Circular rnas encoding car and uses thereof

Abstract

A circular RNA encoding a CAR is provided, as well as its use for the production of immune cells targeting specific diseases, such as lymphomas, multiple myeloma and leukemias, as well as autoimmune diseases, such as systemic lupus erythematosus, lupus nephritis and myasthenia gravis.

Description

Technical Field

[0001] This application relates to circular RNAs encoding CARs and related molecules, and their use in immunotherapy, such as for the treatment of cancer, hyperplastic diseases, fibrosis, and autoimmune diseases. Background Technology

[0002] Gene therapy offers a potential means to enhance immune recognition to target specific diseases. Adoptive transfer therapy of chimeric antigen receptor (CAR)-modified T cells has achieved significant breakthroughs in cancer treatment for B-cell lineage tumors. To date, six CAR-T cell therapies targeting B-cell leukemia, lymphoma, and multiple myeloma have been approved by the FDA. However, the high cost and complex manufacturing processes make this therapy unaffordable for many patients who could potentially benefit. One solution is to program T cells or other immune cells in vivo. Circular RNAs are particularly suitable for in vivo protein expression therapies. Chimeric antigen receptors can be encoded by circular RNAs, which are then delivered in vivo via LNPs and expressed in immune cells. This application relates to chimeric T-cell receptors encoded by circular RNAs, and methods for using these receptors to promote immune responses against selected targets. Our circRNA CAR platform promises to provide an effective, repeatable, and scalable off-the-shelf immunotherapy without the need for lymphocyte depletion. Surprisingly, sustained antitumor effects were observed in PBMC humanized mice with only two doses of circRNA CAR. Significant anti-B cell activity was observed after a single dose in non-human primates. Furthermore, a reduction in disease markers was also achieved in a lupus mouse model. These results demonstrate the clinical potential of our circRNACAR in patients with cancer or autoimmune diseases. Summary of the Invention

[0003] This application relates to a novel circular RNA encoding a chimeric antigen receptor (CAR) or a variant thereof, which is capable of recognizing specific targets and treating diseases through the therapeutic use of the circular RNA.

[0004] In one aspect, this application provides a composition comprising the circular RNA disclosed herein, wherein the composition comprises a pharmaceutically acceptable excipient.

[0005] In one aspect, this application provides a circular RNA for pharmaceutical use, comprising the regulatory and expression elements described herein.

[0006] In one aspect, this application provides a method for administering immunotherapy to a subject, comprising administering circular RNA to the subject. The immunotherapy is selected from: CAR-T cell therapy, CAR-NK cell therapy, CAR-macrophage therapy, or CAR-Treg cell therapy.

[0007] In one area, immunotherapy is selected from treatments for cancer, hyperplastic diseases, fibrosis, or autoimmune diseases. Circular RNAs can produce durable tumor control, almost completely eliminating tumor cells and persisting for a long time after treatment is stopped.

[0008] In one aspect, this application provides linear RNA for preparing the circular RNA disclosed herein.

[0009] In one aspect, this application provides a DNA vector suitable for synthesizing the linear RNA and circular RNA disclosed herein.

[0010] In one aspect, this application provides a population of eukaryotic cells comprising the circular RNA disclosed herein.

[0011] In one aspect, this application provides a method for preparing the circular RNA disclosed herein, wherein the method includes circularization using a substitution intron-exon (PIE) system in combination with class I introns or T4 RNA ligase 2. Attached Figure Description

[0012] Figure 1 This is the general structure of a circular RNA encoding a CAR.

[0013] Figure 2 The in vitro expression of anti-hCD19 CAR and anti-mCD19 CAR in HEK293T and Jurkat cells transfected with the corresponding circular RNA is shown. Figure 2 A-2C shows the expression of the circRNA encoding CAR in HEK293T and Jurkat cells 12 hours after transfection. Figure 2 D-2E shows the duration of CAR expression in vitro. CAR expression was assessed by flow cytometry (FCM) from 6 h to 15 d post-transfection.

[0014] Figure 3 This study demonstrated the antitumor effect of anti-hCD19 CAR circRNA on CD19+ B cell malignancies in humanized mice. Figure 3 A is a schematic diagram outlining an experimental overview of B-cell leukemia treatment in humanized mice. Figure 3 B is a representative image at a specified time point, showing the Nalm6 tumor burden in treated and control mice. Figure 3 C shows the total tumor burden values ​​of the treated and control mice. Figure 3 D-3F demonstrates in vivo expression of anti-CD19 CAR in human T cell populations.

[0015] Figure 4It showed antitumor activity at doses as low as 0.2 mg / kg.

[0016] Figure 5 This study demonstrated the inhibitory effect of anti-hCD19 CAR circRNA on the growth of malignant CD19+ B cells in humanized mice. Treatment with circRNA CAR encapsulated in modified LNPs (fLNPs) effectively delayed the growth of Nalm6 tumors in M-NSG mice. Figure 5 A-5C). CAR expression in vivo and human T cell regeneration were detected by flow cytometry. Figure 5 D-5I).

[0017] Figure 6 This study demonstrated the potent antitumor effect of CAR in vivo in a preclinical mouse model of B-cell leukemia, with controllable cytokine release. Figure 6 A represents the experimental design for studying the anti-tumor effects in the A20-BALB / c mouse model. Figure 6 B showed that anti-mCD19 CAR circRNA treatment delayed tumor progression in the A20 syngeneic tumor model. Figure 6 C shows the changes in body weight after administration of anti-mCD19 CAR circRNA. Figure 6 D-6I showed serum cytokine levels 12 hours after injection of anti-mCD19 CAR circRNA.

[0018] Figure 7 This study demonstrated the efficacy of anti-mCD19 CAR circRNA in treating lupus. Figure 7 A shows an experimental overview of the use of circRNA-CAR to treat systemic lupus erythematosus in MRL-lpr mice. Figure 7 B-7C shows the serological analysis results of anti-dsDNA antibody and total mouse IgG. Figure 7 D-7F shows proteinuria and CREA test results, indicating the extent of kidney damage.

[0019] Figure 8 The study demonstrated that circRNA-CAR induced potent B-cell clearance in non-human primates (NHP). Figure 8 A shows the NHP study design. Figure 8 B cells were shown to represent the structure of a CD20 / CD19 bispecific CAR. Following intravenous injection of the anti-CD20 / 19 bispecific CAR, peripheral blood B cells were specifically eliminated. Figure 8 C). Detailed Implementation

[0020] Unless otherwise defined below, all technical and scientific terms used in this application have the meanings commonly understood by those skilled in the art. The term "technique" as used herein is intended to refer to techniques generally understood in the art, including modifications or equivalent substitutions that are obvious to those skilled in the art. While those skilled in the art should be able to readily understand the following terms, the definitions are provided below for better explanation of this application.

[0021] General definition As used herein, the terms “comprising,” “including,” “having,” or “containing,” and variations thereof, are inclusive or open-ended and do not exclude other elements or method steps not listed. In some embodiments, “comprising,” “including,” “having,” or “containing” may be replaced by “consisting of” or “consists”.

[0022] As used herein, the terms “implementation,” “disclosed herein,” or “disclosure” are not intended to be limiting but are generally applicable to any embodiment as defined in the claims or described herein. These terms may be used interchangeably herein.

[0023] As used herein, the terms "treat / treating / treatment," etc., refer to the elimination, reduction, or improvement of a disease or condition, and / or related symptoms. Although not excluded, treating a disease or condition does not require the complete elimination of the disease, condition, or related symptoms. The term "treatment" and its synonyms refer to the administration of a therapeutically effective amount of the circular RNA or composition disclosed herein to a subject who requires such treatment. Treatment can be directed at symptoms, such as suppressing symptoms. Symptoms can be affected in the short term, treated in the medium term, or treated long-term, such as in a maintenance therapy setting.

[0024] In this disclosure, the term "a / an" refers to one or more of the same entity; for example, "a polynucleotide" is understood to mean one or more polynucleotides. Therefore, the terms "a / an," "one or more," and "at least one" are used interchangeably in this application.

[0025] As used herein, the term "variant" refers to a peptide that differs from the stated peptide due to amino acid substitutions, deletions, insertions, and / or modifications. Variants can be generated using mutagenesis techniques known in the art.

[0026] The term “composition” or “pharmaceutical composition” refers to a composition comprising the circular RNA provided herein, together with, for example, a pharmaceutically acceptable carrier, excipient or diluent, for administration to a subject in need of treatment.

[0027] The term "pharmaceutically acceptable" means that, within reasonable medical judgment, exposure to human and animal tissues is appropriate without causing excessive toxicity or other complications commensurate with a reasonable benefit / risk ratio.

[0028] "Effective dose" refers to the dose of the circular RNA provided herein, which, when administered to a subject as a single dose or as part of a series of doses, is effective in treating the patient. This dose may be a fixed dose for all subjects to be treated; it may also vary depending on the subject's weight, health status and physical condition, desired weight loss or weight maintenance, the formulation of the circular RNA or composition disclosed herein, professional assessment of the medical condition, and other relevant factors.

[0029] The term "subject" refers to any subject who requires treatment with the circular RNA or composition provided herein, specifically, a mammalian subject. Mammal subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, dairy cows, apes, monkeys, orangutans, and chimpanzees. In one embodiment, the subject is a human subject.

[0030] Circular RNA As used herein, the terms "circRNA," "circular polynucleotide," or "circular RNA" are used interchangeably and refer to polynucleotides that form a circular structure through covalent or non-covalent bonds. When referring to circular RNA, those skilled in the art will understand that the polynucleotide in RNA refers to a polynucleotide.

[0031] In one aspect, this application provides a circular RNA encoding a chimeric antigen receptor (CAR) or a variant thereof.

[0032] In some embodiments, the circular RNA encoding a chimeric antigen receptor (CAR) includes regulatory and expression elements, the expression element containing a polynucleotide encoding the chimeric antigen receptor (CAR); and This chimeric antigen receptor (CAR) includes an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular T cell signal transduction domain. Each element is optionally linked by a multinucleotide encoding a linker.

[0033] In some implementations, the light chain variable region and the heavy chain variable region in the CAR are optionally connected by a connector, wherein the connector is selected from or derived from: GA, GS, GG, GGA, GGS, GGG, GGGA, GGGS, GGGG, GGGGA, GGGGG, GGGGA, GGGGS, GGGGGG, GGAG, GGSG, AGGGG, SGGG, GAGA, GSGSGS, GAGAGAGA, GSGSGSGS, GAGAGAGA, GSGSGSGS, GAGAGAGAGA, GSGSGSGS, GSGS GSGSGS, GAGAGAGAGAGA, GAGSGSGSGSGS, GGAGGA, GGSGGS, GGAGGAGGA, GGSGGSGGS, GGAGGAGGAGGA, GGSGGSGGSGGS, GGAGGGAG, GGSGGGSG, GGAGGGAGGGAG, GGSGGGSGGSG, GGGAGGGAGGGA, GGGSGGGSGGGS, (GGGGS)3 or GSTGSGKPGSGEGSTKG.

[0034] In some embodiments, the polynucleotide encoding CAR or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, or 30.

[0035] In some embodiments, the amino acid sequence of the CAR encoded by the polynucleotide or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 29.

[0036] In some embodiments, the circular RNA further comprises a polynucleotide encoding a signal peptide selected from: a CD8a leader sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 18, and a colony-stimulating factor 2 receptor α subunit (CSF2RA) signal peptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 19.

[0037] In some embodiments, the antigen-binding domain is one or more antigen-binding domains specific to CD19, CD20, BCMA, CLDN6, CLDN18.2, ERBB2 (HER2), PSMA, SLAMF3, SLAMF7, CD66c, MSLN, CD38, CD123, GPC3, EGFRvIII, CD171, MUC1 and / or GPRC5D; preferably, it is a single-stranded variable fragment (scFv) specific to CD19, CD20 and / or BCMA; more preferably, the amino acid sequence of the scFv has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity with the sequence shown in SEQ ID NO: 25, 33, 35, 37, 39 or 41. The amino acid sequence of scFv is encoded by a polynucleotide sequence that has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO:26, 34, 36, 38, 40, or 42.

[0038] In some implementations, the antigen-binding domain is one or more antigen-binding domains that are specific to CD19, wherein CD19 may be humanized CD19, partially humanized CD19, or mouse CD19.

[0039] In some implementations, the antigen-binding domain is one or more antigen-binding domains that are specific to CD20, wherein CD20 may be humanized CD20, partially humanized CD20, or mouse CD20.

[0040] In some embodiments, the antigen-binding domain is one or more antigen-binding domains specific to BCMA, wherein the BCMA may be humanized BCMA, partially humanized BCMA, or mouse BCMA. In some embodiments, the co-stimulatory domain is selected from CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and combinations thereof; preferably, it is CD28 or 4-1BB; more preferably, the co-stimulatory domain of CD28 has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 23.

[0041] In some implementations, the transmembrane domain is selected from CD28 and CD8 transmembrane domains.

[0042] In some embodiments, the intracellular T cell signal transduction domain is a CD3ζ signal transduction domain. Preferably, the CD3ζ signal transduction domain has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 21.

[0043] In some embodiments, the circular RNA of this application comprises a regulatory element and an expression element, the expression element comprising a polynucleotide encoding a bispecific CAR specific for CD20 and CD19. The regulatory element comprises a polynucleotide encoding an internal ribosome entry site (IRES) or a fragment thereof as defined in claim 9; Among them, the bispecific CAR includes an antigen-binding domain, a co-stimulatory domain disclosed in this paper, a transmembrane domain disclosed in this paper, and an intracellular T cell signal transduction domain disclosed in this paper. The antigen-binding domain is an antigen-binding domain that is specific to CD19 and CD20; preferably, it includes a single-stranded variable fragment (scFv); more preferably, it is FMC-63 scFv and Leu-16 scFv. Wherein, FMC-63 scFv is an amino acid sequence of scFv having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence selected from SEQ ID NO: 25, 33, or 35; wherein, Leu-16 scFv is an amino acid sequence of scFv having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 41; Each element is optionally linked by a multinucleotide encoding a linker.

[0044] In some embodiments, the polynucleotide encoding CAR or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 32.

[0045] In some embodiments, the amino acid sequence of the CAR encoded by the polynucleotide or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 31.

[0046] In some embodiments, the circular RNA of this application comprises a regulatory element and an expression element, the expression element comprising a polynucleotide encoding a CAR. The regulating element is a CVB3 IRES having the sequence shown in SEQ ID NO: 17; CAR includes an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, an intracellular T cell signal transduction domain, and a signal peptide element. The antigen-binding domain is specific to hCD19 or mCD19, and the antigen-binding domain is an amino acid sequence of scFv having the sequence shown in SEQ ID NO: 25, 33, 35, 37, 39 or 41. The co-stimulatory domain is a CD28 co-stimulatory domain having the sequence shown in SEQ ID NO: 23; Among them, the transmembrane structural domain is a CD28 or CD8 transmembrane structural domain; The intracellular T cell signal transduction domain is a CD3ζ signal transduction domain having the sequence shown in SEQ ID NO: 21; and The signal peptide element is a colony-stimulating factor 2 receptor α subunit (CSF2RA) signal peptide having the sequence shown in SEQ ID NO: 19. Each element is linked by a polynucleotide encoding (GGGGS)3.

[0047] Control element A regulatory element may include a sequence located adjacent to an expression element encoding an expression product. The regulatory element may be operatively linked to a neighboring sequence. The regulatory element may increase the amount of expression product compared to the amount of expression product in the absence of a regulatory element. Furthermore, a regulatory element may increase the amount of product expressed by multiple tandemly linked expression sequences. Therefore, a regulatory element may enhance the expression of one or more expression sequences.

[0048] In some implementations, the regulatory element increases the translation yield of circular RNA. In some implementations, the regulatory element comprises an internal ribosome entry site (IRES) or a fragment thereof.

[0049] A suitable IRES element contained in a cyclic polynucleotide contains an RNA sequence capable of binding to a eukaryotic ribosome. In some embodiments, the length of the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

[0050] In some embodiments, the IRES is selected from: Coxsackievirus B3 (CVB3) IRES, Enterovirus 71 (EV71) IRES, Encephalomyocarditis Virus (EMCV) IRES, Picornavir Virus (PV) IRES, Hepatitis C Virus (HCV) IRES, Adenovirus (AdV) IRES, Human Papillomavirus 31 (HPV31) IRES, Human Herpesvirus (HHV) IRES, Raul's Sarcoma Virus (RSV) IRES, Classical Swine Fever Virus (CSFV) IRES, FGF9 IRES, SLC7A1 IRES, and RUNX1 IRES; preferably, the IRES is a CVB3 IRES or a variant thereof, which has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 17.

[0051] Pharmaceutical Composition In one aspect, this application provides a composition comprising the circular RNA as described in any of the foregoing embodiments, wherein the composition comprises a pharmaceutically acceptable excipient.

[0052] In some embodiments, the composition comprises nanoparticles, such as lipid nanoparticles.

[0053] In some embodiments, the circular RNA is administered either as naked circular RNA or as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. In non-limiting examples, pharmaceutically acceptable excipients are polyethyleneimine (PEI) or lipid nanoparticles (LNP). Other examples of liposomes that can be used to administer the circular RNA or composition include: protamine, cationic nanoemulsions, modified dendritic macromolecular nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid nanoparticles, cationic lipid-cholesterol nanoparticles, cationic lipid-cholesterol PEG nanoparticles, cationic lipid transfection reagents sold under the trademark LIPOFECTAMINE, non-liposome transfection reagents sold under the trademark FUGENE, or any combination thereof, all of which may be used as pharmaceutically acceptable excipients.

[0054] In some embodiments, the lipid nanoparticles comprise at least one of ionizable lipids, structural lipids, and PEG-modified lipids; preferably, the ionizable lipid is SM102 (CAS No.: 2089251-47-6).

[0055] In some embodiments, the pharmaceutical composition may optionally contain one or more other active substances, such as substances with therapeutic and / or preventative activities.

[0056] disease In one aspect, this application provides a method for administering immunotherapy to a subject, comprising administering to the subject a therapeutically effective amount of the circular RNA or composition disclosed herein.

[0057] In one aspect, this application provides the use of the circular RNA or composition disclosed herein in the preparation of a medicament for immunotherapy in a subject.

[0058] In one aspect, this application provides the circular RNA or composition disclosed herein for use in immunotherapy in subjects.

[0059] In some implementations, the immunotherapy is selected from: CAR-T cell therapy, CAR-NK cell therapy, CAR-macrophage therapy, or CAR-Treg cell therapy.

[0060] In some implementations, immunotherapy is selected from: treatments for cancer, treatments for hyperplastic diseases, treatments for fibrosis, or autoimmune diseases. Treatable cancers include unvascularized or not yet sufficiently vascularized tumors, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) or may include solid tumors.

[0061] In some implementations, the cancer is selected from: acute lymphoblastic carcinoma, acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder epithelial carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer or colorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), Hodgkin lymphoma, etc. Hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, fluid-filled tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small bowel cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0062] In some implementation schemes, the autoimmune disease is selected from systemic lupus erythematosus (SLE), lupus nephritis, myasthenia gravis, pemphigus vulgaris, or type I diabetes.

[0063] In some implementations, fibrosis is referred to as cardiac fibrosis.

[0064] linear RNA In one aspect, this application provides a linear RNA for preparing the circular RNA disclosed herein.

[0065] In some embodiments, the linear RNA polynucleotide includes a regulatory element, an expression element, and at least one self-cyclizing element; the regulatory element includes a polynucleotide encoding the internal ribosome entry site (IRES) or a fragment thereof as described in the preceding embodiments, and the expression element encodes the chimeric antigen receptor (CAR) polypeptide as described in any of the preceding embodiments. Each element is optionally linked by a polynucleotide encoding a linker, wherein the linker is selected from or derived from: GA, GS, GG, GGA, GGS, GGG, GGGA, GGGS, GGGG, GGGGA, GGGGG, GGGGA, GGGGS, GGGGG, GGAG, GGSG, AGGGG, SGGG, GAGA, GSGSGS, GAGAGAGA, GSGSGSGS, GAGAGAGAGA, GSGSGSGS, GAGAGAGAGA GSGSGSGSGS, GAGAGAGAGAGA, GAGSGSGSGSGS, GGAGGA, GGSGGS, GGAGGAGGA, GGSGGSGGS, GGAGGAGGAGGA, GGSGGSGGSGGS, GGAGGGAG, GGSGGGSG, GGAGGGAGGGAG, GGSGGGSGGSG, GGGAGGGAGGGA, GGGGSGGGSGGGS or (GGGGS)3.

[0066] In some implementations, the linear RNA polynucleotide comprises, from 5' to 3', the following: Optionally, a 5'-self-looping element; Regulatory elements comprising polynucleotides encoding the internal ribosome entry site (IRES) or a fragment thereof disclosed herein; Optionally, the signal peptide element disclosed herein; The expression element comprises a polynucleotide encoding the chimeric antigen receptor (CAR) as described in any of the foregoing embodiments; Optionally, terminate the codon; Optionally, a 3'-self-circulating element.

[0067] In some embodiments, the 5'-self-cyclizing element comprises a 5'-I class intron fragment and exon 2. Preferably, the 5'-I class intron replacing the intron-exon homologous arm has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 27. In some embodiments, the 3'-self-cyclizing element comprises exon 1 and a 3'-I class intron fragment. Preferably, the nucleotide sequence of the 3'-I class intron replacing the intron-exon homologous arm has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 28. In some embodiments, the 3'-I class intron fragment and the 5'-I class intron fragment are Anabaena class I intron fragments.

[0068] In some embodiments, the self-cyclization element is cyclized by T4 RNA ligase 2, the 5' end of the linear RNA polynucleotide is phosphorylated, preferably monophosphorylated, and the 3' end of the linear RNA polynucleotide is OH. T4 RNA ligase 2 catalyzes the formation of a phosphodiester bond between the 5-phosphate (donor) and 3-hydroxyl (acceptor) terminal groups in RNA in an ATP-dependent reaction.

[0069] In some implementations, the linear RNA polynucleotide comprises, from 5' to 3', the following: 5' Class I introns replace intron-exon homologous arms, having the sequence shown in SEQ ID NO: 27; A regulatory element comprising a polynucleotide encoding an internal ribosome entry site (IRES) or a fragment thereof disclosed herein, wherein the IRES is a CVB3 IRES having the sequence shown in SEQ ID NO: 17; Signal peptide element, wherein the signal peptide element is a colony-stimulating factor 2 receptor α subunit (CSF2RA) signal peptide having the sequence shown in SEQ ID NO: 19; An expression element comprising a polynucleotide encoding a CAR, wherein the expression element comprises an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, an intracellular T cell signal transduction domain, and a signal peptide element; wherein the antigen-binding domain is specific for hCD19 or mCD19, and the antigen-binding domain is an amino acid sequence of scFv having the sequence shown in SEQ ID NO: 25, 33, 35, 37, 39, or 41; wherein the co-stimulatory domain is a CD28 co-stimulatory domain having the sequence shown in SEQ ID NO: 23; wherein the transmembrane domain is a CD28 or CD8 transmembrane domain; wherein the intracellular T cell signal transduction domain is a CD3ζ signal transduction domain having the sequence shown in SEQ ID NO: 21; and wherein the signal peptide element is a colony-stimulating factor 2 receptor α subunit (CSF2RA) signal peptide having the sequence shown in SEQ ID NO: 19; The stop codon; and 3' Class I introns replace intron-exon homologous arms, having the sequence shown in SEQ ID NO: 28; Each element is optionally linked by a polynucleotide encoding (GGGGS)3.

[0070] DNA vectors and eukaryotic cell populations In one aspect, this application provides a DNA vector suitable for synthesizing the linear RNA and circular RNA disclosed herein.

[0071] In one aspect, this application provides a population of eukaryotic cells comprising the circular RNA disclosed herein.

[0072] In some implementations, eukaryotic cell populations express CAR complex proteins encoded by circular RNA polynucleotides on their cell surfaces.

[0073] In some implementations, this cell population is able to kill tumor cells more effectively or more persistently than an equivalent eukaryotic cell population containing linear RNA encoding the same CAR.

[0074] Preparation methods of circular RNA In one aspect, this application provides a method for preparing the circular RNA disclosed herein, wherein the method includes circularization using a substitution intron-exon (PIE) system in combination with class I introns or T4 RNA ligase 2.

[0075] In some embodiments, the method for preparing the circular RNA of this application includes binding class I introns using a substitution intron-exon (PIE) system.

[0076] In some embodiments, the method for preparing the circular RNA of this application includes circularization using T4 RNA ligase 2.

[0077] In some implementations, the circular RNA is formed by cyclizing the linear RNA polynucleotides described in the aforementioned implementations.

[0078] Example To make the objectives and technical solutions of this application clearer, the application will be further described below with reference to specific embodiments. It should be understood that these embodiments are not intended to limit the scope of this application. Furthermore, specific experimental methods not mentioned in the following embodiments are all performed according to conventional experimental methods.

[0079] The sequence numbers used in this application are listed in Table 1. The nucleotide sequences shown in the sequence listing can represent RNA and can be converted into the WIPO standard ST.26 sequence listing.

[0080] Table 1

[0081] Example 1: Vector Construction The open reading frame of the circular RNA encoding anti-hCD19 CAR consists of three functional domains. Figure 1(Top): One is a single-stranded variable fragment (scFv) molecule that recognizes human CD19 (SEQ ID NO: 25), one is a portion of the human CD28 protein (SEQ ID NO: 23), and the remaining portion is the intracellular domain of human CD3ζ (SEQ ID NO: 21). These three functional domains are arranged in tandem and are guided by a signal peptide derived from the α chain of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (i.e., the colony-stimulating factor 2 receptor α subunit (CSF2RA) signal peptide, the amino acid sequence of which is shown in SEQ ID NO: 19). The exact sequence of CD28 begins with the I114 and MYPPPY motifs, and the portions of its extracellular domain that can bind CD80 and CD86 are included in this sequence. The transmembrane domain of CD28 and the entire cytoplasmic tail are also included. The nucleotides encoding the CAR protein were codon-optimized using an internal algorithm and translated via the internal ribosome entry site (IRES) of Coxsackievirus B3 (CVB3) (SEQ ID NO: 17). The CAR targeting mouse CD19 consists of the light chain variable region of the 1D3 antibody, the linker peptide (GGGGS)3, the heavy chain variable region of the 1D3 antibody, a portion of the mouse CD28 molecule, and the cytoplasmic region of the mouse CD3ζ molecule (SEQ ID NO: 29). Figure 1 (Bottom). Insert a BspQ I or Pme I restriction endonuclease digestion site 3' downstream of the element required for plasmid linearization.

[0082] Example 2: Generation of circRNA-CAR via class I intron self-splicing reaction The codon-optimized nucleotide sequence was synthesized by Anshunda Life Sciences. The Anabaena Class I intron-replacement intron-exon (PIE) DNA construct sequence, as shown in SEQ ID NO: 27 or 28, consists of complementary homologous arms at both ends, facilitating the folding of the active ribozyme structure. The synthesized fragment was cloned into the Spe I / Age I site of the pUC57 plasmid and linearized at 37°C for 2 hours using PmeI (NEB, R0560L). The linearized plasmid was purified using the DNA Purification and Concentration Series-25 Kit (ZYMO RESEARCH, D4034) and used for precursor RNA synthesis. The precursor RNA was synthesized in vitro using T7 RNA polymerase (Nanjing Novizan Biotechnology Co., Ltd., DD4101). In the IVT reaction system, the following reaction components were assembled: 400 ng linearized DNA template, 100 U T7 RNA polymerase (Nanjing Novizan Biotechnology Co., Ltd., DD4101), 100 μM NTP mixture, 10× reaction buffer (Nanjing Novizan Biotechnology Co., Ltd., DD4101R), 10 U RNase inhibitor (Nanjing Novizan Biotechnology Co., Ltd., DD4102-PA-01), and 0.008 U pyrophosphatase (Nanjing Novizan Biotechnology Co., Ltd., DD4103-PC-01). The IVT reaction was then performed using a thermal cycler and incubated at 37°C for 2 hours. After in vitro transcription, the IVT reaction solution was treated with 10 U DNase I (Nanjing Novizan Biotechnology Co., Ltd., EN401) and incubated at 37°C for 30 minutes to remove the linearized DNA template. After treatment with DNase I, the reaction solution was heated at 55°C for 15 minutes to allow for as much transesterification as possible. The RNA was then purified by column chromatography using the Monarch® RNA Purification Kit (NEB, T2040L).

[0083] Example 3: Preparation of circRNA-CAR using T4 Rnl2 In summary, the plasmid was linearized by digestion with BspQ I restriction endonuclease (Nanjing Novizan Biotechnology Co., Ltd., DD4302-PC-02), and the reaction was incubated at 50°C for 3 hours. The linearized DNA template was then purified using the DNA Purification and Concentration Series-25 Kit (ZYMO RESEARCH, D4034). After the linearization step, a portion of the linearized DNA template was added to the IVT reaction to obtain precursor RNA. In the IVT reaction system, the reaction components were 400 ng linearized DNA template, 100 U T7 RNA polymerase (Nanjing Novizan Biotechnology Co., Ltd., DD4101-PC-03), NTP mixture (including 100 μM GTP (Nanjing Novizan Biotechnology Co., Ltd., DD4108-PA-02), 600 μM GMP solution, 100 μM ATP (Nanjing Novizan Biotechnology Co., Ltd., DD4106-PA-02), 100 μM CTP (Nanjing Novizan Biotechnology Co., Ltd., DD4107-PA-02), 100 μM UTP (Nanjing Novizan Biotechnology Co., Ltd., DD4105-PA-02)), 10× reaction buffer (Nanjing Novizan Biotechnology Co., Ltd., DD4101R), 10 U RNase inhibitor (Nanjing Novizan Biotechnology Co., Ltd., DD4102-PA-01), and 0.008 U of pyrophosphatase (Nanjing Novizan Biotechnology Co., Ltd., DD4103-PC-01) was used, and the IVT reaction was incubated at 37°C for 2 hours. Subsequently, the linear product was digested with DNase I (Nanjing Novizan Biotechnology Co., Ltd., EN401) to remove template DNA, and the RNA transcripts were purified by column chromatography using the Monarch® RNA Purification Kit (NEB, T2040L). To obtain circular RNA, the RNA transcripts were then circularized using T4 Rnl2 (Kaikai Biotechnology (Shanghai) Co., Ltd., TRL-BE103-C1) for enzymatic ligation. 10 U of T4 RNA ligase II, 10× reaction buffer, and 10 μg of linear RNA were added to 40 μL of reaction mixture, and the reaction vessel was incubated at 25°C for 2 hours. Finally, RNase R exonuclease (Suzhou Nearshore Protein Technology Co., Ltd., GMP-E224-M001) was added to degrade linear RNA and enrich circular products. 1 μg of column-purified RNA product and 1 U of RNase R were assembled into a 6 μl reaction mixture, and the reaction was incubated at 37°C for 30 minutes. The circular RNA product was then purified using the Monarch® RNA Purification Kit (NEB, T2040L).

[0084] Example 4: Preparation of circRNA-LNP / fLNP circRNA enriched with RNase R was formulated into lipid nanoparticles (LNPs) using the ionizable lipid SM102 (catalog number: 06040008800, Xiamen Sinobond Biotechnology Co., Ltd.). In short, an ethanol lipid mixture of the ionizable cationic lipid SM-102, DSPC, cholesterol, and DMG-PEG2000 was mixed with an aqueous solution containing mRNA at an acidic pH using NanoAssemblr. TM Ignite TM The nanoparticle formulation system is used for mixing.

[0085] To prepare the modified LNP (fLNP), circRNA was first encapsulated into the LNP. The LNP was then conjugated with purified anti-human CD5 antibody (clone 5D7). The LNP was prepared using a microfluidic mixing system with the NanoAssemblr™ Ignite™ nanoparticle formulation. Briefly, an ethanol-lipid mixture consisting of ionizable cationic lipids, phosphatidylcholine, cholesterol, polyethylene glycol-lipids, and functionalized maleimide PEG-lipids in a ratio of 50:10:38:1.5:0.5 was prepared and then rapidly mixed with an aqueous solution containing circRNA at an acidic pH. The volume ratio of aqueous phase to ethanol was 3:1. The LNP containing functionalized maleimide PEG-lipids was dialyzed overnight at 4°C in PBS buffer containing 10 mM EDTA. The purified anti-human CD5 antibody (clone 5D7) was first reduced with 5-fold antibody equivalents of TCEP (Sigma-Aldrich) in 5 mM PBS at 37°C with gentle shaking for 1 hour to conjugate with maleimide. Excess TCEP was removed by passing the antibody solution through a Zeba 7 MWCO desalting column (Thermo Fisher). The concentration of the reduced antibody was quantified by absorbance at 280 nm using NanoDrop. The active thiol group on the antibody was then conjugated with functionalized LNPs with gentle shaking at room temperature for 1 hour. The fLNPs were purified and concentrated using an Amicon® Ultra-15 centrifuge filter (MilliporeSigma). The circRNA content was calculated using a RiboGreen RNA assay (Thermo Fisher). Finally, the fLNPs were stored at 4°C before injection.

[0086] Example 5: In vitro expression of circRNA encoding anti-CD19 CAR HEK293T cells were transfected with formulated circRNA-LNP (anti-hCD19 CAR: SEQ ID NO: 9, anti-mCD19 CAR: SEQ ID NO: 29). Twelve hours post-transfection, cell surface staining was performed using FITC-labeled anti-FMC63 monoclonal antibody (Beijing Baipusaisi Biotechnology Co., Ltd., FM3-FY45G0) and PE-labeled recombinant protein L (Beijing Baipusaisi Biotechnology Co., Ltd., RPL-PP2H2) for transient expression analysis. Results showed that approximately 90% of cells expressed CAR on the cell surface. Figure 2 (A-2C). circRNA-LNP was transfected into Jurkat cells or HEK293T cells containing a reduced dose of fetal bovine serum (FBS) (2.5%) to assess the persistence of anti-CD19 CAR. Anti-hCD19 CAR expression persisted for approximately 2 weeks in Jurkat cells and approximately 1 week in HEK293T cells. Anti-mouse CD19 CAR expression persisted for approximately 1 week in both Jurkat and HEK293T cells, and gradually decreased 9 days post-transfection. Figure 2 D-2E).

[0087] Example 6: Anti-hCD19 CAR circRNA therapy showed sustained anti-tumor effects in vivo. To test the function of anti-hCD19 CAR circRNA (SEQ ID NO: 9) in vivo, we constructed a humanized mouse model carrying Nalm6 tumor cells expressing luciferase (Nalm6-luc) to monitor the function of anti-hCD19 CAR circRNA in treating human B-cell leukemia. By using 1x10 7 Human peripheral blood mononuclear cells (PBMCs) were intravenously injected into 6-week-old female NCG mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., Nanjing, China) to generate hPBMC-NCG mice. This severely immunodeficient environment allowed for highly efficient transplantation of human PBMCs. Six days later, 5 × 10⁶ cells were injected intravenously. 5 A hPBMC-NCG-Nalm6-luc tumor model was constructed using Nalm6 cells, a constitutively luciferase-expressing human B-cell leukemia cell line. Figure 3 A). This technology utilizes luciferase and luciferin to detect bioluminescence in tissues and visualize tumor cell growth in living organisms. Ten days after PBMC injection, human immune cells (hCD45) were detected by flow cytometry analysis of blood cells. + The transplantation efficiency was high. A successful mouse model was defined as having more than 1 × 10⁻⁶ cells reconstituted in peripheral blood. 4 Individual T cells, and with 5x10 3 Up to 1.5x104 p / sec / cm 2 The intermediate luciferase photon signal of / sr.

[0088] hPBMC-NCG-Nalm6-luc mice were randomly assigned to a control group and a treatment group. circRNA-LNP (SEQ ID NO: 9) was administered intravenously at a dose of 20 μg every two days (Q2D). All mice received a total of 5 treatments. Tumor growth was monitored weekly using the IVIS® Spectrum in vivo imaging system. Figure 3 A). Mice treated with anti-hCD19 CAR circRNA (SEQ ID NO: 9) showed durable tumor control, with tumor cells almost completely eliminated, and this effect persisted for a long time after treatment cessation, while tumors in the control group showed continuous progression. Figure 3 (B and 3C).

[0089] To investigate the efficiency of CAR-T cell generation in vivo, CAR expression was analyzed in peripheral blood samples from hPBMC-M-NSG humanized mice. Mice were administered 20 μg of anti-hCD19 CAR circRNA, and peripheral blood was analyzed by flow cytometry at specified time points using FITC-labeled anti-FMC63 antibody (Beijing Baipusaisi Biotechnology Co., Ltd., FM3-FY45G0). Results showed that approximately 30% of T cells were stained as CAR-positive cells. A single dose of anti-hCD19 CAR circRNA remained effective 48 hours post-injection and persisted for approximately 4 days, demonstrating successful in vivo CAR-T cell generation. Figure 3 D-3F).

[0090] Example 7: Anti-hCD19 CAR circular RNA is effective at low doses and low frequencies. Based on the potent anti-tumor efficacy of anti-hCD19 CAR circRNA, hPBMC-NCG-Nalm6-luc mice were treated with low doses and low frequencies. Even at doses as low as 0.2 mg / kg with only two administrations, complete tumor control was still observed. Figure 4 This is something that mRNA CAR has never achieved before.

[0091] Example 8: Efficacy of fLNP formulation of hCD19 CAR circRNA against B-cell leukemia The antitumor efficacy of anti-hCD19 CAR circRNA was investigated in the hPBMC-M-NSG-Nalm6-Luc tumor model. Six- to eight-week-old M-NSG mice (Shanghai Southern Model Biotechnology Co., Ltd., Shanghai, China) were vaccinated with 1×10⁻⁶ mmol / L of anti-hCD19 CAR circRNA. 7Human PBMCs were administered intravenously 12 days after injection of human PBMCs, followed by the intravenous injection of Nalm6 cells expressing luciferase. Two days later, mice were randomly assigned to groups and treated with anti-hCD19 CAR circRNA targeting CD5-mediated fLNP. Mice were administered 1 mg / kg twice daily on days 0 and 3, and tumor burden was monitored twice weekly via IVIS. Figure 5 A). Compared with the control group, mice in the fLNP group treated with anti-hCD19 CAR circRNA showed significantly lower tumor luminescence signal (A). Figure 5 B-5C). Flow cytometry was used to analyze peripheral blood immune cell populations and CAR expression. On day 18 after treatment initiation, compared with the control group, the treatment group showed increased immune cell counts, CD3+ T cells, CD4+ T cells, and CD8+ T cells. Figure 5 D-5G indicates that T cells expanded after circRNA treatment. High levels of CAR expression were detected in human T cells in the fLNP treatment group, while very low levels of CAR expression were detected in mouse immune cells. Figure 5 H-5I).

[0092] Example 9: Anti-mouse CAR circRNA therapy delayed tumor progression in the A20 syngeneic model. The antitumor efficacy of anti-mCD19 CAR circRNA was evaluated in an A20 cell line-based orthotopic leukemia mouse model. 2×10 6 A20-luc leukemia cells were intravenously transplanted into female BALB / c mice. Nine days after A20-luc cell inoculation, mice were randomly assigned to groups and administered anti-mCD19 CAR circRNA on days 0, 3, and 6. Tumor burden was measured weekly by in vivo imaging. Figure 6 A). The results showed that anti-mCD19 CAR circRNA treatment inhibited the growth of A20 tumor cells in a dose-dependent manner. Figure 6 B), and there was no significant difference in weight change ( Figure 6 C).

[0093] Cytokine release syndrome is a major safety concern with CAR-T cell therapy. Based on the dose-dependent antitumor effect observed in the BALB / c-A20 syngeneic mouse model, cytokine release was assessed in this immunocompetent mouse model. Serum cytokines were measured on day 4 (12 hours after the last administration). BD was used. TMFlow cytometry microsphere array (CBA) mouse inflammation kit (BD Biosciences, 552364) was used to detect cytokines in the serum of mice treated with anti-mCD19 CAR circRNA. Following injection of anti-hCD19 CAR circRNA, IFN-γ, IL-10, TNF, MCP-1, and IL-6 all increased in a dose-dependent manner, but the increases were within a controllable range. Figure 6 D-6I).

[0094] Example 10: Application of anti-mCD19 CAR circRNA therapy in the treatment of SLE in a mouse disease model Prior to disease onset, LNP-encapsulated anti-mCD19 CAR-circRNA was administered to MRLfas / fas (MRL-lpr) mice to determine its role in SLE disease control. 8-10 week old female MRL-lpr mice were administered anti-mCD19 CAR-circRNA intravenously every three days during the first week at 1 mg / kg or 0.2 mg / kg, followed by three treatments. After this, all mice received circRNA treatment weekly. Figure 7 A). To evaluate the therapeutic efficacy of circRNA-CAR, we used enzyme-linked immunosorbent assay (ELISA) to detect the titers of anti-dsDNA autoantibodies and total IgG in the serum of mice in the circRNA treatment group and the control group on days 63 and 77, respectively. Figure 7 B-7C). Proteinuria and urinary creatinine were also measured on day 70 to monitor for kidney damage. Figure 7 These results indicate that the circRNA-encoded anti-CD19 CAR is effective in the prevention and treatment of SLE in mouse models, suggesting its potential for clinical application.

[0095] Example 11: CircRNA-CAR induces potent B-cell clearance in non-human primates (NHP) The effect of CAR on B cell clearance in vivo was further evaluated in non-human primates. Male rhesus monkeys ( Macaca mulatta The anti-CD20 / CD19 bispecific CAR (amino acid sequence SEQ ID NO: 31, nucleotide sequence SEQ ID NO: 32) was administered intravenously on days 0 and 14. The anti-CD20 / CD19 bispecific CAR is generated by linking the variable region (scFv) of a single-chain fragment of a CD20 antibody (clone Leu-16) and a CD19 antibody (clone FMC-63) to the CD8 hinge region and transmembrane domain, and then linking the intracellular 4-1BB (CD137) and CD3ζ signaling domains. Figure 8B). The percentage of lymphocytes before and after intravenous circRNA injection was determined by flow cytometry. Results showed that on day 1 after circRNA injection, peripheral blood B cells decreased by 84% and remained at a low level until at least day 4. Subsequently, B cell levels slowly increased, reaching 33% of pre-treatment B cell levels by day 12. Figure 8 C). The results showed that very rapid and sustainable B-cell clearance was achieved in NHP.

[0096] Since the methods, compounds and compositions described herein have been fully described, those skilled in the art will understand that the same operations can be performed under a wide range of equivalent conditions, formulations and other parameters without affecting the scope of the methods, compounds and compositions or any embodiments thereof provided herein.

[0097] All patents, patent applications and publications cited in this document are incorporated herein by reference in their entirety.

Claims

1. A circular RNA encoding a chimeric antigen receptor (CAR), in, The circular RNA comprises regulatory and expression elements, the expression elements comprising a polynucleotide encoding a chimeric antigen receptor (CAR) or a variant thereof; and The chimeric antigen receptor (CAR) comprises an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular T cell signal transduction domain. Each element is optionally linked by a polynucleotide encoding a linker.

2. The circular RNA according to claim 1, wherein, The polynucleotide encoding CAR or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, or 30.

3. The circular RNA according to claim 2, wherein, The amino acid sequence of the CAR encoded by the polynucleotide or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 29.

4. The circular RNA according to any one of the preceding claims, wherein, The antigen-binding domain is one or more antigen-binding domains specific to CD19, CD20, BCMA, CLDN6, CLDN18.2, ERBB2 (HER2), PSMA, SLAMF3, SLAMF7, CD66c, MSLN, CD38, CD123, GPC3, EGFRvIII, CD171, MUC1 and / or GPRC5D; preferably, the antigen-binding domain is a single-stranded variable fragment (scFv) specific to CD19, CD20 and / or BCMA; more preferably, the amino acid sequence of the scFv has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity with the sequence shown in SEQ ID NO: 25, 33, 35, 37, 39 or 41. The amino acid sequence of the scFv is encoded by a polynucleotide sequence, which has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO:26, 34, 36, 38, 40, or 42.

5. The circular RNA according to any one of the preceding claims, wherein, The co-stimulatory domain is selected from CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM and combinations thereof; preferably CD28 or 4-1BB; more preferably, the co-stimulatory domain of CD28 has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity with the sequence shown in SEQ ID NO:

23.

6. The circular RNA according to any one of the preceding claims, wherein, The transmembrane domains are selected from CD28 and CD8 transmembrane domains.

7. The circular RNA according to any one of the preceding claims, wherein, The intracellular T cell signal transduction domain is a CD3ζ signal transduction domain. Preferably, the CD3ζ signal transduction domain has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO:

21.

8. The circular RNA according to any one of the preceding claims, wherein, The regulatory element increases the translation yield of the circular RNA, and preferably, the regulatory element comprises a polynucleotide encoding an internal ribosome entry site (IRES) or a fragment thereof.

9. The circular RNA according to any one of the preceding claims, wherein, The IRES is selected from: Coxsackievirus B3 (CVB3) IRES, Enterovirus 71 (EV71) IRES, Encephalomyocarditis Virus (EMCV) IRES, Picornavir Virus (PV) IRES, Hepatitis C Virus (HCV) IRES, Adenovirus (AdV) IRES, Human Papillomavirus 31 (HPV31) IRES, Human Herpesvirus (HHV) IRES, Raul's Sarcoma Virus (RSV) IRES, Classical Swine Fever Virus (CSFV) IRES, FGF9 IRES, SLC7A1 IRES, and RUNX1 IRES; preferably, the IRES is a CVB3 IRES or a variant thereof, which has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO:

17.

10. The circular RNA according to any one of the preceding claims, wherein, The circular RNA further comprises a polynucleotide encoding a signal peptide selected from: a CD8a leader sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 18, and a colony-stimulating factor 2 receptor α subunit (CSF2RA) signal peptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO:

19.

11. The circular RNA according to any one of claims 1 and 4-10, comprising a regulatory element and an expression element, said expression element comprising a polynucleotide encoding a bispecific CAR specific to CD20 and CD19; in, The regulatory element comprises a polynucleotide encoding an internal ribosome entry site (IRES) or a fragment thereof as defined in claim 9; The bispecific CAR includes an antigen-binding domain, a co-stimulatory domain as defined in claim 5, a transmembrane domain as defined in claim 6, and an intracellular T cell signal transduction domain as defined in claim 7. The antigen-binding domain is an antigen-binding domain specific to CD19 and CD20; preferably, it includes a single-stranded variable fragment (scFv); more preferably, it is FMC-63 scFv and Leu-16 scFv. Wherein, the FMC-63 scFv is an amino acid sequence of an scFv having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence selected from SEQ ID NO: 25, 33, or 35; wherein, the Leu-16 scFv is an amino acid sequence of an scFv having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 41; Each element is optionally linked by a multinucleotide encoding a linker.

12. The circular RNA according to claim 11, wherein, The polynucleotide encoding the CAR or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 32; The amino acid sequence of the CAR encoded by the polynucleotide or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO:

31.

13. The circular RNA according to any one of the preceding claims, wherein, The linker is selected from or derived from: GA, GS, GG, GGA, GGS, GGG, GGGA, GGGS, GGGG, GGGGA, GGGGG, GGGGA, GGGGS, GGGGG, GGAG, GGSG, AGGGG, SGGG, GAGA, GSGS, GAGAGA, GSGSGS, GAGAGAGA, GSGSGSGS, GAGAGAGAGA, GSGSGSGSGS, G AGAGAGAGAGA, GAGSGSGSGSGS, GGAGGA, GGSGGS, GGAGGAGGA, GGSGGSGGS, GGAGGAGGAGGA, GGSGGSGGSGGS, GGAGGGAG, GGSGGGSG, GGAGGGAGGGAG, GGSGGGSGGSG, GGGAGGGAGGGA, GGGSGGGSGGGS or (GGGGS)3.

14. A composition comprising the circular RNA of any one of the preceding claims, wherein the composition comprises a pharmaceutically acceptable excipient.

15. The composition according to claim 14, further comprising nanoparticles; preferably lipid nanoparticles.

16. The composition according to claim 15, wherein, The lipid nanoparticles comprise at least one of ionizable lipids, structural lipids, and PEG-modified lipids; preferably, the ionizable lipid is SM102.

17. A method for administering immunotherapy to a subject, comprising administering to the subject a therapeutically effective amount of the circular RNA of any one of claims 1-13 or the composition of any one of claims 14-16; or Use of the circular RNA of any one of claims 1-13 or the composition of any one of claims 14-16 in the preparation of a medicament for immunotherapy in a subject; or The circular RNA of any one of claims 1-13 or the composition of any one of claims 14-16 is used for immunotherapy in a subject; Preferably, the immunotherapy is selected from: CAR-T cell therapy, CAR-NK cell therapy, CAR-macrophage therapy or CAR-Treg cell therapy.

18. The method, use, or circular RNA according to claim 17, wherein, The immunotherapy is selected from: treatment of cancer, treatment of hyperplastic diseases, treatment of fibrosis or autoimmune diseases; Preferably, the cancer comprises non-solid tumors or solid tumors; more preferably, the cancer is selected from: acute lymphoblastic carcinoma, acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, fluid-filled tumor, liver cancer, etc. Lung cancer, lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small bowel cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer and ureteral cancer; preferably, the brain cancer is medulloblastoma, the head and neck cancer is squamous cell carcinoma of the head and neck, and the lung cancer is non-small cell lung cancer or lung adenocarcinoma; Preferably, the autoimmune disease is selected from systemic lupus erythematosus (SLE), lupus nephritis, myasthenia gravis, pemphigus vulgaris, or type I diabetes. Preferably, the fibrosis is cardiac fibrosis.

19. A linear RNA polynucleotide comprising a regulatory element, an expression element, and at least one cyclization element, said regulatory element comprising a polynucleotide encoding an internal ribosome entry site (IRES) or a fragment thereof as defined in claim 9, said expression element encoding a chimeric antigen receptor (CAR) polypeptide as defined in any one of claims 1-7. in, Each element is optionally linked by a polynucleotide encoding a linker, wherein the linker is selected from or derived from: GA, GS, GG, GGA, GGS, GGG, GGGA, GGGS, GGGG, GGGGA, GGGGG, GGGGA, GGGGS, GGGGG, GGAG, GGSG, AGGGG, SGGG, GAGA, GSGS, GAGAGA, GSGSGSGS, GAGAGAGAGA, GSGSGSGSGS, GAGAGAGAGA, GSGSGSGSGS, GAGAGAGAGAGA, GAGSGSGSGSGS, GGAGGA, GGSGGS, GGAGGA, GGSGGAGGA, GGSGGSGSGGS, GGAGGAGGAGGA, GGSGGSGSGSGGS, GGAGGGAG, GGSGGGSG, GGAGGGAGGAGGAG, GGAGGGAGGAGGAG, GGSGGGSGGSGGS, GGGAGGGAGGAGGAG, GGGSGGGSGGSGGS or (GGGGS)3.

20. The linear RNA polynucleotide of claim 19, comprising, from 5' to 3', the following: Optionally, a 5'-self-looping element; A regulatory element comprising a polynucleotide encoding an internal ribosome entry site (IRES) or a fragment thereof as defined in claim 9; Optionally, the signal peptide element as defined in claim 10; An expression element comprising a polynucleotide encoding a chimeric antigen receptor (CAR) as defined in any one of claims 1-7; Optionally, terminate the codon; Optionally, a 3'-self-circulating element.

21. The linear RNA polynucleotide according to claim 19 or 20, wherein... The 5'-self-circularization element comprises a 5'-I type intron fragment and exon 2. Preferably, the 5'-I type intron replacing the intron-exon homologous arm has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO:

27. The 3'-self-cyclized element comprises exon 1 and a 3'-class I intron fragment; preferably, the nucleotide sequence of the 3'-class I intron replacing the intron-exon homologous arm has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 28; More preferably, the 3' I-type intron fragment and the 5' I-type intron fragment are Anabaena I-type intron fragments.

22. The linear RNA polynucleotide according to any one of claims 19-21, wherein, The self-cyclization element is cyclized by T4 RNA ligase 2, and the 5' end of the linear RNA polynucleotide is phosphorylated, preferably monophosphate, and the 3' end of the linear RNA polynucleotide is OH.

23. A DNA vector suitable for synthesizing linear RNA polynucleotides as described in any one of claims 19-22.

24. A population of eukaryotic cells comprising the circular RNA polynucleotide of any one of claims 1-13, wherein the population of eukaryotic cells expresses a CAR complex protein encoded by the circular RNA polynucleotide on its cell surface.

25. A method for preparing circular RNA according to any one of claims 1-13, comprising circularization using a substitution intron-exon (PIE) system with class I introns or T4 RNA ligase 2.

26. The method of claim 25, wherein, The circular RNA is formed by cyclizing the linear RNA polynucleotide as described in claim 21 or 22.

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