Preparation method of CAR-T cell and product thereof

Abstract

The invention relates to a preparation method of CAR-T cells and a product thereof. A method for preparing a CAR-T cell according to the present invention comprises the step of introducing a cyclic mRNA into a T cell that does not contain a chimeric antigen receptor; and, the cyclic mRNA comprises a first cyclic mRNA encoding a chimeric antigen receptor; in addition, the first annular mRNA comprises the following sequence (a) or (b): (a) a translation starting sequence and a coding sequence; and (b) translating the starting sequence, the coding sequence and the insertion element sequence. In the method, the transfection efficiency of the annular mRNA is high, and the positive rate is as high as 90%; compared with preparation methods of viruses, transposon systems and the like, the process is simple, the production cost is greatly reduced, and the safety is also improved; compared with linear mRNA, the protein expression time is greatly prolonged.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering transformation, and specifically relates to a preparation method of CAR-T cells and products thereof. Background Art

[0002] In recent years, immunotherapy has emerged as an important treatment method and is widely used in various disease treatment scenarios including tumors. A series of new immunotherapy methods have gradually entered the clinical stage. Among them, cell therapy represented by chimeric antigen receptor T cell therapy (CAR-T cell therapy) has shined in the treatment of blood tumors and has attracted much attention. Currently, many CAR-T cell therapies have been approved by the FDA for marketing, such as Kymriah, Yescarta, Abecma and Carvykti. As of April 2022, there are 2,756 cell therapy drugs in the global tumor immunotherapy R&D pipeline, of which CAR-T pipelines are as high as 1,432 (Saez- et al., 2022).

[0003] However, the application of cell therapy still has many limitations and faces many challenges. Taking CAR-T cells as an example, the technology currently still faces problems such as antigen escape, off-target effects, poor tumor infiltration ability, tumor immunosuppressive microenvironment and large toxic side effects (Zhang et al., 2020). Application strategies that can be adopted to address the above limitations include: (1) targeting multiple antigens to reduce the possibility of antigen escape; (2) targeting tumor-specific targets, including protein post-transcriptional modifications; (3) local administration instead of systemic administration; (4) modifying cells to express chemokine receptors or improve their ability to penetrate physical barriers; (5) modifying cells to enable them to provide immune activation signals or to enable them to resist immunosuppressive signals; (6) loading molecular switches to regulate cells (Sterner & Sterner, 2021).

[0004] Whether it is to introduce CAR molecules into T cells or to modify their characteristics, it is necessary to rely on gene vector delivery technology. At present, the most mature vector for industrial application is the lentiviral vector system / retrovirus. Lentivirus can integrate the target fragment into the genome of the infected cell, thus achieving high efficiency and long-term expression. By optimizing the plasmid vector packaged by the lentivirus, the virus particles can retain only the infection characteristics without the ability to replicate, which greatly ensures the safety of lentiviral application. However, the application of the lentiviral vector system is limited by two aspects. One is its high preparation and detection costs, and the other is that its random integration process introduces safety issues. Preclinical and clinical studies are also actively exploring non-viral vector gene delivery systems, such as transposon systems and messenger RNA (mRNA). The transposon system only requires a plasmid containing the target gene and a plasmid / mRNA / protein expressing the transposase to achieve long-term gene transduction, which greatly reduces the preparation and detection costs compared to the viral system. From the "Sleeping Beauty" transposon system, the piggyBac transposon system to the Tol2 transposon system, the vector capacity and transduction efficiency are constantly being upgraded and optimized. At present, gene delivery mediated by the transposon system is also limited in two aspects. First, it relies on electroporation for plasmid delivery, resulting in low transduction efficiency. Second, its integration process is also random integration, which introduces safety risks (Morgan & Boyerinas, 2016). As a type of gene delivery method of non-viral vectors, mRNA has advantages that other technical methods do not have: (1) it can be used to transfect quiescent or low-proliferation cells through electroporation or liposome transfection; (2) it does not integrate into the genome, so the probability of mutation caused by insertion is very low; (3) it can deliver multiple mRNAs encoding different proteins at the same time, making it easier to achieve combined application of different targets. The application limitation of mRNA is that the protein expression mediated by it is transient, and the protein expression will gradually decrease and disappear, but this is more beneficial for application scenarios that only require transient protein expression. In 2011, Barrett et al. used classic linear mRNA to encode CAR molecules and prepared CAR-T cells for the treatment of advanced leukemia. Their in vitro data showed that the efficiency of linear mRNA electroporation of T cells was very high (>90%), but its high positive rate (>80%) was only maintained for about 3 days, and the longest time that the positive cell population could be detected was 10 days (Barrett et al., 2011).

[0005] According to the records of the applicant's prior patent applications CN114438127A and CN114574483A, compared with classic linear mRNA, the preparation process of circular mRNA does not require capping and tailing reactions, the process is simpler and the cost is lower; circular mRNA can reduce immunogenicity through sequence optimization and process optimization, without nucleotide modification, further reducing costs; more importantly, circular mRNA can mediate higher and more sustained protein expression. At present, there are literature reports that circular mRNA has been successfully used in intratumoral injection tumor treatment (Yang et al., 2022), infectious disease vaccines (Qu et al., 2022) and tumor vaccines (Li et al., 2022). However, whether the advantages of low cost, long expression time, low immunogenicity and high safety of circular mRNA based on Clean-PIE recorded in the prior patent application can be successfully applied to cell therapy, especially cell transformation, etc., has not yet been disclosed.

[0006] References:

[0007] Saez- AR, Upadhaya, S., Partridge, T., Shah, M., Correa, D., & Campbell, J. (2022). Landscape of cancer cell therapies: trends and real-world data. NatRev Drug Discov, 21(9), 631-632.

[0008] Zhang, Q., Ping, J., Huang, Z., Zhang,

[0009] Sterner, RC, & Sterner, RM (2021). CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J, 11(4), 69.

[0010] Morgan, RA, & Boyerinas, B. (2016). Genetic Modification of TCells. Biomedicines, 4(2).

[0011] Barrett, D.M., Zhao, Y., Liu, X., Jiang, S., Carpenito, C., Kalos, M.,... Grupp, S.A. (2011). Treatment of advanced leukemia in mice with mRNA engineered T cells. Hum Gene Ther, 22(12), 1575 - 1586.

[0012] Yang, J., Zhu, J., Sun, J., Chen, Y., Du, Y., Tan, Y.,... Zuo, C. (2022). Intratumoral delivered novel circular mRNA encoding cytokines for immune modulation and cancer therapy. Mol Ther Nucleic Acids, 30, 184 - 197.

[0013] Qu, L., Yi, Z., Shen, Y., Lin, L., Chen, F., Xu, Y.,... Wei, W. (2022). Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell, 185(10), 1728 - 1744.e1716.

[0014] Li, H., Peng, K., Yang, K., Ma, W., Qi, S., Yu, X.,... Yu, G. (2022). Circular RNA cancer vaccines drive immunity in hard-to-treat malignancies. Theranostics, 12(14), 6422 - 6436. Summary of the Invention

[0015] Problem that the invention aims to solve

[0016] The research on T cell engineering, especially CAR-T cell preparation in the above-mentioned prior art is still not sufficient. Then, the present invention studies the protein expression mediated by the circular mRNA prepared based on the Clean-PIE system in T cells, and finds that the circular mRNA based on Clean-PIE can be used for the engineering transformation of T cells. On this basis, the present invention provides a preparation method and a transformation method of CAR-T cells, which can not only prepare CAR-T cells using circular mRNA based on Clean-PIE; CAR-T cells that have been prepared by non-mRNA pathways (such as lentiviral pathways) can also be encoded by circular mRNA based on Clean-PIE. Proteins (such as cytokines) improve the characteristics of CAR-T cells; At the same time, CAR-T cells can be prepared and transformed simultaneously by a variety of circular mRNAs based on Clean-PIE. In addition, the present invention also finds that CAR-T cells can be prepared by circular mRNA based on Clean-PIE for the delivery of circular mRNA drugs or non-mRNA drugs.

[0017] Solutions for solving problems

[0018] [1] A method for preparing a CAR-T cell, comprising the step of introducing a circular mRNA into a T cell that does not contain a chimeric antigen receptor;

[0019] Furthermore, the circular mRNA includes a first circular mRNA encoding a chimeric antigen receptor;

[0020] Furthermore, the first circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0021] (a) translation initiation sequence and coding sequence;

[0022] (b) Translation initiation sequence, coding sequence and insert element sequence.

[0023] [2] The preparation method according to [1], wherein the chimeric antigen receptor targets the CD19 molecule.

[0024] [3] The preparation method according to [1] or [2], wherein the nucleotide sequence of the first circular mRNA comprises the sequence shown in SEQ ID NO.3, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.3, and encodes a sequence of a protein having or partially having the chimeric antigen receptor activity.

[0025] [4] A method for modifying CAR-T cells, wherein the method comprises the steps shown in (i) or (ii):

[0026] (i) a step of introducing circular mRNA into T cells that do not contain a chimeric antigen receptor;

[0027] (ii) a step of introducing circular mRNA into CAR-T cells;

[0028] In the step (i), the circular mRNA includes a first circular mRNA encoding a chimeric antigen receptor and a second circular mRNA encoding a functional polypeptide;

[0029] In the step (ii), the circular mRNA includes a second circular mRNA encoding a functional polypeptide;

[0030] Furthermore, the first circular mRNA and the second circular mRNA comprise sequences arranged in the order shown in (a) or (b):

[0031] (a) translation initiation sequence and coding sequence;

[0032] (b) Translation initiation sequence, coding sequence and insert element sequence.

[0033] [5] The modification method according to [4], wherein the chimeric antigen receptor targets the CD19 molecule; and the functional polypeptide includes any one or more of the group consisting of IL-2, IL-12sc, CXCR2 and Flt3L.

[0034] [6] The modification method according to [4] or [5], wherein the nucleotide sequence of the first circular mRNA comprises the sequence shown in SEQ ID NO.3, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.3, and encodes a sequence of a protein having or partially having the chimeric antigen receptor activity;

[0035] The nucleotide sequence of the second circular mRNA comprises a sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6, and encoding a protein having or partially having the activity of the functional polypeptide.

[0036] [7] A CAR-T cell, wherein the CAR-T cell is obtained by the preparation method according to any one of [1] to [3] or the modification method according to any one of claims 4 to 6.

[0037] [8] A method for delivering a drug to a target cell, wherein the method comprises: contacting the CAR-T cell according to [7] containing the drug with the target cell;

[0038] Preferably, the drug includes any one or more of a circular mRNA drug, a linear RNA drug, a DNA drug and a small molecule drug.

[0039] [9] The method according to [8], wherein the target cells are tumor cells.

[0040]

[10] The method according to [8] or [9], wherein the method comprises administering the CAR-T cells according to [7] containing the drug to a subject.

[0041] Effects of the Invention

[0042] Through the implementation of the above technical solution, the present invention achieves the following technical effects:

[0043] The present invention finds that circular mRNA based on Clean-PIE can mediate efficient and persistent protein expression on human T cells. On this basis, the present invention provides a method for preparing and modifying CAR-T cells. First, CAR-T cells can be prepared using circular mRNA based on Clean-PIE. Secondly, not only can CAR-T cells prepared by other methods be modified by circular mRNA based on Clean-PIE, but also CAR-T cells can be prepared and modified simultaneously by circular mRNA based on Clean-PIE.

[0044] In the preparation and transformation method of CAR-T cells provided by the present invention, the circular mRNA transfection efficiency is high, and the positive rate is as high as 90%; compared with the preparation methods such as virus and transposon systems, the process is simple, the production cost is greatly reduced, and the safety is also improved; compared with linear mRNA, the protein expression time is greatly extended.

[0045] The present invention verifies that the modification of CAR-T cells by circular mRNA based on Clean-PIE can further optimize the function of engineered T cells and obtain enhanced CAR-T cells. For example, the combination with T cell activation-related cytokines can enhance the killing ability of CAR-T cells on target cells, and the combination with chemokines can enhance the migration ability of CAR-T cells and enhance the infiltration ability of solid tumors.

[0046] At the same time, the present invention proposes the concept of transforming T cells or CAR-T cells through circular mRNA for drug delivery. The delivered drugs include circular mRNA drugs (such as circular mRNA encoding factors that regulate tumor microenvironment) and non-mRNA drugs (such as small molecule drugs). CAR-T cells have the characteristics of tumor cell-specific targeting, so that they can play the role of tumor-targeted drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1A This is a graph showing the percentage of GFP-positive cells detected after T cells were electroporated with circular mRNA encoding GFP in Example 2.

[0048] Figure 1B This is a detection diagram of the average fluorescence brightness of GFP-positive cells after T cells were electroporated with circular mRNA encoding GFP in Example 2.

[0049] Figure 1C This is a detection diagram of the total fluorescence intensity of GFP on the surface of positive cells after the circular mRNA encoding GFP was electroporated into T cells in Example 2.

[0050] Figure 2 This is a graph showing the killing ability of CAR-T cells after modification with different circular mRNAs in Example 3.

[0051] Figure 3A This is a detection diagram of cells killing tumor target cells under different treatment conditions in Example 4.

[0052] Figure 3B This is a graph showing the detection of the intracellular factor IFN-γ during the process of cells under different treatment conditions killing tumor target cells in Example 4.

[0053] Figure 3C This is a graph showing the detection of the intracellular factor TNF-α during the process of cells killing tumor target cells under different treatment conditions in Example 4.

[0054] Figure 4 This is a graph showing the migration ability of cells under different treatment conditions in Example 5.

[0055] Figure 5A This is a graph showing the weight changes of mice in each experimental group in Example 6.

[0056] Figure 5B This is a graph showing the weight change rate of mice in each experimental group in Example 6.

[0057] Fig. 6A This is a statistical diagram of the survival of animals in each experimental group during the test period in Example 6.

[0058] Figure 6B This is a statistical chart of the in vivo imaging results of animals in each experimental group in Example 6.

[0059] Figure 6C These are the results of in vivo imaging of animals in each experimental group in Example 6.

[0060] Figure 7 This is a graph showing the detection of cytokines released into the tumor of mice in each experimental group in Example 7. DETAILED DESCRIPTION

[0061] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0062] definition

[0063] In the present invention, a numerical range expressed using “a numerical value A to a numerical value B” or “a numerical value A-a numerical value B” means a range including the endpoints A and B.

[0064] In the present invention, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0065] In the present invention, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes the situation where the event occurs and the situation where the event does not occur.

[0066] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0067] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0068] In the present invention, the term "about" may mean that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters used to define the present invention are all approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains the standard deviation caused by the aforementioned test device or method. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about".

[0069] In the present invention, the terms "polypeptide", "protein" interchangeably refer to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), which may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. A polypeptide may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0070] In the present invention, the term "amino acid" may include natural amino acids, unnatural amino acids, amino acid analogs, and all their D and L stereoisomers.

[0071] In the present invention, "individual", "object", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (such as cows, sheep, cats, dogs or horses, etc.), primates (such as humans, non-human primates such as monkeys or gorillas, etc.), rabbits, and rodents (such as mice, rats or guinea pigs, etc.).

[0072] In the present invention, the term "circular RNA" refers to a nucleic acid molecule in a closed circular shape. In some embodiments, the circular RNA is a circular mRNA.

[0073] In the present invention, the term "linear RNA" refers to an RNA precursor that can circularize to form a circular RNA, which is generally transcribed from a linear DNA molecule.

[0074] In the present invention, the term "linear RNA" refers to RNA with translation function including a 5' cap structure (5' Cap), a 3' polyadenosine tail (PolyA tail), a 5' untranslational region (5' UTR), a 3' untranslational region (3' UTR) and an open reading frame (Open reading frame, ORF) and other structures.

[0075] In the present invention, "treatment" means: after suffering from a disease, the subject is exposed to (e.g., administered) the CAR-T cells of the present invention, thereby alleviating the symptoms of the disease compared to when the subject is not exposed, and does not necessarily mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means: the body has symptoms of the disease.

[0076] In the present invention, "prevention" means that before a subject develops a disease, by exposing the subject to (e.g., administering) the CAR-T cells of the present invention, the symptoms after the disease is alleviated compared to when the subject is not exposed to the CAR-T cells, etc., and does not necessarily mean that the disease must be completely suppressed.

[0077] In the present invention, the terms "sequence identity" and "percentage of identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). The sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0078] In the present invention, "chimeric antigen receptor (CAR)" is a genetically engineered receptor. These engineered receptors can be inserted into immune cells (including T cells) and expressed by the immune cells through circular mRNA as described in the present invention. For CAR, a single receptor can be programmed to recognize a specific antigen and activate immune cells to attack and destroy cells carrying the antigen when bound to the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells. In some embodiments, the chimeric antigen receptor of the present invention comprises: an antigen binding domain, a transmembrane domain, and an intracellular domain.

[0079] In the present invention, "T cell activation-related cytokines" refer to various cytokines that can promote T cells to enter or maintain an activated state, for example, cytokines such as IL-2 that can promote T cell growth, and cytokines such as IL-12sc that can induce T cell proliferation.

[0080] In the present invention, "T cell chemokine" refers to a cytokine that can promote T cell migration, such as CXCR2.

[0081] In the present invention, "CD19 molecule" is a transmembrane protein on the surface of B cells, which is closely related to B cell activation, signal transduction and growth regulation. It is a functional receptor molecule on the surface of B lymphocytes. When the B cell antigen receptor recognizes the antigen, it constitutes a B cell dual antigen binding model and participates in the Ca 2+ binding in B cells. 2+ CD19 is a highly expressed protein in malignant tumors of B cell lineage origin, and it is not expressed in cells outside the B cell lineage, thus reducing off-target toxicity to a certain extent, making CD19 an ideal tumor-associated antigen target.

[0082] In the present invention, "functional polypeptide" refers to a polypeptide with certain biological activity functions, such as a polypeptide having the function of promoting T cell proliferation or chemotaxis, or having anti-tumor function, etc. In some embodiments, the functional polypeptide of the present invention includes any one or more of IL-2, IL-12sc, CXCR2 and Flt3L.

[0083] IL-2, also known as T cell growth factor (TCRF), is an important factor in immune response, antibody response, hematopoiesis and tumor surveillance. IL-2 can enable T cells cultured in vitro to survive for a long time and can enhance the activity of antigen-induced Tc in vivo. Tc induced by IL-2 has a strong anti-tumor effect. At the same time, IL-2 can induce T cells to secrete cytokines such as IFN-γ, TNF, and CSF.

[0084] IL-12 is a type of proinflammatory cytokine produced by antigen-presenting cells. It is composed of two subunits, p35 and p40. The IL-12sc molecule is a heterodimer p70 connected by a flexible linker. Its main functions include promoting the differentiation of Th1 cells, enhancing the killing ability of T cells and NK cells, and inhibiting immunosuppressive cells such as tumor-associated macrophages.

[0085] CXCR2 is CXC receptor 2, which belongs to the G protein-coupled receptor family. Its ligands are various CXC chemokines such as CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, etc. CXCR2 can recruit neutrophils to migrate to the inflammatory area. At the same time, it is involved in the occurrence, development and treatment of various tumors.

[0086] Flt3L is the Flt3 ligand, which is secreted by bone marrow stromal stem cells and other cells. It can cooperate with other growth factors to promote the proliferation, differentiation and maturation of pre-B lymphocytes, dendritic cells, natural killer cells and cytotoxic T lymphocytes, thus playing an important anti-tumor effect.

[0087] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0088] Circular mRNA

[0089] The circular mRNA described in the present invention is prepared by the Clean-PIE system. For the specific content of the Clean-PIE system, reference may be made to patent applications CN202210200112.6 and CN202210200186.X. The entire contents of the above patents are incorporated into this application by reference.

[0090] In some embodiments, the circular mRNA described in the present invention comprises a sequence arranged in the order shown in (a) or (b):

[0091] (a) translation initiation sequence and coding sequence;

[0092] (b) Translation initiation sequence, coding sequence and insert element sequence.

[0093] In some specific embodiments, the circular mRNA of the present invention consists of sequences arranged in the order shown in (a) or (b):

[0094] (a) translation initiation sequence and coding sequence;

[0095] (b) Translation initiation sequence, coding sequence and insert element sequence.

[0096] In some embodiments, the translation initiation sequence of the present invention can be any type of sequence that can initiate the translation of the coding sequence in the circular mRNA. In some embodiments, the translation initiation sequence includes one or more of the following sequences with translation initiation activity: IRES sequence, 5'UTR sequence, Kozak sequence, m 6A modified (N(6) methyladenosine modified) sequence, a complementary sequence of ribosomal 18S rRNA. In other embodiments, the translation initiation sequence can also be any other type of cap-independent translation initiation sequence.

[0097] In some embodiments, the translation initiation sequence is an IRES sequence, and the source of the IRES sequence includes but is not limited to viruses, mammals, fruit flies, etc. In some optional embodiments, the IRES sequence is derived from a virus. Exemplary, the IRES sequence comprises an IRES sequence from a small RNA virus. Further, the IRES sequence includes but is not limited to an IRES sequence derived from Echovirus, Human poliovirus, Human Enterovirus, Coxsackievirus, Human rhinovirus, Caninepicornavirus, Turdivirus 3, Hepatovirus, Passerivirus, Picornaviridae, TremovirusA, Feline kobuvirus, Murine kobuvirus, Kobuvirus sewage Kathmandu, Ferretkobuvirus, Marmot kobuvirus, Human parechovirus, Chicken picornavirus, Falconpicornavirus, Feline picornavirus, French Guiana picornavirus, etc.

[0098] The translation initiation sequence in the present application includes the sequences with initiation circular mRNA translation activity shown in patent applications CN202011408937.4, CN202111435528.8, CN202210200112.6, and CN202210200186.X. The entire contents of the above patents are incorporated into the present application by reference.

[0099] In some embodiments, the coding sequence of the present invention comprises a coding region encoding at least one target protein. In some specific embodiments, the coding sequence comprises only one target protein coding region. In other embodiments, the coding sequence comprises two or more target protein coding regions, each coding region independently encoding any type of target protein; each coding region can be directly connected or linked by a linker, and the linker can be a polynucleotide encoding a 2A peptide, or other types of polynucleotides for encoding an interval linker peptide.

[0100] In some embodiments, the insertion element sequence of the present invention is selected from at least one of the following elements: transcriptional level regulatory element, translational level regulatory element, purification element. Exemplarily, the insertion element comprises one or a combination of any two or more of the following sequences: untranslated region (UTR) sequence, polyN sequence, aptamer sequence, riboswitch sequence, sequence binding transcriptional regulatory factor; in the polyN sequence, N is selected from at least one of A, T, G, and C.

[0101] First circular mRNA

[0102] The first circular mRNA of the present invention encodes a chimeric antigen receptor.

[0103] In some specific embodiments, the first circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0104] (a) a translation initiation sequence and a coding sequence encoding a chimeric antigen receptor;

[0105] (b) Translation initiation sequence, coding sequence encoding chimeric antigen receptor and insertion element sequence.

[0106] In some preferred embodiments, the first circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0107] (a) a translation initiation sequence and a coding sequence encoding a chimeric antigen receptor;

[0108] (b) Translation initiation sequence, coding sequence encoding chimeric antigen receptor and insertion element sequence.

[0109] In some specific embodiments, the chimeric antigen receptor described in the present invention targets the CD19 molecule.

[0110] In some more preferred embodiments, the nucleotide sequence of the first circular mRNA comprises a sequence as shown in SEQ ID NO.3, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.3, and encodes a sequence of a protein having or partially having the activity of the chimeric antigen receptor.

[0111] Second circular mRNA

[0112] The second circular mRNA of the present invention encodes a functional polypeptide. In some embodiments, the functional polypeptide includes any one or more of the group consisting of IL-2, IL-12sc, CXCR2 and Flt3L.

[0113] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0114] (a) a translation initiation sequence and a coding sequence encoding IL-2;

[0115] (b) Translation initiation sequence, coding sequence encoding IL-2 and insertion element sequence.

[0116] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0117] (a) a translation initiation sequence and a coding sequence encoding IL-2;

[0118] (b) Translation initiation sequence, coding sequence encoding IL-2 and insertion element sequence.

[0119] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO.2, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.2, and encoding a protein having or partially having the IL-2 activity.

[0120] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0121] (a) translation initiation sequence and coding sequence encoding IL-12sc;

[0122] (b) Translation initiation sequence, coding sequence encoding IL-12sc and insertion element sequence.

[0123] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0124] (a) translation initiation sequence and coding sequence encoding IL-12sc;

[0125] (b) Translation initiation sequence, coding sequence encoding IL-12sc and insertion element sequence.

[0126] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO.5, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.5 and encoding a protein having or partially having the IL-12sc activity.

[0127] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0128] (a) a translation initiation sequence and a coding sequence encoding CXCR2;

[0129] (b) Translation initiation sequence, coding sequence encoding CXCR2 and insertion element sequence.

[0130] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0131] (a) a translation initiation sequence and a coding sequence encoding CXCR2;

[0132] (b) Translation initiation sequence, coding sequence encoding CXCR2 and insertion element sequence.

[0133] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO.4, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.4, and encoding a protein having or partially having the CXCR2 activity.

[0134] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0135] (a) Translation initiation sequence and coding sequence encoding Flt3L;

[0136] (b) Translation initiation sequence, coding sequence encoding Flt3L and insertion element sequence.

[0137] In other preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0138] (a) Translation initiation sequence and coding sequence encoding Flt3L;

[0139] (b) Translation initiation sequence, coding sequence encoding Flt3L and insertion element sequence.

[0140] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO.6, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.6 and encoding a protein having or partially having the Flt3L activity.

[0141] CAR-T cell preparation method and product

[0142] The present invention provides a method for preparing a CAR-T cell, the method comprising the step of introducing a circular mRNA into a T cell that does not contain a chimeric antigen receptor; the circular mRNA comprises a first circular mRNA encoding a chimeric antigen receptor.

[0143] The "T cells without chimeric antigen receptors" described in the present invention include wild-type T cells or modified T cells; the wild-type T cells are T cells in a natural state, for example, they can be T cells directly isolated from the target object; the modified T cells are T cells in a non-natural state, for example, they can be T cells that have been genetically modified after being isolated from the target object.

[0144] The "introduction" described in the present invention includes a variety of ways, illustratively, electroporation, liposome-mediated transfection or LNP-mediated transfection, etc. In some preferred embodiments, the method of the present invention includes the step of electroporation of circular mRNA into T cells that do not contain chimeric antigen receptors.

[0145] The CAR-T cell preparation method provided by the present invention uses circular mRNA prepared based on the Clean-PIE system for the first time, with high transfection efficiency and a positive rate of up to 90%. Compared with preparation methods such as virus and transposon systems, the process is simple, the production cost is greatly reduced, and the safety is also improved. Compared with linear mRNA, the protein expression time is greatly extended.

[0146] The present invention also provides CAR-T cells prepared by the above preparation method, which can effectively kill target cells and, after being reinfused into experimental animals, can effectively treat tumors and improve the survival rate of tumor model experimental animals.

[0147] CAR-T cell modification method and its products

[0148] The present invention provides a method for modifying CAR-T cells. By modifying CAR-T cells, enhanced CAR-T cells can be obtained, for example, CAR-T cells with stronger killing ability and / or stronger migration ability, or CAR-T cells containing circular mRNA drugs can be obtained.

[0149] In some embodiments, the method for modifying CAR-T cells of the present invention comprises (i) the step of introducing circular mRNA into T cells without chimeric antigen receptors, wherein the circular mRNA comprises a first circular mRNA encoding a chimeric antigen receptor and a second circular mRNA encoding a functional polypeptide. In this method, the preparation and modification of CAR-T cells are achieved simultaneously.

[0150] In some embodiments, the "T cells without chimeric antigen receptors" include wild-type T cells or modified T cells; the wild-type T cells are T cells in a natural state, for example, they can be T cells directly isolated from the target object; the modified T cells are T cells in a non-natural state, for example, they can be T cells that have been genetically modified after being isolated from the target object.

[0151] In some embodiments, the "introduction" includes a variety of methods, illustratively, electroporation, liposome-mediated transfection or LNP-mediated transfection, etc. are used; preferably, electroporation is used.

[0152] In other embodiments, the CAR-T cell modification method of the present invention includes (ii) the step of introducing a circular mRNA into the CAR-T cell, wherein the circular mRNA includes a second circular mRNA encoding a functional polypeptide.

[0153] In this method, the CAR-T cells can be CAR-T cells prepared by various methods, and exemplary methods include lentiviral / retroviral systems, transposon systems, linear mRNA transfection, or PIE-based circular mRNA transfection. In some embodiments, the CAR-T cells do not contain circular mRNA prepared based on the Clean-PIE system.

[0154] By delivering different cytokines to CAR-T cells, the function of engineered T cells can be further optimized to obtain enhanced CAR-T cells. For example, the combination of T cell activation-related cytokines can enhance the killing ability of CAR-T cells on target cells, and the combination of chemokines can enhance the migration ability of CAR-T cells and enhance the infiltration ability of solid tumors.

[0155] The present invention also provides CAR-T cells obtained by the above-mentioned transformation method.

[0156] Methods for delivering drugs to target cells

[0157] The present invention provides a method for delivering a drug to a target cell, comprising: contacting a drug-containing CAR-T cell with the target cell.

[0158] In some embodiments, the CAR-T cells are obtained by the above-mentioned preparation method or modification method.

[0159] In some embodiments, the target cell is a tumor cell. The present invention has found that the tumor cell-specific targeting property of CAR-T cells can be utilized to enable them to perform the function of tumor-targeted drug delivery.

[0160] The present invention does not specifically limit the drugs contained in specific CAR-T cells. Exemplarily, they may include gene drugs and non-gene drugs. The gene drugs typically include circular mRNA drugs, linear RNA drugs, DNA drugs, etc., and the non-gene drugs typically include small molecule compound drugs.

[0161] The present invention does not specifically limit how the drug is introduced into CAR-T cells. Exemplarily, for gene drugs such as circular mRNA drugs, CAR-T cells can be introduced by electrotransfection, liposome-mediated transfection, or LNP-mediated transfection. In some specific embodiments, the gene drug can be transferred into T cells at the same time as the CAR-T cells are prepared using circular mRNA based on the Clean-PIE system, and the T cells are transformed. In other specific embodiments, the gene drug can be transferred into the CAR-T cells at the same time as the circular mRNA based on the Clean-PIE system is used to transform the CAR-T cells, so that the CAR-T cells contain drugs while the characteristics are transformed. Exemplarily, for small molecule drug compounds, small molecule compound drugs can be delivered into cells by methods such as electrotransfection and microfluidics.

[0162] To facilitate specific operations, in some specific embodiments, the method of delivering a drug to a target cell includes: administering the drug-containing CAR-T cells to a subject.

[0163] The present invention does not specifically limit the specific subject. For example, the subject may be a tumor cell population, a cell population containing tumor cells, or a subject carrying tumor cells.

[0164] In some embodiments, the method of delivering a drug to a target cell is not for the purpose of diagnosing or treating a disease.

[0165] The nucleotide sequence and amino acid sequence of the present invention

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] Example

[0178] The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out under normal conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are all commercially available conventional products.

[0179] Example 1. Preparation of circular mRNA based on Clean-PIE

[0180] This example provides a method for preparing circular mRNA capable of expressing 19-CAR-4-1BB by Clean-PIE.

[0181] 1.1 Experimental methods

[0182] The plasmid (pUC57-NA19-CAR-4-1BB, whose nucleotide sequence is shown in SEQ ID NO.11) was synthesized and cloned by Suzhou Jinweizhi Biotechnology Co., Ltd. After obtaining the plasmid, plasmid amplification and enzyme linearization were performed. The enzyme cutting system is shown in the following table.

[0183] Table 1 Enzyme digestion system

[0184] Reagents volume Plasmids 10 μg Restriction endonuclease BsaI (1000 units) 5μL 10×cutsmart buffer 50μL Nuclease free water Total 500 μL

[0185] The enzyme digestion was carried out at 37°C overnight. The enzyme digestion products were recovered using a universal DNA gel recovery kit (Tiangen Biochemical Technology Co., Ltd.), the OD value was determined, and the enzyme digestion products were identified using 1% agarose gel electrophoresis. The purified linear plasmid template was used for in vitro transcription. The in vitro transcription system is shown in the following table.

[0186] Table 2 In vitro transcription system

[0187] Reagents volume 10× Reaction Buffer 2μL ATP(20mM) 2μL CTP (20 mM) 2μL UTP(20mM) 2μL GTP(20mM) 2μL Linearized DNA template 1 μg

[0188]

[0189] Incubate at 37°C for 2.5h, then digest the linear DNA template with DNase I, purify using silica membrane centrifugal column method (Thermo, GeneJET RNA Purification Kit), measure OD value and identify RNA size by 1% denaturing agarose gel electrophoresis. The purified linear mRNA is used for cyclization, and the cyclization system is shown in the following table.

[0190] Table 3 Cyclization system

[0191] Solution volume mRNA 25 μg mRNA GTP solution (20 mM) 50μL GTP buffer Make up to 500 μL

[0192] The solution in the above system was heated at 55°C for 15 min and then placed on ice. The circularized RNA product was purified using a silica membrane centrifugal column method (Thermo, GeneJET RNA Purification Kit), and the OD value was measured and the RNA size was identified by 1% denaturing agarose gel electrophoresis.

[0193] 1.2 Experimental Results

[0194] Table 4 Experimental results

[0195] concentration volume Plasmids 351 ng / μL 100μL Linearize plasmid template 149.9 ng / μL 50μL Transcription product 3890ng / μL 70μL Linear mRNA purification 1875.1ng / μL 55μL Circular mRNA 275.1 ng / μL 35μL

[0196] The method for preparing circular mRNA provided in this embodiment can be used to prepare circular mRNA encoding other target proteins at the same time. The remaining circular mRNAs disclosed in the present invention are all prepared using the method provided in this embodiment.

[0197] Example 2. Detection of positive rate and expression time after circular mRNA encoding GFP was electroporated into T cells based on Clean-PIE

[0198] This example provides the expression efficiency and duration of circular mRNA-encoded proteins in human T cells based on Clean-PIE.

[0199] 2.1 Experimental methods

[0200] After PBMCs were separated from fresh peripheral blood of healthy donors, they were cultured in cell culture flasks coated with anti-CD3 and anti-CD28 antibodies. Activated T cells and circular mRNA encoding GFP (nucleotide sequence as shown in SEQ ID NO.1) were taken and electroporated at multiple time points using a Yida electroporator. Flow cytometry was used to detect the percentage of GFP-positive cells in electroporated T cells (referred to as GFP-T cells). The GFP-T cell detection method is as follows: 1×10 6 T cells that have been electroporated with circular mRNA encoding GFP (GFP-T cells), and the Fixable Viability Dye eFluor TM 506 was diluted 1:100, GFP-T cells were resuspended in 100 μL 1×PBS, placed in a 4°C refrigerator for staining for 30 min, washed once with 1 mL 1×PBS, centrifuged, resuspended in 400 μL 1×PBS, and filtered through a 70 μm filter. The ratio of GFP-positive cells in the eFlour506-negative cell population was determined by the machine.

[0201] 2.2 Experimental Results

[0202] Figure 1AThe results showed that the positive rate of T cells was as high as 92.83% on the first day after electroporation of T cells with circular mRNA encoding GFP (circular mRNA-GFP), and the high positive rate (>80%) was maintained for at least 8 days. A small number of GFP-positive cells could still be detected 16 days after electroporation. Figure 1B The average fluorescence brightness of GFP-positive cells is shown. It can be seen that the abundance of GFP on the surface of T cells is gradually decreasing over time. Figure 1C The total fluorescence intensity (number of cells × average fluorescence intensity) of all positive cell surface GFP changes over time. It can be observed that the change in overall fluorescence intensity from the beginning of electroporation to 16 days after electroporation does not exceed 2 orders of magnitude. This verifies that circular mRNA can mediate efficient and persistent protein expression on T cells.

[0203] Example 3. After the circular mRNA encoding IL-2 was electroporated into CAR-T cells, the improvement of the killing ability of CAR-T cells on target cells in vitro was detected

[0204] This embodiment provides a method for modifying CAR-T cells that have been prepared by other means using circular mRNA in order to optimize their characteristics.

[0205] 3.1 Experimental methods

[0206] Lentiviral packaging and lentiviral-mediated CAR-T cell preparation: 3×10 6293T cells were inoculated in a 10 cm culture dish at a density of cells / dish. On the second day, Lipofectatime2000 (thermofisher) was used for plasmid transfection (including: viral envelope plasmid pMD2.G, purchased from Wuhan Miaoling Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO.7; viral packaging plasmid pSPAX2, purchased from Wuhan Miaoling Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO.8; and expression plasmid LV19-CAR-4-1BB (nucleotide sequence is shown in SEQ ID NO.12), which uses pLVX-Luc2-puro (purchased from Shanghai Haijihaoge Biotechnology Co., Ltd., and the nucleotide sequence is shown in SEQ ID NO.9) as a vector, and the exogenous gene 19-CAR-4-1BB sequence (as shown in SEQ ID NO.10) is cloned into pLVX-Luc2-puro, thereby constructing it). On the fourth day, the supernatant was collected, filtered through a 40 μm filter membrane, and then used to determine the virus titer. On the day of plasmid transfection, PBMC (peripheral blood mononuclear cell) was isolated and activated. 48 hours after activation, cells were collected and transferred to 24-well plates coated with CD3 and CD28 antibodies. Lentiviral particles were added at MOI = 50, centrifuged at 1000g for 3 hours at room temperature, and then placed in an incubator for continued culture. After 24 hours of infection, the culture medium was replaced, culture was continued, transfection efficiency was detected, and CAR-T cells prepared by lentivirus were obtained.

[0207] The CAR-T cells prepared by lentivirus were divided into three groups: blank group (referred to as CAR-T control), group electroporated with circular mRNA encoding GFP (circular mRNA-GFP) (referred to as CAR-T circular mRNA GFP), and group electroporated with circular mRNA encoding IL-2 (circular mRNA-IL-2) (referred to as CAR-T circular mRNA IL-2). Empty electroporation, electroporation with circular mRNA-GFP (nucleotide sequence as shown in SEQ ID NO.1), and electroporation with circular mRNA-IL-2 (nucleotide sequence as shown in SEQ ID NO.2) were performed respectively. 24 hours after electroporation, appropriate amount of target cells (Raji cells) were taken, stained with CellTrace, washed twice with 1×PBS, and then 1 mL of cell culture medium was added to resuspend the cell pellet, counted and diluted to 2×10 5 / mL for standby use. According to the experimental design, the ratio of CAR-T cells to target cells was 20:1, with 2×10 4 / well plated 96-well (Round-bottom) plate, that is, 100μL / well plated. Then, according to the effect-target ratio of 20:1, 100μL / well of different groups of CAR-T cells were added to the target cells, and the wells containing only target cells were set as the control group. After incubation in a 37°C incubator for 4h, the cells were collected, washed once with 1×PBS, and 100μL of 1×PBS containing cell death dye eFlour780 was added. They were placed in a 4°C refrigerator for staining for 30min, washed once with 200μL 1×PBS, centrifuged, resuspended with 200μL 1×PBS, and filtered through a 70μm filter. The proportion of eFlour780-positive cells in the CellTrace-positive cell population was determined by machine detection.

[0208] 3.2 Experimental Results

[0209] like Figure 2 As shown in the figure, at an effector-target ratio of 20:1, 50% target cell killing was achieved after 4 hours of incubation of CAR-T cells prepared by lentivirus with target cells. When electroporated with circular mRNA encoding GFP, the killing ability of CAR-T cells did not change significantly. However, when electroporated with circular mRNA encoding IL-2, the killing ability of CAR-T cells was significantly enhanced.

[0210] Example 4. Detecting changes in T cell function by co-electroporating circular mRNA encoding IL-12sc and CAR into T cells

[0211] 4.1 Experimental methods

[0212] After PBMCs were isolated from fresh peripheral blood of healthy donors, they were cultured in cell culture flasks coated with anti-CD3 and anti-CD28 antibodies. T cells (CD3 positivity > 95%) on the seventh day after activation were electroporated with circular mRNA encoding CAR-19 molecules (nucleotide sequence as shown in SEQ ID NO.3) and circular mRNA encoding human IL-12sc (nucleotide sequence as shown in SEQID NO.5) (the resulting cells were recorded as CAR-T-IL-12). At the same time, T cells that were only electroporated with circular mRNA encoding CAR-19 molecules (the resulting cells were recorded as CAR-T), and T cells that were only electroporated but without circular mRNA (the resulting cells were recorded as NC-T) were prepared as negative controls. Cells were taken 24 hours after electroporation for killing detection. An appropriate amount of target cells (Raji cells) were taken, stained with CellTrace, washed twice with 1×PBS, and then 1mL of cell culture medium was added to resuspend the cell pellet, counted and diluted to 2×10 5 / mL for standby use. According to the experimental design, the ratios of effector cells to target cells were 20:1, 5:1, and 1:1, respectively, with 2×10 4 / well plated 96-well (Round-bottom) plate, that is, 100μL / well plated. Then, according to different effector-target ratios, 100μL / well of effector cells were added to the target cells, and wells containing only target cells were set as control groups. After incubation in a 37°C incubator for 4 hours, the cells were collected, washed once with 1×PBS, and 100μL of 1×PBS containing cell death dye eFlour780 was added. They were placed in a 4-degree refrigerator for staining for 30 minutes, washed once with 200μL 1×PBS, centrifuged, resuspended in 200μL 1×PBS, and filtered through a 70μm filter. The cells were tested on the machine to determine the proportion of eFlour780-positive cells in the CellTrace-positive cell population.

[0213] For intracellular factor detection, target cells were stained with CellTrace, washed twice with 1×PBS, and then 1 mL of cell culture medium was added to resuspend the cell pellet. After counting, the cell pellet was diluted to 2×10 5 / mL for standby use. According to the experimental design, the ratios of effector cells to target cells were 20:1, 5:1, and 1:1, respectively, with 2×10 4 / well plated 96-well (Round-bottom) plate, i.e. 100 μL / well plated. Then, according to different effector-target ratios, 100 μL / well of effector cells were added to target cells, and wells containing only target cells were set as control groups. After incubation in the incubator for 0.5 h, Golgistop was added to each well, and incubated in the incubator for another 4.5 h. The cells were collected, stained with IFN-γ and TNF-α antibodies, and the positive proportion of IFN-γ and TNF-α was detected by flow cytometry.

[0214] 4.2 Experimental Results

[0215] Figure 3A The results showed that under different effector-target ratios, compared with CAR-T cells prepared from healthy donor Donor1#, the killing ability of CAR-T-IL-12 was significantly enhanced when the effector-target ratio was 5:1 (P < 0.0001). Figure 3B The results showed that during the cell killing process, the secretion of IFN-γ by CAR-T-IL-12 cells was significantly increased compared with CAR-T cells that were not electroporated with circular mRNA encoding human IL-12sc (P < 0.01). Figure 3C The results showed that the secretion of TNF-α in CAR-T-IL-12 cells during cell killing was significantly increased compared with CAR-T cells that were not electroporated with circular mRNA encoding human IL-12sc (P < 0.01). This proves that both IL-12sc and CAR-19 molecules encoded by circular mRNA have active functions, can effectively identify target cells, kill target cells, and IL-12sc improves the killing ability of CAR-T cells.

[0216] Example 5. Detection of changes in T cell migration ability by electroporating circular mRNA encoding CXCR2 into T cells

[0217] 5.1 Experimental Methods

[0218] After PBMCs were separated from fresh peripheral blood of healthy donors, they were cultured in cell culture flasks coated with anti-CD3 and anti-CD28 antibodies. T cells (CD3 positive rate>95%) on the seventh day after activation were electroporated with circular mRNA encoding CAR-19 molecules (nucleotide sequence as shown in SEQ ID NO.3) and circular mRNA encoding human CXCR2 (nucleotide sequence as shown in SEQ ID NO.4) (the obtained cells were recorded as CXCR2-CAR-T), and T cells that were only electroporated with circular mRNA encoding CAR-19 molecules (the obtained cells were recorded as CAR-T), and T cells that were only electroporated without adding circular mRNA (the obtained cells were recorded as NC-T) were prepared. Cells were taken 24 hours after electroporation for cell migration detection.

[0219] Cell migration assay: The chemotaxis of CAR-T cells was measured by migration through a polycarbonate filter with an 8 μm pore size in a 24-well Transwell chamber (purchased from Corning). CXCL1 recombinant protein (brand MCE, catalog number HY-P70508, diluent 1640 culture medium) at a concentration of 1 μg / ml was added to the lower chamber to stimulate the above different cells, and the responding cells pre-labeled with cell trace were incubated in the upper chamber for 3-5 hours. Cells migrating from the upper chamber to the lower chamber were evaluated by flow cytometry.

[0220] 5.2 Experimental Results

[0221] like Figure 4 As shown in the figure, the number of CXCR2-CAR-T cells migrating from the upper chamber to the lower chamber is significantly greater than that of CAR-T cells. This proves that the migration ability of CXCR2-CAR-T cells is greatly improved compared with CAR-T cells.

[0222] Example 6. Simultaneous electroporation of molecules encoding CAR molecules and T cell function regulators into T cells to evaluate their anti-tumor efficacy in vivo

[0223] 6.1 Experimental methods

[0224] After PBMCs were separated from fresh peripheral blood of healthy donors, they were cultured in cell culture bottles coated with anti-CD3 and anti-CD28 antibodies. T cells on the seventh day after activation were subjected to the following operations: electroporation only, electroporation of circular mRNA encoding CAR-19 molecules (nucleotide sequence as shown in SEQ ID NO.3), electroporation of circular mRNA encoding CAR-19 molecules (nucleotide sequence as shown in SEQ ID NO.3) and circular mRNA encoding human IL-12sc (nucleotide sequence as shown in SEQ ID NO.5) (the obtained cells were recorded as Mock T cell, CD19 CAR-T and CD19 CAR-T with IL-12sc, respectively). Cells were taken 24 hours after electroporation for reinfusion.

[0225] Female NCG (purchased from Jiangsu Jicui Yaokang Experimental Animal Co., Ltd.), 6-8 weeks old, weighing between 18 and 22 grams, were adapted in a feeding room (Jima Gene) for at least three days before the study. Mice had free access to food and sterile water. Feeding environment: maintain 12h light and 12h dark day and night alternation, humidity maintained at 40-70% and daily humidity difference <5%; temperature maintained at 20-25°C. Raji-luci cells (luciferase-labeled human lymphoma cells) were purchased from the Chinese Academy of Sciences and cultured in RPMI (Roswell Park Memorial Institute) medium, with 10% FBS (Fatal Bovine Serum) and 1% PS (Penicillin-Streptomycin Solution). RPMI medium was purchased from VivaCell and PBS was purchased from Gibco. Cell counting was performed by mixing Trypan Blue Solution (SIGMA) with the cell suspension in a 1:1 ratio and counting using a Countstar cell counter. When Raji-luci cells were cultured to the cell proliferation growth phase, 2.5E5 / 100 μL of cells were injected into the tail vein of each mouse to construct a xenograft tumor model (CDX model).

[0226] On the third day after Raji-luci cell inoculation (designated as day 0), the model animals were randomly divided into 4 groups, with 6 animals in each group, namely solvent control group (recorded as Untreated), Mock T cell control group, CD19 CAR-T group and CD19 CAR-T with IL-12sc group, and were given tail vein injection treatment once on day 0, day 5 and day 13 respectively (the 4 groups were injected with solvent (solvent composition: 95% (V / V) 0.9wt% sodium chloride injection + 5% (V / V) human albumin), Mock T cell, CD19 CAR-T and CD19 CAR-T with IL-12sc, and the cell injection amount was 7E6 for each treatment). Body weight was measured when grouping, and body weight was measured once a week thereafter. Live imaging was performed once a week during the experiment to record the level of tumor changes.

[0227] The method of in vivo imaging experiment is as follows: experimental animals are intraperitoneally injected with luciferase substrate luciferin aqueous solution and placed in an anesthesia chamber for anesthesia. Luciferin substrate was purchased from Promega (VivoGlo TM Luciferin, P1043), use according to the instructions: dissolve with saline before use, the injection dose for each animal is 50 mg / kg, use about 10 minutes after injection Lumina III live imager was used for imaging. Regions of interest (ROI) were quantified as mean radiance (photons s-1cm-2sr-1, represented by color-scaled images superimposed on grayscale photos of mice using Caliper Life Sciences' Living Image software). For absolute quantification, bioluminescent signals were blotted as total flux (photons s-1).

[0228] 6.2 Experimental Results

[0229] 6.2.1 Results of weight monitoring experiment

[0230] Figure 5A The results showed that during the experiment, the body weight of animals in each group showed a continuous decreasing trend. Figure 5B The results showed that the weight loss of animals in the CD19 CAR-T and CD19CAR-T with IL-12sc groups became very slow after treatment, and the degree of weight loss was significantly reduced compared with the untreated group (solvent control group) and the Mock T cell group.

[0231] 6.2.2 Results of in vivo mouse imaging experiments

[0232] Fig. 6AThe survival curve results showed that CD19 CAR-T could significantly prolong the survival of mice. The CD19CAR-T with IL-12sc group significantly enhanced the efficacy of CD19 CAR-T and further prolonged the survival of mice. Figure 6B and Figure 6C The results showed that the tumor growth of animals in the CD19 CAR-T treatment group was delayed, and the tumor growth rate in the CD19 CAR-T with IL-12sc group was even slower.

[0233] Example 7. Evaluation of the feasibility of using circular mRNA to modify T cells for targeted drug delivery

[0234] 7.1 Experimental methods

[0235] After PBMC was separated from fresh peripheral blood of healthy donors, it was cultured in a cell culture flask coated with anti-CD3 and anti-CD28 antibodies. T cells (CD3 positive rate>95%) on the seventh day after activation were taken and electroporated with circular mRNA encoding CAR-19 molecules (nucleotide sequence as shown in SEQ ID NO.3), circular mRNA encoding human IL-12sc (nucleotide sequence as shown in SEQ ID NO.5) and circular mRNA encoding human Flt3L (nucleotide sequence as shown in SEQ ID NO.6) (the obtained cells were recorded as CAR-T+IL-12+Flt3L), and the group of mice injected with this cell was the 12F-CAR-T group. At the same time, T cells that were only electroporated with circular mRNA encoding CAR-19 molecules (the obtained cells were recorded as CAR-T), and the group of mice injected with this cell was the CAR-T group, and T cells that were only electroporated but not with circular mRNA were prepared (the obtained cells were recorded as NC-T), and the group of mice injected with this cell was the NC-T group. The cells were taken 24 hours after electroporation and injected into the tail vein of mice.

[0236] Thirty NCG mice were randomly divided into three groups: CAR-T group, 12F-CAR-T group, and NC-T group. Each group had 10 mice, and 2×10 6Raji cells were used to construct a tumor model. The specific model construction method is as follows: female NCG (Jiangsu Jicui Yaokang Experimental Animal Co., Ltd.), 6-8 weeks old, weighing between 18 and 22 grams, were adapted in a feeding room (Jima Gene) for at least three days before the study. Mice can freely access food and sterile water. Feeding environment: maintain 12h light and 12h dark day and night alternation, humidity maintained at 40% to 70% and daily humidity difference <5%; temperature maintained at 20-25°C. Raji cells (human lymphoma cells) were purchased from (Chinese Academy of Sciences) and cultured in RPMI (Roswell Park Memorial Institute) medium, with 10% FBS (Fatal Bovine Serum, fetal bovine serum) and 1% PS (Penicillin-Streptomycin Solution, penicillin-streptomycin solution). RPMI medium was purchased from VivaCell and PBS was purchased from Gibco. Cell counting was performed by mixing Trypan Blue Solution (SIGMA) with the cell suspension in a 1:1 ratio and counting using a Countstar cell counter. When collecting cells, the cells were centrifuged to obtain the cell pellet, resuspended in an appropriate amount of 1× PBS for counting, and resuspended in phosphate buffered saline (PBS, Shanghai Yuanpei Biotechnology Co., Ltd.) supplemented with Matrigel (Corning) after centrifugation. Each female NCG mouse was subcutaneously inoculated with 2×10 6 / 100μL of Raji cells.

[0237] After injection, mice were allowed to eat freely, and the tumor volume was measured using a vernier caliper twice a week until it reached 50 mm 3 Cell administration was started by tail vein injection at 1:00 pm, with 2×10 cells injected into each mouse. 6 / 200μL of CAR-T cells, CAR-T+IL-12+Flt3L cells or NC-T cells. The next day, the mice were dissected to obtain tumor tissues and detect the expression of IL-12 and Flt3L proteins in the tumor.

[0238] Analysis of tumor-infiltrating immune cells and IL-12 and Flt3L: Mice were euthanized 1-2 days after administration, and tumor tissue was dissected. The tumor was minced and digested with Collagenase Type IV (purchased from Gibco) at 37°C for 30 min, filtered through a 70μm filter, and centrifuged to collect cells for flow cytometry antibody staining. The supernatant of the tumor tissue was collected in a new tube for ELISA testing.

[0239] Detection of cytokines released from mouse tumors: After euthanasia of mice, tumors were removed, epidermis and connective tissues were removed, 1×PBS was used to wash away surface blood, and the tumors were placed in 2 mL centrifuge tubes. An appropriate amount of 1×PBS and protease inhibitors (Roche) were added for homogenization. After homogenization, the supernatant was centrifuged and aspirated for subsequent cytokine determination. ELISA MAX TM Deluxe Set Human IL-12 (p70) kit was purchased from Biolegend, and Human Flt3L ELISA Kit was purchased from Wuhan Fine Biotechnology Co., Ltd. The corresponding buffer and standard were prepared according to the kit instructions, and the tumor homogenate supernatant was diluted with sample dilution buffer. The standard and diluted samples with different concentration gradient dilutions were added to the well plate pre-coated with antibodies, shaken at room temperature or incubated at 37 degrees for 1.5-2 hours, washed the well plate and added the detection antibody, shaken at room temperature or incubated at 37 degrees for 1 hour, washed the plate, added HRP (Horseradish Peroxidase) cross-linked avidin dye solution, shaken at room temperature or incubated at 37 degrees for 0.5 hours, washed the plate, and added the color development solution and stood at room temperature or 37 degrees for 10-20 minutes for color development. The microplate spectrophotometer detected the absorbance at 450nm, statistically plotted the standard curve, and calculated the sample concentration.

[0240] 7.2 Experimental Results

[0241] like Figure 7 As shown, the cytokines IL-12 and Flt3L released intratumorally in mice in the 12F-CAR-T group were significantly more than those in the CAR-T group and the NC-T group, proving that the CAR molecules encoded by circular mRNA achieved tumor targeting of cells and simultaneously delivered the encoded cytokine drugs IL-12 and Flt3L into the tumor.

Claims

1. A method for preparing CAR-T cells, characterized in that: The method comprises the steps of introducing circular mRNA into T cells that do not contain a chimeric antigen receptor; Furthermore, the circular mRNA includes a first circular mRNA encoding a chimeric antigen receptor; Furthermore, the first circular mRNA comprises a sequence arranged in the order shown in (a) or (b): (a) translation initiation sequence and coding sequence; (b) Translation initiation sequence, coding sequence and insert element sequence.

2. The preparation method according to claim 1, characterized in that: The chimeric antigen receptor targets the CD19 molecule.

3. The preparation method according to claim 1 or 2, characterized in that: The nucleotide sequence of the first circular mRNA comprises the sequence shown in SEQ ID NO.3, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.3, and encodes a sequence of a protein having or partially having the chimeric antigen receptor activity.

4. A method for modifying CAR-T cells, characterized in that: The method comprises the steps shown in (i) or (ii): (i) a step of introducing circular mRNA into T cells that do not contain a chimeric antigen receptor; (ii) a step of introducing circular mRNA into CAR-T cells; In the step (i), the circular mRNA includes a first circular mRNA encoding a chimeric antigen receptor and a second circular mRNA encoding a functional polypeptide; In the step (ii), the circular mRNA includes a second circular mRNA encoding a functional polypeptide; Furthermore, the first circular mRNA and the second circular mRNA comprise sequences arranged in the order shown in (a) or (b): (a) translation initiation sequence and coding sequence; (b) Translation initiation sequence, coding sequence and insert element sequence.

5. The transformation method according to claim 4, characterized in that: The chimeric antigen receptor targets the CD19 molecule; the functional polypeptide includes any one or more of the group consisting of IL-2, IL-12sc, CXCR2 and Flt3L.

6. The transformation method according to claim 4 or 5, characterized in that: The nucleotide sequence of the first circular mRNA comprises the sequence shown in SEQ ID NO.3, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.3, and encodes a sequence of a protein having or partially having the chimeric antigen receptor activity; The nucleotide sequence of the second circular mRNA comprises a sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6, and encoding a protein having or partially having the activity of the functional polypeptide.

7. A CAR-T cell, characterized in that: The CAR-T cells are obtained by the preparation method according to any one of claims 1 to 3 or the transformation method according to any one of claims 4 to 6.

8. A method for delivering a drug to a target cell, characterized in that: The method comprises: contacting the CAR-T cell according to claim 7 containing the drug with a target cell; Preferably, the drug includes any one or more of a circular mRNA drug, a linear RNA drug, a DNA drug and a small molecule drug.

9. The method according to claim 8, characterized in that The target cells are tumor cells.

10. The method according to claim 8 or 9, characterized in that: The method comprises: administering the CAR-T cells according to claim 7 containing the drug to a subject.

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