Nucleic acid form IL-9 combined immune checkpoint medicine preparation combination and application

Abstract

The invention relates to the technical field of research and development of cancer drugs, in particular to a preparation combination of a nucleic acid form IL-9 combined immune checkpoint drug and application. The tumor treatment combination medicine obtained by the invention comprises IL-9 in a nucleic acid form and an immune checkpoint medicine, and the IL-9 in the nucleic acid form comprises DNA (Deoxyribonucleic Acid) and mRNA (Messenger Ribonucleic Acid) for coding the IL-9. Experimental results show that IL-9 in the form of nucleic acid can enhance the response rate of immune checkpoint drugs, increase infiltration of specific T cells in tumors and the proportion of natural immune killer cells such as NK and the like, and enhance the tumor treatment effect; iL-9 in a recombinant protein form does not have the function, and even plays a reverse role; therefore, the nucleic acid coded IL-9 can be combined with the existing immune checkpoint medicine to be used for treating various cancers.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer drug research and development, and specifically to a preparation combination and application of IL-9 in nucleic acid form combined with immune checkpoint drugs. Background Art

[0002] According to statistics, there are about 12 million new cancer patients and more than 8 million cancer deaths worldwide each year. Cancer remains the primary threat to human health. The emergence of immunotherapy in recent years has brought new hope to cancer patients. Immunotherapy includes cell therapy such as CAR-T, TCR-T, TIL, immune checkpoint therapy represented by PD-1 / PD-L1, and cancer vaccine therapy. The main working principle of immune checkpoint inhibitors is to relieve the suppression of tumor cells on the immune system, such as blocking the inhibitory signals transmitted by Treg, M2 macrophages, MDSC cells, etc., enhancing the activity of tumor-specific T cells, and reversing the aging, apoptosis, and exhaustion of tumor-specific T cells. The immune checkpoints discovered so far include PD-1 / PD-L1, CTLA4, LAG3, TIM3, TIGIT, VISTA, 4-1BB, SIP1a, CD47, Siglec15, etc. Among them, immune checkpoint inhibitors represented by PD-1 / PD-L1 have achieved widespread success in clinical applications. Significantly prolong the survival of cancer patients. Indications have been expanded to various types of cancer. A wide range of immune responses have been obtained in various indications. Currently, 10 PD-1 monoclonal antibodies and three PD-L1 monoclonal antibodies have been approved for clinical use. However, due to the complexity of tumors, such as cold tumors with low mutation load and poor immune infiltration, the response rate of immune checkpoint inhibition in patients is less than 30%. How to improve the response rate of immune checkpoint inhibitors in the patient population has become a scientific problem that needs to be solved urgently.

[0003] The success of immune checkpoint inhibitors comes from in-depth research on the mechanism of action between cancer and the immune system. Take PD-1 as an example. When T cells bind to target antigens on tumor cells through T cell receptors (TCR), phosphorylation signal cascades are initiated to promote the proliferation and killing function of T cells. The PD-1 protein expressed on T cells will be bound by PD-L1 expressed by tumor cells and recruit SHP-2 phosphatase to reverse the phosphorylation signal transmission after TCR recognizes the ligand, inhibiting the activation, proliferation and cytokine release of T cells. Therefore, using monoclonal antibodies to block the interaction between PD-1 and PD-L1 can restore the normal recognition and killing process of T cells to tumors. Therefore, it can be used to treat various types of cancer. In addition to PD-1 and PD-L1 signals, there are many other molecules and signal pathways that interfere with immune responses. Therefore, preparing monoclonal antibodies of other immunosuppressive pathways with PD-1 monoclonal antibodies into bispecific antibodies can improve the response rate of PD-1. In addition, some chemotherapy drugs and radiotherapy can promote the immunogenic death of tumor cells. The so-called immunogenic death refers to the release of death-related molecular patterns after tumor cell death, which can activate the immune system. This promotes the formation of an anti-tumor immune microenvironment and makes cold tumors hot. Therefore, this type of chemotherapy and radiotherapy can also enhance the efficacy of immune checkpoint inhibitors. Tumors induce angiogenesis, but the excessive permeability of the formed blood vessels and the role of pro-angiogenic factors are not conducive to the infiltration of immune cells, but promote the infiltration of immunosuppressive cells. Therefore, combined anti-angiogenic drugs can also enhance the therapeutic effect of immune checkpoint inhibitors. For example, in liver cancer, the combination of anti-angiogenic bevacizumab and immune checkpoint atelizumab successfully defeated lenvatinib and became the first-line treatment for advanced liver cancer. In addition, recent clinical trials have also shown that targeted drugs such as EGFR, RAS, ALK, PARP, and intestinal flora transplantation have also shown superiority in combination with immune checkpoint inhibitors.

[0004] Cytokines have the function of dispatching immune cells to kill tumors. Cytokines such as IL-2, IL-15, and IL-12 have shown the ability to enhance the anti-tumor effect of immune checkpoint inhibitors in combination with immune checkpoint inhibitors. Recently, Roche Pharmaceuticals has achieved good preclinical results by fusing PD-1 monoclonal antibodies with IL-2 to form antibody-interleukin fusion proteins.

[0005] Interleukin 9 (IL-9) is a cytokine discovered in recent years. It has the function of promoting lymphocyte proliferation and is widely involved in inflammatory and autoimmune diseases. IL-9 was originally discovered in type 2 helper T cells. Later, it was discovered that IL-9 is mainly secreted by Th9 cells. In addition, mast cells, eosinophils, neutrophils, and natural killer T cells can also secrete it. After IL-9 binds to its receptor, it affects the function of target cells through the activation of the JAK-STAT signaling pathway. These target cells include mast cells, T cells, DC cells, etc. Although the function of IL-9 is well understood, no researchers have yet combined IL-9 with immune checkpoint inhibitors for in vivo treatment to enhance the therapeutic effect of immune checkpoint inhibitors. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a preparation combination and application of IL-9 in nucleic acid form combined with immune checkpoint drugs.

[0007] The present invention provides a nucleic acid encoding interleukin-9,

[0008] The DNA encoding interleukin-9 has the nucleotide sequence shown in SEQ ID NO: 2;

[0009] The mRNA encoding interleukin-9 has the nucleotide sequence shown in SEQ ID NO:3.

[0010] Furthermore,

[0011] The nucleic acid sequence encoding interleukin-9 mRNA is shown in SEQ ID NO:7.

[0012] Furthermore, the nucleic acid further comprises:

[0013] A nucleic acid molecule encoding a protein with the same or similar functions as the nucleotide sequence shown in SEQ ID NO: 2 and / or SEQ ID NO: 3, formed by replacing, deleting or adding nucleotides in multiples of 3; and / or

[0014] A nucleic acid molecule that is complementary or partially complementary to the nucleotide sequence shown in SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0015] The interleukin-9 in the form of nucleic acid of the present invention significantly improves the immunotherapy effect after being used in combination with an immune checkpoint inhibitor, and the interleukin-9 in the form of nucleic acid has a broad spectrum of effects on the treatment of immune checkpoint drugs:

[0016] In some specific embodiments of the present invention, the interleukin-9 DNA vector or lipid nanoparticles containing mRNA combined with the immune checkpoint drug PD-1 can inhibit the proliferation of mouse rectal cancer cells and cause the mouse rectal cancer cells to regress until they completely disappear, while PD-1 alone can only cause a temporary regression of the tumor and cannot completely eliminate the tumor;

[0017] In some other specific embodiments of the present invention, the interleukin-9 DNA vector combined with the immune checkpoint inhibitor PD-L1 or CTLA-4 has a significantly higher therapeutic effect on melanoma and lymphoma than the immune checkpoint drug alone;

[0018] In other specific embodiments of the present invention, IL-9 in the form of a recombinant protein cannot enhance the therapeutic effect of immune checkpoint inhibitors or agonists.

[0019] The present invention experiments found that recombinant protein form of IL-9 in vivo induces the production of a large number of neutralizing antibodies, which in turn antagonizes the therapeutic effect of immune checkpoints; while nucleic acid form of IL-9 can avoid the production of neutralizing antibodies and enhance the efficacy of immune checkpoint inhibitors.

[0020] In the present invention, the DNA sequence encoding interleukin 9 is optimized. Experimental results show that the expression level of the product of the nucleic acid with optimized sequence is significantly higher than that of the nucleic acid with unoptimized sequence. The expression level of the nucleic acid encoding interleukin 9 without sequence optimization is very low after integration into the expression vector.

[0021] In the present invention, the nucleic acid also includes a nucleic acid encoding a signal peptide. The present invention transforms the signal peptide of interleukin-9, and the results show that the expression and secretion level of interleukin-9 is significantly improved after the transformation.

[0022] The nucleic acid described in the present invention can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions, such as CMV or CMV / R promoters, transcription terminators, 5'-UTR, 3'-UTR and 3'-terminal PolyA. In the RNA sequence encoding the nucleic acid described in the present invention, the 5'-UTR is β-globin-2, as shown in SEQ ID NO:5; the 3'-UTR is 2β-globin, as shown in SEQ ID NO:6; the length of the 3'-terminal PolyA is 120bp; the structure of the RNA fragment is 5'UTR-IL-9-3'UTR-A (120). The nucleic acid described in the present invention can be linear or circular in topology. The nucleic acid can be a part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the aid of recombination, enzymatic or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0023] The nucleic acid of the present invention is sequence optimized; the optimization includes but is not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.) and / or restriction sites that may affect cloning, etc.

[0024] The present invention provides an expression unit, which comprises: a CMV-R promoter, intron A and at least one of the nucleic acids described in the present invention.

[0025] Further, the transcription unit refers to a DNA sequence starting from a promoter and ending at a terminator. The promoter and terminator may also include regulatory segments on both sides or between them, and the regulatory segments may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably linked to the nucleic acid sequence, such as an enhancer of the promoter, a poly(A) signal, etc.

[0026] The present invention provides a recombinant vector comprising a vector backbone and at least one of the nucleic acids described in the present invention.

[0027] Furthermore, in the recombinant vector, the vector backbone is selected from at least one of pGEM-3Zf(+) (for transcribing mRNA) and / or pVR (for DNA-based drugs).

[0028] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression of an operably linked coding gene in a specific host organism. The nucleic acid sequence or element necessary for the expression in a model animal or mammalian cell includes a promoter, a ribosome binding site and possible other sequences. Known eukaryotic cells utilize promoters, enhancers and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" can sometimes be interchanged and used, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or only potential genomic insert. In a specific embodiment, the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various eukaryotic expression vectors. For example, its backbone vector can be a pVR series vector (see Chinese Patent ZL202110624820.8).

[0029] The present invention provides a host,

[0030] Its genome is integrated with the nucleic acid described in the present invention; or

[0031] Transfect or transform the recombinant vector of the present invention.

[0032] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, and the like.

[0033] The host provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, which are not limited in the present invention. The present invention uses a vector constructed by recombinant DNA technology to transform or transfect a host, so that the transformed host has the ability to replicate a protein-encoding vector or express a desired protein.

[0034] Furthermore, the host described in the present invention is mainly used for the storage and culture replication of the nucleic acid described in the present invention and / or the recombinant vector described in the present invention.

[0035] The present invention provides a nucleic acid preparation, which comprises an auxiliary material and at least one of the following a) to c):

[0036] a), the nucleic acid of the present invention;

[0037] b) the recombinant vector of the present invention;

[0038] c) After culturing the host of the present invention, the nucleic acid or recombinant vector is obtained by extraction.

[0039] Furthermore, the auxiliary materials described in the present invention include but are not limited to carriers, surfactants, reducing agents, buffer solutions, and the like.

[0040] The present invention provides the use of at least one of the following i) to iv) in the preparation of a tumor immunotherapy drug:

[0041] i), the nucleic acid of the present invention;

[0042] ii), the recombinant vector of the present invention;

[0043] iii), the host of the present invention;

[0044] iv) The nucleic acid preparation of the present invention.

[0045] The present invention provides a tumor immunotherapy drug, the raw materials of which include: an immune checkpoint drug and at least one of the following I) to IV):

[0046] 1), the nucleic acid of the present invention;

[0047] II), the recombinant vector of the present invention;

[0048] III), after culturing the host according to claim 5, the nucleic acid or recombinant vector obtained by extraction;

[0049] IV), the nucleic acid preparation of the present invention.

[0050] Furthermore, the immune checkpoints targeted by the immune checkpoint drug include at least one of PD-1, PD-L1, CTLA-4, LAG3, TIM3, TIGIT, VISTA, 4-1BB, SIP1α, CD47, NKG2A, CD137, TNFRSF4, GITR, CD27, ICOS, siglec7, siglec15, LILBR2 or LILBR2.

[0051] Furthermore,

[0052] The immune checkpoint drugs include: monoclonal antibodies or small molecule drugs targeting immune checkpoints;

[0053] The small molecule drugs include: small molecule drugs targeting cytokines or chemokines or small molecule drugs targeting metabolic pathways; the small molecule drugs targeting cytokines or chemokines include at least one of PSMA-617, ARRY-382, Vimseltinib and / or BLZ945; the small molecule drugs targeting metabolic pathways include at least one of INCB001158, TT-702, TT4, AB680, ORIC-533, BAY2416964 and / or 1K175.

[0054] In the tumor immunotherapy drug described in the present invention, the tumor includes at least one of liver cancer, cervical cancer, ovarian cancer, lung cancer, head and neck cancer, prostate cancer, breast cancer, blood cancer, ovarian cancer and / or colorectal cancer.

[0055] In the present invention, the tumor immunotherapy drug may be in the form of DNA or RNA, which is not limited in the present invention.

[0056] Furthermore, the tumor immunotherapy drug of the present invention further includes an adjuvant or a buffer; the adjuvant is used to maintain the activity of the drug or help the drug to function. The adjuvant includes but is not limited to a carrier, a surfactant, and a reducing agent; the carrier includes a lentiviral auxiliary vector, an adenoviral auxiliary vector, and other vectors that assist in assembly or integration; the buffer includes a PBS buffer, a KCl buffer, a NaCl buffer, a Tris buffer, and the like.

[0057] Furthermore, the tumor immunotherapy drug described in the present invention also includes the transcription mRNA or translation initiation product of the nucleic acid, etc., and the present invention is not limited to this.

[0058] Furthermore, the tumor immunotherapy drug also includes lipid nanoparticles containing the nucleic acid or mRNA. The preparation process of the lipid nanoparticles includes encapsulating the nucleic acid or mRNA using a transfection reagent in an animal body to form a tumor therapy drug in the form of lipid nanoparticles containing the nucleic acid or mRNA for tumor immunotherapy.

[0059] The treatment described in the present invention refers to administering the product for preventing and treating a disease described in the present invention after the disease occurs, which can improve symptoms, inhibit the progression of the disease, and restore the patient to health. For example, the product described in the present invention can be used to prevent and treat tumors, which can cause the tumor to regress or even disappear.

[0060] The present invention provides a method for tumor immunotherapy, which comprises administering the tumor therapeutic drug of the present invention, and the administration method thereof includes oral administration, injection and / or electroporation.

[0061] The present invention obtains a tumor treatment combination drug, which includes IL-9 in the form of nucleic acid and an immune checkpoint drug, wherein the nucleic acid form of IL-9 includes DNA and mRNA encoding IL-9. Experimental results show that IL-9 in the form of nucleic acid can enhance the response rate of immune checkpoint drugs, increase the specific T cell infiltration inside the tumor and the proportion of natural immune killer cells such as NK, and enhance the tumor treatment effect; IL-9 in the form of recombinant protein does not have this function, and even has the opposite effect; therefore, nucleic acid-encoded IL-9 can be used in combination with existing immune checkpoint drugs for the treatment of various types of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The expression level of intracellular IL-9 was detected by western blot after the optimized IL-9 was transfected into 293T cells;

[0063] Figure 2 The expression level of IL-9 in the culture medium supernatant was detected by ELISA after the optimized IL-9 was transfected into 293T cells;

[0064] Figure 3 The results show the therapeutic effect of DNA and mRNA encoded IL-9 combined with immune checkpoint inhibitor PD-1 on mouse colorectal cancer cell line MC38 xenografts;

[0065] Figure 4 The results show the therapeutic effect of DNA-encoded IL-9 combined with immune checkpoint inhibitor PD-L1 on mouse melanoma cell line B16 transplanted tumors;

[0066] Figure 5 The results show the therapeutic effect of DNA-encoded IL-9 combined with immune checkpoint inhibitor CTLA-4 on mouse lymphoma cell line EG-7 xenografts;

[0067] Figure 6 The results show the therapeutic effects of recombinant protein IL-9 combined with immune checkpoint inhibitors, respectively, combined with immune checkpoint inhibitors PD-1 and CTLA-4, on mouse colorectal cancer cell line MC38 xenografts;

[0068] Figure 7 The results show the results of the antibody level test against IL-9 protein induced after mice were immunized with IL-9 in DNA and recombinant protein forms. DETAILED DESCRIPTION

[0069] The present invention provides a combination and application of a preparation of IL-9 in the form of a nucleic acid combined with an immune checkpoint drug. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0070] The present invention provides a nucleic acid-encoded IL-9 as an immune enhancement technology for enhancing the therapeutic effect of immune checkpoint inhibitors or agonists on tumors. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0071] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0072] The IL-9 described in the present invention is an important cytokine in the human body, which has the effect of promoting lymphocyte proliferation. The present invention's research shows that IL-9 can significantly enhance the therapeutic effect of immune checkpoint inhibitors or agonists on tumors.

[0073] In the present invention, the IL-9 may be a human fragment or a fragment of other animal sources, such as mouse, rabbit, monkey, pig, etc. It may be a complete IL-9 or a fragment or mutant having IL-9 activity, which is not limited in this application. In the embodiment of the present application, human IL-9 is used as the experimental subject to verify the enhancing effect of IL-9 on immune checkpoint inhibitors or agonists. The amino acid sequence of the human IL-9 is as shown in SEQ ID NO: 1.

[0074] The number of immune checkpoint inhibitors or agonists of the present invention is at least 1. For example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more. In the research of the present invention, experiments were conducted on the therapeutic effects of 1 or 2 immune checkpoint inhibitors or agonists, and good effects were shown.

[0075] The immune checkpoints of the present invention include but are not limited to PD-1, PD-L1, CTLA-4, LAG3, TIM3, TIGIT, VISTA, 4-1BB, SIP1α, CD47, NKG2A, CD137, TNFRSF4, GITR, CD27, ICOS, siglec7, siglec15, LILBR2, LILBR2. The corresponding agonist or inhibitor is a monoclonal antibody or a corresponding small molecule of the corresponding immune checkpoint. The monoclonal antibody of the corresponding immune checkpoint can be a monoclonal antibody targeting a single immune checkpoint, or a bispecific antibody or a trispecific antibody simultaneously targeting two or three immune checkpoints.

[0076] In some embodiments, the immune checkpoints are PD-1, PD-L1, and CTLA-4.

[0077] In some embodiments, the IL-9 is DNA-encoded, mRNA-encoded or recombinant protein IL-9.

[0078] The present invention utilizes the stimulating effect of IL-9 on the immune system to improve the anti-tumor effect of immune checkpoint inhibitors or agonists. In the embodiments of the present invention, monoclonal antibodies to PD-1, PD-L1 and CTLA-4 are used as examples to demonstrate the immune enhancement effect of nucleic acid-encoded IL-9, and other immune checkpoint agonists or inhibitors combined with nucleic acid-encoded IL-9 will also have a good effect. However, recombinant protein IL-9 will antagonize the therapeutic effects of immune checkpoint agonists and inhibitors.

[0079] In the present invention, in order to improve the expression effect of IL-9, a signal peptide is added to the N-terminus of IL-9 to promote protein secretion to the extracellular space. In some embodiments, the signal peptide is an IgE signal peptide. Specifically, the optimized IL-9 and the entire vector nucleotide sequence are shown in SEQ ID NO: 4.

[0080] The treatment described in the present invention refers to the administration of the drug described in the present invention after the occurrence of a tumor, which can inhibit tumor growth, reduce tumor volume or slow down the growth rate of the tumor. In the examples of the present invention, subcutaneous transplanted tumors of mouse colorectal cancer cell line MC-38, lymphoma cell line EG-7 and melanoma cell line B16 were used as experimental subjects to verify the effect of the product.

[0081] In the embodiments of the present invention, the amino acid sequences of the involved fragments and the encoded nucleic acid fragments are shown in Table 1:

[0082] Table 1. Amino acid sequences and encoded nucleic acid fragments involved in the present invention

[0083] SEQ ID NO:1 Amino acid sequence of IL-9 protein SEQ ID NO:2 DNA sequence of IL-9 SEQ ID NO:3 IL-9 coding mRNA sequence SEQ ID NO:4 DNA vector containing the complete sequence of IL-9 SEQ ID NO:5 5-UTR (β-globin-2) SEQ ID NO:6 3-UTR (2β-globin) SEQ ID NO:7 5-UTR-IL-9mRNA-3-UTR

[0084] The amino acid sequence of IL-9 protein is: MLLAMVLTSALLLCSVAGQGCPTLAGILDINFLINKMQEDPASKCHCSANV TSCLCLGIPSDNCTRPCFSERLSQMTNTTMQTRYPLIFSRVKKSVEVLKNNK CPYFSCEQPCNQTTAGNALTFLKSLLEIFQKEKMRGMRGKI (as shown in SEQ ID NO: 1);

[0085] The DNA sequence of IL-9 is: atggactggacctggatcctgtttctggtggccgctgccacaagagtgcacagccaaggatgtcctacactggccggaatcctggatatcaacttcctgat caacaagatgcaagaggacccgccagcaagtgccactgtagcgccaatgtgaccagctgcctgtgtctgggcatccccagcgacaattgcaccagaccttgcttcagcgagc ggctgagccagatgaccaacaccaccatgcagacccggtatcctctgatcttcagccgcgtgaagaaaagcgtggaagtgctgaagaacaacaagtgcccctacttcagctgc gagcagccctgcaatcagaccacagccggaaatgccctgaccttcctgaagtccctgctggaaatcttccagaaagaaaagatgcggggcatgcgcggcaagatctga (as SEQ ID NO:2);

[0086] The coding mRNA sequence of IL-9 is: auggacuggaccuggauccuguuucugguggccgcugccacaagagugcacagccaaggauguccuacacuggccggaauccuggauaucaacuuccugaucaacaagaugcaagaggaccccgccagcaagugccacuguagcgccaaugugaccagcugccugugucugggcauccccagcgacaauugcaccagaccuugcuucagcgagcggcugagccagaugaccaacaccaccaugcagacccgguauccucugaucuucagccgcgugaagaaaagcguggaagugcugaagaacaacaagugccccuacuucagcugcgagcagcccugcaaucagaccacagccggaaaugcccugaccuuccugaagucccugcuggaaaucuuccagaaagaaaagaugcggggcaugcgcggcaagaucuga (as shown in SEQ ID NO:3), where u in the sequence listing is converted to t;

[0087]

[0088] The sequence of 5-UTR (β-globin-2) is: agagcggccgcuuuuucagcaagauuaagcccagggcagagccaucuauugcuuacauuugcuucugaca caacuguguucacuagcaaccucaaacagacacc (as shown in SEQ ID NO: 5), in which u in the sequence list is converted to t;

[0089] The sequence of 3-UTR (2β-globin) is: agcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugc (as shown in SEQ ID NO: 6), in which u is converted to t in the sequence list;

[0090] The sequence of the completed mRNA (5-UTR-IL-9mRNA-3-UTR) is: (as shown in SEQ ID NO: 7), and u is converted to t in the sequence list.

[0091] 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 construed as limiting the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0092] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0093] The present invention will be further described below in conjunction with embodiments:

[0094] Example 1 Sequence optimization and replacement of the original IL-9 signal peptide with the IgE signal peptide to enhance the expression level and secretion capacity of IL-9

[0095] Considering that nucleic acid form of IL-9 is usually injected locally to express IL-9, which then reaches target sites such as tumors through the circulation. Therefore, there are high requirements for the expression level and secretion capacity at the injection site. In the present invention, the IgE signal peptide replaces the original IL-9 signal peptide to form a new chimeric protein and obtains the DNA and mRNA form IL-9 sequences of SEQ ID NO:2 and SEQ ID NO:3 through codon optimization. Intron A is added before the DNA sequence to construct a pVR vector to obtain the DNA form of IL-9 such as SEQ ID NO:4. After transfection of 293T cells, western blot was performed to detect the intracellular IL-9 expression level and ELISA was performed to detect the IL-9 level in the culture medium supernatant. The results showed that the vector and sequence modification of the present invention significantly improved the expression level and secretion level of IL-9 ( Figure 1 , Figure 2 ).

[0096] Example 2 Therapeutic effect of DNA and mRNA encoded IL-9 combined with immune checkpoint inhibitor PD-1

[0097] Immune checkpoint inhibitors have a wide range of clinical application scenarios and are suitable for the treatment of various types of cancer. The present invention first evaluated whether IL-9 encoded by DNA and mRNA has a combined effect with PD-1, the most widely used immune checkpoint with the most marketed drugs. Since the colorectal cancer cell line MC-38 is a sensitive cell line to PD-1, the present invention preferentially uses the MC-38 cell line to construct a subcutaneous transplant tumor model to verify the combined effect of IL-9 and PD-1. The process is to resuscitate the MC-38 cell line and passage it more than twice, then culture it to a confluence of about 90%, digest it with trypsin, and count it. According to 2 cells per mouse, × 10 5 Finally, resuspend the required total amount of cells into 2 × 10 6The concentration of cells per milliliter was 100 microliters of cell suspension was injected subcutaneously in the armpit of each mouse. After the tumor was fully formed in about 7 days, the measurement and group administration were carried out. The DNA form of IL-9 was directly synthesized by Qingke Biotechnology according to SEQ ID NO:4, which contains the vector backbone and sequence-optimized IL-9. After the synthesis of the plasmid, the company amplified and extracted it for efficacy testing. The company also synthesized the mRNA vaccine form of IL-9 and encapsulated it with nanoparticles. The specific process was to connect 5'UTR-CCL11-IL-9-3'UTR-A(120) into the cloning vector pGEM-3Zf(+)(promega) respectively, and complete the construction of the in vitro transcription expression system, named pGEM-IL-9. The UTR sequences of the in vitro transcription system are shown in SEQ ID NO:5(5-UTR(β-globin-2)) and SEQ ID NO:6(3-UTR(2β-globin)).

[0098] The recombinant plasmid was linearized, and the single enzyme digestion reaction system was as shown in Table 2: the reaction conditions were 37°C, 3h.

[0099] Table 2. Single enzyme digestion reaction system

[0100] composition volume XOt 4μL Recombinant plasmid 12 μg 10×CutSmart Buffer 5μL <![CDATA[dd H2O]]> Supplement Total 50μL

[0101] In vitro transcription reaction:

[0102] 1. Choose T7-Flash Scribe TM Transcription Kit (Cellscript) was used for in vitro transcription. During the preparation of the in vitro transcription system, UTP was replaced with N1-Methylpseudouridine-5'-Triphosphate (Trilink Biotech).

[0103] The reaction system is shown in Table 3, and the reaction conditions are: after the first step, 35°C for 30 min; after the second step, 35°C for 15 min.

[0104] Table 3. In vitro transcription reaction system

[0105]

[0106]

[0107] mRNA capping: Use the ScriptCap kit TM Cap 1 Capping System (Cell script) was used for operation.

[0108] Cap-mRNA purification

[0109] Choose MEGAclear TM The purification reaction was performed using the Purification Kit (Invitrogen).

[0110] Preparation of liposome nanoparticles LNP-Man

[0111] DOTAP ((2,3-dioleoyl-propyl) trimethylammonium chloride), DOPE (dioleoylphosphatidylethanolamine), and DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) were purchased from Shanghai Avite Pharmaceutical Technology Co., Ltd.; DSPE-PEG2000-Man was purchased from Xi'an Haoran Biotechnology Co., Ltd.;

[0112] Nanoparticles were prepared by rotary evaporation. The specific operation process of LNP-Man preparation is as follows:

[0113] 1) Add DOTAP:DOPE:DSPE-PEG2000-Man in a molar ratio of 50:50:1 into a round-bottom flask, and add 6 mL of chloroform until the solid is fully dissolved;

[0114] 2) Water bath ultrasound for 15 min;

[0115] 3) Place the round-bottom flask into a rotary evaporator. The dissolved material in the round-bottom flask must be below the water surface. The speed is 100 rpm and the rotary evaporation is performed for 15 minutes.

[0116] 4) Remove the round-bottom flask, place it in a fume hood, and add 8 mL of HEPES buffer to dissolve the film on the inner wall of the flask;

[0117] 5) Water bath ultrasound for 30 min;

[0118] 6) The sonicated solution was filtered through a 0.22 μm filter membrane for three times to obtain the desired liposome nanoparticles LNP-Man.

[0119] Preparation of LNPs / mRNA

[0120] The prepared cationic lipid nanomaterial LNP-Man was mixed with mRNA according to the set N / P=10:1 (molar ratio), and the required volumes of LNPs and mRNA were calculated; an equal volume of 10mM HEPES buffer was added to each of the LNPs and mRNA before mixing; the mixed LNPs / mRNA was oscillated on a vortex oscillator for 1 min, and allowed to stand at room temperature for 30 min before use.

[0121] After the mice formed tumors and were measured and grouped, PD-1 monoclonal antibody (Bioxcell) was injected intraperitoneally every three days, for a total of four injections, with an injection dose of 300 micrograms per mouse. IL-9 was injected intramuscularly once every seven days, for a total of two injections. The DNA and mRNA doses were both 50 micrograms per mouse. After that, the tumor was measured every other day and the tumor volume was calculated. The tumor growth curve was drawn as shown in the figure. Figure 1 As shown. The results showed that the tumors of all mice in the IL-9 plasmid, IL-9-mRNA combined with PD-1 monoclonal antibody treatment group regressed faster than those in the control group and the PD-1 group alone until they disappeared completely. The tumors in the PD-1 group alone regressed briefly but did not disappear completely and eventually relapsed and continued to grow. This result shows that the combination of nucleic acid form IL-9 and PD-1 enhances the therapeutic effect of PD-1.

[0122] Example 3 The therapeutic effect of DNA-encoded IL-9 combined with immune checkpoint inhibitors PD-L1 or CTLA-4 on melanoma and lymphoma.

[0123] In view of the fact that nucleic acid-encoded IL-9 significantly enhanced the therapeutic effect of the immune checkpoint PD-1 monoclonal antibody in Example 1, in order to exclude the specific effect between IL-9 and PD-1, it is further verified whether nucleic acid-encoded IL-9 has the effect of broadly enhancing the effect of immune checkpoint inhibitors. The present invention continues to use DNA-encoded IL-9 combined with different immune checkpoint inhibitors for pharmacodynamic evaluation in different cancer types.

[0124] The present invention uses melanoma cell line B16 and lymphoma cell line EG-7 to construct subcutaneous transplant tumor models to verify the combined effects of DNA-encoded IL-9 and monoclonal antibodies of immune checkpoints PD-L1 and CTLA-4. The process is to resuscitate the melanoma cell line B16 and passage it more than twice, then culture it to a confluence of about 90%, digest it with trypsin, and count it. × 10 5 Finally, resuspend the required total amount of cells into 5 × 10 6 Since lymphoma cell lines are suspension cell lines, EG-7 cells were revived and passaged twice or more before being cultured to approximately 1 × 10 6 Each milliliter concentration can be directly collected and counted, according to each mouse 5 × 10 4 Finally, resuspend the required total amount of cells into 5 × 10 5The concentration of cells per milliliter. For both cell lines, 100 microliters of cell suspension were injected subcutaneously in the armpit of each mouse. After about 7-10 days when the tumor was fully formed, it was measured and grouped for administration. IL-9 in DNA form was directly synthesized by Qingke Bio. Our company used the Tiangen Company's plasmid extraction kit to extract a small amount for subsequent experiments. Plasmid DNA-encoded IL-9 was delivered by intramuscular injection and electroporation using Shanghai Tarisa's in vivo electroporation instrument. After the mice formed tumors and were measured and grouped, PD-L1 and CTLA-4 monoclonal antibodies (Bioxcell) were injected intraperitoneally every three days, for a total of four injections, with an injection dose of 250 micrograms of PD-L1 per mouse, and a total of 150 micrograms of CTLA-4 per mouse. DNA-encoded IL-9 was injected intramuscularly once every seven days, for a total of two injections. The dose of DNA-encoded IL-9 was 50 micrograms per mouse. After that, the tumor was measured every other day and the tumor volume was calculated. The tumor growth curve is drawn as follows Figure 2 and Figure 3 As shown. The results showed that the tumor growth rate of all mice in the IL-9 plasmid combined with PD-L1 and CTLA-4 monoclonal antibody treatment group was slower than that in the control group and the PD-L1 group and CTLA-4 group alone, and IL-9 alone had almost no activity in the melanoma experiment, which shows that in some cancers, this combined synergistic effect requires the simultaneous appearance of IL-9 and immune checkpoints. In addition, DNA-encoded IL-9 alone for EG-7 also has a certain therapeutic effect, indicating that IL-9 alone for some cancers also has a certain therapeutic effect. The results show that DNA-encoded IL-9 combined with monoclonal antibodies for PD-L1 and CTLA-4 also significantly enhances their therapeutic effects. Therefore, IL-9 can be used in combination with a variety of immune checkpoint inhibitors to enhance the therapeutic effect, and this synergistic effect of IL-9 has a broad spectrum.

[0125] Example 4 Efficacy of IL-9 fusion protein in antagonizing immune checkpoint inhibitors

[0126] In view of the fact that nucleic acid-encoded IL-9 has the combined activity of monoclonal antibodies corresponding to immune checkpoints, and previous studies have found that recombinant protein IL-9 combined with PD-1 has the effect of promoting T cell proliferation and killing in T lymphocytes cultured in vitro, but immunotherapy in vivo is completely different from in vitro. The immune system in the body is composed of complex and diverse immune cells and immune organs, and the interaction between the immune system and tumors is also complex and cannot be simulated in vitro. Therefore, the efficacy of immunotherapy needs to be verified in vivo to be instructive. Therefore, the present invention continues to evaluate whether recombinant IL-9 has the same effect as nucleic acid-encoded IL-9 in enhancing the effect of immune checkpoint drugs.

[0127] Since the colorectal cancer cell line MC-38 is a cell line sensitive to monoclonal antibodies of PD-1 and CTLA-4, the present invention preferentially uses the MC-38 cell line to construct a subcutaneous transplant tumor model to verify the effect of the combination of recombinant protein IL-9 and monoclonal antibodies of PD-1 and CTLA-4. The process is to resuscitate the MC-38 cell line and subculture it more than twice, then culture it to a confluence of about 90%, digest it with trypsin, and count it. × 10 5 Finally, resuspend the required total amount of cells into 2 × 10 6 Each mouse was injected subcutaneously under the armpit with 100 microliters of cell suspension. The tumor was measured and divided into groups for administration after about 7 days of complete tumor formation.

[0128] After the mice formed tumors and were measured and grouped, PD-1 and CTLA-4 monoclonal antibodies (Bioxcell) were injected intraperitoneally every three days, for a total of four injections, with an injection dose of 350 micrograms of PD-1 per mouse and 150 micrograms of CTLA-4 per mouse. Recombinant protein IL-9 was injected intraperitoneally every three days, for a total of four injections. The dose of recombinant protein IL-9 was 10 micrograms per mouse. After that, the tumor was measured every other day and the tumor volume was calculated. The tumor growth curve was drawn as shown in the figure. Figure 4 As shown. The results showed that the tumor growth rate of all mice in the recombinant protein IL-9 combined with PD-1 and CTLA-4 monoclonal antibody treatment group was faster than that in the PD-1 group alone and the CTLA-4 group alone, although the growth rate was still slightly slower than that in the control group. However, it showed a significant antagonistic effect compared with the PD-1 and CTLA-4 monoclonal antibody treatment groups. This result shows that the recombinant protein form of IL-9 combined with the monoclonal antibodies of PD-L1 and CTLA-4 also significantly weakened their therapeutic effects. Therefore, recombinant protein form of IL-9 cannot be used in combination with immune checkpoint inhibitors or agonists.

[0129] Example 5 explores the differences in IL-9 antibodies produced in mice after injection of DNA-encoded IL-9 and recombinant protein IL-9.

[0130] Given that recombinant protein IL-9 plays a completely opposite role to nucleic acid IL-9 when used in combination with immune checkpoint inhibitors, the present invention explores possible mechanisms. According to previous studies, the use of neutralizing antibodies to IL-9 protein to remove IL-9 will weaken the anti-tumor effect of the drug. Therefore, the present invention evaluates the differences in IL-9 antibodies produced in mice after injection of DNA-encoded IL-9 and recombinant protein IL-9.

[0131] We collected the serum of the mice in Example 1 and Example 4 respectively. The method is to collect blood from the inner canthus of the capillary tube to collect about 100 microliters of peripheral blood without adding anticoagulant. The entire sample was centrifuged at 3000rpm for 20 minutes. The supernatant is the serum. One day in advance, the IL-9 protein used in Example 4 purified by the laboratory was diluted with PBS and coated on the ELISA plate. Each well contained 10μg of protein and was placed at 4° overnight. The plate was washed the next day and blocked with blocking solution at room temperature for 2 hours. The mouse serum was diluted 1:100, 100μl was added to the well and placed at 4°C overnight. ELISA was developed on the third day. The results are as follows Figure 5 As shown. The results showed that after the injection of recombinant protein IL-9 in Example 4, the mice produced significantly higher IL-9 antibodies than the control group, while after the injection of DNA-encoded IL-9, the mice did not produce significantly higher IL-9 antibodies than the control group. This shows that the injection of recombinant protein IL-9 produced a large amount of IL-9 antibodies, which antagonized endogenous and exogenous IL-9, resulting in not only failure to enhance the effect of immune checkpoint inhibitors, but also played the opposite role.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention 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 make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid encoding interleukin-9, The DNA encoding interleukin-9 has the nucleotide sequence shown in SEQ ID NO: 2; The mRNA encoding interleukin-9 has the nucleotide sequence shown in SEQ ID NO:

3.

2. The nucleic acid according to claim 1, characterized in that The nucleic acid sequence encoding interleukin-9 mRNA is shown in SEQ ID NO:

7.

3. A recombinant vector comprising a vector backbone and at least one of the nucleic acids described in claim 1 or 2.

4. The recombinant vector according to claim 3, characterized in that The vector backbone is selected from at least one of pGEM-3Zf(+) and / or pVR.

5. A host transfected or transformed with the recombinant vector according to claim 2 or 3.

6. A nucleic acid preparation comprising an auxiliary material and at least one of the following a) to c): a), the nucleic acid according to claim 1 or 2; b) The recombinant vector according to claim 3 or 4; c) After culturing the host according to claim 5, the nucleic acid or recombinant vector is obtained by extraction.

7. Use of at least one of the following i) to iv) in the preparation of tumor immunotherapy drugs: i), the nucleic acid according to claim 1 or 2; ii), the recombinant vector according to claim 3 or 4; iii) the host according to claim 5; iv) The nucleic acid preparation according to claim 6.

8. Tumor immunotherapy drugs, the raw materials of which include: Immune checkpoint drugs and at least one of the following I) to IV): 1), the nucleic acid according to claim 1 or 2; II), the recombinant vector according to claim 3 or 4; III), after culturing the host according to claim 5, the nucleic acid or recombinant vector obtained by extraction; IV) The nucleic acid preparation according to claim 6.

9. The tumor immunotherapy drug according to claim 8, characterized in that: The immune checkpoints targeted by the immune checkpoint drug include at least one of PD-1, PD-L1, CTLA-4, LAG3, TIM3, TIGIT, VISTA, 4-1BB, SIP1α, CD47, NKG2A, CD137, TNFRSF4, GITR, CD27, ICOS, siglec7, siglec15, LILBR2 or LILBR2.

10. The tumor immunotherapy drug according to claim 8 or 9, characterized in that: The immune checkpoint drugs include: monoclonal antibodies or small molecule drugs targeting immune checkpoints; The small molecule drugs include: small molecule drugs targeting cytokines or chemokines or small molecule drugs targeting metabolic pathways; the small molecule drugs targeting cytokines or chemokines include at least one of PSMA-617, ARRY-382, Vimseltinib and / or BLZ945; the small molecule drugs targeting metabolic pathways include at least one of INCB001158, TT-702, TT4, AB680, ORIC-533, BAY2416964 and / or 1K175.

11. The tumor immunotherapy drug according to any one of claims 8 to 10, characterized in that: The tumor includes at least one of liver cancer, cervical cancer, ovarian cancer, lung cancer, head and neck cancer, prostate cancer, breast cancer, blood cancer, ovarian cancer and / or colorectal cancer.

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