified artificial antigen antibody or modified artificial antigen protein, the antibody or protein specifically binds to tumor cells (e.g., specifically recognizes tumor cell surface markers), and the markers targeted by the antibody include, but are not limited to, programmed death ligand 1 (PD-L1), CD276 (a member of the B7 immunomodulatory molecule family), G protein-coupled receptor 65 (GPR65), V-set and immunoglobulin domain-containing protein 4 (VSIG4), γ-aminobutyric acid metabolite (GABA), discoid domain receptor 1 (DDR1), ADP-ribosyltransferase 1 (ART1), basal cell adhesion molecule (BCAM), protein tyrosine phosphatase non-receptor type 2 (PTPN2), chemokine receptor 8 (CCR8), etc.; the protein includes one or more of serum albumin and transferrin.

[0013] Furthermore, the single-chain antibody of the artificial antigen includes αFITC scFv; the coding sequence of said αFITC scFv is shown in SEQ ID NO.1.

[0014] Furthermore, transcription factors, including one or more of Gal4-VP64, LexA-VP64, Gal4-KRAB, and ZFHD1-VP64, are responsible for initiating signal responses to regulate the expression of downstream target genes (activate gene expression). UAS is the binding site of GAL4. When GAL4 binds to UAS, it initiates transcription of downstream promoters, thereby driving the expression of downstream target genes.

[0015] Furthermore, the natural antigens include, but are not limited to, CD19, HER2, Claudin 18.2, EGFR, FAP, TROP2, etc.

[0016] Furthermore, the Notch core sequence is shown in SEQ ID NO.2.

[0017] Furthermore, the CAR-T cells are obtained by transfecting T cells with a first gene expression frame and a second gene expression frame.

[0018] Furthermore, the chimeric antigen receptor contains a single-chain antibody coding sequence of a natural antigen.

[0019] Furthermore, the single-chain antibodies against the natural antigen include single-chain antibodies against CD19 and / or single-chain antibodies against HER2.

[0020] Furthermore, the single-chain antibody sequence of CD19 is shown in SEQ ID NO.3; the single-chain antibody sequence of HER2 is shown in SEQ ID NO.4.

[0021] Furthermore, the chimeric antigen receptor also contains a hinge region coding sequence, a transmembrane region coding sequence, and an intracellular signaling region coding sequence.

[0022] Furthermore, the hinge area includes a CD28 hinge area.

[0023] Furthermore, the transmembrane region includes the CD28 transmembrane region.

[0024] Furthermore, the intracellular signaling region contains a co-stimulatory molecule region and a signal transduction molecule region; the co-stimulatory molecule region includes the CD28 intracellular signaling region, and the signal transduction molecule region includes the CD3zeta intracellular signaling region.

[0025] Furthermore, the chimeric antigen receptor has a tag sequence, such as the Thy1.1 coding sequence, attached after the CD3zeta intracellular signaling region.

[0026] Furthermore, the coding sequence and tag sequence of the CD3zeta intracellular signaling region are linked by a 2A peptide.

[0027] A second object of the present invention is to provide a pharmaceutical composition comprising:

[0028] Tumor-targeting artificial antigens, wherein the tumor-targeting artificial antigens include proteins or antibodies that modify artificial antigens; the artificial antigens are markers not expressed on the cell surface, and the proteins or antibodies can target tumor cells;

[0029] An immune cell containing a first gene expression frame and a second gene expression frame; the first gene expression frame contains a coding sequence for an extracellular recognition region, a coding sequence for a transmembrane core region, and a coding sequence for an intracellular effector region, wherein the extracellular recognition region contains a single-chain antibody sequence of the artificial antigen, the transmembrane core region contains a Notch core sequence, and the intracellular effector region contains a transcription factor sequence; the second gene expression frame contains an upstream activation sequence that specifically binds to the transcription factor, and a chimeric antigen receptor located after the upstream activation sequence, wherein the chimeric antigen receptor targets a natural antigen, and the natural antigen is a marker highly expressed on the surface of tumor cells.

[0030] Furthermore, the immune cells include, but are not limited to, T cells and NK cells.

[0031] Furthermore, the pharmaceutical composition is universal, and can be designed into corresponding pharmaceutical formulations by selecting the corresponding natural antigen targets according to the type of tumor to be treated.

[0032] A third object of the present invention is to provide the use of the medical product or the pharmaceutical composition in the preparation of a pharmaceutical product, wherein the pharmaceutical product is used in any of the following:

[0033] (1) Used to kill tumor cells in vivo or in vitro;

[0034] (2) Used in the preparation of antitumor drugs;

[0035] (3) Used to increase the range of tumor cells that can be killed and reduce immune escape;

[0036] (4) Used to reduce CRS;

[0037] (5) Used to reduce non-tumor-targeted toxicity during tumor treatment.

[0038] A fourth object of the present invention is to provide an antitumor drug, including the medical product or the pharmaceutical composition.

[0039] Furthermore, the antitumor drug is used to treat any solid tumor, including but not limited to melanoma, ovarian cancer, cervical cancer, lung cancer, breast cancer, head and neck cancer, liver cancer, intestinal cancer, colon cancer, kidney cancer, stomach cancer, glioma, bladder cancer, pancreatic cancer, etc.

[0040] By means of the above-described solution, the present invention has at least the following advantages:

[0041] This invention first utilizes N-hydroxysuccinimide-functionalized fluorescent molecule FITC (NHS-PEG-FITC) to modify serum albumin and PD-L1 antibody, respectively, to prepare tumor-targeting artificial antigens BSA-FITC and aPD-L1-FITC. Figure 1 A). On the other hand, using the synNotch receptor that recognizes FITC, logic-gated MANAGE CAR-T cells containing the artificial antigen FITC and the natural antigen (CD19 or HER2) were prepared. Without stimulation by the artificial antigen, the MANAGE CAR-T cells only expressed the synNotch receptor that recognizes the artificial antigen FITC. Figure 1 B). Injection of BSA-FITC or aPD-L1-FITC allows these tumor-targeting artificial antigens to accumulate in tumors and specifically bind to tumor cells, causing the tumor cells to express the artificial antigen FITC (BSA-FITC). Figure 1 C). Subsequently, MANAGECAR-T is injected. The CAR-T recognizes the artificial antigen FITC within the tumor, triggering a logic-gated switch to induce the expression of aCD19 or aHER2CAR, thereby specifically killing tumor cells expressing these targets. Normal cells, lacking artificial antigens or tumor-specific antigens, cannot activate MANAGECAR-T, thus reducing damage to normal cells and OTOT toxicity. Figure 1C). This design also has the following advantages: 1) The adapter can target different sites, and injecting different adapters can enable CAR-T to recognize heterogeneous tumor cells and different tumors. 2) Because FITC is not expressed in vivo, the efficacy of MANAGECAR-T can be regulated in vivo by adjusting the administration time and dosage of artificial antigens, thereby controlling CRS and improving safety. Attached Figure Description

[0042] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0043] Figure 1 Preparation and mechanism of action of modular, artificial-natural antigen dual-gate CAR-T cells. (A) Preparation of tumor-targeting artificial antigens. (B) Schematic diagram of the sequence and construction of the MANAGE CAR-T receptor. (C) Schematic diagram of tumor therapy using MANAGE CAR-T in combination with an adapter, illustrating how combination therapy improves efficacy, reduces non-tumor-targeted toxicity, and reduces CRS.

[0044] Figure 2 To demonstrate that BSA-FITC can specifically bind to tumor cells. (A) Number of FITC linkages on BSA-FITC at different feed ratios. (B) Flow cytometry representations of BSA-FITC binding to tumor cells (B16F10 cells, 4T1 cells, Panc-02 cells) and T cell surfaces. (C) Quantitative analysis of mean fluorescence intensity (MFI) of FITC on different cell surfaces. (D) Representative flow cytometry histograms of FITC expression on tumor cells at different time points and (E) Trends in expression.

[0045] Figure 3The tumor-targeting artificial antigen BSA-FITC can activate MANAGE CAR-T cells in vitro. (A) Schematic diagram showing the promotion of CAR and tag protein Thy1.1 expression after FITC-synNotch receptor activation. (B) Activation of FITC-synNotch receptor on CAR-T cells by BSA-FITC with different numbers of FITCs. (C) Activation effect of FITC-synNotch receptor on CAR-T cells after co-incubation with tumor cells at different concentrations of BSA-FITC. (D) Activation kinetics of FITC-synNotch receptor on CAR-T cells by BSA-FITC. (E) Schematic diagram of the characterization of MANAGE CAR-T cells activated by artificial-natural dual antigens. (F) Flow cytometry representation of CD69 expression in MANAGE CAR-T cells under different activation conditions and (G) quantitative analysis. (H) Killing effect of MANAGE CAR-T cells on B16F10-hCD19 tumor cells at different effector-target ratios and (I) secretion levels of cytokines IFN-γ and IL-2. (J)MANAGE: Killing effect of CAR-T cells on 4T1-HER2 tumor cells and secretion levels of (K)IFN-γ and IL-2 at different effector-to-target ratios. Data are expressed as mean ± standard deviation (n = 3 or 4). ns indicates no statistical significance, ****P < 0.0001.

[0046] Figure 4 The tumor-targeting artificial antigen aPD-L1-FITC can activate MANAGE CAR-T cells in vitro. (A) The number of FITCs linked to aPD-L1-FITC at different feed ratios. (B) The activation effect of different concentrations of aPD-L1-FITC on the FITC-synNotch receptor after incubation. (C) The activation kinetics of aPD-L1-FITC on the FITC-synNotch receptor. (D) The killing effect of MANAGE CAR-T cells on B16F10-hCD19 tumor cells at different effector-target ratios and (E) the secretion levels of cytokines IFN-γ and IL-2. (F) Schematic diagram of the HER2 CAR structure. (G) The killing effect of MANAGE CAR-T cells on 4T1-HER2 tumor cells at different effector-target ratios and (H) the secretion levels of cytokines IFN-γ and IL-2. Data are expressed as mean ± standard deviation (n=4) and analyzed using one-way or two-way ANOVA combined with Tukey post-hoc test. ns indicates no significant difference, ****P<0.0001.

[0047] Figure 5MANAGE CAR-T cells can be specifically activated in tumors. (A) Fluorescence imaging of the biodistribution of tumor-targeting artificial antigens in major organs and (B) fluorescence percentage. (C) Schematic diagram of MANAGE CAR-T cells expressing Luciferase reporter protein. (D) Flowchart of the experiment for detecting CAR-T cell activation in vivo. (E) Representative images of tumor bioluminescence at different time points and (F) change curves. 48 h after CAR-T injection, (F) representative bioluminescence images of major organs and (GH) quantitative analysis. Data are expressed as mean ± standard deviation (n = 4 or 5). ns indicates no significant difference, ****P < 0.0001.

[0048] Figure 6 Synergistic in vivo immune response of MANAGE CAR-T and tumor-targeted artificial antigens. (A) Experimental flowchart. (B) Thy1.1 in tumors, spleen, and lymphoid organs of each treatment group. + CAR-T cells in CD45 + Representative flow cytometry plots showing the proportion of cells in the cell. (C)Thy1.1 + CAR-T cells in tumor CD45 + Quantitative analysis of the percentage of cells and the concentration of Thy1.1 per mg of tumor + Number of CAR-T cells. (Thy1.1) + CAR-T cells in (D) lymph nodes and (E) spleen CD45 + Percentage within cells. (F) Intratumoral Thy1.1 + Representative flow cytometry plots and quantitative analysis of (G) proportions of IFN-γ and TNF-α expression in CAR-T cells. (H) Intratumoral Thy1.1 + Statistical graph of PD-1 and LAG-3 expression in CAR-T cells. Data are expressed as mean ± standard deviation (n=6). ns indicates no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0049] Figure 7Evaluation of the antitumor efficacy of MANAGE CAR-T cells. (A) Flowchart of the experiment examining efficacy in a unilateral B16F10-hCD19 tumor. (B) Tumor growth curve. (C) Mouse survival statistics. (D) Mouse weight change statistics. (E) Flowchart of the experiment examining efficacy in a bilateral B16F10 tumor model. (F) Bilateral tumor growth curve. (G) Flowchart of the experiment examining efficacy in an orthotopic breast cancer tumor model. (H) Tumor growth curve. (I) Statistical analysis of the number of lung metastatic tumor nodules. Data are expressed as mean ± standard deviation (n = 5 or 6). ns indicates no statistically significant difference, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0050] Figure 8 To reduce OTOT toxicity of MANAGE CAR-T cells. (A) Representative and statistical graphs of liver biofluorescence expression after high-pressure tail vein injection of a plasmid encoding Luciferase. (B) Flowchart of the experiment investigating non-tumor-targeting toxicity of MANAGE CAR-T. (C) Detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in blood. (D) Changes in mouse body weight. (E) Representative images of hematoxylin-eosin (H&E) staining of liver tissue from mice in different treatment groups. Data are expressed as mean ± standard deviation (n = 3). ns indicates no statistically significant difference; ***P < 0.001, ****P < 0.0001.

[0051] Figure 9 To reduce CAR-T-induced cytokine release syndrome by regulating the injection cycle of artificial antigens. (A) Schematic diagram of CAR-T cell-induced CRS mouse model construction and different treatment regimens. (B) Tumor growth curve. (C) Monitoring of mouse body weight changes at different time points. Changes in (D) IL-6 and (F) IFN-γ in mouse blood. Changes in (F) white blood cells, (G) red blood cells, (H) platelets, and (I) hemoglobin in mouse blood. Data are expressed as mean ± standard deviation (n = 6). ns indicates no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0053] 1. The materials and methods involved in the embodiments of the present invention are as follows:

[0054] (1) Reagents

[0055] NHS-PEG 2000FITC and BSA were purchased from Xi'an Ruixi Biotechnology Co., Ltd. Mouse PD-L1 monoclonal antibody (10F.9G2) was purchased from BioXCell, and mouse IL-2 and IL-7 were purchased from PeproTech, USA. Matrigel was purchased from Corning Incorporated, USA.

[0056] (2) Experimental cells and animals

[0057] HEK-293T cells were purchased from Zhejiang Zhongchu Biotechnology Co., Ltd. Female SPF-grade C57BL / 6 mice and BALB / c mice (both 6–8 weeks old) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. Mice underwent a 1–3 day acclimatization period before the start of the experiment. All animal experiments were conducted under the strict review and supervision of the Animal Experiment Ethics Committee of Soochow University to ensure that every step of the experimental procedure adhered to the principles of laboratory animal welfare and the laboratory animal ethics guidelines of Soochow University.

[0058] 2. The sequence information involved in the embodiments of the present invention is as follows:

[0059] anti-FITC-scFv(SEQ ID NO.1):

[0060] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDD AKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSS

[0061] The notch core (SEQ ID NO.2):

[0062] ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR

[0063] anti-CD19-scFv(SEQ ID NO.3):

[0064] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0065] anti-HER2-scFv(SEQ ID NO.4):

[0066] QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLGAAAGGGGS

[0067] Example 1

[0068] 1. MANAGE CAR sequence design

[0069] The MANAGE CAR sequence comprises two parts: the first part is the MANAGE CAR structure that recognizes artificial targets, which integrates the coding sequences for anti-FITC-scFv (clone FMC63), the notch core, and the intracellular transcription factor (TF) into a lentiviral vector to form the MANAGE CAR plasmid. The second part is the CAR structure that recognizes natural antigens, which integrates the coding sequences for the transcription factor binding site Gal4-UAS, anti-CD19-CAR (containing αCD19 scFv (extracellular antigen binding region), CD28 hinge, CD28 TM (transmembrane region), CD28 co-stimulatory region, CD3zeta intracellular domain), P2A, and Thy1.1 into a lentiviral vector to form the CAR plasmid.

[0070] 2. Preparation of CAR-T

[0071] The MANAGE CAR plasmid, CAR plasmid, packaging plasmid, and coating plasmid were amplified using E. coli. Hieff was employed. Liposomal Transfection Reagent, MANAGE CAR plasmid (or CAR plasmid), packaging plasmid, and envelope plasmid were transfected into HEK293T cells at a ratio of 3:2:1. Virus-containing supernatant was collected at 48 and 72 h, centrifuged at 1000×g for 5 min at 4°C, and filtered through a 0.45 μm cellulose acetate membrane. The resulting virus solution was then ultracentrifuged at 25000×g for 2 h at 4°C to a concentrated concentration, resuspended in serum-free medium, and stored at -80°C.

[0072] CD8 was extracted from mouse spleen. + T cells. Female C57BL / 6 or BALB / c mice were euthanized with carbon dioxide, and the spleens were harvested. Cells were ground through a 70 μm cell filter, centrifuged at 700 × g for 5 min to collect cell clumps, and incubated at room temperature for 3 min with erythrocyte lysis buffer. Lysis was terminated by dilution with PBS. The cell suspension was filtered through a 40 μm cell filter, centrifuged again (700 × g, 5 min), the supernatant was discarded, and the cells were resuspended in PBS. EasySep was used to lyse the cells. TM mouse CD8 + CD8 cells were purified using a T-cell isolation kit. + T cells. CD8 +T cells were resuspended in a T cell-specific culture medium containing mouse IL-2 (20 ng / mL) and IL-7 (10 ng / mL), transferred to pre-coated anti-CD3 and anti-CD28 culture dishes, and incubated at 37°C for 3 days for activation.

[0073] Lentiviral transfection of CD8 + T cell preparation MANAGE CAR-T cells. Activated CD8 + T cells were mixed with MANAGE CAR virus solution and CAR virus solution, and the cell density was adjusted to 5 × 10⁶ cells / mL using serum-free culture medium. 6 Cells / mL were added, and Polybrene was added to a final concentration of 8 μg / mL. After 12 h of infection, an equal volume of T cell-specific culture medium containing mouse IL-2 and IL-7 was added for further culture.

[0074] 3. Preparation of tumor-targeted artificial antigens

[0075] FITC-modified BSA or aPD-L1 are used to prepare tumor-targeting artificial antigens. NHS-PEG 2000 -FITC was dissolved in anhydrous ethanol to prepare a concentration of 100 mg / mL. Following the instructions for BSA or aPD-L1 and NHS-PEG... 2000 The FITC molar ratios of 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:6, 1:8, and 1:10 were reacted in low-protein adsorption tubes. PBS was added to bring the final protein concentration to 1 mg / mL, and the pH was adjusted to 8.5. After reacting in a four-dimensional rotator at room temperature for 4 hours, the sample was added to a 50 kDa ultrafiltration tube, and free NHS-PEG was removed by ultrafiltration with PBS. 2000 -FITC. The absorbance at A280 of the product was measured using NanoDrop to determine the protein concentration. The absorbance of the FITC group at 495 nm was measured using a UV spectrophotometer, according to NHS-PEG. 2000 -FITC standard curves were constructed to determine FITC concentrations. The number of FITCs linked to each BSA or aPD-L1 was calculated according to Equation 1.

[0076]

[0077] Formula 1: Number of FITCs connected to each BSA or aPD-L1. C FITC NHS-PEG 2000 -FITC concentration; C Biologics The concentration of BSA or aPD-L1.

[0078] Example 2: In vitro activation of MANAGE CAR-T by tumor-targeted artificial antigens

[0079] (1) After digestion, B16F10-hCD19 cells were plated in 24-well plates with 100,000 cells per well. BSA-FITC with artificial target FITC linkages of 0, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, and 4 were added to the B16F10-hCD19 cells and 100,000 MANAGE CAR-T cells were incubated together for 24 h. After centrifugation, the cells were collected, stained with anti-Thy1.1-APC, and the expression efficiency of CAR molecules was detected by flow cytometry.

[0080] Taking a tumor-targeting artificial antigen with FITC molecules linked to a single molecule as an example, B16F10 cells were digested and plated in 24-well plates with 100,000 cells per well. B16F10-hCD19 cells were incubated with BSA-FITC at final concentrations of 0 nM, 4 nM, 6 nM, 8 nM, and 10 nM, along with 100,000 MANAGE CAR-T cells, for 24 h. Cells were collected by centrifugation, stained with anti-Thy1.1-APC, and the expression efficiency of CAR molecules was detected by flow cytometry.

[0081] Here are the results:

[0082] Serum albumin (BSA) and NHS-PEG 2000 When the molar ratio of FITC to BSA was 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:6, 1:8, and 1:10, the number of FITC artificial antigens linked to BSA was 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, and 4, respectively, successfully preparing the tumor-targeting artificial antigen BSA-FITC. Figure 2 A). We then verified that BSA-FITC could successfully bind to tumor cells (B16F10 cells, 4T1 cells, and panc-02 cells) but not to T cells. Figure 2 BC). Using a fluorescent antibody targeting FITC, only FITC on the cell surface was stained. Results showed that BSA-FITC binds to the surface of tumor cells (BC). Figure 2 D), and can maintain FITC on the surface of tumor cells for up to 5 days ( Figure 2 E).

[0083] After the prepared BSA-FITC binds to tumor cells, if it can bind to the FITC-synNotch receptor of MANAGE CAR-T, it can stimulate CAR-T to express CAR targeting the natural antigen CD19 and the CAR tag protein Thy1.1. Figure 3A). MANAGE CAR-T cells, B16F10 tumor cells, and BSA-FITC cells linked with different numbers of FITCs were co-incubated. After 24 hours, the expression level of Thy1.1 in CAR-T cells was detected by anti-Thy1.1 flow cytometry staining. The expression level of Thy1.1 in CAR-T cells gradually increased with the increase of the number of FITCs linked to BSA. Figure 3 (B) When one FITC is attached to each BSA, the Thy1.1 positivity rate of CAR-T is 87%, which is the most cost-effective. Therefore, subsequent experiments will use BSA-FITC with one FITC attached.

[0084] Different concentrations of BSA-FITC were co-incubated with MANAGE CAR-T cells and B16F10 tumor cells. After 24 hours, the expression efficiency of Thy1.1 in MANAGE CAR-T cells was detected by flow cytometry. Thy1.1 expression in CAR-T cells was positively correlated with BSA-FITC concentration. Figure 3 C). Furthermore, we characterized the kinetics of CAR expression. Thy1.1 expression began after 1 hour of co-incubation with BSA-FITC and peaked at 24 hours, maintaining that level. After replacing the culture medium with fresh medium at 36 hours and removing BSA-FITC, the Thy1.1 positivity rate of CAR-T cells gradually decreased, dropping to 18.6% at 96 hours. Figure 3 D).

[0085] FITC-synNotch recognizes the artificial antigen FITC, inducing CAR expression. CAR, upon recognizing natural tumor antigens, can activate CAR-T cells and upregulate the expression of the T cell activation marker CD69. Figure 3 E). MANAGE CAR-T cells and B16F10 tumor cells (hCD19 positive or negative) were co-incubated. In the absence of BSA-FITC (G1 and G2 groups), the artificial antigen FITC did not bind to the FITC-synNotch receptor, thus preventing the expression of CD19 CAR and Thy1.1, thereby failing to activate CAR-T cells and increase their CD69 expression. Figure 3 F, G). In the presence of BSA-FITC (G3 and G4 groups), MANAGE CAR-T cells expressed Thy1.1, indicating that BSA-FITC binding to tumor cells can induce CD19 CAR expression. However, when the tumor cells are B16-F10 cells, due to the lack of the antigen hCD19, they cannot bind to CD19 CAR, thus failing to activate T cells and upregulate their CD69 expression. Figure 3F, G). When the tumor cells were B16-F10-hCD19 (G4 group), MANAGE CAR-T cells highly expressed CD69, indicating that MANAGE CAR-T cells only exhibited activation characteristics when both FITC and hCD19 targets were co-expressed on the surface of tumor cells. Figure 3 F, G).

[0086] (2) We also use NHS-PEG 2000 -FITC-modified PD-L1 antibody to construct aPD-L1-FITC. aPD-L1 with NHS-PEG. 2000 When the FITC molar ratio is 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:6, 1:8, and 1:10, the number of artificial antigen FITCs linked to aPD-L1 is 0.11, 0.19, 0.35, 0.8, 1.2, 1.8, 2.5, 3, and 3.8, respectively. Figure 4 A). Different concentrations of aPD-L1-FITC (a mixture of 50% each of tumor-targeting artificial antigens with FITC molecules linked to 0.8 and 1.2 molecules) were co-incubated with 100,000 MANAGE CAR-T cells and 100,000 B16F10 tumor cells. After 24 hours, the expression efficiency of Thy1.1 in MANAGE CAR-T cells was detected by flow cytometry. The expression of Thy1.1 in CAR-T cells was positively correlated with the concentration of aPD-L1-FITC, and the positive rate of Thy1.1 at 4 nM reached ~90%. Figure 4 B). The activation kinetics of aPD-L1-FITC on MANAGE CAR-T are similar to those of BSA-FITC. Figure 4 C).

[0087] Example 3: In vitro killing of target cells by MANAGE CAR-T

[0088] Ten thousand tumor cells (B16F10-luc or B16F10-hCD19-luc) were seeded in 96-well plates. Mouse spleen cells were also seeded in 96-well plates. MANAGE CAR-T cells (effect cells) and tumor cells (target cells) were co-incubated according to different effector-to-target ratios (E:T). The specific groups were as follows: G1: B16F10; G2: B16F10-hCD19; G3: B16F10 + BSA-FITC; G4: B16F10-hCD19 + BSA-FITC. The effector-to-target ratios were E:T = 20:1, 10:1, 5:1, and 2.5:1, with four replicates. The cells were cultured in an incubator for 48 hours to induce cell death. 100 μL of Bright-Lite was added to the cells. TM After the test reagents are used, the absorbance of the cells is measured using an ELISA reader.

[0089] Here are the results:

[0090] (1) MANAGE CAR-T only killed tumor cells in the presence of BSA-FITC and when tumor cells expressed hCD19 (G4 group), and showed killing effect at different effector-to-target ratios (E:T = 20:1, 10:1, 5:1 or 2.5:1). Figure 3 H). Cell culture supernatant was collected, and the levels of IFN-γ and IL-2 were detected by ELISA. The secretion levels of IFN-γ and IL-2 in the G4 group were significantly higher than those in other groups. Figure 3 I). The above results indicate that MANAGE CAR-T cells can only be activated and specifically exert anti-tumor effects when both FITC and hCD19 are present simultaneously. Next, we verified the killing effect of MANAGE CAR-T cells on tumor cells expressing different natural tumor antigens, HER2. We replaced the hCD19 CAR sequence with the HER2-targeting aHER2 CAR and used 4T1 and 4T1-HER2 breast cancer cell lines. Only when BSA-FITC and 4T1-HER2 cells were present simultaneously (G4) could MANAGE CAR-T cells effectively kill 4T1-HER2 cells at different effector-target ratios. Figure 3 J), and significantly promotes the secretion of cytokines IFN-γ and IL-2 (J). Figure 3 K).

[0091] (2) MANAGE CAR-T only killed tumor cells in the presence of aPD-L1-FITC and in breast cancer tumor cells (4T1) expressing hCD19 (G4 group). This effect was observed at different effector-to-target ratios (E:T = 20:1, 10:1, 5:1, or 2.5:1). Figure 4 D). Cell culture supernatant was collected, and the levels of IFN-γ and IL-2 were detected by ELISA. The secretion levels of IFN-γ and IL-2 in group G4 were significantly higher than those in other groups. Figure 4 E). Next, we verified the killing effect of MANAGE CAR-T cells on tumor cells expressing different natural tumor antigens HER2. We replaced the hCD19 CAR sequence with aHER2-targeting aHER2 CAR. Figure 4 F), and utilizes 4T1 and 4T1-HER2 tumor cells. Tumor-targeted artificial antigens can activate MANAGE CAR-T cells, thereby specifically killing tumor cells expressing HER2. Figure 4 G), and increases the secretion of IFN-γ and IL-2 (G), and enhances the secretion of IFN-γ and IL-2. Figure 4 H).

[0092] Example 4: In vivo biodistribution of artificial antigens

[0093] The distribution of artificial targets in various organs after tail vein injection was verified in a C57BL / 6 mouse model using a small animal imaging system. Since FITC fluorescence is easily affected by background interference, Cy5 was used instead of FITC for in vivo distribution studies. C57BL / 6 mice were subcutaneously injected with 1×10⁻⁶ cytidine after shaving their backs. 6 One B16F10 tumor cell, until the tumor cell size reaches 100mm. 3 C57BL / 6 mice were randomly divided into three groups: G1 (PBS group), G2 (aPD-L1-Cy5 group), and G3 (BSA-Cy5 group). Each mouse was intravenously injected with either 40 μg of BSA-Cy5 or 50 μg of aPD-L1-Cy5 (the amount of quantified Cy5 was the same). Twenty-four hours after injection, the mice were euthanized with CO2, and their hearts, livers, spleens, lungs, kidneys, tumors, and lymph nodes were dissected for small animal imaging (excitation wavelength 620 nm, emission wavelength 680 nm).

[0094] The results showed that BSA-Cy5 and aPD-L1-Cy5 accounted for 32.4% and 17.5% of the total fluorescence signal in the tumor, respectively, and BSA-Cy5 exhibited superior tumor targeting compared to aPD-L1-Cy5. Figure 5 A, B).

[0095] Example 5: Activation of CAR-T cells in various organs.

[0096] The in vivo distribution of MANAGE CAR-T cells was validated in a C57BL / 6 mouse model using a small animal imaging system. We replaced Thy1.1 protein with luciferase protein for in vivo tracking of MANAGE-T cells. Figure 5 C). C57BL / 6 mice were shaved on the back and then subcutaneously injected with 1×10⁻⁶ mice. 6 One B16F10 tumor cell, until the tumor cell size reaches 100mm. 3 C57BL / 6 mice were randomly divided into four groups: G1 (PBS group); G2 (MANAGE CAR-T group); G3 (MANAGE CAR-T + BSA-FITC group); and G4 (MANAGE CAR-T + aPD-L1-FITC group). On day -1, each mouse was intraperitoneally injected with 2 mg of cyclophosphamide for lymphocyte depletion. Simultaneously, the G3 group received a tail vein injection of BSA-FITC, and the G4 group received a tail vein injection of aPD-L1-FITC. On day 0, MANAGE CAR-T cells were collected, and mice in groups G2-G4 were injected with 5 × 10⁵ cells via tail vein injection. 6 MANAGE CAR-T cells. On days 1, 2, 3, 4, and 5 post-injection, 100 μL of 100 mg / mL fluorescein potassium solution was injected intraperitoneally, and the bioluminescence intensity was monitored using an IVIS imaging system. Figure 5 D).

[0097] Here are the results:

[0098] In group G2, MANAGE CAR-T cells were not activated, and only background biofluorescence readings were observed. Groups G3 and G4 showed similar trends: biofluorescence was detectable at the tumor site on day 1, with fluorescence intensity continuously increasing, peaking after day 4, and then decreasing. Figure 5 E, F). These results show that both BSA-FITC and aPD-L1-FITC can specifically activate MANAGE CAR-T cells at the tumor site. However, BSA-FITC showed significantly higher activation of MANAGE CAR-T cells at the tumor site than aPD-L1, inducing more biofluorescence at the tumor site ( Figure 5 E, F). At 48 hours, the animals were dissected, and the biofluorescence of major organs such as the heart, liver, spleen, lungs, kidneys, lymph nodes, and tumor sites was examined. The results showed that only the tumor sites exhibited biofluorescence, while the major organs showed only background biofluorescence, indicating that MANAGE CAR-T was specifically activated only at the tumor site, and that BSA-FITC significantly enhanced the activation effect of MANAGE CAR-T at the tumor site compared to aPD-L1-FITC. Figure 5 G, H).

[0099] Example 6: In vivo immunological effects of MANAGE CAR-T

[0100] A bilateral melanoma model was established by subcutaneously injecting 1×10⁻⁶ cells into the back of C57BL / 6 mice. 6 One B16F10 tumor cell (100 μL), or seeded with 1 × 10 6 Count B16F10-hCD19 cells. Observe the tumor formation in mice daily. Continue until the tumors on both sides of the mice reach 60 mm in size. 3 Mice were randomly divided into the following groups: G1: B16F10-hCD19 + PBS group; G2: B16F10-hCD19 + CAR-T group; G3: B16F10-hCD19 + MANAGE CAR-T group; G4: B16F10 + MANAGE CAR-T + BSA-FITC group; G5: B16F10-hCD19 + MANAGE CAR-T + BSA-FITC group; G6: B16F10-hCD19 + MANAGE CAR-T + aPD-L1-FITC group. Figure 6A). On day -1, each mouse was intraperitoneally injected with 2 mg of cyclophosphamide for lymphocyte clearance. Simultaneously, groups G4 and G5 received tail vein injection of BSA-FITC, and group G6 received tail vein injection of aPD-L1-FITC. MANAGECAR-T cells and CAR-T cells were collected, and 5 × 10⁶ cells were injected into the tail vein of mice. 6 Mice were treated with either MANAGE CAR-T cells or CAR-T cells. BSA-FITC or aPD-L1-FITC was administered every 3 days for a total of three injections. Nine days after cell therapy, mice were euthanized. Tumors, spleens, and lymphocytes were extracted from the mice and subjected to flow cytometry staining. Counting beads were added to each well, and staining was performed using anti-Thy1.1-FITC, anti-CD8-APC / cy7, anti-IFN-γ-PE, and anti-TNF-α-PE / cy7 antibodies. Flow cytometry was used to detect various MANAGE CAR-T parameters.

[0101] The results showed that the artificial antigen significantly proliferated intratumoral MANAGE CAR-T cells and enhanced their function:

[0102] After grinding and collecting the tumors, they were stained with fluorescent antibodies and analyzed by flow cytometry. Group G3 lacked the artificial antigen FITC signal. (Thy1.1) + The cell proportion was similar to that of the G1 group, indicating that MANAGE CAR-T cells were not activated. Figure 6 B, C). In groups G4, G5, and G6, which possess the artificial antigen FITC, compared to group G4 (without natural tumor antigen), Thy1.1 in groups G5 and G6 + Cells within the tumor CD45 + The proportion of Thy1.1 in cells increased twofold, per milligram of tumor tissue. + The number of T cells increased tenfold, indicating that artificial-natural antigen binding effectively activates and promotes the expansion of MANAGE CAR-T cells, and that the number of T cells was greater than that of CAR-T cells targeting natural antigens (G2 group). Figure 6 B, C). We also performed flow cytometry analysis on the spleen and inguinal lymph nodes of mice in each treatment group. Unlike the G2 group, the MANAGE CAR-T cells in the G5 and G6 groups did not express Thy1.1 (i.e., CAR) in non-tumor sites such as the lymph nodes and spleen. Figure 6 D and E) can reduce OTOT.

[0103] Furthermore, the total proportion of IFN-γ and TNF-α positive cells in groups G5 and G6 was significantly higher than that in groups G2 and G4, indicating that the artificial-natural antigen combination can enhance the anti-tumor efficacy of MANAGE CAR-T. Figure 6F, G). Compared with G2 and G4 groups, the expression levels of PD-1 and LAG-3 in MANAGE CAR-T cells and unactivated MANAGE CAR-T cells in groups G5 and G6 decreased by 2.2-fold and 1.7-fold, respectively, indicating that the artificial-natural antigen combination can maintain a low depletion level of MANAGE CAR-T cells. Figure 6 H).

[0104] Example 7: Evaluation of the anti-tumor effect of CAR-T cells in MANAGES

[0105] A melanoma model was established by subcutaneously injecting 1×10⁻⁶ cells into the back of C57BL / 6 mice. 6 One B16F10-hCD19 tumor cell (100 μL) was added, and the mouse tumor was allowed to grow to approximately 60 mm. 3 Mice were randomly divided into the following groups: G1: PBS group; G2: MANAGECAR-T group; G3: MANAGECAR-T+BSA-FITC group; G4: MANAGECAR-T+aPD-L1-FITC group. On day -1, each mouse was intraperitoneally injected with 2 mg cyclophosphamide for lymphocyte depletion. Group G3 received a tail vein injection of BSA-FITC, and group G4 received a tail vein injection of aPD-L1-FITC. On day 0, the prepared MANAGECAR-T cells were collected, and mice in groups G2, G3, and G4 were injected with 5 × 10⁵ cells via tail vein injection. 6 The mice were treated with MANAGE CAR-T cells. BSA-FITC or aPD-L1-FITC was administered every 3 days. Tumor volume and weight were measured every two days, and mouse survival time was recorded.

[0106] A bilateral tumor model was established by subcutaneously injecting 1×10⁻⁶ cells into the left back of C57BL / 6 mice. 6 100 μL of B16F10 tumor cells were subcutaneously injected into the right side of the back. 6 Count B16F10-hCD19 cells. Observe the tumor formation in mice daily. Continue until the tumors on both sides of the mouse reach 60 mm in size. 3 Mice were randomly divided into the following groups: G1: PBS group; G2: MANAGE CAR-T group; G3: MANAGE CAR-T + BSA-FITC group; G4: MANAGE CAR-T + aPD-L1-FITC group. On day -1, each mouse was intraperitoneally injected with 2 mg cyclophosphamide for lymphocyte depletion. Group G3 received a tail vein injection of BSA-FITC, and group G4 received a tail vein injection of aPD-L1-FITC. On day 0, the prepared MANAGE CAR-T cells were collected, and mice in groups G2, G3, and G4 were injected with 5 × 10⁵ cells via tail vein injection. 6The mice were treated with MANAGE CAR-T cells. BSA-FITC or aPD-L1-FITC was administered every 3 days, and tumor size was measured every 2 days.

[0107] In an orthotopic breast cancer tumor model, 1×10⁻⁶ mc cells were injected into the mammary glands of BALB / c mice. 6 One 4T1-hCD19 or 1×10 6 4 T1 tumor cells (100 μL) were added to the mouse tumor until it reached a size of 60 mm. 3 4T1-hCD19 and 4T1 tumor-bearing mice were grouped as follows: G1: 4T1-HER-2+PBS group;

[0108] G2: 4T1+MANAGE CAR-T+BSA-FITC group; G3: 4T1+MANAGE CAR-T+aPD-L1-FITC group; G4: 4T1-HER-2+MANAGE CAR-T group; G5: 4T1-HER-2+MANAGE CAR-T+BSA-FITC group; G6: 4T1-HER-2+MANAGE CAR-T+aPD-L1-FITC group. On day -1, each mouse was intraperitoneally injected with 2 mg of cyclophosphamide for lymphocyte depletion. G2 and G5 groups received tail vein injection of BSA-FITC, while G3 and G6 groups received tail vein injection of aPD-L1-FITC. On day 0, the prepared MANAGE CAR-T cells were collected, and mice in G2-G6 groups received 5 × 10⁵ cells via tail vein injection. 6 The mice were treated with MANAGE CAR-T cells. BSA-FITC or aPD-L1-FITC was administered every 3 days, and tumor size was measured every 2 days. If any mouse's tumor reached 1500 mm², the tumor was treated accordingly. 3 All mice were euthanized, and their lungs were dissected to count the number of tumor nodules.

[0109] Here are the results:

[0110] Tumor inhibition experiments were conducted in B16F10-hCD19 tumor-bearing mice. Figure 7 A). Compared to group G1, infusion of MANAGECAR-T cells alone did not inhibit tumor growth. In groups G3 and G4, in the presence of artificial antigens, MANAGECAR-T cells significantly delayed tumor growth; by day 12, the tumor volume was less than 10% of that in group G2. Figure 7 B). All mice in group G1 died on day 12, all mice in group G2 died on day 13, and at least 60% of mice in groups G3 and G4 were still alive on day 60. Figure 7 C). Furthermore, there was no significant difference in body weight among the mice in each treatment group, indicating that the artificial antigen had no significant toxicity to mice. Figure 7 D).

[0111] Bilateral tumor assays were performed on the left side of the mouse back using tumor-bearing B16F10 cells (without natural antigens) and on the right side using tumor-bearing B16F10-hCD19 cells (without natural antigens). Figure 7 E). The tumor growth trend on the left side was the same in all groups of mice. Figure 7 F). The right-side tumors in G2 group mice grew similarly to those in G1 group, while tumor growth in G3 and G4 group mice was significantly inhibited. Figure 7 F).

[0112] In a mouse mammary gland orthotopic 4T1-hCD19 or 4T1 tumor model, mice were divided into 6 groups ( Figure 7 G), 4T1 model mice were divided into two groups, injected with MANAGE CAR-T and BSA-FITC (G2 group, lacking natural tumor antigen) and MANAGE CAR-T and aPD-L1-FITC (G3 group, lacking natural tumor antigen), respectively; 4T1-hCD19 model mice were divided into four groups, injected with PBS (G1 group), MANAGE CAR-T (G4 group, lacking artificial antigen), MANAGE CAR-T and BSA-FITC (G5 group), and MANAGE CAR-T and aPD-L1-FITC (G6 group), respectively. Groups G2-G4 lacked either natural or artificial antigens and had poor anti-tumor effects. Group G3, due to the therapeutic effect of aPD-L1-FITC itself, showed the slowest tumor growth among these groups. Figure 7 G, H). Both G5 and G6 groups showed complete dual antigen signaling, and MANAGE CAR-T cells significantly delayed tumor growth. At day 27, the mean tumor volume was 24.9% and 18.6% of that in the G1 group, respectively. Figure 7 H). All mice were euthanized on day 27, and their lungs were dissected and counted for metastatic tumor nodules. Group G4 mice had 9 tumor nodules in their lungs, while Group G5 mice had 1 tumor nodule in their lungs. Figure 7 I) aPD-L1-FITC is significantly superior to BSA-FITC in inhibiting lung metastasis.

[0113] In summary, the combined effects of G5 group (BSA-FITC) and G6 group (aPD-L1-FITC) in inhibiting in situ tumors were comparable, while aPD-L1-FITC was significantly better than BSA-FITC in inhibiting tumor metastasis.

[0114] Example 8: Safety Evaluation of MANAGE CAR-T

[0115] To assess OTOT toxicity, C57BL / 6 mice were subcutaneously injected with 1×10⁻⁶ oz. 6100 μL of B16F10-hCD19 tumor cells were collected and the mouse tumors were allowed to grow to 100 mm in size. 3 Mice were randomly divided into the following groups: G1 group: PBS; G2 group: MANAGECAR-T; G3 group: CAR-T. On day -1, each mouse was intraperitoneally injected with 2 mg cyclophosphamide to deplete lymphocytes, and then injected via tail vein hyperbaric injection with 20 μg (1 mL) of plasmid encoding the hCD19 antigen. On day 0, mice in group G2 were injected via tail vein with 5 × 10 6 5 × 10 MANAGECAR-T cells were injected into the tail vein of G3 group mice. 6 CAR-T cells were collected. Mice were weighed daily, and weight changes were recorded. Blood AST and ALT levels were measured every two days. On day 4, all mice were euthanized, and their livers were dissected for H&E staining.

[0116] To assess cytokine storm, subcutaneous injection of 1×10⁻⁶ cytokine gas was performed on the back of C57BL / 6 mice. 6 100 μL of B16F10-hCD19 tumor cells were collected and the mouse tumors were allowed to grow to 200 mm in size. 3 Mice were randomly assigned to the following groups: G1 group: PBS; G2 group: CAR-T; G3 group: BSA-FITC; G4 group: MANAGE CAR-T; G5 group: MANAGE CAR-T + BSA-FITC; G6 group: MANAGE CAR-T + interrupted BSA-FITC. On day -1, each mouse was intraperitoneally injected with 2 mg of cyclophosphamide for lymphocyte depletion, and mice in groups G3, G5, and G6 were injected via tail vein with BSA-FITC. On day 0, mice in group G2 were injected via tail vein with 5 × 10⁵ ppm of cyclophosphamide. 6 Mice in the G4, G5, and G6 groups were treated with CAR-T cells, receiving a tail vein injection of 5 × 10⁸ cells. 6 The mice were treated with MANAGE CAR-T cells. Groups G3 and G5 received a booster injection of BSA-FITC every 3 days, while group G6 received a booster injection every 6 days. Tumor volume and weight were measured every two days, mouse survival time was recorded, and changes in blood IL-6 and IFN-γ levels, as well as routine blood tests, were measured.

[0117] The results show:

[0118] (1) MANAGE CAR-T significantly reduces OTOT toxicity in vivo:

[0119] Bioluminescence in mice was detected by IVIS at different time points after high-pressure injection of a plasmid encoding luciferase via the tail vein. The results showed that the mouse liver exhibited significant bioluminescence, which could last for more than 5 days. Figure 8A) demonstrates that tail vein injection of the antigen plasmid can induce hepatocyte expression of the plasmid-encoded protein. Using this system, we established a subcutaneous B16F10-hCD19 melanoma model. Figure 8 B) Simultaneously, tumor-bearing mice were injected intravenously via high-pressure injection of a plasmid encoding hCD19 into the tail vein, inducing the expression of the tumor antigen hCD19 in the livers of these mice. Subsequently, the tumor-bearing mice were divided into three groups, receiving intravenous injections of PBS (G1 group), MANAGECAR-T cells (G2 group), and CAR-T cells (G3 group), respectively. Peripheral blood was collected every two days to measure AST and ALT, and mouse body weight was measured daily. Compared to the PBS group, the G3 group showed a 73% and 146% increase in AST and ALT after aCD19 CAR-T cell treatment, demonstrating significant OTOT toxicity. Figure 8 C). In the G2 group, AST and ALT increased by only 19% and 23% respectively after MANAGE CAR-T cell therapy, indicating that the combination of MANAGE CAR-T and tumor-targeted artificial antigens can significantly reduce OTOT. After treatment, the body weight of mice in the G2 group remained at the same level as the normal mouse group, while the body weight of mice in the G3 treatment group decreased rapidly, with a decrease of 11% by day 4. Figure 8 D). Analysis of liver H&E staining results on day four showed that hepatocytes in group G2 had regular morphology, abundant cytoplasm, and round, centrally located nuclei, while liver tissue in group G3 exhibited disordered structure, hepatocyte swelling and degeneration, and some hepatocyte nuclei showed pyknosis, fragmentation, or dissolution, suggesting the presence of liver damage. Figure 8 E).

[0120] (2) Regulating the injection cycle of artificial antigens can significantly reduce CAR-T-induced cytokine storm.

[0121] In a subcutaneous model of B16F10-hCD19 melanoma ( Figure 9A) Tumor-bearing mice were divided into six groups: intravenously injected with PBS (G1 group), 15 million CAR-T cells (G2 group), BSA-FITC (G3 group), 15 million MANAGE CAR-T cells (G4 group), 15 million MANAGE CAR-T cells and BSA-FITC (G5 group), and 15 million MANAGE CAR-T cells and BSA-FITC (G6 group). Groups G3 and G5 received BSA-FITC every 3 days, while group G6 received BSA-FITC every 6 days. This experiment used large tumors and three times the dose of CAR-T and MANAGE CAR-T cells to highlight the toxic side effects caused by CAR-T cell killing and overactivation, such as cytokine syndrome, during treatment. Group G5 received tumor-targeting artificial antigens every 3 days, providing a continuous supply of artificial antigens and mimicking the non-modular synNotch CAR-T. Group G6, by regulating the duration of the artificial antigen's presence, highlighted the advantage of the in vivo controllable on / off nature of the modular synNotch CAR-T.

[0122] In terms of efficacy, groups G2, G5, and G6 all significantly inhibited tumor growth, with group G6 showing the best efficacy. Figure 9 B). After administration, mice in groups G2, G5, and G6 experienced a sharp drop in body weight, suggesting a cytokine storm. The body weight of group G2 recovered slowly over time, group G5 returned to normal weight on day 16, and group G6 returned to normal weight by day 12 after administration was discontinued. Figure 9 C). On day 2 after drug administration, the levels of pro-inflammatory cytokines IL-6 and IFN-γ in the blood of G2, G5, and G6 groups rapidly increased. In G2 mice, IL-6 and IFN-γ returned to normal levels by day 22; in G5 mice, they returned to normal levels by day 14; and in G6 mice, they returned to normal levels by day 10, significantly better than the G5 group. Figure 9 D, E). Blood routine analysis showed that after drug administration, the number of white blood cells, red blood cells, platelets, and hemoglobin in the blood of mice decreased rapidly, and the recovery of these cells and molecules in the G6 group mice was significantly faster than that in the G5 group ( Figure 9 (FI). These data demonstrate that regulating the injection cycle of artificial antigens can alleviate CAR-T-induced cytokine storms and reduce toxic side effects, and also significantly reduce toxic side effects compared to the G5 group, which mimics non-modular synNotch CAR-T.

[0123] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A medical product with dual gating of artificial antigens and natural tumor antigens, characterized in that, include: A first formulation contains a tumor-targeting artificial antigen, wherein the tumor-targeting artificial antigen includes a protein that modifies the artificial antigen or an antibody that modifies the artificial antigen; wherein the artificial antigen is a marker not expressed on the cell surface, and the antibody or protein can target tumor cells; The second formulation contains CAR-T cells, which contain a first gene expression cassette and a second gene expression cassette. The first gene expression cassette contains a coding sequence for an extracellular recognition region, a coding sequence for a transmembrane core region, and a coding sequence for an intracellular effector region. The extracellular recognition region contains a single-chain antibody sequence of the artificial antigen, the transmembrane core region contains a Notch core sequence, and the intracellular effector region contains a transcription factor sequence. The second gene expression cassette contains an upstream activation sequence that specifically binds to the transcription factor, and a chimeric antigen receptor located after the upstream activation sequence. The chimeric antigen receptor targets a natural antigen, which is a marker highly expressed on the surface of tumor cells.

2. The medical product according to claim 1, characterized in that, It must contain at least one of the following characteristics: (1) The ratio of artificial antigens to antibodies or proteins connected to the link is (0.1-10):1; (2) The artificial antigen includes one or more of fluorescent molecules, biotin, and artificial peptide chains; (3) The proteins include serum albumin and / or transferrin; (4) The antibody specifically recognizes tumor cell surface markers.

3. The medical product according to claim 2, characterized in that, It must contain at least one of the following characteristics: (1) Fluorescent molecules include one or more of FITC, Cy3, Cy5, and Cy7; (2) Tumor cell surface markers include one or more of the following: programmed death ligand 1, CD276, G protein-coupled receptor 65, V-set and protein 4 containing immunoglobulin domain, γ-aminobutyric acid metabolite, discoid domain receptor 1, ADP-ribosyltransferase 1, basal cell adhesion molecule, protein tyrosine phosphatase non-receptor type 2, and chemokine receptor 8. (4) The single-chain antibody of the artificial antigen includes αFITC scFv; the coding sequence of αFITC scFv is shown in SEQ ID NO.

1.

4. The medical product according to claim 1, characterized in that, It must contain at least one of the following characteristics: (1) Transcription factors include one or more of Gal4-VP64, LexA-VP64, Gal4-KRAB, and ZFHD1-VP64; (2) The natural antigens include one or more of CD19, HER2, Claudin 18.2, EGFR, FAP, and TROP2; (3) The Notch core sequence is shown in SEQ ID NO.2; (4) The chimeric antigen receptor contains a single-chain antibody encoding sequence of a natural antigen; (5) The chimeric antigen receptor contains a hinge region coding sequence, a transmembrane region coding sequence and an intracellular signal region coding sequence.

5. A pharmaceutical composition, characterized in that, contain: Tumor-targeting artificial antigens, wherein the tumor-targeting artificial antigens include proteins or antibodies that modify artificial antigens; the artificial antigens are markers not expressed on the cell surface, and the proteins or antibodies target target cells; An immune cell containing a first gene expression cassette and a second gene expression cassette; the first gene expression cassette contains a coding sequence for an extracellular recognition region, a coding sequence for a transmembrane core region, and a coding sequence for an intracellular effector region, wherein the extracellular recognition region contains a single-chain antibody sequence of the artificial antigen, the transmembrane core region contains a Notch core sequence, and the intracellular effector region contains a transcription factor sequence; the second gene expression cassette contains an upstream activation sequence that specifically binds to the transcription factor, and a chimeric antigen receptor located after the upstream activation sequence, wherein the chimeric antigen receptor targets a natural antigen, and the natural antigen is a marker highly expressed on the surface of the target cell.

6. The pharmaceutical composition according to claim 5, characterized in that, It must contain at least one of the following characteristics: (1) The target cells include tumor cells; (2) The immune cells include T cells and / or NK cells.

7. The use of the medical product according to any one of claims 1-4 or the pharmaceutical composition according to claim 5 or 6 in the preparation of a pharmaceutical product, characterized in that, The pharmaceutical product is used in any of the following: (1) Used to kill tumor cells in vivo or in vitro; (2) Used in the preparation of antitumor drugs; (3) Used to increase the range of tumor cells that can be killed and / or to reduce immune escape; (4) Used to reduce cytokine syndrome; (5) Used to reduce non-tumor-targeted toxicity during tumor treatment.

8. An antitumor drug, characterized in that, This includes the medical product according to any one of claims 1-4 or the pharmaceutical composition according to claim 5 or 6.

9. The antitumor drug according to claim 8, characterized in that, The antitumor drug is used to treat any solid tumor.

10. The antitumor drug according to claim 9, characterized in that, The solid tumors include one or more of the following: melanoma, ovarian cancer, cervical cancer, lung cancer, breast cancer, head and neck cancer, liver cancer, intestinal cancer, colon cancer, kidney cancer, stomach cancer, glioma, bladder cancer, and pancreatic cancer.

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