CN114591988B - Preparation method of genetically modified stem cells for activating tumor immunity - Google Patents

Abstract

The invention provides a mesenchymal stem cell for activating tumor immunity and application thereof, belonging to the field of biological medicine. The invention adopts gene engineering means to make mesenchymal stem cells express anti-OX-40 monoclonal antibody fusion protein, the fusion protein as OX40/OX40L channel agonist can enhance the activation and proliferation of effector T cells, specifically bind to OX-40 high-expression T lymphocytes, and specifically activate CD4 ⁺ T、CD8 ⁺ T cells, thereby inhibiting the immune escape of tumor cells, achieving the purpose of treating tumor-related diseases and providing a more ideal new method for treating diseases such as tumors and the like.

Description

Preparation method of genetically modified stem cells for activating tumor immunity

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a preparation method of a genetically modified stem cell for activating tumor immunity.

Background

Tumor (tumor) refers to a new organism formed by local tissue cell proliferation under the action of various tumorigenic factors, because the new organism mostly presents space-occupying block-shaped protrusions, which are also called neoplasms. According to the cellular characteristics of neoplasms and the degree of harm to the body, tumors are classified into benign tumors and malignant tumors. Malignant tumors can be classified into carcinomas and sarcomas, and carcinomas refer to malignant tumors derived from epithelial tissues. Sarcoma refers to malignant tumor of mesenchymal tissues including fibrous connective tissue, fat, muscle, vessel, bone and cartilage tissue. It has been found that tumor cells exhibit metabolic changes that differ from normal cells, while tumor cells themselves can adapt to changes in the metabolic environment by switching between glycolysis and oxidative phosphorylation. Development of antibodies against immune checkpoints on the surface of T cells has been one of the hot spots in tumor immunotherapy, but in addition to cell surface inhibitory receptors such as CTLA-4 and PD-1, T cell surfaces also have a class of activating receptor molecules, which also play an important role in the immune response of the body, and OX40 is one of them.

OX40 (also called CD134, TNFRSF4, ACT 35) belongs to Tumor Necrosis Factor Receptor Superfamily (TNFRSF), is type I transmembrane glycoprotein with molecular weight of about 50kDa, and contains 277 amino acids (49 cytoplasmic amino acids, 186 extracellular amino acids). OX40 is a relatively conserved molecule whose extracellular domain of ligand binding domain is composed of three complete cysteine-rich regions CRD1, CRD2, CRD3 and one incomplete cysteine-rich region CRD 4.

In vivo, OX40 is predominantly on activated CD4 ⁺ T、CD8 ⁺ T cell surface inducibly expresses, whereas neither naive T cells nor primed memory T cells express OX40. In addition, OX40 is expressed on regulatory T cells, NK cells, NKT cells and neutrophils. TCR signaling can induce OX40 expression, but its high expression on the T cell surface still requires the assistance of additional costimulatory signals and associated cytokines, such as CD28-B7.1/2, IL-2, TNF, and the like. T cells began to express OX40 24 hours after antigen stimulation and peaked 48-72 hours later. The length of expression of OX40 depends on the strength of the TCR signal and the associated costimulatory signal, and usually lasts for 5-6 days. OX40 is expressed much more rapidly on memory T cells than on effector T cells, usually within 1-4 hours after activation. Of the cell lines cultured in vitro, only a few are capable of expressing OX40, and all HTLV-1 infected human T lymphocytes, the vast majority of which are CD4 ⁺ T cells, e.g., hut-102, MT-2, ATL-43T and PHA-stimulated PBMC, while unstimulated PBMC are not expressed. In addition to the above cells, other cells have not been found to express OX40.

Patent No. cn201880050094.X discloses that Treg-depleting antibody molecules, such as Treg-depleting anti-4-1 BB antibodies or Treg-depleting OX-40 antibodies, for cancer treatment can specifically bind to targets belonging to the Tumor Necrosis Factor Receptor Superfamily (TNFRSF). Patent cn201910303942.X discloses a recombinant anti-OX 40 (CD 134) antibody and its use, which antibody is capable of specifically binding to mammalian OX40, preferably having high affinity for human and/or primate OX40 proteins, and stimulating T cell activation and proliferation at certain concentrations.

Enhancement of OX40 signaling using OX40 agonist antibodies may improve survival in several preclinical cancer models, including B16 melanoma, lewis lung cancer, colon cancer, breast cancer and 4T-1 breast cancer, by activating enhanced T cell-mediated anti-tumor immunity, enhancing effector species-specific T cell immune responses, enhancing production of cytokines IL-2, IL-5 and IFN-g, and enhancing production of tumor-specific memory T cells following antigen challenge, contributing to tumor regression and prolonging survival, OX40 agonists may increase T cell infiltration into tumors, and enhance anti-tumor immune responses of conventional CD4 and CD8T cells. Thus, OX40/OX40L pathway agonists can be used in cancer immunotherapy. However, although therapeutic monoclonal antibody drugs have advanced anti-cancer functions, the problems of immunogenicity of antibodies, tolerance of long-term use of tumor targets, simple blocking of long-term effects of signal transduction pathways and the like still need to be further researched, and most monoclonal antibodies have difficulty in realizing long-term effective inhibition and killing of tumor cells.

Based on this, a new method for solving the above-mentioned deficiencies of monoclonal antibodies is needed to prepare a drug for activating tumor immunity.

Disclosure of Invention

Aiming at the defects, the invention provides a mesenchymal stem cell for activating tumor immunity and application thereof. The invention adopts a genetic engineering means to enable the mesenchymal stem cells to express OX-40 monoclonal antibody fusion protein, can enhance the activation and proliferation of effector T cells, and specifically combines Treg cells with high OX-40 expression, thereby inhibiting the immune escape of tumor cells and achieving the purpose of treating tumor-related diseases.

In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:

in one aspect, the invention provides an anti-OX-40 monoclonal antibody fusion protein (hereinafter "OX-40 monoclonal antibody fusion protein" or "fusion protein") encoding nucleotide comprising a nucleotide encoding an OX-40 antibody.

Specifically, the nucleotide sequence encoding OX-40antibody comprises the nucleotide sequence shown in SEQ ID NO. 3 or SEQ ID NO. 5 encoding the VL of the light chain variable region of the OX-40antibody, and the nucleotide sequence shown in SEQ ID NO. 4 or SEQ ID NO. 6 encoding the VH of the heavy chain variable region of the OX-40 antibody.

The nucleotide sequence shown in SEQ ID NO. 3 is as follows:

GACATCCAAATGACTCAGAGTCCAAGTTCTTTGTCCGCCTCAGTTGGCGACAGAGTAACGATCACTTGCAGAGCCAGCCAAGATATCAGTAACTACTTGAATTGGTACCAACAGAAACCTGGGAAGGCACCGAAACTTCTGATCTACTACACATCTAAACTTCATTCAGGCGTCCCTTCACGCTTCTCTGGTTCCGGCTCTGGAACTGACTACACACTGACAATTTCAAGTCTCCAGCCCGAAGATTTTGCAACATATTATTGTCAACAGGGCTCCGCGCTCCCATGGACGTTTGGACAAGGAACGAAGGTAGAAATCAAG；

the nucleotide sequence shown in SEQ ID NO. 4 is:

CAAGTTCAACTTCAGGAATCAGGTCCCGGCTTGGTTAAGCCTTCACAGACACTCTCCCTGACTTGTGCAGTGTATGGCGGTTCTTTTAGTAGTGGGTATTGGAATTGGATAAGGAAGCATCCAGGAAAAGGTCTTGAGTACATCGGGTATATCAGTTACAATGGGATCACCTATCACAACCCGAGCCTGAAGTCCCGAATAACTATTAACAGGGACACCTCTAAAAACCAGTATAGCCTTCAATTGAATAGTGTTACGCCAGAGGATACTGCTGTGTACTACTGTGCTAGATACAAATACGACTATGATGGAGGACATGCGATGGACTACTGGGGACAGGGAACCCTGGTGACCGTCTCCTCC；

the nucleotide sequence shown in SEQ ID NO. 5 is as follows:

GACATACAAATGACCCAGAGTCCTAGCTCACTATCGGCGTCTGTCGGTGACCGGGTGACCATCACATGTCGAGCCTCGCAGGACATATCGAACTACCTTAACTGGTACCAGCAGAAACCTGGGAAAGCACCAAAGCTTTTAATCTACTATACTTCGAAGCTTCATTCTGGTGTCCCATCGCGTTTCAGCGGATCTGGTTCCGGGACAGACTACACCCTGACCATATCAAGCCTGCAACCCGAAGATTTTGCGACTTACTACTGCCAACAGGGATCAGCTCTTCCATGGACGTTCGGGCAAGGCACAAAGGTCGAGATTAAA；

the nucleotide sequence shown in SEQ ID NO. 6 is:

CAGGTCCAATTGCAGGAGTCAGGACCTGGGCTCGTCAAGCCGTCACAGACTTTATCTTTGACATGCGCAGTGTATGGGGGATCGTTCTCAAGCGGTTACTGGAACTGGATTAGAAAGCATCCGGGGAAGGGGCTAGAGTACATTGGGTATATCTCGTATAACGGAATCACGTACCACAATCCCTCGTTGAAGAGCAGGATTACCATCAACAGGGACACGTCAAAAAATCAGTACAGTCTTCAACTGAATTCAGTAACTCCCGAGGATACGGCGGTATATTACTGTGCCCGATACAAATATGACTACGACGGAGGTCATGCGATGGACTACTGGGGGCAGGGTACGCTGGTTACGGTTAGCTCG。

more specifically, the nucleotide sequence encoding OX-40antibody further comprises the nucleotide sequence shown as SEQ ID NO. 1 encoding a signal peptide and the nucleotide sequence shown as SEQ ID NO. 2 encoding a linker.

Specifically, the encoding nucleotide also comprises a nucleotide for encoding an Fc fragment of human immunoglobulin IgG1, and the nucleotide for encoding the Fc fragment of IgG1 is the nucleotide sequence shown in SEQ ID NO. 7.

More specifically, the coding nucleotide is a sequence shown in SEQ ID NO. 8 or SEQ ID NO. 9.

Specifically, the encoding nucleotide codes OX-40 monoclonal antibody fusion protein, and the OX-40 monoclonal antibody fusion protein comprises OX-40antibody and Fc fragment of human immunoglobulin IgG 1.

More specifically, the OX-40antibody comprises a signal peptide of the amino acid sequence shown as SEQ ID NO. 10, a linker of the amino acid sequence shown as SEQ ID NO. 11, an OX-40antibody light chain variable region VL of the amino acid sequence shown as SEQ ID NO. 12, and an OX-40antibody heavy chain variable region VH of the amino acid sequence shown as SEQ ID NO. 13.

More specifically, the Fc fragment of the human immunoglobulin IgG1 is an amino acid sequence shown in SEQ ID NO. 14.

More specifically, the amino acid sequence of the OX-40 monoclonal antibody fusion protein is the amino acid sequence shown in SEQ ID NO. 15.

The OX-40 monoclonal antibody is used as OX40 agonist, has the functions of activating T cells (including T cell subsets such as CD4 and CD 8), and enhancing the generation of cytokines such as IL-2 and IFN-g, so that the effect of improving the antitumor activity is achieved.

In yet another aspect, the present invention provides a vector comprising the above-described coding nucleotide.

Specifically, the vector includes but is not limited to plasmid, virus, phage.

In yet another aspect, the present invention provides a host cell comprising the above-described encoding nucleotide or the above-described vector.

Specifically, the host cell includes but is not limited to a microorganism, a plant or an animal cell, preferably a mesenchymal stem cell.

Specifically, the nucleotide or the vector is introduced into the host cell by a genetic engineering technique.

More specifically, the genetic engineering techniques include, but are not limited to, viral transfection, lipofection, electrotransfer, gene editing, or mRNA transfection, preferably using lentiviral vector systems.

In yet another aspect, the invention provides a method of producing the above-described OX-40 monoclonal antibody fusion protein, the method comprising culturing the above-described host cell under conditions such that the fusion protein is expressed.

In still another aspect, the present invention provides a mesenchymal stem cell comprising the above-described encoding nucleotide or the above-described vector.

Specifically, the mesenchymal stem cells are derived from bone marrow tissue, adipose tissue, umbilical cord tissue or placenta tissue.

In another aspect, the present invention provides a method for preparing the mesenchymal stem cell, the method comprising the steps of:

(1) Constructing a lentivirus expression vector containing the nucleotide sequence coding the OX-40 monoclonal antibody fusion protein;

(2) Infecting host cells with the lentivirus expression vector obtained in the step (1), and packaging to obtain mature lentivirus;

(3) And (3) harvesting the mature lentivirus obtained in the step (2), infecting mesenchymal stem cells, and screening the cells successfully infected.

In some embodiments of the invention, a lentiviral expression plasmid of OX-40 monoclonal antibody fusion protein is firstly constructed by using a lentiviral vector system, wherein the OX-40 monoclonal antibody fusion protein is a fusion protein consisting of an OX-40antibody and an Fc fragment of a heavy chain of IgG1, and the 3' -end of an encoding gene of the OX-40 monoclonal antibody fusion protein is connected with an IRES sequence and an EGFP gene in sequence to form an OX-40 Fc-I-EGPF sequence which is positioned at the downstream of a CMV promoter in a LV-EGFP lentiviral vector; secondly, mixing the lentivirus expression plasmid with lentivirus frame plasmids pGag/Pol, pRev and Pvsv-g, introducing the mixture into HEK293T cells through LTX liposome to complete gene transcription, successfully packaging and releasing the lentivirus vector into supernatant of HEK293T cell culture medium, and then collecting the supernatant containing the recombinant lentivirus vector; finally, infecting the mesenchymal stem cells by the recombinant lentiviruses in the harvested supernatant, and adding puromycin after 24 hours to screen to obtain the successfully infected mesenchymal stem cells. OX-40 monoclonal antibody fusion protein is expressed by mesenchymal stem cell ribosome, is secreted out of cells under the guidance of signal peptide, and is specifically combined with Treg cells with high OX-40, so that the immune escape of tumor cells is inhibited, and the aim of treating tumor-related diseases is fulfilled.

In still another aspect, the present invention provides a pharmaceutical composition comprising the above-described coding nucleotide, a vector, a host cell and/or a mesenchymal stem cell.

Specifically, the pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.

Further specifically, the pharmaceutically acceptable carrier includes, but is not limited to: diluent, excipient, filler, wetting agent, disintegrating agent, flavoring agent and adhesive.

Specifically, the pharmaceutical composition may be administered alone, sequentially or simultaneously in combination with other drugs selected from, but not limited to, penicillin, amoxicillin, benzylpenicillin, ampicillin, azithromycin, erythromycin, clarithromycin, cefadroxil, cefixime, tetracycline, minocycline, rifampin, rifabutin, sulfanilamide, aminoglycosides, bacitracin, polymyxin B, ornidazole, retinol, retinal, isotretinoin, viaminate, adapalene, tazarotene, estrogen, progestin, glucocorticoid, antiandrogen, vitamin D3 analogs such as calcipotriol and tacalcitol, glucocorticoid-like drugs such as prednisone and methylprednisolone, tacrolimus, pimecrolimus, cyclosporine A, mycophenolate esters, leukotriene B4 inhibitors, IL-1 inhibitors, IL-2 inhibitors, IL-1 beta inhibitors, IL-6 inhibitors, IL-8 inhibitors, IL-12 inhibitors, IL-23 inhibitors, IL-17 inhibitors, GM-19 inhibitors, CD-20 inhibitors. The methods of combination therapy also include administration in combination with photodynamic therapy, laser therapy, or fruit acid therapy.

In still another aspect, the present invention provides an application of the above coding nucleotide, vector, host cell and/or mesenchymal stem cell in the preparation of a medicament, a kit and/or a medical device for preventing and/or treating tumor.

Compared with the prior art, the invention has the advantages that:

the modified mesenchymal stem cell can express OX-40 monoclonal antibody fusion protein, and the mesenchymal stem cell can specifically express the OX-40 monoclonal antibody fusion protein to a tumor part to play a role by utilizing the targeting effect of the mesenchymal stem cell on the tumor part, the immunoregulation capability and the repair capability of a damaged part and a gene modification method. The OX-40 monoclonal antibody fusion protein serving as OX40/OX40L pathway agonist can enhance activation and proliferation of effector T cells, specifically bind to OX-40 high-expression T lymphocytes and specifically activate CD4 ⁺ T、CD8 ⁺ T cells, thereby inhibiting the immune escape of tumor cells and achieving the purpose of treating tumor-related diseases. The present invention provides an ideal new method for treating diseases such as tumor, etc.

Drawings

FIG. 1 is a schematic diagram showing LV-OX40 Fc-I-EGFP plasmid information.

FIG. 2 is a result chart of flow cytometry for detecting green fluorescent protein expressed by genetically modified stem cells.

FIG. 3 is a graph showing the results of ELISA detection of the content of fusion protein IgG1 secreted by cells.

FIG. 4 is a graph showing the results of measuring the amount of OX40 mAb secreted from genetically modified stem cells by a binding assay with OX-40 protein.

FIG. 5 is a graph showing the results of an experiment for detecting IFN-. Gamma.secretion by peripheral blood lymphocytes activated by genetically modified stem cells.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The three letter code and the one letter code of the amino acid used in the present invention are as described in J.biol.chem,243, p3558 (1968, IUPAC-IUB Commission).

Exemplary techniques for use in conjunction with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are known in the art. Many such techniques and procedures are described, for example, in Sambrook et al, molecular Cloning: a Laboratory Manual (2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. (1989)), among many others.

The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

A "host cell" as described herein is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the invention. The host cell may be a prokaryote, e.g., e.coli, or it may be a eukaryote, e.g., a unicellular eukaryote (e.g., yeast or other fungus), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma.

The Mesenchymal Stem Cells (MSCs) are derived from mesoderm in early development stage, and have various cell functions, including repair and regeneration of damaged tissues, participation in immune regulation of organisms, promotion of angiogenesis, homing and targeting of inflammation parts, and inhibition of tumor growth. MSCs isolated from tissues such as umbilical cord have better in vitro growth potential and higher regeneration ability than adult tissues, and are defined to have some common basic properties such as in vitro plastic adhesion, expression of surface markers CD73, CD90 and CD105, and differentiation ability of osteogenic, adipogenic and chondrogenic in vitro. In addition to these typical characteristics of MSCs, cord-derived MSCs may produce and release more transforming growth factor-beta (TGF-. Beta.), vascular endothelial factor-alpha (VEGF-. Alpha.), and Epidermal Growth Factor (EGF).

The massive secretion of MSCs in the tumor microenvironment, such as fibroblast growth factor-2 (FGF-2), monocyte chemoattractant protein-1 (MCP-1), proinflammatory factor interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF α), contributes to their accumulation at the tumor site, and the interaction of immune cells with MSCs in the tumor stroma can activate their immunomodulatory capacity. MSCs secrete a variety of immunomodulators, such as Nitric Oxide (NO), prostaglandin E2 (PGE 2), interleukin 6 (IL-6), interleukin 10 (IL-10), metabolites of indoleamine 2, 3-dioxygenase (IDO), and the like, which are involved in the induction of immune tolerance and the shift of T cell Th1 to Th2 immune responses. In addition, MSCs can inhibit the proliferation of effector T cells by activating apoptotic mechanisms by PD-1 and interacting with the corresponding ligands PD-L1 and PD-L2. In addition to addressing the immunosuppressive and anti-inflammatory effects of regulatory T cells, MSCs are involved in the process of transforming macrophages.

In addition to interacting with different types of cells in the tumor stroma, MSCs can interact directly or indirectly with cancer cells, and based on their ability to home to the tumor site, MSCs are considered ideal candidates for delivering anti-tumor drugs, a potential clinical approach.

The "antibody" of the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure formed by linking two identical heavy chains and two identical light chains via interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus in their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes called immunoglobulins, i.e., igM, igD, igG, igA, igE, and their corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, for example, igG can be divided into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

The approximately 110 amino acid sequence of the heavy and light chains of antibodies near the N-terminus varies widely, as the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence-conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each light chain variable region (VL or LCVR) and heavy chain variable region (VH or HCVR) consists of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3, and the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.

The term "complementarity determining region" (CDR) refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding. Typically, there are three CDRs (HCDR 1, HCDR2, HCDR 3) per heavy chain variable region and three CDRs (LCDR 1, LCDR2, LCDR 3) per light chain variable region. The amino acid sequence boundaries of the CDRs are determined using the "Kabat numbering convention" (see Kabat et al (1991)).

The terms "specific binding", "selective binding" and "selective binding" refer to the binding of an antibody to a predetermined epitope of an antigen. Typically, the antibody is present in an amount less than about 10 ^-8 M, e.g. less than about 10 ^-9 M、10 ^-10 M、10 ^-11 M or less affinity (KD) binding. Where "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.

The term "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, which are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the invention is contemplated.

In certain embodiments, the OX-40 monoclonal antibody fusion proteins of the invention can be obtained by culturing a host cell comprising the nucleic acid molecule or vector or a genetically modified cell comprising a nucleotide sequence encoding the OX-40 monoclonal antibody fusion protein.

Example 1 construction of lentivirus expression plasmids in vitro

A fourth generation lentiviral vector system LV-EGFP (from OriGene) was used to construct a lentiviral expression plasmid for the OX-40 mab fusion protein, with an empty LV-EGFP plasmid as control.

Wherein, the OX-40 monoclonal antibody fusion protein is a fusion protein (SEQ ID NO: 15) composed of an OX-40antibody and an IgG1 heavy chain Fc fragment. The OX-40antibody comprises a signal peptide of an amino acid sequence shown as SEQ ID NO. 10, a joint of an amino acid sequence shown as SEQ ID NO. 11, an OX-40antibody light chain variable region VL of an amino acid sequence shown as SEQ ID NO. 12, and an OX-40antibody heavy chain variable region VH of an amino acid sequence shown as SEQ ID NO. 13; the Fc fragment of the human immunoglobulin IgG1 is an amino acid sequence shown in SEQ ID NO. 14.

The encoding gene (shown as SEQ ID NO:8 or SEQ ID NO: 9) of the OX-40 monoclonal antibody fusion protein comprises a nucleotide sequence of an OX-40antibody and a nucleotide sequence of an IgG1 heavy chain Fc fragment, wherein the nucleotide encoding the OX-40antibody comprises a nucleotide sequence shown as SEQ ID NO:1 encoding a signal peptide, a nucleotide sequence shown as SEQ ID NO:2 encoding a linker, a nucleotide sequence shown as SEQ ID NO:3 or SEQ ID NO:5 encoding a light chain variable region VL of the OX-40antibody, and a nucleotide sequence shown as SEQ ID NO:4 or SEQ ID NO:6 encoding a heavy chain variable region VH of the OX-40 antibody; the nucleotide sequence of the IgG1 heavy chain Fc fragment is shown as SEQ ID NO. 7.

The 3' -end of the OX-40 monoclonal antibody fusion protein coding gene (the sequence shown in SEQ ID NO:8 or SEQ ID NO: 9) is connected with an IRES sequence and an EGFP gene in sequence to form OX-40-OS1: fc-I-EGFP or OX-40-OS2: fc-I-EGFP sequence which are positioned at the downstream of a CMV promoter (the vector schematic diagram is shown in figure 1).

Specifically, the nucleotide sequence of SEQ ID NO 3 or SEQ ID NO 5 encoding the light chain variable region of OX-40antibody is optimized from the WT sequence of SEQ ID NO 16, and the nucleotide sequence of SEQ ID NO 4 or SEQ ID NO 6 encoding the heavy chain variable region of OX-40antibody is optimized from the WT sequence of SEQ ID NO 17. Meanwhile, the WT sequence is utilized to construct an OX-40-WT: fc-I-EGFP sequence (the nucleotide sequence of the light chain variable region of the OX-40antibody coded by the sequence is the WT sequence shown in SEQ ID NO:16, the nucleotide sequence of the heavy chain variable region of the OX-40antibody coded by the sequence is the WT sequence shown in SEQ ID NO:17, and the rest sequences are the same as the OX-40-OS1: fc-I-EGFP or OX-40-OS2: fc-I-EGFP sequence).

According to the gene structure, the target gene fragment is inserted into the corresponding site of the LV-EGFP plasmid by using a conventional genetic engineering means to obtain lentivirus expression plasmids LV-OX-40-OS1: fc, LV-OX-40-OS2: fc and LV-OX-40-WT: fc.

Example 2 Virus preparation and harvesting

The LV-OX-40-OS1: fc, LV-OX-40-OS2: fc and LV-OX-40-WT: fc plasmids prepared in example 1 were transfected into HEK293T cells via LTX liposomes together with lentiviral framework plasmids pGag/Pol, pRev, pVSV-G, respectively, and the successfully packaged recombinant lentiviral vectors were released into HEK293T cell culture supernatants, and cell culture supernatants containing the recombinant lentiviral vectors were harvested. Control LV-EGFP viruses were also prepared.

Example 3 preparation of genetically modified mesenchymal Stem cells

And (3) performing ultrafiltration purification on the cell culture supernatant containing the recombinant lentiviral vector harvested in the example 2, adding the cell culture supernatant into P2-P3 generation umbilical cord mesenchymal stem cells in logarithmic phase, changing the solution after the recombinant lentiviral vector infects the mesenchymal stem cells for 24 hours, continuously culturing for 24 hours, changing the solution again, and adding puromycin to screen to obtain successfully infected mesenchymal stem cells EGFP-MSC, OX-40-OS1: fc-MSC, OX-40-OS2: fc-MSC and OX-40-WT: fc-MSC.

Example 4 detection of expression of Green fluorescent protein by genetically modified Stem cells by flow cytometry

The mesenchymal stem cells successfully infected with lentivirus in example 3 are inoculated into a T75 culture flask, the cells are harvested after being cultured to 80 percent, and the green fluorescence emitted by the mesenchymal stem cells after lentivirus infection is detected by a flow cytometry method (the result is shown in figure 2), so that positive signals can be detected in a green fluorescence channel by the mesenchymal stem cells after LV-EGFP and LV-OX40-OS1: fc, LV-OX-40-OS2: fc and LV-OX-40-WT: fc lentivirus infection, while the normal mesenchymal stem cells have no green fluorescence, which indicates that the lentiviruses of LV-EGPF and LV-OX40: fc fusion protein can successfully infect the mesenchymal stem cells, and the infection rate is more than 85 percent.

Example 5 ELISA detection of the amount of fusion protein IgG1 protein secreted by cells

IgG1 ELISA kit (BMS 2092, thermo) is adopted to detect the content of the fusion protein IgG1 secreted in the cell culture supernatant.

After culturing the genetically modified mesenchymal stem cells obtained in example 3 for 72 hours, cell supernatants were harvested and added to an IgG1 ELISA kit to detect the IgG1 protein level in the fusion protein, and as a result, it was found that the mesenchymal stem cells after infection with LV-OX-40-OS1: fc, LV-OX-40-OS2: fc and LV-OX-40-WT: fc lentivirus (OX-40-OS 1: fc-MSC, OX-40-OS2: fc-MSC and OX-40-WT: fc-MSC) all highly expressed IgG1 and the expression was between 27.5 and 35.8ng/mL, and there was no significant difference between the three groups (P > 0.05) compared with normal hUC-MSC and EGFP-MSC (see FIG. 3 for results).

Example 6 ELISA assay for content of OX-40 monoclonal antibody secreted by genetically modified Stem cell

The expression of OX-40antibody in the cell culture supernatant was examined by binding OX-40 recombinant protein to OX-40 antibody.

The enzyme label plate is coated with Human OX40/TNFRSF4/CD134 protein Fc Tag (Acro, OX 0-H5255) at the concentration of 1 mu g/mL, after blocking, anti-OX-40Antibody (SinoBiological 10481-T16) is used as a standard substance to be added into the air of the standard substance, supernatant obtained after culturing the genetically modified mesenchymal stem cells obtained in example 3 for 72H is added into the sample well, after 2H incubation, HRP-conjugated Anti-Human IgG Antibody (Acro, IG 1-S71) and HRP-conjugated Anti-rabbit IgG Antibody (southern Biotech, 4055-05) are used as enzyme-labeled antibodies to be incubated for 1H, then TMB developing liquid is added for 10-20min, stopping liquid, and OD450nm reading is detected by an enzyme label instrument. The results are shown in FIG. 4, OX-40-OS1: fc-MSC, OX-40-OS2: fc-MSC and OX-40-WT: fc-MSC all expressed OX-40antibody bound to recombinant OX-40 protein, and OX-40-OS1: fc-MSC secreted OX-40antibody at the highest concentration of 106.0. + -. 12.3ng/mL, with a significant difference (P < 0.01) compared to OX-40-WT: fc-MSC, whereas normal hUC-MSC and EGFP-MSC did not express OX-40 antibody.

Example 7 detection of the activation of IFN-. Gamma.secretion by genetically modified Stem cells by peripheral blood lymphocytes

Activation function of genetically modified mesenchymal stem cells was examined by detecting inflammatory factor IFN-. Gamma.secreted after PBMC activation, human PBMC cells were isolated, and anti-CD 3 antibody (Biolegend, 317325) (1. Mu.g/mL) and anti-CD 28 antibody (Biolegend, 302933) (1. Mu.g/mL) were added to each group of cells obtained in example 3 (1X 10) ⁴ /cm ² Inoculating 24-well plate and culturing for 24 h) and culturing for 72h, stimulating PBMC with anti-CD 3 antibody (1 μ g/mL) and anti-CD 28 antibody (1 μ g/mL) for 72h as control group, and performing ELISA (R)&D, DIF 50C) the concentration of IFN-. Gamma.in the cell supernatant was determined.

The result shows that CD3/CD28 can stimulate PBMC to secrete IFN-gamma at the concentration of 473.2 +/-68.2 pg/mL, normal hUC-MSC and EGFP-MSC cells can obviously inhibit activated PBMC to secrete IFN-gamma at the concentration of 57.6 +/-4.8 pg/mL, and OX-40-OS1: fc-MSC, OX-40-OS2: fc-MSC and OX-40-WT: fc-MSC cells can obviously enhance the release of IFN-gamma in PBMC at the concentrations of 1462.4 +/-122.8 pg/mL, 1209.6 +/-176.1 pg/mL and 922.2 +/-97.3 pg/mL respectively; OX-40-OS1: fc-MSCs released IFN- γ in PBMCs increased by 58.6% (P < 0.01) compared to OX-40-WT: fc-MSC cells.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

SEQUENCE LISTING

<110> Beijing Beibei Biotech Co., ltd

<120> preparation method of genetically modified stem cell for activating tumor immunity

<130> 20220328

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 60

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60

<210> 2

<211> 60

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

ggcgggggcg gttcaggcgg cgggggctct ggtggagggg gctcaggtgg aggaggctct 60

<210> 3

<211> 321

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

gacatccaaa tgactcagag tccaagttct ttgtccgcct cagttggcga cagagtaacg 60

atcacttgca gagccagcca agatatcagt aactacttga attggtacca acagaaacct 120

gggaaggcac cgaaacttct gatctactac acatctaaac ttcattcagg cgtcccttca 180

cgcttctctg gttccggctc tggaactgac tacacactga caatttcaag tctccagccc 240

gaagattttg caacatatta ttgtcaacag ggctccgcgc tcccatggac gtttggacaa 300

ggaacgaagg tagaaatcaa g 321

<210> 4

<211> 363

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

caagttcaac ttcaggaatc aggtcccggc ttggttaagc cttcacagac actctccctg 60

acttgtgcag tgtatggcgg ttcttttagt agtgggtatt ggaattggat aaggaagcat 120

ccaggaaaag gtcttgagta catcgggtat atcagttaca atgggatcac ctatcacaac 180

ccgagcctga agtcccgaat aactattaac agggacacct ctaaaaacca gtatagcctt 240

caattgaata gtgttacgcc agaggatact gctgtgtact actgtgctag atacaaatac 300

gactatgatg gaggacatgc gatggactac tggggacagg gaaccctggt gaccgtctcc 360

tcc 363

<210> 5

<211> 321

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

gacatacaaa tgacccagag tcctagctca ctatcggcgt ctgtcggtga ccgggtgacc 60

atcacatgtc gagcctcgca ggacatatcg aactacctta actggtacca gcagaaacct 120

gggaaagcac caaagctttt aatctactat acttcgaagc ttcattctgg tgtcccatcg 180

cgtttcagcg gatctggttc cgggacagac tacaccctga ccatatcaag cctgcaaccc 240

gaagattttg cgacttacta ctgccaacag ggatcagctc ttccatggac gttcgggcaa 300

ggcacaaagg tcgagattaa a 321

<210> 6

<211> 363

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

caggtccaat tgcaggagtc aggacctggg ctcgtcaagc cgtcacagac tttatctttg 60

acatgcgcag tgtatggggg atcgttctca agcggttact ggaactggat tagaaagcat 120

ccggggaagg ggctagagta cattgggtat atctcgtata acggaatcac gtaccacaat 180

ccctcgttga agagcaggat taccatcaac agggacacgt caaaaaatca gtacagtctt 240

caactgaatt cagtaactcc cgaggatacg gcggtatatt actgtgcccg atacaaatat 300

gactacgacg gaggtcatgc gatggactac tgggggcagg gtacgctggt tacggttagc 360

tcg 363

<210> 7

<211> 684

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa atga 684

<210> 8

<211> 1488

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60

gacatccaaa tgactcagag tccaagttct ttgtccgcct cagttggcga cagagtaacg 120

atcacttgca gagccagcca agatatcagt aactacttga attggtacca acagaaacct 180

gggaaggcac cgaaacttct gatctactac acatctaaac ttcattcagg cgtcccttca 240

cgcttctctg gttccggctc tggaactgac tacacactga caatttcaag tctccagccc 300

gaagattttg caacatatta ttgtcaacag ggctccgcgc tcccatggac gtttggacaa 360

ggaacgaagg tagaaatcaa gggcgggggc ggttcaggcg gcgggggctc tggtggaggg 420

ggctcaggtg gaggaggctc tcaagttcaa cttcaggaat caggtcccgg cttggttaag 480

ccttcacaga cactctccct gacttgtgca gtgtatggcg gttcttttag tagtgggtat 540

tggaattgga taaggaagca tccaggaaaa ggtcttgagt acatcgggta tatcagttac 600

aatgggatca cctatcacaa cccgagcctg aagtcccgaa taactattaa cagggacacc 660

tctaaaaacc agtatagcct tcaattgaat agtgttacgc cagaggatac tgctgtgtac 720

tactgtgcta gatacaaata cgactatgat ggaggacatg cgatggacta ctggggacag 780

ggaaccctgg tgaccgtctc ctccgacaaa actcacacat gcccaccgtg cccagcacct 840

gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 900

atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 960

gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1200

ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260

tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1380

gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca cgaggctctg 1440

cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatga 1488

<210> 9

<211> 1488

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60

gacatacaaa tgacccagag tcctagctca ctatcggcgt ctgtcggtga ccgggtgacc 120

atcacatgtc gagcctcgca ggacatatcg aactacctta actggtacca gcagaaacct 180

gggaaagcac caaagctttt aatctactat acttcgaagc ttcattctgg tgtcccatcg 240

cgtttcagcg gatctggttc cgggacagac tacaccctga ccatatcaag cctgcaaccc 300

gaagattttg cgacttacta ctgccaacag ggatcagctc ttccatggac gttcgggcaa 360

ggcacaaagg tcgagattaa aggcgggggc ggttcaggcg gcgggggctc tggtggaggg 420

ggctcaggtg gaggaggctc tcaggtccaa ttgcaggagt caggacctgg gctcgtcaag 480

ccgtcacaga ctttatcttt gacatgcgca gtgtatgggg gatcgttctc aagcggttac 540

tggaactgga ttagaaagca tccggggaag gggctagagt acattgggta tatctcgtat 600

aacggaatca cgtaccacaa tccctcgttg aagagcagga ttaccatcaa cagggacacg 660

tcaaaaaatc agtacagtct tcaactgaat tcagtaactc ccgaggatac ggcggtatat 720

tactgtgccc gatacaaata tgactacgac ggaggtcatg cgatggacta ctgggggcag 780

ggtacgctgg ttacggttag ctcggacaaa actcacacat gcccaccgtg cccagcacct 840

gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 900

atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 960

gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1200

ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260

tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1380

gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca cgaggctctg 1440

cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatga 1488

<210> 10

<211> 20

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 10

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 11

<211> 20

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 11

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 12

<211> 107

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 121

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 13

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 227

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 14

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 15

<211> 495

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 15

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp

35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser

100 105 110

Ala Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

145 150 155 160

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe

165 170 175

Ser Ser Gly Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu

180 185 190

Glu Tyr Ile Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro

195 200 205

Ser Leu Lys Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln

210 215 220

Tyr Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr

225 230 235 240

Tyr Cys Ala Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His

260 265 270

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

305 310 315 320

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

485 490 495

<210> 16

<211> 321

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60

atcacctgtc gggccagcca ggacatcagc aactacctga actggtatca gcagaagccc 120

ggcaaggccc ccaagctgct gatctactac accagcaagc tgcacagcgg cgtgcccagc 180

agattcagcg gcagcggctc cggcaccgac tacaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag ggctccgccc tgccctggac ctttggccag 300

ggcaccaagg tggaaatcaa g 321

<210> 17

<211> 363

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 17

caggtgcagc tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg 60

acctgtgccg tgtacggcgg cagcttcagc agcggctact ggaactggat ccggaagcac 120

cccggcaagg gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac 180

cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca gtactccctg 240

cagctgaaca gcgtgacccc cgaggacacc gccgtgtact actgcgcccg gtacaaatac 300

gactacgacg gcggccacgc catggactac tggggccagg gcaccctggt caccgtgtcc 360

tct 363

<210> 18

<211> 1488

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 18

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 120

atcacctgtc gggccagcca ggacatcagc aactacctga actggtatca gcagaagccc 180

ggcaaggccc ccaagctgct gatctactac accagcaagc tgcacagcgg cgtgcccagc 240

agattcagcg gcagcggctc cggcaccgac tacaccctga ccatcagcag cctgcagccc 300

gaggacttcg ccacctacta ctgccagcag ggctccgccc tgccctggac ctttggccag 360

ggcaccaagg tggaaatcaa gggcgggggc ggttcaggcg gcgggggctc tggtggaggg 420

ggctcaggtg gaggaggctc tcaggtgcag ctgcaggaaa gcggccctgg cctggtcaag 480

cccagccaga ccctgagcct gacctgtgcc gtgtacggcg gcagcttcag cagcggctac 540

tggaactgga tccggaagca ccccggcaag ggcctggaat acatcggcta catcagctac 600

aacggcatca cctaccacaa ccccagcctg aagtcccgga tcaccatcaa ccgggacacc 660

agcaagaacc agtactccct gcagctgaac agcgtgaccc ccgaggacac cgccgtgtac 720

tactgcgccc ggtacaaata cgactacgac ggcggccacg ccatggacta ctggggccag 780

ggcaccctgg tcaccgtgtc ctctgacaaa actcacacat gcccaccgtg cccagcacct 840

gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 900

atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 960

gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1200

ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260

tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1380

gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca cgaggctctg 1440

cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatga 1488

Claims (9)

1. A nucleotide encoding an anti-OX-40 monoclonal antibody fusion protein characterized by: the coding nucleotide consists of the following sequences: a nucleotide sequence encoding a light chain variable region VL of an OX-40antibody, a nucleotide sequence encoding a heavy chain variable region VH of an OX-40antibody, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a linker and a nucleotide sequence encoding an Fc fragment of human immunoglobulin IgG 1;

in the coding nucleotide, the nucleotide sequence for coding the light chain variable region VL of the OX-40antibody is SEQ ID NO. 3, and the nucleotide sequence for coding the heavy chain variable region VH of the OX-40antibody is SEQ ID NO. 4;

or in the coding nucleotide, the nucleotide sequence for coding the OX-40antibody light chain variable region VL is SEQ ID NO. 5; the nucleotide sequence of encoding OX-40antibody heavy chain variable region VH is SEQ ID NO 6;

the nucleotide sequence of the coding signal peptide is SEQ ID NO. 1;

the nucleotide sequence of the coding joint is SEQ ID NO. 2;

the nucleotide sequence of the Fc fragment of the encoded human immunoglobulin IgG1 is SEQ ID NO. 7.

2. The coding nucleotide according to claim 1, characterized in that: the coding nucleotide is a sequence shown in SEQ ID NO. 8 or SEQ ID NO. 9.

3. A vector comprising the coding nucleotide of any one of claims 1-2.

4. A host cell comprising the coding nucleotide of any one of claims 1-2 or the vector of claim 3.

5. A method for producing an OX-40 monoclonal antibody fusion protein, comprising: the method comprises culturing the host cell of claim 4 under conditions such that the fusion protein is expressed.

6. A mesenchymal stem cell, characterized by: the mesenchymal stem cell comprises the coding nucleotide of any one of claims 1-2 or the vector of claim 3.

7. A method of preparing the mesenchymal stem cell of claim 6, wherein: the preparation method comprises the following steps:

(1) Constructing a lentiviral expression vector comprising the coding nucleotide sequence of any one of claims 1-2;

(2) Infecting host cells with the lentiviral expression vector obtained in the step (1), and packaging to obtain mature lentiviruses;

(3) And (3) harvesting the mature lentivirus obtained in the step (2), infecting mesenchymal stem cells, and screening the cells successfully infected.

8. A pharmaceutical composition characterized by: the pharmaceutical composition comprises the coding nucleotide of any one of claims 1-2, the vector of claim 3, the host cell of claim 4, and/or the mesenchymal stem cell of claim 6.

9. Use of the coding nucleotide of any one of claims 1-2, the vector of claim 3, the host cell of claim 4, and/or the mesenchymal stem cell of claim 6 in the manufacture of a medicament or kit for treating a tumor.

Images