CN107916255B - PDL-1 gene targeted specific methylation epigenetic modification antitumor molecule and application thereof - Google Patents

Abstract

The invention belongs to the technical field of gene therapy, and particularly relates to a PDL-1 gene targeted specific methylation epigenetic modification anti-tumor molecule and application thereof. The invention designs a novel protein molecule PPLA for carrying out epigenetic modification on a human PD-L1 gene promoter sequence according to the principles of bioinformatics, epigenetic engineering and immunology, and the novel protein molecule PPLA is introduced into human cells through an adenovirus vector, so that the protein molecule PPLA specific to the human PD-L1 gene promoter sequence can be efficiently expressed, the expressed gene can effectively inhibit a PD-L1 gene, the molecule can generate a methylation chemical modification effect on a PDL-1 gene, and an in vitro intracellular experiment proves that the novel protein molecule PPLA has the function of efficiently blocking PDL-1 gene expression, so that the immune tolerance is broken, the effect of treating chronic viral infection and tumor is finally achieved, the anti-tumor and anti-virus immune treatment effect is realized, and the novel protein molecule PPLA has wide application prospects in the aspect of preparing epigenetic engineering tumor and anti-virus medicines.

Description

PDL-1 gene targeted specific methylation epigenetic modification antitumor molecule and application thereof

Technical Field

The invention belongs to the technical field of gene therapy, and particularly relates to a PDL-1 gene targeted specific methylation epigenetic modification anti-tumor molecule and application thereof.

Background

Immunity (immunity) generally refers to the resistance of an organism to infectious diseases, the recognition of self and non-self substances, and the monitoring and clearance of denatured substances produced in the body. Immunization is divided into innate immunity (nature) and acquired immunity (acquired immunity), which in turn can be subdivided into cellular and humoral immunity. Immune regulation is an important link of immunity. Immunomodulation refers to the execution and rational regulation of the immune response process and the level of immune response by an organism to meet the needs of immune defense and to maintain the homeostasis (homeostatis) of the organism. The essence is a regulation process with multi-factor participation under the regulation of genetic genes.

Lymphocyte activation is an important process in cell-mediated immune responses. After the antigen is captured, processed and presented by APC, the information is transmitted to T cells, so that the T cells are promoted to proliferate and differentiate into Th cells of CD4+, the Th cells can secrete cytokines such as IL-2, IL-4, IL-12 and IFN-gamma, and the proliferation, differentiation and maturation of Tc cells of cytotoxic T cells of CD8+ are further promoted. Tc cells can specifically recognize specific antigenic determinants, and can directionally release cytotoxins (proteolytic enzyme, serine lipase and the like) by contacting with target cells containing specific antigenic peptides, which are mediated by MHC-I molecules or acted by non-specific adhesion molecules, so as to perform cytotoxic action on the target cells; or directly has toxic effect on target cells by releasing TNF-alpha and TNF-beta.

T cell activation requires several factors to act simultaneously: (1) binding of the MHC-antigen peptide complex on the surface of the APC to the TCR on the surface of the T cell; (2) APC surface B7 molecules bind to T cell surface CD28 molecules; (3) cytokines such as IL-1 and IL-2 stimulate T cell activation.

Immunodetection points (immunodetection points) are important molecules present in the immune system of an organism and on some pathways that play a limiting role in the functioning of the immune system, and have a key role in immune recognition, the formation of immune tolerance and the regulation of the immune strength of the immune system. Many immunodetection spots have been discovered so far, and most of them are expressed on the surface of immune cells and play a role in limiting the functions of the immune cells. The intracellular segments generally contain a plurality of immunoreceptor tyrosine-based inhibition motifs (ITIMs) and play a role in negative regulation through the ITIM motifs. The immunodetection points now well defined include: CTLA-4, PD-1, PD-L1, and the like.

The PD-1 (CD 279)/PD-L1 (CD 274) signal pathway is an important signal for mediating the negative regulation of T cell immunity and tumor apoptosis and is one of the main signal pathways for tumor cells to realize immune evasion. PD-1 is expressed on the surface of activated T cells, and PD-L1 is expressed on the surface of various tumor cells. Recent studies have shown that by blocking the signaling pathways of PD-1 and PD-L1, T cell responses and their anti-tumor effects can be effectively improved. PD-1 was first discovered by a method of reducing hybridization on apoptotic T-cell hybridoma cells in 1992 by TasukuHonjo of Kyoto university, Japan, and was named as programmed cell death receptor 174 because its function is related to apoptosis of cells. As with other immune negative regulatory receptors, PD-1 is expressed on the surface of activated T cells, tregs, B cells, monocytes, DC cells, NK cells, and NKT cells. The gene of human PD-1 protein is located on 2q37.3 chromosome, the total length is 9028bp, the gene sequence has 23% similarity with CTLA-4, and the gene sequence comprises 5 exons. PD-1 has a molecular size of 55kDa and belongs to type I transmembrane proteins in immunoglobulin family. The extracellular segment contains an IgV structural domain, and 4N connecting sites are glycosylated; the intracellular segment contains an ITIM and an ITSM, which, when signaled, inhibit cytoplasmic phosphorylation and function to antagonize immune function.

PD-L1 is a ligand for PD-1, which is involved in negative regulation in T cell activation. The human PD-L1 gene is located on chromosome 9q24, and has a total length of 20065bp, 6 exons and 290 coded amino acids. PD-L1 is a type I transmembrane protein composed of the extracellular domains of IgV, IgC, a hydrophobic transmembrane domain and a charged intracellular domain of 30 amino acids. The mRNA of human PD-L1 is widely found in various tissues of the human body, but is not expressed on the cell membrane. It is only found to be expressed on the cell surface of a small fraction of cells (e.g., macrophage-like cells in the liver or lung) and of partially immune-privileged tissues (e.g., eye, uterus, etc.). However, PD-L1 is highly expressed selectively on the surface of lung cancer, stomach cancer, colon cancer, breast cancer, cervical cancer, kidney cancer, bladder cancer, liver cancer, glioma, melanoma and other various human tumor cells as well as infiltrating lymphocytes and endothelial cells in the tumor microenvironment, and is a specific, highly effective, low-toxicity anti-tumor target.

PD-L1 can be stimulated by a number of cytokines to increase expression, such as IFN-. gamma., TNF. alpha., VEGE, GM-CSF and IF-10. The expression of PD-L1 molecule is found on the surface of many tumor cells, while in Tumor Infiltrating Lymphocytes (TILs), PD-1 is specifically and highly expressed, thereby binding with PD-L1 and inhibiting the effect. When the tumor cells are attacked by the immune system, the tumor cells begin to over-express the PD-L1 molecule, reducing the effect of T cells and thus successfully evading immune attack.

In the tumor microenvironment, T cells can be down regulated in their immune killing by the PD-1/PD-L1 pathway in a variety of ways. It has now been demonstrated that overexpression of PD-L1 on the surface of tumor cells results in significant limitation of the antitumor effects of macrophages, DCs, MDSCs, etc. Molecules such as phosphorylated Akt, mTOR, S6 and ERK2 are important molecules in the growth and differentiation process of Treg, and PD-L1 can promote the differentiation of Treg cells by regulating the secretion of the molecules, thereby limiting the functions of T cells. On the other hand, PD-1 down-regulates the secretion of the anti-apoptotic protein Bcl-xL by inhibiting the activity of PI3K pathway, thereby inducing apoptosis of activated T cells. PD-1 protein can also inhibit the activity of TCR, thereby reducing secretion of lymphocyte growth-related factor IL-2, and stopping cell division.

In tumor immunotherapy, the specific T cells are killed to the tumor by breaking immune tolerance, so that the purpose of treating the tumor is achieved. Research shows that the expression of PD-1/PD-L1 on the surfaces of lymphocytes and tumor cells can reduce the killing effect of immune cells on tumors and down regulate the response of tumor immunity. The monoclonal antibody is used for blocking a PD-1/PD-L1 passage, a mouse tumor model is treated, the growth of local tumors is obviously inhibited, and the symptom is well relieved. Simultaneously, the channel is blocked, IFN gamma secretion can be up-regulated, and the purpose of resisting tumor is achieved.

According to the invention, core sequence specific binding protein aiming at CpG II of a human PD-L1 gene promoter is designed according to a brand-new epigenetic genetic engineering technology and an immunological principle, and is fused with a catalytic domain of methylase (Dnmts) to construct a methylated molecule PPLA of a targeted PD-L1 gene, and adenovirus is adopted for packaging, so that the expression of cancer cell PD-L1 gene is expected to be inhibited through targeted methylation of the PD-L1 gene promoter, so that immune tolerance is broken, and the therapeutic effect on chronic viral infection and tumor is achieved. The key problem of gene therapy is to select safe and effective vectors and introduce therapeutic genes into target cells. Adenovirus (Ad) vectors are important because they have the advantages of wide host range, high efficiency, stability, safety, and easy operation.

Disclosure of Invention

The invention aims to provide a PDL-1 gene targeted specific methylation epigenetic modification anti-tumor molecule and application thereof.

The invention firstly designs a new protein molecule aiming at human PD-L1 gene promoter, which is prepared by fusing and connecting the active domain of maintenance methylase Dnmt1 and the active domain of de novo methylase Dnmt3a and performing apparent methylation modification, and the protein molecule is used for inhibiting the expression of PD-L1 gene and finally preparing the anti-tumor and anti-virus therapeutic drugs.

In one aspect of the invention, a specific binding protein molecule for identifying a human PD-L1 gene promoter is provided, the amino acid sequence of the specific binding protein molecule is shown in SEQ.ID.No.1 and is named as PPLA. The invention utilizes bioinformatics means, firstly analyzes the promoter sequence of PD-L1 gene through a eukaryotic gene promoter database, analyzes potential methylation sites of the promoter core sequence through CpG island software, and designs a protein structural domain with specific recognition and combination functions aiming at DNA sequence by adopting corresponding software, thus obtaining the specific binding protein molecule for recognizing PD-L1 gene promoter, wherein the amino acid sequence of the specific binding protein molecule is SEQ.ID.No. 1.

In another aspect of the invention, a gene encoding the protein molecule PPLA is provided, and the nucleotide sequence of the gene is shown in seq.id No. 2.

In another aspect of the present invention, the gene expression vector of the above protein molecule PPLA is provided, which comprises selecting human methylase, recombining to plasmid pcdna3.1, and serial expressing PPLA gene expression plasmid at N-terminal, packaging adenovirus, and constructing PDL-1P target specific DNA methylase domain, as shown in fig. 1.

On the other hand, the invention can efficiently express protein molecules specific to the promoter sequence of the human PD-L1 gene by introducing the adenovirus vector into human cells, and can generate the function of methylation epigenetic modification on the PDL-1 gene, and experiments prove that the PPLA has the function of efficiently inhibiting the expression of the PDL-1 gene, namely, the promoter of the PD-L1 gene can be effectively methylated, and the expression of the PD-L1 gene can be obviously reduced, so that the aims of resisting tumor and virus lymphocytes and improving the immunologic function are achieved, the treatment effect of resisting tumor and virus is realized, and new experimental basis and technical means are provided for the prevention and treatment of tumor and virus. Therefore, the protein molecule PPLA, or the gene for coding the protein molecule PPLA, or the expression vector thereof can be used for preparing anti-tumor and anti-virus medicines.

Drawings

FIG. 1 is a PPLA adenovirus expression plasmid map.

FIG. 2 is an expression profile in adenovirus-packaged cells of a PPLA gene expression vector.

FIG. 3 is a Western Blot (Western Blot) analysis of PPLA protein expression.

FIG. 4 is a schematic diagram showing the binding of PPLA protein to the DNA of the PD-L1 gene promoter.

FIG. 5 is a graph showing the effect of PPLA on the methylation rate of PD-L1 gene.

FIG. 6 is an expression profile of PD-L1 molecule encoding mRNA.

FIG. 7 is a graph showing the expression level of PD-L1 in FACS analysis of PPLA-transfected cells.

FIG. 8 is a graph of the expression level of PD-L1 in PPLA-inhibited tumor cells.

Detailed Description

1. Design and synthesis of specific binding protein of human PD-L1 gene promoter

And (3) selecting an optimal sequence and designing a specific binding protein by adopting ZF Tools Ver3.0 software according to CpG sites of the promoter region of the human PD-L1. The amino acid sequence of the selected DNA binding and mediating DNA methylation protein PPLA aiming at the human PD-L1 promoter region is shown in SEQ.ID.No.1, and the nucleotide sequence of each structural domain of the corresponding DNA is shown in SEQ.ID.No. 2.

2. Construction of human PD-L1 gene promoter targeting methylase and adenovirus package

Cloning and recombining the artificially synthesized DNA fragment encoding the PPLA on pcDNA3.1 (shown in figure 1), carrying out adenovirus packaging on an expression plasmid (shown in figure 2) to obtain an expressed gene engineering protein PPLA, confirming that the protein content is 130KDa (shown in figure 3) by Western-blot, simulating a spatial structure by bioinformatics analysis, and combining the intracellularly expressed protein with a PD-L1 gene promoter (shown in figure 4). The protein is proved to induce the PD-L1 gene promoter to generate a specific methylation phenomenon through methylation sequencing, and meanwhile, quantitative PCR is adopted at 24h, 48h and 72h respectively, and a further analysis of an elapsed cell instrument and Westernblot proves that the protein can cause the PD-L1 gene expression to be remarkably reduced (such as figures 6, 7 and 8).

Sequence listing

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atggattaca aggatgacga cgataagccc aagaagaagc gcaaggtgct ggaaccgggc 60

gaaaaaccgt ataaatgccc ggaatgcggc aaaagcttta gcgaacgcag ccatctgcgc 120

gaacatcagc gcacccatac cggcgaaaaa ccgtataaat gcccggaatg cggcaaaagc 180

tttagccgca gcgataaact ggtgcgccat cagcgcaccc ataccggcga aaaaccgtat 240

aaatgcccgg aatgcggcaa aagctttagc cagagcagca gcctggtgcg ccatcagcgc 300

acccataccg gcgaaaaacc gtataaatgc ccggaatgcg gcaaaagctt tagccgcgcg 360

gataacctga ccgaacatca gcgcacccat accggcgaaa aaccgtataa atgcccggaa 420

tgcggcaaaa gctttagccg caacgatgcg ctgaccgaac atcagcgcac ccataccggc 480

gaaaaaccgt ataaatgccc ggaatgcggc aaaagcttta gccataccgg ccatctgctg 540

gaacatcagc gcacccatac cggcgaaaaa ccgtataaat gcccggaatg cggcaaaagc 600

tttagcgatc cgggcgcgct ggtgcgccat cagcgcaccc ataccggcaa aaaaaccagc 660

ggccgcggcg gcggcggcag cggcggcggc ggcagcccag ctgagaagag gaagcccatc 720

cgggtgctgt ctctctttga tggaatcgct acagggctcc tggtgctgaa ggacttgggc 780

attcaggtgg accgctacat tgcctcggag gtgtgtgagg actccatcac ggtgggcatg 840

gtgcggcacc aggggaagat catgtacgtc ggggacgtcc gcagcgtcac acagaagcat 900

atccaggagt ggggcccatt cgatctggtg attgggggca gtccctgcaa tgacctctcc 960

atcgtcaacc ctgctcgcaa gggcctctac gagggcactg gccggctctt ctttgagttc 1020

taccgcctcc tgcatgatgc gcggcccaag gagggagatg atcgcccctt cttctggctc 1080

tttgagaatg tggtggccat gggcgttagt gacaagaggg acatctcgcg atttctcgag 1140

tccaaccctg tgatgattga tgccaaagaa gtgtcagctg cacacagggc ccgctacttc 1200

tggggtaacc ttcccggtat gaacaggccg ttggcatcca ctgtgaatga taagctggag 1260

ctgcaggagt gtctggagca tggcaggata gccaagttca gcaaagtgag gaccattact 1320

acgaggtcaa actccataaa gcagggcaaa gaccagcatt ttcctgtctt catgaatgag 1380

aaagaggaca tcttatggtg cactgaaatg gaaagggtat ttggtttccc agtccactat 1440

actgacgtct ccaacatgag ccgcttggcg aggcagagac tgctgggccg gtcatggagc 1500

gtgccagtca tccgccacct cttcgctccg ctgaaggagt attttgcgtg tgtgggcggc 1560

ggcggcagcg gcggcggcgg cagcaaaatc cgggtcaaca agttctacag gcctgagaac 1620

acccacaagt ccactccagc gagctaccac gcagacatca acctgctcta ctggagcgac 1680

gaggaggccg tggtggactt caaggctgtg cagggccgct gcaccgtgga gtatggggag 1740

gacctgcccg agtgcgtcca ggtgtactcc atgggcggcc ccaaccgctt ctacttcctc 1800

gaggcctata atgcaaagag caaaagcttt gaagatcctc ccaaccatgc ccgtagccct 1860

ggaaacaaag ggaagggcaa gggaaaaggg aagggcaagc ccaagtccca agcctgtgag 1920

ccgagcgagc cagagataga gatcaagctg cccaagctgc ggaccctgga tgtgttttct 1980

ggctgcgggg ggttgtcgga gggattccac caagcaggca tctctgacac gctgtgggcc 2040

atcgagatgt gggaccctgc ggcccaggcg ttccggctga acaaccccgg ctccacagtg 2100

ttcacagagg actgcaacat cctgctgaag ctggtcatgg ctggggagac caccaactcc 2160

cgcggccagc ggctgcccca gaagggagac gtggagatgc tgtgcggcgg gccgccctgc 2220

cagggcttca gcggcatgaa ccgcttcaat tcgcgcacct actccaagtt caaaaactct 2280

ctggtggttt ccttcctcag ctactgcgac tactaccggc cccggttctt cctcctggag 2340

aatgtcagga actttgtctc cttcaagcgc tccatggtcc tgaagctcac cctccgctgc 2400

ctggtccgca tgggctatca gtgcaccttc ggcgtgctgc aggccggtca gtacggcgtg 2460

gcccagacta ggaggcgggc catcatcctg gccgcggccc ctggagagaa gctccctctg 2520

ttcccggagc cactgcacgt gtttgctccc cgggcctgcc agctgagcgt ggtggtggat 2580

gacaagaagt ttgtgagcaa cataaccagg ttgagctcgg gtcctttccg gaccatcacg 2640

gtgcgagaca cgatgtccga cctgccggag gtgcggaatg gagcctcggc actggagatc 2700

tcctacaacg gggagcctca gtcctggttc cagaggcagc tccggggcgc acagtaccag 2760

cccatcctca gggaccacat ctgtaaggac atgagtgcat tggtggctgc ccgcatgcgg 2820

cacatcccct tggccccagg gtcagactgg cgcgatctgc ccaacatcga ggtgcggctc 2880

tcagacggca ccatggccag gaagctgcgg tatacccacc atgacaggaa gaacggccgc 2940

agcagctctg gggccctccg tggggtctgc tcctgcgtgg aagccggcaa agcctgcgac 3000

cccgcagcca ggcagttcaa caccctcatc ccctggtgcc tgccccacac cgggaaccgg 3060

cacaaccact gggctggcct ctatggaagg ctcgagtggg acggcttctt cagcacaacc 3120

gtcaccaacc ccgagcccat gggcaagcag ggccgcgtgc tccacccaga gcagcaccgt 3180

gtggtgagcg tgcgggagtg tgcccgctcc cagggcttcc ctgacaccta ccggctcttc 3240

ggcaacatcc tggacaagca ccggcaggtg ggcaatgccg tgccaccgcc cctggccaaa 3300

gccattggct tggagatcaa gctttgtatg ttggccaaag cccgagagag tgcctcagct 3360

aaaataaagg aggaggaagc tgctaaggac tag 3393

Claims (2)

1. A specific binding protein molecule for identifying a human PD-L1 gene promoter has an amino acid sequence shown in SEQ.ID.No.1 and is named PPLA.

2. A gene encoding the protein molecule of claim 1, having the nucleotide sequence shown in seq.id No. 2.

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