CN111826395A - Recombinant oncolytic virus expression anti-immune checkpoint fusion antibody and immunostimulatory molecule - Google Patents

Recombinant oncolytic virus expression anti-immune checkpoint fusion antibody and immunostimulatory molecule Download PDF

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CN111826395A
CN111826395A CN201910312308.2A CN201910312308A CN111826395A CN 111826395 A CN111826395 A CN 111826395A CN 201910312308 A CN201910312308 A CN 201910312308A CN 111826395 A CN111826395 A CN 111826395A
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黄雪芬
陈思毅
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Isonmingxu Corp
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Abstract

The invention provides a recombinant oncolytic virus expressing an anti-immune checkpoint fusion antibody and an immunostimulating molecule and application thereof. An oncolytic virus construct comprising: a first nucleic acid molecule encoding a secreted anti-PD-L1 fusion antibody molecule for specifically inhibiting PD-L1, said anti-PD-L1 fusion antibody comprising: an anti-PD-L1 single chain antibody, an IgG1 hinge region, and an IgG1Fc region, said anti-PD-L1 single chain antibody being linked to said IgG1Fc region by an IgG1 hinge region, said IgG1Fc region having the T250Q and M248L amino acid mutations; and a second nucleic acid molecule encoding an immunostimulatory molecule. The constructs according to embodiments of the invention encode antibodies and immunostimulatory molecules that inhibit PD-L1, and are capable of inhibiting PD-1: the immunosuppression mechanism mediated by PD-L1 and the specific immune response of individual tumor are caused, so that the tumor cells are killed systemically and effectively.

Description

Recombinant oncolytic virus expression anti-immune checkpoint fusion antibody and immunostimulatory molecule
Technical Field
The invention relates to the field of biomedicine, in particular to an oncolytic virus construct, an oncolytic virus, a recombinant cell and a pharmaceutical composition, and further relates to application of the oncolytic virus construct, the oncolytic virus and the recombinant cell in preparation of a medicament for treating or preventing cancer and inhibiting malignant cell proliferation of mammals.
Background
The oncolytic virus refers to a virus which has the capability of selectively replicating and packaging in tumor cells and realizes the selective killing of the tumor cells. At present, most researches achieve the oncolytic effect by modifying some virulent strains which exist in the nature and are weak in pathogenicity to enable the virulent strains to be specifically expressed and packaged in tumor cells. The principle of the oncolytic virus to achieve selective killing of tumor cells is mainly divided into two principles: first, selectively infecting tumor cells using inactivation or a defect of a tumor suppressor gene in target cells; secondly, the tumor-specific promoter is selected to regulate the expression of the virus key gene, so that the oncolytic virus can be massively replicated in the tumor cell and expresses toxic protein to destroy the tumor cell, and/or simultaneously secrete cytokine to stimulate an autoimmune system to attack the tumor cell. The corresponding oncolytic virus cannot or reduces replication in normal body cells without or with reduced killing effect, so the oncolytic virus has high antitumor effect and low side effect. In recent decades, oncolytic virus therapy has attracted much attention and related research has progressed. Currently, adenovirus (adenovirus), herpes simplex virus-1 (HSV-1), Newcastle disease virus, etc. are sequentially transformed into oncolytic virus. In 2006, oncolytic adenovirus products (oncorine) have been used in clinical treatment in china, mainly for the treatment of nasopharyngeal carcinoma and the like. According to the principle of Oncorine, the E1B-55kD region of human adenovirus 5 is deleted, so that the virus can propagate in cancer cells with p53 gene mutation and kill host cells, and an oncolytic therapeutic effect is generated. However, clinical data show that the therapeutic effect of such oncolytic adenovirus based on the mutation of the E1B gene is not very ideal. JX-594 of Jennerex, U.S. biotherapeutics, is a modified vaccinia virus. In a secondary clinical trial completed in 2013, it was found that the life extension time of primary liver cancer patients after high-dose injection of virus could reach 14.1 months, while those receiving low-dose injection had a life extension of only 6.7 months. The genetically engineered herpes simplex virus Oncovex GM-CSF (T-Vec) developed by BioVex, a biotechnology company, has passed FDA approval in 2015 10 months and has become the first oncolytic virus product on the market in the world. OncVex can selectively kill tumor cells and express and secrete GM-CSF to start the immune response of the organism to generate a system to kill residual local tumor cells and metastatic tumors thereof. The results of a metastatic melanoma phase II trial published by BioVex in 2009 showed that 26% of 50 patients responded to treatment and 8 patients had complete recovery. This company was purchased from Amgen in 2011 for advancing phase three clinical trials. In 2013, am en (Amgen) published the treatment data for OncoVex, and clinically demonstrated that it successfully reduced tumors in advanced patients, and Amgen's drug performed better than the other drugs of the same class in a phase III study in over 400 patients tested.
The research results are combined to discover that the oncolytic virus is really a sharp instrument for treating tumors, but the oncolytic virus still has weak killing effect on locally injected tumors and non-injected metastatic tumor lesions, and still needs further development and improvement by researchers to enhance local and systemic anti-tumor effects.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention provides an oncolytic virus which can be massively replicated in tumor cells and finally destroys the tumor cells, can secrete an anti-PD-L1 fusion antibody to inhibit an immune escape mechanism of the tumor cells, and can secrete an immune stimulating molecule to stimulate an immune response and attract and activate more immune cells to continuously kill local residual cancer cells and cancer cells which are transferred and spread systemically.
In a first aspect of the invention, the invention features an oncolytic viral construct. According to an embodiment of the invention, the oncolytic viral construct comprises: a first nucleic acid molecule encoding a secreted anti-PD-L1 fusion antibody molecule for specifically inhibiting PD-L1, said anti-PD-L1 fusion antibody comprising: an anti-PD-L1 single chain antibody, an IgG1 hinge region, and an IgG1Fc region, said anti-PD-L1 single chain antibody being linked to said IgG1Fc region by an IgG1 hinge region, said IgG1Fc region having the T250Q and M248L amino acid mutations; and a second nucleic acid molecule encoding an immunostimulatory molecule. The oncolytic virus construct according to the embodiment of the invention encodes an anti-PD-L1 fusion antibody which is secreted extracellularly and specifically binds to PD-L1 on the surface of tumor cells, effectively blocking PD-1: the PD-L1 mediated immune escape mechanism further induces tumor specific immune response, and meanwhile, the immunostimulating molecules coded by the oncolytic virus construct according to the embodiment of the invention can induce systemic immune response to effectively kill local residual cancer cells and cancer cells metastasized and spread systemically.
According to an embodiment of the present invention, the above-mentioned oncolytic virus construct may further comprise at least one of the following additional technical features:
according to the embodiment of the invention, the anti-PD-L1 single-chain antibody has an amino acid sequence shown in SEQ ID NO. 1.
MLLLVTSLLLCELPHPAFLLIPDIVLTQSPASLALSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEEEDAAMYFCQQSRRVPYTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQAWGYPWGQGTLVTVSS(SEQ ID NO:1)。
According to an embodiment of the invention, the IgG1 hinge region has the amino acid sequence shown in SEQ ID NO. 2.
DKTHTCP(SEQ ID NO:2)。
According to an embodiment of the present invention, the IgG1Fc region has the amino acid sequence shown in SEQ ID NO. 3.
PCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)。
According to an embodiment of the present invention, the anti-PD-L1 fusion antibody has an amino acid sequence shown in SEQ ID NO. 4.
MLLLVTSLLLCELPHPAFLLIPDIVLTQSPASLALSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEEEDAAMYFCQQSRRVPYTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQAWGYPWGQGTLVTVSSAAADKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK(SEQ ID NO:4)。
According to an embodiment of the invention, the first nucleic acid molecule has a nucleotide sequence shown as SEQ ID NO 5-7.
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGACATTGTGCTCACCCAATCTCCAGCTTCTTTGGCTCTGTCTCCCGGGGAGAGAGCCACCCTCTCCTGCAGAGCCACTGAAAGTGTTGAATACTATGGCACAAGTTTAGTGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAGCGTAGATTCTGGGGTCCCTTCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAATTCTCTGGAGGAGGAGGATGCTGCAATGTATTTCTGTCAGCAAAGTAGGAGGGTTCCGTACACGTTCGGACAGGGGACCAAGCTGGAGATAAAAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAAGCAGGCCCCTGGGCAGCGCCTTGAGTGGATTGGATATGTTAATCCTTTCAATGATGGTACTAAGTACAATGAGATGTTCAAAGGCAGGGCCACACTGACTTCAGACAAATCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGGTCTGAGGACACTGCGGTCTATTACTGTGCAAGACAGGCTTGGGGTTACCCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCTGCGGCCGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACCAACTGATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(SEQ ID NO:5)。
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGAAATCGTCCTGACTCAAAGCCCAGCGACTTTGTCCCTCTCCCCAGGCGAGCGGGCCACGCTGTCTTGCAGGGCCACCGAGTCTGTGGAGTATTATGGTACAAGCCTGGTGCAATGGTATCAACAAAAGCCTGGTCAGCCGCCTAAACTCCTCATCTACGCTGCCTCTTCAGTAGATTCAGGCGTTCCATCTCGATTCTCTGGCAGCGGAAGCGGAACAGACTTCACCCTCACGATTAATAGCCTGGAAGCAGAAGACGCTGCAACCTATTATTGCCAGCAGTCAAGAAGGGTTCCATATACGTTTGGCGGCGGAACCAAACTTGAGATAAAAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAAGTACAACTGGTGCAGAGTGGAGCGGAGGTTAAGAAACCGGGGGCAACAGTGAAAATATCTTGCAAGGTTTCTGGTTATACGTTTACGAGTTATGTCATGCATTGGGTCCGACAGGCCCCTGGGCAGGGCCTGGAATGGATGGGTTATGTGAACCCCTTCAATGATGGGACGAAATACAATGAAATGTTCAAAGGTAGAGTGACAATTACACGGGATACGTCCGCAAGCACGGCATATATGGAGCTTAGTTCACTCCGCAGTGAAGATACTGCTGTCTACTATTGTGCGAGACAAGCCTGGGGGTATCCATGGGGGCAAGGCACGCTTGTAACGGTGAGTGCGGCGGCCGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACCAACTGATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(SEQ ID NO:6)。
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGATATAGTGCTCACACAAAGCCCGGCCTCTCTCGCCGTAAGTCTGGGGCAACGAGCTACTATCAGTTGCCGCGCTACGGAGAGCGTGGAATACTATGGAACGAGTCTGGTGCAGTGGTATCAGCAAAAACCGGGGCAACCACCGAAACTGCTGATATACGCCGCTTCATCTGTTGACTCTGGAGTGCCAGCAAGGTTTAGTGGTAGCGGCTCTGGCACTGACTTCTCACTTACAATACATCCTGTGGAGGAGGATGACATAGCCATGTACTTCTGTCAGCAATCCAGGCGAGTCCCATACACGTTTGGTGGGGGGACGAAGTTGGAAATAAAAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAAGTTCAGTTGCAACAGTCTGGTCCAGAGCTTGTTAAACCGGGGGCAAGCGTTAAAATGAGCTGCAAAGCCTCAGGGTACACCTTTACAAGTTATGTAATGCACTGGGTTAAACAGAAACCCGGCCAGGGTCTGGAGTGGATTGGCTACGTCAACCCCTTTAATGACGGTACCAAGTACAATGAGATGTTCAAGGGCAAAGCCACACTTACGTCCGATAAGAGTAGTAGCACCGCCTACATGGAACTTTCTAGCTTGACTTCCGAAGACAGTGCATGGTACTATTGTGCGAGACAAGCGTGGGGTTATCCTTGGGGCCAAGGTACTCTTGTGACGGTATCAGCGGCGGCCGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACCAACTGATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(SEQ ID NO:7)。
According to an embodiment of the invention, the immunostimulatory molecule comprises at least one member selected from the group consisting of human GM-CSF, Flt-3L, IL-2, IL-12, IL-15, IL-18, IL-24, TNF, IFN α, IFN β, IFN γ, MIP-I, MIP-I β, MCP-I, RANTES. The immunostimulating molecules can cause a systemic immune response of a human, and further, can be more effective in killing tumor cells by immunity.
According to an embodiment of the invention, the immunostimulatory molecule is GM-CSF. Granulocyte-macrophage colony stimulating factor (GM-CSF) can recruit dendritic cells and natural killer cells and mediate tumor-specific CD8+Induction of cytotoxic T lymphocytes. The insertion of the human GM-CSF gene into HSV enhances the anti-tumor response of the FDA-approved oncolytic virus T-vec. When GM-CSF gene is inserted into HSV, it has inhibitory action on tumor, either by in situ injection or remote injection, which shows that GM-CSF can induce systemic anti-tumor immune response.
The inventor finds that GM-CSF can better induce systemic immune response of organisms, and the synergistic effect of GM-CSF and the anti-PD-L1 fusion antibody according to the embodiment of the invention in vivo is more effective, so that more effective immune killing on systemic tumor cells can be induced.
According to an embodiment of the invention, the vector of the construct is a vaccinia virus vector, the vaccinia virus is a WR strain vaccinia virus, and the thymidine kinase gene and growth factor gene in the vaccinia virus are inactivated. Vaccinia virus is used in the form of a live vaccine to prevent smallpox and more recently as a vaccine to treat tumors. Vaccinia viruses have been engineered to carry a variety of antigens, cytokines and immunostimulatory molecules including carcinoembryonic antigen, gp100, MART-1, granulocyte/macrophage colony stimulating factor, B7-1, IL3-1 β, IL-2, and IL-12, as tumor vaccines to activate anti-tumor immune responses. Compared with wild viruses, the inactivated thymidine kinase gene (TK gene) or growth factor gene (VGF gene) can obviously reduce the toxicity of the viruses. Viruses carrying the TK-gene require the synthesis of DNA by drawing TTP from the nucleotide pool of dividing cells. This results in advantageous viral replication in dividing cells to selectively kill tumor cells. VGF is a secreted protein that is produced early in viral infection and acts as a mitogen to promote peripheral cell division, receiving vaccinia virus infection. Removal of this growth factor causes a decrease in viral replication in resting normal cells. Removal of the TK and VGE genes together can further increase specific selective killing of tumors. Absent the TK gene, viral replication requires the withdrawal of TTP from dividing cells. While the phenomenon of stimulating the division of surrounding cells does not occur in the absence of VGF. Thus, viral replication can only occur selectively in dividing tumor cells.
According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are located after the 240 th base and before the 308 th base of the thymidine kinase gene.
According to an embodiment of the invention, the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; a second promoter operably linked to the first nucleic acid molecule. The first nucleic acid molecule and the second nucleic acid molecule can independently start expression under the control of the first promoter and the second promoter, and have no interference with each other and higher expression control efficiency.
According to an embodiment of the invention, said first promoter and said second promoter are each independently selected from at least one of pSel, p 7.5. The p7.5 promoter, controlled by vaccinia-specific polymerase, is a traditional early and late promoter and is widely used for recombinant gene expression. pSel is a synthetic vaccinia virus promoter that is based on sequence engineering that increases the activity of the P7.5 early/late promoter. These two promoters are widely used for expression of foreign genes to generate a protective immune response in a host immunized with vaccinia virus.
In a second aspect of the invention, the invention features an oncolytic viral construct. The oncolytic virus construct according to the embodiment of the invention carries the nucleotide sequence shown in SEQ ID NO: 5-7, and the secretory type three-strain anti-PD-L1 single-chain antibody and the immune stimulating molecule (GM-CSF) coded by the construct can specifically inhibit the expression of PD-1: PD-L1 mediated immunosuppression mechanism and systemic immune response, thereby realizing effective and specific killing of tumor cells.
According to an embodiment of the invention, the construct carries SEQ ID NO: 8-10.
TCATTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTGTAGTGGTTGTGCAGAGCCTCATGCAGCACGGAGCATGAGAAGACGTTCCCCTGCTGCCACCTGCTCTTGTCCACGGTGAGCTTGCTGTAGAGGAAGAAGGAGCCGTCGGAGTCCAGCACGGGAGGCGTGGTCTTGTAGTTGTTCTCCGGCTGCCCATTGCTCTCCCACTCCACGGCGATGTCGCTGGGATAGAAGCCTTTGACCAGGCAGGTCAGGCTGACCTGGTTCTTGGTCATCTCCTCCCGGGATGGGGGCAGGGTGTACACCTGTGGTTCTCGGGGCTGCCCTTTGGCTTTGGAGATGGTTTTCTCGATGGGGGCTGGGAGGGCTTTGTTGGAGACCTTGCACTTGTACTCCTTGCCATTCAGCCAGTCCTGGTGCAGGACGGTGAGGACGCTGACCACACGGTACGTGCTGTTGTACTGCTCCTCCCGCGGCTTTGTCTTGGCATTATGCACCTCCACGCCGTCCACGTACCAGTTGAACTTGACCTCAGGGTCTTCGTGGCTCACGTCCACCACCACGCATGTGACCTCAGGGGTCCGGGAGATCATCAGTTGGTCCTTGGGTTTTGGGGGGAAGAGGAAGACTGACGGTCCCCCCAGGAGTTCAGGTGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCTGCGGCCGCAGAAGAGACAGTGACCAGAGTCCCTTGGCCCCAGGGGTAACCCCAAGCCTGTCTTGCACAGTAATAGACCGCAGTGTCCTCAGACCTCAGGCTGCTGAGCTCCATGTAGGCTGTGCTGGTGGATTTGTCTGAAGTCAGTGTGGCCCTGCCTTTGAACATCTCATTGTACTTAGTACCATCATTGAAAGGATTAACATATCCAATCCACTCAAGGCGCTGCCCAGGGGCCTGCTTCACCCAGTGCATAACATAGCTAGTGAATGTGTATCCAGAAGCCTTGCAGGACATCTTCACTGAAGCCCCAGGCTTTTTCACCTCAGCTCCAGACTGCACCAGCTGGACCTCGCCCTTGGTGGATCCCTCGCCAGATCCGGGCTTGCCGGATCCAGAGGTGGAGCCTTTTATCTCCAGCTTGGTCCCCTGTCCGAACGTGTACGGAACCCTCCTACTTTGCTGACAGAAATACATTGCAGCATCCTCCTCCTCCAGAGAATTGATGGTGAGGGTGAAGTCTGTCCCAGACCCACTGCCACTAAACCTGGAAGGGACCCCAGAATCTACGCTGGATGCAGCATAGATGAGGAGTTTGGGTGGCTGTCCTGGTTTCTGTTGGTACCACTGCACTAAACTTGTGCCATAGTATTCAACACTTTCAGTGGCTCTGCAGGAGAGGGTGGCTCTCTCCCCGGGAGACAGAGCCAAAGAAGCTGGAGATTGGGTGAGCACAATGTCTGGGATCAGGAGGAATGCTGGGTGTGGTAACTCACAGAGCAGAAGGCTTGTCACCAGGAGAAGCATTCTAGAATCTAGATGCATTCGCGAGGTACCGTCGACTTCGAGCTTATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTTAAGCTTTCACTAATTCCAAACCCACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCGTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGATCATAACGATCTCTATAATCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATAAACAGGCTGGGACACTTCACCTCGAGATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTGA(SEQ ID NO:8)。
In the present application, the nucleotide sequence shown in SEQ ID NO. 8 is designated PDL1scFv (I) -Fc-pSel-p7.5-GMCSF insert DNA SEQ.
TCATTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTGTAGTGGTTGTGCAGAGCCTCATGCAGCACGGAGCATGAGAAGACGTTCCCCTGCTGCCACCTGCTCTTGTCCACGGTGAGCTTGCTGTAGAGGAAGAAGGAGCCGTCGGAGTCCAGCACGGGAGGCGTGGTCTTGTAGTTGTTCTCCGGCTGCCCATTGCTCTCCCACTCCACGGCGATGTCGCTGGGATAGAAGCCTTTGACCAGGCAGGTCAGGCTGACCTGGTTCTTGGTCATCTCCTCCCGGGATGGGGGCAGGGTGTACACCTGTGGTTCTCGGGGCTGCCCTTTGGCTTTGGAGATGGTTTTCTCGATGGGGGCTGGGAGGGCTTTGTTGGAGACCTTGCACTTGTACTCCTTGCCATTCAGCCAGTCCTGGTGCAGGACGGTGAGGACGCTGACCACACGGTACGTGCTGTTGTACTGCTCCTCCCGCGGCTTTGTCTTGGCATTATGCACCTCCACGCCGTCCACGTACCAGTTGAACTTGACCTCAGGGTCTTCGTGGCTCACGTCCACCACCACGCATGTGACCTCAGGGGTCCGGGAGATCATCAGTTGGTCCTTGGGTTTTGGGGGGAAGAGGAAGACTGACGGTCCCCCCAGGAGTTCAGGTGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCTGCGGCCGCTACGAAGAGGACCACTGTTCGGAAGACGAGACACTCAATGGTGTGGGTCGTAAGGAGGACTAGGGTCTTTAGCAGGACTGAGTTTCGGGTCGCTGAAACAGGGAGAGGGGTCCGCTCGCCCGGTGCGACAGAACGTCCCGGTGGCTCAGACACCTCATAATACCATGTTCGGACCACGTTACCATAGTTGTTTTCGGACCAGTCGGCGGATTTGAGGAGTAGATGCGACGGAGAAGTCATCTAAGTCCGCAAGGTAGAGCTAAGAGACCGTCGCCTTCGCCTTGTCTGAAGTGGGAGTGCTAATTATCGGACCTTCGTCTTCTGCGACGTTGGATAATAACGGTCGTCAGTTCTTCCCAAGGTATATGCAAACCGCCGCCTTGGTTTGAACTCTATTTTCCGAGGTGGAGACCTAGGCCGTTCGGGCCTAGACCGCTCCCTAGGTGGTTCCCGCTTCATGTTGACCACGTCTCACCTCGCCTCCAATTCTTTGGCCCCCGTTGTCACTTTTATAGAACGTTCCAAAGACCAATATGCAAATGCTCAATACAGTACGTAACCCAGGCTGTCCGGGGACCCGTCCCGGACCTTACCTACCCAATACACTTGGGGAAGTTACTACCCTGCTTTATGTTACTTTACAAGTTTCCATCTCACTGTTAATGTGCCCTATGCAGGCGTTCGTGCCGTATATACCTCGAATCAAGTGAGGCGTCACTTCTATGACGACAGATGATAACACGCTCTGTTCGGACCCCCATAGGTACCCCCGTTCCGTGCGAACATTGCCACTCACGCTCTAGAATCTAGATGCATTCGCGAGGTACCGTCGACTTCGAGCTTATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTTAAGCTTTCACTAATTCCAAACCCACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCGTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGATCATAACGATCTCTATAATCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATAAACAGGCTGGGACACTTCACCTCGAGATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTGA(SEQ ID NO:9)。
In the present application, the nucleotide sequence shown in SEQ ID NO. 9 is designated PDL1scFv (II) -Fc-pSel-p7.5-GMCSF insert DNA SEQ.
TCATTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTGTAGTGGTTGTGCAGAGCCTCATGCAGCACGGAGCATGAGAAGACGTTCCCCTGCTGCCACCTGCTCTTGTCCACGGTGAGCTTGCTGTAGAGGAAGAAGGAGCCGTCGGAGTCCAGCACGGGAGGCGTGGTCTTGTAGTTGTTCTCCGGCTGCCCATTGCTCTCCCACTCCACGGCGATGTCGCTGGGATAGAAGCCTTTGACCAGGCAGGTCAGGCTGACCTGGTTCTTGGTCATCTCCTCCCGGGATGGGGGCAGGGTGTACACCTGTGGTTCTCGGGGCTGCCCTTTGGCTTTGGAGATGGTTTTCTCGATGGGGGCTGGGAGGGCTTTGTTGGAGACCTTGCACTTGTACTCCTTGCCATTCAGCCAGTCCTGGTGCAGGACGGTGAGGACGCTGACCACACGGTACGTGCTGTTGTACTGCTCCTCCCGCGGCTTTGTCTTGGCATTATGCACCTCCACGCCGTCCACGTACCAGTTGAACTTGACCTCAGGGTCTTCGTGGCTCACGTCCACCACCACGCATGTGACCTCAGGGGTCCGGGAGATCATCAGTTGGTCCTTGGGTTTTGGGGGGAAGAGGAAGACTGACGGTCCCCCCAGGAGTTCAGGTGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCTGCGGCCGCTACGAAGAGGACCACTGTTCGGAAGACGAGACACTCAATGGTGTGGGTCGTAAGGAGGACTAGGGTCTATATCACGAGTGTGTTTCGGGCCGGAGAGAGCGGCATTCAGACCCCGTTGCTCGATGATAGTCAACGGCGCGATGCCTCTCGCACCTTATGATACCTTGCTCAGACCACGTCACCATAGTCGTTTTTGGCCCCGTTGGTGGCTTTGACGACTATATGCGGCGAAGTAGACAACTGAGACCTCACGGTCGTTCCAAATCACCATCGCCGAGACCGTGACTGAAGAGTGAATGTTATGTAGGACACCTCCTCCTACTGTATCGGTACATGAAGACAGTCGTTAGGTCCGCTCAGGGTATGTGCAAACCACCCCCCTGCTTCAACCTTTATTTTCCGAGGTGGAGACCTAGGCCGTTCGGGCCTAGACCGCTCCCTAGGTGGTTCCCGCTTCAAGTCAACGTTGTCAGACCAGGTCTCGAACAATTTGGCCCCCGTTCGCAATTTTACTCGACGTTTCGGAGTCCCATGTGGAAATGTTCAATACATTACGTGACCCAATTTGTCTTTGGGCCGGTCCCAGACCTCACCTAACCGATGCAGTTGGGGAAATTACTGCCATGGTTCATGTTACTCTACAAGTTCCCGTTTCGGTGTGAATGCAGGCTATTCTCATCATCGTGGCGGATGTACCTTGAAAGATCGAACTGAAGGCTTCTGTCACGTACCATGATAACACGCTCTGTTCGCACCCCAATAGGAACCCCGGTTCCATGAGAACACTGCCATAGTCGCTCTAGAATCTAGATGCATTCGCGAGGTACCGTCGACTTCGAGCTTATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTTAAGCTTTCACTAATTCCAAACCCACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCGTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGATCATAACGATCTCTATAATCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATAAACAGGCTGGGACACTTCACCTCGAGATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTGA(SEQ ID NO:10)。
In the present application, the nucleotide sequence shown in SEQ ID NO. 10 is designated PDL1scFv (III) -Fc-pSel-p7.5-GMCSF insert DNA SEQ.
In a third aspect of the invention, an oncolytic virus is provided. According to an embodiment of the invention, the oncolytic virus carries an oncolytic virus construct as described previously. The oncolytic virus provided by the embodiment of the invention can specifically inhibit PD-1: the immunosuppression mechanism mediated by PD-L1 and the systemic immune response are caused, thereby realizing the effective and specific killing of tumor cells.
According to an embodiment of the present invention, the above oncolytic virus may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the virus is an intracellular maturation virus, an intracellular packaging virus, a cell-associated packaging virus or an extracellular packaging virus, preferably the virus is an extracellular packaging virus or an intracellular maturation virus, more preferably the virus is an extracellular packaging virus. Intracellular mature viruses form a lipid bilayer shell and are mostly localized within infected cells until the cells rupture. Cell-associated packaging viruses and extracellular packaging viruses have an additional lipid bilayer and are able to bud from host cells without lysing the host cells. The extracellularly packaged virus has a higher infection efficiency on target cells, such as tumor cells, and can be more easily produced and purified for therapeutic use.
According to an embodiment of the invention, the virus is a vaccinia virus, a herpes simplex virus, an adenovirus, a vesicular stomatitis virus, a newcastle disease virus, a retrovirus, a reovirus, a measles virus, a Sinbis virus or an influenza virus, preferably the virus is a vaccinia virus. Vaccinia virus is a member of the poxvirus family, has double-stranded DNA, and is packaged in brick granules with a size of 300X 240X 120 nm. Vaccinia virus particles include a number of virally encoded enzymes, such as DNA-dependent RNA polymerase, transcription factors, capping and methylating enzymes, and poly (a) polymerase. These enzymes allow the virus to independently synthesize translatable mRNAs in a host cell under the control of viral factors produced (present in the cytoplasm of the infected cell). Vaccinia virus has been widely regarded as an effective vaccine in the worldwide smallpox eradication program, and because of its function as a vaccine against smallpox, vaccinia virus has been the longest and most widely studied in various viruses in humans. Both the physiological and pathological characteristics of vaccinia virus have been studied in detail and there is a large amount of available fundamental, preclinical and clinical data on vaccinia virus. Vaccinia viruses have several unique characteristics that continue to develop into biological therapeutics, particularly cancer therapeutics, that are extremely attractive. Vaccinia viruses have a wide host range and are capable of infection and replication in almost all humans and many other cell types. Therefore, it is widely used and studied in many animal models to assist in planning meaningful preclinical and conversion to clinical trials. Vaccinia virus infection and secondary gene expression occur at a highly efficient rate, and a number of viral promoters are available to control the timing and level of gene expression. Because the entire replication cycle occurs in the cytoplasm, the vaccinia virus gene is not integrated into the host cell chromosomal nucleic acid. An advantage of vaccinia viruses is that antiviral drugs, such as vaccinia immunoglobulins, can be used to limit the toxicity and spread of the virus. Furthermore, using standard DNA manipulation techniques available, vaccinia viruses can be efficiently recombined with large and/or numerous transcribed genes, up to 25Kb, for insertion into the vaccinia virus genome. Vaccinia virus can be produced simply at relatively high titers and the vaccinia virus particles are stable and can be frozen in solution or dry powder form for extended periods of time without significant loss of activity. Finally, vaccinia virus, an immunogenic virus, is able to elicit robust immune responses, providing the basis for vaccinia virus as a tumor vaccine. The PD-L1scFv-Fc fusion antibody according to the example of the present invention was expressed in oncolytic vaccinia virus, and based on the above characteristics of oncolytic vaccinia virus, the expression of PDL1scFv-Fc fusion antibody in oncolytic vaccinia virus was better than that of PDL1scFv-Fc fusion antibody in other oncolytic viruses.
In a fourth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, the recombinant cell is obtained by introducing the oncolytic virus construct described above into a recipient cell, or by transfecting the recipient cell with the oncolytic virus described above. The recombinant cell of the embodiment of the invention can provide necessary conditions for rapid replication and maturation of the oncolytic virus, and further can specifically inhibit the expression of PD-1: the immunosuppression mechanism mediated by PD-L1 and the systemic immune response are caused, thereby realizing the effective and specific killing of tumor cells.
According to an embodiment of the present invention, the recombinant cell may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the recipient cell comprises at least one selected from an immune cell or a tumor cell. The recombinant cell obtained by taking the immune cell or the tumor cell as a receptor cell can more safely and specifically inhibit an immune suppression mechanism mediated by an immune checkpoint molecule and cause a systemic immune response, thereby realizing effective and specific killing of the tumor cell and having higher safety.
In a fifth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises the oncolytic virus as described above and a pharmaceutically acceptable carrier. The inventor finds that the pharmaceutical composition can effectively inhibit the proliferation of tumor cells.
In a sixth aspect, the present invention provides the use of an oncolytic viral construct as hereinbefore described, an oncolytic virus as hereinbefore described or a recombinant cell as hereinbefore described in the manufacture of a medicament for the treatment or prophylaxis of cancer, inhibiting malignant cell proliferation in a mammal. The medicine prepared by the oncolytic virus construct, the oncolytic virus or recombinant cell provided by the embodiment of the invention can effectively inhibit the proliferation of malignant cells of mammals, and has remarkable curative effects on prevention and treatment of tumors.
Drawings
FIG. 1 is a schematic structural diagram of a recombinant vaccinia virus shuttle vector with a series of genes encoding secreted anti-PD-L1 fusion antibodies according to an embodiment of the invention;
FIG. 2 is a graph showing the results of in situ intratumoral injection of recombinant vaccinia virus VV-PDL1scFv-Fc/GM effective in controlling in situ tumor growth in accordance with an embodiment of the invention;
FIG. 3 is a graph showing the results of systemic anti-tumor activity of the in situ intratumoral injection of recombinant vaccinia virus VV-PDL1scFv-Fc/GM according to an embodiment of the present invention;
FIG. 4 is a graph showing the results of recombinant vaccinia virus VV-PDL1scFv-Fc/GM infected tumor cells expressing and secreting anti-PD-L1 scFv-Fc fusion antibody that inhibits the interaction of PD-1 with PD-L1 according to the present example;
FIG. 5 is a graph showing the results of in situ intratumoral injection of recombinant vaccinia virus VV-PDL1scFv-Fc/GM expressing anti-PD-L1 scFv-Fc fusion antibody and anti-PD-L1 scFv-Fc fusion protein antibody in mouse serum inhibiting the interaction of PD-1 with PD-L1, according to an embodiment of the present invention; and
FIG. 6 is a graph of the results of intratumoral injection of recombinant oncolytic virus VV-PDL1scFv-Fc/GM according to an embodiment of the invention more effective against the growth of distant tumors than oncolytic vaccinia virus VV-PD1 Fc/GM.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Human antibodies comprise two immunoglobulin light chains and two immunoglobulin heavy chains, which are linked covalently or non-covalently, resulting in the formation of three separate protein regions-two Fab regions and an Fc region. The Fab region and the Fc region are connected by a flexible connecting portion as a hinge region. The Fab region of the antibody is of a similar structure, having a specific antigen binding site, and the Fc region has a ligand interaction site that induces effector functions, including cellular Fc receptors and the C1q complement component. The physiological activity of therapeutic antibodies is mediated by two independent native immunoglobulin mechanisms: the efficacy of a therapeutic antibody results from its specificity and bivalent binding to the antigen of interest (e.g., blocking or neutralizing the target antigen or inducing apoptosis), and also from effector functions activated by the immune complex formed by Fc and effector ligands (Fc receptor and C1q components).
A single chain antibody (scFv) is a genetically engineered antibody in which VH and VL domains are linked to a flexible polypeptide linker. Single chain antibodies exhibit better tissue penetration pharmacokinetics than the Fab region of whole antibodies and have full antigen binding specificity since the antigen binding surface is unaltered. However, the half-life of the single-chain antibody in blood is short because the single-chain antibody lacks an Fc molecule fragment and lacks effector functions of Fc fragment. The PD-L1 single-chain antibody in the anti-PD-L1 fusion antibody according to the embodiment of the present invention is linked to the IgG1Fc region via the IgG1 hinge region, and thus the half-life of the PD-L1 fusion antibody in blood is significantly prolonged compared to the PD-L1 single-chain antibody alone.
The Fc region of an antibody mediates its serum half-life and effector functions such as Complement Dependent Cytotoxicity (CDC), Antibody Dependent Cellular Cytotoxicity (ADCC) and Antibody Dependent Cellular Phagocytosis (ADCP). The Fc fragment is used for linking with the single-chain antibody to increase the half-life of the single-chain antibody in blood and has Fc effector function. Five classes of immunoglobulins (IgM, IgD and IgE, IgG, IgA, IgG subclass) and four IgG subclasses (IgG1, IgG2, IgG3, IgG4) are present in humans. The highest IgG content was found in human serum. Subclasses four IgG1, IgG2, IgG3 and IgG4 are highly conserved, differing in their constant regions, particularly in the hinge CH2 domain. These regions are involved in binding to IgG Fc receptors (Fc γ R) and complement C1 q. As a result, different subclasses have different effector functions in triggering Fc γ r-expressing cells, thereby causing phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. IgG1 and IgG3 were effective in triggering this classical pathway of complement, but IgG2 and IgG4 were not as effective.
The PD-L1 immune checkpoint molecule is often highly expressed in tumor cells. According to the embodiment of the invention, the single-chain antibody of PD-L1 expressed aiming at the tumor cells is fused with IgG1Fc, the half-life of the single-chain antibody is prolonged, and the binding capacity of the single-chain antibody and PD-L1 is enhanced, so that the interaction between PD-1 and PD-L1 is blocked, and the killing of CTL (cytotoxic lymphocyte) on the tumor cells is enhanced. IgG1Fc can effectively trigger effector functions such as Complement Dependent Cytotoxicity (CDC), Antibody Dependent Cellular Cytotoxicity (ADCC) and Antibody Dependent Cellular Phagocytosis (ADCP) to further kill tumor cells.
Specifically, the inventors found through experiments that the PD-L1 fusion antibody (PDL1-scFv-Fc fusion antibody) according to the embodiment of the present invention has a significant advantage in inhibiting PD-1: PD-L1 interaction compared to the PD-L1scFv single-chain antibody (results are shown in fig. 4, fig. 5). This is because the PD-L1scFv-Fc fusion antibody can form multimers by IgG Fc disulfide bond, and the binding force of the PD-L1scFv-Fc fusion antibody is stronger than that of a single-chain antibody; and the PD-L1scFv-Fc fusion antibody has a large molecular weight, the half-life period in blood is longer than that of the single-chain antibody, the concentration for effectively inhibiting the effect of PD-1: PD-L1 can be achieved in blood, and the concentration for effectively inhibiting the effect of PD-1: PD-L1 can not be achieved in blood by the single-chain antibody.
Meanwhile, the inventor also finds that the PD-L1scFv-Fc fusion antibody is expressed in the oncolytic vaccinia virus, and according to the characteristics of the oncolytic vaccinia virus, the PD-L1scFv-Fc fusion antibody is better expressed in the oncolytic vaccinia virus than the PD-L1scFv-Fc fusion antibody is expressed in other oncolytic viruses.
Vaccinia Virus (VV) has many inherent properties and is a desirable choice for oncolytic virus therapy. VV has a natural tendency to cancer cells. Unlike other viruses, VV does not have a specific cell surface receptor so that its entry into cells causes a wide range of cell infections. They rely on a certain amount of membrane fusion pathways to enter cells.
Vaccinia viruses can utilize active molecular pathways in tumor cells to aid in their replication. VV has a short life cycle of 8 hours, the entire life cycle occurring in the cytoplasm, eliminating the risk of genomic integration. Replication usually begins 2 hours after infection, at which time host cell nucleic acid synthesis ceases, as all cellular resources are directed to viral replication. Cell lysis occurs between 12 and 48 hours, releasing the packaged virus particles.
The presence of various antigenic forms of mature viruses enables them to evade the host immune system. Extracellular envelope of the virus the viral form is encapsulated in a host-derived envelope containing several host complement control proteins. In addition, VV infected cells secrete complement control proteins to bind to inactivated C4b and C3B to inhibit the classical and alternative complement activation pathways. Thus, VV can spread relatively undamaged to distant tumors in the bloodstream, thereby enabling more systemic spread of the virus.
The hypoxic nature of large tumors brings about an aggressive and therapeutically resistant phenotype. In contrast to adenovirus, the inventors found that hypoxic conditions did not affect VV replication, viral protein production, cytotoxicity and transgene expression. These results indicate that VV can be used for the treatment of large volume hypoxic tumors.
Finally, VV has been used as a vaccine for over a century with good safety records. Mild and non-severe side effects include fever and rash. Moderate to severe side effects include vaccinal eczema and post-vaccination encephalitis. Side effects are rare, the incidence rate is lower than 1:10000, and particularly serious side effects are extremely rare. The gene-modified recombinant VV has high safety because of its tumor selectivity. Recent clinical trials of the JX-594 virus in hepatocellular carcinoma have shown that the treatment is well tolerated, mainly manifested as an influenza-like symptom side effect in all patients.
Many genetic engineering approaches are now aimed at the pharmacokinetic/pharmacodynamic properties of the engineered antibodies. Improving the pharmacokinetic properties of antibodies, a relatively intensive field of research involves studying the interaction of the Fc region with the neonatal Fc receptor (FcRn). FcRn binds to immunoglobulin (IgG) in an endosomal acidic (pH-6) environment, and releases IgG into the circulation when exposed to physiological pH. FcRn, which does not bind IgG within the endosome, undergoes protein degradation in lysosomes. The ratio of IgG entering the circulatory system and entering the degradation pathway is critical to determining the half-life of IgG in the circulatory system. There has been evidence from studies that the half-life of antibodies can be significantly increased by mutating residues in the CH2 and CH3 Fc regions to improve the binding properties of IgG to FcRn. The inventor finds that the half-life of IgG in vivo can be remarkably improved by mutating Met at 428 to Leu (M428L) and Thr at 250 to Gln (T250Q) in the Fc region of the IgG1 antibody.
The anti-PD-L1 fusion antibody comprises: a PD-L1 single chain antibody, an IgG1 hinge region, and an IgG1Fc region, said PD-L1 single chain antibody being linked to said IgG1Fc region by an IgG1 hinge region, said IgG1Fc region having T250Q and M248L mutations. The oncolytic virus construct according to the embodiment of the invention encodes anti-PD-L1 fusion antibody and immune stimulating molecule, can inhibit immune suppression mechanism mediated by immune checkpoint molecules and cause individual tumor specific immune response, and further realizes systemic and effective killing of tumor cells.
The construction of the oncolytic virus constructs and the construction process of the oncolytic virus of the present application will be described in detail below, and the experimental data is used to demonstrate the tumor killing effect of the oncolytic virus.
Example 1 materials and methods
The cell line African green monkey kidney fibroblast (CV-1) is from the American germplasm collection center. CV-1 cells were cultured in DMEM medium supplemented with antibiotic-antifungal solution (100U/mL penicillin G, 250ng/mL amphotericin B, 100units/mL streptomycin) and 10% fetal bovine serum (FBS; Invitrogen Corporation). The culture environment is 37 ℃ and 5% CO2. Human 143 TK-cells (ATCC) were cultured in basal medium BSS supplemented with 0.015mg/ml 5-bromo-2' -deoxyuridine (BUdR), 10% fetal bovine serum and Earle.
Human melanoma cell A375, human pancreatic cancer cell Panc1, human brain tumor cell U87, human lung cancer cell H226, human head and neck cancer cell ATCC TCP-1012 were taken from ATCC. Normal cell lines, including human normal cells PDF (human primary dermal fibroblasts), MRC5 (normal human lung fibroblasts), IMR-90(ATCC CCL-186, normal human lung fibroblasts) and BJ (ATCC CRL-2522, normal human skin fibroblasts) were from ATCC.
Virus replication and titration in cell culture
CV-1(1×105) Cells were seeded in 12 or 24-well plates. After 24h of culture, the cells were infected with a small amount of virus. Cells were incubated at 37 ℃ for 1 hour with brief shaking every 10 minutes to accelerate virus infection of cells. After 1 hour, the infection fluid was removed and the medium was replaced with fresh one. Cells were harvested at 24h, 48h, 72h or 96h, respectively. Virus particles that infected the cells were released by rapid freeze-thaw cycling and the virus titer in the medium (pfu/mL) was determined twice by CV-1 monolayer plaque assay. The same procedure was performed for the remaining CV-1 cells. The remaining CV-1 cells were cultured in DMEM containing 5% FBS in a dense monolayer state for 6 days prior to viral infection.
Determination of cytopathic Effect (CPE)
Tumor cells were added at 1X10 per well4The density of individual cells was plated in 96-well plates with 100. mu.L of medium per well and incubated overnight at 37 ℃. The virus infected tumor cells at the indicated titers, and the first 2 hours post-infection were cultured in medium containing 2.5% FBS, after which the complete medium was replaced. All samples were tested in triplicate. The viability of the tumor cells was determined by MTS formalzan viability assay kit (Promega, Madison, Wis.). The optical density of the wells was determined at 490nm on a read plate (molecular devices VERSAmax, Sunnyvale, CA (molecular devices)). The average viability of tumor cells treated for each virus dilution was ± SEM calculated by relative percentage (viability of cells in control wells set as 100% viability relative to control wells treated with medium alone). Each test was performed in triplicate.
Animal research
NSG-SGM3 mice express human IL3, GM-CSF and SCF triple transgenic NSG-SGM3(NSGS) mice combine the features of highly immunodeficient NOD Scid Gamma (NSG) mice with cytokines that support stable engraftment of myeloid lineage cell populations. Hu-CD34+ NSG-SGM3 mice were purchased from Jackson laboratories and bred in specific pathogen free environments at the southern California university animal facility and at the Beller college. All animal studies were approved by the animal protection and use committee of the university of southern california and the animal protection and use committee of the baylor college of medicine.
Hu-CD34+
NSG-SGM3 mice were implanted with autologous tumor cells (right or left flank) subcutaneously (number of implantation was 0.5-1X 10)6Individual cells/mouse). For example, human pancreatic cancer cells PD-L1+Panc1, human brain tumor cell PD-L1+U87, human Lung cancer cell PD-L1+H226 cell implantation Hu-CD34+NSG-SGM3 mice. All mice were aged 8-10 weeks and gender matched. When tumors grew to 5-10mm (diameter), mice were randomized into groups and received intratumoral injection of different recombinant oncolytic viruses. Tumor size was measured by caliper every three or four days.
Statistical analysis
All data are expressed as mean and standard deviation (s.e.). Analysis of variance was used to measure the level of difference between different groups. The differential comparisons between the different groups were performed using either Student-Newman-Keuls analysis or Chi-square analysis using SigmaStat2.03 software (SPSS, Inc.). P values less than 0.05 are considered to be significantly different.
Example 2 construction of recombinant vaccinia oncolytic virus (VV-PDL1scFv-Fc/GM) co-expressing anti-human PD-L1 fusion antibody and human GM-CSF
A nucleotide sequence encoding an anti-human PD-L1 fusion antibody (comprising a human PD-L1 single-chain antibody and a human IgG Fc fragment) was synthesized with a contiguous restriction enzyme site (Not1 and SalI). The synthetic DNA fragment was cloned into vaccinia virus shuttle vector (pSEL-DsRed) (RFP: red fluorescent protein), thereby constructing pVV-PDL1scFv-Fc shuttle vector (shown in FIG. 1). The human GM-CSF gene was ligated with restriction enzyme sites (XhoI and EcoRV) and synthesized and cloned into pVV-PDL1scFv-Fc shuttle vector to construct shuttle vector pVV-PDL1 scFv-Fc/GM. In the shuttle vector pVV-PDL1scFv-Fc/GM obtained, the PDL1scFv-Fc fusion gene was under the control of the pSEL promoter, and the GM-CSF gene was under the control of the p7.5 promoter. The human GM-CSF gene can also be cloned into the pSEL-DsRed vector to construct shuttle vector pVV-GM (shown in FIG. 1). The PDL1scFv gene can also be cloned into the pSEL-DsRed vector to construct shuttle vector pVV-PDL1scFv (shown in FIG. 1).
Western Reserve (WR) Vaccinia Virus (VV) (vSC20) with deleted viral growth factors was used to construct recombinant double-deletions (VGF)-TK-) The vaccinia virus (JA.McCart, DL.Bartlett, B.Moss.US7208313B2.Combined VGF/TK-deleted vaccinia virus vector.2000), simultaneously expressing PDL1scFv-Fc fusion gene and GM-CSM, the specific construction method is referred to "systematic cancer therapy with a tumor-selective vacucinia virus mutant deletion gene and vaccinia growth factor. cancer Res.2001; 61: 8751-. Briefly, vSC20, a vgf gene-deleted WR strain VV was used as the parental virus for homologous recombination. To obtain recombinant double deletions (VGF)-TK-) VV-PDL1scFv-Fc/GM-CSF, inventors infected CV-1 cells with vSC20 at multiple titers (0.1-0.5), and then transfected CV-1 cells with shuttle plasmid DNA pVV-PDL1 scFv-Fc/GM-CSF. Recombinant viruses were selected in 143 Tk-cells in the presence of BUdR and the successful insertion of PDL1scFv-Fc and GM-CSF genes into recombinant vaccinia virus was determined by PCR and sequence analysis of the recombinant virus DNA. These recombinant oncolytic vaccinia viruses (VV-PDL1scFv-Fc/GM) (co-expressing anti-PD-L1 fusion antibody and GM-CSF) and control oncolytic vaccinia viruses, VV-PDL1scFv (expressing only anti-PD-L1 single chain antibody), VV-PDL1scFv-Fc (expressing only anti-PD-L1 fusion antibody), VV-GM (expressing only GM-CSF), and VV-RFP (expressing only RFP) (the genes of which are shown in FIG. 1) were produced and titrated for further study. Expression of PDL1scFv-Fc fusion antibody and GM-CSF protein in VV-PDL1scFv-Fc/GM infected tumor cells was detected by Western blotting and ELISA.
Example 3 oncolytic vaccinia virus VV-PDL1scFv-Fc/GM has potent direct tumor-solubilizing activity
The inventors first examined whether recombinant oncolytic (VV-PDL1scFv-Fc/GM) can maintain the killing activity of lytic tumors during cell culture using MTS cell proliferation assay kit (Promega, Madison, Wis.). The specific operation method refers to the instruction of the kit. Human melanoma cells A375, human pancreatic cancer cells Panc1, human brain tumor cells U87, and human lung cancer cells H226 were plated in tissue culture dishes and infected with VV-PDL1scFv-Fc/GM, VV-PDL1scFv, VV-GM, VV-RFP, or mock (PBS), respectively. The inventors found that recombinant TK-VGF-vaccinia virus VV-PDL1scFv-Fc/GM has comparable effects to VV-PDL1scFv, VV-PDL1scFv-Fc, VV-GM or VV-RFP in killing various tumor cells. VV-PDL1scFv-Fc/GM and VV-RFP viruses are not as potent in killing normal human cells, including PDF cells (human primary skin fibroblasts), MRC5 (normal human lung fibroblasts), IMR-90 (normal human lung fibroblasts), and BJ (normal human skin fibroblasts), as compared to human tumor cells. This result shows that: the insertion of the PDL1scFv-Fc fusion protein antibody gene and GM-CSF gene did not impair the oncolytic activity of the recombinant oncolytic vaccinia virus VV-PDL1 scFv-Fc/GM.
Example 4 oncolytic vaccinia virus VV-PDL1scFv-Fc/GM can effectively destroy local tumors by tumor in situ injection
Tumors grow in the host, creating a tumor microenvironment in which tumor cells are symbiotic with many other types of cells, including fibroblasts and immune cells that may express the immune checkpoint PD-L1. The inventors used Hu-CD34+ NSG-SGM3 mice to test the anti-tumor potential of oncolytic vaccinia virus VV-PDL1 scFv-Fc/GM. To examine whether local intratumoral injection of VV-PDL1scFv-Fc/GM could inhibit tumor growth, Hu-CD34+ NSG-SGM3 mice were injected subcutaneously with 5x105PD-L1+H226 tumor cells, which were then divided into several groups (5-8 mice/group). When the tumor size reached about-4-5 mm in diameter, mice received intratumoral injection of VV-PDL1scFv-Fc/GM or VV-PDL1scFv, VV-GM, VV-RFP (1X 10)6pfu/mouse) or PBS twice. The size of the tumor was monitored every 3-4 days. The results are shown in FIG. 2. FIG. 2 shows that recombinant oncolytic virus VV-PDL1scFv-Fc/GM can rapidly destroy PD-L1+H226 human lung cancer cells. Recombinant oncolytic viruses VV-PDL1scFv, VV-PDL1scFv-Fc, and VV-GM are capable of inhibiting tumor growth, but take longer to destroy PD-L1+H226. The experimental result shows that the recombinant oncolytic virus VV-PDL1scFv-Fc/GM has more enhanced anti-local tumor activity.
Example 5 oncolytic vaccinia virus VV-PDL1scFv-Fc/GM has a more potent ability to elicit a systemic anti-tumor response
The inventors have examined that local intratumoral injection of VV-PDL1scFv-Fc/GM is able to induce a systemic anti-tumor response. PD-L1 with a diameter of about 4-5mm at the right rib+Hu-CD34 of H226 tumor+NSG-SGM3 mice were locally intratumorally injected with VV-PDL1scFv-Fc/GM or VV-PDL1scFv-Fc, VV-PDL1scFv-Fc, VV-GM-CSF, VV-RFP (1X 10)6pfu/mouse), injected twice at the indicated times. One week later, intratumorally injected mice were injected subcutaneously at the left costal region with PD-L1+H226 tumor cell (5x 10)5Mice). Tumor size at the left and right ribs was monitored every 3-4 days. The results are shown in FIG. 3. The results in FIG. 3 show that intratumoral injection of recombinant oncolytic viruses VV-PDL1scFv-Fc/GM is able to resist the growth of distant (left flank) tumors, whereas intratumoral injection of recombinant oncolytic vaccinia viruses VV-PDL1scFv-Fc and VV-GM only weakly inhibits the growth of these distant, threatened tumors. This result demonstrates that the recombinant oncolytic vaccinia virus VV-PDL1scFv-Fc/GM-CSF is more potent in inducing systemic anti-tumor activity.
Example 6 infection of tumor cells by recombinant vaccinia virus VV-PDL1scFv-Fc/GM expression of anti-PDL 1scFv-Fc fusion antibody inhibits the interaction of PD-1 with PD-L1
In this example, human lung carcinoma cells H226 were infected with recombinant oncolytic vaccinia viruses VV-PDL1scFv-Fc/GM, VV-PDL1scFv, VV-PDL1scFv-Fc, VV-GM or VV-RFP (MOI0.5), and after 48 hours, culture supernatants were harvested for PD-1: PD-L1 inhibition experiments. 100 ng/well of PD-L1 protein (Aerobiosystems, Boston, Mass.) was used to coat 96-well ELISA plates. 10ng of biotinylated PD1(Aerobiosystems, Boston, Mass.) was mixed with 100uL of each CAR-T supernatant or 50ng of commercially available anti-hPD-L1 (Aerobiosystems, Boston, Mass.), supplemented to 200uL, and the mixture was added to coated 96-well plates. The 96-well plate to which the mixture solution was added was left at room temperature for 2 hours, after which washing was sufficiently performed. Diluted streptavidin-HRP was added to the well plate and allowed to stand at room temperature for 1 hour while shaking slowly. After 1 hour, the well plate was washed 6-times and 100 μ L TMB HRP substrate was added when the blue color was positiveWhen present in the control wells, 100uL of 1N sulfamic acid was added to stop the reaction. The OD of the well plate at 450nm was measured. Inhibitory Activity (%) - (OD)450of Mock-OD450of sample)/(OD450ofMock-OD450of background)x 100％.
The results of the experiment are shown in FIG. 4. The results in FIG. 4 show that the recombinant oncolytic vaccinia virus VV-PDL1scFv-Fc/GM or VV-PDL1scFv-Fc infected tumor cell cultures significantly inhibited PD-1: PD-L1 effects, whereas oncolytic vaccinia virus VV-GM-CSF or VV-RFP infected tumor cell cultures did not inhibit PD-1: PD-L1 effects (P < 0.01; VV-PDL1scFv-Fc/GM vs. VV-GM or VV-RFP). The culture solution of tumor cells infected by the oncolytic vaccinia virus VV-PDL1scFv has a certain effect of inhibiting PD-1: PD-L1, but is obviously weaker than the culture solution of tumor cells infected by the oncolytic vaccinia virus VV-PDL1scFv-Fc or VV-PDL1scFv-Fc/GM (P < 0.01; VV-PDL1scFv-Fc or VV-PDL1scFv-Fc/GM vs. This indicates that recombinant oncolytic vaccinia virus VV-PDL1scFv-Fc/GM infected tumor cells can secrete IgG Fc fusion anti-PD-L1 antibody containing amino acid mutation and have strong effect of inhibiting PD-1: PD-L1.
Example 7 in situ intratumoral injection of recombinant vaccinia virus VV-PDL1scFv-Fc/GM generates PDL1scFv-Fc fusion antibodies against PD-L1, anti-PDL 1scFv-Fc fusion antibodies in mouse serum inhibiting the interaction of PD-1 with PD-L1
In this embodiment, Hu-CD34+NSG-SGM3 mice were injected subcutaneously with 5x105PD-L1+H226 tumor cells, which were then divided into several groups (5-8 mice/group). When the tumor size reaches about-100 mm3When mice received intratumoral injection of VV-PDL1scFv-Fc/GM or VV-PDL1scFv-Fc, VV-PDL1scFv, VV-GM-CSF, VV-RFP (1X 10)6pfu/mouse), or PBS twice (2 consecutive days). After 2 days, mouse sera were collected and separated and mixed equally from 4 mice per group. A96-well ELISA plate was coated with 100 ng/well of commercially available PD-L1 protein (Aerobiosystems, Boston, Mass.). 10ng of biotinylated PD1(Aerobiosystems, Boston, Mass.) was mixed with 100uL of each CAR-T supernatant or 50ng of commercially available anti-hPD-L1 (Aerobiosystems, Boston, Mass.), made up to 200uL after mixing, and added to the coated plates. BoardIncubate at room temperature for 2 hours and wash well. Diluted streptavidin-HRP was then added to the plate and incubated at room temperature for 1 hour with slow shaking. Thereafter, the plate was washed 5 times and 100uL of TMB HRP substrate was added, and the reaction was stopped by adding 100uL of 1N sulfuric acid when the positive control wells turned blue. The OD of the plate at 450nm was measured. The inhibition ratio (The inhibition activity (%))) (OD450 of Mock-OD450of sample)/(OD450 of Mock-OD450of background) x 100%.
The results of the experiment are shown in FIG. 5. The results in FIG. 5 show that serum from mice intratumorally injected with recombinant oncolytic vaccinia virus VV-PDL1scFv-Fc/GM significantly inhibited PD-1: PD-L1 action, while serum from mice intratumorally injected with oncolytic vaccinia virus VV-GM, or VV-RFP, did not inhibit PD-1: PD-L1 action (. about.P < 0.01; VV-PDL1scFv-Fc/GM-CSF vs. VV-GM or VV-PDL1 scFv). The serum of an oncolytic vaccinia virus VV-PDL1scFv intratumorally injected mouse has a certain effect of inhibiting PD-1: PD-L1, but is obviously weaker than the serum of an oncolytic vaccinia virus VV-PDL1scFv-Fc or VV-PDL1scFv-Fc/GM intratumorally injected mouse to inhibit PD-1: PD-L1 (P < 0.01; VV-PDL1scFv-Fc or VV-PDL1scFv-Fc/GM vs.VV-PDL1 scFv). This indicates that the recombinant oncolytic vaccinia virus VV-PDL1scFv-Fc/GM can secrete IgGFc fusion anti-PD-L1 antibody containing amino acid mutation in blood by intratumoral injection, and has strong in vivo inhibition effect on PD-1: PD-L1. Although the recombinant oncolytic vaccinia virus VV-PDL1scFv can secrete anti-PD-L1 single-chain antibody by intratumoral injection, the concentration for effectively inhibiting PD-1: PD-L1 in vivo cannot be reached probably due to short half-life in blood.
Example 8 oncolytic vaccinia virus VV-PDL1scFv-Fc/GM has a greater ability to elicit a systemic anti-tumor response than oncolytic vaccinia virus VV-PD1Fc/GM (co-expressing PD1-IgG Fc fusion protein and GM-CSF)
The inventors have examined that local intratumoral injection of VV-PDL1scFv-Fc/GM is able to induce a systemic anti-tumor response. PD-L1 having a diameter size of about-4-5 mm at the right rib+Hu-CD34 of H226 tumor+NSG-SGM3 mice were locally intratumorally injected to co-express PDL1scFv-Fc fusion antibodies and GM-CSF oncolytic vaccinia virus (VV-PDL1scFv-Fc/GM) or co-express PD1-Fc fusion proteins and GM-CSF oncolytic vaccinia virus (VV-PD1-Fc/GM) (1x 10)6pfu/mouse) And injected twice within the indicated time. One week later, intratumorally injected mice were injected subcutaneously at the left costal region with PD-L1+H226 tumor cell (5x 10)5Mice). Tumor size at the left and right ribs was monitored every 3-4 days. The results are shown in FIG. 6. The results in FIG. 6 show that intratumoral injection of recombinant oncolytic virus VV-PDL1scFv-Fc/GM is more effective against the growth of distant (left-flank) tumors than oncolytic vaccinia virus VV-PD1 Fc/GM. This result demonstrates that recombinant oncolytic vaccinia virus VV-PDL1scFv-Fc/GM induces systemic anti-tumor activity more efficiently than oncolytic vaccinia virus VV-PD1 Fc/GM.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Life sequence Co
<120> recombinant oncolytic virus expressing anti-immune checkpoint fusion antibody and immunostimulatory molecule
<130>PIDC3172016
<160>10
<170>PatentIn version 3.3
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Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
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20 25 30
Leu Ala Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Thr
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Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Val Gln Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Val
65 70 75 80
Asp Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Asn Ser Leu Glu Glu Glu Asp Ala Ala Met Tyr
100 105 110
Phe Cys Gln Gln Ser Arg Arg Val Pro Tyr Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser
130 135 140
Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val Gln Ser Gly Ala
145 150 155 160
Glu Val Lys Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser
165 170 175
Gly Tyr Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Ala Pro
180 185 190
Gly Gln Arg Leu Glu Trp Ile Gly Tyr Val Asn Pro Phe Asn Asp Gly
195 200 205
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210 215 220
Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
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Gly Gln Gly Thr Leu Val Thr Val Ser Ser
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20 25 30
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35 40 45
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
50 55 60
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
65 70 75 80
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
85 90 95
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
100 105 110
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
115 120 125
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
130 135 140
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
145 150 155 160
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
165 170 175
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
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Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His
195 200 205
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210215 220
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<223> amino acid sequence of anti-PD-L1 fusion antibody
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Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
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20 25 30
Leu Ala Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Thr
35 40 45
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Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Val
65 70 75 80
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Phe Cys Gln Gln Ser Arg Arg Val Pro Tyr Thr Phe Gly Gln Gly Thr
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Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val Gln Ser Gly Ala
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Gly Gln Arg Leu Glu Trp Ile Gly Tyr Val Asn Pro Phe Asn Asp Gly
195 200 205
Thr Lys Tyr Asn Glu Met Phe Lys Gly Arg Ala Thr Leu Thr Ser Asp
210 215 220
Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
225 230 235 240
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Ala Trp Gly Tyr Pro Trp
245 250 255
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Asp Lys Thr
260 265 270
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
275 280 285
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg
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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
305 310 315 320
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
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Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
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Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
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Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
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Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
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Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala
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Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
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<210>5
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<223> nucleotide sequence of first nucleic acid molecule
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atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60
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gccaccctct cctgcagagc cactgaaagt gttgaatact atggcacaag tttagtgcag 180
tggtaccaac agaaaccagg acagccaccc aaactcctca tctatgctgc atccagcgta 240
gattctgggg tcccttccag gtttagtggc agtgggtctg ggacagactt caccctcacc 300
atcaattctc tggaggagga ggatgctgca atgtatttct gtcagcaaag taggagggtt 360
ccgtacacgt tcggacaggg gaccaagctg gagataaaag gctccacctc tggatccggc 420
aagcccggat ctggcgaggg atccaccaag ggcgaggtcc agctggtgca gtctggagct 480
gaggtgaaaa agcctggggc ttcagtgaag atgtcctgca aggcttctgg atacacattc 540
actagctatg ttatgcactg ggtgaagcag gcccctgggc agcgccttga gtggattgga 600
tatgttaatc ctttcaatga tggtactaag tacaatgaga tgttcaaagg cagggccaca 660
ctgacttcag acaaatccac cagcacagcc tacatggagc tcagcagcct gaggtctgag 720
gacactgcgg tctattactg tgcaagacag gcttggggtt acccctgggg ccaagggact 780
ctggtcactg tctcttctgc ggccgcagac aaaactcaca catgcccacc gtgcccagca 840
cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggaccaactg 900
atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 960
gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1020
cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1080
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atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1200
cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1260
ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1320
aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1380
gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgct gcatgaggct 1440
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<210>6
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<223> nucleotide sequence of first nucleic acid molecule
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atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60
atcccagaaa tcgtcctgac tcaaagccca gcgactttgt ccctctcccc aggcgagcgg 120
gccacgctgt cttgcagggc caccgagtct gtggagtatt atggtacaag cctggtgcaa 180
tggtatcaac aaaagcctgg tcagccgcct aaactcctca tctacgctgc ctcttcagta 240
gattcaggcg ttccatctcg attctctggc agcggaagcg gaacagactt caccctcacg 300
attaatagcc tggaagcaga agacgctgca acctattatt gccagcagtc aagaagggtt 360
ccatatacgt ttggcggcgg aaccaaactt gagataaaag gctccacctc tggatccggc 420
aagcccggat ctggcgaggg atccaccaag ggcgaagtac aactggtgca gagtggagcg 480
gaggttaaga aaccgggggc aacagtgaaa atatcttgca aggtttctgg ttatacgttt 540
acgagttatg tcatgcattg ggtccgacag gcccctgggc agggcctgga atggatgggt 600
tatgtgaacc ccttcaatga tgggacgaaa tacaatgaaa tgttcaaagg tagagtgaca 660
attacacggg atacgtccgc aagcacggca tatatggagc ttagttcact ccgcagtgaa 720
gatactgctg tctactattg tgcgagacaa gcctgggggt atccatgggg gcaaggcacg 780
cttgtaacgg tgagtgcggc ggccgcagac aaaactcaca catgcccacc gtgcccagca 840
cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggaccaactg 900
atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 960
gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1020
cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1080
gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1140
atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1200
cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1260
ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1320
aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1380
gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgct gcatgaggct 1440
ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa 1488
<210>7
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<220>
<223> nucleotide sequence of first nucleic acid molecule
<400>7
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60
atcccagata tagtgctcac acaaagcccg gcctctctcg ccgtaagtct ggggcaacga 120
gctactatca gttgccgcgc tacggagagc gtggaatact atggaacgag tctggtgcag 180
tggtatcagc aaaaaccggg gcaaccaccg aaactgctga tatacgccgc ttcatctgtt 240
gactctggag tgccagcaag gtttagtggt agcggctctg gcactgactt ctcacttaca 300
atacatcctg tggaggagga tgacatagcc atgtacttct gtcagcaatc caggcgagtc 360
ccatacacgt ttggtggggg gacgaagttg gaaataaaag gctccacctc tggatccggc 420
aagcccggat ctggcgaggg atccaccaag ggcgaagttc agttgcaaca gtctggtcca 480
gagcttgtta aaccgggggc aagcgttaaa atgagctgca aagcctcagg gtacaccttt 540
acaagttatg taatgcactg ggttaaacag aaacccggcc agggtctgga gtggattggc 600
tacgtcaacc cctttaatga cggtaccaag tacaatgaga tgttcaaggg caaagccaca 660
cttacgtccg ataagagtag tagcaccgcc tacatggaac tttctagctt gacttccgaa 720
gacagtgcat ggtactattg tgcgagacaa gcgtggggtt atccttgggg ccaaggtact 780
cttgtgacgg tatcagcggc ggccgcagac aaaactcaca catgcccacc gtgcccagca 840
cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggaccaactg 900
atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 960
gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1020
cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1080
gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1140
atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1200
cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1260
ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1320
aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1380
gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgct gcatgaggct 1440
ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa 1488
<210>8
<211>2231
<212>DNA
<213>Artificial
<220>
<223> sequence of PDL1scFv (I) -Fc-pSel-p7.5-GMCSF insert DNA
<400>8
tcatttaccc ggagacaggg agaggctctt ctgcgtgtag tggttgtgca gagcctcatg 60
cagcacggag catgagaaga cgttcccctg ctgccacctg ctcttgtcca cggtgagctt 120
gctgtagagg aagaaggagc cgtcggagtc cagcacggga ggcgtggtct tgtagttgtt 180
ctccggctgc ccattgctct cccactccac ggcgatgtcg ctgggataga agcctttgac 240
caggcaggtc aggctgacct ggttcttggt catctcctcc cgggatgggg gcagggtgta 300
cacctgtggt tctcggggct gccctttggc tttggagatg gttttctcga tgggggctgg 360
gagggctttg ttggagacct tgcacttgta ctccttgcca ttcagccagt cctggtgcag 420
gacggtgagg acgctgacca cacggtacgt gctgttgtac tgctcctccc gcggctttgt 480
cttggcatta tgcacctcca cgccgtccac gtaccagttg aacttgacct cagggtcttc 540
gtggctcacg tccaccacca cgcatgtgac ctcaggggtc cgggagatca tcagttggtc 600
cttgggtttt ggggggaaga ggaagactga cggtcccccc aggagttcag gtgctgggca 660
cggtgggcat gtgtgagttt tgtctgcggc cgcagaagag acagtgacca gagtcccttg 720
gccccagggg taaccccaag cctgtcttgc acagtaatag accgcagtgt cctcagacct 780
caggctgctg agctccatgt aggctgtgct ggtggatttg tctgaagtca gtgtggccct 840
gcctttgaac atctcattgt acttagtacc atcattgaaa ggattaacat atccaatcca 900
ctcaaggcgc tgcccagggg cctgcttcac ccagtgcata acatagctag tgaatgtgta 960
tccagaagcc ttgcaggaca tcttcactga agccccaggc tttttcacct cagctccaga 1020
ctgcaccagc tggacctcgc ccttggtgga tccctcgcca gatccgggct tgccggatcc 1080
agaggtggag ccttttatct ccagcttggt cccctgtccg aacgtgtacg gaaccctcct 1140
actttgctga cagaaataca ttgcagcatc ctcctcctcc agagaattga tggtgagggt 1200
gaagtctgtc ccagacccac tgccactaaa cctggaaggg accccagaat ctacgctgga 1260
tgcagcatag atgaggagtt tgggtggctg tcctggtttc tgttggtacc actgcactaa 1320
acttgtgcca tagtattcaa cactttcagt ggctctgcag gagagggtgg ctctctcccc 1380
gggagacaga gccaaagaag ctggagattg ggtgagcaca atgtctggga tcaggaggaa 1440
tgctgggtgt ggtaactcac agagcagaag gcttgtcacc aggagaagca ttctagaatc 1500
tagatgcatt cgcgaggtac cgtcgacttc gagcttattt atattccaaa aaaaaaaaat 1560
aaaatttcaa tttttaagct ttcactaatt ccaaacccac ccgcttttta tagtaagttt 1620
ttcacccata aataataaat acaataatta atttctcgta aaagtagaaa atatattcta 1680
atttattgca cggtaaggaa gtagatcata acgatctcta taatctcgcg caacctattt 1740
tcccctcgaa cactttttaa gccgtagata aacaggctgg gacacttcac ctcgagatgt 1800
ggctgcagag cctgctgctc ttgggcactg tggcctgcag catctctgca cccgcccgct 1860
cgcccagccc cagcacgcag ccctgggagc atgtgaatgc catccaggag gcccggcgtc 1920
tcctgaacct gagtagagac actgctgctg agatgaatga aacagtagaa gtcatctcag 1980
aaatgtttga cctccaggag ccgacctgcc tacagacccg cctggagctg tacaagcagg 2040
gcctgcgggg cagcctcacc aagctcaagg gccccttgac catgatggcc agccactaca 2100
agcagcactg ccctccaacc ccggaaactt cctgtgcaac ccagattatc acctttgaaa 2160
gtttcaaaga gaacctgaag gactttctgc ttgtcatccc ctttgactgc tgggagccag 2220
tccaggagtg a 2231
<210>9
<211>2231
<212>DNA
<213>Artificial
<220>
<223> nucleotide sequence of PDL1scFv (II) -Fc-pSel-p7.5-GMCSF insert DNA
<400>9
tcatttaccc ggagacaggg agaggctctt ctgcgtgtag tggttgtgca gagcctcatg 60
cagcacggag catgagaaga cgttcccctg ctgccacctg ctcttgtcca cggtgagctt 120
gctgtagagg aagaaggagc cgtcggagtc cagcacggga ggcgtggtct tgtagttgtt 180
ctccggctgc ccattgctct cccactccac ggcgatgtcg ctgggataga agcctttgac 240
caggcaggtc aggctgacct ggttcttggt catctcctcc cgggatgggg gcagggtgta 300
cacctgtggt tctcggggct gccctttggc tttggagatg gttttctcga tgggggctgg 360
gagggctttg ttggagacct tgcacttgta ctccttgcca ttcagccagt cctggtgcag 420
gacggtgagg acgctgacca cacggtacgt gctgttgtac tgctcctccc gcggctttgt 480
cttggcatta tgcacctcca cgccgtccac gtaccagttg aacttgacct cagggtcttc 540
gtggctcacg tccaccacca cgcatgtgac ctcaggggtc cgggagatca tcagttggtc 600
cttgggtttt ggggggaaga ggaagactga cggtcccccc aggagttcag gtgctgggca 660
cggtgggcat gtgtgagttt tgtctgcggc cgctacgaag aggaccactg ttcggaagac 720
gagacactca atggtgtggg tcgtaaggag gactagggtc tttagcagga ctgagtttcg 780
ggtcgctgaa acagggagag gggtccgctc gcccggtgcg acagaacgtc ccggtggctc 840
agacacctca taataccatg ttcggaccac gttaccatag ttgttttcgg accagtcggc 900
ggatttgagg agtagatgcg acggagaagt catctaagtc cgcaaggtag agctaagaga 960
ccgtcgcctt cgccttgtct gaagtgggag tgctaattat cggaccttcg tcttctgcga 1020
cgttggataa taacggtcgt cagttcttcc caaggtatat gcaaaccgcc gccttggttt 1080
gaactctatt ttccgaggtg gagacctagg ccgttcgggc ctagaccgct ccctaggtgg 1140
ttcccgcttc atgttgacca cgtctcacct cgcctccaat tctttggccc ccgttgtcac 1200
ttttatagaa cgttccaaag accaatatgc aaatgctcaa tacagtacgt aacccaggct 1260
gtccggggac ccgtcccgga ccttacctac ccaatacact tggggaagtt actaccctgc 1320
tttatgttac tttacaagtt tccatctcac tgttaatgtg ccctatgcag gcgttcgtgc 1380
cgtatatacc tcgaatcaag tgaggcgtca cttctatgac gacagatgat aacacgctct 1440
gttcggaccc ccataggtac ccccgttccg tgcgaacatt gccactcacg ctctagaatc 1500
tagatgcatt cgcgaggtac cgtcgacttc gagcttattt atattccaaa aaaaaaaaat 1560
aaaatttcaa tttttaagct ttcactaatt ccaaacccac ccgcttttta tagtaagttt 1620
ttcacccata aataataaat acaataatta atttctcgta aaagtagaaa atatattcta 1680
atttattgca cggtaaggaa gtagatcata acgatctcta taatctcgcg caacctattt 1740
tcccctcgaa cactttttaa gccgtagata aacaggctgg gacacttcac ctcgagatgt 1800
ggctgcagag cctgctgctc ttgggcactg tggcctgcag catctctgca cccgcccgct 1860
cgcccagccc cagcacgcag ccctgggagc atgtgaatgc catccaggag gcccggcgtc 1920
tcctgaacct gagtagagac actgctgctg agatgaatga aacagtagaa gtcatctcag 1980
aaatgtttga cctccaggag ccgacctgcc tacagacccg cctggagctg tacaagcagg 2040
gcctgcgggg cagcctcacc aagctcaagg gccccttgac catgatggcc agccactaca 2100
agcagcactg ccctccaacc ccggaaactt cctgtgcaac ccagattatc acctttgaaa 2160
gtttcaaaga gaacctgaag gactttctgc ttgtcatccc ctttgactgc tgggagccag 2220
tccaggagtg a 2231
<210>10
<211>2231
<212>DNA
<213>Artificial
<220>
<223> nucleotide sequence of PDL1scFv (III) -Fc-pSel-p7.5-GMCSF insert DNA
<400>10
tcatttaccc ggagacaggg agaggctctt ctgcgtgtag tggttgtgca gagcctcatg 60
cagcacggag catgagaaga cgttcccctg ctgccacctg ctcttgtcca cggtgagctt 120
gctgtagagg aagaaggagc cgtcggagtc cagcacggga ggcgtggtct tgtagttgtt 180
ctccggctgc ccattgctct cccactccac ggcgatgtcg ctgggataga agcctttgac 240
caggcaggtc aggctgacct ggttcttggt catctcctcc cgggatgggg gcagggtgta 300
cacctgtggt tctcggggct gccctttggc tttggagatg gttttctcga tgggggctgg 360
gagggctttg ttggagacct tgcacttgta ctccttgcca ttcagccagt cctggtgcag 420
gacggtgagg acgctgacca cacggtacgt gctgttgtac tgctcctccc gcggctttgt 480
cttggcatta tgcacctcca cgccgtccac gtaccagttg aacttgacct cagggtcttc 540
gtggctcacg tccaccacca cgcatgtgac ctcaggggtc cgggagatca tcagttggtc 600
cttgggtttt ggggggaaga ggaagactga cggtcccccc aggagttcag gtgctgggca 660
cggtgggcat gtgtgagttt tgtctgcggc cgctacgaag aggaccactg ttcggaagac 720
gagacactca atggtgtggg tcgtaaggag gactagggtc tatatcacga gtgtgtttcg 780
ggccggagag agcggcattc agaccccgtt gctcgatgat agtcaacggc gcgatgcctc 840
tcgcacctta tgataccttg ctcagaccac gtcaccatag tcgtttttgg ccccgttggt 900
ggctttgacg actatatgcg gcgaagtaga caactgagac ctcacggtcg ttccaaatca 960
ccatcgccga gaccgtgact gaagagtgaa tgttatgtag gacacctcct cctactgtat 1020
cggtacatga agacagtcgt taggtccgct cagggtatgt gcaaaccacc cccctgcttc 1080
aacctttatt ttccgaggtg gagacctagg ccgttcgggc ctagaccgct ccctaggtgg 1140
ttcccgcttc aagtcaacgt tgtcagacca ggtctcgaac aatttggccc ccgttcgcaa 1200
ttttactcga cgtttcggag tcccatgtgg aaatgttcaatacattacgt gacccaattt 1260
gtctttgggc cggtcccaga cctcacctaa ccgatgcagt tggggaaatt actgccatgg 1320
ttcatgttac tctacaagtt cccgtttcgg tgtgaatgca ggctattctc atcatcgtgg 1380
cggatgtacc ttgaaagatc gaactgaagg cttctgtcac gtaccatgat aacacgctct 1440
gttcgcaccc caataggaac cccggttcca tgagaacact gccatagtcg ctctagaatc 1500
tagatgcatt cgcgaggtac cgtcgacttc gagcttattt atattccaaa aaaaaaaaat 1560
aaaatttcaa tttttaagct ttcactaatt ccaaacccac ccgcttttta tagtaagttt 1620
ttcacccata aataataaat acaataatta atttctcgta aaagtagaaa atatattcta 1680
atttattgca cggtaaggaa gtagatcata acgatctcta taatctcgcg caacctattt 1740
tcccctcgaa cactttttaa gccgtagata aacaggctgg gacacttcac ctcgagatgt 1800
ggctgcagag cctgctgctc ttgggcactg tggcctgcag catctctgca cccgcccgct 1860
cgcccagccc cagcacgcag ccctgggagc atgtgaatgc catccaggag gcccggcgtc 1920
tcctgaacct gagtagagac actgctgctg agatgaatga aacagtagaa gtcatctcag 1980
aaatgtttga cctccaggag ccgacctgcc tacagacccg cctggagctg tacaagcagg 2040
gcctgcgggg cagcctcacc aagctcaagg gccccttgac catgatggcc agccactaca 2100
agcagcactg ccctccaacc ccggaaactt cctgtgcaac ccagattatc acctttgaaa 2160
gtttcaaaga gaacctgaag gactttctgc ttgtcatccc ctttgactgc tgggagccag 2220
tccaggagtg a 2231

Claims (12)

1. An oncolytic viral construct comprising:
a first nucleic acid molecule encoding a secreted anti-PD-L1 fusion antibody molecule for specifically inhibiting PD-L1, said anti-PD-L1 fusion antibody comprising: an anti-PD-L1 single chain antibody, an IgG1 hinge region, and an IgG1Fc region, said anti-PD-L1 single chain antibody being linked to said IgG1Fc region by an IgG1 hinge region, said IgG1Fc region having the T250Q and M248L amino acid mutations; and
a second nucleic acid molecule encoding an immunostimulatory molecule.
2. The oncolytic virus construct of claim 1, wherein the anti-PD-L1 single chain antibody has the amino acid sequence shown in seq id No. 1;
optionally, the IgG1 hinge region has the amino acid sequence shown in SEQ ID NO. 2;
optionally, the IgG1Fc region has the amino acid sequence shown in SEQ ID NO. 3;
optionally, the anti-PD-L1 fusion antibody has the amino acid sequence shown in SEQ ID NO. 4.
3. The oncolytic viral construct of claim 1, wherein the first nucleic acid molecule has a nucleotide sequence shown in SEQ id nos 5-7.
4. The oncolytic viral construct according to claim 1, wherein the immunostimulatory molecule comprises at least one member selected from the group consisting of human GM-CSF, Flt-3L, IL-2, IL-12, IL-15, IL-18, IL-24, TNF, IFN α, IFN β, IFN γ, MIP-I β, MCP-I, RANTES;
preferably, the immunostimulatory molecule is GM-CSF.
5. The oncolytic viral construct of claim 1, wherein the construct is a vaccinia virus that is a WR strain vaccinia virus in which thymidine kinase gene and growth factor gene are inactivated;
optionally, the first nucleic acid molecule and the second nucleic acid molecule are disposed after the 240 base and before the 308 base of the thymidine kinase gene;
optionally, the construct further comprises:
a first promoter operably linked to the first nucleic acid molecule;
a second promoter operably linked to the second nucleic acid molecule;
optionally, the first promoter and the second promoter are each independently selected from at least one of pSel, p 7.5.
6. An oncolytic viral construct carrying the sequence of SEQ ID NO: 8-10.
7. An oncolytic virus carrying the oncolytic virus construct of any one of claims 1 to 6.
8. The oncolytic virus of claim 7, wherein the virus is an intracellular maturation virus, an intracellular packaging virus, a cell-associated packaging virus, or an extracellular packaging virus, preferably the virus is an extracellular packaging virus or an intracellular maturation virus, more preferably the virus is an extracellular packaging virus;
optionally, the virus is a vaccinia virus, herpes simplex virus, adenovirus, vesicular stomatitis virus, newcastle disease virus, retrovirus, reovirus, measles virus, Sinbis virus, or influenza virus, preferably, the virus is a vaccinia virus.
9. A recombinant cell obtained by introducing the oncolytic virus construct according to any one of claims 1 to 6 into a recipient cell or by transfecting the oncolytic virus according to any one of claims 7 to 8 into a recipient cell.
10. The recombinant cell of claim 9, wherein the recipient cell comprises at least one selected from an immune cell or a tumor cell.
11. A pharmaceutical composition comprising the oncolytic virus of any one of claims 7 to 8 and a pharmaceutically acceptable carrier.
12. Use of an oncolytic viral construct according to any one of claims 1 to 6, an oncolytic virus according to any one of claims 7 to 8 or a recombinant cell according to any one of claims 9 to 10 for the manufacture of a medicament for treating or preventing cancer, inhibiting malignant cell proliferation in a mammal.
CN201910312308.2A 2019-04-18 2019-04-18 Recombinant oncolytic virus expression anti-immune checkpoint fusion antibody and immunostimulatory molecule Pending CN111826395A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022122026A1 (en) * 2020-12-11 2022-06-16 Genesail (Shanghai) Co., Ltd. A modified oncolytic virus, composition and use thereof
WO2023226942A1 (en) * 2022-05-23 2023-11-30 上海药明康德新药开发有限公司 Recombinant hsv-1 vector for encoding immunostimulatory factor and anti-immune checkpoint antibody

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102264762A (en) * 2008-09-26 2011-11-30 达纳-法伯癌症研究公司 Human anti-pd-1, pd-l1, and pd-l2 antibodies and uses therefor
US20160311903A1 (en) * 2015-03-13 2016-10-27 Cytomx Therapeutics, Inc. Anti-pdl1 antibodies, activatable anti-pdl1 antibodies, and methods of use thereof
CN108642070A (en) * 2018-04-11 2018-10-12 沈阳金石生物制药有限公司 Recombined human Fc antibody of specific inducing apoptosis of tumour cell and preparation method thereof, purposes
CN108728488A (en) * 2017-04-19 2018-11-02 生命序有限公司 Oncolytic virus construct, oncolytic virus and its application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102264762A (en) * 2008-09-26 2011-11-30 达纳-法伯癌症研究公司 Human anti-pd-1, pd-l1, and pd-l2 antibodies and uses therefor
US20160311903A1 (en) * 2015-03-13 2016-10-27 Cytomx Therapeutics, Inc. Anti-pdl1 antibodies, activatable anti-pdl1 antibodies, and methods of use thereof
CN108728488A (en) * 2017-04-19 2018-11-02 生命序有限公司 Oncolytic virus construct, oncolytic virus and its application
CN108642070A (en) * 2018-04-11 2018-10-12 沈阳金石生物制药有限公司 Recombined human Fc antibody of specific inducing apoptosis of tumour cell and preparation method thereof, purposes

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022122026A1 (en) * 2020-12-11 2022-06-16 Genesail (Shanghai) Co., Ltd. A modified oncolytic virus, composition and use thereof
WO2023226942A1 (en) * 2022-05-23 2023-11-30 上海药明康德新药开发有限公司 Recombinant hsv-1 vector for encoding immunostimulatory factor and anti-immune checkpoint antibody

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