Application of chimeric antigen receptor macrophages in immunotherapy of solid tumors

Abstract

This application relates to the use of chimeric antigen receptor macrophages in the immunotherapy of solid tumors. Specifically, it relates to a chimeric antigen receptor targeting VEGFR2, its encoding nucleic acid, an expression vector, immune response cells (such as macrophages) expressing the chimeric antigen receptor, and its use in the treatment of breast cancer.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology and relates to a novel chimeric antigen receptor (CAR) more suitable for activating macrophages, a chimeric antigen receptor-macrophage (CAR-M) and its preparation method, as well as its pharmaceutical applications. Background Technology

[0002] In 2020, there were approximately 2.26 million new cases of breast cancer globally, surpassing lung cancer to become the leading cause of cancer death worldwide. In the same year, 685,000 people died from breast cancer. In China, the incidence of breast cancer is also rising annually; in 2020, it ranked fourth in new cases, after lung cancer, colorectal cancer, and stomach cancer, making it the leading cause of death for Chinese women.

[0003] Breast cancer is a heterogeneous tumor. Based on the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), breast cancer is clinically classified into four molecular subtypes: Luminal A, Luminal B, HER2-positive, and triple-negative.

[0004] In the early stages of breast cancer, surgery, adjuvant chemotherapy, and radiotherapy remain the preferred treatment methods. Compared to chemotherapy, targeted drugs have the advantages of high specificity, high efficacy, and low toxicity, and they target a wider range of targets, including HER2, epidermal growth factor receptor, vascular endothelial growth factor, and tyrosine kinase inhibitors. However, many cancer patients develop resistance to these treatments. Immunotherapy, including antibodies, vaccines, immune checkpoint inhibitors, and adoptive cell therapy, offers hope for patients.

[0005] Chimeric antigen receptor-T (CAR-T) cell therapy is one of the most innovative immunotherapies, achieving significant breakthroughs in the treatment of hematologic malignancies, such as demonstrating a 92% complete recovery rate in the treatment of acute lymphoblastic leukemia (ALL). Currently, six CAR-T products have been approved by the US FDA, and two have been approved by the China National Medical Products Administration for the treatment of hematologic malignancies such as diffuse large B-cell lymphoma (DLBCL), acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), adult mantle cell lymphoma (MCL), and multiple myeloma (MM) under specific conditions. CAR-T technology involves genetically modifying a patient's or donor's T cells to express chimeric antigen receptors, thereby enhancing the targeting, killing, and persistence of effector T cells. CAR-T cell therapy has been explored for the treatment of solid tumors, with several clinical trials targeting breast cancer underway, such as NCT01022138, NCT02713984, and NCT03740256. However, early trials have shown that CAR-T therapy is less effective in solid tumors, primarily due to low CAR-T cell infiltration rates and the immunosuppressive state of the tumor microenvironment promoting T cell dysfunction. Furthermore, CAR-T therapy can trigger severe adverse reactions such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), limiting its efficacy and widespread application.

[0006] Macrophages are innate immune cells that play a crucial role in immune defense and possess a wide range of biological functions. They can trigger both innate immune responses and initiate adaptive immune regulation. Human macrophages can differentiate into various phenotypes depending on the composition of cytokines in the surrounding environment and the influence of the tissue microenvironment. However, they are generally simplified into two types based on their immune function: M1 type (pro-inflammatory, classical activated macrophages) and M2 type (anti-inflammatory, alternative activated macrophages). M1 macrophages can be stimulated by lipopolysaccharide (LPS) and IFN-gamma. They induce inflammatory responses by secreting pro-inflammatory cytokines such as TNF-α and IL-6, thereby clearing pathogens and tumor cells from the body. M2 macrophages secrete high levels of anti-inflammatory cytokines such as IL-10, PGE2, and TGF-β, primarily participating in the clearance of parasites and the maintenance of homeostasis. Tumor-associated macrophages (TAMs) reside in the tumor microenvironment (TME) and are often considered to possess M2-type characteristics. In the tumor microenvironment, TAMs suppress endogenous anti-tumor immunity through various mechanisms, including upregulating immunosuppressive surface proteins, secreting reactive oxygen species, producing cytokines that inhibit T cell function, and secreting chemokines that recruit Treg cells. Furthermore, TAMs have been shown to directly or indirectly participate in several key processes related to malignant tumor development, including promoting tumor angiogenesis, promoting tumor invasion and metastasis, regulating the tumor microenvironment, and enhancing tumor treatment resistance. Studies have shown that macrophages possess strong plasticity, and the different phenotypes mentioned above can transform into each other under certain conditions. In addition, unlike the low infiltration rate of T cells in tumors, macrophages account for 30% to 50% of all immune cells in various solid tumors. These characteristics make TAMs potential effector cells or targets for tumor therapy.

[0007] Currently, Dr. Saar Gill of the University of Pennsylvania has successfully constructed the first-generation chimeric antigen receptor macrophage (CARMA), which combines the variable region (scFv) of a single-chain antibody against CD19, mesothelin, or HER2 with the intracellular CD3 domain. This CARMA has demonstrated potent killing activity in preclinical models, and the antigen receptor macrophage targeting HER2 has completed a phase I clinical trial (CT-0508). Researchers at Zhejiang University have developed chimeric antigen receptor-expressing macrophages (CAR-iMac) derived from induced pluripotent stem cells (iPSCs) for tumor immunotherapy. CAR-iMacs targeting CD19 or mesothelin have demonstrated the ability to inhibit tumor cell growth in mouse models of hematologic malignancies and solid tumors.

[0008] Most solid tumors require angiogenesis for growth, survival, and metastasis. Among all cytokines that regulate angiogenesis, vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and tumor metastasis in human tumorigenesis. Studies have shown that blocking the VEGF signaling pathway can delay tumor angiogenesis and inhibit tumor growth. VEGF exerts its effects by binding to and activating its receptors, VEGFRs. In tumor angiogenesis, VEGFR2 is the main receptor for VEGF-stimulated changes in cell viability and vascular permeability. It has been reported that the rational combination of VEGFR2 inhibitors and anticancer drugs can synergistically kill tumor cells.

[0009] Therefore, there is still a need in the field for a novel design of CARs, chimeric antigen receptor macrophages (CAR-M) and their preparation methods that are more suitable for activating macrophages, as well as pharmaceutical applications targeting VEGFR2 as an example. Summary of the Invention

[0010] Some embodiments of this application provide a chimeric antigen receptor comprising an antigen-binding domain that binds to VEGFR2, wherein the VEGFR2 is human VEGFR2 or non-human animal VEGFR2, preferably human VEGFR2.

[0011] In some implementation schemes, the non-human animal is selected from: mice, rats, guinea pigs, canines, lagodons, primates, bovids, and sheep; mice and rats are preferred.

[0012] In some implementations, the antigen-binding domain includes a heavy chain variable region and a light chain variable region.

[0013] In some implementations, a linker peptide is included between the heavy chain variable region and the light chain variable region.

[0014] In some implementations, the linker peptide is (G x S) y As shown, x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6 (1, 2, 3, 4, 5, or 6).

[0015] In some specific implementations, the linker peptide is (G4S). n As shown, n is 2, 3, 4, or 5; more preferably n is 3. As an example, the linker peptide is shown in SEQ ID NO: 13.

[0016] In some embodiments, the chimeric antigen receptor of this application further comprises any one or a combination of the following: a hinge region, a transmembrane domain, and an intracellular domain.

[0017] In some embodiments, the hinge region is located at the carboxyl terminus of the antigen-binding domain.

[0018] In some embodiments, the transmembrane domain is located at the carboxyl terminus of the hinge region.

[0019] In some implementations, the intracellular domain is located at the carboxyl terminus of the transmembrane domain.

[0020] In some embodiments, the hinge region is selected from any of the following: the hinge region of TLR4, the hinge region of IFNGR1, the CD8α domain, the CD28 domain, the IgG Fc domain, the CH2 / CH3 domain of Fc, and DAP12.

[0021] In some implementations, the transmembrane domain is selected from any of the following: the transmembrane domain of TLR4, the transmembrane domain of IFNGR1.

[0022] In some implementations, the intracellular domain comprises any one or a combination of the following: at least one signal transduction region, at least one co-stimulatory signal transduction region, and optional adapters.

[0023] In some implementations, the intracellular domain is selected from any of the following structures:

[0024] i) First intracellular domain-connector-second intracellular domain-connector-third intracellular domain;

[0025] ii) First intracellular domain - Second intracellular domain - Third intracellular domain;

[0026] iii) First intracellular domain;

[0027] iv) Second intracellular domain - connector - third intracellular domain;

[0028] v) Second intracellular domain - Third intracellular domain;

[0029] - indicates a covalent bond.

[0030] In some implementations, the first intracellular domain, the second intracellular domain, and the third intracellular domain are each independently selected from or include any one of the following: the intracellular domain of TLR4, the TIR domain of TLR4, the intracellular domain of IFNGR1, the intracellular domain of IFNGR2, and the type III fibronectin domain of IFNGR2.

[0031] In some specific implementations, the intracellular domain comprises any one of the following:

[0032] i) TLR4 intracellular domain-connector-IFNGR1 intracellular domain-connector-IFNGR2 intracellular domain;

[0033] ii) TLR4 intracellular domain - IFNGR1 intracellular domain - IFNGR2 intracellular domain;

[0034] iii) TLR4 intracellular domain;

[0035] iv) IFNGR1 intracellular domain-connector-IFNGR2 intracellular domain;

[0036] v) IFNGR1 intracellular domain - IFNGR2 intracellular domain.

[0037] In some specific implementations, the signal transduction region is selected from any one or a combination of the following: phosphorylation sites of downstream signals of IFNGR1, and type III fibronectin domains of IFNGR2.

[0038] In some specific implementations, the co-stimulatory signal transduction region is selected from any one or a combination of the following: the TIR domain of TLR4.

[0039] In some specific implementations, the connector is (G x S) y As shown, x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6; preferably, the connector is (G4S). n As shown, n is 2, 3, 4, or 5; more preferably, n is 3.

[0040] In some specific implementations, the TLR4 is a human or non-human animal TLR4.

[0041] In some specific implementations, the IFNGR1 is a human or non-human animal IFNGR1.

[0042] In some specific implementations, the IFNGR2 is human or non-human animal IFNGR2.

[0043] In some specific embodiments, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5 or 17 (90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any range between two values, including integers and decimals), and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 or 16 (90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any range between two values, including integers and decimals).

[0044] In some specific implementations, the antigen-binding domain is scFv.

[0045] In some specific implementations, the antigen-binding domain is a human or non-human animal scFv.

[0046] In some specific embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 4 or 15.

[0047] In some embodiments, the antigen-binding domain is also covalently bound to a signal peptide at its N-terminus.

[0048] In some specific implementations, the signal peptide is the signal peptide of IFNGR1.

[0049] In some specific embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2.

[0050] In some implementations, the chimeric antigen receptor has the structure shown in formula (I) from the amino terminus to the carboxyl terminus:

[0051] Signal peptide-antigen binding domain-hinge region-transmembrane domain-intracellular domain, formula (I);

[0052] Where - represents a covalent bond.

[0053] According to some implementation schemes, an immune-response cell is provided that expresses the chimeric antigen receptor of this application.

[0054] In some embodiments, the chimeric antigen receptor is constitutively expressed on the surface of the immune-responding cells. In some embodiments, the immune-responding cells are macrophages.

[0055] According to some implementation schemes, a nucleic acid molecule is provided that encodes the chimeric antigen receptor of this application.

[0056] In some specific implementations, the nucleic acid molecule encoding the scFv contains a polynucleotide as shown in SEQ ID NO:3 or 14.

[0057] In some specific implementations, the nucleic acid molecule encoding the signal peptide comprises a polynucleotide as shown in SEQ ID NO:1.

[0058] In some specific implementations, the nucleic acid molecule encoding the hinge region comprises a polynucleotide as shown in SEQ ID NO:24 or 30.

[0059] In some specific implementations, the nucleic acid molecule encoding the transmembrane domain comprises a polynucleotide as shown in SEQ ID NO:26 or 32.

[0060] In some specific implementations, the nucleic acid molecule encoding the intracellular domain comprises a polynucleotide as shown in SEQ ID NO:28, 34 or 36.

[0061] According to some implementation schemes, an expression vector is provided that contains the nucleic acid molecule of this application.

[0062] In some specific implementations, the expression vector further comprises any one or a combination of the following: promoter, start sequence, origin of replication, terminator, enhancer, restriction endonuclease site, label, internal ribosome entry site, and polyA.

[0063] In some specific implementations, the marker is a screening marker, an resistance marker, or a fluorescent marker.

[0064] In some embodiments, the expression vector is selected from any of the following: lentiviral vector, adenovirus vector, adeno-associated virus vector, plasmid, Sendai virus vector, adenovirus vector, adeno-associated virus vector, retroviral vector, gamma retroviral vector, herpesvirus vector.

[0065] In some specific implementations, the expression vector is a lentiviral vector or an adenovirus vector.

[0066] According to some implementation schemes, a host cell is provided which contains the expression vector of this application.

[0067] The host cell cannot develop into a complete plant or animal individual.

[0068] In some specific implementations, the host cell is a macrophage.

[0069] According to some embodiments, a pharmaceutical composition is provided comprising a therapeutically effective amount of the immune-responding cells or host cells of the present application; optionally, a pharmaceutically acceptable carrier.

[0070] According to some embodiments, a kit for treating cancer (preferably breast cancer) is provided, comprising a therapeutically effective amount of the immune-responding cells or host cells of this application. In some embodiments, the breast cancer is selected from any of the following: Luminal A, Luminal B, HER2-positive, and triple-negative.

[0071] According to some implementation schemes, use in the preparation of a medicament for treating breast cancer is provided from any of the following: the chimeric antigen receptor of this application, the immune response cell of this application, the nucleic acid molecule of this application, the expression vector of this application, and the host cell of this application.

[0072] According to some implementation schemes, use of any of the following in the preparation of a medicament for improving the survival rate of breast cancer subjects is provided: the chimeric antigen receptor of this application, the immune response cells of this application, the nucleic acid molecules of this application, the expression vector of this application, and the host cells of this application.

[0073] In some specific implementations, the breast cancer is selected from any of the following: Luminal A, Luminal B, HER2 positive, or triple negative. Attached Figure Description

[0074] Figure 1 Exemplary plasmid map.

[0075] Figures 2A to 2E The study investigated the phagocytosis of tumor cells by CAR-M and the phenotype of CAR-M after co-culturing CAR-M with HUVEC (highly expressing target VEGFR2) and 4T1 breast cancer cell lines.

[0076] Figures 3A to 3F Detection of TNF-α cytokine in the supernatant of co-cultured CAR-M, HUVEC and 4T1 breast cancer cell lines.

[0077] Figures 4A to 4E In vivo validation of the antitumor effect of CAR-M (small animal in vivo imaging experiment).

[0078] Figures 5A to 5C In vivo validation of the anti-tumor effect of CAR-M (immune-intelligent mouse breast cancer model).

[0079] Figures 6A to 6D In vivo validation of the antitumor effect of CAR-M (immunodeficient nude mouse breast cancer model).

[0080] Figures 7A to 7D Safety evaluation of CAR-M cell therapy. Detailed Implementation

[0081] Chimeric antigen receptor

[0082] This application provides a chimeric antigen receptor.

[0083] "Chimeric antigen receptor" or "CAR" refers to an artificial T-cell receptor engineered to express and specifically bind to an antigen (or its epitope) on immune effector cells. CARs can be used as a therapy employing adoptive cell transfer. T cells are removed from a subject and modified so that they express receptors specific to a particular antigen, such as a tumor-associated antigen. As an example, the extracellular antigen-binding domain of a CAR is engineered in the form of a single-chain antibody scFv that specifically targets and recognizes tumor-associated antigens (such as VEGFR2).

[0084] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to a molecule in which a heavy chain variable domain (VH) and a light chain variable domain (VL) are linked by a linker peptide. Such scFv molecules may have the general structure: NH₂-VL-linker peptide-VH-COOH or NH₂-VH-linker peptide-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof (e.g., Holliger et al. 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linker peptides that can be used in this application can be found in Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001) Cancer Immunol. Although the constant region is removed and linker peptides are introduced in scFv, scFv retains the antigen-binding specificity of the original immunoglobulin.

[0085] "CDR" refers to one of the hypervariable regions in the variable domains of an antibody that facilitates antigen binding. The most commonly used definition of CDR is provided by Kabat EA et al. (1991, Sequences of proteins of immunological interest. NIH 91-3242). Other well-known schemes can also be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat" numbering rule, the "Chothia" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. It should be understood that when applied to scFv, those skilled in the art are capable of determining the corresponding CDR numbering rule.

[0086] According to some implementation schemes, a chimeric antigen receptor is provided that includes an antigen-binding domain that binds to VEGFR2.

[0087] The term “VEGFR2” should be interpreted broadly to encompass all forms of VEGFR2 molecules at various stages in the animal body, including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the VEGFR2 gene (e.g., precursor VEGFR2, mature VEGFR2, membrane-expressed VEGFR2, VEGFR2 splice variants, modified VEGFR2, or fragments thereof). The term also covers artificially prepared or in vitro expressed VEGFR2.

[0088] In the specific implementation plan, VEGFR2 refers to human VEGFR2 or homologous VEGFR2 from non-human animals. Non-human animals refer to mice, rats, guinea pigs, canines, lagodons, primates, bovids, and sheep; mice and rats are preferred.

[0089] When used for therapeutic purposes in humans, especially human VEGFR2.

[0090] Technicians can obtain the amino acid and nucleotide sequence information of VEGFR2 from public databases.

[0091] "Binding VEGFR2" refers to the specific binding of VEGFR2 or its epitope. "Specific binding" means that the chimeric antigen receptor (or scFv, or CAR-M) in this application recognizes VEGFR2 or its epitope, but essentially does not recognize other molecules. Typically, "specific binding" means that the chimeric antigen receptor (or scFv, or CAR-M) binds at approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 The equilibrium dissociation constant (KD) (M or smaller) binds to the antigen or its epitope. KD can be measured using known methods, such as by... Measured by surface plasmon resonance assay. It should be understood that chimeric antigen receptors (or scFv, or CAR-M) that specifically bind to an antigen or its epitope may be cross-reactive to related antigens from other species, for example, to corresponding antigens from monkeys (e.g., cynomolgus monkeys, chimpanzees, or marmosets).

[0092] In some implementations, the antigen-binding domain includes a heavy chain variable region and a light chain variable region.

[0093] "Variable region" refers to the domain in the heavy chain or light chain that participates in binding antigens. The heavy chain variable region (VH) and the light chain variable region (VL) each contain four frame regions (FR) and three complementarity-determining regions (CDR).

[0094] In some embodiments, a linker peptide is included between the heavy chain variable region and the light chain variable region.

[0095] "Linker," "linker peptide," or "connector" refers to a linker unit that connects two polypeptide fragments. It typically possesses a degree of flexibility, and the use of a linker peptide does not result in the loss of the original function of the domain. Linker peptides appearing in the same chemical entity can be identical or different. A linker peptide contains one or more amino acids, typically about 1 to 30, 2 to 24, or 3 to 15 amino acids. The linker peptides used in this application can be the same or different.

[0096] In some implementations, the linker peptide is (G x S) y As shown, x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6.

[0097] In some implementations, the chimeric antigen receptor has a signal peptide covalently bound to the amino terminus of the antigen-binding domain (such as scFv).

[0098] In some specific implementations, the antigen-binding domain includes a leader sequence or signal peptide that guides the nascent protein into the endoplasmic reticulum. The leader sequence or signal peptide is essential if the CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader sequence is a peptide sequence (approximately 5, 10, 15, 20, 25, or 30 amino acids in length) located at the N-terminus of the newly synthesized protein that guides the protein into the secretory pathway.

[0099] In a non-limiting example, the signal peptide is covalently linked to the N-terminus of the antigen-binding domain. In some embodiments, the signal peptide comprises a signal peptide that is IFNGR1. In some specific embodiments, the signal peptide comprises the amino acids of the sequence shown in SEQ ID NO: ...

[0100] In some implementations, the chimeric antigen receptor also includes a hinge region.

[0101] The hinge region is an extracellular structural region connecting the scFv (cell ferrite core) and the transmembrane domain. The hinge region typically maintains stable CAR expression and activity in effector cells. Most CAR constructions utilize derivatives of the CD8α or CD28 extracellular domains or IgG-based hinge regions. Comparisons of CD28 and CD8α hinge regions have shown that CD28 promotes CAR molecule dimerization more effectively. Therefore, CARs with CD28 hinge regions produce a stronger activation stimulus, but may also lead to side effects. IgG-based hinge regions are also widely used in CARs. The advantage of IgG-based hinge regions is structural flexibility; they typically contain the Fc or CH2 / CH3 domains of IgG1 or IgG4. The length of the hinge region can be adjusted to accommodate antigen recognition.

[0102] In some embodiments, the hinge region is selected from any of the following: the hinge region of TLR4, the hinge region of IFNGR1, the CD8α domain, the CD28 domain, the IgG Fc domain, the CH2 / CH3 domain of Fc, and DAP12.

[0103] In some specific implementations, the hinge region contains amino acids of the sequence shown in SEQ ID NO: ...

[0104] In some implementations, the chimeric antigen receptor also includes a transmembrane domain.

[0105] The choice of transmembrane domain affects the degree of activation of the CAR structure in cellular function. In some embodiments, the transmembrane domain is selected from: the transmembrane domain of TLR4, the transmembrane domain of IFNGR1, or a combination thereof.

[0106] In some specific implementations, the transmembrane domain contains amino acids of the sequence shown in SEQ ID NO: ...

[0107] In some implementations, the chimeric antigen receptor also includes an intracellular domain.

[0108] In some implementations, the intracellular domain includes at least one signal transduction region and / or at least one co-stimulatory signal transduction region.

[0109] In some embodiments, the intracellular domain is selected from any one or a combination of the following: the intracellular domain of TLR4, the TIR domain of TLR4, the intracellular domain of IFNGR1, the intracellular domain of IFNGR2, and the type III fibronectin domain of IFNGR2.

[0110] In a non-limiting embodiment, the intracellular domains of the CAR can activate or stimulate macrophages.

[0111] In some embodiments, the signal transduction region is selected from any one or a combination of the following: phosphorylation sites of downstream IFNGR1 signals, the type III fibronectin domain of IFNGR2, lymphocyte receptor chains, TCR / CD3 complex protein, Fc receptor subunit, IL-2 receptor subunit, signal transduction regions of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD278, FcεRI, DAP10, and DAP12.

[0112] Intracellular signal transduction regions trigger the activation of at least one effector function of an immune cell. "Effective function" refers to a specialized cellular function. Therefore, a "signal transduction region" refers to a protein portion that transduces effector signals and directs the cell to perform its specialized function. While the entire signal transduction region can often be used, in many cases it is not necessary to use the entire strand. To the extent that a truncated portion of the signal transduction region is used, this truncated portion can be used in place of the complete strand, as long as it transduces effector signals.

[0113] Signals generated through a single signal transduction zone may not be sufficient to fully activate immune cells, and secondary or co-stimulatory signals may also be required.

[0114] The co-stimulatory signal transduction region refers to a portion of the CAR (cellular cellular receptor) that contains the intracellular domains of co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are required for macrophages to respond to antigens.

[0115] Various costimulatory domains confer different properties. For example, CARs that bind to DAP10 are associated with reduced persistence in vivo (Barber et al., Gene Ther 2011; 18:509-516). Different costimulatory domains produce different cytokine profiles, which may in turn affect target cell-mediated activity and the tumor microenvironment.

[0116] In view of this, in some embodiments, the intracellular domain further includes a co-stimulatory signal transduction region selected from any one or a combination of the following: the TIR domain of TLR4, MHC class I molecules, TNF receptors, cytokine receptors, integrins, signal transduction lymphocyte activating molecules, NK cell activating receptors, Toll ligand receptors, B7-H3, BAFFR, BTLA, BLAME, CD2, CD4, CD5, CD7, CD8α, CD8β, CD11a, LFA-1, CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96, CD100, CD103, OX40, 4-1BB, SLAM, CD160, SELPLG, DNAM1, Ly9, SLAMF4, I COS, CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM, IA4, ICAM-1, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, K IRDS2, LAT, LFA-1, LIGHT, LTBR, ​​NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80, PAG / Cbp, PD-1, PSGL1, SLAMF6, SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6.

[0117] The elements within the intracellular structural domains of the CAR in this application (such as co-stimulatory signal transduction regions and signal transduction regions) can be connected to each other in a random or specified order. Optionally, the elements are connected by linkers. Optionally, short oligopeptide or polypeptide linkers with a length between 2 and 10 amino acids are preferred; for example, glycine-serine provides a particularly suitable linker.

[0118] In some specific implementations, the chimeric antigen receptor has the following structure (N-terminus to C-terminus):

[0119] Signal peptide + scFv + hinge + transmembrane domain + intracellular domain.

[0120] In some specific embodiments, the chimeric antigen receptor comprises an amino acid sequence having at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) sequence identity with the amino acid sequence shown in SEQ ID NO: ...

[0121] "Identity" refers to the sequence identity between two polymer molecules, such as two amino acid molecules. Two amino acid sequences are identical at the same position when they have the same residue. In alignments, the degree of identity or similarity between two amino acid sequences is often expressed as a percentage.

[0122] Encoding nucleic acids

[0123] This application provides a nucleic acid molecule encoding the CAR of this application.

[0124] The term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, with double-stranded DNA being preferred.

[0125] Unless otherwise specified, “nucleic acid molecules encoding CAR” are also intended to include all polynucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.

[0126] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, the first nucleic acid sequence is operably linked to the second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, the operably linked DNA sequences are adjacent and, if necessary, in the same reading frame.

[0127] expression carrier

[0128] This application provides an expression vector for expressing the CAR of this application.

[0129] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0130] In some implementations, the expression vector is suitable for eukaryotes.

[0131] A "vector" includes isolated nucleic acids that can be used to deliver the isolated nucleic acids to target cells or their interior. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.

[0132] Viral vector technology is well known in the art and is described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory, New York. Numerous virus-based systems have been developed for the transfer of genes into mammalian cells.

[0133] Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gamma retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, lentiviral vectors, etc. For example, a gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art; the recombinant virus can then be isolated and delivered to the subject's in vivo or ex vivo cells. In some embodiments, the retroviral vector is pFSG or pFB. In other embodiments, an adenovirus vector is used; adenovirus vectors are known in the art.

[0134] The expression vector also includes sufficient functional elements for expression; other elements for expression may be supplied by the host cell or by the in vitro expression system.

[0135] In some implementations, expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operatively linking the nucleic acid encoding the CAR to a promoter and incorporating the construct into an expression vector.

[0136] Exemplary vectors contain any one or a combination of the following: transcription and translation terminators, start sequences, promoters, origin of replication, restriction endonuclease sites, markers, internal ribosome entry sites, and polyA.

[0137] In some implementations, immune cells expressing CAR can express CAR only transiently. For example, cells can be transduced using mRNA containing a nucleic acid sequence encoding the CAR. In this case, the method for generating mRNA for transfection includes in vitro transcription of a template with primers, followed by the addition of a polyA tail to produce a construct containing 3' and 5' untranslated sequences, a 5' cap and / or an internal ribosome entry site (IRES), the CAR-encoding nucleic acid to be expressed, and a polyA tail. The RNA thus generated can be efficiently transfected into different cell types. In one implementation, the CAR vector is transduced into cells via electroporation.

[0138] To assess the expression of CAR peptides or their components, the expression vector to be introduced into cells may also contain a marker gene or a reporter gene or both (such as selection markers, fluorescent markers, antibiotic markers, etc.) to facilitate the identification and selection of expressing cells from the cell population.

[0139] Reporter genes are used to identify transfected cells and to assess the function of regulatory sequences. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, alkaline phosphatase, or green fluorescent protein. In a preferred embodiment, the marker contains a nucleic acid sequence encoding GFP.

[0140] Various promoters may be used, including but not limited to the CMV promoter, elongation growth factor-1α, simian virus 40 early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (LTCR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rouss sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the use of constitutive promoters should not be limited. Inducible promoters are also considered part of this application. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0141] Cells expressing CAR (especially CAR-M)

[0142] This application also provides cells, cell populations, and compositions containing said cells, such as cells containing a nucleic acid sequence encoding a CAR.

[0143] This application provides an immune response cell or host cell that contains or expresses the CAR of this application.

[0144] In a specific implementation, the CAR is constitutively expressed on the surface of the cell.

[0145] Methods for generating cells containing expression vectors and / or exogenous nucleic acids are well known in the art. In the case of expression vectors, the expression vectors of this application can be introduced into host cells (e.g., mammalian macrophages) by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means. Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems (such as macromolecular complexes, nanocapsules, microspheres, beads); and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery vector in vitro and in vivo is a liposome.

[0146] The cells are typically eukaryotic cells, such as mammalian cells, and are often human cells, such as allogeneic or autologous donor cells. The cells used for CAR delivery can be isolated from a sample, for example, a sample obtained from or derived from a subject.

[0147] In some implementations, the subject is a subject with a disease, a subject who needs cell therapy, or a subject who will receive cell therapy. In some implementations, the subject is a person who needs a therapeutic intervention.

[0148] Prior to amplification and genetic modification, cells can be obtained from the subject using various non-restrictive methods. Cells can be obtained from many non-restrictive sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, the cells may be a mixed cell population. In the case of cell therapy (e.g., adoptive cell therapy), the sample includes samples from both autologous and allogeneic sources.

[0149] In some specific implementations, the cells are macrophages or macrophage populations.

[0150] In the context of this application, macrophages that express the CAR of this application on their cell surface or that contain the expression vector of this application are referred to as CAR-M.

[0151] Pharmaceutical Composition

[0152] This application provides a pharmaceutical composition comprising: a therapeutically effective amount of the aforementioned immune-response cells or the aforementioned host cells; and optionally, a pharmaceutically acceptable carrier.

[0153] Pharmaceutical compositions are generally intended for parenteral administration. As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized as follows: for example, administration into the bloodstream, into muscles, or into internal organs.

[0154] Therefore, parenteral administration includes, but is not limited to, administration of the drug composition by means of: injection, administration through surgical incision, administration through non-surgical wounds that penetrate tissue. Specifically, parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrasheath, intravenous, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion.

[0155] Pharmaceutical compositions suitable for parenteral administration typically contain an active ingredient and a pharmaceutically acceptable carrier. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, etc., in oily or aqueous media. As an example, aqueous solutions may contain carriers (such as salts, carbohydrates, and buffers). As yet another example, parenteral formulations are prepared as sterile non-aqueous solutions or as dry forms for use with a suitable medium (such as sterile, pyrogen-free water).

[0156] The pharmaceutical composition of this application may also be a formulation of liposomes. In the case of using a non-viral delivery system, an exemplary delivery carrier is a liposome. Nucleic acids can associate with lipids. The nucleic acids associated with lipids can be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, linked to the liposome via a linker, embedded in the liposome, complexed with the liposome, or contained in the lipid as a suspension.

[0157] Suitable lipids can be obtained from commercial sources. Examples include dimyristyl phosphatidylcholine (DMPC), diceryl phosphate, cholesterol, and dimyristyl phosphatidylglycerol.

[0158] In some implementations, the pharmaceutical composition contains a therapeutically effective amount of CAR cells.

[0159] In some implementations, the pharmaceutical composition may be prepared to contain a unit dose of CAR cells.

[0160] In some specific implementation schemes, the unit dose contains 1×10 6 Up to 5×10 6 Macrophages expressing CAR at high concentrations.

[0161] medicine box

[0162] This application provides a medicine box containing materials that can be used to treat or prevent disease. The medicine box includes one or more containers. Suitable containers include, for example, bottles, vials, syringes, IV bags, etc. The containers independently contain macrophages expressing CARs or other active compounds of this application.

[0163] The kit also includes any one or a combination of the following: a label or insert (indicating the indication for the cell), and other necessary materials (e.g., buffers, diluents, filters, needles, syringes).

[0164] Therapeutic uses and treatment methods

[0165] This application provides for the use of chimeric antigen receptors, nucleic acid molecules, expression vectors, or host cells (or immune response cells) according to this application for the treatment of cancer.

[0166] This application provides the use of the chimeric antigen receptor, nucleic acid molecule, expression vector, or host cell (or immune response cell) according to this application for the preparation of a medicament for the treatment of cancer.

[0167] This application provides the use of the chimeric antigen receptor, nucleic acid molecule, expression vector, or host cell (or immune response cell) according to this application for the preparation of a medicament for improving the survival rate of breast cancer subjects.

[0168] This application provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the host cells (or immune response cells) of this application.

[0169] In some implementations, the cancer is breast cancer. In some specific implementations, the breast cancer is selected from any of the following: Luminal A, Luminal B, HER2-positive, or triple-negative.

[0170] "Treatment" refers to eliminating disease, stopping disease progression, slowing disease progression, reducing the duration of one or more symptoms associated with the disease, improving or reversing at least one measurable parameter associated with the disease, or increasing the survival rate of a subject with the disease.

[0171] "Effective dose" refers to the amount of an active ingredient (such as the CAR-M of this application) that elicits the desired effect in a subject. In specific implementations, those skilled in the art can determine the selection of an effective dose based on considerations of a variety of factors (e.g., through clinical trials), including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the patient's weight, the patient's immune status, and other factors known to those skilled in the art.

[0172] The effective dosage in the specific implementation plan can be obtained from the dose-response curve derived from the animal model testing system, and can be determined based on the doctor's judgment and the condition of each patient. The relationship between the dosage in animals and humans is described in Freireich et al. 1966, Cancer Chemother Rep 50:219, and the human body surface area can be approximately determined by the patient's height and weight. The amount of drug required for a single administration to a patient can be conveniently obtained by calculating the product of the patient's weight and the unit body weight dose required for a single administration. For example, in the process of drug preparation, it is generally believed that the weight of an adult is 50-70 kg, and the dosage can initially be determined by the equivalent dose conversion relationship between the unit body weight dose of experimental animals and humans. For example, it can be determined according to the guidelines issued by drug regulatory agencies such as the FDA and SFDA, or by referring to ("Equivalent dose conversion between animals and between animals and humans in pharmacological experiments", Chinese Journal of Clinical Pharmacology and Therapeutics, 2004; 9(9): 1069-1072). In some implementations, the dosage for humans and mice can be converted using a conversion factor of 0.0026 based on the body surface area of ​​humans and mice.

[0173] In some implementations, for the case of genetically engineered macrophages expressing CAR, the number of cells administered to the subject ranges from approximately 1 million to approximately 100 billion; for example, 1 million to approximately 50 billion cells; approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or any range between any two of the aforementioned values. In some implementations, the number of cells administered to the subject ranges from approximately 10 million to approximately 100 billion; for example, approximately 20 million cells, approximately 30 million cells, approximately 40 million cells, approximately 50 million cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 20 billion cells, approximately 50 billion cells, or any range between any two of the aforementioned values. In a specific implementation plan, 300 to 500 million cells are administered to the subject, for example, 400 million ± 10% of cells (equivalent to a human dose).

[0174] CAR-expressing macrophages can be administered in a single dose or divided into many smaller unit doses administered at regular time intervals. It should be understood that the exact dose, duration, and interval of treatment are functions of the disease being treated and can be inferred from animal or clinical trial data. The administration may include a single dose or two or more doses at appropriate time intervals. The interval between two consecutive administrations may be 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0175] In the context, ifngr1, IFNGR1 and ifngr2, IFNGR2 represent the two subunits of the type II interferon receptor: the ligand adsorption chain and the accessory chain, respectively; tlr4 and TLR4 represent Toll-like receptor 4.

[0176] Those skilled in the art understand that although non-human animal (e.g., mouse) homologs of the target gene / protein are used in subsequent embodiments, corresponding human homologs will be used when applied to humans.

[0177] Example 1. Construction of CAR and its expression vector

[0178] 1. CAR is represented by the following general formula (I):

[0179] Signal peptide + antigen-binding domain (e.g., scFv) + hinge + transmembrane domain + intracellular domain.

[0180] 2. An exemplary CAR expression vector is shown in equation (II) below:

[0181] Signal peptide + antigen-binding domain (e.g., scFv) + hinge + transmembrane domain + intracellular domain + action element (e.g., IRES) (optional) + label (optional).

[0182] The intracellular domains are selected from any of the following structures:

[0183] i) First intracellular domain + connector + second intracellular domain + connector + third intracellular domain;

[0184] ii) First intracellular domain + Second intracellular domain + Third intracellular domain;

[0185] iii) First intracellular domain;

[0186] iv) Second intracellular domain + adapter + third intracellular domain;

[0187] v) Second intracellular domain + Third intracellular domain;

[0188] The first intracellular domain, the second intracellular domain, and the third intracellular domain are each independently selected from the intracellular domains of TLR4, IFNGR1, and IFNGR2.

[0189] 3. Exemplary expression carriers:

[0190] (1) mmA: ifngr1 signal peptide + mouse scFv + tlr4 hinge + tlr4 transmembrane domain + intracellular (tlr4 intracellular domain + adapter + ifngr1 intracellular domain + adapter + ifngr2 intracellular domain) + IRES + GFP.

[0191] (2) mmB: ifngr1 signal peptide + mouse scFv + tlr4 hinge + tlr4 transmembrane domain + intracellular (tlr4 intracellular domain + ifngr1 intracellular domain + ifngr2 intracellular domain) + IRES + GFP.

[0192] (3) mmC: ifngr1 signal peptide + mouse scFv + tlr4 hinge + tlr4 transmembrane domain + intracellular (tlr4 intracellular domain) + IRES + GFP;

[0193] (4) mmD: ifngr1 signal peptide + mouse scFv + ifngr1 hinge + ifngr1 transmembrane domain + intracellular (ifngr1 intracellular domain + adapter + ifngr2 intracellular domain) + IRES + GFP;

[0194] (5) mmE: ifngr1 signal peptide + mouse scFv + ifngr1 hinge + ifngr1 transmembrane domain + intracellular (ifngr1 intracellular domain + ifngr2 intracellular domain) + IRES + GFP;

[0195] (6)hmA: ifngr1 signal peptide + human scFv + tlr4 hinge + tlr4 transmembrane domain + intracellular (tlr4 + adapter + ifngr1 intracellular domain + adapter + ifngr2 intracellular domain) + IRES + GFP.

[0196] (7)hmB: ifngr1 signal peptide + human scFv + tlr4 hinge + tlr4 transmembrane domain + intracellular (tlr4 + ifngr1 intracellular domain + ifngr2 intracellular domain) + IRES + GFP;

[0197] (8)hmC: ifngr1 signal peptide + human scFv + tlr4 hinge + tlr4 transmembrane domain + intracellular (tlr4 intracellular domain) + IRES + GFP;

[0198] (9)hmD: ifngr1 signal peptide + human scFv + ifngr1 hinge + ifngr1 transmembrane domain + intracellular (ifngr1 intracellular domain + adapter + ifngr2 intracellular domain) + IRES + GFP;

[0199] (10)hmE: ifngr1 signal peptide + human scFv + ifngr1 hinge + ifngr1 transmembrane domain + intracellular (ifngr1 intracellular domain + ifngr2 intracellular domain) + IRES + GFP.

[0200] 4. The coding sequence of the vector is as follows (from the 5' to 3' end, and from the amino terminus to the carboxyl terminus):

[0201] (1) The coding sequence of the mouse-derived ifngr1 signal peptide:

[0202] atgggcccgcaggcggcagctggcaggatgattctgctggtggtcctgatgctgtctgcgaaggtcggggagtgga (SEQ ID NO: 1);

[0203] Mouse-derived ifngr1 signal peptide:

[0204] MGPQAAAGRMILLVVLMLSAKVGSG (SEQ ID NO: 2);

[0205] (2) Human IFNGR1 signal peptide coding sequence:

[0206] atggctctcctctttctcctaccccttgtcatgcagggtgtgagcagggct (SEQ ID NO: 38)

[0207] Human IFNGR1 signal peptide:

[0208] MALLFLLPLVMQGVSRA (SEQ ID NO: 39).

[0209] (3) Encoding sequence of murine VEGFR2 antibody scFv:

[0210] atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcactctgaggtgcagctggtggagaccggcggcggactggtgcagcctggaaactccctgaaactgtcctgtgctacaagcggcttcatcttctccaccacctggatgaactggattaggcagacacctggcaagaggctggaatggctggctcagattgaagacaaaagcaacaactacttcatcagctatagcgagtctgtgaagggcaggttcaccatcagcagggacgacagcaaaagcagcgtgtacctgcagatgaacaacctgaaggaggaggacacagccatctactactgcagctggaagtacaggagcaattactacttcgactactggggacagggggtgatggtgaccgtgagcagcggcggcgggggaagtggaggaggaggaagcggcggcggcggatctgacatcgtgctgacccagagccccgccctggctgtgagcctggaacagagggcaaccatcagctgcaagacaagccagaacgtggactactacggaattagctacctgcattggtaccagcagaagcctggccagcagcctaaactgctgatttacgaagggagcaacctggccagcggaatccccgccaggttcagcgggagcggcagcggaaccgacttcaccctgaccatcgacccagtggaggccgacgacatcgtgacctactactgccagcagtccaaggattacccctacaccttcggcgccggcaccaagctggagctgaaa(SEQ ID NO:3);

[0211] Table 1. Murine VEGFR2 antibody scFv

[0212]

[0213] (4) Human VEGFR2 antibody scFv:

[0214] gacattcagatgacccagagccccagcagcgtgagcgccagcatcggcgacagagtgaccatcacctgcagagcaagccagggaatcgacaactggctgggatggtaccagcagaaacctggcaaagccccaaaactgctgatctacgatgccagtaacctggacaccggcgtgccctcccgctttagcggcagcggaagcggcacctactttaccctgactatcagctccctgcaggccgaggacttcgccgtgtacttctgccagcaggccaaagcttttcctcctaccttcggcggcggcaccaaggtggacatcaagcgcaccgtgggcggcggaggcagcggaggaggaggatctggcggcggagggagcgaggtgcagctggtgcagtccgggggcggactggtgaagcctggcggaagcctgaggctgagttgtgccgctagtggatttacattcagtagttatagtatgaactgggtgaggcaggctcctggaaaggggctggagtgggtgagcagcattagcagcagtagtagctacatctattacgctgattccgtgaagggcagattcaccatctctagggacaacgctaagaacagcctgtacctgcagatgaacagcctgagggccgaggacaccgccgtgtactactgcgccagggtgactgacgcctttgacatttggggccagggcaccatggtgaccgtgagcagc(SEQ ID NO: 14);

[0215] Table 2. Human VEGFR2 antibody scFv

[0216]

[0217] (5) Mouse TLR4 hinge region coding sequence:

[0218] cagaagcagttcttggtgaatgttgaacaaatgacatgtgcaacacctgtagagatgaatacctccttagtgttggatttta ataattctacctgttatatgtacaagacaatcatcagtgtgtcagtggtcagt (SEQ IDNO: 24);

[0219] Mouse-derived TLR4 hinge area:

[0220] QKQFLVNVEQMTCATPVEMNTSLVLDFNNSTCYMYKTIISVSVVS(SEQ ID NO: 25);

[0221] Human-derived TLR4 hinge region encoding sequence:

[0222] cagaggcagctcttggtggaagttgaacgaatggaatgtgcaacaccttcagataagcagggcatgcctgtgctgagt ttgaatatcacctgtcagatgaataagaccatcattggtgtgtcggtcctcagt (SEQ ID NO: 40);

[0223] Human-made TLR4 hinge area:

[0224] QRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTIIGVSVLS (SEQ ID NO: 41).

[0225] (6) Mouse-derived TLR4 transmembrane domain coding sequence:

[0226] gtgattgtggtatccactgtagcatttctgatataccacttctattttcacctgatacttatt(SEQID NO: 26);

[0227] Mouse-derived TLR4 transmembrane domain:

[0228] VIVVSTVAFLIYHFYFHLILI (SEQ ID NO: 27);

[0229] Human TLR4 transmembrane domain coding sequence:

[0230] gtgcttgtagtatctgttgtagcagttctggtctataagttctattttcacctgatgcttctt(SEQID NO: 42);

[0231] Human TLR4 transmembrane domain:

[0232] VLVVSVVAVLVYKFYFHLMLL (SEQ ID NO: 43).

[0233] (7) Mouse TLR4 intracellular domain coding sequence:

[0234] gctggctgtaaaaagtacagcagaggagaaagcatctatgatgcatttgtgatctactcgagtcagaatgaggactgggtgagaaatgagctggtaaagaatttagaagaaggagtgccccgctttcacctctgccttcactacagagactttattcctggtgtagccattgctgccaacatcatccaggaaggcttccacaagagccggaaggttattgtggtagtgtctagacactttattcagagccgttggtgtatctttgaatatgagattgctcaaacatggcagtttctgagcagccgctctggcatcatcttcattgtccttgagaaggttgagaagtccctgctgaggcagcaggtggaattgtatcgccttcttagcagaaacacctacctggaatgggaggacaatcctctggggaggcacatcttctggagaagacttaaaaatgccctattggatggaaaagcctcgaatcctgagcaaacagcagaggaagaacaagaaacggcaacttggacc (SEQ ID NO: 28);

[0235] Mouse TLR4 intracellular domain:

[0236] AGCKKYSRGESIYDAFVIYSSQNEDWVRNELVKNLEEGVPRFHLCLHYRDFIPGVAIAANIIQEGFHKSRKVIVVVSRHFIQSRWCIFEYEIAQTWQFLSSRSGIIFIVLEKVEKSLLRQQVELYRLLSRNTYLEWEDNPLGRHIFWRRLKNALLDGKASNPEQTAEEEQETATWT (SEQ ID NO: 29);

[0237] Human TLR4 intracellular domain coding sequence:

[0238] gctggctgcataaagtatggtagaggtgaaaacatctatgatgcctttgttatctactcaagccaggatgaggactgggtaaggaatgagctagtaaagaatttagaagaaggggtgcctccatttcagctctgccttcactacagagactttattcccggtgtggccattgctgccaacatcatccatgaaggtttccataaaagccgaaaggtgattgttgtggtgtcccagcacttcatccagagccgctggtgtatctttgaatatgagattgctcagacctggcagtttctgagcagtcgtgctggtatcatcttcattgtcctgcagaaggtggagaagaccctgctcaggcagcaggtggagctgtaccgccttctcagcaggaacacttacctggagtgggaggacagtgtcctggggcggcacatcttctggagacgactcagaaaagccctgctggatggtaaatcatggaatccagaaggaacagtgggtacaggatgcaattggcaggaagcaacatctatc(SEQ ID NO: 44);

[0239] Human TLR4 intracellular domain:

[0240] AGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI(SEQ ID NO: 45).

[0241] (8) Mouse ifngr1 hinge region coding sequence:

[0242] tctttctggcaagttagaacagaaaaatcgaaagacgtctgtatccctcctttccatgatgacagaaaggattca (SEQ ID NO: 30);

[0243] Mouse source ifngr1 hinge area:

[0244] SFWQVRTEKSKDVCIPPFHDDRKDS (SEQ ID NO: 31);

[0245] Human IFNGR1 hinge region coding sequence:

[0246] attttcaatagcagt(SEQ ID NO: 46);

[0247] Human-derived IFNGR1 hinge area:

[0248] IFNSS (SEQ ID NO: 47).

[0249] (9) Mouse-derived ifngr1 transmembrane domain coding sequence:

[0250] atttggattctggtggttgctcctcttaccgtctttacagtagttatcctggtatttgcgtat(SEQID NO: 32);

[0251] Mouse-derived ifngr1 transmembrane domain:

[0252] IWILVVAPLTVFTVVILVFAY (SEQ ID NO: 33);

[0253] Human IFNGR1 transmembrane domain coding sequence:

[0254] tctctttggattccagttgttgctgctttactactctttctagtgcttagcctggtattcatc (SEQ ID NO: 48);

[0255] Human IFNGR1 transmembrane domain:

[0256] SLWIPVVAALLLFLVLSLVFI (SEQ ID NO: 49).

[0257] (10) Intracellular domain coding sequence of mouse-derived ifngr1:

[0258] tggtatactaagaagaattcattcaagagaaaaagcataatgttacctaagtccttgctctctgtggtaaaaagtgccacgttagagacaaaacctgaatcgaagtattcacttgtcacaccgcaccagccagctgtcctagagagtgagacggtgatctgtgaagagcccctgtccacagtgacagctccagacagccccgaagcagcagaacaggaagaactttcaaaagaaacaaaggctctggaggctggaggaagcacgtctgccatgaccccagacagccctccaactccgacacaaagacgcagcttttccctgttaagtagtaaccagtcaggcccttgtagcctcaccgcctatcactcccgaaacggctctgacagtggcctcgtgggatcgggcagctccatatcggacttggaatctctcccaaacaacaactcagaaacaaagatggcagagcacgaccctccacccgtgagaaaggcccccatggcctccggttatgacaaaccgcacatgttggtggacgtgcttgtggatgttggggggaaggagtctctcatggggtatagactcacaggagaggcccaggagctgtcc(SEQ ID NO:34);

[0259] Mouse IFNGR1 intracellular domain:

[0260] WYTKKNSFKRKSIMLPKSLLSVVKSATLETKPESKYSLVTPHQPAVLESETVICEEPLSTVTAPDSPEAAEQEELSKETKALEAGGSTSAMTPDSPPTPTQRRSFSLLSSNQSGPCSLTAYHSRNGSDSGLVGSGSSISDLESLPNNNSETKMAEHDPPPVRKAPMASGYDKPHMLVDVLVDVGGKESLMGYRLTGEAQELS(SEQ ID NO:35);

[0261] Human IFNGR1 intracellular domain coding sequence:

[0262] tgtttttatattaagaaaattaatccattgaaggaaaaaagcataatattacccaagtccttgatctctgtggtaagaagtgctactttagagacaaaacctgaatcaaaatatgtatcactcatcacgtcataccagccattttccttagaaaaggaggtggtctgtgaagagccgttgtctccagcaacagttccaggcatgcataccgaagacaatccaggaaaagtggaacatacagaagaactttctagtataacagaagtggtgactactgaagaaaatattcctgacgtggtcccgggcagccatctgactccaatagagagagagagttcttcacctttaagtagtaaccagtctgaacctggcagcatcgctttaaactcgtatcactccagaaattgttctgagagtgatcactccagaaatggttttgatactgattccagctgtctggaatcacatagctccttatctgactcagaatttcccccaaataataaaggtgaaataaaaacagaaggacaagagctcataaccgtaataaaagcccccacctcctttggttatgataaaccacatgtgctagtggatctacttgtggatgatagcggtaaagagtccttgattggttatagaccaacagaagattccaaagaattttca(SEQ IDNO:50);

[0263] Human IFNGR1 intracellular domain:

[0264] CFYIKKINPLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSLEKEVVCEEPLSPATVPGMHTEDNPGKVEHTEELSSITEVVTTEENIPDVVPGSHLTPIERESSSPLSSNQSEPGSIALNSYHSRNCSESDHSRNGFDTDSSCLESHSSLSDSEFPPNNKGEIKTEGQELITVIKAPTSFGYDKPHVLVDLLVDDSGKESLIGYRPTEDSKEFS(SEQ ID NO:51).

[0265] (11) Intracellular domain coding sequence of mouse-derived ifngr2 cells:

[0266] aaataccaaagccgagtgaagtactggtttcaggctccgccaaacatcccggaacaaatcgaagagtatctaaaggac ccagaccaattcatcttagaggtcttggacaaggacggttcaccgaaggaggactcctgggactccgtgtcaattatttcttctc cagaaaaggagcgagatgatgtgctccaaacaccg (SEQ ID NO: 36);

[0267] Intracellular domains of mouse-derived ifngr2:

[0268] KYQSRVKYWFQAPPNIPEQIEEYLKDPDQFILEVLDKDGSPKEDSWDSVSIISSPEKERDDVLQTP (SEQ ID NO: 37).

[0269] Human IFNGR2 intracellular domain coding sequence:

[0270] ctggtcctgaaatatagaggcctgattaaatactggtttcacactccaccaagcatcccattacagatagaagagtattta aaagacccaactcagcccatcttagaggccttggacaaggacagctcaccaaaggatgacgtctgggactctgtgtccattat ctcgtttccggaaaaggagcaagaagatgttctccaaacgctt (SEQ ID NO: 52);

[0271] Intracellular domains of human IFNGR2:

[0272] LVLKYRGLIKYWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSISFPEKEQEDVLQTL (SEQ ID NO: 53).

[0273] Example 2. Method for constructing chimeric antigen receptor macrophages (CAR-M)

[0274] 1.D0: Lay two six-hole boards using RAW264.7;

[0275] 2. Day 1 (D1): When the confluence of RAW264.7 is approximately 30%-50%, discard the supernatant and replace it with DMEM medium containing 10% FBS and no antibiotics, 1 ml / well. Thaw the lentivirus at room temperature, 1 ml / well. The contents of each well are as follows:

[0276] 1) No virus added, 2) Plenti-GFP virus added, 3) hmA, 4) hmB, 5) hmC, 6) hmD, 7) hmE, 8) mmA, 9) mmB, 10) mmD, 11) mmE. Additionally, polybrene was added to each well to a final concentration of 8 μg / ml.

[0277] 3. Gently shake the above liquid to mix well, and centrifuge at 37°C and 1600 rpm for 60 min;

[0278] 4. D2: After incubation at 37℃ for 24 hours, replace with fresh culture medium (observe cell condition during this period; if cell condition is poor, shorten the incubation time and replace with fresh complete culture medium in time).

[0279] 5. D6: If cell density allows, replace the medium with fresh puromycin 96 hours after viral infection. If cell density is extremely high, replace with fresh puromycin at least 48 hours after viral infection. The concentration of puromycin varies depending on the cell line and should be determined in advance; the optimal concentration should be such that non-resistant cells die within 2-3 days.

[0280] 6. Infection efficiency was determined based on fluorescence, and positive cells were enriched by flow cytometry.

[0281] Example 3. Phagocytosis of tumor cells by CAR-M

[0282] CAR-M and PKH26-labeled breast cancer cells were co-cultured to detect CAR-M phagocytosis of tumor cells and macrophage phenotype.

[0283] I. Method:

[0284] 1. Take 2 × 10 7 4 T1 cells (mouse breast cancer cells) were prepared into a single-cell suspension, placed in a 1.5 ml centrifuge tube, and washed once with serum-free culture medium;

[0285] Centrifuge at 2400g for 5 minutes to obtain a relatively loose cell pellet;

[0286] 3. Carefully aspirate the supernatant to ensure that the remaining liquid is less than 25 μl;

[0287] 4. Add 1 ml of Diluent C to prepare a 2× cell suspension, and gently pipette to mix and obtain a single-cell suspension;

[0288] 5. Prepare 2× staining solution (4×10⁻⁶)-6 M): Add 1 ml of Diluent C to a polypropylene centrifuge tube, then add 4 μl of PKH26 ethanolic staining solution (#P9691, sigma) to the 1 ml of Diluent C and mix thoroughly.

[0289] 6. Quickly add 1 ml of 2× cell suspension to 1 ml of 2× staining solution and immediately mix by pipetting. The final cell concentration at this point is 1×10⁻⁶. 7 cells / ml and 2×10 -6 M.

[0290] 7. Incubate at room temperature for 1-5 minutes, mixing occasionally. Prolonged staining time does not significantly improve staining results; in fact, due to the lack of physiological salts in Diluent C, excessively long staining times can negatively impact cell viability. A control group can be set up that uses only diluent and one that metabolizes the dye with ethanol.

[0291] 8. Add an equal volume (2 ml) of serum to stop staining, and incubate for another 1 min to complete the binding of excess dye.

[0292] Centrifuge at 400g, 20-25℃ for 10 min, carefully aspirate the supernatant and resuspend the cells in 10 ml of complete culture medium, transfer to a new sterile conical polypropylene tube, centrifuge at 400g, 20-25℃ for 5 min, replace with a new centrifuge tube, and wash twice with 10 ml of complete culture medium to completely remove unbound dye.

[0293] 10. Resuspend the cell pellet in complete culture medium and adjust the cell concentration to 1×10⁻⁶. 6 Cells / ml

[0294] 11. CAR-M macrophages, human umbilical vein endothelial cells (HUVECs), and PKH26-labeled 4T1 tumor cells were mixed at a ratio of 5 × 10⁻⁶. 5 5×10 5 1×10 6 Mix in the specified proportions and incubate at 37°C in a 5% CO2 incubator for 2 hours.

[0295] 12. Collect cells and perform flow cytometry staining. Dilute the CD45 / CD80 / MHCII flow cytometry antibody using antibody dilution buffer at a ratio of 1:100. (Per 10 cells) 6 Add 100 μl of diluted flow cytometry antibody to the cells and incubate at 4°C in the dark for 30 min.

[0296] 13. Flow cytometry detection and analysis: After incubation, wash cells twice with pre-chilled PBS, centrifuge at 500g for 3 min to collect cells, add 500μl of pre-chilled PBS, mix gently, and place all samples on ice in the dark. Detect the cells using a flow cytometer within 2 h. Analyze the results.

[0297] II. Results Explanation:

[0298] Figure 2A The results showed that Western blotting detected VEGFR2 expression in RAW264.7, HUVEC, mouse hepatocellular carcinoma cells Hepa1-6, breast cancer cell line 4T1, and lung cancer cell line LLC. The antibody could recognize human and mouse VEGFR2. This indicates that HUVEC highly expresses VEGFR2, while other cell lines express VEGFR2 at low levels or not at all.

[0299] Figure 2B and Figure 2C The results showed that after co-culture, the expression of CD86 and MHCII on macrophages was significantly increased in the four CAR-M groups (HmA, HmB, HmC, and HmD) compared to the original RAW264.7 cells.

[0300] Figure 2D The results showed that, according to flow cytometry analysis of tumor cell apoptosis after co-culture, the proportion of early apoptosis in the three groups co-cultured with HmB, HmC, and HmD cells was significantly higher than that in the control group co-cultured with RAW264.7 cells.

[0301] Figure 2E The results showed that flow cytometry analysis was used to detect the phagocytosis of tumor cells by macrophages after co-culture. Compared with the control group co-cultured with RAW264.7 cells, the proportion of tumor cells phagocytosed was significantly increased in the three groups co-cultured with HmA, HmB, and HmD cells.

[0302] Example 4. Detection of TNF-α cytokine in the supernatant of CAR-M and tumor cell co-culture. I. Detection of CAR-M expressing anti-mouse VEGFR2 scFv by ELISA.

[0303] Concentration of TNF-α in the supernatant of co-culture with 4T1 tumor cells

[0304] I. Steps:

[0305] 1. The cell concentrations of chimeric antigen receptor macrophages (mmA, mmB, mmC, mmD, mmE) expressing anti-mouse VEGFR2 scFv and the control Plenti were adjusted to 2 × 10⁻⁶ cells. 6 cells / ml, then 2×10 5 The density of cells per well: Various CAR-M cells were seeded in 6-well cell culture plates, with 6 replicates for each cell type.

[0306] 2. Adjust the concentration of 4T1 breast cancer cells to 2×10⁻⁶. 62 × 10⁻⁶ cells / ml 5 The cells were seeded into 6-well cell culture plates from step 1, such that 3 out of 6 replicates for each CAR-M were co-cultured with 4T1 cells, while the other 3 replicates contained only CAR-M cells. The culture medium volume in each well was 2 ml, and the culture plates were then incubated at 37°C with 5% CO2 for 48 h.

[0307] 3. Collect cell culture supernatant: After culturing for 48 hours, centrifuge at 300g for 10 minutes to collect the supernatant. The collected supernatant should be tested immediately or aliquoted and stored at -20℃.

[0308] 4. The concentration of TNF-α factor in the culture supernatant was detected using the Linko Bio TNF-α Detection Kit (EK282 / 4).

[0309] 5. After conducting the tests according to the instructions, analyze and compile the test results.

[0310] II. Results Explanation:

[0311] Figures 3A to 3B The control group consisted of empty vector macrophages that did not express anti-mouse VEGFR2 scFv, named the Plenti group, consistent with the name of the vector plasmid. The experimental groups were named according to the CAR-M cells used in that group. Compared to the control group, after co-culturing with 4T1, the TNF-α secreted by CAR-M cells in the mmC and mmD groups was significantly increased, while there was no significant difference in TNF-α secretion among the other three groups. The results indicate that mmC and mmD cells can secrete more TNF-α after tumor stimulation.

[0312] II. ELISA detection of TNF-α concentration in the supernatant of co-cultured CAR-M, HUVEC, and 4T1 tumor cells expressing anti-human VEGFR2 scFv

[0313] I. Steps:

[0314] 1. The cell concentrations of chimeric antigen receptor macrophages expressing anti-human VEGFR2 scFv (HmA, HmB, HmC, HmD, HmE) and the control group Plenti were adjusted to 2 × 10⁻⁶ cells. 6 cells / ml, at 2×10 5 The density of cells per well: Various CAR-M cells were seeded into 6-well cell culture plates, with 6 replicates for each cell type.

[0315] 2. Adjust the concentration of HUVEC cells to 2 × 10⁻⁶. 6 cells / ml, at 2×10 5Add HUVEC cells to each well of the 6-well plate in step 1 at a density of 1 cell per well.

[0316] 3. Adjust the concentration of 4T1 breast cancer cells to 2×10⁻⁶. 6 2 × 10⁻⁶ cells / ml 5 The cells were seeded into 6-well cell culture plates from step 1, resulting in 6 replicates for each CAR-M cell type: 3 replicates containing CAR-M cells, HUVEC cells, and 4T1 cells; and 3 replicates containing CAR-M cells and HUVEC cells. Each well contained 2 ml of culture medium. The culture plates were then incubated at 37°C with 5% CO2 for 48 hours.

[0317] 4. Collect cell culture supernatant: After co-culturing for 48 hours, centrifuge at 300g for 10 minutes to collect the supernatant. The collected supernatant should be tested immediately or aliquoted and stored at -20℃.

[0318] 5. The concentration of TNF-α factor in the culture supernatant was detected using the Linko Bio TNF-α Detection Kit (EK282 / 4).

[0319] 6. After conducting the tests according to the instructions, analyze and compile the test results.

[0320] II. Results Explanation:

[0321] Figures 3C to 3D The control group consisted of macrophages stably transfected with the empty vector plasmid, named the Plenti group, consistent with the name of the vector plasmid. The experimental groups were named after the CAR-M cells used in each group. After co-culturing CAR-M cells with VEGFR2-expressing HUVEC cells, TNF-α secretion from all CAR-M cell groups was significantly altered. The HmC and HmD groups showed significantly different increases in TNF-α compared to the Plenti control group, while the other three CAR-M cell groups showed decreased TNF-α levels relative to the control group. Furthermore, when CAR-M, HUVEC, and 4T1 cells were co-cultured, even with potential inhibitory signals from tumor cells, the HmC and HmD groups showed significantly increased TNF-α secretion. Notably, compared to CAR-M cells not stimulated by VEGFR2-expressing HUVEC cells, the addition of HUVEC cells increased TNF-α secretion by approximately one order of magnitude. These results indicate that HmC and HmD cells can be activated by VEGFR2 expression in HUVEC cells and secrete large amounts of the inflammatory factor TNF-α.

[0322] III. Flow cytometry detection of CD45 in a co-culture system of CAR-M cells expressing human anti-VEGFR2 scFv, human umbilical vein endothelial cells (HUVECs), and 4T1 tumor cells. + TNF-α + proportion

[0323] I. Steps:

[0324] 1. After collecting the co-culture supernatant, add 2 ml of fresh complete culture medium to the co-culture system, and add 2 μl of Golgi inhibitor to each culture well at a ratio of 1:1000. Then, incubate at 37°C in an incubator containing 5% CO2 for 3 hours.

[0325] After 2.3 hours, all cells were collected, rinsed twice with PBS, and then 100 μl of cell permeabilization solution was added. The cells were incubated at 4°C for 30 minutes.

[0326] 3. After 30 minutes, rinse the cells twice with washing buffer, then stain with flow cytometry antibody. Detect CD45 saturation 40 minutes after staining. + TNF-α + The proportion of cells.

[0327] II. Results Explanation:

[0328] Figures 3E to 3F The results showed that when CAR-M cells were co-cultured with HUVECs, compared with the control group Plenti, the TNF-α levels in the other four CAR-M cell types (except HmE) were significantly lower. + The proportions of all three groups showed a significant increase; when CAR-M, HUVEC, and 4T1 were co-cultured, the TNF-α levels of HmA, HmB, HmC, HmD, and HmE were significantly increased. + The proportion of cells in both groups was significantly higher than that in the control group, with HmC showing the highest TNF-α levels. + The proportion of cells increased most significantly. The experimental results suggest that HmA, HmB, HmC, and HmD, which target VEGFR2, can be activated by VEGFR2 expressed on HUVECs and secrete the pro-inflammatory factor TNF-α; after encountering tumor cells, HmC can maintain its characteristic of high TNF-α secretion.

[0329] Example 5. In vivo verification of the antitumor effect of CAR-M (small animal in vivo imaging experiment)

[0330] I. Steps:

[0331] 1. Construction of an orthotopic breast cancer mouse model: 4T1-luc breast cancer cells were digested with trypsin to adjust the cell concentration to 5 × 10⁻⁶. 6100 μl of 4T1-luc cells / ml were injected into the penultimate pair of mammary fat pads of healthy female babl / c mice at 5 × 10⁻⁶ cells / ml. 5 One cell per animal, a total of 35 animals were injected.

[0332] 2. Treatment grouping: After the tumors grew in the mice, 35 tumor-bearing mice were randomly divided into 7 groups. The group names were consistent with the names of the CAR-M cells used to treat the group, namely mmA, mmB, mmC, mmD, mmE, and the control group Plenti and PBS.

[0333] 3. CAR-M therapy in mice with cancer: On days 6 (D6), 12 (D12), and 18 (D18) after inoculation with 4T1-luc breast cancer cells, 1×10⁻⁶ CAR-M cells were injected via tail vein. 6 200 μl of each group of CAR-M cells were infused into the corresponding tumor-bearing mice.

[0334] 4. In vivo imaging of small animals: In vivo fluorescence images of mice in each group were taken on day 5 (D5) and day 20 (D20) after inoculation with 4T1-luc breast cancer cells. The specific operation steps are as follows:

[0335] 1) Prepare an appropriate amount of 1×luciferase substrate (protect from light) and inject 200 μl of the substrate into each tumor-bearing mouse via intraperitoneal injection.

[0336] 2) After injecting the substrate, allow the mice to react for about 8 minutes, then use isoflurane to induce coma in the mice. Once the mice are completely comatose, perform in vivo imaging of the small animals.

[0337] 3) Standardize the obtained images, analyze the images, and statistically analyze the differences.

[0338] II. Results Explanation:

[0339] The Plenti group and the PBS group served as control groups. The Plenti group received macrophages transfected with an empty plasmid, while the PBS group received an equal volume of cell buffer PBS. The remaining groups were experimental groups, and their naming conventions were consistent with those of the transfused CAR-M cells. Figure 4A , Figure 4B ).

[0340] On day 5 of tumor bearing, except for the mmA and mmB groups where the tumor fluorescence intensity was significantly higher than that of the control group, the tumor fluorescence intensity of the other groups of mice was not significantly different from that of the control group. Figure 4C ). Figure 4DThe results showed that on day 20 of tumor bearing, the tumor fluorescence intensity of mmA, mmB, and mmC groups was significantly lower than that of the control group, while the fluorescence intensity of mmD and mmE groups was not significantly different from that of the control group. Figure 4E The results showed that when the tumor fluorescence intensity on day 20 was divided by the tumor fluorescence intensity on day 5, the tumor fluorescence intensity in groups mmA, mmB, mmC, and mmD was significantly lower than that in the control group (with the mmA and mmB groups being more significantly lower), while the tumor fluorescence intensity in group mmE was not significantly different from that in the control group.

[0341] Small animal in vivo imaging experiments showed that intravenous injection of four types of CAR-M cells (mmA, mmB, mmC, and mmD) into the tail vein of mice with breast cancer could inhibit tumor growth and effectively reduce the volume of breast cancer in tumor-bearing mice.

[0342] Example 6. In vivo validation of the antitumor effect of CAR-M (immune-intact mouse breast cancer model)

[0343] I. Steps:

[0344] 1. Establishment of an immunocompetent mouse breast cancer model: 4T1 breast cancer cells were removed from the incubator, digested with trypsin, and the cell concentration was adjusted to 5 × 10⁻⁶. 6 100 μl of 4 T1 cells / ml was injected into the penultimate pair of mammary fat pads of healthy female babl / c mice at 5 × 10⁻⁶ cells / ml. 5 One cell per animal, a total of 25 animals were injected.

[0345] 2. Treatment grouping: When the tumors grew to a suitable size, all tumor-bearing mice were randomly divided into 5 groups, named in the same way as the infused CAR-M, namely mmA, mmB, mmC, mmD and control group PBS.

[0346] 3. Tumor measurement: Starting from day 5 of tumor bearing in mice, the tumor size and mouse weight were measured every other day, and the tumor volume was calculated (volume = length × width × width / 2).

[0347] 4. CAR-M therapy: On days 6 (D6), 12 (D12), and 18 (D18) after tumor bearing, 1×10⁻⁶ CAR-M was administered via tail vein injection. 6 Each group of CAR-M cells was infused with 200 μl of PBS into the corresponding tumor-bearing mice, while the control group was infused with 200 μl of PBS.

[0348] 5. On day 21 of tumor-bearing mice, blood was collected from the orbital cavity, and the mice were euthanized by cervical dislocation. The tumor tissue, spleen tissue, liver tissue, and lung tissue were completely removed, photographed, and weighed. The tissues were then preserved in 4% paraformaldehyde.

[0349] II. Results Explanation:

[0350] Figure 5A Tumor growth curves for each group of mice are shown, with the PBS group serving as the control group receiving only PBS buffer infusion; compared to the control group, the group showed a slower tumor growth rate in mmB, mmC, and mmD. Figure 5B and Figure 5C The results showed that, compared to the control group, the tumor size and weight of mice in the mmB and mmC infusion groups were significantly smaller than those of the control group.

[0351] This indicates that in immunocompetent mice with breast cancer, infusion of CAR-M cells from the mmB and mmC groups can kill tumors, inhibit tumor growth, and reduce the tumor burden in mice.

[0352] Example 7. In vivo validation of the antitumor effect of CAR-M (immunodeficient nude mouse breast cancer model)

[0353] I. Steps:

[0354] 1. Construction of an immunodeficient nude mouse breast cancer model: Well-cultured 4T1 breast cancer cells were removed from the incubator, digested with trypsin, and counted. The cell concentration was then adjusted to 5 × 10⁻⁶ cells / year. 6 Cells / ml, 100 μl of which was injected into the penultimate pair of mammary fat pads of 6-week-old female nude mice (D0), i.e., 5 × 10⁶ cells / ml. 5 One cell per animal, a total of 25 animals were injected.

[0355] 2. Treatment grouping: When the tumors grew to a suitable size, all tumor-bearing mice were randomly divided into 5 groups, named in the same way as the infused CAR-M, namely mmA, mmB, mmC, mmD and control group PBS.

[0356] 3. Tumor measurement: Starting from day 5 of tumor bearing in mice, the tumor size and mouse weight were measured every other day, and the tumor volume was calculated (volume = length × width × width / 2).

[0357] 4. CAR-M therapy: On days 6 (D6), 12 (D12), and 18 (D18) after tumor bearing, 1×10⁻⁶ CAR-M was administered via tail vein injection. 6 Each group of CAR-M cells was infused with 200 μl of PBS into the corresponding tumor-bearing mice, while the control group was infused with 200 μl of PBS.

[0358] 5. When nude mice died, blood was drawn from their orbits, and all mice were euthanized by cervical dislocation. Tumor tissue, spleen tissue, liver tissue, lung tissue, kidney tissue, and heart tissue were completely removed, photographed, and weighed. Except for a portion of the tumor tissue from the central part used for subsequent flow cytometry experiments, all remaining tissues were preserved in 4% paraformaldehyde.

[0359] 6. Flow cytometry experiment: to detect the infiltration of macrophages in tumor tissue of nude mice. The specific operation steps are as follows.

[0360] 1) Prepare type IV collagenase solution: Prepare an appropriate amount of type IV collagenase digestion solution with a concentration of 0.5 mg / ml using 1640 medium.

[0361] 2) Tumor digestion: After cutting all tumor tissues from the treatment group into small pieces, transfer them into 50ml centrifuge tubes and add 10ml of type IV collagenase digestion solution to the tubes. Place the centrifuge tubes on a shaker at 37℃ and 200rpm for about 1 hour.

[0362] 3) Tumor grinding: Take the digested tumor tissue from the shaker, grind the tumor tissue appropriately, and then filter it through a 70um filter membrane to obtain a cell suspension.

[0363] 4) Red blood cell lysis: Centrifuge the obtained cell suspension at 500g for 5 minutes to obtain a cell pellet. Add 5 ml of red blood cell lysis buffer to the cell pellet, mix thoroughly, and incubate at room temperature for 5 minutes. After incubation, wash 2 to 3 times with 30 ml of cell buffer PBS. Centrifuge at 500g for 5 minutes each time. Finally, resuspend the cell pellet obtained by centrifugation in PBS and filter it again.

[0364] 5) Prepare flow cytometry antibody dilution buffer: Dilute CD45 / CD11b / F4-80 flow cytometry antibody with antibody dilution buffer at a dilution ratio of 1:100.

[0365] 6) Flow cytometry antibody staining: Take an appropriate amount of cell pellet obtained from step 4, add 100 μl of diluted flow cytometry antibody, and incubate at 4°C in the dark for 30 min.

[0366] 7) Flow cytometry detection and analysis: After incubation, wash cells twice with pre-chilled PBS, centrifuge at 500g for 3 min to collect cells, add 500μl of pre-chilled PBS, mix gently, and place all samples on ice in the dark. Detect the cells using a flow cytometer within 2 h. Analyze the results.

[0367] II. Results Explanation:

[0368] Figure 6A Tumor growth curves for each group of mice are shown, with the PBS group serving as the control group receiving only PBS buffer infusion; groups mmA, mmB, mmC, and mmD all showed different tumor growth rates. Figure 6B and Figure 6C This indicates that, compared with the control group, only nude mice infused with mmC showed significantly smaller tumor growth volume and weight. Figure 6DThis indicates that infusion of mmC and mmD into tumor-bearing nude mice can increase the proportion of macrophages infiltrating the tumor tissue, while infusion of mmA and mmB cannot.

[0369] Therefore, the results indicate that in immunodeficient nude mice with breast cancer, CAR-M cells infused with mmC can recruit more macrophages to infiltrate tumor tissue, inhibit tumor growth, and achieve a therapeutic effect. Although infusion of mmD can also recruit more macrophages to reach the tumor, it may not exert an anti-tumor effect.

[0370] Example 8. Safety assessment of CAR-M cell therapy

[0371] The liver and lung tissues of the tumor-bearing nude mice in Example 7 were removed, weighed, and photographed.

[0372] The control group consisted of mice injected with an equal volume of PBS buffer via the tail vein. Figure 7A The image shows liver tissue from nude mice in all groups. The appearance and volume of liver tissue from groups mmA, mmB, mmC, and the control group (PBS) were basically the same. However, after infusing CAR-M cells from group mmD into nude mice, the liver tissue of this group was significantly larger. Upon magnification, compared with the control, the surface of the liver tissue in this group was covered with irregular protrusions, and the liver surface showed redness, swelling, and erosion. Figure 7B The weight of liver tissue in nude mice across all groups was significantly higher than that in the control group (mmD), while the liver tissue in the mmC group was lighter than that in the control group. Figure 7C The weight of lung tissue in nude mice was measured in all groups. Compared with the control group, only the lung tissue weight of group mmD was significantly increased. Figure 7D The weight of all nude mice was measured at the last time before they were sacrificed. Compared with the control group, the weight of nude mice in the mmD group was significantly lower, and the difference was statistically significant.

[0373] The above results indicate that in immunodeficient nude mice with breast cancer, infusion of CAR-M cells from the mmD group damaged the liver and lung tissues, exhibiting hepatotoxicity and pulmonary toxicity, and causing weight loss in the mice. CAR-M cells from other groups showed no significant toxicity.

Claims

1. A chimeric antigen receptor, from amino terminus to carboxy terminus, is a structure of the following formula: IFNGR1 signal peptide - antigen binding domain - TLR4 hinge region - TLR4 transmembrane domain - TLR4 intracellular domain - IRES - GFP; wherein: the antigen binding domain is a scFv of SEQ ID NO: 15 or SEQ ID NO: 4; - represents a covalent bond.

2. The chimeric antigen receptor of claim 1, wherein: the TLR4 is a human or murine TLR4.

3. The chimeric antigen receptor of claim 1, wherein: the hinge region is of any one sequence selected from the group consisting of SEQ ID NO: 41, 25.

4. The chimeric antigen receptor of claim 1, wherein: the transmembrane domain is of any one sequence selected from the group consisting of SEQ ID NO: 43, 27.

5. The chimeric antigen receptor of claim 1, wherein: the TLR4 intracellular domain is of SEQ ID NO: 45 or 29.

6. The chimeric antigen receptor of claim 1, wherein: the signal peptide is of SEQ ID NO: 39 or 2.

7. An immunoresponsive cell expressing the chimeric antigen receptor of any one of claims 1 to 6, the immunoresponsive cell being a macrophage.

8. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1 to 6.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. The expression vector of claim 9, further comprising any one or a combination thereof selected from the group consisting of: a promoter, a start sequence, an origin of replication, a terminator, an enhancer, a restriction endonuclease site, a marker, an internal ribosome entry site, a polyA; the marker is a selection marker, a resistance marker, or a fluorescent marker.

11. The expression vector of claim 9, selected from the group consisting of any one of: a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a plasmid, a Sendai virus vector, a retrovirus vector, a herpes virus vector.

12. The expression vector of claim 11, the retrovirus vector is a gamma retrovirus vector.

13. A host cell comprising the expression vector of claim 9; the host cell is a macrophage.

14. A pharmaceutical composition comprising: a therapeutically effective amount of the immunoresponsive cell of claim 7, or the host cell of claim 13.

15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable carrier.

16. A kit for treating cancer, comprising: a therapeutically effective amount of the immunoresponsive cell of claim 7, or a therapeutically effective amount of the host cell of claim 13; the cancer is breast cancer; the breast cancer is of any one selected from the group consisting of: Luminal A, Luminal B, HER2 positive, triple negative. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 17. Use of any one selected from the group consisting of: a chimeric antigen receptor according to any one of claims 1 to 6, a nucleic acid molecule according to claim 8, an expression vector according to claim 9; in the manufacture of a medicament for the treatment of breast cancer or for increasing survival of a subject with breast cancer.

18. Use according to claim 17, wherein the breast cancer is selected from the group consisting of: Luminal A, Luminal B, HER2-positive, triple-negative.

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