Adenovirus vector and use thereof

Abstract

Provided in the present disclosure are an adenovirus vector and the use thereof. A promoter of the adenovirus vector carrying a chimeric antigen receptor (CAR) of the present disclosure contains one or more of a CMV promoter, a CAG promoter, a CBh promoter, a CMVIntron promoter and an EF1A promoter. The adenovirus vector increases the expression intensity and duration of CAR molecules in modified immunocytes, while maintaining the M1 phenotype.

Description

Adenoviral vector and its application TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an adenoviral vector and its application. BACKGROUND

[0002] Macrophages are immune cells derived from monocytes, which exist in almost all tissues of the human body and participate in a large number of biological processes including development, bone remodeling and wound healing. The main function of macrophages is to phagocytose and digest pathogens and cell fragments in body fluids and tissues, and at the same time, it can initiate and enhance adaptive immune responses by antigen presentation, recruitment of T cells and secretion of cytokines. Chimeric antigen receptor macrophage (CAR-Macrophage) or monocyte (CAR-Monocyte) therapy (hereinafter collectively referred to as CAR-M) has become an important direction in the field of cellular immunotherapy. This therapy is to introduce foreign genes into monocytes or macrophages by viral means, and to produce CAR-M by inducing CD14-positive monocytes from peripheral blood or macrophages.

[0003] At present, there are mainly two vectors for producing chimeric antigen receptor macrophages / monocytes (CAR-M) by viral infection: lentivirus and adenovirus. However, due to the phagocytosis of viruses by macrophages / monocytes, the transduction efficiency is less than 20%. Moreover, due to the risk of random integration of lentivirus infection, there is a risk of causing genetic mutation of macrophages / monocytes, and more often, non-genetically integrated adenovirus is selected as a transduction vector, but at the same time, non-genetic integration also causes problems such as short expression duration and low abundance of CAR molecules.

[0004] The initiation of transcription is a key stage of gene expression, and an important problem at this stage is the interaction of RNA polymerase with the promoter: the structure of the promoter affects its affinity with RNA polymerase, thereby affecting the level of gene expression. The promoter is a DNA sequence located upstream of the gene, usually near the transcription start site. It is an important regulatory element for controlling gene transcription, i.e. converting DNA-encoded information into mRNA, and further synthesizing proteins. Selecting the right promoter when constructing a vector is more conducive to high-intensity and stable expression of CAR molecules and maintenance of M1 phenotype. SUMMARY

[0005] In view of the deficiencies in the prior art, the present disclosure provides a series of promoters for modifying adenovirus, which improves the expression intensity and duration of CAR molecules in modified immune cells, especially macrophages or monocytes, while maintaining the M1 phenotype.

[0006] According to one aspect of the present disclosure, there is provided an adenoviral vector carrying a chimeric antigen receptor (CAR) encoding nucleic acid sequence, the adenoviral vector comprising one or more of a CMV promoter, a CAG promoter, a CBh promoter, a CMV Intron promoter, and an EF1A promoter, the CAR encoding nucleic acid sequence operably linked to the promoter.

[0007] In some embodiments, the CMV promoter has a nucleotide sequence as set forth in SEQ ID NO: 1, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 1.

[0008] In some embodiments, the CAG promoter has a nucleotide sequence as set forth in SEQ ID NO: 2, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 2.

[0009] In some embodiments, the CBh promoter has a nucleotide sequence as set forth in SEQ ID NO: 3, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 3.

[0010] In some embodiments, the CMV Intron promoter has a nucleotide sequence as set forth in SEQ ID NO: 4, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 4.

[0011] In some embodiments, the EFlA promoter has a nucleotide sequence as set forth in SEQ ID NO: 5, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 5.

[0012] In some embodiments, the adenoviral vector further comprises one or more of a reporter gene, a polyadenylation tail, an untranslated region, an enhancer, and a terminator.

[0013] In some embodiments, the adenoviral vector comprises a backbone from adenovirus serotype 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24-30, 31, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, or a functional variant thereof. In some preferred embodiments, the adenoviral vector comprises an Ad5F35 adenoviral backbone.

[0014] In some embodiments, the CAR molecule has an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0015] In some embodiments, the antigen binding domain binds a tumor associated antigen.

[0016] In some embodiments, the tumor-associated antigen comprises CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); TNF receptor family member B-cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine-protein kinase orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD 117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); testisin (PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); Mucin 1,O-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein coupled receptor class C group 5 member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD 179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of the glycosphingolipid globoH (GloboH); breast cancer differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6 on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Mesothelin (MSLN); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (Melan A or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1);CCCTC-binding factor (Zinc finger protein) like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous cell carcinoma antigen recognized by T cells 3 (SART3); Paired box protein Pax-5 (PAX5); Proacrosin binding protein sp32 (OY-TES1); Lymphocyte cell-specific protein-tyrosine kinase (LCK); Kinase-anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); Receptor for advanced glycation end products (RAGE-1); Renal ubiquitous 1 (RU1); Renal ubiquitous 2 (RU2); legumain; Human papilloma virus E6 (UPV E6); Human papilloma virus E7 (UPV E7); Intestinal carboxyl esterase; Mutated heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like protein 5 (FCRL5); and Immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0017] In some embodiments, the antigen binding domain comprises an anti-CD19 antibody, an anti-HER2 antibody, an anti-PMSA antibody, and fragments thereof.

[0018] In some embodiments, the antigen binding domain comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragments thereof.

[0019] In some embodiments, the antigen binding domain comprises a Fab, a F(ab')2, a scFv, a Fab', a dsFv, a single domain antibody (VHH), a nanobody.

[0020] In some embodiments, the antigen binding domain comprises a Fab, a F(ab')2, a scFv, a Fab', a dsFv, a single domain antibody (VHH), a nanobody.

[0021] In some embodiments, the hinge domain comprises at least one or more hinge regions of CD8a, CD28, 4-1BB, OX40, CD3-zeta, T cell receptor alpha or beta chain, CD3-zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154.

[0022] In some embodiments, the transmembrane domain comprises at least one or more transmembrane regions of alpha, beta, or zeta chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.

[0023] In some embodiments, the intracellular signal transduction domains include TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, universal FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, and lymphocyte function-associated antigen-1 (LFA-1). CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80, CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6 , CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96, CEACAM1, CRTAM, One or more of the following receptors: Ly9, CD160, PSGL1, CD100, CD69, SLAMF6 (NTB-A, Ly108), SLAM, BLAME, SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.

[0024] In some embodiments, the CAR is expressed by modified immune cells. In some specific embodiments, the CAR is expressed by modified monocytes, modified macrophages, or modified T lymphocytes.

[0025] According to another aspect of this disclosure, a host cell is provided, the host cell comprising the adenovirus vector or the nucleic acid molecule described herein.

[0026] In some embodiments, the host cell is a mammalian cell expressing an adenovirus vector.

[0027] In some embodiments, the cells may be isolated cells that are not present in a living animal. Mammalian cells may be cells from any organ or tissue, including those from humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cattle, and apes. In some embodiments, the cells are human cells. In some embodiments, the cells may be primary or immortalized cells.

[0028] In some embodiments, the host cell is selected from immune cells, including but not limited to monocytes, macrophages, or T lymphocytes.

[0029] In some embodiments, the host cell is a monocyte.

[0030] In some preferred embodiments, the host cell is a CD14+ mononuclear cell.

[0031] In some embodiments, the host cell is a macrophage.

[0032] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising the adenovirus vector or the host cell described herein, and a pharmaceutically acceptable excipient or carrier.

[0033] According to another aspect of this disclosure, a kit is provided comprising the adenovirus vector, optionally a cell line that allows the adenovirus vector to infect cells and replicate the viral genome, and optionally a DNA plasmid for constructing the adenovirus vector.

[0034] According to another aspect of this disclosure, the use of the adenovirus vector, the host cell, the pharmaceutical composition, or the kit described herein is provided in the preparation of a medicament for the prevention, treatment, or reduction of inflammatory diseases and / or tumors.

[0035] In some embodiments, the drug is used to inhibit the growth of tumor cells and / or induce tumor cell apoptosis.

[0036] In some embodiments, the inflammatory disease includes, but is not limited to, chronic and acute inflammatory diseases. Examples of inflammatory diseases include Alzheimer's disease, asthma, atopic dermatitis, allergies, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, and ventilator-induced lung injury.

[0037] In some embodiments, the tumor includes tumors that are not vascularized or have not yet been substantially vascularized, as well as vascularized tumors. Tumors can include non-solid tumors (such as hematologic malignancies, e.g., leukemia and lymphoma) or solid tumors. Tumor types include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, as well as certain leukemias or lymphomas, benign and malignant tumors such as sarcomas, carcinomas, and melanomas. Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors (such as sarcomas and carcinomas) include fibrosarcoma, myxosarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, rectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas, such as brainstem gliomas and mixed gliomas). Gliomas (also known as gliomas) and glioblastomas (also known as multimorphic glioblastomas). Hematologic malignancies are tumors of the blood or bone marrow. Examples of hematologic (or blood-borne) malignancies include leukemias such as acute leukemias (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, and monocytic leukemia), chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (painless and high-grade forms), multiple myeloma, myelodysplastic syndromes, hairy cell leukemia, and myelodystrophy.

[0038] In some specific embodiments, the tumor includes gastric cancer, breast cancer, or esophageal cancer.

[0039] In some specific embodiments, the tumor includes esophageal cancer.

[0040] In some specific embodiments, the tumor includes prostate cancer.

[0041] According to another aspect of this disclosure, a method for improving adenovirus transfection efficiency is provided, the method comprising transfecting the adenovirus vector into immune cells.

[0042] In some embodiments, the immune cells are selected from monocytes, macrophages, or T lymphocytes. Attached Figure Description

[0043] Figure 1 shows a schematic diagram of the AD5 / F35 adenovirus plasmid structure.

[0044] Figure 2A shows a schematic diagram of CAR-macrophage preparation, and Figure 2B shows a schematic diagram of CAR-monocyte preparation.

[0045] Figure 3A shows the design pattern of HER2 CAR sequences based on different promoters; Figure 3B shows the design pattern of EGFP adenovirus sequences based on different promoters.

[0046] Figure 4A shows the percentage of CAR molecules expressed on the macrophage membrane surface after transduction with HER2 CAR adenoviruses from different promoter sources; Figure 4B shows the percentage of EGFP molecules expressed in macrophages after transduction with EGFP adenoviruses from different promoter sources; Figure 4C shows the percentage of CAR molecules expressed on the 293T cell membrane surface after transduction with HER2 CAR adenoviruses from different promoter sources.

[0047] Figure 5A shows the expression levels of CAR molecules on the macrophage membrane surface after transduction with HER2 CAR adenoviruses from different promoter sources; Figure 5B shows the expression levels of EGFP molecules within macrophages after transduction with EGFP adenoviruses from different promoter sources; Figure 5C shows the expression levels of CAR molecules on the 293T cell membrane surface after transduction with HER2 CAR adenoviruses from different promoter sources.

[0048] Figure 6A shows that macrophages infected with HER2 CAR adenovirus containing different promoters can all express CAR molecules; among them, CMV Intron, SFFV, CAG, and CBh promoters still express CAR molecules 7 days after infection, and the expression lasts until day 18 post-infection; Figure 6B shows that macrophages transduced with EGFP adenovirus from different promoters can all express EGFP molecules, and all of them can be stably expressed until day 18 post-infection.

[0049] Figure 7A shows the percentage of CAR molecules expressed on monocytes after transduction with HER2 CAR adenoviruses from different promoters; Figure 7B shows the persistent expression levels of HER2 CAR adenoviruses with different promoters after infection with monocytes, where CAG, CMV, CMV Intron, and CBh can persist until day 21 post-transfection.

[0050] Figure 8 shows CAR molecules with different structures.

[0051] Figure 9A shows the percentage of CAR molecules expressed in adenovirus-transduced macrophages with different CAR structures between the CMV and CAG promoters; Figure 9B shows the expression levels of CAR molecules in adenovirus-transduced macrophages with different CAR structures between the CMV and CAG promoters.

[0052] Figure 10 shows the phagocytosis of OE19 by CAR-macrophages from different promoter sources 2 days after infection.

[0053] Figure 11 shows the phagocytosis of OE19 by CAR-macrophages from different promoter sources after 7 and 15 days of culture following adenovirus infection.

[0054] Figure 12 shows the expression rate of CD80 molecules on days 2, 4, 7, 10, and 14 after macrophages were infected with adenoviruses from different promoter sources.

[0055] Figure 13 shows the expression rate of CD86 molecules on days 2, 4, 7, 10, and 14 after macrophages were infected with adenoviruses from different promoter sources.

[0056] Figure 14 shows the in vitro killing results of macrophages infected with adenoviruses from different promoter sources.

[0057] Figure 15 shows a schematic diagram of the HER2 CAR structure based on different hinge regions.

[0058] Figure 16A shows the percentage of CAR molecules expressed on the macrophage membrane surface after transduction of macrophages with HER2 CAR adenoviruses from different promoter sources based on different hinge regions; Figure 16B shows the expression level of CAR molecules on the macrophage membrane surface after transduction of macrophages with HER2 CAR adenoviruses from different promoter sources based on different hinge regions.

[0059] Figure 17 shows the phagocytosis of OE19 by CAR-macrophages from different promoter sources based on different hinge regions 2 days after infection.

[0060] Figure 18A shows a schematic diagram of the PSMAVH-VL CAR structure based on different promoter sources in different hinge regions; Figure 18B shows a schematic diagram of the PSMAVL-VH CAR structure based on different promoter sources in different hinge regions.

[0061] Figure 19A shows the percentage of CAR molecules expressed on the macrophage membrane surface after transduction of macrophages with PSMAVH-VL CAR adenoviruses from different promoter sources; Figure 19B shows the expression level of CAR molecules on the macrophage membrane surface after transduction of macrophages with PSMAVH-VL CAR adenoviruses from different promoter sources; Figure 19C shows the percentage of PSMAVH-VH CAR molecules expressed with different promoter sources; Figure 19D shows the expression level of PSMAVH-VH CAR molecules expressed with different promoter sources.

[0062] Figure 20A shows the in vitro phagocytosis results of LnCap cells by PSMAVH-VL CAR macrophages from different promoter sources. Figure 20B shows the in vitro phagocytosis results of LnCap cells by PSMAVH-VL CAR macrophages from different promoter sources. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0064] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0065] Unless the context clearly indicates otherwise, references to a specific quantity herein include their plural forms. For example, references to "cells" include one or more such cells and equivalents known to those skilled in the art, etc.

[0066] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0067] Adenoviruses are double-stranded DNA viruses. Adenoviruses are small (approximately 90 nm in diameter) non-enveloped viruses with an icosahedral capsid from which 12 spikes extend. The virus consists of 240 hexagonal trimers, 12 pentaagonal pentamers, 60 IIIa monomers, 12 spike trimers, 60 VII hexamers, 80 IX trimers, two terminal repeat proteins, and multiple core proteins.

[0068] The sequential process of gene transcription from the adenovirus genome reflects the protein requirements of the virus at each stage of replication. Therefore, depending on the timing of transcriptional initiation at each viral promoter, transcription of the adenovirus genome is divided into early and late events. The first protein produced from the viral genome is the E1A protein. E1A proteins have two main functions in infected cells. First, they induce the cell to enter the S phase of the cell cycle, allowing efficient replication of the viral genome. Second, they induce transcription of other early promoters within the viral genome through transactivation. These promoters control the production of E1B, E2, E3, E4, and E5 proteins. Immediately following E1A expression is the production of VARNA and E1B and E3 proteins. These proteins and RNA molecules help to inhibit the development of antiviral responses. These early events in viral replication help shape the intracellular environment, allowing the viral genome to replicate before packaging. Subsequent transcriptional events involve the production of structural proteins and proteins essential for cell lysis, which are primarily derived from major late promoters that transcribe late regions 1–5.

[0069] Adenovirus genes are divided into early (E1-4) and late (L1-5) transcripts, with multiple protein isoforms driven by a series of splicing events. The early region is divided into E1, E2, E3, and E4. E1 is essential for transitioning the cell to a cell cycle stage that favors viral replication, inhibits apoptosis, and promotes cell division. The E2 region is primarily responsible for DNA genome replication and contains DNA-binding proteins (E2A), terminal proteins, and DNA polymerase (E2B). E3 contains genes involved in regulating the host immune response, and E4 contains a series of genes involved in regulating cellular pathways, such as non-homologous end joining (NHEJ) and binding to E1B-55K to mediate p53 degradation. Late adenovirus genes are all transcribed from the same major late promoter and share the same 5' mRNA end, which contains three exons that together form a triplet leader sequence. Late genes are expressed through a series of splicing events that allow the expression of about 13 proteins that form part of the viral particle (e.g., hexagonal and spike) or participate in its assembly (e.g., 100K protein).

[0070] The adenovirus vector used herein may be an adenovirus vector derived from an adenovirus genome, as conventionally used in the art. In some embodiments, the adenovirus vector comprises a backbone derived from adenovirus serotypes 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24-30, 31, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad 3, canine Ad 2, sheep Ad, or porcine Ad 3, or functional variants thereof. In a specific embodiment, the adenovirus vector used herein comprises the AD5 / F35 adenovirus vector backbone. The vector backbone is primarily based on adenovirus type 5 (Ad5, adenovirus serotype-5), with its fibrin replaced by that of adenovirus type 35 (Ad35). The Ad5F35 chimeric adenovirus backbone is a chimeric vector modified from the conventional Ad5 adenovirus vector, with modifications to the receptor-binding spike region. Adenoviruses include 7 species (AG) and 57 serotypes. Commonly used adenovirus vectors are derived from adenovirus types 2 and 5, with type 5 (Ad5) being the most prevalent. Ad5 exhibits broad-spectrum tropism; studies have shown that Ad5 containing chimeric F5 / F35 spikes significantly increases tropism for hematopoietic cells.

[0071] The term "chimeric antigen receptor" or "CAR" refers to an artificial T-cell surface receptor that has been engineered to be expressed on immune effector cells and specifically bind to antigens. CARs can be used as a treatment via adoptive cell transfer. Monocytes are taken from a patient (blood, tumor, or ascites) and modified to express a specific receptor for a particular antigen form. In some embodiments, for example, a CAR specific to tumor-associated antigens has been expressed. CARs may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain containing a tumor-associated antigen-binding region. The specificity of the CAR design may be derived from a ligand of the receptor (e.g., a peptide). In some embodiments, a CAR can target cancer by redirecting the expression of the CAR to monocytes / macrophages specific for tumor-associated antigens.

[0072] A promoter is a nucleotide sequence that binds to RNA polymerase and directs the transcription of a gene. Typically, promoters are located in the 5' uncoding region of a gene, near the transcription start site. The sequence components within the promoter that initiate transcription are usually characterized by shared nucleotide sequences.

[0073] The CMV promoter belongs to the early enhancer / promoter of human cytomegalovirus (CMV) and may have varying strengths in different cell types. In some embodiments, the CMV promoter has a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO:1.

[0074] The CAG promoter is a modified artificially constructed promoter, a CMV early enhancer fused to the chicken β-actin promoter, used to drive high-level gene expression in mammalian vectors. In some embodiments, the CAG promoter has a nucleotide sequence as shown in SEQ ID NO:2, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO:2.

[0075] The CBh promoter is a modified artificially constructed promoter, a CMV early enhancer fused to the chicken β-actin promoter. In some embodiments, the CBh promoter has a nucleotide sequence as shown in SEQ ID NO:3, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO:3. The CMV Intron promoter is a human cytomegalovirus immediate early enhancer / promoter fused to the splicing signal of human globin intron 2; it may have variable strength in certain cell types; the presence of the globin intron promotes nuclear export of mRNA through splicing and is expected to enhance gene expression in eukaryotes. In some embodiments, the CMV Intron promoter has a nucleotide sequence as shown in SEQ ID NO:4, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO:4.

[0076] The EF1α promoter belongs to the human eukaryotic translation elongation factor 1α1 promoter. In some embodiments, the EF1A promoter has a nucleotide sequence as shown in SEQ ID NO:5, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO:5.

[0077] The hPGK promoter belongs to the human phosphoglyceric kinase (hPGK)1 promoter. In some embodiments, the hPGK promoter has a nucleotide sequence as shown in SEQ ID NO:6, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO:6.

[0078] The SFFV promoter belongs to the spleen focus-forming virus promoter family and can drive high levels of gene expression, particularly in cell types of the bone marrow lineage. In some embodiments, the SFFV promoter has a nucleotide sequence as shown in SEQ ID NO:7, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO:7.

[0079] In this article, examples of cell surface markers that can serve as antigens binding to the antigen-binding domain of a CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells. The selection of the antigen-binding domain depends on the type and quantity of antigens present on the target cell surface. For example, the antigen-binding domain may be selected to recognize antigens that serve as cell surface markers on target cells associated with a specific disease state.

[0080] In some embodiments, the antigen-binding domain binds to tumor antigens, such as specific antigens of a target tumor or cancer. In one embodiment, the tumor antigens described herein comprise one or more antigenic cancer epitopes. Non-limiting examples of tumor-associated antigens include CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD 117); Interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin-11 receptor α (IL-11Ra); Prostate stem cell antigen (PSCA); Protease serine 21 (Testisin or PRSS21); Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor α; Receptor tyrosine protein kinase ERBB2 (Her2 / neu); Mucin 1, Cell surface associated compound (MUC1); Epidermal growth factor receptor (EGFR); Neural cell adhesion molecule (NCAM); Prostase; Prostatic acid phosphatase (PAP); Mutated elongation factor 2 (ELF2M); Ephrin B2; Fibroblast activating protein α (FAP); Insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Macropain) subunit β-type 9 (LMP2); Glycoprotein 100 (gp100); Oncogene fusion protein (bcr-abl) composed of the breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homologue 1 (Abl); Tyrosinase; Ephrin type A receptor 2 (EphA2); Fucose GM1; Sialic acid Lewis adhesion molecule (sLe);Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 related compound (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C5 group D member (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD 179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); GloboH (the hexasaccharide moiety of glycoceramide); Breast differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus receptor 1 (HAVCR1); Adrenaline receptor β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); Sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); mesothelin MSLN; Fos-associated antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8) 8) Melanoma antigen 1 (MelanA or MART1) recognized by T cells; Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); Pax-3 (PAX3) pairing box protein; Androgen receptor; Cyclin B1; v-myc avian myeloma virus oncogene neuroblastoma-derived homologue (MYCN);Ras homologues include: C (RhoC); tyrosinase-associated protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like protein (BORIS or Brother of the Regulator of Imprinted Sites); Squamous cell carcinoma antigen 3 recognized by T cells (SART3); Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase-anchored protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous protein 1 (RU1); renal ubiquitous protein 2 (RU2); legumain; and human papillomavirus E6 (UPV). E6); Human papillomavirus E7 (UPV E7); Intestinal carboxyl esterase; Mutant heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecular-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); Mucin-like hormone receptor-like protein 2 containing EGF-like modules (EMR2); Lymphocyte antigen 75 (LY75); Phosphatidylinositol proteoglycan-3 (Glypican-3, GPC3); Fc receptor-like protein 5 (FCRL5); and Immunoglobulin λ-like polypeptide 1 (IGLL1).

[0081] The antigen-binding domain may include any domain that binds to an antigen, and may include, but is not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. Therefore, in one embodiment, the antigen-binding domain partially comprises a mammalian antibody or a fragment thereof. In another embodiment, the antigen-binding domain of the CAR is selected from anti-CD19 antibodies, anti-HER2 antibodies, and fragments thereof. HER2, or human epidermal growth factor receptor 2, is responsible for regulating cell growth and division and is associated with the invasiveness and poor prognosis of various cancers (such as breast cancer and gastric cancer). In another embodiment, the antigen-binding domain of the CAR is selected from anti-PSMA antibodies. PSMA, or prostate-specific membrane antigen, is highly expressed in prostate cancer cells but rarely expressed in other tumors or normal tissues.

[0082] In some implementations, the antigen-binding domain is derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR comprises a human antibody, a humanized antibody, or a fragment thereof.

[0083] In some embodiments, the antigen-binding domain is operatively linked to another domain of the CAR, such as a transmembrane domain or an intracellular signaling domain, for expression in the cell. In one embodiment, the nucleic acid encoding the antigen-binding domain is operatively linked to both a nucleic acid encoding the transmembrane domain and a nucleic acid encoding the intracellular signaling domain.

[0084] In some embodiments, the antigen-binding domain is operatively linked to one or more "hinge domains" or hinge regions of the CAR, which function in positioning the antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation. A CAR typically includes one or more hinge domains between the antigen-binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may include amino acid sequences of naturally occurring immunoglobulin hinge domains or modified immunoglobulin hinge domains. Exemplary hinge domains suitable for use in the CARs described herein include hinge domains of human proteins, such as CD8α, CD28, 4-1BB, OX40, CD3-ζ, T cell receptor α or β chains, CD3ζ chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, their functional derivatives, and any combination thereof. In some embodiments, the hinge domain includes CD8α. In some embodiments, the hinge domain includes CD28. In some embodiments, the hinge domain includes, but is not limited to, any one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD).

[0085] Regarding transmembrane domains, CARs can be designed to include transmembrane domains that link the antigen-binding domain of the CAR to an intracellular signaling domain. In one embodiment, the transmembrane domain is naturally associated with one or more domains in the CAR. In some cases, the transmembrane domain may be selectively modified, or modified by amino acid substitution, to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0086] The transmembrane domain can be derived from natural or synthetic sources. In the case of a natural source, the domain can originate from any membrane-binding or transmembrane protein. The transmembrane region specifically used in this disclosure can comprise at least one or more of the following transmembrane regions: the α, β, or ζ chain of a T-cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; Toll-like receptor 1 (TLR1); TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some cases, various human hinges, including human Ig (immunoglobulin) hinges, can also be used.

[0087] In some embodiments, the transmembrane domain may be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0088] Regarding intracellular signal transduction domains, the intracellular signal transduction domains or otherwise cytoplasmic domains of the CAR include intracellular signal transduction domains similar to or identical to those of chimeric intracellular signal transduction molecules described elsewhere herein, and are responsible for the activation of cells expressing the CAR. In some embodiments, the intracellular signal transduction domains of the CAR include domains responsible for signal activation and / or transduction.

[0089] Examples of intracellular signal transduction domains used in this disclosure include, but are not limited to, the cytoplasmic portion of surface receptors, co-stimulatory molecules, and any molecules that work together to initiate signal transduction in monocytes or macrophages, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capacity.

[0090] Examples of intracellular signal transduction domains include fragments or domains from one or more molecules or receptors, including but not limited to TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, universal FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NK G2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LF A-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2 B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108) SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequence of costimulatory molecules with the same activity, and any combination thereof.

[0091] In some embodiments, the intracellular signal transduction domains of the CAR include dual signal transduction domains, such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor β chain, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, dectin 1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, together with any combination of any of the signal transduction domains listed in the preceding paragraphs. In another embodiment, the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD3, the FcεRIγ chain, any derivative or variant thereof, any synthetic sequence thereof having the same functional ability, and any combination thereof.

[0092] A spacer domain may be incorporated between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular signaling domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that functions to link the transmembrane domain to the antigen-binding domain or the intracellular signaling domain in a polypeptide chain. In one embodiment, the spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In another embodiment, short oligopeptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, may form a link between the transmembrane domain and the intracellular signaling domain of the CAR. Examples of linkers include glycine-serine duplexes.

[0093] Operable linkage refers to the connection of at least the first and second elements such that the constituent elements are in a relationship that allows them to function in their intended manner. For example, if a regulatory sequence (e.g., a promoter sequence) and a coding sequence are linked in a manner that allows the expression of the coding sequence to be controlled by the regulatory sequence, then the nucleic acid regulatory sequence is “operably linked” to the nucleic acid coding sequence. In some embodiments, the “operably linked” regulatory sequence is covalently bound to the coding sequence directly or indirectly (e.g., in a single nucleic acid molecule). In some embodiments, the regulatory sequence controls the expression of the coding sequence in a trans-regulatory manner, and including the regulatory sequence in the same nucleic acid as the coding sequence is not a requirement for operable linkage.

[0094] As used herein, "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. Antibodies are generally tetramers of immunoglobulin molecules. The antibodies in this invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies.

[0095] As used herein, “antibody fragment” refers to a portion of a complete antibody and specifically to the antigenic determinant variable region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments. As used herein, “antibody heavy chain (VH)” refers to the larger of the two types of polypeptide chains present in the native conformation of all antibody molecules. As used herein, “antibody light chain (VL)” refers to the smaller of the two types of polypeptide chains present in the native conformation of all antibody molecules.

[0096] As used herein, “coding” refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, serving as a template for other polymers and macromolecules having defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequences or defined amino acid sequences used in synthetic biological processes, and the resulting biological properties. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is provided in the sequence listing) and the non-coding strand that serve as a transcription template for a gene or cDNA can be referred to as the protein or other product encoding that gene or cDNA.

[0097] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0098] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0099] As used in this article, “transfection,” “transformation,” or “transduction” refers to the process of transferring or introducing exogenous nucleic acids into host cells. Cells that are “transfected,” “transformed,” or “transduced” are cells that have already been transfected, transformed, or transduced with exogenous nucleic acids. These cells include the original target cell and its progeny. Exemplary transfection methods include calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polyethylene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biological munitions.

[0100] In some embodiments, the host cells are immune cells. Immune cells include monocytes, macrophages, and / or T lymphocytes. Non-limiting examples of subjects from which immune cells are obtained include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is a human. Cells can be obtained from a variety of sources, including peripheral blood monocytes, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In some embodiments, any number of monocytes, macrophages, or T lymphocytes available in the art can be used. In some embodiments, cells can be obtained using any technique known to those skilled in the art, such as Ficoll isolation. In one embodiment, cells from an individual's circulating blood are obtained by blood separation or leukocyte extraction. Blood separation products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells (white blood cells), red blood cells (red blood cells), and platelets. In some embodiments, the M1 or M2 markers of the cells are measured by FACS. M1 markers are selected from, for example, HLADR, CD86, CD80, and PDL1. M2 markers are selected from, for example, CD206 and CD163.

[0101] Macrophages are phenotypic immune cells that play a crucial role during both the onset and resolution of inflammation. Macrophages can polarize into two phenotypes upon stimulation: (1) the classical activated (inflammation) phenotype M1, which can be induced by lipopolysaccharide (LPS) or interferon-γ (IFN-γ) to produce pro-inflammatory cytokines such as TNFα and IL-1β; and (2) the alternative activated (wound healing) phenotype M2, which can be induced by IL-4 and IL-13 to produce anti-inflammatory cytokines such as IL-10, IL-13, and Arg1. The balance of M1 / M2 macrophage polarization determines the fate of an organ in inflammation or injury. M1 exerts a pro-inflammatory effect in the early stages of inflammation, counteracting stimuli, but its continued activity can cause tissue damage; M2 exerts an anti-inflammatory effect, promoting tissue repair and angiogenesis.

[0102] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0103] Example

[0104] Example 1. Construction of adenovirus vectors from different promoter sources

[0105] Adenovirus vectors containing different promoters were constructed, namely: CMV (SEQ ID NO:1), CAG (SEQ ID NO:2), CBh (SEQ ID NO:3), CMV Intron (SEQ ID NO:4), EF1A (SEQ ID NO:5), hPGK (SEQ ID NO:6), and SFFV (SEQ ID NO:7). The vector backbone was AD5 / F35 adenovirus (purchased from Yunzhou Biotechnology; a schematic diagram of the adenovirus vector structure is shown in Figure 1). Nucleic acid sequences targeting the HER2 CAR molecule (SEQ ID NO:8, VH-VL) or EGFP (SEQ ID NO:10) were inserted into the constructed vectors containing different promoters. The amino acid sequences of the signal peptide, hinge region (CD28 hinge), transmembrane region (CD8 TM), and intracellular region (FCE ICD) are shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively; the linker sequence is shown in SEQ ID NO:24.

[0106] Example 2. Detection of CAR molecule expression in macrophages

[0107] CAR-macrophage preparation is shown in Figure 2A.

[0108] Apheresis blood obtained from a commercial company (with relevant ethical approvals) was centrifuged at 200g for 10 minutes to remove platelets and some red blood cells. The supernatant was discarded, and the cell pellet was mixed with PBS, then added to Ficoll (lymphocyte separation medium, purchased from Sigma), and centrifuged at 1200 rpm for 30 minutes. After centrifugation, mononuclear cells (PBMCs) were recovered and washed once with phosphate-buffered saline (PBS) by centrifugation. PBMCs and CD14... + Magnetic beads (purchased from Miltenyi) were incubated for 15 minutes, and CD14 sorting columns (purchased from Miltenyi) were used for CD14 sorting. + Cell isolation and CD14 harvest + Monocytes were induced to differentiate into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0109] After monocytes were induced to differentiate into macrophages, they were divided into 7 experimental groups (see Figure 3A for grouping), with 3 × 10⁶ cells per well. 6Cells were evenly seeded into 6-well plates. Adenovirus vectors containing different promoters and carrying HER2-targeting CAR molecules (encoding DNA sequence as shown in SEQ ID NO:8, amino acid sequence as shown in SEQ ID NO:9), constructed in Example 1, were added to each well at an MOI of 1000 for transfection. The promoters were CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV. One negative control group (UTD group) consisted of untransfected macrophages. Cells were harvested 48 hours after transfection, with at least 1 × 10⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and 2 μL of FCR Blocking (Mitteni, 130-059-901) was added. After incubation at room temperature in the dark for 10 minutes, the antibody mixture in Table 1 was added, and the cells were incubated at 4°C in the dark for 30 minutes. After centrifugation at 400g for 5 minutes, the cells were washed once with PBS and then analyzed by flow cytometry. The percentage of HER2-targeting CAR molecules expressed on the macrophage membrane surface was detected by flow cytometry (Figure 4A) and the CAR molecule expression level (Figure 5A). Table 1 shows the antibodies used in the flow cytometry and their manufacturers.

[0110] Table 1. Antibody information detected by flow cytometry

[0111] The results showed that all seven different promoters could express CAR molecules on the macrophage membrane surface, and the promoters CMV, CAG, CBh, CMV Intron and EF1A expressed CAR molecules significantly better than the promoters hPGK and SFFV (Figures 4A and 5A).

[0112] Example 3. Detection of EGFP molecular expression in macrophages

[0113] Following the method in Example 2, monocytes were induced to differentiate into macrophages and then divided into 7 experimental groups (grouping shown in Figure 3B). Each group was transfected with adenovirus vectors containing different promoters and carrying EGFP molecules (encoding DNA sequence as shown in SEQ ID NO:10, amino acid sequence as shown in SEQ ID NO:11), constructed in Example 1. The MOI for all groups was 1000. The promoters were CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV, respectively. One negative control group (UTD group) consisted of untransfected macrophages. Cells were harvested 48 hours after transfection, with at least 1 × 10⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, the cell pellet was resuspended in PBS, and the cells were subjected to flow cytometry to detect EGFP protein, and the percentage and intensity of EGFP protein expression in macrophages were analyzed.

[0114] The results showed that all seven different promoters could express EGFP protein in macrophages, and the promoters CMV, CAG, CBh, CMVIntron and EF1A expressed EGFP protein significantly better than the promoters hPGK and SFFV (Figures 4B and 5B).

[0115] Example 4.293T cell CAR molecular expression detection

[0116] 293T cells were plated in 6-well plates at a density of 3 × 10⁻⁶ cells per well. 6 Cells were evenly seeded into 6-well plates. They were divided into 7 experimental groups (see Figure 3A for grouping), and each group was transfected with an adenovirus vector containing different promoters and carrying a HER2-targeting CAR molecule (encoding DNA sequence as shown in SEQ ID NO:8, amino acid sequence as shown in SEQ ID NO:9), constructed in Example 1. The MOI for all groups was 5. The promoters were CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV. One negative control group (UTD group) consisted of untransfected 293T cells. Cells were harvested 24 hours after transfection, with at least 1 × 10⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and the cell pellet was mixed with PBS. HER2 protein (purchased from Acro) (4μl / test) was added and incubated at room temperature for 20 minutes. After centrifugation at 400g for 5 minutes to remove unbound HER2 protein, the cells were washed and then subjected to flow cytometry detection of HER2-targeting CAR molecules. The percentage and intensity of CAR molecule expression on the surface of 293T cell membranes were analyzed.

[0117] The results showed that all seven different promoters could express CAR molecules on the surface of 293T cells (see Figures 4C and 5C). Compared with the macrophage system, the corresponding promoters showed a significant advantage in expressing the same CAR molecules on the surface of 293T cells, indicating that macrophages have a selective preference for CAR molecules in terms of promoters, and that the CMV, CAG, EF1A, CBh and CMV Intron promoters are more advantageous in macrophage expression.

[0118] Example 5. Detection of CAR molecule expression intensity and persistence in macrophages

[0119] CAR-macrophages were prepared according to the method in Example 2. After monocytes were induced to differentiate into macrophages, they were divided into 42 experimental groups. Adenoviruses carrying HER2-targeting CAR molecules with different promoters prepared in Example 1 were added to each group. The promoters were CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV, respectively. The negative control group (UTD group) consisted of macrophages that were not transfected with adenovirus. Cells were harvested on Day 2, Day 4, Day 7, Day 10, Day 14, and Day 18 after transfection, with at least 1 × 10⁻⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and the cell pellet was mixed with PBS. HER2 protein (4μl / test) was added and incubated. The cells were centrifuged at 400g for 5 minutes to remove unbound HER2 protein. After washing, the cells were subjected to flow cytometry analysis to detect the intensity and duration of CAR molecule expression targeting HER2.

[0120] The results showed that CAR molecule expression was detected on macrophages by all seven different promoters 2 days after transfection (see Figure 6A). 18 days after transfection, CMV, CAG, CBh, CMV Intron, and SFFV still showed high levels of CAR expression. + Rate.

[0121] Example 6. Detection of EGFP molecule expression intensity and persistence in macrophages

[0122] EGFP-macrophages were prepared according to the method in Example 3. After monocytes were induced to differentiate into macrophages, they were divided into 42 experimental groups. Adenoviruses carrying EGFP molecules and containing different promoters prepared in Example 1 were added to each group. The promoters were CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV, respectively. The negative control group (UTD group) consisted of macrophages that were not transfected with adenovirus. Cells were harvested on Day 2, Day 4, Day 7, Day 10, Day 14, and Day 18 after transfection, with at least 1 × 10⁻⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and the cell pellet was resuspended in PBS. The cells were then subjected to flow cytometry to detect the intensity and duration of EGFP expression.

[0123] The results showed that macrophages transduced with EGFP adenoviruses from different promoter sources could stably express EGFP molecules until day 18 post-infection, with CMV, CAG, CBh, CMV Intron, EF1A and SFFV showing better expression (see Figure 6B).

[0124] Example 7. Detection of CAR molecule expression intensity and persistence in monocytes

[0125] CAR-monocyte preparation is shown in Figure 2B.

[0126] Apheresis blood obtained from a commercial company (with relevant ethical approvals) was centrifuged at 200g for 10 minutes to remove platelets and some red blood cells. The supernatant was discarded, and the cell pellet was mixed with PBS, then added to Ficoll (lymphocyte separation medium, purchased from Sigma), and centrifuged at 1200 rpm for 30 minutes. After centrifugation, mononuclear cells (PBMCs) were recovered and washed once with phosphate-buffered saline (PBS) by centrifugation. PBMCs and CD14... + Magnetic beads (purchased from Miltenyi) were incubated for 15 minutes, and CD14 sorting columns (purchased from Miltenyi) were used for CD14 sorting. + Cell isolation and CD14 harvest + Mononuclear cells.

[0127] After sorting, monocytes were divided into 28 experimental groups, each inoculated with adenovirus containing different promoters targeting the HER2 CAR molecule, prepared in Example 1. The promoters were CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV. The negative control group (UTD group) consisted of untransfected monocytes. Cells were harvested on Day 2, Day 6, Day 17, and Day 21 post-transfection, with at least 1 × 10⁻⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and the cell pellet was mixed with PBS. HER2 protein (4μl / test) was added and incubated. The cells were centrifuged at 400g for 5 minutes to remove unbound HER2 protein. After washing, the cells were subjected to flow cytometry analysis to detect the intensity and duration of CAR molecule expression targeting HER2.

[0128] The results showed that CAR molecule expression was detectable on monocytes in the CMV, CAG, CBh, CMV Intron, and EF1A groups two days after transfection (see Figure 7A). Among them, CAG, CMV, and CMV Intron showed the highest CAR expression. + The highest CAR rate was observed. Furthermore, regarding persistent expression: within the 21-day detection range, CAG, CMV, CMV Intron, and CBh all exhibited high CAR rates.+ The rate was high, and CAG and CBh showed that they could maintain the sustained expression of CAR molecules in monocytes (see Figure 7B).

[0129] Example 8. Detection of transfection efficiency of different CAR molecules in macrophages

[0130] CAR-macrophages were prepared according to the method in Example 2. After monocytes were induced to differentiate into macrophages, they were divided into four experimental groups. Adenoviruses containing CMV and CAG promoters prepared in Example 1, along with CAR molecules targeting HER2 with different structures, were added to each group (see Figure 8; the encoding DNA sequence of HER2 CAR (VH-VL) is shown in SEQ ID NO:8, and the amino acid sequence is shown in SEQ ID NO:9; the encoding DNA sequence of HER2 CAR (VL-VH) is shown in SEQ ID NO:12, and the amino acid sequence is shown in SEQ ID NO:13, where the amino acid sequences of HER2 scFv VH and HER2 scFv VL are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively). The negative control group (UTD group) consisted of macrophages not transfected with adenovirus. Cells were harvested on day 7 after transfection, with at least 1 × 10⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and the cell pellet was mixed with PBS. HER2 protein (4μl / test) was added and incubated. The cells were centrifuged at 400g for 5 minutes to remove unbound HER2 protein. After washing, the expression level and intensity of HER2-targeting CAR molecules were detected by flow cytometry.

[0131] The results showed that different CAR molecular structures regulated by CMV and CAG promoters could achieve better transfection efficiency (positive rate >80%) in macrophages (see Figures 9A and 9B).

[0132] Example 9. In vitro phagocytosis experiment

[0133] OE19 cells (human esophageal cancer cells, purchased from COBIOER BIOSCIENCES) were suspended in PBS. DiR dye (5 μM) was added to the cell suspension, and the cells were incubated at 37°C for 20 minutes. The cells were centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in preheated X-VIVO15 medium (purchased from Lonza, hereinafter referred to as "complete medium") containing 10% FBS (purchased from Gibco) to a density of 1×10⁶ cells / mL. 6 Cells / mL.

[0134] CAR-macrophages were prepared according to the method in Example 2. Seven groups of macrophages, infected for 2 days with adenoviruses derived from CMV, CAG, CBh, CMVIntron, EF1A, hPGK, and SFFV promoters, were suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. Preheated complete culture medium was added to the cell pellet to resuspend the cells until the density reached 1×10⁻⁶. 6 Cells / mL.

[0135] The negative macrophage group (UTD group) consisted of macrophages untransfected with adenovirus, suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in pre-warmed complete culture medium until the density was 1×10⁶. 6 Cells / mL.

[0136] Adenovirus-infected macrophages and negative macrophages are collectively referred to as effector cells, while OE19 cells are referred to as target cells. Effector cells and target cells were added to culture dishes at a 1:1 ratio and co-cultured for 2 hours. Cells were then collected, stained with anti-CD11b-FITC (purchased from eBioscience), and CD11b was analyzed by flow cytometry. + Cells in the population containing the DiR label are considered to be macrophages that have engulfed OE19.

[0137] The results showed that the phagocytic levels of the CAG group, CMV group, CBh group, CMV intron group, and EF1A group were all better than those of the UTD group (see Figure 10).

[0138] Example 10. In vitro phagocytosis experiment of CAR macrophages at different culture times

[0139] OE19 cells (human esophageal cancer cells) were suspended in PBS. DiR dye (5 μM) was added to the cell suspension, and the cells were incubated at 37°C for 20 minutes, followed by centrifugation at 400g for 5 minutes. The supernatant was aspirated, and the cells were resuspended in preheated complete culture medium until the density reached 1 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0140] CAR-macrophages were prepared according to the method in Example 2. A total of 14 groups of macrophages were collected on days 7 and 15 post-infection with adenoviruses derived from CMV, CAG, CBh, CMVIntron, EF1A, hPGK, and SFFV promoters. These macrophages were suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in preheated complete culture medium until a density of 1×10⁻⁶ cells / mL was reached. 6 Cells / mL.

[0141] The negative macrophage group (UTD group) consisted of macrophages untransfected with adenovirus, suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in pre-warmed complete culture medium until the density was 1×10⁶. 6 Cells / mL.

[0142] Adenovirus-infected macrophages and negative macrophages are collectively referred to as effector cells, while OE19 cells are referred to as target cells. Effector cells were harvested on days 7 and 15 post-infection, and then added to culture dishes at a 1:1 ratio with target cells. After co-culturing for 2 hours, cells were harvested, stained with anti-CD11b-FITC, and analyzed by flow cytometry for CD11b. + Cells labeled with DiR in the population are considered to be macrophages that have engulfed OE19.

[0143] The results showed that CMV, CAG, CBh and CMVIntron all had strong phagocytic ability as the post-infection culture time was extended, and CAG, CBh and CMVIntron showed stronger phagocytic ability than other promoters 15 days after infection (see Figure 11).

[0144] Example 11. Macrophage M1 phenotype detection

[0145] CAR-macrophages were prepared according to the method in Example 2. After monocytes were induced to differentiate into macrophages, they were divided into 35 experimental groups. Adenoviruses carrying HER2-targeting CAR molecules, prepared in Example 1 and containing different promoters (CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV), were added to each group. The negative control group (UTD group) consisted of macrophages not transfected with adenovirus. Cells were harvested on days 2, 4, 7, 10, and 14 post-transfection, with at least 1 × 10⁻⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and the cell pellet was mixed with PBS. CD80 antibody (purchased from BD) and CD86 antibody (purchased from BD) (5μL / test) were added and incubated at room temperature for 30 minutes. After centrifugation at 400g for 5 minutes to remove unbound antibodies, the cells were washed and then analyzed by flow cytometry.

[0146] The results showed that, compared with UTD, macrophages infected with adenoviruses from different sources all exhibited good expression rates and intensities of the M1 type characterization molecules CD80 and CD86 (see Figures 12-13), indicating that promoters from different sources all have good M1 type maintenance effects.

[0147] Example 12. In vitro killing experiment of HER2 CAR macrophages

[0148] OE19 cells (human esophageal cancer cells) were seeded in 6-well plates and modified using a commercially available luciferase lentivirus (purchased from Genomics Inc.) at an MOI of 10. The modified cells were then screened using puromycin to obtain a stable OE19 modified cell line expressing luciferase (hereinafter referred to as OE19-luciferase), which was used for in vitro killing assays.

[0149] OE19-luciferase cells were harvested and resuspended in preheated complete culture medium at a cell density of 1×10⁻⁶. 5 Cells / mL. After mixing, dispense 100 μL per well (1 × 10⁻⁶ cells / mL). 4 ), and lay 96-well plates (Corning / Cat: 3610) and incubate at 37°C for 2 hours.

[0150] HER2 CAR macrophages harvested on day 7 post-infection with adenoviruses derived from CMV, CAG, CBh, CMV Intron, and EF1A promoters were divided into 21 groups, suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium until a density of 1×10⁻⁶ cells / mL was reached. 6 Cells / mL.

[0151] The negative macrophage group (UTD group) consisted of macrophages untransfected with adenovirus, suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in pre-warmed complete culture medium until the density was 1×10⁶. 6 Cells / mL. Effector cells were prepared at effector-to-target ratios of 5:1, 2:1, and 1:1 (100 μL added to each well). After incubation at 37°C for 48 hours, the cells were centrifuged at 500g for 5 min to precipitate the cells. 100 μL of the supernatant was discarded, and 50 μL of the prepared solution was added to each well. Luciferase Assay reagent (Promega / Cat: E2520), after 5 minutes of reaction, read the value on the microplate reader.

[0152] The results showed that, based on OE19-luciferase target cells, promoters CMV, CAG, CBh, CMV Intron, and EF1A all had high killing rates (see Figure 14).

[0153] Example 13. Detection of HER2 CAR molecular expression in different hinge regions

[0154] Figure 15 shows a schematic diagram of the HER2 CAR structure (including the promoter) in different hinge regions. The amino acid sequence of the hinge region (CD8 hinge) is shown in SEQ ID NO:27, and the sequences of other regions are the same as in Example 1.

[0155] CAR-macrophage preparation is described in Example 2. After monocytes were induced to differentiate into macrophages, they were divided into 7 experimental groups (grouping shown in Figure 15). Each group was injected with an adenovirus vector containing different promoters (the DNA sequence of which is shown in SEQ ID NO:15, and the amino acid sequence is shown in SEQ ID NO:14), constructed in Example 1. The promoters were CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV, respectively. One negative control group (UTD group) consisted of untransfected macrophages. Cells were harvested 48 hours after transfection, with at least 1 × 10⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and the cell pellet was mixed with PBS. HER2 protein (purchased from Acro) (4μl / test) was added and incubated at room temperature for 20 minutes. After centrifugation at 400g for 5 minutes to remove unbound HER2 protein, the cells were washed and then subjected to flow cytometry detection of CAR molecules targeting HER2. The percentage and intensity of CAR molecule expression on the macrophage membrane surface were analyzed.

[0156] The results showed that all seven different promoters could express CAR molecules, and the percentage of CAR expression is shown in Figure 16A and the CAR expression level (MFI) is shown in Figure 16B. Among them, the CMV, CAG, and CMV Intron promoters had the highest expression levels.

[0157] Example 14. In vitro phagocytic function detection of HER2 CAR molecules in different hinge regions

[0158] CAR-macrophages were prepared according to the method in Example 2. Seven groups of macrophages were infected with adenoviruses derived from CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV promoters for 2 days. These macrophages were suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. Preheated complete culture medium was added to the cell pellet to resuspend the cells until the density reached 1×10⁻⁶. 6 Cells / mL.

[0159] The negative macrophage group (UTD group) consisted of macrophages untransfected with adenovirus, suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in pre-warmed complete culture medium until the density was 1×10⁶. 6 Cells / mL.

[0160] Adenovirus-infected macrophages and negative macrophages are collectively referred to as effector cells, while OE19 cells are referred to as target cells. Effector cells and target cells were added to culture dishes at a 1:1 ratio and co-cultured for 2 hours. Cells were then collected, stained with anti-CD11b-FITC (purchased from eBioscience), and CD11b was analyzed by flow cytometry. + Cells in the population containing the DiR label are considered to be macrophages that have engulfed OE19.

[0161] The results showed that the phagocytosis of OE19 target cells by the CAG group, CMV group and CMV intron group was significantly better than that by the UTD group (see Figure 17).

[0162] Example 15. PSMACAR Molecular Expression Detection

[0163] The PSMACAR structure (including the promoter) is shown in Figures 18A and 18B. The PSMACAR structure of VH-VL is shown in Figure 18A, and the PSMACAR structure of VL-VH is shown in Figure 18B.

[0164] CAR-macrophage preparation is described in Example 2. After monocytes were induced to differentiate into macrophages, they were prepared at a rate of 3 × 10⁶ cells per well. 6 Cells were evenly seeded into 6-well plates. They were divided into 7 experimental groups (see Figure 18A or Figure 18B for grouping), and each group was transfected with an adenovirus vector containing different promoters and targeting PSMACAR molecules (encoding DNA sequences as shown in SEQ ID NO:17 and 19, amino acid sequences as shown in SEQ ID NO:16 and 18, with the amino acid sequences of PSMAscFv VH and PSMAscFv VL as shown in SEQ ID NO:28 and SEQ ID NO:29, respectively), with an MOI of 1000. The promoters were CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV, respectively. One negative control group (UTD group) consisted of untransfected macrophages. Cells were harvested 48 h after transfection, with at least 1 × 10⁶ cells per group. 6 Cells were mixed with PBS, centrifuged at 400g for 5 minutes, and 2 μL of FCR Blocking (Mitteni, 130-059-901) was added. After incubation at room temperature in the dark for 10 minutes, the antibody mixture in Table 2 was added, and the cells were incubated at 4°C in the dark for 30 minutes. After centrifugation at 400g for 5 minutes, the cells were washed once with PBS and then analyzed by flow cytometry. The percentage and intensity of PSMA CAR molecule expression on the macrophage membrane surface were analyzed by flow cytometry.

[0165] Table 2. Antibody information detected by flow cytometry

[0166] Results: All seven different promoters could express the PSMACAR molecule. The percentage of PSMAVH-VL CAR expression is shown in Figure 19A and the CAR expression level (MFI) is shown in Figure 19B; the percentage of PSMAVL-VH CAR expression is shown in Figure 19C and the CAR expression level (MFI) is shown in Figure 19D. Among them, the CMV, CAG, and CMV Intron promoters showed the highest expression levels.

[0167] Example 16. In vitro phagocytic function detection of PSMACAR macrophages

[0168] LnCap cells (human prostate cancer cells, purchased from Kebai Biotechnology) were suspended in PBS. DiR dye (5 μM) was added to the cell suspension, and the cells were incubated at 37°C for 20 minutes, followed by centrifugation at 400g for 5 minutes. The supernatant was aspirated, and the cells were resuspended in preheated complete culture medium until the density reached 1 × 10⁶ cells / mL. 6 Cells / mL.

[0169] PSMACAR-macrophages were prepared according to the method in Example 2. Seven groups of macrophages were infected for 2 days with adenoviruses derived from CMV, CAG, CBh, CMV Intron, EF1A, hPGK, and SFFV promoters. The cells were suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. Preheated complete culture medium was added to the cell pellet to resuspend the cells until the density reached 1×10⁻⁶. 6 Cells / mL.

[0170] The negative macrophage group (UTD group) consisted of macrophages untransfected with adenovirus, suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in pre-warmed complete culture medium until the density was 1×10⁶. 6 Cells / mL.

[0171] Adenovirus-infected macrophages and negative macrophages are collectively referred to as effector cells, while LnCap cells are called target cells. Effector cells and target cells were added to culture dishes at a 1:1 ratio and co-cultured for 2 hours. Cells were then collected, stained with anti-CD11b-FITC (purchased from eBioscience), and CD11b was analyzed by flow cytometry. + Cells in the population containing the DiR label are considered to be macrophages that have phagocytosed LnCap.

[0172] The results showed that among PSMAVH-VL CAR macrophages or PSMAVL-VH CAR macrophages from different promoter sources, the phagocytic levels of LnCap target cells in the CAG group, CMV group, and CMV intron group were significantly better than those in the UTD group (see Figures 20A and 20B).

[0173] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An adenoviral vector carrying a chimeric antigen receptor (CAR) encoding nucleic acid sequence, the adenoviral vector comprising one or more of a CMV promoter, a CAG promoter, a CBh promoter, a CMV Intron promoter, and an EF1A promoter, the CAR encoding nucleic acid sequence operably linked to the promoter.

2. The adenoviral vector of claim 1, wherein, the CMV promoter has a nucleotide sequence as set forth in SEQ ID NO: 1, or a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 1; the CAG promoter has a nucleotide sequence as set forth in SEQ ID NO: 2, or a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 2; the CBh promoter has a nucleotide sequence as set forth in SEQ ID NO: 3, or a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 3; the CMV Intron promoter has a nucleotide sequence as set forth in SEQ ID NO: 4, or a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 4; the EF1A promoter has a nucleotide sequence as set forth in SEQ ID NO: 5, or a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence as set forth in SEQ ID NO:

5.

3. The adenoviral vector of claim 1, wherein, the adenoviral vector further comprises one or more of a reporter gene, a polyadenylation tail, an untranslated region, an enhancer, and a terminator, preferably, the adenoviral vector comprises an AD5 / F35 adenoviral vector backbone.

4. The adenoviral vector of claim 1, wherein, the CAR molecule has an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain; preferably, the CAR is expressed by a modified immune cell, preferably by a modified monocyte, a modified macrophage, or a modified T lymphocyte, preferably, the antigen binding domain binds a tumor associated antigen.

5. A host cell comprising the adenoviral vector of any one of claims 1-4.

6. The host cell of claim 5, wherein, the cell is a mammalian cell expressing the adenoviral vector; preferably, the host cell is selected from an immune cell, preferably from a monocyte, a macrophage, or a T lymphocyte, preferably, the host cell is a CD14+ monocyte, preferably, the host cell is a M1 type macrophage.

7. A pharmaceutical composition comprising the adenoviral vector of any one of claims 1-4 or the host cell of claim 5 or 6, and a pharmaceutically acceptable excipient or carrier.

8. A kit comprising the adenoviral vector of any one of claims 1-4, optionally a cell line allowing the adenoviral vector to infect cells and replicate the viral genome, and optionally a DNA plasmid used to construct the adenoviral vector.

9. Use of the adenoviral vector of any one of claims 1 to 4, the host cell of claim 5 or 6, the pharmaceutical composition of claim 7 or the kit of claim 8 for the manufacture of a medicament for the prevention, treatment or alleviation of an inflammatory disease and / or a tumor, preferably for the inhibition of the growth of tumor cells and / or the induction of apoptosis in tumor cells, preferably the tumor is selected from the group consisting of esophageal cancer or prostate cancer.

10. A method for increasing the transfection efficiency of an adenovirus, the method comprising transfecting an immune cell with the adenoviral vector of any one of claims 1 to 4, preferably the immune cell is selected from the group consisting of a monocyte, a macrophage or a T lymphocyte.

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