Chimeric antigen receptor suitable for modulating car-t signaling

Abstract

Provided is a chimeric antigen receptor suitable for modulating CAR-T signaling. Provided is an intracellular domain for constructing a chimeric antigen receptor, said intracellular domain comprising a costimulatory signaling region and a CD3ζ intracellular region which are connected in sequence, the CD3ζ intracellular region being connected to a PD1-ITSM motif. Also provided is the use of the intracellular domain in the preparation of a chimeric antigen receptor. Compared with the prior art, the chimeric antigen receptor (BBiz-CAR) has a lower signal, which can alleviate the problem that CAR-T cells with excessively strong background signals are prone to exhaustion, and can also alleviate the activation-induced death (AICD) generated due to the excessive activation of CAR-T cells when attacking tumor cells, thereby endowing the CAR-T cell with better persistence; and the BBzi-CAR has a higher signal, which can help CAR-T cells to respond to low-antigen tumors when encountering with heterogeneous tumors or tumors achieving immune escapes via downregulation of surface antigen expression, thereby improving effector functions.

Description

A chimeric antigen receptor suitable for regulating CAR-T signaling Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and in particular to a chimeric antigen receptor suitable for regulating CAR-T signaling. Background Technology

[0002] CAR-T therapy, a novel cancer immunotherapy, has garnered significant attention since its inception in the late 20th century. After isolating T cells from a patient's peripheral blood, genetic engineering is used to express a chimeric antigen receptor (CAR) on the T cell surface, representing a representative of adoptive cell therapy. CAR design primarily comprises four regions: an antigen recognition region, a hinge region, a transmembrane region, and an intracellular signal transduction region. In the iterative development of CAR-T, there have been antibody optimization designs based on target changes, as well as optimizations of the hinge region from a biophysical perspective. Generally, the generational classification of CAR-T is based on the composition of its intracellular signal transduction domain. First-generation CARs only contain CD3ζ or FcRγ intracellular segments, with CD3ζ being superior due to its better activation of signals under weak antigen stimulation, but overall response levels and cell proliferation capacity are relatively poor. Second-generation CARs introduce co-stimulatory molecules, such as CD28 and 4-1BB, to enhance the maintenance and proliferation capacity of CAR-T cells, and products such as Kymriah and Yescarta have been approved by the FDA. Third-generation CARs tandem with co-stimulatory molecules based on second-generation CARs in order to obtain stronger cell proliferation and killing capabilities. Fourth-generation CARs increase the synthesis and secretion of cytokines such as IL-2 by CAR-T cells through transcription factors such as NFAT. More novel CARs, such as those that add membrane receptors like IL-2R to induce activated CAR-T cells, are being continuously reported.

[0003] Currently, several CAR-T therapies have been approved for marketing by the FDA or NMPA. They target CD19 and are mainly used for acute lymphoblastic leukemia, non-Hodgkin's lymphoma, and mantle cell lymphoma; or they target BCMA and are mainly used for multiple myeloma.

[0004] Although CAR-T therapy for hematological malignancies currently boasts good complete response rates, approximately 50% of patients still experience relapse in multiple clinical trials and follow-ups. Current clinical analyses of hematological malignancies categorize relapse into two types: antigen-positive / T-cell-dependent relapse, primarily due to poor T-cell persistence and memory, closely related to scFv, co-stimulatory domain selection, and the patient's own T-cell status; and relapse based on target antigen changes. Clinical studies have found that CAR-T cell therapy exerts immune selection pressure on target cells (i.e., B cells), which tend to modify or lose their surface CD19 to evade CAR-T cell recognition and killing. Research analyzing clinical samples has revealed a significant downregulation of CD19 on the surface of tumor cells before and after treatment. Further analysis of patient prognosis and relapse showed a significant negative correlation between the degree of antigen downregulation during treatment and prognosis, indicating that antigen escape is indeed one of the main reasons why, despite the high response rate of CAR-T therapy for hematological malignancies, it is also accompanied by a high relapse rate. This suggests that we need CAR-T cells that are more sensitive to low antigens to respond to changing tumor cells, thereby reducing the probability of recurrence.

[0005] Aside from its relatively successful clinical application in hematologic malignancies, the use of CAR-T therapy in solid tumors is currently relatively limited. Besides the fact that solid tumor antigens are less specific than those in hematologic malignancies, the physical barrier formed by solid tumors affects T cell infiltration, and the more immunosuppressive microenvironment within them makes CAR-T cells prone to exhaustion. Some reports suggest that continuous antigen stimulation causes T cells to express Fas and FasL, entering an apoptosis-sensitive state. Furthermore, in the immunosuppressive microenvironment, pathways such as PD-1 / PD-L1 can downregulate the anti-apoptotic molecule Bcl-XL and upregulate the pro-apoptotic molecule Bim, leading to T cell AICD and thus affecting its persistence in solid tumor treatment. The different efflux of metabolic products between solid tumor cells and hematologic malignancies also subject T cells to different survival and functional pressures. In addition, excessively strong binding between scFv and antigens can over-activate CAR-T cells, thus triggering the braking mechanisms of T cell function and proliferation. Currently widely accepted scFvs targeting solid tumor antigens differ from FMC63 targeting CD19; they appear to have stronger charge aggregation distribution, leading to strong antigen-independent self-activation. When CAR-T cells enter the tumor microenvironment, they enter a state of exhaustion due to their prior over-activation. This suggests that a more moderate CAR design might help improve its durability in solid tumor treatment.

[0006] Whether it's insufficient CAR-T activation due to antigen escape or the excessive self-activation often seen in solid tumor CAR-T cells, it can all be summarized as CAR-T cells not reaching their optimal activation level. Currently, most optimizations of CAR-T cell therapies are primarily based on receptors or intracellular signal transduction molecules, transcription factors, and metabolism-related genes, which significantly increases the complexity of CAR-T cell preparation. Therefore, a new CAR molecule optimization strategy is needed to controllably regulate CAR-T cell signaling and function. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a chimeric antigen receptor suitable for regulating CAR-T signaling and its uses, in order to solve the problems in the prior art.

[0008] To achieve the above and other related objectives, the present invention provides an intracellular domain for constructing a chimeric antigen receptor, comprising a co-stimulatory signal transduction region and a CD3ζ intracellular region connected in sequence, wherein the CD3ζ intracellular region is connected to a PD1-ITSM motif.

[0009] A second aspect of the present invention provides the use of the aforementioned intracellular domain in the preparation of chimeric antigen receptors.

[0010] This invention provides a BBz chimeric antigen receptor suitable for regulating CAR-T signaling, comprising:

[0011] The extracellular domain, transmembrane domain, and intracellular domain are connected sequentially.

[0012] The extracellular domain includes an antigen recognition region and a hinge region;

[0013] The intracellular domains include the aforementioned intracellular regions.

[0014] A third aspect of the present invention provides a polynucleotide sequence selected from:

[0015] (1) A multinucleotide sequence encoding the aforementioned intracellular domain or the aforementioned chimeric antigen receptor; and / or

[0016] The complementary sequence of the polynucleotide sequence described in (2)(1).

[0017] A fourth aspect of the present invention provides a nucleic acid construct containing the aforementioned polynucleotide sequence.

[0018] The fifth aspect of the present invention provides a lentiviral vector system comprising the aforementioned nucleic acid construct and lentiviral vector auxiliary components.

[0019] The sixth aspect of the present invention provides a genetically modified T cell, wherein the T cell contains the aforementioned polynucleotide sequence, or contains the aforementioned nucleic acid construct, or is infected with the aforementioned lentiviral vector system.

[0020] The seventh aspect of the present invention provides the use of the aforementioned intracellular domain, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, or the aforementioned lentiviral vector system in the preparation of products for any one or more of the following purposes: (1) preparing CAR-T cells; (2) regulating the secretion of cytokines IFN-γ, IL-2, and TNF from CAR-T cells; (3) regulating the degranulation ability of CAR-T cells; (4) promoting the in vivo antitumor ability of CAR-T cells; (5) enhancing the sustained proliferation ability of CAR-T cells after stimulation by target cells; (6) reducing activation-induced cell death of CAR-T cells; and (7) regulating the activation signaling ability of CAR-T cells.

[0021] The eighth aspect of the present invention provides the application of the aforementioned intracellular domain, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construction, the aforementioned lentiviral vector system, or the aforementioned gene-modified T cells in the preparation of tumor therapeutic products.

[0022] As described above, the chimeric antigen receptor of the present invention, suitable for regulating CAR-T signaling, has the following beneficial effects:

[0023] (1) The modified BBiz-CAR has a lower signal than BBz-CAR, which can alleviate the problem of CAR-T cells with excessive background signal being prone to exhaustion. It can also reduce the overactivation of CAR-T cells when attacking tumor cells, resulting in activation-induced death (AICD), thus giving CAR-T cells better persistence.

[0024] (2) The modified BBzi-CAR has a higher signal than BBz-CAR. For CAR-T cells with insufficient background signal, BBzi can help increase the background signal, thereby enhancing their in vitro expansion capacity. At the same time, when faced with heterogeneous tumors or when tumor immune escape downregulates surface antigen expression, traditional BBz-CAR-T may not be fully activated by antigens. Using BBzi design can help CAR-T cells respond better to low antigen tumors and enhance effector function.

[0025] (3) Using CAR as a research method, we found that PD1-ITSM can have a positive function under certain scenarios. Under different ambient charge environments, PD1-ITSM tends to recruit different kinases and phosphatases, thereby transducing different signals. This is a new mechanism for its function. Attached Figure Description

[0026] Figure 1 shows the results of detecting the surface level, CD4 and CD8 subset ratio, degranulation ability after in vitro stimulation by tumor cells, in vitro killing ability and proliferation ability after stimulation, and in vivo antitumor ability in mice of α-CD133-BBzi CAR-T in Example 1. a: Flow cytometry results of α-CD133-BBzi CAR-T cell membrane surface level; b: CD4+ in α-CD133-BBzi and α-CD133-BBz CAR-T cells. + and CD8 + c: Flow cytometry plot of cell proportions; d: Flow cytometry plot of cytokine secretion in α-CD133-BBzi and α-CD133-BBz CAR-T cells after in vitro stimulation by tumor cells; e-f: Flow cytometry plot of degranulation in α-CD133-BBzi and α-CD133-BBz CAR-T cells after in vitro stimulation by tumor cells; g: Flow cytometry plot of α-CD133-BBzi and α-CD133-BBz CAR-T cells after antigen stimulation; h: Flow cytometry plot of apoptosis in α-CD133-BBzi CAR-T cells after antigen stimulation; i: Flow cytometry plot of cell exhaustion level in α-CD133-BBzi CAR-T cells after antigen stimulation; j: Flow cytometry plot of α-CD133-BBzi and α-CD133-BBz CAR-T cells. Figure 1: In vivo antitumor activity of CAR-T cells after subcutaneous implantation of Huh7 to induce tumor formation in mice (n=3-4); k: α-CD133-BBzi and α-CD133-BBz CAR-T cells after subcutaneous implantation of Huh7 to induce tumor formation in mice (n=4-7); l: α-CD133-BBzi and α-CD133-BBz CAR-T cells after subcutaneous implantation of HCT116 to induce tumor formation in mice (n=3-4); m: α-CD133-BBzi and α-CD133-BBz CAR-T cells after subcutaneous implantation of HCT116 to induce tumor formation in mice (n=3-4); Figure 1 shows the results of CAR-T cell infusion therapy for anti-tumor activity (n = 4-7). In this figure, BBzi represents α-CD133-BBzi, BBz represents α-CD133-BBz, and YF-BBzi represents α-CD133-YF-BBzi.

[0027] Figure 2 shows the results of detecting the surface level, CD4 and CD8 subset ratios, and in vivo antitumor activity of α-CD19-BBzi CAR-T cells in Example 2. a: Flow cytometry results of α-CD19-BBzi CAR-T cell surface level; b: CD4+ in α-CD19-BBzi and α-CD19-BBz CAR-T cells. + and CD8 + c: Flow cytometry plot of cell proportions; d: Flow cytometry plot of basal activation and proliferation levels of α-CD19-BBzi CAR-T cells; e: Flow cytometry plot of cytokine secretion of α-CD19-BBzi and α-CD19-BBz CAR-T cells after stimulation by target cells with different antigen densities; f: Flow cytometry plot of tumor killing ability and antigen-induced amplification ability of α-CD19-BBzi CAR-T cells; YF-BBzi plot of anti-tumor detection results of α-CD19-BBzi and α-CD19-BBz CAR-T cells in mice. (Note: BBzi represents α-CD19-BBzi, BBz represents α-CD19-BBz, and YF-BBzi represents α-CD19-YF-BBzi.)

[0028] Figure 3 shows the detection results of the surface level, CD4 and CD8 subset ratio, and in vitro tumor killing of α-CD22-BBzi CAR-T cells in Example 3. a: Flow cytometry detection results of α-CD22-BBzi CAR-T cells at the cell membrane surface level; b: CD4+ in α-CD22-BBzi and α-CD22-BBz CAR-T cells. + and CD8 + c: Flow cytometry plot of cell proportions; d: Flow cytometry statistical plot of cytokine secretion of α-CD22-BBzi and α-CD22-BBz CAR-T cells after stimulation by target cells with different antigen densities; d: Killing efficiency of α-CD22-BBzi and α-CD22-BBz CAR-T cells against tumor cells with different antigen densities, where BBzi represents α-CD22-BBzi, BBz represents α-CD22-BBz, and YF-BBzi represents α-CD22-YF-BBzi.

[0029] Figure 4 shows the results of detecting the surface level, CD4 and CD8 subset ratios, and in vitro and in vivo antitumor activity of α-HER2-BBiz CAR-T cells in Example 4. a: Flow cytometry results of α-HER2-BBiz CAR-T cell membrane surface level; b: CD4+ in α-HER2-BBiz and α-HER2-BBz CAR-T cells. + and CD8 +c: Flow cytometry results of basal activation and apoptosis levels in α-HER2-BBiz and α-HER2-BBz CAR-T cells; d-e: Flow cytometry results of α-HER2-BBiz and α-HER2-BBz CAR-T cells after in vitro stimulation by tumor cells and their degranulation ability; f: Flow cytometry results of α-HER2-BBiz and α-HER2-BBz CAR-T cells after in vitro stimulation by target cells; g: Flow cytometry results of α-HER2-BBiz and α-HER2-BBz CAR-T cells after in vitro antigen-induced proliferation signal detection; h: Results of α-HER2-BBiz and α-HER2-BBz CAR-T cells in clearing tumor cells stimulated by K562-HER2 cells and their own proliferation ability; i: α-HER2-BBiz and α-HER2-BBz Figure 1: Results of CAR-T cell exhaustion level detection after target cell stimulation; j: Results of α-HER2-BBiz and α-HER2-BBz CAR-T cell tumor control in mice MDA-MB-231-HER2-luciferase; k: Results of α-HER2-BBiz and α-HER2-BBz CAR-T cell tumor control in mice HCC1954-luciferase. BBiz represents α-HER2-BBiz, BBz represents α-HER2-BBz, and YF-BBiz represents α-HER2-YF-BBiz.

[0030] Figure 5 shows the results of detecting the surface level, CD4 and CD8 subset ratio, basal activation level, cytokine secretion level, and target cell killing and proliferation capabilities of α-GD2-BBiz CAR-T cells in Example 5. a: Flow cytometry results of α-GD2-BBiz CAR-T cell membrane surface level; b: CD4+ in α-GD2-BBiz and α-GD2-BBz CAR-T cells. + and CD8 +c: Flow cytometry plot of cell proportions; d: Flow cytometry results of basal activation levels in α-GD2-BBiz and α-GD2-BBz CAR-T cells; e: Flow cytometry results of basal cell death and proliferation signals in α-GD2-BBiz and α-GD2-BBz CAR-T cells; f: Detection results of cytokine secretion levels in α-GD2-BBiz and α-GD2-BBz CAR-T cells; g: Detection results of activation-induced cell death levels in α-GD2-BBiz and α-GD2-BBz CAR-T cells; h: Detection results of proliferation signals in α-GD2-BBiz and α-GD2-BBz CAR-T cells after target cell stimulation; i: Flow cytometry results of α-GD2-BBiz and α-GD2-BBz CAR-T cells. Results of CAR-T cell killing ability against target cells and self-proliferation ability. Among them, BBiz all represent α-GD2-BBiz, BBz all represent α-GD2-BBz, and YF-BBiz all represent α-GD2-YF-BBiz.

[0031] Figure 6 shows the mechanism of bidirectional regulatory effect of Jurkat-α-CD19-BBiz / BBzi in Example 6. a: Western blotting (WB) identification of phosphorylation levels of CAR molecules and juxtamembrane signaling molecules; b: ITSM-CAR activation level after mutating threonine or tyrosine of PD1-ITSM to alanine and phenylalanine, respectively; c: WB detection of recruitment of Jurkat-α-CD19-BBz / BBiz / BBzi kinases and phosphatases; d: WB detection of differential recruitment of kinases and phosphatases after tyrosine mutation in PD1-ITSM; e: WB detection of the collaboration between Jurkat-α-CD19-BBz / BBiz / BBzi and surrounding charged motifs; f: WB detection of differential recruitment of kinases and phosphatases after mutation in the charge-rich region surrounding PD1-ITSM. Detailed Implementation

[0032] The present invention provides an intracellular domain for constructing a chimeric antigen receptor, comprising a co-stimulatory signal transducer and a CD3ζ intracellular region connected in sequence, wherein the CD3ζ intracellular region is connected to a PD1-ITSM motif.

[0033] In some embodiments of the present invention, the PD1-ITSM motif is attached to the anterior or posterior end of the CD3ζ intracellular region. PD1-ITSM can induce alterations in CAR-T cell signaling by differentially recruiting kinases or phosphatases from its surrounding charged motifs. Furthermore, the amino acid sequence of the PD1-ITSM motif is as shown in SEQ ID NO: 1; specifically: EQTEYATIV.

[0034] In one embodiment, the co-stimulatory signal transduction region is selected from one or more intracellular regions of CD27, CD28, CD134, 4-1BB, OX40, or ICOS.

[0035] In a preferred embodiment, the co-stimulatory signal transduction region is selected from the intracellular region of 4-1BB.

[0036] The intracellular amino acid sequence of 4-1BB is shown in SEQ ID NO: 2. Specifically: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0037] In one embodiment, the amino acid sequence of the CD3ζ intracellular region is shown in SEQ ID NO: 3. Specifically, it is: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0038] Preferably, when the above-mentioned PD1-ITSM motif is attached to the front end of the CD3ζ intracellular region, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO: 4, and is named BBiz.

[0039] SEQ ID NO: 4

[0040] When the PD1-ITSM motif is attached to the posterior end of the CD3ζ intracellular region, the amino acid sequence of the intracellular domain is shown in SEQ ID NO: 5 and is named BBzi.

[0041] SEQ ID NO: 5

[0042] This application also provides a chimeric antigen receptor suitable for regulating CAR-T signaling, comprising:

[0043] The extracellular domain, transmembrane domain, and intracellular domain are connected sequentially.

[0044] The extracellular domain includes an antigen recognition region and a hinge region;

[0045] The intracellular domains include the aforementioned intracellular regions.

[0046] The chimeric antigen receptor provided in this application, which is suitable for regulating CAR-T signaling, has a PD1-ITSM motif linked to the CD3ζ intracellular region compared to the original CAR molecule. Through the difference in the linkage position, the regulation of CAR-T activation signal is achieved, so that CAR-T cells that do not yet have suitable signals under different scenarios can reach a relatively optimal activation level.

[0047] In some embodiments of the present invention, the regulation of CAR-T signaling refers to either weakening or enhancing the activation signal of CAR-T cells. Furthermore, when the PD1-ITSM motif is attached to the front end of the CD3ζ intracellular region, i.e., BBiz-CAR, BBiz-CAR exhibits a weaker CAR-T cell activation signal compared to BBz-CAR without the PD1-ITSM motif; when the PD1-ITSM motif is attached to the rear end of the CD3ζ intracellular region, i.e., BBzi-CAR, BBzi-CAR exhibits a stronger CAR-T cell activation signal compared to BBz-CAR without the PD1-ITSM motif.

[0048] In one embodiment, the antigen recognition region is selected from single-chain antibodies against tumor surface antigens, wherein the single-chain antibodies are selected from one or more of CD19, Glypican-3 (GPC3), mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD133, CD138, BCMA, Fibroblast activation protein (FAP), CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, and GD2.

[0049] In some implementations, the single-chain antibody contains a light chain variable region and a heavy chain variable region.

[0050] In some implementations, the light chain variable region and the heavy chain variable region are connected by a connector sequence.

[0051] In one specific embodiment, the single-chain antibody is selected from CD133, CD19, CD22, HER2, and GD2. Among them, the corresponding monoclonal antibodies of CD133, CD19, CD22, HER2, and GD2 are AC133.1, FMC63, m971, 4D5, and 14g2a, respectively. These antibodies have a stronger ability to enhance or weaken CAR-T signaling, so that CAR-T cells that do not yet have suitable signals under different scenarios can reach a relatively optimal activation level.

[0052] In one embodiment, the single-chain antibody contains a light chain variable region and a heavy chain variable region of the monoclonal antibody AC133.1, the light chain variable region and the heavy chain variable region optionally being linked by a linker sequence.

[0053] In one embodiment, the single-chain antibody contains a light chain variable region and a heavy chain variable region of the monoclonal antibody FMC63, the light chain variable region and the heavy chain variable region optionally being linked by a linker sequence.

[0054] In one embodiment, the single-chain antibody contains a light chain variable region and a heavy chain variable region of the monoclonal antibody m971, the light chain variable region and the heavy chain variable region optionally being linked by a linker sequence.

[0055] In one embodiment, the single-chain antibody contains a light chain variable region and a heavy chain variable region of monoclonal antibody 4D5, the light chain variable region and the heavy chain variable region optionally being linked by a linker sequence.

[0056] In one embodiment, the single-chain antibody contains a light chain variable region and a heavy chain variable region of monoclonal antibody 14g2a, the light chain variable region and the heavy chain variable region optionally being linked by a linker sequence.

[0057] The scFv amino acid sequence of AC133.1 is shown in SEQ ID NO: 6. Specifically:

[0058] The scFv amino acid sequence of FMC63 is shown in SEQ ID NO: 7. Specifically:

[0059] The amino acid sequence of m971 scFv is shown in SEQ ID NO: 8. Specifically:

[0060] The scFv amino acid sequence of 4D5 is shown in SEQ ID NO: 9. Specifically:

[0061] The scFv amino acid sequence of 14g2a is shown in SEQ ID NO: 10. Specifically:

[0062] The hinge region is selected from one or more of the CD28 hinge region, CD8α hinge region, CD4 hinge region, the hinge region of immunoglobulin IgG, or the hinge region of immunoglobulin IgG coupled to the CH2CH3 region. That is, it can be IgG1 hinge or IgG1 hinge-CH2CH3.

[0063] Optionally, the hinge region sequence is a CD8α hinge region (CD8αhinge). The amino acid structure is shown in SEQ ID NO: 11. Specifically, it is: TTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0064] In one embodiment, the extracellular domain further includes a signal peptide, forming a sequentially connected signal peptide-antigen recognition region-hinge region.

[0065] Furthermore, the transmembrane domains mentioned above are selected from one or more transmembrane regions of CD8α, CD4, CD28, OX40 or H2-Kb.

[0066] Preferably, the transmembrane domain is selected from the transmembrane region of CD8α. Specifically, the amino acid sequence of the transmembrane region of CD8α is shown in SEQ ID NO: 12, specifically: IYIWAPLAGTCGVLLLSLVITLYC.

[0067] In some embodiments, the amino acid sequences of the chimeric antigen receptor are as shown in SEQ ID NO: 13 (α-CD133-BBzi CAR-T), SEQ ID NO: 14 (α-CD19-BBzi CAR-T), SEQ ID NO: 15 (α-CD22-BBzi CAR-T), SEQ ID NO: 16 (α-HER2-BBiz CAR-T), and SEQ ID NO: 17 (α-GD2-BBiz CAR-T).

[0068] SEQ ID NO: 13 (α-CD133-BBzi CAR-T):

[0069] SEQ ID NO: 14 (α-CD19-BBzi CAR-T):

[0070] SEQ ID NO: 15 (α-CD22-BBzi CAR-T):

[0071] SEQ ID NO: 16 (α-HER2-BBiz CAR-T):

[0072] SEQ ID NO: 17 (α-GD2-BBiz CAR-T):

[0073] The nucleotide sequence corresponding to SEQ ID NO: 13 (α-CD133-BBzi CAR-T) is SEQ ID NO: 18 (α-CD133-BBzi CAR-T), SEQ ID NO: 18:

[0074] The nucleotide sequence corresponding to SEQ ID NO: 14 (α-CD19-BBzi CAR-T) is SEQ ID NO: 19 (α-CD19-BBzi CAR-T). SEQ ID NO: 19:

[0075] The nucleotide sequence corresponding to SEQ ID NO: 15 (α-CD22-BBzi CAR-T) is SEQ ID NO: 20 (α-CD22-BBzi CAR-T), SEQ ID NO: 20:

[0076] The nucleotide sequence corresponding to SEQ ID NO: 16 (α-HER2-BBiz CAR-T) is SEQ ID NO: 21 (α-HER2-BBiz CAR-T), SEQ ID NO: 21:

[0077] The nucleotide sequence corresponding to SEQ ID NO: 17 (α-GD2-BBiz CAR-T) is SEQ ID NO: 22 (α-GD2-BBiz CAR-T), SEQ ID NO: 22:

[0078] The various portions forming the chimeric antigen receptor of the present invention can be directly linked to each other or linked via adapter sequences. The adapter sequence can be a known antibody-compatible adapter sequence, such as a G and S-containing adapter sequence. Typically, the adapter contains one or more repeating motifs. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent in the adapter sequence, with no inserted amino acid residues between the repeats. The adapter sequence can consist of 1, 2, 3, 4, or 5 repeating motifs. The length of the adapter can be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In some embodiments, the adapter sequence is a polyglycine adapter sequence. The number of glycine residues in the adapter sequence is not particularly limited, typically 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc.

[0079] It should be understood that in gene cloning, it is often necessary to design suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed amino acid sequence, without affecting the activity of the target sequence. To construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, such as, but not limited to, suitable adaptor peptides, signal peptides, leader peptides, and terminal extensions. Therefore, the amino or carboxyl terminus of the fusion protein (i.e., CAR) of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0080] This application also provides a polynucleotide sequence selected from:

[0081] (1) A multinucleotide sequence encoding the aforementioned chimeric antigen receptor; and / or

[0082] The complementary sequence of the polynucleotide sequence described in (2)(1).

[0083] The polynucleotide sequence of this application can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. This invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0084] The polynucleotide sequences described herein can generally be obtained using PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0085] The present invention also provides a nucleic acid construct containing the aforementioned polynucleotide sequence.

[0086] The nucleic acid construct also includes one or more regulatory sequences operatively linked to the aforementioned polynucleotide sequence. The coding sequence of the CAR of this application can be manipulated in various ways to ensure protein expression. The nucleic acid construct can be manipulated according to the expression vector or requirements before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0087] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is typically operatively linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0088] The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention.

[0089] The regulatory sequence can also be a suitable leader sequence, or an untranslated region of mRNA important for translation in the host cell. The leader sequence is operatively linked to the 5′ end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention.

[0090] In a preferred embodiment of this application, the nucleic acid construct is a vector.

[0091] Expression of the CAR-encoding polynucleotide sequence is typically achieved by operatively linking the polynucleotide sequence to a promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0092] The polynucleotide sequence encoding the CAR of this application can be cloned into many types of vectors. For example, it can be cloned into plasmids, phage particles, phage derivatives, animal viruses, and granules. Further, the vector is an expression vector. The expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers.

[0093] In one specific embodiment of this application, the nucleic acid construct is a lentiviral vector containing a replication start site, a 3'LTR, a 5'LTR, and the aforementioned polynucleotide sequence.

[0094] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMμLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the use of inducible promoters may also be considered. The use of inducible promoters provides a molecular switch that enables expression of the polynucleotide sequence operatively linked to the inducible promoter during time-limited expression and disables expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0095] To assess the expression of CAR peptides or portions thereof, the expression vector introduced into cells may also contain one or both of optional marker genes or reporter genes to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. Alternatively, the optional marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may have appropriate regulatory sequences flanking them to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes such as neo.

[0096] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. After DNA has been introduced into recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well-known and can be prepared using known techniques or are commercially available.

[0097] Methods for introducing genes into cells and expressing genes into cells are known in the art. Vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0098] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and others. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0099] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, lentiviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into lentiviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0100] This application also provides a lentiviral vector system, which includes the aforementioned nucleic acid construct and lentiviral vector auxiliary components.

[0101] Lentiviral auxiliary components include lentiviral packaging plasmids and cell lines. A lentiviral vector system is constructed by viral packaging of the aforementioned nucleic acid constructs with the assistance of lentiviral packaging plasmids and cell lines. The method for constructing lentiviral vector systems is a commonly used method in this field.

[0102] This application also provides a method for regulating T cell signaling, including the step of infecting the aforementioned T cells with the aforementioned lentivirus.

[0103] The CAR-T cells described in this application can alter signaling by recruiting kinases or phosphatases through differential recruitment of PD1-ITSM with its surrounding charged motifs. Tyrosine residues in PD1-ITSM are essential amino acids for its function. When PD1-ITSM is attached to the anterior end of the CD3ζ intracellular region, it recruits phosphatases SHP1 and SHP2 in cooperation with the adjacent C-terminal negatively charged amino acid-rich region of 4-1BB, resulting in a weaker activation signal in BBiz-CAR-T cells. When ITSM is attached to the posterior end of the CD3ζ intracellular region, it recruits and activates kinase Lck in cooperation with multiple basic amino acid-rich regions of the adjacent CD3z, resulting in a stronger activation signal in CAR-T cells.

[0104] The CAR-T cells of this invention tend to recruit different kinases and phosphatases, thereby transducing different signals and regulating CAR signals. This allows CAR-T cells that do not yet have suitable signals in different scenarios to reach a relatively optimal activation level, avoiding insufficient activation or excessive self-activation of CAR-T cells.

[0105] This application also includes a class of cell therapies in which T cells are genetically modified to express the CAR described herein, and CAR-T cells are infused into recipients in need of them. The infused cells are able to kill the recipient's tumor cells by modulating optimal activation levels. Unlike antibody therapies, CAR-T cells can replicate in vivo, producing long-lasting efficacy that can lead to sustained tumor control.

[0106] Antitumor immune responses induced by CAR-T cells can be active or passive. Furthermore, CAR-mediated immune responses can be part of adoptive immunotherapy procedures, in which CAR-T cells induce a specific immune response against the antigen-binding portion of the CAR.

[0107] The treatable cancers can be non-solid tumors, such as hematologic malignancies like leukemia and lymphoma; or solid tumors, such as colon and rectal cancer. In particular, diseases can be treated using the CAR of this invention, its coding sequence, nucleic acid constructs, expression vectors, viruses, and CAR-T cells. Specifically, these diseases can be CD19, CD133, CD22, HER2, and GD2-mediated, especially CD19, CD133, CD22, HER2, and GD2-mediated tumors.

[0108] Specifically, in this article, "CD19-mediated diseases" include, but are not limited to, leukemia and lymphoma, such as B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0109] Specifically, in this article, "CD133-mediated diseases" include, but are not limited to, leukemia, colon cancer, rectal cancer, lung cancer, liver cancer, and glioblastoma.

[0110] Specifically, in this article, "CD22-mediated diseases" include, but are not limited to, leukemia and lymphoma, such as acute lymphoblastic leukemia, non-Hodgkin's lymphoma, and hairy cell leukemia.

[0111] Specifically, in this article, "HER2-mediated diseases" include, but are not limited to, breast cancer, non-small cell lung cancer, gastric cancer, colorectal cancer, ovarian cancer, and bladder cancer.

[0112] Specifically, in this article, "GD2-mediated diseases" include, but are not limited to, neuroblastoma, osteosarcoma, small cell lung cancer, and diffuse endophytic glioma.

[0113] Another aspect of this application provides a genetically modified T cell containing the aforementioned polynucleotide sequence, or containing the aforementioned nucleic acid construct, or infected with the aforementioned lentiviral vector system.

[0114] The CAR-modified T cells of this application can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as associated cytokines or cell populations. In simple terms, the pharmaceutical compositions of this application may include CAR-T cells as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0115] The pharmaceutical composition of this application can be administered in a manner suitable for treating (or preventing) a disease. The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0116] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-inhibitory effective amount," "therapeutic effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that: pharmaceutical compositions comprising T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6The dosage is 1 cell / kg body weight. The T-cell composition can also be administered at these dosages multiple times. Cells can be administered using infusion techniques known in immunotherapy. The optimal dosage and treatment regimen for a specific patient can be readily determined by medical professionals by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0117] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0118] In some embodiments of this application, the CAR-T cells or compositions thereof of the present invention can be combined with other therapies known in the art. These therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, treatment may be administered in combination with various radiotherapy agents, including cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, FK506, fludarabine, rapamycin, and mycophenolic acid. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, T-cell ablation therapy using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH (e.g., before, simultaneously with, or after).

[0119] In this article, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various cancer-related physiological symptoms.

[0120] The terms "patient," "subject," "individual," and "subject" are used interchangeably herein to refer to a living organism capable of evoking an immune response, such as a mammal or a non-mammal. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The non-mammals include, for example, non-mammal vertebrates such as birds (e.g., chickens or ducks) or fish, as well as non-mammal invertebrates. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and their transgenic species. In one specific embodiment, the subject may be a human, such as an immunocompromised patient or a patient with cancer.

[0121] This application also includes the use of the aforementioned intracellular domain, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, and the aforementioned lentiviral vector system in the preparation of products for any one or more of the following purposes: (1) preparing CAR-T cells; (2) regulating the secretion of cytokines IFN-γ, IL-2, and TNF from CAR-T cells; (3) regulating the degranulation ability of CAR-T cells; (4) promoting the in vivo antitumor ability of CAR-T cells; (5) enhancing the sustained proliferation ability of CAR-T cells after stimulation by target cells; (6) reducing activation-induced cell death of CAR-T cells; and (7) regulating the activation signaling ability of CAR-T cells.

[0122] This application also includes the use of the aforementioned intracellular domain, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, the aforementioned lentiviral vector system, or the aforementioned genetically modified T cells in the preparation of tumor therapeutic products.

[0123] In one specific embodiment of this application, the tumor is selected from one or more of leukemia or solid tumors.

[0124] In a preferred embodiment of this application, the tumor is selected from hepatocellular carcinoma, colorectal cancer, B-cell lymphoma, breast cancer, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, acute myeloid leukemia, clear cell carcinoma of the ovary, or squamous cell carcinoma of the lung, etc.

[0125] The present invention also provides a treatment method for tumors, the treatment method comprising administering a therapeutic amount of the genetically modified T cells to a subject in need.

[0126] "Treatment" or "therapy" for a condition includes preventing or alleviating the condition, slowing the onset or progression of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, achieving complete or partial reversal of the condition, curing the condition, or a combination of the above. For cancer, "treatment" or "therapy" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" or "therapy" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination of the above.

[0127] When administering the medication to subjects, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. Animal study results and other factors may be considered, but the total dosage should not exceed a certain range.

[0128] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0129] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0130] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0131] Example 1:

[0132] α-CD133-BBzi is a modified version of the α-CD133-BBz sequence. α-CD133-BBz includes AC133.1 scFv, a CD8α hinge region, a CD8α transmembrane region, 4-1BB, and a CD3ζ intracellular region. Since α-CD133-BBz exhibits a weak CAR-T antigen signal, we used gene synthesis technology to insert PD-ITSM (EQTEYATIV) into the C-terminus of the CD3ζ intracellular region, naming it α-CD133-BBzi. The amino acid sequence of α-CD133-BBzi is shown in SEQ ID NO: 13, specifically:

[0133] The nucleotide sequence of α-CD133-BBzi is shown in EEQ ID NO: 18, specifically:

[0134] Preparation method: α-CD133-BBzi, α-CD133-BBz, and α-CD133-YF-BBzi (YF being a tyrosine mutation in ITSM) were cloned into a lentiviral expression pHAGE vector and identified as correct. The CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2.G at a ratio of 10:7.5:3.5 and transfected into 293FT cells using calcium phosphate transfection to produce lentiviral particles. After 48 hours, the viral supernatant was collected, filtered through a 0.45 μm filter membrane, centrifuged at 27,000 rpm for 2 hours, and the supernatant was discarded. The virus was dissolved in serum-free medium, and human peripheral blood mononuclear cells activated for 24 hours with α-CD3 / 28-beads were added. After 48 hours, the virus and beads were removed. At this point, the culture system should only contain human primary T cells. Two days later, fluorescent protein-positive T cells were sorted by flow cytometry, thus obtaining CAR-T cells, with α-CD133-BBz CAR-T and α-CD133-YF-BBzi CAR-T cells serving as controls. The sorted cells exhibited stable surface CAR molecule expression. A series of indicators were used to identify CAR-T cells, including CAR membrane level, CD4 and CD8 subset ratio, in vitro cytokine secretion, in vitro killing ability, in vitro proliferation ability, in vitro apoptosis level, in vitro survival ability, and in vivo anti-tumor effect.

[0135] CAR surface level and CD4 and CD8 subset ratio detection: Anti-HA-Rabbit-IgG was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. Then, anti-Rabbit-IgG-AF647 was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. Flow cytometry was used to detect the signal intensity of AF647. Anti-CD4-APC and anti-CD8-PE-cy7 antibodies were used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS, and then flow cytometry analysis was performed. The flow cytometry results are shown in Figures 1a-b. The prepared α-CD133-BBzi CAR-T cells had similar surface CAR levels and similar CD4 and CD8 subset ratios to α-CD133-BBz CAR-T cells and α-CD133-YF-BBzi CAR-T cells.

[0136] In vitro cytokine secretion capacity assay: For adherent tumor cells, target cells were seeded into wells at an appropriate density 24 hours in advance. An appropriate density meant that the target cells occupied approximately 70% of the wells after 24 hours. An appropriate number of CAR-T cells were taken, and the CAR-T cells and target cells in the wells were washed once with x-vivo15 medium (without any serum or cytokine additives). The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing target cells. After 20 hours, cytokine efflux was blocked using BFA for 4 hours. Cells were then collected, fixed, and punched into wells. The cells were stained overnight with anti-IFNg-APC, anti-IL2-BV421, and anti-TNFα-PE antibodies. Excess antibodies were then washed away with PBS, and flow cytometry was performed. Alternatively, CAR-T cells were stimulated using the same method for 24 hours, the culture supernatant was collected, diluted appropriately, and the levels of IFNg, IL2, and TNFα were detected using an ELISA kit. The flow cytometry results are shown in Figure 1c. When CAR-T cells were stimulated with HCT116 and Huh7 cells, α-CD133-BBzi CAR-T cells had a significantly higher cytokine secretion capacity.

[0137] In vitro degranulation ability assay after tumor cell stimulation: For adherent tumor cells, HCT116 or Huh7 cells were seeded in wells at an appropriate density 24 hours in advance. An appropriate density meant that the HCT116 or Huh7 cell density in the wells was approximately 70% after 24 hours. An appropriate number of CAR-T cells were taken, and the CAR-T cells and target cells or suspended tumor cells in the wells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing HCT116 or Huh7 cells. In addition, monensin (final concentration 1 μg / mL) and anti-CD107a-APC were added to the system. The cells were centrifuged at 300g for 1 min to quickly contact the tumor cells with the CAR-T cells. After stimulation at 37℃ for 1–4 h, the cells were harvested, washed once with PBS, and analyzed by flow cytometry. The flow cytometry results are shown in Figure 1d. When CAR-T cells were stimulated with HCT116 and Huh7 cells, α-CD133-BBzi CAR-T cells showed significantly higher degranulation ability.

[0138] In vitro killing ability and post-stimulation proliferation capacity assay: For adherent tumor cells, HCT116 or Huh7 cells were seeded in wells at an appropriate density 24 hours in advance. An appropriate density meant that the HCT116 or Huh7 cell density in the wells was approximately 70% after 24 hours. An appropriate number of CAR-T cells (α-CD133-BBzi CAR-T, α-CD133-BBz CAR-T, and α-CD133-YF-BBzi CAR-T) were taken, and the CAR-T cells and HCT116 or Huh7 cells in the wells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing HCT116 or Huh7 cells. At specific time points (24h, 48h, 72h, and 96h), cells were collected for flow cytometry counting to calculate the target cell killing efficiency and the CAR-T cell proliferation capacity at the corresponding time points. As shown in Figures 1e-f, when HCT116 and Huh7 cells were used in the killing experiment, α-CD133-BBzi CAR-T cells showed significantly higher killing ability at 24 h, and α-CD133-BBzi CAR-T cells showed better amplification ability after stimulation.

[0139] Detection of activation-induced cell death, exhaustion, and proliferation levels in vitro: For adherent tumor cells, Huh7 or HCT116 cells were seeded in wells at an appropriate density 24 hours in advance. An appropriate density meant that the target cell density in the wells was approximately 70% after 24 hours. An appropriate number of CAR-T cells (α-CD133-BBzi CAR-T, α-CD133-BBz CAR-T, and α-CD133-YF-BBzi CAR-T) were taken, and the CAR-T cells and Huh7 or HCT116 cells in the wells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing Huh7 or HCT116 cells. At specific time points, such as 24h, cells were incubated with Annexin V at room temperature in the dark for 10 min, washed once with 1*binding buffer, and then analyzed by flow cytometry to obtain CAR-T cell death signals. At 72h, cells were stained with anti-PD1-APC, anti-TIM3-PE, and anti-TIGIT-BV421, incubated on ice for 30 min, washed once, and then analyzed by flow cytometry to characterize the CAR-T cell depletion level. At 96h, immediately after tumor cells were removed, cells were transnuclearized, stained with anti-Ki67-APC, incubated on ice overnight, washed once, and then analyzed by flow cytometry to characterize the CAR-T cell proliferation level. The results are shown in Figures 1g-i. α-CD133-BBzi CAR-T showed a stronger proliferation signal than α-CD133-BBz-CAR-T under antigen stimulation (Figure 1g); α-CD133-BBzi CAR-T did not exhibit stronger activation-induced cell death than α-CD133-BBz-CAR-T under antigen stimulation (Figure 1h); and α-CD133-BBzi CAR-T did not induce a higher level of exhaustion under antigen stimulation (Figure 1i).

[0140] In vivo antitumor activity assay in mice: Tumor formation was achieved by subcutaneous implantation of Huh7 cells, with a specific cell count of 2*103. 6 / mouse / , using B-NDG mice for tumor formation experiment, including 3 mice in the α-CD133-BBz group, 3 mice in the α-CD133-BBzi group, and 4 mice in the PBS group. After 10 days, the tumors grew to a suitable size, and then 3×10 6CAR-T (α-CD133-BBzi CAR-T, α-CD133-BBz CAR-T) infusion therapy, as shown in Figure 1j, resulted in better control of tumor growth and prolonged survival in mice after BBzi CAR-T infusion. Further tumorigenesis experiments were conducted in B-NDG mice (n=7 in the α-CD133-BBz group, n=7 in the α-CD133-BBzi group, and n=4 in the PBS group). The results, as shown in Figure 1k, confirmed that α-CD133-BBzi CAR-T infusion resulted in better control of liver tumor growth and prolonged survival in mice.

[0141] In vivo antitumor activity assay in mice: Tumor formation was achieved by subcutaneous implantation of HCT116 cells, with a specific cell count of 2*102. 6 / mouse / , using B-NDG mice for tumor formation experiment, including 3 mice in the α-CD133-BBz group, 3 mice in the α-CD133-BBzi group, and 4 mice in the PBS group. After 5 days, the tumors grew to a suitable size, and then 4×10 6 CAR-T (α-CD133-BBzi CAR-T, α-CD133-BBz CAR-T) infusion therapy, as shown in Figure 11, resulted in better control of tumor growth and prolonged survival in mice after BBzi CAR-T infusion. Further validation was achieved using tumorigenesis experiments with 4-7 B-NDG mice (7 in the α-CD133-BBz group, 7 in the α-CD133-BBzi group, and 4 in the PBS group). The results, as shown in Figure 1m, indicate that α-CD133-BBzi CAR-T infusion controlled colon tumor growth and prolonged survival in mice.

[0142] Example 2:

[0143] α-CD19-BBzi is based on the α-CD19-BBz sequence. α-CD19-BBz includes FMC63 scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB, and CD3ζ intracellular region. The α-CD19-BBz CAR-T base and antigen signal are relatively weak. Based on α-CD19-BBz, we used gene synthesis technology to insert PD-ITSM (EQTEYATIV) into the C-terminus of the CD3ζ intracellular region, and named it α-CD19-BBzi.

[0144] The amino acid sequence of α-CD19-BBzi is shown in EEQ ID NO: 14, specifically:

[0145] The nucleotide sequence of α-CD19-BBzi is shown in EEQ ID NO: 19, specifically:

[0146] α-CD19-BBzi was cloned into a lentiviral expression pHAGE vector and confirmed to be correct. The CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2.G at a ratio of 10:7.5:3.5 and transfected into 293FT cells using calcium phosphate transfection to produce lentiviral particles. After 48 hours, the viral supernatant was collected, filtered through a 0.45 μm filter, centrifuged at 27,000 rpm for 2 hours, and the supernatant was discarded. The virus was then dissolved in serum-free medium, and human peripheral blood mononuclear cells activated with α-CD3 / 28-beads for 24 hours were added. After 48 hours, the virus and beads were removed. At this point, the culture system should contain only human primary T cells. Two days later, fluorescent protein-positive T cells were sorted by flow cytometry, thus obtaining CAR-T cells. α-CD19-BBzi CAR-T cells and α-CD19-YF-BBzi CAR-T cells were used as controls. A series of indicators were used to identify CAR-T cells, including CAR membrane level, CD4 and CD8 subset ratio, in vitro cytokine secretion, in vitro killing ability, in vitro proliferation ability, in vitro apoptosis level, in vitro survival ability, and in vivo anti-tumor effect.

[0147] CAR surface level and CD4 and CD8 subset ratio detection: Anti-HA-Rabbit-IgG was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. Then, anti-Rabbit-IgG-AF647 was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. Flow cytometry was used to detect the signal intensity of AF647. Anti-CD4-APC and anti-CD8-PE-cy7 antibodies were used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS, and then flow cytometry analysis was performed. The flow cytometry results are shown in Figures 2a-b. The prepared α-CD19-BBzi CAR-T cells had similar surface CAR levels and similar CD4 and CD8 subset ratios to α-CD19-BBz CAR-T cells and α-CD19-YF-BBzi CAR-T cells.

[0148] Detection of CAR-T cell basal activation and proliferation signals: CAR-T cells (α-CD19-BBzi CAR-T, α-CD19-BBz CAR-T, and α-CD19-YF-BBzi CAR-T) were stained at 4℃ for 30 min using anti-CD25-ef450 antibody. Excess antibody was then washed away with PBS, and flow cytometry was performed to obtain basal activation signals. After CAR-T cell fixation and nuclear translocation, CAR-T cells were stained overnight with anti-Ki67-APC, washed once with PBS, and flow cytometry was performed to obtain basal proliferation signals. The results are shown in Figure 2c. During culture, α-CD19-BBzi CAR-T cells exhibited stronger background and proliferation signals compared to α-CD19-BBz CAR-T cells.

[0149] Cytokine secretion capacity assay: For suspended tumor cells, target cells do not need to be pre-plated; they can be treated simultaneously with CAR-T cells. Take an appropriate number of CAR-T cells (α-CD19-BBzi CAR-T, α-CD19-BBz CAR-T, and α-CD19-YF-BBzi CAR-T), wash the CAR-T cells and suspended tumor cells once with x-vivo15 medium without any serum or cytokine additives, then resuspend the CAR-T cells and K562 cells in the same medium to an appropriate density. Mix them in an appropriate ratio and add them to 96-well plates. After 20 hours, block cytokine efflux using BFA for 4 hours, then collect the cells, fix them, punch them in wells, and stain overnight with anti-IFNg-APC, anti-IL2-BV421, and anti-TNFa-PE antibodies. Excess antibodies were then washed away with PBS, and flow cytometry was performed. Alternatively, 24 hours after CAR-T cell stimulation using the same method, the culture supernatant was collected, diluted appropriately, and the levels of IFNg, IL2, and TNFα were detected using an ELISA kit. In this embodiment, flow cytometry analysis was used, and the results are shown in Figure 2d. When CAR-T cells were stimulated with K562 cells at different antigen expression levels, α-CD19-BBzi CAR-T cells exhibited significantly higher cytokine secretion capacity, especially when stimulated by target cells with low antigen expression.

[0150] Detection of CAR-T cell killing ability and post-stimulation proliferation: For suspended tumor cells, target cells do not need to be pre-plated; they can be processed simultaneously with CAR-T cells. Appropriate numbers of CAR-T cells (α-CD19-BBzi CAR-T, α-CD19-BBz CAR-T cells, and α-CD19-YF-BBzi CAR-T cells) were taken, and the CAR-T cells and suspended K562 tumor cells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing K562 cells. Cell counts were performed at specific time points. The results, as shown in Figure 2e, indicate that CD19-BBzi CAR-T cells exhibited stronger tumor cell killing ability and antigen-stimulated proliferation. When killing tumor cells with low antigen expression, α-CD19-BBzi CAR-T cells showed better tumor control and CAR-T cell proliferation capacity (Figure 2e).

[0151] In in vivo experiments, tumorigenesis was induced by tail vein injection of Raji-luciferase cells (cells with knocked-down CD19, i.e., cells with low CD19 levels on their surface), specifically at a cell count of 5 × 10⁶. 5 / each, after 5 days, the tumor grew to a suitable size, and then 2*10 were administered. 6 / CAR-T infusion treatment. As shown in Figure 2f, tumor growth was recorded by biofluorescence imaging. After α-CD19-BBzi CAR-T infusion, tumor growth in mice was better controlled and the survival time of mice was prolonged.

[0152] Example 3

[0153] α-CD22-BBzi is based on the α-CD22-BBz sequence, which includes m971 scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB and CD3ζ intracellular region. Based on α-CD22-BBz, we used gene synthesis technology to insert PD-ITSM (EQTEYATIV) into the C-terminus of the CD3ζ intracellular region, and named it α-CD22-BBzi.

[0154] The amino acid sequence of α-CD22-BBzi is shown in EEQ ID NO: 15, specifically:

[0155] The nucleotide sequence of α-CD22-BBzi is shown in SEQ ID NO: 20, specifically:

[0156] Preparation method of α-CD22-BBzi-CAR-T: α-CD22-BBzi was cloned into a lentiviral expression pHAGE vector and identified as correct. The CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2.G at a ratio of 10:7.5:3.5, and then transfected into 293FT cells using calcium phosphate transfection to produce lentiviral particles. After 48 hours, the viral supernatant was collected, filtered through a 0.45 μm filter membrane, centrifuged at 27,000 rpm for 2 hours, and the supernatant was discarded. The virus was dissolved in serum-free medium, and human peripheral blood mononuclear cells activated with α-CD3 / 28-beads for 24 hours were added. After 48 hours, the virus and beads were removed. At this point, the culture system should only contain human primary T cells. Two days later, fluorescent protein-positive T cells were sorted by flow cytometry, yielding α-CD22-BBzi CAR-T cells, with α-CD22-BBz CAR-T cells and α-CD22-YF-BBzi CAR-T cells serving as controls. A series of indicators were evaluated for CAR-T cells, including CAR membrane level, CD4 and CD8 subset ratio, in vitro cytokine secretion, in vitro killing ability, in vitro proliferation ability, in vitro apoptosis level, in vitro survival rate, and in vivo antitumor effect.

[0157] CAR surface level and CD4 and CD8 subset ratio detection: Anti-HA-Rabbit-IgG was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. Then, anti-Rabbit-IgG-AF647 was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. Flow cytometry was used to detect the signal intensity of AF647. Anti-CD4-APC and anti-CD8-PE-cy7 antibodies were used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS, and then flow cytometry analysis was performed. The flow cytometry results are shown in Figures 3a-b. The prepared α-CD22-BBzi CAR-T cells had similar surface CAR levels and similar CD4 and CD8 subset ratios to α-CD22-BBz CAR-T cells and α-CD22-YF-BBzi CAR-T cells.

[0158] Cytokine secretion assay: For suspension tumor cells, K562 cells do not need to be pre-plated; they can be treated simultaneously with CAR-T cells. Take an appropriate number of CAR-T cells, wash both CAR-T and K562 cells once with x-vivo15 medium (without any serum or cytokine additives), then resuspend the CAR-T cells in the same medium to an appropriate density and add them to wells containing K562 cells. After 20 hours, block cytokine efflux using BFA for 4 hours, then collect the cells, fix them, punch them in wells, and stain overnight with anti-IFNg-APC, anti-IL2-BV421, and anti-TNFα-PE antibodies. Afterward, wash away excess antibodies with PBS and perform flow cytometry. Alternatively, after stimulating CAR-T cells using the same method for 24 hours, collect the culture supernatant, dilute it appropriately, and use an ELISA kit to detect the levels of IFNg, IL2, and TNFα. As shown in Figure 3c, when CAR-T cells with low antigen expression were used to stimulate α-CD22-BBzi CAR-T cells, α-CD22-BBzi CAR-T cells had a significantly higher cytokine secretion capacity.

[0159] In in vitro killing experiments, as shown in Figure 3d, α-CD22-BBzi showed better killing ability against K562 cells with low antigen expression levels, and exhibited stronger effector function.

[0160] Example 4

[0161] α-HER2-BBiz is based on the α-HER2-BBz sequence, which includes the 4D5 scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB, and CD3ζ intracellular region. α-HER2-BBz CAR-T has a strong antigen-induced signal. Based on α-HER2-BBz, we used gene synthesis technology to insert PD-ITSM (EQTEYATIV) between 4-1BB and CD3ζ, and named it α-HER2-BBiz.

[0162] The amino acid sequence of α-HER2-BBiz is shown in EEQ ID NO: 16, specifically:

[0163] The nucleotide sequence of α-HER2-BBiz is shown in SEQ ID NO: 21, specifically:

[0164] Preparation method of α-HER2-BBiz-CAR-T: α-HER2-BBiz was cloned into a lentiviral expression pHAGE vector and identified as correct. The CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2.G at a ratio of 10:7.5:3.5, and then transfected into 293FT cells using calcium phosphate transfection to produce lentiviral particles. After 48 hours, the viral supernatant was collected, filtered through a 0.45 μm filter membrane, centrifuged at 27,000 rpm for 2 hours, and the supernatant was discarded. The virus was dissolved in serum-free medium, and human peripheral blood mononuclear cells activated with α-CD3 / 28-beads for 24 hours were added. After 48 hours, the virus and beads were removed. At this point, the culture system should only contain human primary T cells. Two days later, fluorescent protein-positive T cells were sorted by flow cytometry, thus obtaining α-HER2-BBiz CAR-T cells, with α-HER2-BBz CAR-T cells and α-HER2-YF-BBiz CAR-T cells serving as controls. A series of indicators were evaluated for CAR-T cells, including CAR membrane level, CD4 and CD8 subset ratio, in vitro cytokine secretion, in vitro killing ability, in vitro proliferation ability, in vitro apoptosis level, in vitro survival rate, and in vivo antitumor effect.

[0165] CAR surface level detection: Anti-HA-Rabbit-IgG was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. Then, anti-Rabbit-IgG-AF647 was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. The signal intensity of AF647 was detected by flow cytometry. The flow cytometry results are shown in Figure 4a. Compared with the control α-HER2-BBz CAR-T cells, the surface level of the prepared α-HER2 BBiz CAR-T cells was similar or slightly lower. Meanwhile, the other control cell line, α-HER2YF-BBiz CAR-T, had the lowest surface level (Figure 4a).

[0166] CD4 and CD8 subset ratio detection: Anti-CD4-APC and anti-CD8-PE-cy7 antibodies were used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS and flow cytometry analysis. The flow cytometry results are shown in Figure 4b. The prepared α-HER2-BBiz CAR-T cells had similar CD4 and CD8 subset ratios to α-HER2-BBz CAR-T cells and α-HER2-YF-BBiz CAR-T cells.

[0167] Detection of basal activation and basal apoptosis levels: A suitable number of unstimulated CAR-T cells were stained with anti-CD25-ef450 antibody at 4°C for 30 min. Excess antibody was then washed away with PBS, and the ef450 signal intensity was detected by flow cytometry to obtain the basal activation signal. A suitable number of unstimulated CAR-T cells were stained with Annexin V-APC in 1* binding buffer at room temperature in the dark for 10 min. After washing once with 1* binding buffer, the cells were resuspended in 1* binding buffer, and PI was added. The Annexin V and PI signals were detected by flow cytometry to obtain the basal apoptosis level. The flow cytometry results are shown in Figure 4c. α-HER2-BBiz CAR-T cells showed lower basal activation levels compared to α-HER2-BBz CAR-T cells and α-HER2-YF-BBiz CAR-T cells, and lower basal apoptosis levels compared to α-HER2-BBz CAR-T cells.

[0168] Cytokine secretion capacity assay: For adherent tumor cells such as HCC1954 and Mcf7, target cells were seeded into wells at an appropriate density 24 hours in advance. An appropriate density meant that the target cell density in the wells was approximately 70% after 24 hours. For suspension tumor cells K562-Her2, target cells did not need to be seeded in advance and could be processed simultaneously with CAR-T cells. An appropriate number of CAR-T cells were taken, and the CAR-T cells and target cells or suspension tumor cells in the wells were washed once with x-vivo15 medium (without any serum or cytokine additives). The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing target cells. After 20 hours, cytokine efflux was blocked using BFA for 4 hours. Cells were then collected, fixed, and punched into wells. The cells were stained overnight with anti-IFNg-APC, anti-IL2-BV421, and anti-TNFα-PE antibodies. Excess antibodies were then washed away with PBS, and flow cytometry was performed. The results of flow cytometry analysis are shown in Figure 4d. When CAR-T cells were stimulated with target cells, α-HER2-BBiz CAR-T cells showed significantly lower cytokine secretion.

[0169] Degranulation ability assay: For adherent tumor cells such as HCC1954 and Mcf7, target cells were seeded into wells at an appropriate density 24 hours in advance. An appropriate density meant that the target cell density in the wells was approximately 70% after 24 hours. For suspension tumor cells K562-Her2, target cells did not need to be seeded in advance and could be treated simultaneously with CAR-T cells. An appropriate number of CAR-T cells were taken and washed once with x-vivo15 medium (without any serum or cytokines) to the target cells or suspended tumor cells in the wells. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing target cells. In addition, monensin (final concentration 1 μg / mL) and anti-CD107a-APC were added to the system. Centrifugation at 300g for 1 min allowed rapid contact between tumor cells and CAR-T cells. After stimulation at 37℃ for 1–4 h, the cells were harvested, washed once with PBS, and analyzed by flow cytometry. As shown in Figure 4e, the flow cytometry results show that α-HER2-BBiz CAR-T has a significantly weaker degranulation ability.

[0170] In vitro apoptosis and proliferation level detection: For adherent tumor cells, MDA-MB-453 cells were seeded in wells at an appropriate density 24 hours in advance. An appropriate density meant that the MDA-MB-453 cell density in the wells was approximately 70% after 24 hours. Appropriate numbers of CAR-T cells (α-HER2-BBiz CAR-T cells, α-HER2-BBz CAR-T cells, and α-HER2-YF-BBiz CAR-T cells) were taken, and the CAR-T cells and MDA-MB-453 cells in the wells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to the wells containing MDA-MB-453 cells. At a specific time point, such as 24 hours, cells were stained with Annexin V-APC, incubated at room temperature in the dark for 10 minutes, washed once, and then PI was added for flow cytometry analysis. As shown in Figure 4f, when CAR-T cells were stimulated with target cells, α-HER2-BBiz CAR-T cells exhibited a significantly lower level of apoptosis (this trend was consistent when different target cells were used for stimulation; only MDA-MB-453 target cells were selected for representation here). Cells were pernuclear fixed, stained with anti-Ki67-APC, and after washing away excess antibody overnight, flow cytometry analysis showed that α-HER2-BBiz CAR-T cells did not significantly reduce antigen-induced proliferation signals compared to α-HER2-BBz CAR-T cells (4g).

[0171] In vitro killing ability, post-stimulation proliferation capacity, and exhaustion level detection: For adherent tumor cells, target cells were seeded into wells at an appropriate density 24 hours in advance. An appropriate density meant that the target cell density in the wells was approximately 70% after 24 hours. For suspension tumor cells, target cells did not need to be seeded in advance and could be processed simultaneously with CAR-T cells. In this example, K562-HER2 cells were suspension cells. Appropriate numbers of CAR-T cells (α-HER2-BBiz CAR-T, α-HER2-BBz CAR-T, and α-HER2-YF-BBiz CAR-T) were taken, and the CAR-T cells and target cells or suspension tumor cells in the wells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing target cells. At specific time points (24h, 48h, 72h, 96h, etc.), cells were collected for flow cytometry counting to calculate the target cell killing efficiency and the proliferation capacity of CAR-T cells during this process. The results are shown in Figure 4h. When stimulated by K562-HER2 cells, α-HER2-BBiz CAR-T cells exhibited a significant proliferation advantage, ensuring sufficient CAR-T cells to perform effector functions and achieve tumor cell clearance. In contrast, control cells α-HER2-BBz CAR-T and α-HER2-YF-BBiz CAR-T cells almost lost their proliferation capacity after K562-HER2 stimulation, making it difficult to control the rapid proliferation of tumor cells. Cells were stained with anti-PD1-APC, anti-TIM3-PE, or anti-TIGIT-BV421, incubated on ice for 30 min, washed once, and then analyzed by flow cytometry to determine cell exhaustion levels. As shown in Figure 4i, BBiz CAR-T exhibits a significantly weaker level of exhaustion during tumor cell killing. During long-term in vitro stimulation of tumor cells, α-HER2-BBiz CAR-T exhibits lower activation-induced cell death due to milder signaling, maintains more cellular effector functions, and faces less tumor stress, thus showing weaker exhaustion.

[0172] In vivo antitumor capacity assay: MDA-MB-231-HER2-luciferase tumors were implanted in situ into the mammary fat pads of mice, with a specific cell count of 2*102 6 / each, 14 days later, injected 4*10 via tail vein 5CAR-T cells (α-HER2-BBiz CAR-T or α-HER2-BBz CAR-T) were used, and the growth of the in situ tumor was recorded using calipers. Mice treated with BBiz CAR-T showed better control of in situ tumor growth. Around 45 days after tumor formation, the tumor had metastasized to multiple sites throughout the body (including but not limited to the axilla and liver). At this time, in vivo fluorescence imaging was used to record the tumor metastasis in the mice. Mice treated with BBiz CAR-T showed fewer tumor metastases (Figure 4j).

[0173] In vivo antitumor activity assay: HCC1954-luciferase tumors were implanted in situ into the mammary fat pads of mice, with a specific cell count of 2*102. 6 / each, 14 days later, injected via tail vein with 1*10 5 Each mouse received one CAR-T cell (α-HER2-BBiz CAR-T or α-HER2-BBz CAR-T), and the growth of the in situ tumor was recorded using calipers. Approximately 25 days after CAR-T infusion therapy, tumors in all CAR-T treatment groups completely regressed; approximately 14 days later, 2*10 cells were reloaded into the contralateral mammary fat pad of the mice. 6 Mice were treated with HCC1954 to simulate the tumor recurrence process. The growth of the recurrent tumor was then recorded using calipers. Mice that had previously received α-HER2-BBiz CAR-T therapy showed better control of recurrent tumors, which may be due to the better in vivo persistence of α-HER2-BBiz (Figure 4k).

[0174] Example 5

[0175] α-GD2-BBiz is based on the α-GD2-BBz sequence. α-GD2-BBz includes 14g2a scFv, the IgG and CD8α hinge region, the CD8α transmembrane region, and the intracellular regions of 4-1BB and CD3ζ. α-GD2-BBz CAR-T has strong antigen-induced activation signals and background signals. Based on α-GD2-BBz, we used gene synthesis technology to insert PD-ITSM (EQTEYATIV) between 4-1BB and CD3ζ, and named it α-GD2-BBiz.

[0176] The amino acid sequence of α-GD2-BBiz is shown in EEQ ID NO: 17, specifically:

[0177] The nucleotide sequence of α-GD2-BBiz is shown in SEQ ID NO: 22, specifically:

[0178] Preparation method of α-GD2-BBiz-CAR-T: α-GD2-BBiz was cloned into a lentiviral expression pHAGE vector and identified as correct. The CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2.G at a ratio of 10:7.5:3.5, and then transfected into 293FT cells using calcium phosphate transfection to produce lentiviral particles. After 48 hours, the viral supernatant was collected, filtered through a 0.45 μm filter membrane, centrifuged at 27,000 rpm for 2 hours, and the supernatant was discarded. The virus was dissolved in serum-free medium, and human peripheral blood mononuclear cells activated with α-CD3 / 28-beads for 24 hours were added. After 48 hours, the virus and beads were removed. At this point, the culture system should only contain human primary T cells. Two days later, fluorescent protein-positive T cells were sorted by flow cytometry, thus obtaining α-GD2-BBiz CAR-T cells, with α-GD2-BBz CAR-T cells and α-GD2-YF-BBiz CAR-T cells serving as controls. A series of indicators were evaluated for CAR-T cells, including CAR membrane level, CD4 and CD8 subset ratio, in vitro cytokine secretion, in vitro killing ability, in vitro proliferation ability, in vitro apoptosis level, in vitro survival rate, and in vivo antitumor effect.

[0179] CAR cell surface level detection: Anti-HA-Rabbit-IgG was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. Then, anti-Rabbit-IgG-AF647 was used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS. The signal intensity of AF647 was detected by flow cytometry. As shown in Figure 5a, the surface level of the prepared α-GD2-BBiz CAR-T cells was similar to or slightly lower than that of the control α-GD2-BBz CAR-T cells. Meanwhile, the other control cell line, α-GD2 YF-BBiz CAR-T, had the lowest surface level.

[0180] CD4 and CD8 subset ratio analysis: Anti-CD4-APC and anti-CD8-PE-cy7 antibodies were used for staining at 4℃ for 30 min, followed by washing away excess antibody with PBS and flow cytometry analysis. As shown in Figure 5b, the prepared α-GD2-BBiz CAR-T cells had similar CD4 and CD8 subset ratios to α-GD2-BBz CAR-T cells and α-GD2-YF-BBiz CAR-T cells.

[0181] Basal activation level detection: The basal activity levels of α-GD2-BBiz CAR-T, α-GD2-BBz CAR-T, and α-GD2-YF-BBiz CAR-T cells were measured before antigen exposure. As shown in Figure 5c, α-GD2-BBiz CAR-T cells exhibited lower basal activation levels than α-GD2-BBz CAR-T cells and α-GD2-YF-BBiz CAR-T cells.

[0182] Detection of CAR-T cell basal cell death and proliferation signals, and exhaustion levels: CAR-T cells (α-GD2-BBiz CAR-T, α-GD2-BBz CAR-T, and α-GD2-YF-BBiz CAR-T) were incubated with Annexin V at room temperature in the dark for 10 min. After washing once with 1* binding buffer, PI was added, and flow cytometry was performed to obtain basal cell death signals. After CAR-T cell fixation and nuclear transfection, anti-Ki67-APC was used for overnight staining, followed by washing with PBS, and flow cytometry was performed to obtain basal cell proliferation signals. After CAR-T cell fixation and nuclear transfection, anti-PD1-APC, anti-TIM3-PE, or anti-TIGIT-BV421 was used for staining, followed by washing with PBS, and flow cytometry was performed to obtain basal cell exhaustion levels. As shown in Figures 5d-e, during the culture process, α-GD2-BBiz CAR-T cells exhibited lower basal cell death and similar proliferation signals compared to α-GD2-BBz CAR-T cells (Figure 5d); α-GD2-BBiz CAR-T cells had a lower basal cell exhaustion level compared to α-GD2-BBz CAR-T cells (Figure 5e).

[0183] In vitro cytokine secretion level detection: For adherent tumor cells, target cells were seeded into wells at an appropriate density 24 hours in advance. An appropriate density meant that the target cell density in the wells was approximately 70% after 24 hours. For suspension tumor cells, target cells did not need to be seeded in advance and could be processed simultaneously with CAR-T cells. An appropriate number of CAR-T cells were taken, and the CAR-T cells and target cells or suspension tumor cells in the wells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing target cells. After 20 hours, cytokine efflux was blocked using BFA for 4 hours. Cells were then collected, fixed, and punched into wells. The cells were stained overnight with anti-IFNg-APC, anti-IL2-BV421, and anti-TNFα-PE antibodies. Excess antibodies were then washed away with PBS, and flow cytometry was performed. Alternatively, 24 hours after CAR-T cell stimulation using the same method, the culture supernatant was collected, diluted appropriately, and the levels of IFNg, IL2, and TNFα were detected using an ELISA kit. As shown in Figure 5f, BBiz CAR-T cells exhibited lower cytokine secretion levels after stimulation with target cells.

[0184] In vitro apoptosis level detection: For adherent tumor cells, target cells were seeded into wells at an appropriate density 24 hours in advance. An appropriate density meant that the target cell density in the wells was approximately 70% after 24 hours. For suspension tumor cells, target cells did not need to be seeded in advance and could be treated simultaneously with CAR-T cells. An appropriate number of CAR-T cells were taken, and the CAR-T cells and target cells or suspension tumor cells in the wells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing target cells. At specific time points, such as 24 hours, 48 ​​hours, 72 hours, and longer, cells were stained with Annexin V-APC, incubated at room temperature in the dark for 10 minutes, washed once, and PI was added for flow cytometry analysis. As shown in Figure 5g, after stimulation with target cells, α-GD2-BBiz CAR-T cells exhibited a lower activation-induced apoptosis level.

[0185] In vitro proliferation and killing capacity assays: For adherent tumor cells, target cells were seeded into wells at an appropriate density 24 hours in advance. An appropriate density meant that the target cell density in the wells was approximately 70% after 24 hours. For suspension tumor cells, target cells did not need to be seeded in advance and were treated simultaneously with CAR-T cells. Appropriate numbers of CAR-T cells (α-GD2-BBiz CAR-T cells, α-GD2-BBz CAR-T cells, and α-GD2-YF-BBiz CAR-T cells) were taken, and the CAR-T cells and Nalm6 cells in the wells were washed once with x-vivo15 medium without any serum or cytokine additives. The CAR-T cells were then resuspended in the same medium to an appropriate density and added to wells containing Nalm6 cells. At specific time points (24h, 48h, 72h, 96h), cells were collected for flow cytometry counting to calculate the target cell killing efficiency and the CAR-T cell proliferation capacity during this process. Cells were fixed and transnuclearized, then stained overnight with anti-Ki67-APC. After washing once, the samples were analyzed by flow cytometry to detect the proliferation signal level of CAR-T cells. As shown in Figures 5h-i, α-GD2-BBiz CAR-T cells did not show a significantly defective proliferation signal after stimulation with target cells (5h). After stimulation with target cells, α-GD2-BBiz CAR-T cells showed a significant advantage in expansion and a stronger killing ability against target cells (5i).

[0186] Example 6

[0187] Preparation method of Jurkat-α-CD19-BBz / BBiz / BBzi: α-CD19-BBz / BBiz / BBzi was constructed into a pHAGE vector. The CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2.G at a ratio of 10:7.5:3.5 and transfected into 293FT cells using calcium phosphate transfection to produce lentiviral particles. After 48 h, the viral supernatant was collected. The virus did not need to be concentrated. 3 mL of the viral stock solution was used to infect 1 million Jurkat cells for 24 h. Afterward, the virus was removed, and the cells were cultured normally. GFP-positive cells, i.e., Jurkat-CAR cells, were sorted by flow cytometry. Jurkat-CAR cells were stimulated with Raji cells at a 1:1 ratio and incubated at 37°C for the appropriate time. Cells were then lysed using RIPA, and protein samples were collected. Western blotting was used to identify the phosphorylation levels of CAR molecules and juxtamembranous signaling molecules. As shown in Figure 6a, based on the phosphorylation levels of each activated molecule, the BBiz signal was weaker than BBz, while the BBzi signal was stronger than BBz.

[0188] When the threonine or tyrosine residues of PD1-ITSM were mutated to alanine and phenylalanine, respectively, Jurkat-CAR was stimulated 1:1 with Raji cells, as shown in Figure 6b. In this case, the activation level of ITSM-CAR after the tyrosine mutation was essentially the same as that of BBz, while the threonine mutation did not affect the signal difference. This indicates that PD1-ITSM functions as a signaling element in the CAR molecule, rather than merely as a random linker effect, and that its function is tyrosine-dependent.

[0189] Raji cells were stimulated 1:1 with Jurkat-CAR cells, and protein samples were collected after lysis and subjected to Co-IP experiments with added antibodies. Western blotting was then used to detect the recruitment of kinases and phosphatases. As shown in Figure 6c, BBiz recruited more phosphatases SHP1 and SHP2 than BBz; BBzi recruited more kinase Lck than BBz, especially its active form. This differential recruitment of kinases and phosphatases may explain the different signaling patterns mediated by BBiz and BBzi.

[0190] Raji cells were stimulated 1:1 with tyrosine-mutated Jurkat-CAR cells. After lysis, protein samples were collected and antibody was added for Co-IP experiments. Western blotting was then used to detect the recruitment of kinases and phosphatases. As shown in Figure 6d, after the tyrosine mutation in PD1-ITSM, the differential recruitment of kinase Lck and phosphatases SHP1 and SHP2 disappeared.

[0191] Using Raji cells to stimulate Jurkat-CAR at a 1:1 ratio, as shown in Figure 6e, after mutating the C-terminal acidic amino acid-rich region of 4-1BB to neutral amino acids (C-terminal D / E mutation to G / S), the activation levels of BBiz and BBz were comparable or even stronger. After mutating the basic amino acid-rich region of CD3ζ to neutral amino acids (R mutation to G / S among the ITMs), the activation levels of BBzi and BBz were essentially the same. When the C-terminal acidic amino acid-rich region of 4-1BB and the basic amino acid-rich region of CD3ζ were mutated simultaneously, the activation levels of BBz, BBiz, and BBzi were comparable. This suggests that PD1-ITSM may regulate CAR signaling by differentially recruiting kinases and phosphatases through cooperation with surrounding charged motifs. Specifically, PD1-ITSM weakens CAR activation through cooperation with the adjacent 4-1BB-ARS motif; after mutating 4-1BB-ARS, the activation levels of BBiz and BBz are comparable or stronger. PD1-ITSM enhances CAR activation through cooperation with the adjacent CD3z-BRS motif; after mutating CD3z-BRS, the activation levels of BBzi and BBz are comparable. When both the charged motifs of 4-1BB and CD3z are mutated, the signaling order is: BBz = BBiz = BBzi. As shown in Figure 6f, when the charged enrichment region surrounding PD1-ITSM in the CAR molecule is mutated, the differential recruitment of kinase Lck and phosphatase SHP2 almost disappears.

[0192] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An intracellular domain for constructing a chimeric antigen receptor, comprising a co-stimulatory signal transduction region and a CD3ζ intracellular region connected in sequence, wherein the CD3ζ intracellular region is connected to a PD1-ITSM motif.

2. The intracellular domain as described in claim 1, characterized in that, The PD1-ITSM motif is linked to the front or back end of the CD3ζ intracellular region; And / or, the amino acid sequence of the PD1-ITSM motif is as shown in SEQ ID NO: 1; And / or, the co-stimulatory signal transduction region is selected from one or more intracellular regions of CD27, CD28, CD134, 4-1BB, OX40 or ICOS.

3. The intracellular domain as described in claim 1, characterized in that, The co-stimulatory signal transduction region is selected from the intracellular region of 4-1BB; preferably, the amino acid sequence of the intracellular region of 4-1BB is shown in SEQ ID NO: 2; And / or, the amino acid sequence of the CD3ζ intracellular region is shown in SEQ ID NO:

3.

4. Use of the intracellular domain as described in any one of claims 1 to 3 in the preparation of chimeric antigen receptors.

5. A chimeric antigen receptor suitable for regulating CAR-T signaling, comprising: The extracellular domain, transmembrane domain, and intracellular domain are connected sequentially. The extracellular domain includes an antigen recognition region and a hinge region; The intracellular domain includes the intracellular region domain as described in any one of claims 1 to 3.

6. The chimeric antigen receptor as described in claim 5, characterized in that, It also includes one or more of the following features: a. The antigen recognition region is selected from single-chain antibodies against tumor surface antigens, wherein the single-chain antibodies are selected from one or more of CD19, Glypican-3 (GPC3), mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD133, CD138, BCMA, Fibroblast activation protein (FAP), CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, and GD2; b. The transmembrane domain is selected from one or more transmembrane regions of CD8α, CD4, CD28, OX40 or H2-Kb.

7. The chimeric antigen receptor as described in claim 6, characterized in that, It also includes one or more of the following features: c. The amino acid sequence of the single-chain antibody is shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; d. The transmembrane domain is selected from the transmembrane region of CD8α, and the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 12; e. The regulation of CAR-T signaling refers to either weakening or enhancing CAR-T cell activation signals.

8. The chimeric antigen receptor as described in claim 5, characterized in that, The amino acid sequences of the chimeric antigen receptor are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:

17.

9. A polynucleotide sequence selected from: (1) Encoding the intracellular domain of any one of claims 1-3 or the chimeric antigen receptor of any one of claims 5-8; and / or The complementary sequence of the polynucleotide sequence described in (2)(1).

10. A nucleic acid construct comprising the polynucleotide sequence of claim 9; Preferably, the nucleic acid construct is a vector; more preferably, the nucleic acid construct is a lentiviral vector containing a replication start site, a 3'LTR, a 5'LTR, and the polynucleotide sequence of claim 9.

11. A lentiviral vector system comprising the nucleic acid construct and lentiviral vector auxiliary components as described in claim 10.

12. A genetically modified T cell, characterized in that, The T cells contain the polynucleotide sequence of claim 9, or the nucleic acid construct of claim 10, or are infected with the lentiviral vector system of claim 11.

13. The use of any one of the intracellular domains of claims 1 to 3, the chimeric antigen receptor of any one of claims 5 to 8, or the polynucleotide sequence of claim 9, or the nucleic acid construct of claim 10, or the lentiviral vector system of claim 11 in the preparation of products for any one or more of the following purposes: (1) preparing CAR-T cells; (2) regulating the secretion of cytokines IFN-γ, IL-2, and TNF from CAR-T cells; (3) regulating the degranulation ability of CAR-T cells; (4) promoting the in vivo antitumor ability of CAR-T cells; (5) enhancing the sustained proliferation ability of CAR-T cells after stimulation by target cells; (6) weakening the activation-induced cell death of CAR-T cells; (7) regulating the activation signaling ability of CAR-T cells.

14. The use of any of the intracellular domains of claims 1 to 3, the chimeric antigen receptor of any one of claims 5 to 8, the polynucleotide sequence of claim 9, the nucleic acid construct of claim 10, the lentiviral vector system of claim 11, or the gene-modified T cells of claim 12 in the preparation of tumor therapeutic products.

15. A treatment method for tumors, characterized in that, The treatment method includes administering a therapeutic amount of the genetically modified T cells of claim 12 to a subject in need.

16. The application as described in claim 14 or the treatment method as described in claim 15, wherein the tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, acute myeloid leukemia, hepatocellular carcinoma, melanoma, clear cell carcinoma of the ovary, or squamous cell carcinoma of the lung.

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