RNA cancer therapeutics expressing immune cell engager molecules

Abstract

A RNA cancer therapeutic agent expressing an immune cell engagement molecule, wherein the RNA cancer therapeutic agent has a therapeutic effect on tumors and can achieve a highly effective inhibitory effect on tumor cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an RNA cancer therapeutic agent expressing a cell-engaging molecule. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Immune cell engager molecules are molecular structures that can connect immune cells and target cells. They are usually bispecific antibodies or other molecules with dual binding functions. One end of the molecules can bind to activation receptors on the surface of immune cells (such as T cells, NK cells, etc.), and the other end can bind to antigens on the surface of target cells (such as tumor cells, virus-infected cells), thereby shortening the distance between immune cells and target cells and initiating the killing effect of immune cells on target cells.

[0004] Natural killer (NK) cells are a unique type of innate immune cell that mediates antiviral and antitumor responses. Therefore, NK cells may have great potential in tumor immunotherapy.

[0005] NK cell activating receptors include activating killer cell Ig-like receptors (KIR), activating receptors (NKG2D) and natural cytotoxicity receptors (NCR). Activating KIR can recognize classical MHC-I molecules, while NKG2D recognizes non-classical MHC-I molecules. Some activated NK cell receptors, such as NKG2D, NKp30, NKp46 and NKp44, can recognize stress-induced ligands expressed on target cells, thereby providing positive signals for NK cells to kill target cells, thereby turning on the killing mode.

[0006] The tumor microenvironment (TME) is highly complex and constantly evolving. It can sustain tumor growth and influence immune escape and malignancy progression. In addition to stromal cells, fibroblasts and endothelial cells, the TME also includes innate and adaptive immune cells.

[0007] NK cells are commonly found in the TME of human tumors, including primary tumors, metastases, and tumor-infiltrating lymph nodes. When NK cells are recruited by integrins, chemokine receptors, and selectins, they extravasate from the blood, traverse the ECM and tumor stroma, and reach the tumor bed. In the TME, NK cells eliminate tumors by degranulation, ADCC, or FASL / TRAIL-induced apoptosis. In addition, NK cells can secrete cytokines or chemokines to recruit other immune cells and upregulate their anti-tumor responses. The responsiveness of NK cells is often hampered by inhibitory factors secreted by tumor cells or other cells or by direct cell-cell interactions.

[0008] In recent years, research on NK cell-related immunotherapy has flourished, and the latest progress has mainly focused on cytokine therapy, adoptive NK cell therapy, genetically engineered NK cell therapy, monoclonal antibodies and NK cell engagement molecules. In addition, NK cell-based therapy has achieved good results whether used alone or in combination with other treatments, indicating that it has a broad and effective application prospect in malignant tumors. Summary of the invention

[0009] The purpose of the present invention is to provide an RNA cancer therapeutic agent expressing an immune cell engager molecule, wherein the RNA cancer therapeutic agent has a therapeutic effect on tumors and can achieve a highly effective inhibitory effect on tumor cells.

[0010] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0011] The present invention provides an RNA cancer therapeutic agent expressing an immune cell engagement molecule, wherein the RNA cancer therapeutic agent expressing an immune cell engagement molecule comprises an RNA molecule and a delivery formulation encapsulating the RNA, wherein the RNA molecule expresses the immune cell engagement molecule.

[0012] In an optional embodiment, the RNA molecule comprises: a molecular fragment (A) and a molecular fragment (C). In an optional embodiment, the molecular fragment (A) encodes an NK cell activating receptor, and the NK cell activating receptor is selected from NKG2D receptor, KIR2DL4 receptor, NKp44 receptor, NKp46 receptor, NKp30 receptor, and NKG2C receptor; the molecular fragment (C) encodes an antibody that specifically binds to immune cell surface membrane proteins, and the antibody that specifically binds to immune cell surface membrane proteins is selected from anti-CD3 antibodies or anti-CD16 antibodies.

[0013] In an optional embodiment, the amino acid sequence of the NKG2D receptor is shown in SEQ ID NO.9.

[0014] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.11 or SEQ ID NO.17, and the amino acid sequence of the light chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.12 or SEQ ID NO.19.

[0015] In an optional embodiment, the RNA molecule includes a molecular fragment (B), and the molecular fragment (B) connects the molecular fragment (A) and the molecular fragment (C); the amino acid sequence encoded by the molecular fragment B is selected from at least one of a GS linker sequence, a CD3 antibody Fc region, and a 2A peptide.

[0016] In an optional embodiment, the molecular fragment B encodes a GS linker sequence, and the GS linker sequence is selected from one of (GnS)m, ((GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q; wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; q is an integer from 1 to 5.

[0017] In an optional embodiment, the amino acid sequence of the GS linker sequence is shown in SEQ ID NO.13.

[0018] In an optional embodiment, the open reading frame sequence of the nucleotide molecule encoding the extracellular domain of the NKG2D receptor is shown as SEQ ID NO.10.

[0019] In an optional embodiment, the nucleotide sequence encoding the heavy chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.15 or SEQ ID NO.18, and the nucleotide sequence encoding the light chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.16 or SEQ ID NO.20.

[0020] In an optional embodiment, the antibody that specifically binds to an immune cell surface membrane protein comprises an Fc region.

[0021] In an optional embodiment, the amino acid sequence of the anti-CD3 antibody Fc region is shown in SEQ ID NO.21.

[0022] In an optional embodiment, the RNA molecule includes a molecular fragment (B) encoding a cytokine, and the cytokine is selected from one or more of cytokine IL12, cytokine IL2, cytokine IL36, cytokine IL7, cytokine IL15, and cytokine IL23.

[0023] In an optional embodiment, the RNA molecule encoding the NK cell activating receptor further comprises a molecular fragment encoding a marker protein (D), and / or a molecular fragment encoding a signal peptide (E).

[0024] In an alternative embodiment, the marker protein is selected from the group consisting of a His tag, a FLAG tag, a HA tag and a MYC tag.

[0025] In an optional embodiment, the RNA cancer therapeutic agent further comprises an antibody selected from anti-PD-1 antibody, anti-CTLA4 antibody, anti-TIGIT antibody, anti-TIM3 antibody, and anti-LAG3 antibody.

[0026] In an alternative embodiment, the RNA molecule is mRNA.

[0027] In an alternative embodiment, the mRNA consists of a sequence including a 5' cap, a 5' UTR, an ORF, a 3' UTR and a 3' poly (A) tail in sequence from the 5' end to the 3' end.

[0028] In an alternative embodiment, the RNA cancer therapy further comprises a delivery formulation encapsulating the RNA molecule.

[0029] In alternative embodiments, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0030] In an alternative embodiment, the components of the lipid nanoparticles include at least one of a protonable cationic lipid, a structural lipid, an auxiliary lipid and a surfactant.

[0031] In an optional embodiment, the components of the lipid nanoparticles include, by weight: 40 to 60 parts of protonable cationic lipids, 20 to 40 parts of structural lipids, 10 to 30 parts of auxiliary lipids and 0.5 to 5 parts of surfactants.

[0032] In an optional embodiment, the protonable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200 and DlinDMA.

[0033] In alternative embodiments, the structured lipid comprises cholesterol, cholesterol ester, steroid hormone, steroid vitamin or bile acid.

[0034] In an alternative embodiment, the helper lipid comprises at least one of DSPC, DOPE, DOPC, DOPG and DOPS.

[0035] In an alternative embodiment, the surfactant includes at least one of PEG-DMG, PEG-DSPE and TPGS.

[0036] In an alternative embodiment, the liposome nanoparticles comprise, by mole percentage, 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol and 1.5% PEG-DMG.

[0037] Another object of the present invention is to provide a method for preparing the aforementioned RNA cancer therapeutic agent, which comprises: mixing the RNA molecule and a delivery preparation to form an RNA cancer therapeutic agent; the delivery preparation comprises lipid nanoparticles or cationic liposomes.

[0038] In an optional embodiment, in the method for preparing the RNA cancer therapeutic agent, mRNA is dissolved in a buffer to obtain an aqueous phase, each lipid component of the liposome nanoparticle is measured and dissolved in an organic solvent to obtain an organic phase, and the aqueous phase and the organic phase are mixed and then the organic phase is removed to obtain the RNA cancer therapeutic agent.

[0039] Another object of the present invention is to provide a biomaterial, characterized in that the biomaterial comprises at least one of the following (x1) to (x4);

[0040] (x1) a DNA molecule or RNA molecule encoding any of the aforementioned RNA cancer therapeutic agents;

[0041] (x2) an expression cassette or vector containing (x1);

[0042] (x3) a microorganism comprising at least one of (x1) and (x2);

[0043] (x4) A cell comprising at least one of (x1) and (x2). BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 This is one of the schematic diagrams of the RNA molecule structure that contains the RNA encoding the NK cell activating receptor and the RNA encoding the antibody that specifically binds to the immune cell surface membrane protein.

[0046] Figure 2 This is the second schematic diagram of the RNA molecule structure that contains the RNA encoding the NK cell activating receptor and the RNA encoding the antibody that specifically binds to the immune cell surface membrane protein.

[0047] Figure 3 This is the third schematic diagram of the RNA molecular structure that contains the RNA encoding the NK cell activating receptor and the RNA encoding the antibody that specifically binds to the immune cell surface membrane protein.

[0048] Figure 4 This is the third schematic diagram of the RNA molecular structure that contains the RNA encoding the NK cell activating receptor and the RNA encoding the antibody that specifically binds to the immune cell surface membrane protein. Figure 5 Schematic diagram of the tumor inoculation position in each group of mouse experiments in the examples of the present invention. Figure 6This is the killing of HepG2 / LUC by PBMC induced by the sample in Example 2 of the present invention. Figure 7 This is the activation of Jurkat T cells induced by the sample in Example 3 of the present invention. Figure 8 This is a curve diagram of tumor volume change in Example 4 of the present invention. Fig. 9 This is the killing of HepG2 / LUC by PBMC induced by the sample in Example 5 of the present invention. Fig.10 This is the expression of CD69 in Jurkat cells induced by the sample in Example 6 of the present invention. Fig.11 This is a curve diagram of tumor volume change in Example 7 of the present invention. Fig.12 This is the expression of CD69 in Jurkat cells induced by the sample in Example 8 of the present invention. Fig.13 This is the expression of CD69 in Jurkat cells induced by the sample in Example 8 of the present invention. In the accompanying drawings, “antibody VH sequence” is the antibody heavy chain variable region sequence; “antibody VL sequence” is the antibody light chain variable region sequence; and “antibody Fc” is the sequence of the antibody Fc region. DETAILED DESCRIPTION

[0049] The two arms of the cell engagement molecule are generally bispecific antibody structures, one of which can specifically recognize tumor-associated antigens and the other arm recognizes immune cells. When the immune cell engagement molecule binds to tumor cells and immune cells, it can bring the two cells closer together, allowing the immune cell receptors of the immune cells to be more effectively activated. The activated immune cells will release cytotoxic substances such as perforin and granzymes to directly kill tumor cells, and can also further activate the body's anti-tumor immune response by secreting cytokines such as interferon-γ.

[0050] The production process of protein drugs that are cell-engaging molecules is complex, including protein expression and purification, which requires strict quality control.

[0051] The purpose of the present invention is to provide an RNA cancer therapeutic agent that expresses an immune cell engager molecule, wherein the RNA cancer therapeutic agent expresses an immune cell engager molecule, has a therapeutic effect on tumors, and can achieve a highly effective inhibitory effect on tumor cells.

[0052] The RNA cancer therapeutic agent expressing the immune cell engager molecule of the present invention comprises an RNA molecule encoding the immune cell engager molecule.

[0053] The functional region of the immune cell engagement molecule of the present invention that specifically recognizes tumor-associated antigens is selected from NK cell activating receptors.

[0054] NK cell activating receptors can recognize ligands on the surface of target cells, thereby activating the killing activity of NK cells. NK cell activating receptors are structurally diverse, and they play a key role in the process of NK cells identifying and clearing abnormal cells such as virus-infected cells and tumor cells. Common NK cell activating receptors include the immunoglobulin-like activating receptor (KIR) family and C-type lectin-like activating receptors (NKG2C, etc.).

[0055] The NK cell activating receptor of the present invention is selected from the group consisting of NKG2D receptor, KIR2DL4 receptor, NKp44 receptor, NKp46 receptor, NKp30 receptor and NKG2C receptor.

[0056] The NK cell activating receptor that specifically recognizes tumor-associated antigens in the immune cell engagement molecule of the present invention is specifically selected from the extracellular domain of the NK cell activating receptor.

[0057] The extracellular segment of KIR family receptors has an immunoglobulin (Ig)-like domain, generally with 2 or 3 Ig-like domains. The sequence and structure of these Ig-like domains determine their specificity for binding to ligands. For example, KIR2DL and KIR3DL mainly recognize HLA-I class molecules, and they interact with specific regions on HLA-I class molecules through their Ig-like domains.

[0058] C-type lectin-like receptors include NK cell lectin-like receptor K1, also known as NK cell family 2 member D (NKG2D), and CD94 / NKG2C. CD94 / NKG2A mainly binds to non-classical MHC-ⅠB molecules, such as human leukocyte antigen E (HLA-E), etc., to exert autonomous control of NK cells and counteract excessive inflammatory responses caused by cytolysis mediated by own NK cells. NKG2D is a homodimer and an activating receptor for NK cells that can recognize several tumor-related and stress-induced ligands, such as MICA, MICB, and ULBP (UL16-binding protein) 1-6 in humans, and Rae1 family members H60 and MULT-1 / ULBP-1 in mice.

[0059] The functional region of the other arm of the immune cell engagement molecule of the present invention that recognizes immune cells is selected from at least one of recognizing T cell antibodies and recognizing NK cell antibodies.

[0060] The T cell recognition antibody of the present invention is selected from anti-CD3 antibodies.

[0061] The CD3 molecule is an important component of the T cell receptor (TCR) complex and is expressed on the surface of all mature T cells. Anti-CD3 antibodies can recognize T cells and can be used to detect the number of T cells in peripheral blood or tissues by binding to CD3 molecules. In flow cytometry analysis, anti-CD3 antibodies are used in combination with other marker antibodies (such as anti-CD4 or anti-CD8 antibodies) to distinguish different subpopulations of T cells, such as CD3+CD4+ helper T cells and CD3+CD8+ cytotoxic T cells.

[0062] The NK cell recognition antibody of the present invention is selected from anti-CD16 antibody.

[0063] CD16, or FcγRIII, is an Fc receptor on the surface of NK cells, and is mainly involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Anti-CD16 antibodies can be used to identify CD16 molecules on the surface of NK cells, and have important applications in studying the ADCC function of NK cells and developing NK cell-based immunotherapy strategies (such as bispecific antibody-mediated NK cell activation).

[0064] The RNA molecule encoding the immune cell engagement molecule of the present invention is a messenger RNA (mRNA molecule), which includes sequences of multiple coding functional regions.

[0065] In an optional embodiment, the RNA molecule includes at least three sequences encoding functional regions, namely molecular fragment (A), molecular fragment (B) and molecular fragment (C), and the molecular fragment (B) connects the molecular fragment (A) and the molecular fragment (C).

[0066] In the accompanying drawings, “antibody VH sequence” is the antibody heavy chain variable region sequence; “antibody VL sequence” is the antibody light chain variable region sequence; and “antibody Fc” is the sequence of the antibody Fc region.

[0067] In the figure, the linker sequences 1-10 are selected from at least one of a GS linker sequence and a 2A peptide.

[0068] In the attached Figure 1 In the present invention, the molecular fragment B is the connecting sequence 2. Figure 2 In the present invention, the molecular fragment B is the connecting sequence 3. Figure 3 In the present invention, the molecular fragment B is a combination of connecting sequence 5+antibody Fc+connecting sequence 6. Figure 4 In the embodiment, the molecular fragment B described in the present invention is a combination of connecting sequence 9+antibody Fc+connecting sequence 10.

[0069] Attached Figure 1-4 The RNA molecule shown above, from left to right, can be from the 5' end to the 3' end or from the 3' end to the 5' end.

[0070] In an optional embodiment, the molecular fragment B encodes a GS linker sequence, and the GS linker sequence is selected from one of (GnS)m, ((GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q; wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; q is an integer from 1 to 5.

[0071] In an optional embodiment, the amino acid sequence of the GS linker sequence is shown in SEQ ID NO.13.

[0072] In an optional embodiment, the molecular fragment (A) encodes the extracellular domain of an NK cell activating receptor of one arm of an immune cell engagement molecule; in an optional embodiment, the NK cell activating receptor is selected from NKG2D receptor, KIR2DL4 receptor, NKp44 receptor, NKp46 receptor, NKp30 receptor, NKG2C receptor. In an optional embodiment, the amino acid sequence encoded by the molecular fragment B is selected from at least one of a GS linker sequence, a CD3 antibody Fc region, and a 2A peptide. In an optional embodiment, the molecular fragment (C) encodes an antibody that specifically binds to an immune cell surface membrane protein in one arm of an immune cell engagement molecule; in an optional embodiment, in an optional embodiment, the antibody that specifically binds to an immune cell surface membrane protein includes a heavy chain variable region and a light chain variable region; in an optional embodiment, the antibody that specifically binds to an immune cell surface membrane protein is selected from an anti-CD3 antibody or an anti-CD16 antibody.

[0073] In an optional embodiment, the amino acid sequence of the NKG2D receptor is shown in SEQ ID NO.9.

[0074] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.11 or SEQ ID NO.17, and the amino acid sequence of the light chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.12 or SEQ ID NO.19; the amino acid sequence of the heavy chain variable region of the anti-CD16 antibody is shown as SEQ ID NO.24, and the amino acid sequence of the light chain variable region of the anti-CD16 antibody is shown as SEQ ID NO.23.

[0075] In an optional embodiment, the open reading frame sequence of the nucleotide molecule encoding the NKG2D receptor is shown as SEQ ID NO.16;

[0076] In an optional embodiment, the nucleotide sequence encoding the heavy chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.17 or SEQ ID NO.18, and the nucleotide sequence encoding the light chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.16 or SEQ ID NO.20.

[0077] In an optional embodiment, the GS linker sequence is selected from one of (GnS)m, ((GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q; wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; q is an integer from 1 to 5. In an optional embodiment, the amino acid sequence of the GS linker sequence is shown in SEQ ID NO.13.

[0078] In an optional embodiment, the RNA molecule further comprises a sequence encoding a cytokine functional region, and the cytokine is selected from one or more of cytokine IL12, cytokine IL2, cytokine IL36, cytokine IL7, cytokine IL15, and cytokine IL23.

[0079] In an optional embodiment, the RNA molecule encoding the NK cell activating receptor further comprises a sequence encoding a marker protein functional region and / or a sequence encoding a signal peptide functional region.

[0080] In an optional embodiment, the marker protein is selected from the group consisting of a His tag, a FLAG tag, a HA tag, a MYC tag, a GST tag, and a SUMO tag.

[0081] In an optional embodiment, the signal peptide is selected from IL-2 signal peptide, VSV-G signal peptide, human OSM signal peptide, Albumin (HSA) signal peptide, BM40 signal peptide, influenza hemagglutinin signal peptide, human insulin signal peptide, silkworm fibrin LC signal peptide, human trypsinogen-2 signal peptide, Gaussia luc signal peptide, human chymotrypsinogen signal peptide.

[0082] In an optional embodiment, the amino acid sequence of the IL-2 signal peptide is shown in SEQ ID NO.25.

[0083] In an optional embodiment, the nucleotide sequence encoding the IL-2 signal peptide is shown as SEQ ID NO.28.

[0084] In an optional embodiment, the RNA molecule encodes the immune cell engagement molecule amino acid sequence as shown in 37.

[0085] In an optional embodiment, the nucleotide sequence of the RNA molecule is as shown in 38.

[0086] In an alternative embodiment, the RNA molecule is selected from mRNA or circular RNA.

[0087] In an alternative embodiment, the mRNA described in the present disclosure consists of a sequence including a 5' cap, a 5' UTR, an ORF, a 3' UTR and a 3' poly (A) tail in sequence from the 5' end to the 3' end.

[0088] In an optional embodiment, the mRNA described in the present disclosure also carries a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase, RdRP), and specifically introduces the sequence design content of Chinese patent CN202110424124.2 regarding self-replicating mRNA into the present disclosure.

[0089] In an optional embodiment, the 5' end cap is selected from ARCA, mCAP, dmCAP, m7G(5"")ppp(5"")(2""OMeA)pG, tmCAP, m7(3""OMeG)(5"")ppp(5"")(2""OMeA)pG, m7(3""OMeG)(5"")ppp(5"")(2""OMeG)pG, dmCAP or m7G(5"")ppp(5"")(2""OMeG)pG;

[0090] In an optional embodiment, the 5' end cap is m7Gppp(5')(2'-OMeA)pG.

[0091] In an optional embodiment, the length of the 5'UTR is preferably 10 to 200 nucleotides.

[0092] In an alternative embodiment, the length of the 5'UTR is 15 to 100 nucleotides.

[0093] In an optional embodiment, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1-3.

[0094] In an optional embodiment, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1.

[0095] In an optional embodiment, the 3'UTR sequence is shown in Seq ID NO. 4-6.

[0096] In an optional embodiment, the 3'UTR sequence is shown in Seq ID NO.4.

[0097] In an alternative embodiment, one or more uridines in the mRNA are replaced with modified nucleosides.

[0098] In an alternative embodiment, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U).

[0099] In an alternative embodiment, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0100] The 5' untranslated region (UTR) described in the present disclosure refers to a sequence located immediately upstream of the start codon of an mRNA that does not encode a polypeptide. When an RNA transcript is produced, the 5'UTR may include a promoter sequence. Such promoter sequences are known in the art. The RNA sequence of the 5'UTR is shown in one of Seq ID NO.1, Seq ID NO.2 or Seq ID NO.3, or a 5'UTR sequence disclosed before the filing date of the present disclosure may also be introduced into the present disclosure.

[0101] The 3' untranslated region (UTR) described in the present disclosure refers to a sequence located downstream of the stop codon of an mRNA that does not encode a polypeptide. The RNA sequence of the 3'UTR is shown in one of Seq ID NO.4, Seq ID NO.5 or Seq ID NO.6, or a 3'UTR sequence disclosed before the filing date of the present disclosure may be introduced into the present disclosure.

[0102] The poly (A) tail described in the present disclosure is a sequence located downstream of the 3'UTR of mRNA containing multiple consecutive adenosine monophosphates. The poly (A) tail may contain 10 to 300 adenosine monophosphates. In cells and / or in vivo, the poly (A) tail is used to protect mRNA from enzymatic degradation and to help transcription termination, and / or mRNA export and translation from the nucleus.

[0103] In an optional embodiment, based on the mRNA open reading frame sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding circular RNA open reading frame sequence, and according to the contents of the public documents such as CN202180048567.4, a circular RNA complete sequence capable of encoding the same amino acid sequence is prepared. In an optional embodiment, based on the mRNA sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding DNA sequence (such as uracil is converted to thymine). Similarly, based on the DNA sequence provided, a person of ordinary skill in the art will obtain the corresponding RNA sequence (such as thymine is converted to uracil). In an optional embodiment, based on the RNA or DNA sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding amino acid sequence.

[0104] The open reading frame (ORF) described in the present disclosure is a continuous stretch of DNA or RNA that begins with a start codon (such as ATG or AUG) and ends with a stop codon (such as TAA, TAG or TGA, or UAA, UAG or UGA).

[0105] In alternative embodiments, the mRNA molecule comprises a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

[0106] In an optional embodiment, the mRNA described in the present disclosure is a self-replicating mRNA, which carries a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase, RdRP). Specifically, the sequence design content of Chinese patent CN202110424124.2 regarding self-replicating mRNA is introduced into the present disclosure.

[0107] The RNA cancer therapeutic agent expressing the immune cell engagement molecule provided by the present invention also comprises a delivery preparation encapsulating the aforementioned RNA molecule.

[0108] In an optional embodiment, the RNA cancer therapeutic agent expressing an immune cell engagement molecule further comprises an antibody selected from anti-PD-1 antibody, anti-CTLA4 antibody, anti-TIGIT antibody, anti-TIM3 antibody, anti-LAG3 antibody.

[0109] In alternative embodiments, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0110] In an alternative embodiment, the components of the lipid nanoparticles include at least one of a protonable cationic lipid, a structural lipid, an auxiliary lipid and a surfactant.

[0111] In an optional embodiment, the components of the lipid nanoparticles include, by weight: 40 to 60 parts of protonable cationic lipids, 20 to 40 parts of structural lipids, 10 to 30 parts of auxiliary lipids and 0.5 to 5 parts of surfactants.

[0112] In an optional embodiment, the protonable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200 and DlinDMA.

[0113] In alternative embodiments, the structured lipid comprises cholesterol, cholesterol ester, steroid hormone, steroid vitamin or bile acid.

[0114] In an alternative embodiment, the helper lipid comprises at least one of DSPC, DOPE, DOPC, DOPG and DOPS.

[0115] In an alternative embodiment, the surfactant includes at least one of PEG-DMG, PEG-DSPE and TPGS.

[0116] In an optional embodiment, the liposome nanoparticles include, by molar percentage, 20% to 50% of cationic lipids, such as but not limited to 20%, 25%, 30%, 35%, 40%, 45% or 50%; 10% to 50% of DSCP, such as but not limited to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%; 5% to 20% of cholesterol, such as but not limited to 5%, 10%, 15% or 20%; and 1% to 5% of PEG-DMG, such as but not limited to 1%, 1.5%, 2%, 3%, 4% or 5%.

[0117] In an alternative embodiment, the liposome nanoparticles comprise, by mole percentage, 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol and 1.5% PEG-DMG.

[0118] In an optional embodiment, the present disclosure provides a method for preparing the RNA cancer therapeutic agent described in any of the aforementioned embodiments, characterized in that it includes: mixing the RNA molecule and a delivery formulation to form an RNA cancer therapeutic agent; the delivery formulation includes lipid nanoparticles or cationic liposomes.

[0119] In an optional embodiment, in the method for preparing the RNA cancer therapeutic agent, the RNA molecule is dissolved in a buffer to obtain an aqueous phase, each lipid component of the liposome nanoparticle is measured and dissolved in an organic solvent to obtain an organic phase, and the aqueous phase and the organic phase are mixed and then the organic phase is removed to obtain the RNA cancer therapeutic agent.

[0120] In an optional embodiment, the volume ratio of the aqueous phase to the organic phase is 1:2 to 4, preferably 1:3. In an optional embodiment, the buffer comprises a citrate buffer or sodium acetate, preferably a citrate buffer. In an optional embodiment, the pH of the buffer is 3 to 7, preferably 4. In an optional embodiment, the concentration of RNA molecules in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL.

[0121] In an optional embodiment, the organic solvent is selected from C1 to C4 low-carbon alcohols, preferably anhydrous ethanol. In an optional embodiment, the concentration of the lipid component in the organic phase is 5 mg / mL to 7 mg / mL, preferably 6 mg / mL. In an optional embodiment, a microfluidic system is used to mix the aqueous phase and the organic phase, and a tangential flow filter is used to filter the organic solvent. Preferably, the flow rate of the microfluidic system is >3 ml / min, and more preferably 12 ml / min.

[0122] In an optional embodiment, a concentration step is further included after mixing, wherein the concentration step makes the final concentration of the RNA molecule 50 μg / mL to 200 μg / mL, preferably 100 μg / mL.

[0123] In an alternative embodiment, the diameter of the lipid nanoparticle is less than about 200 nm. In an alternative embodiment, the diameter of the lipid nanoparticle is less than about 150 nm. In an alternative embodiment, the diameter of the lipid nanoparticle is less than 100 nm. In an alternative embodiment, the diameter of the lipid nanoparticle is about 55 nm to about 90 nm.

[0124] In an optional embodiment, the present disclosure provides a biomaterial, characterized in that the biomaterial comprises at least one of the following (x1) to (x4);

[0125] (x1) a DNA molecule or RNA molecule encoding any of the aforementioned RNA cancer therapeutic agents;

[0126] (x2) an expression cassette or vector containing (x1);

[0127] (x3) a microorganism comprising at least one of (x1) and (x2);

[0128] (x4) A cell comprising at least one of (x1) and (x2).

[0129] In an optional embodiment, the RNA cancer therapeutic agent described in the present disclosure may further include at least one cryoprotectant. In an optional embodiment, the cryoprotectant is selected from at least one of sucrose and glycerol.

[0130] The RNA cancer therapeutic agent disclosed herein can be administered by the following routes, but is not limited to: intramuscular injection, subcutaneous injection, intravenous injection, oral inhalation administration, nasal drops, nasal spray administration, and oral and nasal inhalation administration.

[0131] The RNA cancer therapeutics described herein can be formulated into the following dosage forms, but are not limited to the following: injection, aerosol, dry powder inhaler / powder spray, spray and / or nebulized inhalation solution.

[0132] The dosage of the active pharmaceutical ingredient of the RNA cancer therapeutic agent of the present invention is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day.

[0133] In the present invention, preferably, the amount of the RNA cancer therapeutic agent administered is an effective amount, which is an amount that can inhibit the growth of a tumor or eliminate a tumor. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the results sought. A person skilled in the art can determine the effective amount by using the above factors such as individual basis and using experiments that do not exceed routine.

[0134] When using RNA cancer therapeutics, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dose is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0135] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0136] 1. Construction of PBMC-Huh7 tumor model

[0137] Selection of experimental animals (1) Species: mouse; (2) Strain: C-NKG; (3) Grade: SPF grade; (4) Age: 6 weeks (age at the time of inoculation); (5) Gender: female.

[0138] Establishment of humanized mouse model: 11 days before administration (Day-11), human PBMC cells were resuscitated, washed with PBS, and then injected into C-NKG mice through the tail vein at an injection volume of 5×10 6 cells / 100μL / cell.

[0139] Tumor inoculation: Huh7 cells were revived and subcultured. Huh7 cells in the logarithmic growth phase were collected 7 days before administration (Day-7), the culture medium was removed and the cells were washed twice with PBS before inoculation. The inoculation volume was 5×10 6 cells / 100μL / cell (Matrigel 1:1), inoculation position as Figure 1 Position 3 shown.

[0140] Drug administration: When the tumor volume grows to 100mm 3 Around (Day 0), that is, 7 days after tumor inoculation, the mRNA drug was injected into the tail vein once a week for a total of 3 times.

[0141] 2. Construction of A20 tumor model in BALB / c mice

[0142] Selection of experimental animals (1) Species: mouse; (2) Strain: BALB / c; (3) Grade: SPF grade; (4) Age: 6 weeks (age at the time of inoculation); (5) Gender: female.

[0143] Tumor inoculation: A20 tumor cells were revived and subcultured. A20 tumor cells in the logarithmic growth phase were collected 5 days before administration (Day-5), the culture medium was removed and the cells were washed twice with PBS before inoculation. The inoculation volume was 5×10 5 cells / 100μL / cell (Matrigel 1:1), inoculation position as Figure 1 Position 3 shown.

[0144] Drug administration: When the tumor volume grows to 100mm 3 Around (Day 0), that is, 5 days after tumor inoculation, mRNA drugs were injected into the tumor once a week for a total of 3 times.

[0145] 3. Animal welfare

[0146] During the experiment, the tumor size, mouse weight, and clinical pathology were observed. Clinical scores (weight, mouse posture, activity, hair, skin, etc.) were evaluated based on clinical observations. If the experimental mice showed the following indicators, the animals were euthanized: (1) When the tumor volume of a single mouse exceeded 3000 mm 3 , euthanize each mouse; (2) continuous loose stools; (3) slow movement (inability to eat or drink); (4) hunched back, lying on the side; (5) reduced activity, symptoms of muscle atrophy; (6) difficulty breathing; (7) progressive hypothermia; (8) paralysis, convulsions; (9) continuous bleeding; (10) the animal is unable to move normally due to large tumor or other reasons; (11) the animal is unable to move normally due to severe ascites or increased abdominal girth.

[0147] 4. Indicator calculation formula

[0148] Tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 .

[0149] TGITV (relative tumor inhibition rate) calculation formula:

[0150]

[0151]

[0152] V nt : The tumor volume of the mouse numbered n on day t;

[0153] V n0 : Tumor volume of mice numbered n on day 0;

[0154] RTV n : Relative tumor volume of mouse numbered n on day t;

[0155] meanRTV treat : Mean RTV value of drug-treated groups;

[0156] meanRTV vehicle : Average RTV value of Vehicle group;

[0157] TGITW (tumor weight change) calculation formula:

[0158]

[0159] meanTW treat : The average tumor weight of mice in the drug-treated group at the end point of treatment;

[0160] meanTW vehicle :The average tumor weight of mice in the Vehicle group at the end point of treatment.

[0161] 5. Statistical analysis

[0162] The experimental results are expressed as mean ± standard error (Mean ± SEM). The independent sample T test (T-Test) was used to compare the two groups of samples, and the data were analyzed using SPSS. P < 0.05 was considered to be significantly different, and the graphics software was Graphpad prism.

[0163] Example 1

[0164] A lipid nanoparticle containing an RNA molecule, wherein the lipid nanoparticle comprises Dlin-MC3-DMA 50%, DSPC 10%, cholesterol 38.5% and PEG-DMG 1.5% by mole percentage.

[0165] The preparation method is as follows:

[0166] (a) RNA molecules were dissolved in a pH 4 citrate buffer solution and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0167] (b) Dlin-MC3-DMA, DSCP, cholesterol and PEG-DMG were dissolved in anhydrous ethanol according to the formula amount, and the concentration of the lipid component in the organic phase was adjusted to 6 mg / mL to obtain an organic phase.

[0168] (c) The aqueous phase of step (a) and the organic phase of step (b) are mixed at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 ml / min, and the mixed solution is immediately diluted 100 times with a pH 7.4 PBS solution, and the ethanol component in the solution is removed by tangential flow filtration (TFF), and then concentrated to a concentration of 100 ug / ml of mRNA in the system to obtain lipid nanoparticles containing RNA molecules, that is, the RNA cancer therapeutic agent of the present invention.

[0169] Example 2

[0170] Jurkat cells were transfected with lipid nanoparticles containing RNA molecules, and the supernatant was harvested after 24 hours. MC38 / LUC cells (MC38 cells overexpressing the luciferase gene) were plated in a 96-well plate, mouse spleen cells were added, and 2%-50% of the supernatant of Jurkat cells transfected with mRNA-LNP was added. After 24 hours of co-culture, the supernatant of the co-cultured cells was taken, the LDH (lactate dehydrogenase) content was detected, and the killing effect of spleen cells induced by the immune cell engagement molecules encoded by the RNA cancer therapeutic agent on MC38 / LUC was calculated.

[0171] The amino acid sequence of the immune cell engagement molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 1 is shown in 29, and the nucleotide sequence of the RNA molecule is shown in 30.

[0172] The amino acid sequence of the immune cell engaging molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 2 is shown in 31, and the nucleotide sequence of the RNA molecule is shown in 32.

[0173] The amino acid sequence of the immune cell engager molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 3 is shown in 33, and the nucleotide sequence of the RNA molecule is shown in 34.

[0174] In addition to the open reading frame, the RNA molecule also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced by a 5' pseudouracil.

[0175] The experimental results are as follows Figure 6 As shown. Jurkat cells transfected with lipid nanoparticles containing RNA molecules secrete immune cell engagement molecules into the supernatant. In the co-culture system of spleen cells and tumor cells without the addition of supernatant, spleen cells have no obvious tumor killing effect. With the addition of supernatant containing immune cell engagement molecules to the co-culture system, especially when the supernatant content reaches 10% and 50%, spleen cells significantly kill tumor cells MC38 / LUC, and the killing level reaches 35%-50%. Among them, sample 2 induced the strongest killing effect. This result shows that the immune cell engagement molecules encoded by RNA cancer therapeutics significantly induce the in vitro anti-tumor activity of spleen cells.

[0176] Example 3

[0177] Jurkat cells were transfected with lipid nanoparticles containing RNA molecules, and the supernatant was harvested 24 hours later. MC38 / LUC cells (MC38 cells overexpressing the luciferase gene) were plated in a 12-well plate, mouse spleen cells were added, and 50% of the supernatant of Jurkat cells transfected with lipid nanoparticles containing RNA molecules was added. After 24 hours of co-culture, the expression of CD69 in spleen T cells was detected by flow cytometry.

[0178] The amino acid sequence of the immune cell engagement molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 1 is shown in 29, and the nucleotide sequence of the RNA molecule is shown in 30.

[0179] The amino acid sequence of the immune cell engaging molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 2 is shown in 31, and the nucleotide sequence of the RNA molecule is shown in 32.

[0180] The amino acid sequence of the immune cell engager molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 3 is shown in 33, and the nucleotide sequence of the RNA molecule is shown in 34.

[0181] In addition to the open reading frame, the RNA molecule also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced by a 5' pseudouracil.

[0182] The experimental results are as follows Figure 7 As shown. Jurkat cells transfected with lipid nanoparticles containing RNA molecules secrete immune cell engagement molecules into the supernatant. In the co-culture system of spleen cells and tumor cells without the addition of supernatant, spleen T cells were not activated and the expression level of CD69 was low. With the addition of supernatant containing immune cell engagement molecules to the co-culture system, the level of CD69 expressed by spleen T cells increased significantly, reaching 40%-70%, among which sample 2 induced the highest level. This result shows that the immune cell engagement molecules encoded by RNA cancer therapeutics can significantly induce T cell activation.

[0183] Example 4

[0184] This example investigates the effects of different RNA cancer therapeutic agents on tumor growth. The RNA cancer therapeutic agents of groups G1, G2, G3 and G4 were prepared according to the method of Example 1.

[0185] Four groups of experiments were conducted using the above-mentioned BALB / c mouse A20 tumor model as the experimental model, with 6 mice in each group.

[0186] The G1 group was administered with empty LNP at a dose of 100ul.

[0187] The amino acid sequence of the immune cell engagement molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of group G2 is shown in 29, the nucleotide sequence of the RNA molecule is shown in 30, and the dosage is 30ug.

[0188] The amino acid sequence of the immune cell engagement molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of group G3 is shown in 31, the nucleotide sequence of the RNA molecule is shown in 32, and the dosage is 30ug.

[0189] The amino acid sequence of the immune cell engagement molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of group G4 is shown in 33, the nucleotide sequence of the RNA molecule is shown in 34, and the dosage is 30ug. The above dosage is calculated according to the weight of the lipid nanoparticles containing the RNA molecule.

[0190] In addition to the open reading frame, the RNA molecule also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced by a 5' pseudouracil.

[0191] The RNA cancer therapeutic agent was administered once a week for a three-week period, and all four groups of mice were administered intratumorally. The experimental endpoint was set at 17 days after group administration, and the tumor volume was measured 2-3 times a week after administration to test the effects of the four groups of experiments on tumor growth.

[0192] The experimental results are as follows Figure 8 As shown. In the control group G1, the tumor continued to grow; while the drug-treated group G3 significantly inhibited tumor growth, with tumor inhibition rates of 60.5% and 57.2% at 12 and 17 days after administration, respectively. The drug-treated group G4 had a certain tumor inhibition effect, with tumor inhibition rates of 32.3% and 20.5% at 12 and 17 days after administration, respectively. The drug-treated group G2 had no obvious tumor inhibition effect. It can be seen that the immune cell engagement molecules encoded by RNA cancer therapeutics, especially sample 2, have significant in vivo anti-tumor efficacy.

[0193] Example 5

[0194] Jurkat cells were transfected with RNA cancer therapeutics, and the supernatant was harvested 24 hours later. HepG2 / LUC cells (HepG2 cells overexpressing the luciferase gene) were plated in 96-well plates, human PBMCs were added, and 2%-50% of the supernatant of Jurkat cells transfected with RNA cancer therapeutics were added. After 24 hours of co-culture, the supernatant of the co-cultured cells was taken, the LDH (lactate dehydrogenase) content was detected, and the killing effect of PBMCs induced by the immune cell engagement molecules encoded by the RNA molecules in the RNA cancer therapeutics on HepG2 / LUC was calculated.

[0195] The amino acid sequence of the immune cell engaging molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 058 is shown in 35, and the nucleotide sequence of the RNA molecule is shown in 36.

[0196] The amino acid sequence of the immune cell engaging molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 106 is shown in 37 , and the nucleotide sequence of the RNA molecule is shown in 38 .

[0197] In addition to the open reading frame, the RNA molecule also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced by a 5' pseudouracil.

[0198] The experimental results are as follows Fig. 9As shown. Jurkat cells transfected with lipid nanoparticles containing RNA molecules secrete immune cell engager molecules into the supernatant. In the co-culture system of PBMC cells and HepG2 / LUC tumor cells without the addition of supernatant, PBMC cells only have about 20% background tumor killing effect. With the addition of supernatant containing immune cell engager molecules to the co-culture system, PBMC cells significantly kill tumor cells HepG2 / LUC, and the killing level increases with the increase of supernatant dose. After adding 50% supernatant, the killing effect of PBMC cells can reach 60%. This result shows that the immune cell engager molecules encoded by RNA cancer therapeutics have significant in vitro anti-tumor activity.

[0199] Example 6

[0200] Jurkat cells were transfected with RNA cancer therapeutic agents, and the supernatant was harvested 24 hours later. Caski or HepG2 was plated in a 12-well plate, and Jurkat cells were added. 50% of the supernatant of Jurkat cells transfected with RNA cancer therapeutic agents was added. After 24 hours of co-culture, the expression of CD69 in Jurkat cells was detected by flow cytometry.

[0201] The amino acid sequence of the immune cell engaging molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 058 is shown in 35, and the nucleotide sequence of the RNA molecule is shown in 36.

[0202] The amino acid sequence of the immune cell engaging molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 106 is shown in 37 , and the nucleotide sequence of the RNA molecule is shown in 38 .

[0203] In addition to the open reading frame, the RNA molecule also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced by a 5' pseudouracil.

[0204] The experimental results are as follows Fig.10 As shown. Jurkat cells transfected with lipid nanoparticles containing RNA molecules secrete immune cell engager molecules into the supernatant. In the co-culture system of Jurkat cells and tumor cells without the addition of supernatant, Jurkat T cells were not activated and CD69 was not expressed. With the addition of supernatant containing immune cell engager molecules to the co-culture system, the level of CD69 expressed by Jurkat T cells increased significantly, reaching between 70% and 99%. In addition, under the conditions of using two different tumor cells, the results showed that the activation level of T cells by sample 106 was significantly higher than that of sample 058. It can be seen that the immune cell engager molecules encoded by RNA cancer therapeutics significantly induce the activation of T cells.

[0205] Example 7

[0206] This example investigates the effects of different RNA cancer therapeutic agents on tumor growth. The RNA cancer therapeutic agents of group G1, group G2 and group G3 were prepared according to the method of Example 1.

[0207] The above PBMC-Huh7 mouse tumor model was used as the experimental model for 3 groups of experiments, with 6 mice in each group, and the drug was administered intravenously on days 0, 7, and 14. The tumor volume was measured 2-3 times a week after administration. Dose: 25ug per mouse.

[0208] The G1 group was given empty LNPs.

[0209] The G2 group and the G3 group were given RNA cancer therapeutic agents. The amino acid sequence of the immune cell engagement molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of the G2 group is shown in 35, and the nucleotide sequence of the RNA molecule is shown in 36; the amino acid sequence of the immune cell engagement molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of the G3 group is shown in 37, and the nucleotide sequence of the RNA molecule is shown in 38.

[0210] In addition to the open reading frame, the RNA molecule also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced by a 5' pseudouracil.

[0211] The experimental results are as follows Fig.11 As shown. In the control group G1, the tumor continued to grow; while the drug-treated group G3 significantly inhibited tumor growth, with tumor inhibition rates of 56.7% and 63.8% on days 12 and 15 after administration, respectively; the drug-treated group G2 had a certain tumor inhibition effect, with tumor inhibition rates of 24.4% and 28.5% on days 12 and 15 after administration, respectively. It can be seen that the immune cell engagement molecules encoded by RNA cancer therapeutics have significant in vivo anti-tumor efficacy.

[0212] Example 8

[0213] Samples 015 and 106 were prepared according to the method described in Example 1, and the efficacy of the two samples was tested according to the methods described in Examples 5 and 6.

[0214] The amino acid sequence of the immune cell engager molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 015 is shown in 7 , and the nucleotide sequence of the RNA molecule is shown in 8 .

[0215] The amino acid sequence of the immune cell engagement molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 102 is shown in 39, and the nucleotide sequence of the RNA molecule is shown in 40.

[0216] The amino acid sequence of the immune cell engaging molecule encoded by the RNA molecule in the RNA cancer therapeutic agent of sample 106 is shown in 37 , and the nucleotide sequence of the RNA molecule is shown in 38 .

[0217] In addition to the open reading frame, the RNA molecule also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced by a 5' pseudouracil.

[0218] The experimental results of the killing effect of PBMC on HepG2 / LUC induced by the immune cell engagement molecules encoded by RNA molecules in RNA cancer therapeutics are as follows Fig.12 shown.

[0219] The experimental results of CD69 expression in Jurkat cells are as follows Fig.13 shown.

[0220] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. An RNA cancer therapeutic agent expressing an immune cell engager molecule, characterized in that: The RNA cancer therapeutic comprises an RNA molecule that expresses an immune cell engager molecule; The RNA molecule comprises: a molecular fragment (A) and a molecular fragment (C); The molecular fragment (A) encodes the extracellular domain of an NK cell activating receptor, and the NK cell activating receptor is selected from NKG2D receptor, KIR2DL4 receptor, NKp44 receptor, NKp46 receptor, NKp30 receptor, and NKG2C receptor; the molecular fragment (C) encodes an antibody that specifically binds to an immune cell surface membrane protein, and the antibody that specifically binds to an immune cell surface membrane protein is selected from an anti-CD3 antibody or an anti-CD16 antibody.

2. The RNA cancer therapeutic agent according to claim 1, characterized in that The amino acid sequence of the NKG2D receptor is shown in SEQ ID NO.9; Preferably, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.11 or SEQ ID NO.17, and the amino acid sequence of the light chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.12 or SEQ ID NO.

19.

3. The RNA cancer therapeutic agent according to claim 1, characterized in that The RNA molecule comprises a molecular fragment (B), and the molecular fragment (B) connects the molecular fragment (A) and the molecular fragment (C); the amino acid sequence encoded by the molecular fragment B is selected from at least one of a GS linker sequence, a CD3 antibody Fc region, and a 2A peptide; Preferably, the molecular fragment B encodes a GS linker sequence, and the GS linker sequence is selected from one of (GnS)m, ((GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q; wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; q is an integer from 1 to 5; Preferably, the amino acid sequence of the GS linker sequence is shown as SEQ ID NO.

13.

4. The RNA cancer therapeutic agent according to claim 2, characterized in that The open reading frame sequence of the nucleotide molecule encoding the NKG2D receptor extracellular domain is shown in SEQ ID NO.10; Preferably, the nucleotide sequence encoding the heavy chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.15 or SEQ ID NO.18, and the nucleotide sequence encoding the light chain variable region of the anti-CD3 antibody is shown as SEQ ID NO.16 or SEQ ID NO.

20.

5. The RNA cancer therapeutic agent according to claim 4, characterized in that The antibody that specifically binds to immune cell surface membrane proteins includes an Fc region; Preferably, the amino acid sequence of the anti-CD3 antibody Fc region is shown in SEQ ID NO.

21.

6. The RNA cancer therapeutic agent according to claim 1, characterized in that The RNA molecule includes a molecular fragment (B) encoding a cytokine, wherein the cytokine is selected from one or more of cytokine IL12, cytokine IL2, cytokine IL36, cytokine IL7, cytokine IL15, and cytokine IL23; Preferably, the RNA molecule encoding the NK cell activating receptor further comprises a molecular fragment encoding a marker protein (D), and / or a molecular fragment encoding a signal peptide (E); Preferably, the marker protein is selected from the group consisting of a His tag, a FLAG tag, a HA tag and a MYC tag; Preferably, the RNA cancer therapeutic agent further comprises an antibody selected from anti-PD-1 antibody, anti-CTLA4 antibody, anti-TIGIT antibody, anti-TIM3 antibody, and anti-LAG3 antibody.

7. The RNA cancer therapeutic agent according to claims 1 to 6, characterized in that The RNA molecule is mRNA; Preferably, the mRNA consists of a sequence including a 5' cap, a 5' UTR, an ORF, a 3' UTR and a 3' poly (A) tail in sequence from the 5' end to the 3' end.

8. The RNA cancer therapeutic agent according to claims 1 to 6, characterized in that The RNA cancer therapy also includes a delivery formulation encapsulating the RNA molecule; Preferably, the delivery formulation comprises lipid nanoparticles or cationic liposomes; Preferably, the components of the lipid nanoparticles include: at least one of a protonable cationic lipid, a structural lipid, an auxiliary lipid and a surfactant; Preferably, the components of the lipid nanoparticles include, by weight: 40 to 60 parts of protonable cationic lipids, 20 to 40 parts of structural lipids, 10 to 30 parts of auxiliary lipids and 0.5 to 5 parts of surfactants; Preferably, the protonable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200 and DlinDMA; Preferably, the structural lipids include cholesterol, cholesterol ester, steroid hormones, steroid vitamins or bile acid; Preferably, the helper lipid comprises at least one of DSPC, DOPE, DOPC, DOPG and DOPS; Preferably, the surfactant comprises at least one of PEG-DMG, PEG-DSPE and TPGS; Preferably, the liposome nanoparticles comprise 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol and 1.5% PEG-DMG in molar percentage.

9. The method for preparing the RNA cancer therapeutic agent according to claim 8, characterized in that: It includes: Mixing the RNA molecule with a delivery formulation to form an RNA cancer therapeutic; the delivery formulation includes lipid nanoparticles or cationic liposomes; Preferably, in the method for preparing the RNA cancer therapeutic agent, mRNA is dissolved in a buffer to obtain an aqueous phase, each lipid component of the liposome nanoparticle is measured and dissolved in an organic solvent to obtain an organic phase, and the aqueous phase and the organic phase are mixed and then the organic phase is removed to obtain the RNA cancer therapeutic agent.

10. Biomaterial, characterized in that The biomaterial includes at least one of the following (x1) to (x4); (x1) a DNA molecule or RNA molecule encoding the RNA cancer therapeutic agent according to any one of claims 1 to 7; (x2) an expression cassette or vector containing (x1); (x3) a microorganism comprising at least one of (x1) and (x2); (x4) A cell comprising at least one of (x1) and (x2).

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