Preparation and application of a gene encoding anti-PD-1 antibody and stem cells modified by the gene

Abstract

The present invention provides a gene encoding an anti-PD-1 antibody and the preparation and application of modified stem cells thereof, belonging to the field of genetic engineering technology. The anti-PD-1 antibody gene includes: a nucleotide sequence encoding a light chain variable region SEQ ID NO.1, a nucleotide sequence encoding a linker SEQ ID NO.2, and a nucleotide sequence encoding a heavy chain variable region SEQ ID NO.3. The lentiviral vector prepared by the aforementioned gene of the present invention has an infection rate of more than 80% for mesenchymal stem cells, a high expression level of IgG4 in gene-modified mesenchymal stem cells, a high content of anti-PD-1 antibodies, and can significantly block the binding of PD-1 / PD-L1. Compared with WT, the PD-1 / PD-L1 blocking ability increased by 32.8%, and has a good prospect for clinical application.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to the preparation and application of a gene encoding an anti-PD-1 antibody and stem cells modified therefrom. Background Art

[0002] PD-1, or programmed death receptor 1, also known as CD279 (cluster of differentiation 279), is an important immunosuppressive molecule that can regulate the immune system and prevent autoimmune diseases, but it can also prevent the immune system from killing cancer cells. Therefore, inhibiting the PD-1 signaling pathway can be used as a treatment for diseases such as tumors.

[0003] PD-1 is the main immune checkpoint of tumor immune response, and PD-1 inhibitors can enhance immune function and exert anti-tumor effects by targeting PD-1, which can regulate T cell exhaustion and Treg dysfunction, and play an important role in tumor treatment. There are many types of PD-1 inhibitors, most of which are monoclonal antibody drugs, such as nivolumab, nivolumab, pembrolizumab and tislelizumab, and their targets are all PD-1.

[0004] In addition, some studies focus on the function of PD1 antibody fusion protein as PD-inhibitor.

[0005] Chinese patent 201910224044.5 discloses a combination of T cells that specifically recognize NY-ESO-1 and a high-affinity PD-1 fusion protein. The combination realizes the combination of TCR-T adoptive cell therapy and immune checkpoint therapy, thereby solving the problem that high-affinity TCR-T adoptive immunotherapy cannot overcome the inhibitory effect mediated by the PD-1 signaling pathway, achieving excellent therapeutic effects that cannot be achieved by monotherapy and significantly limiting the growth of tumor cells.

[0006] Chinese patent 202110323845.4 discloses an immune cell that secretes IL-15 and anti-PD1 fusion protein. Gene-edited immune cells are used to secrete IL-15 and anti-PD1 fusion proteins to improve the activity of immune cells and the killing effect on tumors. The immune cells that secrete IL-15 and anti-PD1 fusion proteins include: fusion proteins expressed in series in the order of anti-PD1, G4S*4Linker, IL-15N72D, G4S*4Linker and IL-15RaSu, and gene-edited cells that express the fusion protein and are affected by the autocrine fusion protein. The superkinetic protein of IL-15 and IL15Ra and the fusion protein of anti-PD1 are combined, and the fusion protein is successfully secreted by CART cells to enhance the proliferation ability, anti-apoptosis ability and killing ability of CarT cells for tumors. The killing effect is accurate, the safety is higher, and it is not easy to relapse, which improves the quality of life of patients.

[0007] However, there is still a lot of room for research on gene-modified stem cells that inhibit the PD-1 signaling pathway in the existing technology. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for preparing gene-modified stem cells that inhibit the PD-1 signaling pathway, and specifically provides genes encoding anti-PD-1 antibodies, and the preparation and application of stem cells modified therewith.

[0009] the term:

[0010] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0011] The "expression vector" of the present invention refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises a cis-acting element for expression; other elements for expression may be provided by a 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) incorporated with a recombinant polynucleotide.

[0012] The "antibody" described in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have a κ chain or a λ chain.

[0013] 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. Unless any conventional media or agents are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated.

[0014] First, the present invention provides a gene encoding an anti-PD-1 antibody.

[0015] The gene encoding the anti-PD-1 antibody comprises: a nucleotide sequence encoding the light chain variable region of SEQ ID NO.1, a nucleotide sequence encoding the linker of SEQ ID NO.2, and a nucleotide sequence encoding the heavy chain variable region of SEQ ID NO.3.

[0016] SEQ ID NO.1:

[0017] gagatagtccttactcagagtcctgctacgctcagtctgagtcctggagagagggcaactctttcttgcagggcaagccaatcagtgagcagctatttgggtggtatcaaaaagcccggccaggcaccgcgacttcttatttacgatgcgagtaatc gagcaactgggatacccgcacgattccggctcagggtccggcaccgatttcacacttacaatttcaagtcggccggaggactttgccgtgtactgtcagcagagcagtaattggcctcgaacgttcggacaggaactaaggttgagattaaa

[0018] SEQ ID NO.2:

[0019] ggaggaggtg ggtctggggg cggagggtct ggcggcgggg gatcaggtgg cgggggaagt

[0020] SEQ ID NO.3:

[0021] caggtgcaacttgttgaaagtggagggggtgtagttcaacctggtaggtctttgcggctcgactgcaaagcttcaggaataaccttttcaaacgtggtatgcattgggttcgacaggcaggggaaaggtttggagtgggtggcagttatctggtacgacggatcaa agaggtactacgccgatagtgtaaaaggtcgcttcacttatttctcggataatagtaaataacgttgttcctgcaaatctctttgcgacggacacggctgtctattattgtgcgaccaatgatgactggccaagggaccctcgtgactgtgtcatca

[0022] Furthermore, based on the aforementioned gene encoding the anti-PD-1 antibody, the present invention provides a gene encoding an anti-PD-1 antibody fusion protein, which includes the aforementioned gene encoding the anti-PD-1 antibody; in addition, it may also include genes encoding other fragments that need to be expressed in the fusion protein, such as a gene encoding a human immunoglobulin heavy chain Fc fragment, specifically a gene encoding the heavy chain Fc fragment of IgG4.

[0023] Preferably, the amino acid sequence of the heavy chain Fc fragment may be SEQ ID NO.18.

[0024] Further preferably, the nucleotide sequence of the gene encoding the heavy chain Fc fragment of IgG4 may be SEQ ID NO.4, or a nucleotide sequence having more than 80% identity with the nucleotide sequence shown in SEQ ID NO.4 and encoding the same amino acid sequence.

[0025] SEQ ID NO.4:

[0026] cccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtg gatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaacca tctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaatga

[0027] The present invention further provides an expression vector comprising the aforementioned gene encoding the anti-PD-1 antibody or the gene encoding the anti-PD-1 antibody fusion protein.

[0028] The vector backbone of the expression vector includes, but is not limited to, cosmids, plasmids or viruses incorporated with the recombinant polynucleotide.

[0029] The expression vector may be a lentiviral expression vector, and in some specific embodiments may be selected from a fourth generation lentiviral vector system.

[0030] In order to achieve expression smoothly, the expression vector may generally also include a gene encoding a signal peptide.

[0031] For example, the amino acid sequence of the signal peptide includes SEQ ID NO.8, and its encoding gene includes: SEQ ID NO.9, or a nucleotide sequence that has more than 80% identity with the nucleotide sequence shown in SEQ ID NO.9 and encodes the same amino acid sequence.

[0032] The present invention also provides a cell comprising the aforementioned expression vector.

[0033] Based on the aforementioned genetic engineering elements, the present invention also provides the use of the gene encoding the anti-PD-1 antibody or the gene encoding the anti-PD-1 antibody fusion protein or the expression vector in stem cell modification.

[0034] Preferably, the stem cells are gene-modified stem cells that inhibit the PD-1 signaling pathway.

[0035] The sources of the genetically modified stem cells include but are not limited to umbilical cord tissue, adipose tissue, bone marrow, and placenta; preferably, the genetically modified stem cells can be selected from any one or more of amniotic mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and adult stem cells.

[0036] Specifically, the present invention also provides a method for preparing gene-modified stem cells that inhibit the PD-1 signaling pathway.

[0037] The preparation method includes constructing a vector that expresses the aforementioned gene encoding the anti-PD-1 antibody or the gene encoding the anti-PD-1 antibody fusion protein.

[0038] As known to those skilled in the art, the preparation method may include the following steps:

[0039] S1, artificial synthesis of target gene;

[0040] S2, target gene and vector enzyme ligation to construct recombinant vector;

[0041] S3. After transforming cells with the recombinant vector, screen for positive clones.

[0042] The present invention also provides the use of the aforementioned gene encoding the anti-PD-1 antibody or the gene encoding the anti-PD-1 antibody fusion protein or the expression vector or cell in the preparation of a drug, a kit or a medical device for inhibiting the PD-1 signaling pathway.

[0043] The present invention also provides a drug selected from any one or more of the following:

[0044] (1) comprising or expressing the aforementioned gene encoding the anti-PD-1 antibody or the gene encoding the anti-PD-1 antibody fusion protein;

[0045] (2) comprising the aforementioned expression vector;

[0046] (3) comprising the aforementioned cells.

[0047] The drug has the effect of inhibiting the PD-1 signaling pathway.

[0048] The medicine further comprises an optional pharmaceutically acceptable carrier or excipient.

[0049] Specifically, the pharmaceutical composition can be administered alone, sequentially or simultaneously in combination with other drugs, and the other drugs administered in combination can be selected from penicillin, amoxicillin, benzylpenicillin, ampicillin, azithromycin, erythromycin, clarithromycin, cefadroxil, cefixime, tetracycline, minocycline, rifampicin, rifabutin, sulfonamide, aminoglycoside, bacitracin, polymyxin B, ornidazole, retinol, retinal, isotretinoin, vitaminosis A, adapa, tazarotene, estrogen, progesterone, glucocorticoid, Anti-androgens, vitamin D3 analogs such as calcipotriol and tacalcitol, glucocorticoids such as prednisone and methylprednisone, tacrolimus, pimecrolimus, cyclosporine A, mycophenolate mofetil, leukotriene B4 inhibitors, IL-1 inhibitors, IL-2 inhibitors, IL-1β inhibitors, IL-6 inhibitors, IL-8 inhibitors, IL-12 inhibitors, IL-23 inhibitors, IL-17 inhibitors, TNF inhibitors, GM-CSF inhibitors, CD19 inhibitors, CD20 inhibitors, etc. The combined administration method also includes combined administration with photodynamic therapy, laser therapy, and fruit acid therapy.

[0050] Beneficial effects of the present invention:

[0051] The present invention optimizes the gene encoding the anti-PD-1 antibody fusion protein (specifically the gene of the anti-PD-1 antibody therein), provides a genetic engineering product related to the gene, and verifies the gene-modified stem cells prepared using a lentiviral vector as an example, wherein the infection rate of the prepared lentiviral vector on mesenchymal stem cells reaches more than 80%, the gene-modified mesenchymal stem cells highly express IgG4, with an expression level as high as 1232.96±102.62ng / mL, and an anti-PD-1 antibody content as high as 785±71.3ng / mL, and can significantly block the binding of PD-1 / PD-L1, with a binding inhibition rate of 78.65±8.94%. Compared with WT, the PD-1 / PD-L1 blocking ability increased by 32.8% (P<0.01), and has a good prospect for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a schematic diagram of LV-PD1:Fc plasmid information.

[0053] Figure 2 This is a graph showing the fluorescence expression detection results of Example 4.

[0054] Figure 3 This is a graph showing the results of fusion protein IgG4 protein expression detection.

[0055] Figure 4 This is the result of detecting the content of PD1 antibody secreted by mesenchymal stem cells.

[0056] Figure 5 This is the standard curve of the inhibition of PD-1 / PD-L1 binding by anti-PD-1 antibodies

[0057] Figure 6 This is a graph showing the results of the binding inhibition test of mesenchymal stem cells on PD-1 / PD-L1. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0059] The "αPD-1:Fc" mentioned in the present invention generally refers to a fusion protein containing an anti-PD-1 single-chain antibody and an Fc sequence, and its coding sequence is represented by "αPD-1:Fc". When different coding sequences are involved, the representation may be accompanied by a number, such as in "αPD-1-OS1:Fc", OS1 represents the number.

[0060] Example 1 In vitro construction of αPD-1:Fc lentiviral expression plasmid

[0061] The fourth-generation lentiviral vector system LV-EGFP (purchased from addGene, catalog number: 122848) was used to construct a lentiviral expression plasmid of αPD-1:Fc fusion protein (hereinafter referred to as αPD-1:Fc), and the LV-EGFP empty plasmid was used as a control.

[0062] Among them, αPD-1:Fc is a fusion protein composed of anti-PD-1 antibody and the heavy chain Fc fragment of IgG4, with a length of 500 amino acid residues and a molecular weight of 60kD. The anti-PD-1 antibody sequence includes the light chain variable region SEQ ID NO.15, linker SEQID NO.16, and heavy chain variable region SEQ ID NO.17; the heavy chain Fc fragment of IgG4 is SEQ ID NO.18.

[0063] SEQ ID NO.15:

[0064] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK;

[0065] SEQ ID NO.16:

[0066] GGGGSGGGGSGGGGSGGGGS;

[0067] SEQ ID NO.17:

[0068] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS;

[0069] SEQ ID NO.18:

[0070] PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0071] A gene encoding αPD-1:Fc is hereinafter referred to as αPD1-OS1:Fc, comprising a nucleotide sequence encoding an anti-PD-1 single-chain antibody and a nucleotide sequence encoding an IgG4 heavy chain Fc fragment, wherein the nucleotide sequence encoding the anti-PD-1 single-chain antibody is composed of a nucleotide sequence encoding an anti-PD-1 antibody light chain variable region (SEQ ID NO.1), a nucleotide sequence encoding a linker (SEQ ID NO.2) and a nucleotide sequence encoding an anti-PD-1 antibody heavy chain variable region (SEQ ID NO.3) connected in sequence; the nucleotide sequence encoding an IgG4 heavy chain Fc fragment SEQ ID NO.4 is composed of a nucleotide sequence encoding a hinge region of a heavy chain constant region of a human immunoglobulin IgG4 (SEQ ID NO:5), a nucleotide sequence encoding a heavy chain constant region CH2 of a human immunoglobulin IgG4 (SEQ ID NO:6), a nucleotide sequence encoding a heavy chain constant region CH3 of a human immunoglobulin IgG4 (SEQ ID NO:7) and three parts connected in sequence.

[0072] SEQ ID NO:5:

[0073] cccccatgcccatcatgccca;

[0074] SEQ ID NO:6:

[0075] gcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaa;

[0076] SEQ ID NO:7:

[0077] gggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacg cctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaatga;

[0078] When constructing a lentiviral expression plasmid, a sequence encoding a signal peptide is connected before αPD-1:Fc. The amino acid sequence of the signal peptide is SEQ ID NO.8, and its encoding gene is SEQ ID NO.9 as an example.

[0079] SEQ ID NO.8:

[0080] MYRMQLLSCIALSLALVTNS;

[0081] SEQ ID NO.9:

[0082] atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg;

[0083] The 3'-end of αPD-1:Fc is successively connected to the IRES sequence SEQ ID NO.10 and the EGFP gene SEQ ID NO.11 to form the αPD1-OS1:Fc-EGFP or αPD1-OS2:Fc-EGFP sequence (the part encoding the PD-1 single-chain antibody is SEQ ID NO.12, and the rest is the same as αPD1-OS1:Fc-EGFP), which are both located downstream of the CMV promoter.

[0084] SEQ ID NO.10:

[0085] Cccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaacc;

[0086] SEQ ID NO.11:

[0087] Atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa;

[0088] SEQ ID NO.12:

[0089] Gagatcgtcctgactcagagtccggcgactctttcactttcacctggcgaaagagctacgctaagttgccgggctagtcagtcggtgtcgtcgtacctagcatggtatcagcagaaaccaggacaagccccgcgactgctgatttacgacgcttcgaaccgagcgaccggtattcccgcgcgattcagcggctccggatccggcacagatttcacccttacaatatcttccctagagcctgaggacttcgcggtctactactgtcagcagtcgtctaattggcctcgcacctttgggcaaggaaccaaggtagaaattaaaggtgggggcggctcgggtgggggcggcagcggcggaggaggttccgggggagggggctcacaagtccagctggttgagtcaggcggcggggttgtccagccgggacgttctttacgattggactgtaaggccagtggaatcacattttccaattccggaatgcattgggtccgccaagcaccagggaagggcctagagtgggtagctgtcatatggtatgatggatccaaacgctattacgcagacagcgttaaggggcggttcactatatcacgagacaattccaagaatacactcttcctccagatgaactctttacgtgcggaggataccgcggtctattattgcgcgacaaacgacgactattggggtcaagggactctcgtaaccgtcagttca;

[0090] The nucleotide sequence encoding the heavy chain variable region of the anti-PD-1 antibody shown in SEQ ID NO:3 was optimized by the WT sequence shown in SEQ ID NO:13, and the nucleotide sequence encoding the light chain variable region of the anti-PD-1 antibody shown in SEQ ID NO:1 was optimized by the WT sequence shown in SEQ ID NO:14. At the same time, the WT sequence was used to construct the αPD1-WT:Fc-EGFP sequence (the nucleotide sequence encoding the heavy chain variable region of the anti-PD-1 antibody is the WT sequence shown in SEQ ID NO:10, the nucleotide sequence encoding the light chain variable region of the anti-PD-1 antibody is the WT sequence shown in SEQ ID NO:11, and the rest of the sequence is the same as the αPD1-OS1:Fc-EGFP or αPD1-OS2:Fc-EGFP sequence).

[0091] SEQ ID NO:13:

[0092] Caggtgcagctggtggaaagcggcggcggcgtggtgcagccgggccgcagcctgcgcctggattgcaaagcgagcggcattacctttagcaacagcggcatgcattgggtgcgccaggcgccgggcaaaggcctggaatgggtggcggtgatttggtatgatggcagcaa acgctattatgcggatagcgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtttctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgaccaacgatgattattggggccagggcaccctggtgaccgtgagcagc;

[0093] SEQ ID NO:14:

[0094] Gaaattgtgctgacccagagcccggcgaccctgagcctgagcccgggcgaacgcgcgaccctgagctgccgcgcgagccagagcgtgagcagctatctggcgtggtatcagcagaaaccgggccaggcgccgcgcctgctgatttatgatgcgagcaacc gcgcgaccggcattccggcgcgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctggaaccggaagattttgcggtgtattattgccagcagagcagcaactggccgcgcacctttggccagggcaccaaagtggaaattaa;

[0095] According to the above gene structure, the target gene fragment was inserted into the open reading frame site of the LV-EGFP plasmid using conventional genetic engineering methods to obtain the lentiviral expression plasmids LV-αPD1-OS1:Fc, LV-αPD1-OS2:Fc and LV-αPD1-WT:Fc. Schematic diagram of LV-PD1:Fc plasmid information is shown in Figure 1 .

[0096] Example 2 A method for preparing genetically modified stem cells that inhibit PD-1 signaling pathway

[0097] This example uses the recombinant vector prepared in Example 1.

[0098] The LV-αPD1-OS1:Fc, LV-αPD1-OS2:Fc or LV-αPD1-WT:Fc plasmids prepared in Example 1 were respectively combined with the lentiviral framework plasmids pGag / Pol (purchased from addGene, catalog number: 12251), pRev (purchased from addGene, catalog number: 12253), and pVSV-G (purchased from addGene, catalog number: 12259), and transfected into HEK293T cells (purchased from ATCC, catalog number CRL-3216) through LTX liposomes (purchased from Thermo, catalog number 15338-100). The successfully packaged recombinant lentiviral vector was released into the HEK293T cell culture supernatant, and the cell culture supernatant containing the recombinant lentiviral vector was harvested.

[0099] Example 3 Preparation of Genetically Modified Stem Cells

[0100] The HEK293T cell culture supernatant containing the packaged virus prepared in Example 2 was collected, and cultured mesenchymal stem cells with a fusion degree of 70-80% (isolated from the umbilical cord of a newborn using an enzymatic method, and P2 mesenchymal stem cells were obtained by passage amplification and purification) were added. After culturing at 37° C. and CO2 for 24 h, the culture supernatant of the mesenchymal stem cells that had completed the virus infection was discarded and replaced with fresh culture medium (DF12 culture medium + 10% serum, DF12 culture medium manufacturer: Gibco, product number: 11320-033; serum manufacturer: FND-500) containing 10 μg / mL puromycin (manufacturer: InvivoGen, product number: ant-pr-1) for continued cultivation. After 48 h, the successfully infected mesenchymal stem cells EGFP-MSC, αPD1-OS1:Fc-MSC, αPD1-OS2:Fc-MSC, and αPD1-WT:Fc-MSC were screened out.

[0101] Example 4 Detection of Green Fluorescent Protein Expression in Genetically Modified Stem Cells by Flow Cytometry

[0102] The cells obtained in Example 3 were divided into 1×10 4 Pieces / cm 2 Cultured in T25 cell culture flasks, the green fluorescence channel signal of mesenchymal stem cells was detected by flow cytometry after 24 hours (see the results). Figure 2 ), and found that the positive rate of green fluorescence channel signal of mesenchymal stem cells infected with LV-EGFP, LV-αPD1-OS1:Fc, LV-αPD1-OS2:Fc and LV-αPD1-WT:Fc lentiviruses exceeded 80%, while normal mesenchymal stem cells (hUC-MSC) had no signal in the green fluorescence channel. This shows that LV-EGPF, LV-αPD1-OS1:Fc, LV-αPD1-OS2:Fc and LV-αPD1-WT:Fc lentiviruses can successfully infect mesenchymal stem cells, with an infection rate of more than 80%.

[0103] Example 5 Detection of IgG4 expression in gene-modified mesenchymal stem cells by ELISA

[0104] The Human IgG4 ELISA Kit (BMS2092) from Invitrogen was used to detect the amount of fusion protein IgG4 secreted in the cell culture supernatant. The kit uses a human IgG4 solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) to detect the amount of target bound between matching antibody pairs. IgG4-specific antibodies have been pre-coated in the ELISA plate, and then cell supernatant samples, standards, or controls are added to these wells and bound to the fixed (capture) antibody. A sandwich structure is formed by adding a secondary antibody, and a substrate solution is added to react with the enzyme-antibody-target complex to produce a measurable signal. The intensity of the signal is proportional to the target concentration present in the original sample.

[0105] The gene-modified mesenchymal stem cells obtained in Example 3 were cultured for 72 hours and the cell supernatant was harvested. The cell culture supernatant was added to the IgG4 ELISA kit to detect the IgG4 protein content in the fusion protein. The results showed that the mesenchymal stem cells infected with the LV-αPD1-OS1:Fc lentivirus (αPD1-OS1:Fc-MSC) highly expressed IgG4, with an expression level of up to 1232.96±102.62ng / mL. The IgG4 expression levels of LV-αPD1-OS2:Fc (PD1-OS2:Fc-MSC) and LV-PD1-WT:Fc (PD1-WT:Fc-MSC) were not significantly different from those of LV-PD1-OS1:Fc (P>0.05), while normal hUC-MSC and EGFP-MSC did not express IgG4 (see the results). Figure 3 ).

[0106] Example 6 Detection of αPD-1 Antibody Content

[0107] The PD-1 [Biotinylated]: PD-L1 Inhibitor Screening ELISA Assay Pair kit from ACRO Biosystems was used to detect the content of αPDL1: Fc in the cell supernatant. The gene-modified mesenchymal stem cells obtained in Example 3 were cultured for 72 h and the cell supernatant was harvested. Referring to the instructions of the kit, the cell culture supernatant was added to the ELISA kit. The concentration of the anti-PD-L1 neutralizing antibody (standard) was used as the abscissa and the OD450 value was used as the ordinate. The standard curve was prepared by curve fitting. The experimental results are shown in Figure 2. Figure 4 As shown, the monoclonal antibody content in the culture supernatant of normal hUC-MSC and EGFP-MSC was 0 ng / mL, the antibody content of αPD1-OS1:Fc-MSC was 785±71.3 ng / mL, the antibody content of αPD1-OS2:Fc-MSC was 652.78±67.26 ng / mL, and the antibody content of αPD1-WT:Fc-MSC was 562.44±60.28 ng / mL.

[0108] Example 7 Detection of the function of gene-modified mesenchymal stem cells in blocking the binding of PD-L1 and PD-1

[0109] The PD-1 [Biotinylated]: PD-L1 Inhibitor Screening ELISA Assay Pair Kit from ACRO Biosystems was used to detect the ability of genetically modified stem cells to block PD-1 / PD-L1 binding. The standard in the kit is an anti-PD-1 antibody, which can block the binding of PD-L1 to biotinylated PD-1 protein, thereby reducing the OD450 detection value. Figure 5 As shown, as the concentration of the standard anti-PD-1 antibody increases, the ability to block the binding of PD-L1 to biotinylated PD-1 increases, and the OD450 value decreases. Therefore, the higher the function of blocking PD-1 / PD-L1 binding, the inverse relationship between the blocking ability and the OD450 value. The gene-modified mesenchymal stem cells obtained in Example 3 were cultured for 72 hours and the cell supernatant was harvested. The ability of the αPD-1 antibody in the cell supernatant to block the binding of PD-1 / PD-L1 was detected by the PD-1 / PD-L1 blocking reagent detection kit.

[0110] The PD-1 / PD-L1 binding inhibition rate was calculated using the following formula:

[0111] Binding inhibition rate (%) = [OD450 (blank well) - OD450 (sample well)] / OD450 (blank well) × 100%;

[0112] The results are as follows Figure 6 As shown in the results, normal hUC-MSC and EGFP-MSC cells could not block PD-1 / PD-L1, with binding inhibition rates of 0.06±0.09% and 0.09±0.12%, respectively; while αPD1-OS1:Fc-MSC cells could significantly block the binding of PD-1 / PD-L1, with a binding inhibition rate of 78.65±8.94%, αPD1-OS2:Fc-MSC cell binding inhibition rate of 73.82±8.20%, and αPD1-WT:Fc-MSC cell binding inhibition rate of 59.22±5.98%. Compared with WT, the PD-1 / PD-L1 blocking ability of OS1 group cells increased by 32.8% (P<0.01) (results see Figure 6 ).

Claims

1. A gene encoding an anti-PD-1 antibody, characterized in that: include: The nucleotide sequence encoding the light chain variable region is SEQ ID NO.1, the nucleotide sequence encoding the linker is SEQ ID NO.2, and the nucleotide sequence encoding the heavy chain variable region is SEQ ID NO.3; The gene also includes a gene encoding the heavy chain Fc fragment of IgG4; the nucleotide sequence of the gene encoding the heavy chain Fc fragment of IgG4 is SEQ ID NO.4; the amino acid sequence of the heavy chain Fc fragment is SEQ ID NO.18; The gene also includes a gene encoding a signal peptide, and the amino acid sequence of the signal peptide includes SEQ ID NO.8; The coding gene thereof includes: SEQ ID NO.

9.

2. An expression vector comprising the gene encoding the anti-PD-1 antibody according to claim 1.

3. The expression vector according to claim 2, characterized in that Including lentiviral vectors.

4. Use of the gene encoding the anti-PD-1 antibody according to claim 1 or the expression vector according to any one of claims 2 to 3 in stem cell modification.

5. The use according to claim 4, characterized in that: The stem cells are gene-modified stem cells that inhibit the PD-1 signaling pathway.

6. The use according to claim 5, characterized in that: The gene-modified stem cells are derived from umbilical cord tissue, adipose tissue, bone marrow and placenta.

7. The use according to claim 6, characterized in that: The gene-modified stem cells are any one or more of amniotic mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and adult stem cells.

8. A cell comprising the expression vector according to any one of claims 2 to 3.

9. A method for preparing gene-modified stem cells that inhibit PD-1 signaling pathway, characterized in that: The method comprises constructing a vector for expressing the gene encoding the anti-PD-1 antibody according to claim 1.

10. The preparation method according to claim 9, characterized in that: The following steps are involved: S1, artificial synthesis of target gene; S2, target gene and vector enzyme ligation to construct recombinant vector; S3. After transforming cells with the recombinant vector, screen for positive clones.

11. Use of the gene encoding the anti-PD-1 antibody according to claim 1, the expression vector according to any one of claims 2 to 3, or the cell according to claim 8 in the preparation of a drug, a kit, or a medical device for inhibiting the PD-1 signaling pathway.

12. A drug, characterized in that Select one or more of the following: (1) comprising or expressing the gene encoding the anti-PD-1 antibody according to claim 1; (2) comprising the expression vector according to any one of claims 2 to 3; (3) The cell according to claim 8.

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