TCR-T cell preparation method and product thereof

Abstract

The invention relates to a TCR-T cell preparation method and a TCR-T cell product. The preparation method of the TCR-T cell provided by the invention comprises a step of introducing annular mRNA (messenger Ribonucleic Acid) into a T cell modified by a non-T cell receptor gene; and, the cyclic mRNA comprises a first cyclic mRNA encoding a T cell 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; compared with a virus system and the like, the process is extremely simple, and the production cost and the detection cost are greatly reduced; compared with linear mRNA, the protein expression time is greatly prolonged; compared with a virus system, a transposon system and a CRISPR system, the safety risk is greatly reduced.

Description

Technical Field

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

[0002] Adoptive-cell therapy (ACT) using in vitro amplification of tumor-infiltrating T lymphocytes (TILs) has achieved very remarkable results in clinical practice, but because it is difficult for most patients with solid tumors to prepare sufficient TILs cells for treatment, its application is greatly limited. It is particularly urgent to genetically engineer normal T cells to enable them to have the ability to kill target cells. The two main types of engineered T cells are chimeric antigen receptor T cells (CAR-T cells) and transgenic T-cell receptor T cells (TCR-T cells). The differences between these two types of engineered T cells are mainly shown in the following table (Watanabe & Nishikawa, 2021):

[0003] TCR-T CAR-T Receptor structure Dimer Single Chain Identify the target MHC and peptide complex Cell surface antigens Major histocompatibility complex (MHC) restriction have none Number of immunoreceptor tyrosine-based inhibitory motifs (ITAMs) 10 3 Co-stimulatory signal Requires other ligands After ligation of CD3ζdomain Affinity <![CDATA[K d 1-100μM]]> <![CDATA[K d 1 nM to 1μM]]>

[0004] Since TCR-T does not rely on antigens on the cell surface to function and can target proteins from any part of the cell, it has different application scenarios from CAR-T cells. Currently, TCR-T trials entering the clinical stage target a variety of disease types, including solid tumors, HIV infection, CMV infection, EBV infection, and hematological tumors. Target selection includes tumor-associated antigens, tumor-testis antigens, viral antigens, and new antigens. Although the application of TCR-T is limited by human leukocyte antigens (HLA), its characteristics of targeting HLA and peptide polymers still make it have a wide range of application scenarios (Shafer, Kelly, & Hoyos, 2022).

[0005] At present, the delivery of TCR molecules or their modification mainly relies on gene vector delivery technology, such as lentivirus / retrovirus system, adenovirus system, transposon system, CRISPR gene knock-in, etc. Virus-related gene delivery technology has the disadvantages of high preparation and detection costs, and safety risks caused by random gene integration. Transposon system-mediated gene delivery faces problems such as low plasmid delivery efficiency and safety risks caused by random gene integration. CRISPR technology-mediated gene integration can reduce the risk of random integration through site-specific integration, but its knock-in process involves the process of gene double-strand breakage and chromosomal translocation (Morgan & Boyerinas, 2016). Compared with the above technical means, mRNA as a non-viral vector gene delivery method has advantages that other methods do not have: (1) It can be used to transfect quiescent or low-proliferation cells by electroporation or liposome transfection, with high transfection efficiency, simple method and low cost; (2) There is no gene integration process, so the safety risk is extremely low; (3) Multiple mRNAs encoding different proteins can be delivered simultaneously to achieve combined application of different targets.

[0006] According to the records of the prior patent applications CN114438127A and CN114574483A of the applicant of this application, compared with the classic linear mRNA, the preparation process of circular mRNA based on Clean-PIE is very simple, without the need for capping reaction and tailing reaction, the process is simpler and the cost is lower; the circular mRNA based on Clean-PIE has no other exogenous structures except IRES and coding region, and has lower immunogenicity. Circular mRNA based on Clean PIE can mediate higher and more sustained protein expression. At present, there are literature reports on the application of circular mRNA in tumor injection therapy (Yang et al., 2022), infectious disease treatment (Qu et al., 2022) and tumor vaccines (Li et al., 2022). However, whether the circular mRNA based on Clean PIE can apply its advantages of low cost, long expression time, low immunogenicity and high safety to the preparation and transformation of TCR-T cells still needs to be verified.

[0007] References:

[0008] Watanabe,K.,&Nishikawa,H.(2021).Engineering strategies for broad application of TCR-T-and CAR-T-cell therapies.Int Immunol,33(11),551-562.

[0009] Shafer, P., Kelly, L. M., & Hoyos, V. (2022). Cancer Therapy With TCR-Engineered T Cells: Current Strategies, Challenges, and Prospects. Front Immunol, 13, 835762.

[0010] Morgan, R. A., & Boyerinas, B. (2016). Genetic Modification of T Cells. Biomedicines, 4(2).

[0011] Yang, J., Zhu, J., Sun, J., Chen, Y., Du, Y., Tan, Y.,... Zuo, C. (2022). Intratumoral delivered novel circular mRNA encoding cytokines for immunemodulation and cancer therapy. Mol Ther Nucleic Acids, 30, 184 - 197.

[0012] 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.

[0013] 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

[0014] Problem that the invention aims to solve

[0015] The research on T cell engineering, especially TCR-T cell preparation in the above-mentioned prior art is still not sufficient, and the present invention studies the engineering transformation of T cells by circular mRNA prepared based on the Clean-PIE system. On this basis, the present invention provides a preparation method and a transformation method of TCR-T cells, which can not only prepare TCR-T cells using circular mRNA based on Clean-PIE; TCR-T cells that have been prepared by non-mRNA pathways (such as lentiviral pathways) can also be transformed by circular mRNA encoding proteins (such as cytokines) to obtain more functions or optimize existing functions; At the same time, TCR-T cells can also be prepared and transformed simultaneously by a variety of circular mRNAs based on Clean-PIE. In addition, the present invention also found that TCR-T cells can be prepared by circular mRNA based on Clean-PIE for the delivery of circular mRNA drugs or non-mRNA drugs.

[0016] Solutions for solving problems

[0017] [1] A method for preparing a TCR-T cell, wherein the method comprises the step of introducing a circular mRNA into a non-T cell receptor gene-modified T cell;

[0018] Furthermore, the circular mRNA includes a first circular mRNA encoding a T cell receptor;

[0019] Furthermore, the first circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0020] (a) translation initiation sequence and coding sequence;

[0021] (b) Translation initiation sequence, coding sequence and insert element sequence.

[0022] [2] The preparation method according to [1], wherein the T cell receptor targets NY-ESO-1.

[0023] [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.2, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.2, and encodes a sequence of a protein having or partially having the T cell receptor activity.

[0024] [4] A method for modifying TCR-T cells, wherein the method comprises the steps shown in (i) or (ii):

[0025] (i) a step of introducing circular mRNA into non-T cell receptor gene-modified T cells;

[0026] (ii) a step of introducing circular mRNA into TCR-T cells;

[0027] In the step (i), the circular mRNA includes a first circular mRNA encoding a T cell receptor and a second circular mRNA encoding a functional polypeptide;

[0028] In the step (ii), the circular mRNA includes a second circular mRNA encoding a functional polypeptide;

[0029] Furthermore, the first circular mRNA and the second circular mRNA comprise sequences arranged in the order shown in (a) or (b):

[0030] (a) translation initiation sequence and coding sequence;

[0031] (b) Translation initiation sequence, coding sequence and insert element sequence.

[0032] [5] The modification method according to [4], wherein the T cell receptor targets NY-ESO-1; and the functional polypeptide comprises any one or more of IL-2, IL-12sc, CCL5, XCL1, Flt3L-Fc, GM-CSF, IFN-α and anti-PD-1 nanoantibodies.

[0033] [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.2, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.2, and encodes a sequence of a protein having or partially having the T cell receptor activity;

[0034] The nucleotide sequence of the second circular mRNA comprises a sequence as shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.18, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQID NO.9, SEQ ID NO.10 or SEQ ID NO.18, and encodes a protein having or partially having the activity of the functional polypeptide.

[0035] [7] A TCR-T cell, wherein the TCR-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 [4] to [6].

[0036] [8] A method for delivering a drug to a target cell, wherein the method comprises: contacting the TCR-T cell according to [7] containing the drug with the target cell;

[0037] 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.

[0038] [9] The method according to [8], wherein the target cells are tumor cells.

[0039]

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

[0040] Effects of the Invention

[0041] Through the implementation of the above technical solution, the present invention achieves the following technical effects:

[0042] The present invention provides a method for preparing and modifying TCR-T cells. First, TCR-T cells can be prepared using Clean-PIE-based circular mRNA. Second, not only can TCR-T cells prepared by other methods be modified using Clean-PIE-based circular mRNA, but the preparation and modification of TCR-T cells can also be achieved simultaneously using Clean-PIE-based circular mRNA.

[0043] In the preparation and transformation methods of TCR-T cells provided by the present invention, the circular mRNA has a high transfection efficiency and can efficiently transfect cells in a quiescent state or with a low proliferation rate; compared with the virus system, etc., the process is extremely simple, and the production cost and detection cost are greatly reduced; compared with linear mRNA, the protein expression time is greatly extended; compared with the virus system, transposon system, and CRISPR system, the safety risk is greatly reduced.

[0044] The present invention verifies that the modification of TCR-T cells by circular mRNA based on Clean-PIE can further optimize the function of engineered T cells and obtain enhanced TCR-T cells. For example, the combination with T cell activation-related cytokines can enhance the killing ability of TCR-T cells on target cells, and the combination with chemokines can enhance the migration ability of TCR-T cells and enhance the infiltration ability of solid tumors.

[0045] At the same time, the present invention proposes the concept of transforming T cells or TCR-T cells through circular mRNA for drug delivery. The delivered drugs include circular mRNA drugs and non-mRNA drugs (such as small molecule drugs), and the characteristics of TCR-T with tumor cell-specific targeting are utilized to enable it to play the function of drug targeted delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1A This is a curve chart showing the detection of tumor target cell killing by TCR-T cells prepared from the healthy donor Donor1# in Example 2.

[0047] Figure 1B This is a detection curve of the killing of tumor target cells by TCR-T cells prepared from the healthy donor Donor2# in Example 2.

[0048] Figure 2A This is a statistical chart of the survival of each group of animals in Example 3 during the test period.

[0049] Figure 2B This is a statistical chart of the bioluminescence values ​​of the living imaging of each group of animals in Example 3.

[0050] Figure 2C These are the in vivo imaging images of each group of animals in Example 3.

[0051] Figure 3 This is a graph showing the evaluation results of the killing ability of target cells at different effector-target ratios after the TCR-T cells prepared by lentivirus were transformed using Clean-PIE-based circular mRNA in Example 4.

[0052] Figure 4A This is a detection diagram of cells killing tumor target cells under different treatment conditions in Example 5.

[0053] Figure 4B This is a detection diagram of the intracellular factor IFN-γ during the process of cells killing tumor target cells under different treatment conditions in Example 5.

[0054] Figure 4C 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 5.

[0055] Figure 5 This is a graph showing the migration ability of cells under different treatment conditions in Example 6.

[0056] Fig. 6A This is a detection diagram of Flt3L-Fc released into the tumor of mice in different experimental groups in Example 7.

[0057] Figure 6B This is a detection diagram of XCL1 released into the tumor of mice in different experimental groups in Example 7.

[0058] Figure 6C This is a graph showing the detection of GM-CSF released into the tumor of mice in different experimental groups in Example 7.

[0059] Fig.6D This is a detection diagram of anti-PD-1 nanobody released into the tumor of mice in different experimental groups in Example 7.

[0060] Fig. 6E This is a detection diagram of IFN-α released into the tumor of mice in different experimental groups 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, "RNA composition" is not limited to a combination method that must include at least two RNAs with different sequences, but also covers a combination method that only includes RNAs with the same sequence. In some embodiments, the RNA composition includes two RNAs; in other embodiments, the RNA composition consists of one RNA.

[0076] As used in the present disclosure, "treatment" means that after suffering from a disease, a subject is exposed to (e.g., administered) the TCR-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 are completely suppressed. Suffering from a disease means that the body has symptoms of the disease.

[0077] As used in the present disclosure, "prevention" means that before a subject develops a disease, the symptoms after developing the disease are alleviated compared to when the subject has not developed the disease by contacting (e.g., administering) the TCR-T cells of the present invention, but does not necessarily mean that the disease must be completely suppressed.

[0078] 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.

[0079] In the present invention, "T cell receptor (TCR)" refers to a specific receptor on the surface of T cells, which can recognize antigens presented by the major histocompatibility complex (MHC). Natural TCR is a heterodimer composed of two different subunits, namely, an α subunit and a β subunit, or a γ subunit and a δ subunit. In some embodiments, the TCR described in the present invention also includes different subunit chains, namely, α chains and β chains, and the different chains are connected by P2A and encoded by the same circular mRNA, and each chain contains a variable region and a constant region.

[0080] 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.

[0081] In the present invention, "T cell chemokine" refers to a cytokine that can promote T cell migration, such as CCL5.

[0082] In the present invention, "NY-ESO-1" is a tumor-testis antigen (CTA), which is a good candidate target for immunotherapy. It is limitedly expressed in normal tissues, but widely expressed in various tumors. It is a limited off-target toxicity target and can be widely used in the treatment of various tumors. Tumors rich in CTA include melanoma, ovarian cancer, lung cancer, and bladder cancer.

[0083] 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 polypeptides of the present invention include any one or more of IL-2, IL-12sc, CCL5, XCL1, Flt3L-Fc, GM-CSF, IFN-α and anti-PD-1 nanoantibodies.

[0084] 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.

[0085] 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.

[0086] CCL5 is chemokine ligand 5, also known as RANTES, a chemotactic cytokine that can chemotactic T cells, eosinophils and basophils. With the help of cytokines released by T cells such as IL-2 and IFN-γ, CCL5 can induce the proliferation and activation of certain natural killer cells to form CC chemokine-activated killer cells.

[0087] XCL1 is a class C chemokine that is primarily expressed by activated CD8 + Produced by T cells and natural killer cells, XCL1 triggers their chemotactic function by binding to a receptor called XCR1.

[0088] Flt3L is a Flt3 ligand, which is secreted by bone marrow stromal stem cells and other cells, and 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, thereby having an important anti-tumor effect. In the present invention, Flt3L is fused with the immunoglobulin Fc fragment to form a Flt3L-Fc polypeptide.

[0089] GM-CSF is granulocyte-macrophage colony-stimulating factor, an important immunostimulatory factor that can induce the proliferation and differentiation of bone marrow dendritic cells and M1 macrophages, enhance antigen presentation, and induce the body's anti-tumor immune effect.

[0090] IFN-α is α-interferon produced by leukocytes. It can exert anti-tumor effects by inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and other pathways.

[0091] Anti-PD-1 nanoantibodies have the function of blocking the binding between PD-1 and its ligand PD-L1, thereby blocking the binding between cancer cells and T cells, so that T cells can restart their killing function and eliminate tumor cells.

[0092] 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.

[0093] Circular mRNA

[0094] 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.

[0095] In some embodiments, the circular mRNA described in the present invention comprises a sequence arranged in the order shown in (a) or (b):

[0096] (a) translation initiation sequence and coding sequence;

[0097] (b) Translation initiation sequence, coding sequence and insert element sequence.

[0098] In some specific embodiments, the circular mRNA of the present invention consists of sequences arranged in the order shown in (a) or (b):

[0099] (a) translation initiation sequence and coding sequence;

[0100] (b) Translation initiation sequence, coding sequence and insert element sequence.

[0101] 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 6 A 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] First circular mRNA

[0107] The first circular mRNA of the present invention encodes a T cell receptor.

[0108] In some specific embodiments, the first circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0109] (a) a translation initiation sequence and a coding sequence encoding a T cell receptor;

[0110] (b) Translation initiation sequence, coding sequence encoding T cell receptor and insertion element sequence.

[0111] In some preferred embodiments, the first circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0112] (a) a translation initiation sequence and a coding sequence encoding a T cell receptor;

[0113] (b) Translation initiation sequence, coding sequence encoding T cell receptor and insertion element sequence.

[0114] In some specific embodiments, the T cell receptor described herein targets NY-ESO-1.

[0115] In some more preferred embodiments, the nucleotide sequence of the first circular mRNA comprises the sequence shown in SEQ ID NO.2, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.2, and encodes a sequence of a protein having or partially having the T cell receptor activity.

[0116] Second circular mRNA

[0117] The second circular mRNA of the present invention encodes a functional polypeptide. In some embodiments, the functional polypeptide includes any one or more of IL-2, IL-12sc, CCL5, XCL1, Flt3L-Fc, GM-CSF, IFN-α and anti-PD-1 nanoantibodies.

[0118] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0119] (a) a translation initiation sequence and a coding sequence encoding IL-2;

[0120] (b) Translation initiation sequence, coding sequence encoding IL-2 and insertion element sequence.

[0121] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0122] (a) a translation initiation sequence and a coding sequence encoding IL-2;

[0123] (b) Translation initiation sequence, coding sequence encoding IL-2 and insertion element sequence.

[0124] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO.3, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.3, and encoding a protein having or partially having the IL-2 activity.

[0125] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0126] (a) translation initiation sequence and coding sequence encoding IL-12sc;

[0127] (b) Translation initiation sequence, coding sequence encoding IL-12sc and insertion element sequence.

[0128] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0129] (a) translation initiation sequence and coding sequence encoding IL-12sc;

[0130] (b) Translation initiation sequence, coding sequence encoding IL-12sc and insertion element sequence.

[0131] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO.18, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.18 and encoding a protein having or partially having the IL-12sc activity.

[0132] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0133] (a) a translation initiation sequence and a coding sequence encoding CCL5;

[0134] (b) Translation initiation sequence, coding sequence encoding CCL5 and insertion element sequence.

[0135] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0136] (a) a translation initiation sequence and a coding sequence encoding CCL5;

[0137] (b) Translation initiation sequence, coding sequence encoding CCL5 and insertion element sequence.

[0138] 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 CCL5 activity.

[0139] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0140] (a) a translation initiation sequence and a coding sequence encoding XCL1;

[0141] (b) Translation initiation sequence, coding sequence encoding XCL1 and insertion element sequence.

[0142] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0143] (a) a translation initiation sequence and a coding sequence encoding XCL1;

[0144] (b) Translation initiation sequence, coding sequence encoding XCL1 and insertion element sequence.

[0145] 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 XCL1 activity.

[0146] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0147] (a) a translation initiation sequence and a coding sequence encoding Flt3L-Fc;

[0148] (b) Translation initiation sequence, coding sequence encoding Flt3L-Fc and insertion element sequence.

[0149] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0150] (a) a translation initiation sequence and a coding sequence encoding Flt3L-Fc;

[0151] (b) Translation initiation sequence, coding sequence encoding Flt3L-Fc and insertion element sequence.

[0152] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO.7, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.7 and encoding a protein having or partially having the Flt3L-Fc activity.

[0153] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0154] (a) a translation initiation sequence and a coding sequence encoding GM-CSF;

[0155] (b) Translation initiation sequence, coding sequence encoding GM-CSF and insertion element sequence.

[0156] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0157] (a) a translation initiation sequence and a coding sequence encoding GM-CSF;

[0158] (b) Translation initiation sequence, coding sequence encoding GM-CSF and insertion element sequence.

[0159] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO. 8, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO. 8 and encoding a protein having or partially having the GM-CSF activity.

[0160] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0161] (a) a translation initiation sequence and a coding sequence encoding IFN-α;

[0162] (b) Translation initiation sequence, coding sequence encoding IFN-α and insertion element sequence.

[0163] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0164] (a) a translation initiation sequence and a coding sequence encoding IFN-α;

[0165] (b) Translation initiation sequence, coding sequence encoding IFN-α and insertion element sequence.

[0166] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises the sequence shown in SEQ ID NO.10, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.10 and encoding a protein having or partially having the IFN-α activity.

[0167] In some specific embodiments, the second circular mRNA comprises a sequence arranged in the order shown in (a) or (b):

[0168] (a) a translation initiation sequence and a coding sequence encoding an anti-PD-1 nanobody;

[0169] (b) Translation initiation sequence, coding sequence of anti-PD-1 Nanobody and insertion element sequence.

[0170] In some preferred embodiments, the second circular mRNA consists of a sequence arranged in the order shown in (a) or (b):

[0171] (a) a translation initiation sequence and a coding sequence encoding an anti-PD-1 nanobody;

[0172] (b) Translation initiation sequence, coding sequence of anti-PD-1 Nanobody and insertion element sequence.

[0173] In some more preferred embodiments, the nucleotide sequence of the second circular mRNA comprises a sequence as shown in SEQ ID NO.9, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.9, and encoding a protein having or partially having the anti-PD-1 nanobody activity.

[0174] Preparation method of TCR-T cells and products thereof

[0175] The present invention provides a method for preparing a TCR-T cell, the method comprising the step of introducing a circular mRNA into a non-T cell receptor gene-modified T cell; the circular mRNA comprises a first circular mRNA encoding a T cell receptor.

[0176] The "non-T cell receptor gene-modified T cells" mentioned in the present invention refer to T cells that have not been modified by the T cell receptor gene, including wild-type T cells or T cells that have undergone other modifications; 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 T cells that have undergone other modifications are T cells in a non-natural state, for example, they can be T cells that have been isolated from the target object and then genetically modified in other aspects.

[0177] The "introduction" described in the present invention includes a variety of methods, for example, electroporation, liposome-mediated transfection or LNP-mediated transfection, etc. In some preferred embodiments, the method of the present invention includes the step of electroporating circular mRNA into non-T cell receptor gene-modified T cells.

[0178] The TCR-T cell preparation method provided by the present invention uses for the first time a circular mRNA prepared based on the Clean-PIE system, which has a high transfection efficiency and can efficiently transfect cells in a quiescent state or with a low proliferation rate; compared with the virus system, etc., the process is extremely simple, and the production cost and detection cost are greatly reduced; compared with linear mRNA, its protein expression time is greatly extended; compared with the virus system, transposon system, and CRISPR system, the safety risk is greatly reduced.

[0179] The present invention also provides TCR-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.

[0180] TCR-T cell transformation method and product thereof

[0181] The present invention provides a method for modifying TCR-T cells. By modifying TCR-T cells, enhanced TCR-T cells can be obtained, for example, TCR-T cells with stronger killing ability and / or stronger migration ability, or TCR-T cells containing circular mRNA drugs can be obtained.

[0182] In some embodiments, the method for modifying TCR-T cells of the present invention comprises (i) the step of introducing circular mRNA into non-T cell receptor gene modified T cells; the circular mRNA comprises a first circular mRNA encoding a T cell receptor and a second circular mRNA encoding a functional polypeptide. In this method, the preparation and modification of TCR-T cells are achieved simultaneously.

[0183] In some embodiments, the "non-T cell receptor gene-modified T cells" refer to T cells that have not undergone T cell receptor gene modification, including wild-type T cells or T cells that have undergone other modifications; 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 T cells that have undergone other modifications are T cells in a non-natural state, for example, they can be T cells that have been isolated from the target object and then genetically modified in other aspects.

[0184] 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.

[0185] In other embodiments, the method for modifying TCR-T cells of the present invention comprises (ii) the step of introducing circular mRNA into TCR-T cells; the circular mRNA comprises a second circular mRNA encoding a functional polypeptide.

[0186] In this method, the TCR-T cells can be TCR-T cells prepared by various methods, and exemplary methods include lentiviral / retroviral systems, adenoviral systems, transposon systems, CRISPR gene knock-in, linear mRNA transfection, or PIE system-based circular mRNA transfection to prepare TCR-T cells. In some embodiments, the TCR-T cells do not contain circular mRNA prepared based on the Clean-PIE system.

[0187] By delivering different cytokines to TCR-T cells, the function of engineered T cells can be further optimized to obtain enhanced TCR-T cells. For example, the combination of T cell activation-related cytokines can enhance the killing ability of TCR-T cells on target cells, and the combination of chemokines can enhance the migration ability of TCR-T cells and enhance the infiltration ability of solid tumors.

[0188] The present invention also provides TCR-T cells obtained by the above transformation method.

[0189] Methods for delivering drugs to target cells

[0190] The present invention provides a method for delivering a drug to a target cell, comprising: contacting a TCR-T cell containing the drug with the target cell.

[0191] In some embodiments, the TCR-T cells are obtained by the above-mentioned preparation method or modification method.

[0192] In some embodiments, the target cell is a tumor cell. The present invention has found that the TCR-T cell can be used to specifically target tumor cells, so that it can play the role of tumor-targeted drug delivery.

[0193] The present invention does not specifically limit the drugs contained in specific TCR-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.

[0194] The present invention does not specifically limit how the drug is introduced into TCR-T cells. Exemplarily, for gene drugs such as circular mRNA drugs, etc., they can be introduced into TCR-T cells by electrotransfer, 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 TCR-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 TCR-T cells at the same time as the circular mRNA based on the Clean-PIE system is used to transform TCR-T cells, so that the TCR-T cells contain drugs while the characteristics are transformed. Exemplarily, for small molecule drug compounds, etc., small molecule compound drugs can be delivered into cells by electrotransfer, microfluidics, etc.

[0195] In order to facilitate specific operations, in some specific embodiments, the method of delivering a drug to a target cell comprises: administering the TCR-T cell containing the drug to a subject.

[0196] 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.

[0197] In some embodiments, the method of delivering a drug to a target cell is not for the purpose of diagnosing or treating a disease.

[0198] The nucleotide sequence and amino acid sequence of the present invention

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[0217] Example

[0218] 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.

[0219] Example 1. Preparation of circular mRNA based on Clean-PIE

[0220] 1.1 Experimental methods

[0221] The plasmid pUC57-NY-ESO-1TCR (nucleotide sequence as shown in SEQ ID NO.15) 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.

[0222] Table 1 Enzyme digestion system

[0223] Reagents volume Plasmids 10 μg Restriction endonuclease BsaI (1000 units) 5μL 10×cutsmart buffer 50μL Nuclease free water Total 500 μL

[0224] 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.

[0225] Table 2 In vitro transcription system

[0226] Reagents volume 10× Reaction Buffer 2μL ATP(20mM) 2μL CTP (20 mM) 2μL UTP(20mM) 2μL GTP(20mM) 2μL Linearized DNA template 1 μg T7 RNA Polymerase Mix 2μL <![CDATA[RNA Nuclease free, H2O]]> Total 20 μL

[0227] 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.

[0228] Table 3 Cyclization system

[0229] Solution volume mRNA 25 μg mRNA GTP solution (20 mM) 50μL GTP buffer Make up to 500 μL

[0230] 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.

[0231] 1.2 Experimental Results

[0232] Table 4 Experimental results

[0233]

[0234] 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.

[0235] Example 2. Detection of the killing effect of T cells on target cells in vitro after transfection of T cells with circular mRNA encoding TCR

[0236] 2.1 Experimental methods

[0237] PBMCs were isolated from the peripheral blood of healthy donors and cultured in cell culture flasks coated with anti-CD3 and anti-CD28 antibodies. T cells were taken on the 7th day after activation, and circular mRNA encoding TCR targeting NY-ESO-1 (nucleotide sequence as shown in SEQ ID NO.2) was electroporated into T cells using Yida electroporator. After 24 hours of electroporation, the TCR expression rate was detected. An appropriate amount of target cells K562-NY-ESO-1 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 ratios of TCR-T cells to target cells were 20:1, 5:1, and 1:1, respectively, with 2×10 4 / well of a 96-well (Round-bottom) plate, i.e. 100 μL / well of the plate. Then, according to different effector-target ratios, 100 μL / well of TCR-T cells were added to the target cells, and wells containing only target cells were set as the control group. After incubation in a 37°C incubator for 4 hours, the cells were collected, washed once with 1×PBS, and 100 μL of 1×PBS containing the cell death dye eFlour780 was added. The cells were placed in a 4°C refrigerator for staining for 30 minutes, washed once with 200 μL of 1×PBS, and centrifuged. The cells were resuspended in 200 μL of 1×PBS and filtered through a 70 μm filter. The cells were tested on the machine to determine the proportion of eFlour780-positive cells in the CellTrace-positive cell population.

[0238] 2.2 Experimental Results

[0239] Figure 1A The results showed that at different effector-target ratios, the killing ability of TCR-T cells prepared from healthy donor Donor1# was significantly enhanced compared with T cells (NC-T) that were not electroporated with circular mRNA encoding TCR targeting NY-ESO-1 (P < 0.0001), and the killing ratio increased exponentially with the increase of effector-target ratio. This verified that TCR-T prepared by circular mRNA has a strong killing function against tumor cells in vitro and has strong targeting. Figure 1B The results showed that at different effector-target ratios, TCR-T cells prepared from healthy donor Donor2# had significantly enhanced killing ability (P < 0.0001) compared with T cells (NC-T) that were not electroporated with circular mRNA encoding TCR targeting NY-ESO-1, and the killing ratio increased exponentially with the increase in the effector-target ratio. This proved that TCR molecules encoded by circular mRNA have active functions, can effectively identify target cells, and kill target cells, which has been verified on T cells from at least two donors.

[0240] Example 3. Detection of the killing effect of T cells on target cells in vivo after transfection of T cells with circular mRNA encoding TCR

[0241] 3.1 Experimental methods

[0242] PBMCs were isolated from the peripheral blood of healthy donors and cultured in cell culture flasks coated with anti-CD3 and anti-CD28 antibodies. T cells were taken on the 7th day after activation, and circular mRNA encoding NY-ESO-1 TCR (nucleotide sequence as shown in SEQ ID NO.2) was electroporated into T cells using Yida electroporator. After 24 hours of electroporation, the TCR expression rate was detected.

[0243] NCG mice (purchased from Jiangsu Jicui Yaokang Experimental Animal Co., Ltd.), 6-8 weeks old, weighing between 18 and 22 grams, were acclimated 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% to 70% and daily humidity difference <5%; temperature maintained at 20-25°C. K562-luci cells were injected into mice to construct an animal model.

[0244] On the third day after K562-luci cell inoculation (designated as day 0), the model animals were randomly divided into 6 groups, 6 in each group, namely solvent control group (untreated), NC-T-2E7 group and TCR-T-2E7 group. The solvent control group was injected with a resuspension of the preparation of TCR-T (95% of 0.9% sodium chloride injection + 5% human albumin) through the tail vein, the NC-T-2E7 group was injected with T cells electroporated without mRNA solution through the tail vein, and the TCR-T-2E7 group was injected with T cells electroporated with circular mRNA encoding TCR targeting NY-ESO-1 through the tail vein. The specific treatment plan is shown in the table below. During the experiment, live imaging was performed once a week to record the level of tumor changes.

[0245] Table 5 Treatment options

[0246] Group cell dose frequency way Untreated NA NA q4d,3 times iv NC-T-2E7 Mock-T 2E7 q4d,3 times iv TCR-T-2E7 TCR-T 2E7 q4d,3 times iv

[0247] The in vivo imaging experiment method is as follows: the experimental animals were intraperitoneally injected with luciferase substrate luciferin aqueous solution and placed in an anesthesia chamber for anesthesia. The 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).

[0248] 3.2 Experimental Results

[0249] TCR-T cells transformed by electroporating T cells with circular mRNA encoding TCR that recognizes target cells can recognize target cells at the in vivo level, exert killing function, and effectively curb the proliferation of tumor cells in the body. Figure 2A As shown in the figure, by the 43rd day after tumor inoculation, only 40% and 20% of mice in the vehicle control group and NC-T control group were alive, while 87.5% of mice in the TCR-T group were still alive. TCR-T cells modified by circular mRNA significantly improved the survival rate of tumor-bearing mice. Figure 2B and 2CThe bioluminescence values ​​of the NC-T group showed a time course in which the tumor growth rate approached that of the vehicle control group, and on the 30th day after tumor inoculation, the total bioluminescence value was close to 10 9 However, the tumor growth of the TCR-T group was always suppressed, and its total bioluminescence value hardly increased from the start of drug administration to 43 days after tumor inoculation, indicating that the improved survival rate of tumor-bearing mice caused by TCR-T cells modified by circular mRNA was due to the recognition and killing effect of TCR-T on target cells.

[0250] Example 4. TCR-T cells prepared by using Clean-PIE-based circular mRNA-modified lentivirus

[0251] 4.1 Experimental methods

[0252] Lentivirus packaging and lentivirus-mediated TCR-T cell preparation: 3×10 6 293T 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: envelope plasmid pMD2.G (nucleotide sequence as shown in SEQ ID NO.11, purchased from Wuhan Miaoling Biotechnology Co., Ltd.); packaging plasmid pSPAX2 (nucleotide sequence as shown in SEQ ID NO.12, purchased from Wuhan Miaoling Biotechnology Co., Ltd.); expression plasmid LV-NY-ESO-1TCR (nucleotide sequence as shown in SEQ ID NO.16), expression plasmid pLVX-Luc2-puro (nucleotide sequence as shown in SEQ ID NO.13, purchased from Shanghai Haijihaoge Biotechnology Co., Ltd.) as a vector, cloned and inserted into the NY-ESO-1TCR sequence (nucleotide sequence as shown in SEQ ID NO.1)), and the supernatant was collected on the fourth day, filtered through a 40 μm filter membrane, and then used to determine the virus titer. PBMCs were isolated and activated on the day of plasmid transfection. Cells were collected 48 hours after activation and transferred to 24-well plates coated with CD3 and CD28 antibodies. Lentiviral particles were added at an MOI of 50. After centrifugation at 1000g for 3 hours at room temperature, the cells were placed in an incubator for further culture. After 24 hours of infection, the culture medium was replaced, culture was continued, and transfection efficiency was tested.

[0253] The TCR-T cells prepared by lentivirus were divided into three groups: blank group, electroporated circular mRNA-GFP group and electroporated circular mRNA-IL-12 group. They were electroporated without any cells, electroporated with circular mRNA encoding GFP (nucleotide sequence as shown in SEQ ID NO.17) and electroporated with circular mRNA encoding IL-12sc (nucleotide sequence as shown in SEQ ID NO.18), respectively. After electroporation for 24 hours, appropriate amount of target cells K562-NY-ESO-1 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 TCR-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 effector-target ratio of 20:1, 100μL / well of TCR-T cells of different groups 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 and viability 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 cells were tested on the machine to determine the proportion of eFlour780-positive cells in the CellTrace-positive cell population.

[0254] 4.2 Experimental Results

[0255] like Figure 3 As shown, at different incubation ratios of effector T cells to target cells, TCR-T cells prepared by lentivirus achieved 13%, 21% and 26% target cell killing, respectively. When electroporated with circular mRNA encoding GFP, the killing ability of TCR-T cells on target cells did not change significantly, but when electroporated with circular mRNA encoding IL-12sc, the killing ability of TCR-T cells increased to 21%, 34% and 42%, respectively.

[0256] Example 5. Simultaneous preparation and modification of TCR-T cells using Clean-PIE-based circular mRNA

[0257] 5.1 Experimental Methods

[0258] 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 positive rate>95%) on the seventh day after activation were electroporated with circular mRNA encoding TCR molecules (nucleotide sequence as shown in SEQ ID NO.2) and circular mRNA encoding IL-2 (nucleotide sequence as shown in SEQ ID NO.3) (the obtained cells were recorded as TCR-T+IL-2), while empty electroporation (the obtained cells were recorded as NC-T) and cells electroporated only with circular mRNA encoding TCR molecules (nucleotide sequence as shown in SEQ ID NO.2) (the obtained cells were recorded as TCR-T) were used as control groups. Cells electroporated for 24 hours were taken for killing detection. An appropriate amount of target cells K562-NY-ESO-1 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. Design different ratios of effector cells to target cells according to the experimental design. Collect cells after incubation in a 37°C incubator for 4 hours, wash once with 1×PBS, add 100μL of 1×PBS containing cell death dye eFlour780, put in a 4°C refrigerator for staining for 30 minutes, add 200μL of 1×PBS to wash once, centrifuge, resuspend in 200μL of 1×PBS, and filter through a 70μm filter. Detect on the machine to determine the proportion of eFlour780-positive cells in the CellTrace-positive cell population.

[0259] 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 later use. Different ratios of effector cells to target cells were designed according to the experiment, and wells containing only target cells were set as control groups. After incubation in the incubator for 0.5h, Golgistop was added to each well, and the cells were incubated in the incubator for another 4.5h. The cells were collected, stained with IFN-γ and TNF-α antibodies, and the positive proportions of IFN-γ and TNF-α were detected by flow cytometry.

[0260] 5.2 Experimental Results

[0261] Figure 4A The results showed that at different effector-target ratios, the specific killing ability of T cells simultaneously electroporated with circular mRNA encoding TCR molecules and circular mRNA encoding IL-2 was significantly enhanced compared with TCR-T cells. Figure 4B It was shown that during the cell killing process, the secretion of IFN-γ by TCR-T+IL-2 cells was significantly increased compared with TCR-T cells that were not electroporated with circular mRNA encoding IL-2. Figure 4CThe results showed that the secretion of TNF-α during cell killing was significantly increased in TCR-T+IL-2 cells compared to TCR-T cells that were not electroporated with circular mRNA encoding IL-2. This proved that both IL-2 and TCR molecules encoded by circular mRNA have active functions, can effectively identify target cells, kill target cells, and IL-2 improves the killing ability of TCR-T cells.

[0262] Example 6. Enhancement of TCR-T migration ability using Clean-PIE-based circular mRNA

[0263] 6.1 Experimental methods

[0264] PBMCs were isolated from fresh peripheral blood of healthy donors and 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 TCR molecules (nucleotide sequence as shown in SEQ ID NO.2) and circular mRNA encoding CCL5 (nucleotide sequence as shown in SEQ ID NO.5) (the resulting cells were recorded as TCR-T+CCL5), while empty electroporation (the resulting cells were recorded as NC-T) and cells electroporated only with circular mRNA encoding TCR molecules (nucleotide sequence as shown in SEQ ID NO.2) (the resulting cells were recorded as TCR-T) were used as control groups. The chemotaxis of TCR-T cells was measured by migration through a polycarbonate filter with a pore size of 8μm in a 24-well Transwell chamber. Cells migrating from the upper chamber to the lower chamber were evaluated by flow cytometry.

[0265] T cell migration assay specific method: In a 24-well Transwell chamber (purchased from Corning), the chemotaxis of TCR-T cells was measured by the migration rate of a polycarbonate filter with a pore size of 8 μm. The target cells K562-NY-ESO-1 were added to the lower chamber to stimulate the above different cells, and the responder cells pre-labeled with cell trace (purchased from thermo fisher) were incubated in the upper chamber for 3-5 hours. The cells that migrated from the upper chamber to the lower chamber were evaluated by flow cytometry.

[0266] 6.2 Experimental Results

[0267] like Figure 5 As shown, the number of TCR-T+CCL5 cells that migrated from the upper chamber to the lower chamber was significantly greater than that of TCR-T cells, which proved that the migration ability of TCR-T+CCL5 cells was greatly improved compared with that of TCR-T cells.

[0268] Example 7. Preparation of TCR-T cells using Clean-PIE-based circular mRNA to enable them to have drug delivery capabilities

[0269] 7.1 Experimental methods

[0270] Lymphocytes from mouse spleen were cultured in 24-well plates coated with anti-mouse CD3 and anti-mouse CD28 antibodies. T cells were taken 7 days after activation for electroporation, and the experimental groups were as follows:

[0271] Table 6 Experimental grouping

[0272]

[0273] 70 female C57BL / J mice aged 6-8 weeks were subcutaneously inoculated with 5E5 B16F10-OVA cells. After 7-10 days, the subcutaneous tumors were about 50 mm 3 At the same time, the tumor-bearing mice were randomly divided into 7 groups. 5E6 T cells with the above serial numbers 1-6 were injected through the tail vein respectively. One group was a control group that received tail vein injection of solvent (95% of 0.9% sodium chloride injection + 5% human albumin). 24 hours after administration, 5 mice in each group were euthanized, tumor tissues were taken, the epidermis and connective tissues were removed, the surface blood was washed with 1×PBS, and the cells were placed in a 2mL centrifuge tube. An appropriate amount of 1×PBS and protease inhibitors (Roche) were added for homogenization. After homogenization, the supernatant was centrifuged and the content of the corresponding delivered drug inside the tumor was detected. The tumor volume and survival rate of the remaining mice in each group were observed. When the tumor size reached about 2000mm 3 Or when there were animal health problems (ulceration of 20% of the tumor area), the animals were euthanized.

[0274] 7.2 Experimental Results

[0275] like Figure 6A to Figure 6E As shown, compared with the mice in the first experimental group (injected with the above-mentioned T cell No. 1), higher levels of the corresponding secreted protein drugs were detected in the tumors of the mice in other experimental groups, proving that circular mRNA can be used to transform T cells to achieve targeted drug delivery.

Claims

1. A method for preparing TCR-T cells, characterized in that: The method comprises the steps of introducing circular mRNA into non-T cell receptor gene-modified T cells; Furthermore, the circular mRNA includes a first circular mRNA encoding a T cell 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 T cell receptor targets NY-ESO-1.

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.2, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.2, and encodes a sequence of a protein having or partially having the T cell receptor activity.

4. A method for transforming TCR-T cells, characterized in that: The method comprises the steps shown in (i) or (ii): (i) a step of introducing circular mRNA into non-T cell receptor gene-modified T cells; (ii) a step of introducing circular mRNA into TCR-T cells; In the step (i), the circular mRNA includes a first circular mRNA encoding a T cell 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 T cell receptor targets NY-ESO-1; the functional polypeptide includes any one or more of IL-2, IL-12sc, CCL5, XCL1, Flt3L-Fc, GM-CSF, IFN-α and anti-PD-1 nanoantibodies.

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.2, or has at least 80% sequence identity with the sequence shown in SEQ ID NO.2, and encodes a sequence of a protein having or partially having the T cell receptor activity; The nucleotide sequence of the second circular mRNA comprises a sequence as shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.18, or comprises a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.18, and encodes a protein having or partially having the activity of the functional polypeptide.

7. A TCR-T cell, characterized in that: The TCR-T cell is 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 TCR-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 TCR-T cell according to claim 7 containing the drug to a subject.

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