Engineered immune cells, methods of making and using the same

Abstract

The present application provides engineered immune cells, methods of making the same, and uses thereof. The engineered immune cells of the present application have enhanced resistance to immune suppression by the tumor microenvironment and have superior immune efficacy, e.g., superior tumor killing ability.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and immunotherapy, and more specifically, to an engineered immune cell, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease that seriously threatens human health. The continuous rise in the incidence and mortality of malignant tumors results in more than 220 billion yuan in medical expenses annually, creating a severe social and familial burden. Furthermore, the significant urban-rural disparities and uneven regional distribution of cancer types and treatment in my country exacerbate the already challenging situation for prevention and control. Despite continuous advancements in traditional treatments such as surgery, radiotherapy, and chemotherapy, their effectiveness remains limited for recurrent, refractory, or metastatic tumors. In recent years, adoptive cell transfer therapy (ACT), represented by chimeric antigen receptor T-cell (CAR-T) therapy, has made groundbreaking progress in the treatment of hematologic malignancies (such as B-cell lymphoma and leukemia), bringing hope for long-term survival to some patients.

[0003] However, CAR-T and other immunotherapy methods have shown significantly poor efficacy in treating solid tumors. The core obstacle to this predicament lies in the complex tumor microenvironment (TME). The TME, composed of tumor cells, stromal cells, immune cells, and the extracellular matrix, exhibits a series of physical and chemical characteristics distinct from normal tissues. Specifically, the rapid proliferation and abnormal metabolism of tumor cells lead to a series of conditions known as "metabolic stress" within the TME, including severe depletion of oxygen and key nutrients (such as glucose and glutamine), accumulation of metabolic waste products (such as lactic acid), acidic pH, and elevated levels of reactive oxygen species (ROS). Existing research indicates that the effector functions of immune cells (including conventional CAR-T cells) entering the TME are severely suppressed. For example, a nutrient-deprived environment cannot meet the high energy demands required for immune cell activation, proliferation, and cytotoxicity; acidic pH and oxidative stress directly induce apoptosis or functional exhaustion of immune cells. Therefore, the metabolic inhibitory nature of the TME is currently a key bottleneck limiting the application of immunotherapy in solid tumors.

[0004] To address this challenge, existing technologies attempt to use combination therapies (such as immune checkpoint inhibitors) or simple genetic modifications to immune cells, but the results are often unsatisfactory or fail to achieve comprehensive adaptation to the complex metabolic adversity of the tumor microenvironment (TME). Therefore, there is an urgent need in this field to develop a novel, engineered immune cell that can actively resist or even utilize the metabolic inhibitory characteristics of the TME, maintaining normal survival, proliferation, and anti-tumor activity in the tumor local area, thereby effectively improving the immunotherapy efficacy for solid tumors. Summary of the Invention

[0005] This invention aims to overcome the technical shortcomings of existing immunotherapy (especially CAR-T therapy for solid tumors), where immune cells suffer functional impairment due to metabolic inhibition by the tumor microenvironment (TME) (such as nutritional deficiencies and oxidative stress). To this end, this invention provides an engineered immune cell capable of actively adapting to and resisting TME metabolic adversity, its preparation method, and its applications.

[0006] Specifically, the inventors of this application, through inventive experimental research, have for the first time revealed that the content of vitamin B2 (riboflavin) in the tumor microenvironment is significantly lower than that in peripheral tissues. Further research found that vitamin B2 deficiency is one of the key factors inhibiting T cell proliferation and cytotoxic function, and inducing T cell exhaustion. Based on this novel discovery, this invention proposes a strategy to engineer immune cells to enhance their vitamin B2 uptake capacity, thereby giving them the advantage of maintaining normal metabolism and function even in a low-vitamin B2 microenvironment. This leads to the present invention.

[0007] Engineered immune cells

[0008] Specifically, in a first aspect, this application provides an engineered immune cell comprising a modification that endows the immune cell with an enhanced ability to take up vitamin B2. The ability of the cell to take up vitamin B2 can be assessed by detecting intracellular VB2 levels using, for example, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS).

[0009] The ability of immune cells to take up vitamin B2 can be enhanced by increasing the level of vitamin B2 transporter proteins in the immune cells. Therefore, in some embodiments, the modification is used to increase the expression level of vitamin B2 transporter proteins in the immune cells (e.g., upregulating the expression level of endogenous vitamin B2 transporter proteins in the immune cells, and / or causing the immune cells to express exogenous vitamin B2 transporter proteins).

[0010] In some embodiments, the modification includes one or more selected from the following:

[0011] (i) Enhance the promoter of the gene encoding the vitamin B2 transporter.

[0012] (ii) Linking or introducing the vitamin B2 transporter encoding gene into an enhancer,

[0013] (iii) Increase the copy number of the gene encoding the vitamin B2 transporter.

[0014] (iv) Introduce exogenous polynucleotides encoding vitamin B2 transporter proteins.

[0015] It will be readily understood by those skilled in the art that increasing the copy number of the vitamin B2 transporter encoding gene does not mean that each copy of the gene must have the exact same nucleotide sequence, as long as each copy can perform its original functional activity (e.g., achieve protein expression).

[0016] In some embodiments, the modification includes the exogenous introduction of a polynucleotide encoding a vitamin B2 transporter. In a second aspect, this application provides an engineered immune cell comprising an exogenously introduced polynucleotide encoding a vitamin B2 transporter.

[0017] In certain embodiments of the first or second aspect of this application, the vitamin B2 transporter is selected from: SLC52A1, SLC52A2, SLC52A3, their orthologs, homologs, functional variants, and functional fragments, and any combination thereof. In certain embodiments, the vitamin B2 transporter is selected from: SLC52A3 and its orthologs, homologs, variants, and functional fragments. In certain embodiments, the functional variant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to its derived sequence, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions of 1, 2, 3, 4, or 5 amino acids (preferably conserved substitutions) with respect to its derived sequence, and having vitamin B2 transport activity.

[0018] In some embodiments of the first or second aspect of this application, the vitamin B2 transporter comprises an amino acid sequence as shown in SEQ ID NO: 17 or 24, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 17 or 24.

[0019] In some embodiments, the exogenously introduced polynucleotide encoding the vitamin B2 transporter protein is derived from the same species as the immune cells. In some embodiments, the immune cells are murine, and the vitamin B2 transporter protein comprises the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 17. In some embodiments, the immune cells are human, and the vitamin B2 transporter protein comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 24.

[0020] In some embodiments of the first or second aspect of this application, the immune cells are selected from the group consisting of: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδ T cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof. In other embodiments, the immune cells are selected from: chimeric antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), chimeric antigen receptor macrophages (CAR-M), chimeric antigen receptor NK cells, chimeric antigen receptor γδ T cells, tumor-infiltrating lymphocytes (TILs) (e.g., tumor-infiltrating T lymphocytes), and any combination thereof. In some embodiments, the immune cells comprise an antigen receptor (e.g., T cell receptor (TCR), chimeric antigen receptor (CAR)) that specifically binds to disease-associated antigens, or a polynucleotide encoded thereon.

[0021] In some embodiments, the immune cells comprise a CAR or its encoded polynucleotide, the CAR comprising:

[0022] (a) An extracellular antigen-binding domain that specifically binds to disease-associated antigens;

[0023] (b) Transmembrane domains; and

[0024] (c) Intracellular signal transduction domains.

[0025] In some embodiments, the extracellular antigen-binding domain specifically binds to tumor-associated antigens, autoimmune disease-associated antigens, or viral antigens. In some embodiments, the extracellular antigen-binding domain specifically binds to antigens selected from the group consisting of: CD19, BCMA, CD22, CD20, CD30, CD33, CD123, GD2, HER2, MSLN, Claudin 18.2, GPC3, PSMA, TROP2, and any combination thereof.

[0026] In some embodiments, the extracellular antigen-binding domain specifically binds to TROP2. In some embodiments, the extracellular antigen-binding domain comprises: VH CDR1, VH CDR2, and VH CDR3 contained in VH as shown in SEQ ID NO: 3, and / or VL CDR1, VL CDR2, and VL CDR3 contained in VL as shown in SEQ ID NO: 4. In some embodiments, the CDRs are defined by the Kabat, IMGT, AbM, or Chothia numbering system. In some embodiments, the extracellular antigen-binding domain comprises VH CDR1 shown in SEQ ID NO: 5, VH CDR2 shown in SEQ ID NO: 6, and VH CDR3 shown in SEQ ID NO: 7; and / or VL CDR1 shown in SEQ ID NO: 8, VLCDR2 shown in SEQ ID NO: 9, and VL CDR3 shown in SEQ ID NO: 10. In some embodiments, the CDRs are defined by the Kabat numbering system. In some embodiments, the extracellular antigen-binding domain comprises VH as shown in SEQ ID NO: 3 and / or VL as shown in SEQ ID NO: 4. In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 2.

[0027] In some embodiments, the transmembrane structural domain is derived from the group consisting of: CD3, CD4, CD137, CD80, CD86, CD152, PD-1, CD8 (e.g., CD8α), and CD28.

[0028] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0029] In some embodiments, the intracellular signal transduction domain further comprises a co-stimulatory signal transduction domain. In some embodiments, the co-stimulatory domain is derived from a co-stimulatory molecule selected from the group consisting of: ligands of CD27, CD137, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CD28, 4-1BB, OX-40, and any combination thereof.

[0030] In some embodiments, the CAR further includes a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8 (e.g., CD8α).

[0031] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of its polypeptide. In some embodiments, the signal peptide is a signal peptide derived from IgGκ, CD8α, or GM-CSF. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 27.

[0032] In some embodiments, the CAR comprises: an extracellular antigen-binding domain (e.g., scFv) that specifically binds to TROP2, a hinge domain derived from CD8, a transmembrane domain and a co-stimulatory domain derived from CD28, and a primary intracellular signaling domain derived from CD3ζ.

[0033] In some implementations, the hinge domain, transmembrane domain, co-stimulatory domain, and primary intracellular signal transduction domain are derived from the same species as the immune cell.

[0034] In some embodiments, the immune cells are murine. In some embodiments, the hinge domain, transmembrane domain, co-stimulatory domain, and primary intracellular signaling domain are murine. In some embodiments, the hinge domain comprises the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the primary intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO: 18 or 29.

[0035] In some embodiments, the immune cells are human. In some embodiments, the hinge domain, transmembrane domain, co-stimulatory domain, and primary intracellular signaling domain are human. In some embodiments, the hinge domain comprises the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, the primary intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO: 25 or 31.

[0036] In some embodiments of the first or second aspect of this application, the engineered immune cells comprise a first polynucleotide encoding the CAR and a second polynucleotide exogenously introduced encoding a vitamin B2 transporter. In some embodiments, the vitamin B2 transporter is as defined in any of the foregoing embodiments.

[0037] In some implementations, the first polynucleotide and the second polynucleotide are located in different expression cassettes or in the same expression cassette.

[0038] In some embodiments, the first polynucleotide and the second polynucleotide are co-located in the same expression cassette and are linked by a self-cleaving adapter (e.g., a 2A adapter, such as P2A) encoding a polynucleotide. In some embodiments, the engineered immune cells comprise a polynucleotide encoding the amino acid sequence shown in any one of SEQ ID NO: 1, 19, 28, or 30. In some embodiments, the engineered immune cells are murine and comprise a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 1 or 28. In some embodiments, the engineered immune cells are human and comprise a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 19 or 30.

[0039] In some implementations, the first polynucleotide and / or the second polynucleotide are present in a form that is integrated into or not integrated into the genome.

[0040] Methods for preparing engineered immune cells

[0041] In a third aspect, this application provides a nucleic acid molecule or group of nucleic acid molecules comprising: a first nucleotide sequence encoding a chimeric antigen receptor (CAR), and a second nucleotide sequence encoding a vitamin B2 transporter. The first nucleotide sequence and the second nucleotide sequence are located on the same or different nucleic acid molecules.

[0042] In some embodiments, the second nucleotide sequence is not naturally occurring. In some embodiments, the second nucleotide sequence comprises the coding region (CDS) of the vitamin B2 transporter.

[0043] In some embodiments, the first nucleotide sequence and the second nucleotide sequence are located on the same nucleic acid molecule. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are located in the same expression cassette. In some embodiments, the first nucleotide sequence is operatively linked to the second nucleotide sequence by a nucleotide sequence encoding a self-cleaving adapter (such as a 2A adapter, such as P2A) or a nucleotide sequence encoding an internal ribosome entry site (IRES). In some embodiments, the nucleic acid molecule or group of nucleic acid molecules comprises a nucleotide sequence encoding an amino acid sequence shown in any one of SEQ ID NO: 1, 19, 28, 30.

[0044] In some embodiments, the CAR is as defined in any of the embodiments of the preceding aspect. In some embodiments, the vitamin B2 transporter is as defined in any of the embodiments of the preceding aspect.

[0045] In the fourth aspect, this application provides one or more vectors that contain the nucleic acid molecules or nucleic acid molecule groups of the third aspect.

[0046] In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus (AAV) vectors. In some embodiments, the viral vector is a retroviral vector (e.g., a gamma-retroviral vector).

[0047] In a fifth aspect, this application provides a method for constructing engineered immune cells according to the first or second aspect of this application, comprising:

[0048] (1) Provide immune cells; and,

[0049] (2) Introducing the immune cells with the modifications defined in the first aspect, or introducing the immune cells with a polynucleotide containing a sequence encoding a vitamin B2 transporter.

[0050] Those skilled in the art are familiar with various methods for introducing polynucleotides into cells. The polynucleotide can be introduced into the immune cells by any suitable method, such as using stable transformation methods to integrate the polynucleotide into the cell's genes, or using transient transformation methods (e.g., microinjection, electroporation, or particle bombardment) to transiently express the polynucleotide in the cells. In some embodiments, the polynucleotide or a vector containing the polynucleotide can be delivered into the immune cells using viruses or liposomes.

[0051] In some implementations, the immune cells are as defined in the first or second aspect.

[0052] In some embodiments, the vitamin B2 transporter is as defined in the first or second aspect.

[0053] In some embodiments, the engineered immune cells comprise (i) a CAR or its encoding polynucleotide, and (ii) the modification described in the first aspect or the exogenously introduced polynucleotide encoding the vitamin B2 transporter as described in the second aspect. Such cells can be obtained by further modifying CAR-containing immune cells, or by simultaneously modifying native immune cells for both the CAR and the vitamin B2 transporter.

[0054] For example, in some embodiments, the method includes:

[0055] (1) Provide immune cells containing CAR (e.g., CAR-T cells, CAR-M cells); and

[0056] (2) Introducing the modifications defined in the first aspect into the immune cells described in (1), or introducing into the immune cells a polynucleotide containing a sequence encoding a vitamin B2 transporter.

[0057] In other embodiments, the immune cells described in step (1) (e.g., immune cells isolated from a donor) are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδ T cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof. In some embodiments, the immune cells are activated. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are macrophages, such as M1 macrophages.

[0058] In some embodiments, the method further includes introducing a polynucleotide encoding a CAR into the immune cells. For example, in some embodiments, the method includes:

[0059] (1) Provide the said immune cells, and,

[0060] (2) Introducing a polynucleotide comprising a sequence encoding a CAR sequence and a sequence encoding a vitamin B2 transporter into the immune cells described in (1). In some embodiments, step (2) includes introducing a nucleic acid molecule or group of nucleic acid molecules of the third aspect, or one or more vectors of the fourth aspect, into the immune cells. In another aspect, this application provides a method for constructing engineered immune cells of the first or second aspect of this application, comprising:

[0061] (1) Provide immune cells; and,

[0062] (2) Introducing the immune cells with a first polynucleotide encoding a chimeric antigen receptor sequence and a second polynucleotide encoding a vitamin B2 transporter sequence;

[0063] The chimeric antigen receptor is capable of specifically binding to disease-associated antigens.

[0064] In some embodiments, the first polynucleotide and the second polynucleotide are each located in different expression cassettes, or are located together in the same expression cassette. In some embodiments, the first polynucleotide and the second polynucleotide are located together in the same expression cassette, and are linked by a self-cleaving adapter (e.g., a 2A adapter, such as P2A) encoding a polynucleotide. In some embodiments, the first polynucleotide and / or the second polynucleotide are present in a form that is either non-integrated or integrated into the immune cell genome.

[0065] In some embodiments, the method includes introducing a polynucleotide encoding an amino acid sequence shown in any one of SEQ ID NO: 1, 19, 28, or 30 into the immune cells. In some embodiments, the immune cells are murine, and the method includes introducing a polynucleotide encoding an amino acid sequence shown in SEQ ID NO: 1 or 28 into the immune cells. In some embodiments, the immune cells are human, and the method includes introducing a polynucleotide encoding an amino acid sequence shown in SEQ ID NO: 19 or 30 into the immune cells.

[0066] In some implementations, the chimeric antigen receptor is as defined above.

[0067] In some embodiments, the vitamin B2 transporter is as defined above.

[0068] In some embodiments, the immune cells described in step (1) are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδT cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof. In some embodiments, the immune cells are immune cells isolated from a donor.

[0069] Application of engineered immune cells

[0070] In a sixth aspect, this application provides a pharmaceutical composition comprising engineered immune cells as described in the first or second aspect of the invention, nucleic acid molecules or groups of nucleic acid molecules as described in the third aspect, or one or more carriers as described in the fourth aspect, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises additional pharmaceutically active agents; in some embodiments, the additional pharmaceutically active agents are selected from: immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies), additional engineered immune cells (e.g., TCR-T, CAR-T, CAR-M), and any combination thereof.

[0071] In a seventh aspect, this application provides the use of engineered immune cells as described in the first or second aspect, nucleic acid molecules or groups of nucleic acid molecules as described in the third aspect, one or more carriers as described in the fourth aspect, or pharmaceutical compositions as described in the sixth aspect in the preparation of a medicament for use in a subject to enhance immune cell activity, strengthen immune response, and / or prevent and / or treat tumors or infections or autoimmune diseases.

[0072] In some embodiments, the immune cells are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδT cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof.

[0073] In some embodiments, the enhancement of immune cell activity includes: promoting immune cell differentiation (e.g., memory differentiation), enhancing immune cell function, and / or inhibiting immune cell depletion.

[0074] In some embodiments, the immune response includes cellular immune response and / or humoral immune response.

[0075] In some embodiments, the tumor is selected from solid tumors or hematologic malignancies. In some embodiments, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer), melanoma, colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and any combination thereof.

[0076] In some embodiments, the autoimmune disease is selected from: systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease, ulcerative colitis, and any combination thereof.

[0077] In some embodiments, the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections;

[0078] In some implementations, the subject is a mammal, such as a human.

[0079] In some embodiments, the engineered immune cells, nucleic acid molecules or groups of nucleic acid molecules, one or more carriers, or pharmaceutical compositions are administered in combination with an additional pharmaceutically active agent (e.g., simultaneously, separately, or sequentially). In some embodiments, the additional pharmaceutically active agent is selected from: immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies), additional engineered immune cells (e.g., TCR-T, CAR-T, CAR-M), and any combination thereof.

[0080] In an eighth aspect, this application provides a method for enhancing the activity of immune cells in a subject, strengthening immune responses, and / or preventing and / or treating tumors, infections, or autoimmune diseases in a subject, comprising providing to a subject in need an engineered immune cell as described in the first or second aspect, a nucleic acid molecule or nucleic acid molecule group as described in the third aspect, one or more carriers as described in the fourth aspect, or a pharmaceutical composition as described in the sixth aspect.

[0081] In some embodiments, the immune cells are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδT cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof.

[0082] In some embodiments, the enhancement of immune cell activity includes: promoting immune cell differentiation (e.g., memory differentiation), enhancing immune cell function, and / or inhibiting immune cell depletion.

[0083] In some embodiments, the immune response includes cellular immune response and / or humoral immune response.

[0084] In some embodiments, the tumor is selected from solid tumors or hematologic malignancies. In some embodiments, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer), melanoma, colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and any combination thereof.

[0085] In some embodiments, the autoimmune disease is selected from: systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease, ulcerative colitis, and any combination thereof.

[0086] In some implementations, the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections.

[0087] In some implementations, the subject is a mammal, such as a human.

[0088] In some embodiments, the engineered immune cells, nucleic acid molecules or groups of nucleic acid molecules, one or more carriers, or pharmaceutical compositions are administered in combination with an additional pharmaceutically active agent (e.g., simultaneously, separately, or sequentially). In some embodiments, the additional pharmaceutically active agent is selected from: immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies), additional engineered immune cells (e.g., TCR-T, CAR-T, CAR-M), and any combination thereof.

[0089] The immune cells, nucleic acids or nucleic acid molecules, one or more carriers, or pharmaceutical compositions can be formulated into dosage forms compatible with their intended route of administration. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the necessary dose of the reagents described herein into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. The pharmaceutical preparation can be formulated in dosage units for ease of administration. Dosage unit form refers to physically discrete units suitable for use as a single dose on the subject to be treated; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect when combined with the desired pharmaceutical carrier.

[0090] Application of Vitamin B2 in Enhancing Immune Cell Activity and / or Strengthening Immune Response

[0091] In a ninth aspect, the present invention provides the use of vitamin B2, or a pharmaceutically acceptable salt thereof, its stereoisomers, its crystals, solvates, hydrates or derivatives thereof, in the preparation of a medicament for enhancing immune cell activity and / or strengthening immune responses in a subject.

[0092] In some embodiments, the immune cells are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδT cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof.

[0093] In some embodiments, the immune cells are selected from: chimeric antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), chimeric antigen receptor macrophages (CAR-M), chimeric antigen receptor NK cells, chimeric antigen receptor γδ T cells, tumor-infiltrating lymphocytes (TILs) (e.g., tumor-infiltrating T lymphocytes), and any combination thereof.

[0094] In some implementations, the immune cells are tumor-infiltrating immune cells.

[0095] In some embodiments, the enhancement of immune cell activity includes: promoting immune cell differentiation (e.g., memory differentiation), enhancing immune cell function, and / or inhibiting immune cell depletion.

[0096] In some embodiments, the immune response includes cellular immune response and / or humoral immune response.

[0097] In some implementations, the subject is a mammal, such as a human.

[0098] In some implementations, the subject is a cancer patient, an autoimmune disease patient, or an infected patient.

[0099] In some embodiments, the tumor, autoimmune disease, or infection is as described in any embodiment of the seventh aspect. In some embodiments, the drug further includes immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody), for example, administered simultaneously, separately, or sequentially.

[0100] In a tenth aspect, the present invention provides a method for enhancing the activity of immune cells in a subject and / or strengthening the immune response, comprising administering to a subject in need an effective amount of vitamin B2, or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates thereof, or derivatives thereof.

[0101] In some embodiments, the immune cells are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδT cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof.

[0102] In some embodiments, the immune cells are selected from antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), chimeric antigen receptor macrophages (CAR-M), chimeric antigen receptor NK cells, chimeric antigen receptor γδ T cells, tumor-infiltrating lymphocytes (TILs) (e.g., tumor-infiltrating T lymphocytes), and any combination thereof.

[0103] In some implementations, the immune cells are tumor-infiltrating immune cells.

[0104] In some embodiments, the enhancement of immune cell activity includes: promoting immune cell differentiation (e.g., memory differentiation), enhancing immune cell function, and / or inhibiting immune cell depletion.

[0105] In some embodiments, the immune response includes cellular immune response and / or humoral immune response.

[0106] In some implementations, the subject is a mammal, such as a human.

[0107] In some implementations, the subject is a cancer patient, an autoimmune disease patient, or an infected patient.

[0108] In some embodiments, the tumor, autoimmune disease, or infection is as described in any embodiment of the seventh aspect.

[0109] In some embodiments, the vitamin B2 is applied to a specific site on the subject, such as a lesion or its surrounding area. In some embodiments, the subject is a cancer patient, and the vitamin B2 is applied to the tumor or adjacent tissue.

[0110] In some embodiments, the method further includes administering an immune checkpoint inhibitor (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody) to the subject, for example, simultaneously, separately, or sequentially.

[0111] On the other hand, this application provides a composition comprising:

[0112] (a) Vitamin B2, or a pharmaceutically acceptable salt thereof, its stereoisomers, its crystals, solvates, hydrates, or derivatives thereof,

[0113] as well as,

[0114] (b) Immunostimulants selected from: immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies), engineered immune cells, and any combination thereof.

[0115] In some embodiments, the engineered immune cells are selected from chimeric antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), chimeric antigen receptor macrophages (CAR-M), chimeric antigen receptor NK cells, chimeric antigen receptor γδ T cells, tumor-infiltrating lymphocytes (TILs) (e.g., tumor-infiltrating T lymphocytes), and any combination thereof.

[0116] In some embodiments, the composition is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

[0117] In some implementations, (a) and (b) are provided in a mixed or separate form.

[0118] In an eleventh aspect, the present invention provides the use of a composition in the preparation of a medicament comprising: (i) vitamin B2, or a pharmaceutically acceptable salt thereof, its stereoisomers, its crystals, solvates, hydrates thereof, or derivatives thereof, and (ii) an immune enhancer selected from: immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies), engineered immune cells, and any combination thereof, the medicament being used in the prevention and / or treatment of tumors or infections or autoimmune diseases in a subject.

[0119] In some embodiments, the engineered immune cells are selected from antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), chimeric antigen receptor macrophages (CAR-M), chimeric antigen receptor NK cells, chimeric antigen receptor γδ T cells, tumor-infiltrating lymphocytes (TILs) (e.g., tumor-infiltrating T lymphocytes), and any combination thereof.

[0120] In some embodiments, the tumor is selected from solid tumors or hematologic malignancies. In some embodiments, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer), melanoma, colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and any combination thereof.

[0121] In some embodiments, the autoimmune disease is selected from: systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease, ulcerative colitis, and any combination thereof.

[0122] In some embodiments, the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections;

[0123] In some implementations, the subject is a mammal, such as a human.

[0124] In a twelfth aspect, the present invention provides a method for preventing and / or treating tumors, infections, or autoimmune diseases, comprising administering an effective amount of a composition as defined in the eleventh aspect to a subject in need.

[0125] In some embodiments, the tumor is selected from solid tumors or hematologic malignancies. In some embodiments, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer), melanoma, colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and any combination thereof.

[0126] In some embodiments, the autoimmune disease is selected from: systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease, ulcerative colitis, and any combination thereof.

[0127] In some embodiments, the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections;

[0128] In some implementations, the subject is a mammal, such as a human.

[0129] In some embodiments, the vitamin B2, or a pharmaceutically acceptable salt, stereoisomer, crystal, solvate, hydrate, or derivative thereof, is administered simultaneously, separately, or sequentially with the engineered immune cells. In some embodiments, the vitamin B2 and the engineered immune cells are administered to the same or different sites on the subject. In some embodiments, the method is a way of treating a tumor, in which the vitamin B2 is administered to the tumor or adjacent tissue.

[0130] In some embodiments, the vitamin B2, or a pharmaceutically acceptable salt thereof, its stereoisomers, its crystals, solvates, hydrates or derivatives thereof, and the engineered immune cells are administered in combination with other pharmaceutically active agents (e.g., simultaneously, separately or sequentially).

[0131] Terminology Definition

[0132] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the biochemical and immunological laboratory procedures used herein are standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0133] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0134] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0135] As used in this article, vitamin B2, also known as riboflavin, is a type of B vitamin with the molecular formula C. 17 H 20 N4O6, molecular weight 376.37, CAS number 83-88-5.

[0136] As used herein, the term vitamin B2 transporter includes any functional protein capable of localizing to the cell membrane and actively transporting vitamin B2, which may be naturally occurring or artificially modified. Common naturally occurring vitamin B2 transporters include SLC52A1 (Solute Carrier Family 52 Member 1, also known as RFVT1), SLC52A2 (also known as RFVT2), and SLC52A3 (also known as RFVT3). These proteins may be of human origin or derived from other species (e.g., non-human mammals, fish, reptiles, or birds, such as rodents like mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, primates, etc.). Those skilled in the art are capable of obtaining the amino acid sequences of these proteins from public databases (e.g., NCBI, UniProtKB). For example, exemplary amino acid coding sequences of human SLC52A1, SLC52A2, and SLC52A3 proteins can be obtained from the UniProtKB database using accession numbers Q9NWF4, Q9HAB3, and Q9NQ40, respectively; exemplary amino acid coding sequences of mouse SLC52A2 and SLC52A3 proteins can be obtained from the UniProtKB database using accession numbers UniProtKB: Q9D8F3 and Q9D6X5, respectively.

[0137] As used herein, the term "engineered immune cell" refers to immune cells obtained through human intervention (e.g., genetic modification). In some embodiments, the engineered immune cells are modified to upregulate the expression level of the endogenous vitamin B2 transporter protein. In some embodiments, the engineered immune cells are modified to express an exogenous vitamin B2 transporter protein.

[0138] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0139] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0140] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0141] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0142] The twenty common amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0143] As used herein, the term "pharmaceuticalally acceptable carrier" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

[0144] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0145] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention is an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0146] Beneficial effects of the invention

[0147] Compared with existing technologies, this invention, based on the discovery of a novel mechanism of vitamin B2 deficiency in the tumor microenvironment, enhances the ability of immune cells (such as T cells) to actively take up vitamin B2 by engineering them (overexpressing vitamin B2 transporters) or by directly administering vitamin B2, achieving one or more of the following beneficial effects:

[0148] (1) Improve the survival ability of immune cells in TME.

[0149] (2) Enhance the effector function of immune cells, for example, significantly enhance the killing efficacy of immune cells (such as T cells) against tumor cells, and enhance the function of M1 macrophages.

[0150] (3) Promote the memory differentiation of immune cells and the polarization of macrophages to the M1 type; for example, the optimization of vitamin B2 metabolism is conducive to the differentiation of T cells into central memory T cells (Tcm), thereby achieving a more durable anti-tumor response in vivo; vitamin B2 can promote the polarization of macrophages to the M1 type and enhance the function of M1 macrophages.

[0151] (4) Inhibit the exhaustion of immune cells: By improving the metabolic stress state in the TME, the expression of immunosuppressive receptors (such as PD-1, TIM-3, LAG-3) is reduced, thus delaying or reversing the functional exhaustion of immune cells.

[0152] (5) Resistance to metabolic inhibition in the tumor microenvironment: This invention enables immune cells to actively take up essential vitamin B2, fundamentally improving their adaptability and functional durability in a TME characterized by low vitamin B2, high oxidative stress, and high metabolic competition. Experimental evidence shows that the modified immune cells constructed in this application (such as CAR-T cells overexpressing SLC52A2) exhibit significantly superior anti-tumor efficacy compared to unmodified control cells in both in vitro co-culture models and in vivo animal models, including stronger tumor clearance and longer survival.

[0153] In summary, this invention provides a novel and effective technical solution for overcoming the bottlenecks of immunotherapy in the treatment of solid tumors, and has extremely high clinical application value and commercial development potential.

[0154] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0155] Figure 1 This study revealed vitamin B2 (VB2) deficiency in the peripheral blood and tumor microenvironment of cancer patients. Specifically:

[0156] (A) The levels of VB2 in the serum of healthy individuals and lung cancer patients were compared, and the results showed that the level of VB2 in the serum of lung cancer patients was significantly lower.

[0157] (B) The levels of VB2 in peripheral blood and tumor pleural effusion of lung cancer patients were compared. The results showed that the VB2 level in tumor pleural effusion was significantly lower than that in peripheral blood.

[0158] Figure 2 The study showed that VB2 deficiency inhibited T cell proliferation, viability, and mitochondrial function. Specifically:

[0159] (A) By adding different concentrations of VB2 to a culture medium lacking VB2, the results showed that VB2 deficiency significantly inhibited T cell proliferation, while VB2 supplementation promoted T cell proliferation in a dose-dependent manner.

[0160] (B) Cell viability was detected by 7-AAD staining, and the results showed that VB2 supplementation increased T cell viability in a dose-dependent manner.

[0161] (C) The results of mitochondrial quality and membrane potential assays showed that VB2 deficiency significantly reduced the proportion of healthy mitochondria, while VB2 supplementation promoted the recovery of mitochondrial function in a dose-dependent manner.

[0162] (D) The proportion of functional mitochondria was statistically analyzed by measuring mitochondrial quality and membrane potential. The results showed that VB2 supplementation increased the proportion of functional mitochondria in CD8 T cells in a dose-dependent manner.

[0163] (E) The proportion of dysfunctional mitochondria was statistically analyzed by measuring mitochondrial quality and membrane potential. The results showed that VB2 supplementation reduced the proportion of dysfunctional mitochondria in CD8 T cells in a dose-dependent manner.

[0164] Figure 3 The study demonstrated that vitamin B2 supplementation significantly enhanced the antitumor efficacy of CAR-T and immune checkpoint inhibitors. Specifically:

[0165] (A) In an LLC lung cancer model, VB2 supplementation significantly promoted the therapeutic effect of CAR-T cells.

[0166] (B) In the B16 melanoma model, VB2 supplementation also significantly promoted the therapeutic effect of CAR-T cells.

[0167] (CD) In ​​the MC38 colorectal cancer model, VB2 supplementation alone showed a certain anti-tumor effect, while when VB2 was used in combination with immune checkpoint inhibitors such as anti-PD-L1 antibodies, a synergistic and enhanced anti-tumor effect was produced (the combination therapy group was significantly better than either single-agent therapy group). Tumor growth was inhibited after PD-L1 antibody combined with VB2 treatment (C), and the tumor quality at the experimental endpoint was significantly reduced (D).

[0168] Figure 4 The expression of SLC52A3 (VB2 transporter) in different types of T cells is shown. Among them:

[0169] (A) In mouse T cells, SLC52A3 was significantly upregulated after activation, maintained high expression in effector T cells and memory T cells, and significantly downregulated in exhausted T cells.

[0170] (B) In human T cells, SLC52A3 is significantly overexpressed in memory T cell subsets such as central memory T cells (Tcm) and effector memory T cells (Tem).

[0171] (C) The MFI values ​​of different CD8 T cell subsets SLC52A3 in (B) were analyzed by flow cytometry. The results showed that the expression level of SLC52A3 in Tcm cells of human CD8 T cells was significantly higher than that in other subsets.

[0172] (D) By analyzing tumor-infiltrating T cells from colorectal cancer (CRC) patients, it was found that SLC52A3 was significantly downregulated in PD-1-overexpressing exhausted T cells.

[0173] Figure 5 The diagram shows a schematic of the scaffold of the CAR-T cells expressing SLC52A3 constructed according to the present invention. The nucleic acid sequence encoding the chimeric antigen receptor (CAR) and the nucleic acid sequence encoding the VB2 transporter SLC52A3 are linked via a 2A peptide (P2A) and cloned into a viral expression vector to achieve co-expression of CAR and SLC52A3 in the same T cell.

[0174] Figure 6The construction and expression validation results of CAR-T cells overexpressing SLC52A3 are shown. Among them:

[0175] (A) Flow cytometry was used to detect the transduction efficiency of control CAR-T cells and CAR-T cells overexpressing SLC52A3 (SLC52A3-OE-CAR-T). The results showed that the viral transduction efficiency of both cell types was as high as about 90%.

[0176] (B) Real-time quantitative PCR (qRT-PCR) was used to detect the expression level of SLC52A3 mRNA. The results showed that the expression level of SLC52A3 mRNA in SLC52A3-OE-CAR-T cells was significantly higher than that in control CAR-T cells.

[0177] (C) Western blot analysis of SLC52A3 protein expression level showed that SLC52A3 protein expression in SLC52A3-OE-CAR-T cells was significantly higher than that in control CAR-T cells.

[0178] Figure 7 Construction and expression validation results of human CAR-T cells overexpressing SLC52A3.

[0179] (A) Flow cytometry was used to detect the transduction efficiency of control human CAR-T cells and human CAR-T cells overexpressing SLC52A3 (hSLC52A3-OE-CAR-T). The results showed that the viral transduction efficiency of the two cell lines was approximately 16%.

[0180] (B) The expression level of human hSLC52A3 mRNA was detected by real-time quantitative PCR (qRT-PCR). The results showed that the expression level of SLC52A3 mRNA in hSLC52A3-OE-CAR-T cells was significantly higher than that in control CAR-T cells.

[0181] (C) Flow cytometry was used to detect the expression level of human SLC52A3 protein. The results showed that the SLC52A3 protein in hSLC52A3-OE-CAR-T cells was significantly higher than that in control CAR-T cells.

[0182] (D) The average fluorescence intensity of SLC52A3 detected by flow cytometry in (C) was statistically analyzed. The results showed that the average fluorescence intensity (MFI) of SLC52A3 protein in hSLC52A3-OE-CAR-T cells was significantly higher than that in control CAR-T cells.

[0183] Figure 8The results show the ability of CAR-T cells overexpressing SLC52A3 to take up VB2. Intracellular VB2 levels were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results indicated that, compared with control CAR-T cells, CAR-T cells overexpressing SLC52A3 (SLC52A3-OE-CAR-T) showed significantly increased VB2 uptake.

[0184] Figure 9 The proliferation capacity and cell viability of CAR-T cells overexpressing SLC52A3 (SLC52A3-OE-CAR-T) under different VB2 conditions are shown. SLC52A3 overexpression significantly enhanced the resistance of CAR-T cells to VB2-deficient environments.

[0185] (A) Under VB2 deficiency conditions, SLC52A3-OE-CAR-T cells were able to maintain cell viability comparable to that under VB2 sufficiency conditions, while control CAR-T cells showed a significant decrease in viability under VB2 deficiency conditions.

[0186] (B) Under VB2 deficiency conditions, SLC52A3-OE-CAR-T cells were able to maintain cell proliferation capacity comparable to that under VB2 sufficiency conditions, while control CAR-T cells showed significant proliferation inhibition under VB2 deficiency conditions.

[0187] Figure 10 The study demonstrated the effect of SLC52A3 overexpression on CAR-T cell function. Results showed that SLC52A3 overexpression significantly inhibited the expression of exhaustion molecules in CAR-T cells and promoted memory differentiation and the secretion of effector factors. Specifically:

[0188] (A) Flow cytometry detection of PD-1 + TIM-3 + The proportion of double-positive cells showed that the proportion of exhausted CAR-T cells in SLC52A3-OE-CAR-T cells was significantly lower than that in control CAR-T cells.

[0189] (B) Flow cytometry analysis of memory differentiation showed that CD44 in SLC52A3-OE-CAR-T cells... + CD62L + The proportion of memory cells was significantly higher than that of control CAR-T cells.

[0190] (C) IFN-γ expression detection showed that the proportion of IFN-γ positive cells in SLC52A3-OE-CAR-T cells was significantly higher than that in control CAR-T cells.

[0191] Figure 11This study demonstrated the effect of SLC52A3 overexpression on the tumor-killing ability of CAR-T cells. CAR-T cells were co-cultured with tumor cells at different effector-target ratios (E:T), and the tumor-killing rate was assessed. The results showed that, under different effector-target ratios, CAR-T cells overexpressing SLC52A3 (SLC52A3-OE-CAR-T) exhibited significantly stronger tumor-killing ability than control CAR-T cells.

[0192] Figure 12 This study demonstrates the in vivo antitumor efficacy of CAR-T cells overexpressing SLC52A3 (SLC52A3-OE-CAR-T). The therapeutic effects of SLC52A3-OE-CAR-T cells compared to control CAR-T cells were evaluated using an LLC (Lewis lung cancer) tumor-bearing mouse model.

[0193] (A) Tumor growth curves show that the tumor volume growth in the SLC52A3-OE-CAR-T treatment group was significantly slower than that in the control CAR-T group.

[0194] (B) Tumor weight, showing that the tumor weight in the SLC52A3-OE-CAR-T treatment group was significantly lighter than that in the control CAR-T group.

[0195] (C, D) The number of CAR-T cells infiltrating the tumor tissue shows that the number of CAR-T cells infiltrating the tumor in the SLC52A3-OE-CAR-T group was significantly higher than that in the control CAR-T group.

[0196] (E) Expression of effector factors in tumor-infiltrating CAR-T cells showed that the proportion of TNF-α and IFN-γ positive cells in the SLC52A3-OE-CAR-T group was significantly higher than that in the control CAR-T group.

[0197] (F) The expression of exhaustion molecules in tumor-infiltrating CAR-T cells showed that the proportion of PD-1 and TIM-3 positive cells in the SLC52A3-OE-CAR-T group was significantly lower than that in the control CAR-T group.

[0198] Figure 13 The in vitro tumor-killing effect of human hSLC52A3-OE-CAR-T.

[0199] (A) By setting different effector-target ratios (E:T), human CAR-T cells were co-cultured with tumor cells, and the tumor cell killing rate was detected. The results showed that under different effector-target ratio conditions, the tumor killing ability of human CAR-T cells overexpressing SLC52A3 (hSLC52A3-OE-CAR-T) was significantly stronger than that of control CAR-T cells.

[0200] (B) Memory differentiation analysis showed that CCR7 in human hSLC52A3-OE-CAR-T cells... + CD45RA - The proportion of memory cells was significantly higher than that of control CAR-T cells.

[0201] (C) IFN-γ expression detection showed that the proportion of IFN-γ positive cells in human hSLC52A3-OE-CAR-T cells was significantly higher than that in control CAR-T cells.

[0202] (D) IL-2 expression detection showed that the proportion of IL-2 positive cells in human hSLC52A3-OE-CAR-T cells was significantly higher than that in control CAR-T cells.

[0203] Figure 14 This study demonstrated the antitumor efficacy of human CAR-T cells overexpressing SLC52A3 (hSLC52A3-OE-CAR-T) in vivo. The therapeutic effects of hSLC52A3-OE-CAR-T cells compared to control human CAR-T cells were evaluated using PC-9 and Calu3 CDX (Cell Line Derived Xenograft) tumor-bearing mouse models. Results showed that:

[0204] (A) Flowchart of PC-9 tumor inoculation and CAR-T therapy.

[0205] (B) PC-9 tumor growth curves show that the hSLC52A3-OE-CAR-T treatment group significantly inhibited tumor growth.

[0206] (C) PC-9 tumor weight, showing that the tumor weight in the hSLC52A3-OE-CAR-T treatment group was significantly lighter than that in the control group.

[0207] (D) Flowchart of Calu3 tumor inoculation and CAR-T therapy.

[0208] (E) Calu3 tumor growth curves show that the hSLC52A3-OE-CAR-T treatment group significantly inhibited tumor growth.

[0209] (F) Calu3 tumor weight, showing that the tumor weight in the hSLC52A3-OE-CAR-T treatment group was significantly lighter than that in the control group.

[0210] Figure 15 The study showed that VB2 supplementation significantly promoted macrophage polarization towards M1 macrophages, while having little effect on M2 macrophage polarization. Specifically:

[0211] (A, B) Different concentrations of VB2 were added to a VB2-deficient culture medium to observe the effect of VB2 on IFNγ-induced polarization of M1 macrophages. The results showed that VB2 upregulated iNOS expression in a dose-dependent manner, indicating that VB2 can promote the polarization of M1 macrophages.

[0212] (C, D) Different concentrations of VB2 were added to a VB2-deficient culture medium to observe the effect of VB2 on IL-4-induced polarization of M2 macrophages. The results showed that VB2 had no significant effect on Arg-1 (Arginase 1) expression, suggesting that VB2 has a limited effect on the polarization of M2 macrophages.

[0213] Figure 16 The effect of vitamin B2 supplementation on M1 macrophage function was demonstrated. Different concentrations of vitamin B2 were added to a vitamin B2-deficient culture medium, and their effect on IFNγ-induced polarization of M1 macrophages was observed. Among them:

[0214] (A) VB2 upregulates CD86 expression in a dose-dependent manner.

[0215] (B) VB2 upregulates MHC-I expression in a dose-dependent manner.

[0216] Figure 17 The expression of VB2-associated transporters in M0 and M1 macrophages is shown. Among them:

[0217] (A) shows the expression levels of SLC52A2 in M0 and M1 macrophages.

[0218] (B) shows the expression level of SLC52A3 in M0 and M1 macrophages. Detailed Implementation

[0219] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0220] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples are described by way of illustration and are not intended to limit the scope of the invention.

[0221] Unless otherwise specified, the C57BL / 6 wild-type mice used in the following examples were all purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0222] Example 1: VB2 deficiency in the tumor microenvironment.

[0223] In this embodiment, the inventors of this application, through testing clinical samples, used high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) to detect that the level of VB2 in the peripheral blood of cancer patients (e.g., lung cancer) was significantly lower than that in healthy controls. Figure 1 A). Further analysis of VB2 levels in the pleural effusion microenvironment of lung cancer patients showed that VB2 was further reduced in the tumor microenvironment ( Figure 1 B). The above results indicate that VB2 is deficient in the tumor microenvironment.

[0224] Example 2: Effect of VB2 on T cell activity.

[0225] In this embodiment, the inventors treated mouse CD8 T cells with different concentrations of VB2 (Harveybio, Cat# VIT1313): C57BL / 6 wild-type mice were used, and spleens were aseptically isolated to prepare spleen single-cell suspensions. CD8-positive T cells were obtained using a STEMCELL mouse CD8+ T cell negative sorting kit (STEMCELL Technologies, Cat#19853) via magnetic bead sorting. The sorted CD8+ T cells were then processed at 1×10⁻⁶... 6 T cells were seeded at a density of 1 cell / mL in pretreated 24-well plates. The 24-well plates were pretreated by coating them overnight at 4°C with anti-CD3 antibody (2 μg / mL) and anti-CD28 antibody (1 μg / mL). The T cells were cultured in RPMI 1640 Deficit Medium (Coolaber, Cat# CM0011X) lacking vitamin B2, supplemented with 10% dialyzed fetal bovine serum (Gibco, Cat# 30067334), 1% penicillin-streptomycin (Gibco, Cat# 15140-122), and interleukin-2 (IL-2, final concentration 50 U / mL). Cells were cultured for 48 hours at 37°C in a 5% CO2 incubator in medium supplemented with different concentrations of vitamin B2. The final concentration gradients of VB2 were set at 200 ng / mL, 50 ng / mL, 12.5 ng / mL, 3.125 ng / mL, and 0.8 ng / mL. The initial concentration of 200 ng / mL was used to simulate normal T cell culture conditions (consistent with the concentration in standard RPMI 1640 medium), while subsequent lower concentration gradients were used to simulate VB2 deficiency in the tumor microenvironment.

[0226] Cell proliferation assays (CFSE, carboxyfluorescein succinimide ester cell proliferation assay) and the detection of the apoptosis marker 7-AAD showed that VB2 deficiency (below 12.5 ng / mL) significantly inhibited T cell proliferation and viability. Figure 2 A, 2B). Further analysis of mitochondrial function by flow cytometry of mitochondrial number (MitoTracker™ Green FM, Thermo Fisher Scientific, Cat# M46750) and staining of mitochondrial membrane potential (MitoTracker™ Red CMXRos, Thermo Fisher Scientific, Cat# M46752) showed that VB2 deficiency significantly induced mitochondrial dysfunction in T cells. Figure 2 C-2E).

[0227] Furthermore, VB2 supplementation therapy was performed using an in vivo tumor animal model. Six- to eight-week-old female C57BL / 6 wild-type mice were subcutaneously inoculated with LLC-Trop2 tumor cells or BC16-Trop2 tumor cells (1×10⁻⁶) on the right back. 6 A subcutaneous tumor-bearing model was established (number of mice per mouse). On day 4 after tumor implantation, all mice were pretreated with lymphocyte clearance by intraperitoneal injection of cyclophosphamide (80 mg / kg); on day 7, 1 × 10⁻⁶ mg / kg of cyclophosphamide was reinfused into each tumor-bearing mouse via the tail vein. 6 A control CAR-T cell group was used, and tumor-bearing mice were injected daily via peritumoral injection of VB2 (0.6 mg / kg) and a control saline group. Tumor growth curves were plotted, and the tumor-suppressing effects among the groups were compared. VB2 supplementation significantly promoted the therapeutic effect of control CAR-T cells (the amino acid sequence of CAR is shown in SEQ ID NO: 18) on solid tumors such as lung cancer. Figure 3 A, 3B). Six- to eight-week-old female C57BL / 6 wild-type mice were subcutaneously inoculated with MC38 tumor cells (1×10⁻⁶) on the right back. 6 Subcutaneous tumor-bearing models were established using 1 mouse / mouse. Subsequently, anti-mouse PD-L1 antibody (Anti-mouse PD-L1 in vivo, Selleckchem, Cat# A2115; RRID: AB_3675704) and isotype control antibody (Rat IgG2b isotype control-In Vivo, Selleckchem, Cat# A2116; RRID: AB_3662740) were injected intraperitoneally every three days. Additionally, tumor-bearing mice were injected daily via peritumoral injection with VB2 (0.6 mg / kg) and control saline, respectively. Tumor size was measured twice weekly, and growth curves were plotted. The results showed that VB2 also synergistically enhanced the ability of immune checkpoint inhibitors such as PD-L1 to inhibit tumor growth. Figure 3 C, 3D).

[0228] Example 3: SLC52A3 protein expression was significantly upregulated in peripheral T cells during activation and memory differentiation.

[0229] This embodiment first analyzed the changes in SLC52A3 during mouse T cell development. The results showed that SLC52A3 was significantly upregulated after T cell activation, and SLC52A3 maintained high expression in memory T cells (Tmem). However, in exhausted T cells (Tex), the expression level of SLC52A3 was significantly downregulated. Figure 4 A). Correspondingly, the expression of human T cell SLC52A3 was also analyzed, and the results showed that SLC52A3 was highly expressed in effector memory T cells (Tem) and central memory T cells (Tcm). Figure 4 B, 4C). Further analysis of tumor-infiltrating T cells in clinical colorectal cancer (CRC) patients revealed that SLC52A3 plays a role in PD-1. high Significantly downregulated in exhausted T cells ( Figure 4 D).

[0230] Example 4: Preparation of mouse SLC52A3-OE-CAR-T cells.

[0231] CAR-T cells overexpressing SLC52A3 were constructed from mice to obtain SLC52A3-OE-CAR-T.

[0232] Construction of control CAR plasmid and SLC52A3 overexpression CAR plasmid:

[0233] Using the mouse retroviral vector MIGR1 plasmid (Addgene #27490), the Ctrl-CAR sequence (Trop2scFv-mCD8-mCD28-mCD3ζ, the corresponding amino acid sequence is shown in SEQ ID NO: 18) or the CAR sequence of SLC52A3 overexpression (Trop2scFv-mCD8-mCD28-mCD3ζ-P2A-SLC52A3, the corresponding amino acid sequence is shown in SEQ ID NO: 1) was inserted by enzyme digestion and ligation.

[0234] Retrovirus preparation:

[0235] The retroviral vector and packaging vector pCL-ECO plasmid were transfected into 293T-17 cells using liposome transfection. The viral supernatant was collected 48 h after transfection and frozen at -80°C for later use.

[0236] CAR-T construction method:

[0237] 1) C57BL / 6 mice were euthanized by cervical dislocation. Spleens were harvested in a clean bench, ground and filtered through a 40 μm filter using a 5 mL syringe, then lysed with erythrocyte lysis buffer and washed once with 1×PBS. Mouse CD8 T cells were sorted using a Stem Cell negative selection kit and sorting buffer under a magnetic field. 200 μL of 2 μg / mL anti-mouse CD3 antibody and 1 μg / mL anti-mouse CD28 antibody were added to 24-well plates and incubated overnight at 4°C. Mouse T cells were resuspended in RPMI 1640 complete medium at a density of 1×10⁻⁶ cells / well. 6 The cells were incubated in 24-well plates for 24 h with 10 ng / mL of recombinant mouse IL-2 protein. Activated mouse CD8 T lymphocytes were collected, centrifuged, and the supernatant was discarded. Cells were resuspended in freshly collected retroviral solution and transferred to 12-well plates pretreated with retrovirus. 8 μg / mL of Polybrene and 5 ng / mL of recombinant mouse IL-2 protein were added, and the cells were centrifuged at 800 g for 90 min, then incubated at 37°C in a 5% CO2 incubator for 12 h. Infected lymphocytes were collected in 50 mL EP tubes, centrifuged, and the supernatant was discarded. The cells were then expanded using RPMI 1640 complete medium, and 10 ng / mL of recombinant mouse IL-2 protein was added. The cells were incubated at 37°C in a 5% CO2 incubator for 48 h, and the transduction efficiency was detected by flow cytometry.

[0238] like Figure 5 As shown, the SLC52A3-CAR (amino acid sequence as shown in SEQ ID NO: 1) nucleic acid sequence simultaneously encodes a murine chimeric antigen receptor (CAR) targeting Trop2 and the murine SLC52A3 protein, with the coding nucleic acid sequences of the two linked by the P2A coding sequence.

[0239] The amino acid sequence of the CAR is shown in SEQ ID NO: 18, and it is composed of the following elements in series from the N-terminus to the C-terminus: a signal peptide, a single-chain antibody (scFv) targeting Trop2 as an antigen-binding domain, a mouse CD8 hinge region, a mouse CD28 transmembrane domain (TM) and an intracellular costimulatory domain (costi), and a mouse CD3ζ intracellular signal transduction domain. The sequence numbers of the amino acid sequences of the involved proteins or elements are summarized in Table 1.

[0240] Table 1: Component sequence information contained in mouse SLC52A3-CAR

[0241] By flow cytometry ( Figure 6A) RT-PCR ( Figure 6 B) and western blot ( Figure 6 C) The overexpression of SLC52A3 was verified; wherein the control used was T cells expressing the CAR but not expressing the SLC52A3 protein.

[0242] Example 5: Preparation of human SLC52A3-OE-CAR-T cells.

[0243] Human CAR-T cells overexpressing SLC52A3, SLC52A3-OE-CAR-T, were prepared according to the method described in Example 2, and analyzed by RT-PCR ( Figure 7 B) and flow cytometry ( Figure 7 A, 7C) verified the overexpression of SLC52A3; wherein the control used was T cells expressing the CAR but not expressing SLC52A3 protein.

[0244] The structure of the human SLC52A3-CAR (amino acid sequence as shown in SEQ ID NO: 19, CAR portion amino acid sequence as shown in SEQ ID NO: 25) is similar to that of the mouse SCLC523-CAR. The only differences are that the mouse CD8 hinge region, mouse CD28 transmembrane (TM) domain, mouse costimulatory domain (costi), and mouse CD3ζ intracellular signal transduction domain are replaced with the corresponding human domains, and the mouse SLC52A3 is replaced with the human SLC52A3. The amino acid sequence information of each element is summarized in Table 2.

[0245] Table 2: Component serial numbers contained in the human SLC52A3-CAR

[0246] Example 6: SLC52A3-OE-CAR-T enhances the ability to absorb VB2.

[0247] The ability of mouse CAR-T cells overexpressing SLC52A3 to take up vitamin B2 was investigated. Prepared CAR-T cells were cultured in 1640 medium completely lacking vitamin B2 for 24 h, centrifuged at 300 g for 5 min to remove the supernatant, and then cultured in normal 1640 medium for 2 h. CAR-T cells were then collected, and intracellular vitamin B2 levels were detected by HPLC-MS. The results showed that, compared to control CAR-T cells (Ctrl-CAR-T, which expressed the CAR but did not express SLC52A3), overexpression of SLC52A3 significantly promoted the uptake of vitamin B2 by T cells, resulting in correspondingly higher intracellular vitamin B2 levels in T cells. Figure 8 ).

[0248] Example 7: Overexpression of SLC52A3 promotes the survival and proliferation of mouse CAR-T cells.

[0249] The study investigated the effect of SLC52A3 overexpression on the survival and proliferation of CAR-T cells under different vitamin B2 conditions. Mouse control CAR-T cells and SLC52A3-overexpressing CAR-T cells were pre-stained with CFSE. Then, CAR-T cells were reactivated with anti-CD3 / CD28 antibody at different vitamin B2 concentrations. After 72 hours, the number of proliferating cells was calculated using CFSE (Divided cells, the proportion of proliferation peaks detected by flow cytometry, with the peak of freshly stained T cells used as a standard). The viability of activated T cells was assessed using 7-AAD staining. Figure 9 The results showed that under conditions of sufficient VB2 (e.g., VB2 concentration of 50 ng / mL), overexpression of SLC52A3 did not affect the survival and proliferation of CAR-T cells; however, under conditions of insufficient VB2 (e.g., VB2 concentration of 0.8 ng / mL), overexpression of SLC52A3 significantly promoted the survival and proliferation of CAR-T cells. Figure 9 A, 9B).

[0250] Example 8: Effect of SLC52A3 overexpression on mouse CAR-T cells.

[0251] Flow cytometry was used to detect CAR-T cells and mouse LLC-Trop2. + Functional analysis of CAR-T cells, including PD1, after 6 days of co-culture with tumor cells (3 rounds of co-culture, 48 hours per round, CAR-T cell to tumor cell ratio of 1:2). + TIM3 + The proportion of exhausted CAR-T cells ( Figure 10 A), CD44 + CD62L +The proportion of memory CAR-T cells ( Figure 10 B) and the expression of the effector factor IFN-γ ( Figure 10 C). The results showed that overexpression of SLC52A3 inhibited mouse CAR-T cell exhaustion (C). Figure 10 A), and promotes the differentiation of mouse CAR-T cells into memory cells (A), and promotes the differentiation of mouse CAR-T cells into memory cells. Figure 10 B), enhanced mouse CAR-T cell function, including promoting the expression of effector factors (such as IFN-γ). Figure 10 C).

[0252] Example 9: Validation of the in vitro tumor-killing function of mouse SLC52A3-OE-CAR-T.

[0253] In vitro tumor-killing experiments were conducted using control CAR-T (Ctrl-CAR-T) and mouse SLC52A3-OE-CAR-T cells overexpressing SLC52A3 to validate tumor killing effects. LLC-Trop2 cells (mouse LLC tumor cells overexpressing human Trop2 protein constructed via lentivirus) in logarithmic growth phase were used, and the target cell density was adjusted to 1×10⁶ cells / year using 1640 medium with 10% fetal bovine serum. 5 cells / mL (i.e., 1 × 10⁻⁶ cells / mL per well) 4 (100 μL / well of target cells). It is recommended to optimize the target cell density based on the preliminary experimental results; a commonly used range is 5 × 10⁶ cells / well. 4 ~2×10 5Cells / mL. Mouse control CAR-T cells and mouse SLC52A3-OE-CAR-T cells were collected, resuspended in 1640 medium with 10% fetal bovine serum, counted, and their densities were adjusted. The required effector cell density was calculated based on the set effector-to-target ratio (E:TRatio). (The effector-to-target ratio gradient was set to 1:4, 1:2, and 1:1. Following the detection method of the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, Cat.# G1780) LDH kit, 100 μL of effector cell suspension was added to each well of the effector cell spontaneous release well (the effector cell type corresponded to the experimental group, with control CAR-T and SLC52A3-CAR-T spontaneous release wells respectively). The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 8 hours. 10 μL of 10× lysis buffer (provided with the kit) was added to each well of the maximum target cell release well and incubated for 45 minutes. The co-culture supernatant was collected, and the absorbance (OD value) of each well was measured at 490 nm using a microplate reader. The reference wavelength was set to 650 nm or 690 nm (to subtract background interference from the plate bottom and non-specific data). Specific Lysis (%) = [(OD_Experimental Group - OD_Target Cell Spontaneous Release - OD_Effective Cell Spontaneous Release + ...] [OD_culture medium background) / (OD_target cell maximum release - OD_target cell spontaneous release)] × 100%. Experimental results showed that, compared to mouse control CAR-T, overexpression of SLC52A3 significantly promoted the tumor-killing ability of CAR-T cells. Figure 11 ).

[0254] Example 10: Validation of SLC52A3-OE-CAR-T tumor-killing function in vivo.

[0255] In vivo killing experiments were conducted on lung cancer using control CAR-T and SLC52A3-OE-CAR-T cells overexpressing SLC52A3 in mice, respectively. Six- to eight-week-old female C57BL / 6 wild-type mice were subcutaneously inoculated with LLC-Trop2 tumor cells (1×10⁻⁶) on the right back. 6 A subcutaneous tumor-bearing model was established (number of mice per mouse). On day 7 after tumor implantation, all mice were pretreated with lymphocyte clearance by intraperitoneal injection of cyclophosphamide (80 mg / kg); on day 10, 1 × 10⁻⁶ mg / kg was reinfused into each tumor-bearing mouse via the tail vein. 6 Each mouse was given an experimental group of SLC52A3-OE-CAR-T cells or a control group of Ctrl-CAR-T cells. A separate control group of tumor-bearing mice that did not receive CAR-T cell infusion was also included. From the date of infusion, the long and short diameters of the tumor were measured twice weekly using electronic calipers, and the tumor volume was calculated (volume = long diameter × short diameter). 2 / 2), continuous monitoring until the experimental endpoint (e.g., tumor volume exceeding 2000 mm). 3 (Or mice were in a near-death state), tumor growth curves were plotted, and the tumor-suppressing effects among the groups were compared. Results showed that, compared to the control CAR-T, overexpression of SLC52A3 significantly promoted the tumor-killing ability of CAR-T. SLC52A3-OE-CAR-T significantly inhibited tumor growth ( Figure 12 (A and B). Finally, tumor tissue was minced and digested with collagenase II, collagenase IV, and hyaluronidase to prepare a single-cell suspension. Flow cytometry was then used to detect the proportion and function of CAR-T cells within the tumor. Notably, compared to control CAR-T, overexpression of SLC52A3 significantly promoted CAR-T infiltration in the tumor (A and B). Figure 12 (C, D), and promoted the function of corresponding tumor-infiltrating CAR-T cells, including a significantly enhanced ability to secrete effector factors IFN-γ and TNF-α (C, D). Figure 12 E). Furthermore, overexpression of SLC52A3 significantly inhibited the depletion of tumor-infiltrating CAR-T cells (E). Figure 12 F).

[0256] Example 11: Validation of the in vitro tumor-killing function of human SLC52A3-OE-CAR-T.

[0257] Human CAR-T cells overexpressing SLC52A3 (hSLC52A3-OE-CAR-T) were co-cultured with Trop2-positive PC-9 lung cancer cells in vitro. The LDH assay was used to assess the cytotoxic function of CAR-T cells; all experimental conditions were the same as in Example 9, except for the cell type used. Flow cytometry was used to detect memory cell differentiation and cytokine expression levels in CAR-T cells co-cultured with PC9 tumor cells. The results showed that, compared to the control human CAR-T (hCtrl-CAR-T, which expresses the same human CAR fraction as hSLC52A3-OE-CAR-T but does not express SLC52A3), overexpression of hSLC52A3 significantly promoted the tumor-killing ability of CAR-T cells. Figure 13 (A). Correspondingly, hSLC52A3-OE-CAR-T exhibits a greater number of memory T cell differentiation phenotypes (A). Figure 13 B), which secretes cytokines including IFN-γ ( Figure 13 C) and IL-2 ( Figure 13 The ability of D) is significantly enhanced.

[0258] Example 12: Validation of the tumor-killing function of human SLC52A3-OE-CAR-T in vivo.

[0259] In this embodiment, in vivo killing experiments were conducted on human lung cancer tumors such as PC-9 and Calu3 using both control CAR-T (Ctrl-CAR-T) and CAR-T overexpressing SLC52A3 (SLC52A3-OE-CAR-T). Six- to eight-week-old female NCG-immunodeficient mice were subcutaneously inoculated with PC-9 tumor cells (1×10⁻⁶) on their right back. 6 (each / animal) or Calu-3 (4×10) 5 A subcutaneous tumor-bearing model was established (each mouse carrying one tumor). On day 7 after tumor implantation, 1×10⁻⁶ mcg was infused into each tumor-bearing mouse via the tail vein. 6 Each group of hSLC52A3-OE-CAR-T cells (experimental group) or control hCtrl-CAR-T cells (control group) was used, with tumor-bearing mice that did not receive CAR-T cell infusion serving as blank controls (UTD). From the date of infusion, the long and short diameters of the tumor were measured every 2-3 days using electronic calipers, and the tumor volume was calculated (volume = long diameter × short diameter). 2 / 2), continuous monitoring until the experimental endpoint (e.g., tumor volume exceeding 2000 mm). 3 (Or mice were in a near-death state), tumor growth curves were plotted and the tumor-suppressing effects among the groups were compared. Results showed that, compared to the control hCAR-T, overexpression of SLC52A3 significantly promoted the tumor-killing ability of human CAR-T. hSLC52A3-OE-CAR-T significantly inhibited tumor growth ( Figure 14 ).

[0260] Example 13: Effect of VB2 on macrophage polarization.

[0261] In this embodiment, the inventors treated mouse macrophages with different concentrations of VB2 (Harveybio, Cat# VIT1313): C57BL / 6 wild-type mice were used, and bone marrow was isolated under aseptic conditions to prepare single-cell suspensions. Bone marrow differentiation into macrophages was induced using M-CSF (PeproTech, Cat#315-02). The macrophage cells were then cultured at a concentration of 5 × 10⁻⁶ cells / mL. 5Macrophages were seeded at a density of cells / mL in pretreated 24-well plates. M1 macrophages were induced using RPMI 1640 Deficit Medium (Coolaber, Cat# CM0011X) lacking vitamin B2, supplemented with 10% dialyzed fetal bovine serum (Gibco, Cat# 30067334), 1% penicillin-streptomycin (Gibco, Cat# 15140-122), and IFN-γ (20 ng / mL, PeproTech, Cat# 315-05), or M2 macrophages were induced with IL-4 (20 ng / mL, PeproTech, Cat# 214-14). Cells were cultured at 37°C in a 5% CO2 incubator for 48 hours in medium supplemented with different concentrations of vitamin B2. The final concentration gradients of VB2 were set at 200 ng / mL, 50 ng / mL, 12.5 ng / mL, 3 ng / mL, and 0 ng / mL. The initial concentration of 200 ng / mL was used to simulate normal macrophage culture conditions (consistent with the concentration in standard RPMI 1640 medium), while subsequent lower concentration gradients were used to simulate VB2 deficiency in the tumor microenvironment.

[0262] The effects of vitamin B2 on macrophage polarization were investigated by targeting nitric oxide synthase (iNOS), a polarization marker in M1 macrophages, and arginase 1 (Arg-1), a polarization marker in M2 macrophages. The results showed that vitamin B2 deficiency significantly inhibited the polarization of M1 macrophages. Figure 15 A, 15B), with limited effect on M2 macrophages ( Figure 15 C, 15D).

[0263] Example 14: Effects of VB2 on macrophage function.

[0264] In this embodiment, the inventors treated mouse macrophages with different concentrations of VB2. The final concentration gradient of VB2 was set to 200 ng / mL, 50 ng / mL, 12.5 ng / mL, 3 ng / mL, and 0 ng / mL. Macrophages were then treated at a concentration of 5 × 10⁻⁶ mg / mL. 5 M1 macrophages were seeded at a density of [number] cells / mL in pretreated 24-well plates. The macrophage culture medium was RPMI 1640 deficient medium lacking vitamin B2, supplemented with 10% dialyzed fetal bovine serum, 1% penicillin-streptomycin antibiotics, and IFN-γ to induce M1 macrophage function. The expression of molecules related to M1 macrophage function was then detected. Results showed that VB2 deficiency significantly inhibited M1 macrophage function, including the expression of effector molecules such as CD86 and MCH-I. Figure 16 This indicates that VB2 plays an important role in the function of M1 macrophages.

[0265] Example 15: Macrophages overexpress VB2 transporter to maintain VB2 requirements.

[0266] In this embodiment, macrophage cells were prepared at a rate of 5 × 10⁻⁶. 5 M1 macrophages were seeded at a density of 10 cells / mL in pretreated 24-well plates, and M1 macrophages were induced by adding complete medium containing 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, and IFN-γ. The expression of the VB2 transporter on the macrophages was analyzed. Results showed that SLC52A2 and SLC52A3 had high expression levels in polarized M1 macrophages. Figure 17 This indicates that macrophages need to express VB2 transporters to maintain VB2 uptake, suggesting the great potential of overexpressing related transporters to promote VB2 uptake and enhance macrophage function.

[0267] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

[0268] Sequence information

[0269] The amino acid sequence of the mouse-derived SLC52A3-CAR (SEQ ID NO: 1)

[0270] MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQASNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYLELFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPRGSGATNFSLLKQAGDVEENPGPMAFLTHLLVCVFGMGSWVAINGLWVELPLLVTELPEAWYLPSYLTVVIQLANIGPLLVTLMHRFRPGCLSEVPVIFLILCVGTAACILLAFLWNVTSWIQGGQHSVAFIVLTFFLALVDCTSSVTFLPFMSQLPTYYLTTFFIGEGLSGLLPALVALVQGSGITTCVNVTETPGTTLNTMETPITQGNLSPSLPSPSWHQESRYLAPRFSPLLFFLLLSFLTGCCLVAFFLLQRQPWGRQGSIEDLLHSQVTLHSIRPRDTEDTSSLGAPVSSPGKGSVEASVASLRPAQLAFIYSVVAFVNALTNGVLPSVQTYSCLPYGPVAYHLSATLSSVASPLACFLPIFLPNRSLLFLGVLTVLGTGFGAYNMAMAAMSPCPVLQGHWGGEVLIVLSWVLFAACLSYVKVMLGVILRDRSRSALLWCGAAVQLGSLIGALLMFPLVNVLKLFSSADYCSLDCSV

[0271] anti-Trop scFv amino acid sequence (SEQ ID NO: 2)

[0272] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQAS

[0273] anti-Trop VH amino acid sequence (SEQ ID NO: 3)

[0274] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQAS

[0275] anti-Trop VL amino acid sequence (SEQ ID NO: 4)

[0276] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK

[0277] anti-Trop VH CDR1 (Kabat) (SEQ ID NO: 5)

[0278] NYGMN

[0279] anti-Trop VH CDR2 (Kabat) (SEQ ID NO: 6)

[0280] WINTYTGEPTYTDDFKG

[0281] anti-Trop VH CDR3 (Kabat) (SEQ ID NO: 7)

[0282] GGFGSSYWYFDV

[0283] anti-Trop VL CDR1(Kabat) (SEQ ID NO: 8)

[0284] KASQDVSIAVA

[0285] anti-Trop VL CDR2(Kabat) (SEQ ID NO: 9)

[0286] SASYRYT

[0287] anti-Trop VL CDR3(Kabat) (SEQ ID NO: 10)

[0288] QQHYITPLT

[0289] mus-CD8-hinge amino acid sequence (SEQ ID NO: 11)

[0290] NSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY

[0291] mus-CD28-TM amino acid sequence (SEQ ID NO: 12)

[0292] LELFWALVVVAGVLFCYGLLVTVALCVIWT

[0293] mus-CD28 costi amino acid sequence (SEQ ID NO: 13)

[0294] NSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRP

[0295] mus-CD3-ζ-intracellular amino acid sequence (SEQ ID NO: 14)

[0296] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR

[0297] Standard P2A amino acid sequence (SEQ ID NO: 15)

[0298] ATNFSLLKQAGDVEENPGP

[0299] Optimized P2A amino acid sequence (SEQ ID NO: 16)

[0300] GSGATNFSLLKQAGDVEENPGP

[0301] Mouse SLC52A3 amino acid sequence (SEQ ID NO: 17)

[0302] MAFLTHLLVCVFGMGSWVAINGLWVELPLLVTELPEAWYLPSYLTVVIQLANIGPLLVTLMHRFRPGCLSEVPVIFLILCVGTAACILLAFLWNVTSWIQGGQHSVAFIVLTFFLALVDCTSSVTFLPFMSQLPTYYLTTFFIGEGLSGLLPALVALVQGSGITTCVNVTETPGTTLNTMETPITQGNLSPSLPSPSWHQESRYLAPRFSPLLFFLLLSFLTGCCLVAFFLLQRQPWGRQGSIEDLLHSQVTLHSIRPRDTEDTSSLGAPVSSPGKGSVEASVASLRPAQLAFIYSVVAFVNALTNGVLPSVQTYSCLPYGPVAYHLSATLSSVASPLACFLPIFLPNRSLLFLGVLTVLGTGFGAYNMAMAAMSPCPVLQGHWGGEVLIVLSWVLFAACLSYVKVMLGVILRDRSRSALLWCGAAVQLGSLIGALLMFPLVNVLKLFSSADYCSLDCSV

[0303] Amino acid sequence of a mouse - derived CAR targeting Trop2 (SEQ ID NO: 18)

[0304] MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQASNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYLELFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR

[0305] Human SLC52A3-CAR Amino Acid Sequence (SEQ ID NO: 19)

[0306] MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQASNSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLEFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMAFLMHLLVCVFGMGSWVTINGLWVELPLLVMELPEGWYLPSYLTVVIQLANIGPLLVTLLHHFRPSCLSEVPIIFTLLGVGTVTCIIFAFLWNMTSWVLDGHHSIAFLVLTFFLALVDCTSSVTFLPFMSRLPTYYLTTFFVGEGLSGLLPALVALAQGSGLTTCVNVTEISDSVPSPVPTRETDIAQGVPRALVSALPGMEAPLSHLESRYLPAHFSPLVFFLLLSIMMACCLVAFFVLQRQPRCWEASVEDLLNDQVTLHSIRPREENDLGPAGTVDSSQGQGYLEEKAAPCCPAHLAFIYTLVAFVNALTNGMLPSVQTYSCLSYGPVAYHLAATLSIVANPLASLVSMFLPNRSLLFLGVLSVLGTCFGGYNMAMAVMSPCPLLQGHWGGEVLIVASWVLFSGCLSYVKVMLGVVLRDLSRSALLWCGAAVQLGSLLGALLMFPLVNVLRLFSSADFCNLHCPA

[0307] Human-CD8-hinge amino acid sequence (SEQ ID NO: 20)

[0308] NSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0309] Human-CD28-TM amino acid sequence (SEQ ID NO: 21)

[0310] LEFWVLVVVGGVLACYSLLVTVAFIIFWV

[0311] Human-CD28 costi amino acid sequence (SEQ ID NO: 22)

[0312] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0313] Human-CD3-ζ-intracellular amino acid sequence (SEQ ID NO: 23)

[0314] KLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0315] Human SLC52A3 amino acid sequence (SEQ ID NO: 24)

[0316] MAFLMHLLVCVFGMGSWVTINGLWVELPLLVMELPEGWYLPSYLTVVIQLANIGPLLVTLLHHFRPSCLSEVPIIFTLLGVGTVTCIIFAFLWNMTSWVLDGHHSIAFLVLTFFLALVDCTSSVTFLPFMSRLPTYYLTTFFVGEGLSGLLPALVALAQGSGLTTCVNVTEISDSVPSPVPTRETDIAQGVPRALVSALPGMEAPLSHLESRYLPAHFSPLVFFLLLSIMMACCLVAFFVLQRQPRCWEASVEDLLNDQVTLHSIRPREENDLGPAGTVDSSQGQGYLEEKAAPCCPAHLAFIYTLVAFVNALTNGMLPSVQTYSCLSYGPVAYHLAATLSIVANPLASLVSMFLPNRSLLFLGVLSVLGTCFGGYNMAMAVMSPCPLLQGHWGGEVLIVASWVLFSGCLSYVKVMLGVVLRDLSRSALLWCGAAVQLGSLLGALLMFPLVNVLRLFSSADFCNLHCPA

[0317] Amino acid sequence of human CAR targeting Trop2 (SEQ ID NO: 25)

[0318] MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQASNSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLEFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0319] Linker amino acid sequence (SEQ ID NO: 26)

[0320] GGGGSGGGGSGGGGS

[0321] IgGκ signal peptide amino acid sequence (SEQ ID NO: 27)

[0322] MVLQTQVFISLLLWISGAYG

[0323] Amino acid sequence of murine SLC52A3-CAR (without signal peptide) (SEQ ID NO: 28)

[0324] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQASNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYLELFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPRGSGATNFSLLKQAGDVEENPGPMAFLTHLLVCVFGMGSWVAINGLWVELPLLVTELPEAWYLPSYLTVVIQLANIGPLLVTLMHRFRPGCLSEVPVIFLILCVGTAACILLAFLWNVTSWIQGGQHSVAFIVLTFFLALVDCTSSVTFLPFMSQLPTYYLTTFFIGEGLSGLLPALVALVQGSGITTCVNVTETPGTTLNTMETPITQGNLSPSLPSPSWHQESRYLAPRFSPLLFFLLLSFLTGCCLVAFFLLQRQPWGRQGSIEDLLHSQVTLHSIRPRDTEDTSSLGAPVSSPGKGSVEASVASLRPAQLAFIYSVVAFVNALTNGVLPSVQTYSCLPYGPVAYHLSATLSSVASPLACFLPIFLPNRSLLFLGVLTVLGTGFGAYNMAMAAMSPCPVLQGHWGGEVLIVLSWVLFAACLSYVKVMLGVILRDRSRSALLWCGAAVQLGSLIGALLMFPLVNVLKLFSSADYCSLDCSV

[0325] Amino acid sequence of murine CAR targeting Trop2 (without signal peptide) (SEQ ID NO: 29)

[0326] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQASNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYLELFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR

[0327] Amino acid sequence of human SLC52A3-CAR (without signal peptide) (SEQ ID NO: 30)

[0328] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQASNSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLEFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMAFLMHLLVCVFGMGSWVTINGLWVELPLLVMELPEGWYLPSYLTVVIQLANIGPLLVTLLHHFRPSCLSEVPIIFTLLGVGTVTCIIFAFLWNMTSWVLDGHHSIAFLVLTFFLALVDCTSSVTFLPFMSRLPTYYLTTFFVGEGLSGLLPALVALAQGSGLTTCVNVTEISDSVPSPVPTRETDIAQGVPRALVSALPGMEAPLSHLESRYLPAHFSPLVFFLLLSIMMACCLVAFFVLQRQPRCWEASVEDLLNDQVTLHSIRPREENDLGPAGTVDSSQGQGYLEEKAAPCCPAHLAFIYTLVAFVNALTNGMLPSVQTYSCLSYGPVAYHLAATLSIVANPLASLVSMFLPNRSLLFLGVLSVLGTCFGGYNMAMAVMSPCPLLQGHWGGEVLIVASWVLFSGCLSYVKVMLGVVLRDLSRSALLWCGAAVQLGSLLGALLMFPLVNVLRLFSSADFCNLHCPA

[0329] Amino acid sequence of human CAR targeting Trop2 (without signal peptide) (SEQ ID NO: 31)

[0330] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSQASNSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLEFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. Engineered immune cells comprising modifications that endow the immune cells with enhanced vitamin B2 uptake capacity.

2. The engineered immune cells of claim 1, wherein, The modification is used to increase the expression level of vitamin B2 transporter protein in the immune cells (e.g., upregulate the expression level of endogenous vitamin B2 transporter protein in the immune cells, and / or cause the immune cells to express exogenous vitamin B2 transporter protein). Preferably, the modification includes one or more selected from the following: (i) Enhance the promoter of the gene encoding the vitamin B2 transporter. (ii) Linking or introducing the vitamin B2 transporter encoding gene into an enhancer, (iii) Increase the copy number of the gene encoding the vitamin B2 transporter. (iv) Introduce exogenous polynucleotides encoding vitamin B2 transporter proteins.

3. The engineered immune cells according to claim 1 or 2, wherein, The vitamin B2 transporter is selected from: SLC52A1, SLC52A2, SLC52A3, their orthologs, homologs, variants and functional fragments, and any combination thereof; Preferably, the vitamin B2 transporter is selected from SLC52A3 and its orthologs, homologs, variants and functional fragments.

4. Engineered immune cells containing exogenously introduced polynucleotides encoding vitamin B2 transporters; Preferably, the vitamin B2 transporter is selected from: SLC52A1, SLC52A2, SLC52A3, their orthologs, homologs, variants and functional fragments, and any combination thereof; Preferably, the vitamin B2 transporter is selected from: SLC52A3 and its orthologs, homologs, variants and functional fragments; Preferably, the vitamin B2 transporter encoded by the exogenously introduced polynucleotide is derived from the same species as the immune cells.

5. The engineered immune cells according to any one of claims 1-4, wherein, The immune cells are selected from the following groups: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδ T cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof. Preferably, the immune cells are selected from: chimeric antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), chimeric antigen receptor macrophages (CAR-M), chimeric antigen receptor NK cells, chimeric antigen receptor γδ T cells, tumor-infiltrating lymphocytes (TILs) (e.g., tumor-infiltrating T lymphocytes), and any combination thereof.

6. The engineered immune cells according to any one of claims 1-5, wherein, The immune cells contain antigen receptors (e.g., T-cell receptors (TCRs), chimeric antigen receptors (CARs)) or their encoded polynucleotides that specifically bind to disease-associated antigens; Preferably, the antigen receptor specifically binds to tumor-associated antigens, autoimmune disease-associated antigens, or viral antigens; Preferably, the antigen receptor specifically binds to antigens selected from the group consisting of: CD19, BCMA, CD22, CD20, CD30, CD33, CD123, GD2, HER2, MSLN, Claudin 18.2, GPC3, PSMA, TROP2, and any combination thereof; Preferably, the antigen receptor specifically binds to TROP2.

7. The engineered immune cells according to any one of claims 1-6, wherein, The immune cells contain a CAR or its encoded polynucleotide, the CAR comprising: (a) An extracellular antigen-binding domain that specifically binds to disease-associated antigens; (b) Transmembrane domains; and (c) Intracellular signal transduction domains; Preferably, the extracellular antigen-binding domain specifically binds to tumor-associated antigens, autoimmune disease-associated antigens, or viral antigens; Preferably, the extracellular antigen-binding domain specifically binds to antigens selected from the group consisting of: CD19, BCMA, CD22, CD20, CD30, CD33, CD123, GD2, HER2, MSLN, Claudin 18.2, GPC3, PSMA, TROP2, and any combination thereof. Preferably, the extracellular antigen-binding domain specifically binds to TROP2; preferably, the extracellular antigen-binding domain comprises: HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 3, and / or, LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:

4.

8. The engineered immune cell of claim 7, wherein it possesses one or more of the following characteristics: (1) The transmembrane structural domain is derived from the group consisting of: CD3, CD4, CD137, CD80, CD86, CD152, PD-1, CD8 (e.g., CD8α) and CD28; (2) The intracellular signal transduction domain includes the primary intracellular signal transduction domain of immune effector cells; preferably, the primary intracellular signal transduction domain is derived from CD3ζ; (3) The intracellular signal transduction domain further includes a co-stimulatory signal transduction domain; preferably, the co-stimulatory domain is derived from a co-stimulatory molecule, which is selected from the group consisting of: ligands of CD27, CD137, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CD28, 4-1BB, OX-40 and any combination thereof; (4) The CAR further includes a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain; preferably, the hinge domain is derived from CD8 (e.g., CD8α). (5) The CAR further comprises a signal peptide located at the N-terminus of its polypeptide; preferably, the signal peptide is a signal peptide derived from IgGκ, CD8α or GM-CSF.

9. The engineered immune cells according to claim 7 or 8, wherein, The CAR comprises: an extracellular antigen-binding domain that specifically binds to TROP2 (e.g., scFv), a hinge domain derived from CD8, a transmembrane domain and a co-stimulatory domain derived from CD28, and a primary intracellular signal transduction domain derived from CD3ζ.

10. The engineered immune cell according to any one of claims 7-9, comprising a first polynucleotide encoding the CAR and a second polynucleotide exogenously introduced encoding a vitamin B2 transporter; preferably, the vitamin B2 transporter is as defined in claim 4; Preferably, the first polynucleotide and the second polynucleotide are each located in different expression cassettes, or are located together in the same expression cassette; Preferably, the first polynucleotide and the second polynucleotide are located in the same expression cassette and are linked by a self-cleaving adapter (e.g., a 2A adapter, such as P2A) encoding a polynucleotide. Preferably, the first polynucleotide and / or the second polynucleotide are present in a form that is integrated into or not integrated into the genome.

11. A nucleic acid molecule or group of nucleic acid molecules comprising: a first nucleotide sequence encoding a chimeric antigen receptor (CAR), and a second nucleotide sequence encoding a vitamin B2 transporter; in, The first nucleotide sequence and the second nucleotide sequence are located on the same or different nucleic acid molecules; preferably, the second nucleotide sequence is not naturally occurring. Preferably, the first nucleotide sequence and the second nucleotide sequence are located on the same nucleic acid molecule; Preferably, the first nucleotide sequence and the second nucleotide sequence are located in the same expression cassette; Preferably, the first nucleotide sequence is operatively linked to the second nucleotide sequence by a nucleotide sequence encoding a self-cutting adapter (such as a 2A adapter, such as P2A) or a nucleotide sequence encoding an internal ribosome entry site (IRES).

12. The nucleic acid molecule or group of nucleic acid molecules according to claim 11, wherein, The CAR is as defined in any one of claims 7-9, and / or the vitamin B2 transporter is as defined in claim 4.

13. One or more vectors comprising the nucleic acid molecule or nucleic acid molecule group as described in claim 11 or 12; Preferably, the vector is a plasmid vector or a viral vector; Preferably, the viral vector is selected from retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus (AAV) vectors.

14. A method for constructing engineered immune cells according to any one of claims 1-10, comprising: (1) Provide immune cells; and, (2) Introducing the immune cells with the modification defined in any one of claims 1-3, or introducing the immune cells with a polynucleotide containing a sequence encoding a vitamin B2 transporter; Preferably, the immune cells are as defined in any one of claims 5-9; Preferably, the vitamin B2 transporter is as defined in claim 4; Preferably, the method further comprises: introducing a polynucleotide encoding a chimeric antigen receptor into the immune cells; wherein the chimeric antigen receptor is capable of specifically binding to disease-associated antigens; preferably, the chimeric antigen receptor is as defined in any one of claims 7-9.

15. A method for constructing engineered immune cells according to any one of claims 1-10, comprising: (1) Provide immune cells; and, (2) Introducing the immune cells with a first polynucleotide encoding a chimeric antigen receptor sequence and a second polynucleotide encoding a vitamin B2 transporter sequence; The chimeric antigen receptor is capable of specifically binding to disease-associated antigens; Preferably, the first polynucleotide and the second polynucleotide are located in different expression cassettes or in the same expression cassette; preferably, the first polynucleotide and the second polynucleotide are located in the same expression cassette and are linked by a coding polynucleotide of a self-cleaving adapter (e.g., a 2A adapter, such as P2A); preferably, the first polynucleotide and / or the second polynucleotide are present in a form that is non-integrated or integrated into the immune cell genome. Preferably, the chimeric antigen receptor is as defined in any one of claims 7-9; Preferably, the vitamin B2 transporter is as defined in claim 4; Preferably, the immune cells mentioned in step (1) are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδT cells, peripheral blood mononuclear cells (PBMCs) and any combination thereof; preferably, the immune cells are immune cells isolated from the donor.

16. A pharmaceutical composition comprising an engineered immune cell as described in any one of claims 1-10, a nucleic acid molecule or group of nucleic acid molecules as described in claim 11 or 12, or one or more carriers as described in claim 13, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; preferably, the additional pharmaceutically active agent is selected from: immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody), additional engineered immune cells (e.g., TCR-T, CAR-T, CAR-M), and any combination thereof.

17. Use of the engineered immune cells of any one of claims 1-10, the nucleic acid molecule or nucleic acid molecule group of claims 11 or 12, one or more carriers of claim 13, or the pharmaceutical composition of claim 16 in the preparation of a medicament for use in a subject to enhance immune cell activity, enhance immune response, and / or prevent and / or treat tumors or infections or autoimmune diseases; Preferably, the immune cells are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδT cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof; Preferably, the enhancement of immune cell activity includes: Promote immune cell differentiation (e.g., memory differentiation), enhance immune cell function, and / or inhibit immune cell depletion; Preferably, the immune response includes cellular immune response and / or humoral immune response; Preferably, the tumor is selected from solid tumors or hematologic malignancies; Preferably, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer), melanoma, colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and any combination thereof; Preferably, the autoimmune disease is selected from: systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease, ulcerative colitis, and any combination thereof; Preferably, the infection is selected from viral infection, bacterial infection, fungal infection, and parasitic infection; Preferably, the subject is a mammal, such as a human; Preferably, the engineered immune cells, nucleic acid molecules or groups of nucleic acid molecules, one or more carriers or pharmaceutical compositions are administered in combination with an additional pharmaceutically active agent (e.g., simultaneously, separately or sequentially); preferably, the additional pharmaceutically active agent is selected from: immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody), additional engineered immune cells (e.g., TCR-T, CAR-T, CAR-M), and any combination thereof.

18. A composition comprising: (a) Vitamin B2, or a pharmaceutically acceptable salt thereof, its stereoisomers, its crystals, solvates, hydrates, or derivatives thereof, as well as, (b) Immunostimulants selected from: immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies), engineered immune cells, and any combination thereof; Preferably, the engineered immune cells are selected from chimeric antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), chimeric antigen receptor macrophages (CAR-M), chimeric antigen receptor NK cells, chimeric antigen receptor γδ T cells, tumor-infiltrating lymphocytes (TILs) (e.g., tumor-infiltrating T lymphocytes), and any combination thereof; Preferably, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier; Preferably, (a) and (b) are provided in a mixed or separate form.

19. Use of vitamin B2, or a pharmaceutically acceptable salt thereof, its stereoisomers, its crystals, solvates, hydrates or derivatives thereof, in the preparation of a medicament for use in subjects to enhance immune cell activity and / or enhance immune responses; Preferably, the immune cells are selected from: T cells, macrophages (e.g., M1 macrophages), natural killer (NK) cells, natural killer T (NKT) cells, γδT cells, peripheral blood mononuclear cells (PBMCs), and any combination thereof; Preferably, the immune cells are selected from: chimeric antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), chimeric antigen receptor macrophages (CAR-M), chimeric antigen receptor NK cells, chimeric antigen receptor γδ T cells, tumor-infiltrating lymphocytes (TILs) (e.g., tumor-infiltrating T lymphocytes), and any combination thereof; Preferably, the immune cells are tumor-infiltrating immune cells; Preferably, the enhancement of immune cell activity includes: Promote immune cell differentiation (e.g., memory differentiation), enhance immune cell function, and / or inhibit immune cell depletion; Preferably, the immune response includes cellular immune response and / or humoral immune response; Preferably, the subject is a mammal, such as a human; Preferably, the drug further includes immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody).

20. Use of the composition of claim 18 in the preparation of a medicament for the prevention and / or treatment of tumors, infections, or autoimmune diseases in a subject; Preferably, the tumor is selected from solid tumors or hematologic malignancies; Preferably, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer), melanoma, colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and any combination thereof; Preferably, the autoimmune disease is selected from: systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, type 1 diabetes, psoriasis Crohn's disease, and ulcerative colitis; Preferably, the infection is selected from viral infection, bacterial infection, fungal infection, and parasitic infection; Preferably, the subject is a mammal, such as a human.

Images