WO2022037020A1 - 构建体、溶瘤病毒及其应用 - Google Patents

构建体、溶瘤病毒及其应用 Download PDF

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WO2022037020A1
WO2022037020A1 PCT/CN2021/074592 CN2021074592W WO2022037020A1 WO 2022037020 A1 WO2022037020 A1 WO 2022037020A1 CN 2021074592 W CN2021074592 W CN 2021074592W WO 2022037020 A1 WO2022037020 A1 WO 2022037020A1
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gene
antibody
construct
virus
scfv
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张旭辉
颜青青
何亮亮
冯翠娟
陈小锋
李文佳
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广东东阳光药业有限公司
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
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    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
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    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the fields of biotechnology and gene therapy, in particular, the present invention relates to a construct, an oncolytic virus and its pharmaceutical use.
  • Oncolytic virus has good anti-tumor effect. It selectively replicates on tumor cells and directly lyses and kills tumor cells. The released tumor-related antigens activate the body's anti-tumor immunity through antigen presentation, and achieve the goal of inhibiting tumor growth or even tumor regression. Effect. Antitumor immune responses elicited by infected tumor cell debris can be amplified by the addition of immunostimulatory factors. In order to further improve the therapeutic effect of oncolytic viruses, the virus can be replicated on tumor cells and at the same time express immune checkpoint regulators or inhibitors, which can block the inhibitory pathway of tumor cells on the immune system.
  • Inserting the PD1 antibody gene into the oncolytic virus to construct a new oncolytic virus will help to find new tumor treatment methods and genetic vaccines.
  • the present application aims to solve one of the above technical problems in the background art at least to a certain extent.
  • the present invention proposes a construct.
  • the vector of the construct is derived from HSV
  • the construct carries an immune checkpoint regulator gene coding frame
  • the immune checkpoint regulator gene coding frame is set at the position of the UL23 gene.
  • the above-mentioned construct may further include at least one of the following additional technical features:
  • ICP47 and double copy ICP34.5 of the construct are knocked out
  • Knockout of the replication non-essential gene ICP47 in the HSV genome can improve the expression of MHC-I on the surface of virus-infected tumor cells and the ability to present cellular antigens; knockout of double-copy ICP34.5 can make herpes simplex virus (HSV) in The replication in normal cells is limited, and it selectively replicates in tumor cells, thereby improving the drug safety of HSV virus.
  • HSV herpes simplex virus
  • the coding frame of the immune checkpoint regulator gene is set at the position between the 387th and 521st nucleotides of the UL23 gene sequence.
  • the UL23 gene sequence coding described in this application is coded with the first nucleotide of the UL23 gene initiation codon as the first nucleotide sequence.
  • the coding frame of the immune checkpoint regulator gene is set between the 387-521 nucleotides of the UL23 gene sequence by knocking out the 388-520 nucleotides of the UL23 gene sequence, and then Obtained by inserting the immune checkpoint regulator gene coding frame between nucleotides 387-521 of the UL23 gene sequence.
  • the immune checkpoint regulator gene coding frame is operably linked to a promoter comprising a sequence selected from the group consisting of CMV, CAG, EF1 ⁇ , Rous Sarcoma Virus Long Terminal Repeat (RSV LTR), At least one of Metallothionein I (MTI).
  • a promoter comprising a sequence selected from the group consisting of CMV, CAG, EF1 ⁇ , Rous Sarcoma Virus Long Terminal Repeat (RSV LTR), At least one of Metallothionein I (MTI).
  • the immune checkpoint regulator is selected from at least one of cytokines, chemokines, cytotoxic peptides, immunomodulatory polypeptides, soluble domains of natural receptors, RNAi, antisense molecules, antibodies or protein scaffolds.
  • the cytokine is selected from the group consisting of interleukins (eg, IL-2, IL-7, IL-12, and IL-15), interferons (eg, IFN ⁇ , IFN ⁇ , IFN ⁇ ), tumor necrosis factors (eg, TNF ⁇ , TNF ⁇ ). ), colony stimulating factors (eg, GM-CSF), and any combination thereof.
  • interleukins eg, IL-2, IL-7, IL-12, and IL-15
  • interferons eg, IFN ⁇ , IFN ⁇ , IFN ⁇
  • tumor necrosis factors eg, TNF ⁇ , TNF ⁇
  • colony stimulating factors eg, GM-CSF
  • the chemokine is selected from the group consisting of CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1, and any combination thereof.
  • the cytotoxic peptide is selected from the group consisting of thymidine kinase TK (TK/GCV), TRAIL, FasL, and any combination thereof.
  • the immunomodulatory polypeptide is selected from the group consisting of CD40L, OX40L, inducible costimulatory molecule (ICOS), FTL3L, LIGHT, CD137L, CD70, 4-1BB, GITR, CD28, and any combination thereof.
  • CD40L CD40L
  • OX40L inducible costimulatory molecule
  • ICOS inducible costimulatory molecule
  • FTL3L LIGHT
  • CD137L CD70
  • 4-1BB 4-1BB
  • GITR GITR
  • CD28 any combination thereof.
  • the antibody is selected from anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-Tim-3 antibody, anti-Lag-3 antibody, anti-CD137 antibody , anti-OX40 antibody, anti-GITR antibody, anti-CD73 antibody, anti-KIR antibody, anti-ICOS antibody, anti-CSF1R antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-HER2 antibody, anti-PDGFR antibody, and any combination thereof.
  • the immune checkpoint regulator comprises an antibody or a binding fragment thereof that specifically binds to PD1.
  • the promoter is CMV.
  • the antibody is a single-chain antibody scFv-PD1.
  • the above construct can continuously express scFv-PD1 at a high level, which not only propagates and infects tumor cells with high selectivity, but also has an immunomodulatory effect. , blocking the combination of PD1 and PD-L1, ensuring that tumor cells are completely removed by the body's own immune response, showing high sensitivity to tumor cells.
  • the HSV is HSV1 or HSV2, or an HSV-1/HSV-2 chimeric virus.
  • HSV-1 is selected from any of the existing HSV strains, including the F strain, the HF strain, the KOS strain and the 17 strain.
  • the coding frame of the scFv-PD1 gene has the nucleotide sequence shown below, 1) the nucleotide sequence shown in SEQ ID NO: 1; 2) compared with 1), it has at least 70%, Nucleotide sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical.
  • ggaggaggaggatccggcggaggaggctctggcggcggcggcagc is the nucleotide sequence of the linker peptide linker
  • caccaccaccaccac is the nucleotide sequence of the His-tag.
  • the single-chain antibody scFv-PD1 has the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 95% identity to SEQ ID NO: 2.
  • QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS is a heavy chain variable region, GGGGSGGGGSGGGGS of connecting peptide linker, EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK for the light chain variable region, HHHHHH to His- tag.
  • the construct has the nucleotide sequence shown below, a) the nucleotide sequence shown in SEQ ID NO: 3; b) compared with a), it has at least 70%, at least 75% %, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical nucleotide sequences.
  • the construct may also comprise one or more non-essential genes deleted or mutated.
  • the non-essential genes are selected from the group consisting of UL3 gene, UL4 gene, UL14 gene, UL16 gene, UL21 gene, UL24 gene, UL31 gene, UL32 gene, US3 gene, UL51 gene, UL55 gene, UL56 gene, US2 gene, LAT Genes, and any combination thereof; preferably, one or more of the UL55 gene, the US2 gene, and the LAT gene are deleted or mutated (eg, comprise a loss-of-function mutation, or be replaced with an exogenous nucleotide sequence).
  • the essential genes of the construct are not deleted and do not contain loss-of-function mutations; preferably, one or more essential genes (eg, ICP27 gene, ICP4 gene, VP5 gene, gL gene, gH gene Gene, gD gene, gK gene, gB gene, gN gene, UL5 gene, UL6 gene, UL8 gene, UL9 gene, UL12 gene, UL25 gene, UL26 gene, UL28 gene, UL29 gene, UL30 gene, UL33 gene, UL36 gene,
  • the native promoters of the UL38 gene, UL42 gene, UL48 gene and/or UL52 gene are replaced with tumor-specific promoters such as hTERT, CMV, CAG, EF1 ⁇ , Rous Sarcoma Virus Long Terminal Repeat (RSV LTR), At least one of metallothionein I (MTI) and Egr.
  • one or more essential genes eg, ICP27 gene, ICP4 gene,
  • the present invention provides an oncolytic virus.
  • the oncolytic virus carries the aforementioned construct.
  • the modified virus compared with the wild-type virus, the modified virus often has a significantly reduced sensitivity to tumors.
  • researches such as Mckie E A show that it can be reduced by 100 times (Mckie E A, Maclean A R, Lewis A D, et al.Selective in vitro replication of herpes simplex virus type 1(HSV-1)ICP34.5 null mutants in primary human CNS tumors--evaluation of a potentially effective clinical therapy.[J].British Journal of Cancer,1996,74 (5):745-752.).
  • HSV1 carries scFv-PD1, not only the replication ability in tumor cells and the cytotoxicity to tumor cells are not significantly weakened, but the sensitivity of the virus strain to tumor cells is enhanced, and the The expression of scFv-PD1 through exogenous scFv-PD1 gene activates the body's anti-tumor immune response.
  • the oncolytic virus according to the embodiment of the present invention expressing scFv-PD1 can make the virus replicate in the tumor by intratumoral injection and continuously and highly express the antibody that can bind to PD1 locally, so as to avoid systemic toxicity caused by intravenous administration. side effect.
  • the oncolytic virus can continuously express scFv-PD1 at a high level.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the aforementioned construct or the aforementioned oncolytic virus.
  • the pharmaceutical composition according to the embodiments of the present invention can take effect at a low dose, and has a good killing effect on tumor cells.
  • the present invention proposes the use of the aforementioned construct, the aforementioned oncolytic virus or the aforementioned pharmaceutical composition in the preparation of a medicament for treating or preventing tumors.
  • the drug is used to selectively kill tumor cells.
  • the cells are tumor cells, such as lung cancer cells, liver cancer cells, nasopharyngeal cancer cells, breast cancer cells, osteosarcoma cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, glioma cells, melanoma cells cells, colorectal cancer cells, esophageal cancer cells and pancreatic cancer cells.
  • tumor cells such as lung cancer cells, liver cancer cells, nasopharyngeal cancer cells, breast cancer cells, osteosarcoma cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, glioma cells, melanoma cells cells, colorectal cancer cells, esophageal cancer cells and pancreatic cancer cells.
  • the pharmaceutical composition is used for the treatment of tumors, eg, lung cancer, liver cancer, nasopharyngeal cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer and Pancreatic cancer.
  • tumors eg, lung cancer, liver cancer, nasopharyngeal cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer and Pancreatic cancer.
  • the pharmaceutical composition is an injection or a lyophilized powder.
  • the pharmaceutical composition is in unit dosage form; for example, 10 ⁇ 2-10 ⁇ 11 pfu of recombinant HSV virus is contained in each unit dosage of the pharmaceutical composition.
  • Fig. 1 is the pMD18-HOM-scFv-PD1 plasmid map according to the embodiment of the present invention
  • Fig. 2 is the PCR gel electrophoresis detection diagram of the recombinant virus constructed according to the embodiment of the present invention
  • FIG. 3 is a sequence diagram of a recombinant virus constructed according to an embodiment of the present invention.
  • FIG. 4 is a PCR gel electrophoresis detection diagram of the target site of the recombinant virus constructed according to the embodiment of the present invention.
  • FIG. 5 is a graph showing the expression results of scFv-PD1 in Vero cells according to an embodiment of the present invention.
  • Fig. 6 is the inhibition curve of HSV1-scFv-hPD1, 2K on different tumor cells according to an embodiment of the present invention
  • FIG. 7 is a graph showing the results of proliferation of HSV1-scFv-hPD1, 2K in different tumor cells according to an embodiment of the present invention.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
  • immune checkpoint in the present application refers to a protein directly or indirectly involved in an immune pathway.
  • the “immune checkpoint regulator” refers to a molecule that can modulate the function of immune checkpoint proteins in a positive or negative manner. This term encompasses immune checkpoint modulators (antagonists) capable of at least partially downregulating inhibitory immune checkpoint function and or immune checkpoint modulators (agonists) capable of at least partially upregulating stimulatory immune checkpoint function.
  • immune checkpoint modulators include, but are not limited to, polypeptides, soluble domains of natural receptors, RNAi, antisense molecules, antibodies (eg, PD1 antibodies, PD-L1 antibodies, CTLA4 antibodies), or protein scaffolds, and the like .
  • the "PD1” mentioned in this application is also called programmed death receptor 1, which is mainly expressed on the surface of activated T cells. It binds to the corresponding receptors PD-L1 or PD-L2 and transmits negative regulatory signals to shut down the immune response of T cells.
  • a variety of tumor cells highly express PD-L1, and these tumor cells bind to PD1 on the surface of T cells through their expressed PD-L1, induce tumor-specific T cell apoptosis and immune anergy, and ultimately lead to tumor cells escape from T cell immunity. Surveillance and kill.
  • hPD1 is PD1 of human origin or a fragment thereof.
  • scFv refers to a single-chain antibody, which is an antibody formed by linking the variable region of the heavy chain and the variable region of the light chain of the antibody through an appropriate linker peptide.
  • scFv-PD1 refers to anti-PD1 single-chain antibody.
  • the "scFv-PD1 gene coding frame" described in this application refers to a nucleic acid sequence capable of expressing scFv-PD1, that is, the scFv-PD1 segment encoded by the nucleic acid sequence is a segment capable of realizing the function of the scFv-PD1 , or in other words, the scFv-PD1 segment encoded by the nucleic acid sequence is an essential functional region of scFv-PD1.
  • ICP47 and ICP34.5 are knocked out means that the ICP47 and ICP34.5 genes are silenced. significantly reduced.
  • the "UL23 gene inactivation" mentioned in this application refers to insertion inactivation, that is, after inserting an exogenous gene at the position of the UL23 gene, the original function of the UL23 gene is lost.
  • operably linked in this application refers to linking an exogenous gene to a vector, so that control elements in the vector, such as promoter sequences, etc., can exert their intended functions to regulate the transcription and translation of the exogenous gene. Function.
  • the following examples utilize the CRISPR/Cas9 system to construct recombinant HSV-1 virus, specifically including recombinant construction, recombinant screening, and virus identification.
  • the biological activity of the recombinant HSV-1 virus specifically includes the expression of scFv-PD1, the ability of the virus to infect and kill tumor cells, etc.
  • the KOS virus strain used in this example has 99% nuclear weight compared with the wild type KOS virus strain. nucleotide sequence similarity.
  • the P2 generation recombinant virus 2k-HSV-1-UL23-scFv-hPD1 (wherein, 2k represents the knockout of ICP47 and ICP34.5, and P2 represents the virus generation).
  • Virus infection (6-well plate, dilution of virus stock solution is 5 ⁇ 10 7 )
  • virus name HSV-1-UL23-scFv-hPD1-mAb-s11-1-1 Pick the number of p3 virus plaques 5
  • P3 generation recombinant virus 2k-HSV-1-UL23-scFv-hPD1 (S11-1-1-1#, S11-1-1-3#) (wherein, 2k means knockout ICP47 and ICP34.5, S11 means targeting The code name of the plasmid, 1-1 is the code name of the virus plaque, and p3 represents the virus generation). (Note: 1-1-1 and 1-1-3 are only one spot per well)
  • the primer is UL23HOM1 left-F2/B48431UL24R;
  • PCR products were detected by gel electrophoresis.
  • two p3-generation recombinant viruses HSV-1-UL23-scFv-hPD1 S11-1-1-1#, S11-1-1-3 #
  • sequencing verified that the sequence of UL23HOM1 from the left 618bp to the right 500bp of UL23HOM2 was complete without mutation, which was consistent with the results of the p1 generation, indicating that scFv-hPD1 had been successfully inserted into HSV- 1 - Virus genome.
  • Examples 4-5 verify the protein expression of HSV-1-scFv-PD1 from a single viral plaque after multiple rounds of purification, and evaluate the cytotoxicity of HSV1-scFv-PD1 in different tumor cells.
  • a His-tag tag was inserted before the scFv-hPD1 cDNA during design, so an anti-His-tag antibody can be used to bind to scFv-hPD1 for protein expression identification.
  • the experimental group HSV1-scFv-hPD1 refers to 2K-scFv-hPD1, and the negative control is HSV-1/2K.
  • Vero African green monkey kidney cells
  • HSV1-scFv-hPD1 HSV-1/2K infection MOI (ratio of virus to cell number) is 0.01
  • the culture virus culture supernatant is collected at 24, 48 and 72 hours, respectively.
  • protein loading buffer was added together with the supernatant, heated at 98°C for 10 minutes, and centrifuged for use.
  • Western blot Load the sample on a 12% PAGE gel for electrophoresis. After the sample runs to the bottom of the gel, the sample is electrophoresed to PVDF membrane at 80V for 60 minutes. After transfer, 5% BSA was added to block for 1-2 hours. Then, the His-tag primary antibody was added to bind to the target protein, and then the HRP-tagged secondary antibody was added for incubation. Finally, ECL developer solution is added, and the appropriate exposure time is selected for development.
  • scFv-hPD1 The expression and secretion results of scFv-hPD1 in Vero cells are shown in Figure 5. 24 hours after infection of Vero cells with HSV1-scFv-hPD1, scFv-hPD1 began to be expressed in Vero cells and successfully secreted into the supernatant. The accumulated scFv-hPD1 concentration in the supernatant increased after 48 hours compared to 24 hours.
  • HSV1-scFv-hPD1, HSV1/2K, SKOV3 human ovarian cancer cells
  • Fadu human pharyngeal squamous cell carcinoma cells
  • Hep3B2.1-7 human liver cancer cells
  • Skmel-28 human melanoma cells
  • tumor cells Skmel-28 (melanoma cells).
  • Table 1 shows the toxicity of HSV1-scFv-hPD1, HSV-1/2K (abbreviated as 2K) to different tumor cells.
  • HSV1-scFv-hPD1 virus has a good killing effect on Skmel-28 melanoma cells, Fadu nasopharyngeal carcinoma cells, and Hep3B2.1-7 liver cancer cells at lower doses, and has a killing effect on Hep3B2.1-7 tumor cells at 24 hours. It is obviously stronger than 2K virus, and the killing ability to HepG2 liver cancer cells at 24h is weaker than that of 2K virus. It can be concluded that the recombinant oncolytic virus HSV1-scFv-hPD1 constructed by the present invention selectively has a certain sensitivity on certain tumor cells. increased, as shown in Figure 6.
  • HSV1-scFv-hPD1, HSV-1/2k (abbreviated as 2K)
  • cells are Fadu nasopharyngeal carcinoma cells, Hep3B2.1-7 liver cancer cells and Skmel-28 melanoma cells.
  • Virus titer virus dilution factor*(1000/300)*number of plaques.
  • the results of proliferation of HSV1-scFv-hPD1 and HSV-1/2k viruses on cells of different tumor lines are shown in FIG. 7 .
  • the experimental results show that the replication ability of HSV1-scFv-hPD1 virus on Hep3B2.1-7 liver cancer cells is slightly stronger than that of 2K; the replication ability of HSV1-scFv-hPD1 virus on FaDu nasopharyngeal carcinoma cells and Skmel-28 melanoma cells is comparable to that of 2K, with no significant difference. (P ⁇ 0.05).

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Abstract

提供了一种构建体、溶瘤病毒及其应用。通过将I型单纯疱疹病毒的ICP34.5基因、ICP47基因敲除，并在UL23基因上插入免疫检查点调节因子表达序列，构建得到对肿瘤细胞敏感性提高的重组病毒。

Description

构建体、溶瘤病毒及其应用 技术领域
本发明涉及生物技术和基因治疗领域，具体地，本发明涉及一种构建体、溶瘤病毒以及其制药用途。
背景技术
目前国内外已有多家PD1抗体成功上市，作为一种广谱性的免疫检查点调节因子已经在各种癌症临床前研究中取得巨大成功。但是，在临床试验中，由于肿瘤的异质性，单一PD1抗体疗法适用人群有限，仍有很大部分患者未能从中获益。其中，对黑色素瘤的治疗效果最好，也只有30％多的病人可长期生存。
溶瘤病毒具有良好的抗肿瘤效果，其选择性在肿瘤细胞上复制直接裂解杀伤肿瘤细胞，释放的肿瘤相关抗原通过抗原呈递作用，激活了机体抗肿瘤免疫，达到抑制肿瘤生长甚至肿瘤完全消退的效果。由受感染的肿瘤细胞碎片引起的抗肿瘤免疫响应可通过加入免疫刺激因子而被放大。为了进一步提升溶瘤病毒治疗效果，使病毒在肿瘤细胞上复制的同时表达免疫检查点调节因子或抑制剂，可以阻断肿瘤细胞对免疫系统的抑制途径。
将PD1抗体基因插入溶瘤病毒中，构建新型溶瘤病毒，有助于找到新的肿瘤治疗手段和基因疫苗。
发明内容
本申请旨在至少在一定程度上解决上述背景技术中的技术问题之一。
为此，在本发明的第一方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体的载体来源于HSV，所述构建体携带免疫检查点调节因子基因编码框，所述免疫检查点调节因子基因编码框设置于UL23基因位置。发明人在实验中意外而惊喜发现，当免疫检查点调节因子基因编码框设置于UL23基因位置时，构建体可持续高表达免疫检查点调节因子。
根据本发明的实施例，上述构建体还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述构建体的ICP47和双拷贝ICP34.5被敲除，
敲除HSV基因组中的复制非必需基因ICP47，可提高被病毒感染的肿瘤细胞表面MHC-I的表达和细胞抗原呈递的能力；敲除双拷贝ICP34.5，可使单纯疱疹病毒(HSV)在正常细胞内复制受限，选择性地在肿瘤细胞中复制，进而提高HSV病毒的用药安全性。
根据本发明的实施例，所述免疫检查点调节因子基因编码框设置于UL23基因序列的第387-521位核苷酸之间位置。需要说明的是，本申请所述的UL23基因序列编码是以UL23基因起始密码子的第一位核苷酸为第1位进行顺序编码的，UL23基因的序列可参考 https://www.ncbi.nlm.nih.gov/nuccore/NC_001806.2？report＝genbank&from＝46609 &to＝47803&strand＝true。根据本发明的实施例，免疫检查点调节因子基因编码框设置于UL23基因序列的第387-521位核苷酸之间是通过将UL23基因序列的第388-520位核苷酸敲除，之后将免疫检查点调节因子基因编码框插入UL23基因序列的第387-521位核苷酸之间获得的。
根据本发明的实施例，免疫检查点调节因子基因编码框与启动子可操作地连接，所述启动子包括选自CMV、CAG、EF1α、劳斯肉瘤氏病毒长末端重复序列(RSV LTR)、金属硫蛋白I(MTI)的至少之一。
所述免疫检查点调节因子选自细胞因子、趋化因子、细胞毒性肽、免疫调节多肽、天然受体的可溶结构域、RNAi、反义分子、抗体或蛋白质支架的至少之一。
任选地，所述细胞因子选自白介素(例如IL-2、IL-7、IL-12和IL-15)、干扰素(例如IFNα、IFNβ、IFNγ)、肿瘤坏死因子(例如TNFα、TNFβ)、集落刺激因子(例如GM-CSF)，及其任意组合。
任选地，所述趋化因子选自CCL2、RANTES、CCL7、CCL9、CCL10、CCL12、CCL15、CCL19、CCL21、CCL20、XCL-1，及其任意组合。
任选地，所述细胞毒性肽选自胸苷激酶TK(TK/GCV)、TRAIL、FasL，及其任意组合。
任选地，所述免疫调节多肽选自CD40L、OX40L、可诱导共刺激分子(ICOS)、FTL3L、LIGHT、CD137L、CD70、4-1BB、GITR、CD28，及其任意组合。
任选地，所述抗体选自抗PD-1抗体、抗PD-L1抗体、抗TIGIT抗体、抗BTLA抗体、抗CTLA-4抗体、抗Tim-3抗体、抗Lag-3抗体、抗CD137抗体、 抗OX40抗体、抗GITR抗体、抗CD73抗体、抗KIR抗体、抗ICOS抗体、抗CSF1R抗体、抗EGFR抗体、抗VEGFR抗体、抗HER2抗体、抗PDGFR抗体，及其任意组合。
根据本发明的实施例，所述免疫检查点调节因子包含特异性结合PD1的抗体或其结合片段。
根据本发明的实施例，所述启动子为CMV。
根据本发明的实施例，所述抗体为单链抗体scFv-PD1。
经试验验证，当scFv-PD1基因编码框设置于HSV载体的UL23基因位置时，上述构建体可持续高表达scFv-PD1，不仅高选择地在肿瘤细胞内繁殖感染，而且还可具有免疫调节作用，阻断PD1和PD-L1的结合，确保肿瘤细胞被机体自身的免疫反应彻底清除，显现出对肿瘤细胞的高敏感性。
根据本发明的实施例，所述HSV为HSV1或HSV2，或HSV-1/HSV-2嵌合病毒。
其中所述HSV-1选自任何现有HSV毒株，包括F毒株、HF毒株、KOS毒株和17毒株。
根据本发明的实施例，scFv-PD1基因编码框具有如下所示的核苷酸序列，1)SEQ ID NO:1所示的核苷酸序列；2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的核苷酸序列。
Figure PCTCN2021074592-appb-000001
其中，在SEQ ID NO:1中， ggaggaggaggatccggcggaggaggctctggcggcggcggcagc为连接肽linker的核苷酸序列， caccaccaccaccaccac为His-标签的核苷酸序列。
根据本发明的实施例，所述单链抗体scFv-PD1具有SEQ ID NO:2所示的氨 基酸序列或与SEQ ID NO:2具有至少95％同一性的氨基酸序列。
Figure PCTCN2021074592-appb-000002
其中，在SEQ ID NO:2中，QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS为重链可变区， GGGGSGGGGSGGGGS为连接肽linker，EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK为轻链可变区， HHHHHH为His-标签。
根据本发明的实施例，所述构建体具有如下所示的核苷酸序列，a)SEQ ID NO:3所示的核苷酸序列；b)与a)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的核苷酸序列。
Figure PCTCN2021074592-appb-000003
Figure PCTCN2021074592-appb-000004
在一些实施例中，所述构建体还可包含一个或多个非必需基因被缺失或突变。优选地，所述非必需基因选自UL3基因，UL4基因，UL14基因，UL16基因，UL21基因，UL24基因，UL31基因，UL32基因，US3基因，UL51基因，UL55基因，UL56基因，US2基因，LAT基因，及其任何组合；优选地，UL55基因、US2基因和LAT基因中的一个或多个被缺失或突变(例如包含功能丧失性突变，或者被置换为外源核苷酸序列)。
在一些实施例中，所述构建体的必需基因未被缺失，且不包含功能丧失性突变；优选地，一个或多个必需基因(例如，ICP27基因、ICP4基因、VP5基因、gL基因、gH基因、gD基因、gK基因、gB基因、gN基因、UL5基因、UL6基因、UL8基因、UL9基因、UL12基因、UL25基因、UL26基因、UL28基因、UL29基因、UL30基因、UL33基因、UL36基因、UL38基因、UL42基因、UL48基因和/或UL52基因)的原生启动子被替换为肿瘤特异性启动子，例如hTERT、CMV、CAG、EF1α、劳斯肉瘤氏病毒长末端重复序列(RSV LTR)、金属硫蛋白I(MTI)、Egr的至少之一。
在本发明的第二方面，本发明提出了一种溶瘤病毒。
根据本发明的实施例，所述溶瘤病毒携带前面所述的构建体。
现有技术中，相较于野生型病毒，改造后的病毒往往对肿瘤敏感性会明显降低，例如Mckie E A等的研究显示可降低100倍(Mckie E A,Maclean A R,Lewis  A D,et al.Selective in vitro replication of herpes simplex virus type 1(HSV-1)ICP34.5 null mutants in primary human CNS tumours--evaluation of a potentially effective clinical therapy.[J].British Journal of Cancer,1996,74(5):745-752.)。
然而，根据本发明实施例的溶瘤病毒，HSV1携带scFv-PD1，不仅在肿瘤细胞中的复制能力和对肿瘤细胞的杀伤毒性没有明显减弱，增强了病毒株对肿瘤细胞的敏感性，还可通过外源scFv-PD1基因表达scFv-PD1，激活机体抗肿瘤免疫反应。表达scFv-PD1的根据本发明实施例的溶瘤病毒，可通过瘤内注射的方式使病毒在肿瘤复制并在局部持续高表达可结合PD1的抗体，避免通过静脉给药带来的全身性毒副作用。
根据本发明的实施例，所述溶瘤病毒可持续高表达scFv-PD1。
在本发明的第三方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括前面所述的构建体或前面所述的溶瘤病毒。根据本发明的实施例的药物组合物在低剂量就能起效，对于肿瘤细胞杀伤效果佳。
在本发明的第四方面，本发明提出了前面所述的构建体、前面所述的溶瘤病毒或前面所述的药物组合物在制备用于治疗或预防肿瘤的药物中的用途。
根据本发明的实施例，所述药物用于选择性杀伤肿瘤细胞。
优选地，所述细胞为肿瘤细胞，例如肺癌细胞，肝癌细胞，鼻咽癌细胞，乳腺癌细胞，骨肉瘤细胞，卵巢癌细胞，宫颈癌细胞，前列腺癌细胞，神经胶质瘤细胞，黑色素瘤细胞，结直肠癌细胞，食管癌细胞和胰腺癌细胞。
优选地，所述药物组合物用于治疗肿瘤，例如，肺癌，肝癌，鼻咽癌，乳腺癌，骨肉瘤，卵巢癌，前列腺癌，神经胶质瘤，黑色素瘤，结直肠癌，食管癌和胰腺癌。
优选地，所述药物组合物为注射液或冻干粉剂。
所述药物组合物以单位剂量形式存在；例如每单位剂量的药物组合物中包含10^2-10^11pfu的重组HSV病毒。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将 变得明显和容易理解，其中：
图1是根据本发明实施例的pMD18-HOM-scFv-PD1质粒图谱；
图2是根据本发明实施例的构建的重组病毒的PCR凝胶电泳检测图；
图3是根据本发明实施例的构建的重组病毒的测序结构图；
图4是根据本发明实施例的构建的重组病毒的打靶位点的PCR凝胶电泳检测图；
图5是根据本发明实施例的scFv-PD1在Vero细胞中的表达结果图；
图6是根据本发明实施例的HSV1-scFv-hPD1、2K对不同肿瘤细胞的抑制曲线；
图7是根据本发明实施例的HSV1-scFv-hPD1、2K在不同肿瘤细胞中的增殖结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
需要说明的是，本申请术语“免疫检查点”是指直接或间接参与免疫路径的蛋白质。所述的“免疫检查点调节因子”是指能够以正或负的方式调节免疫检查点蛋白的功能的分子。此术语涵盖能够至少部分地下调抑制性免疫检查点功能的免疫检查点调节因子(拮抗剂)和或能至少部分地上调刺激性免疫检查点功能的免疫检查点调节因子(激动剂)。“免疫检查点调节因子”的实例包括但不限于，多肽、天然受体的可溶结构域、RNAi、反义分子、抗体(例如PD1抗体、PD-L1抗体、CTLA4抗体)或蛋白质支架等等。
本申请所述的“PD1”又称程序性死亡受体1，主要表达于活化的T细胞表面。它与相应受体PD-L1或PD-L2结合后传递负调控信号导致T细胞的免疫应答关闭。多种肿瘤细胞均高表达PD-L1，这些肿瘤细胞通过其表达的PD-L1与T细 胞表面的PD1结合，诱导肿瘤特异性T细胞凋亡和免疫无能，最终导致肿瘤细胞逃脱T细胞的免疫监视和杀伤。其中“hPD1”是人来源的PD1或其片段。
本申请所述的“scFv”是指单链抗体，由抗体的重链可变区和轻链可变区通过适当的连接肽linker连接而成的抗体。本申请所述的“scFv-PD1”是指抗PD1的单链抗体。
本申请所述的“scFv-PD1基因编码框”是指能够表达scFv-PD1的核酸序列，也就说，该核酸序列所编码的scFv-PD1区段是能够实现该scFv-PD1功能的区段，或者说，该核酸序列所编码的scFv-PD1区段是scFv-PD1必要功能区。
本申请所述的“ICP47和ICP34.5被敲除”是指ICP47和ICP34.5基因被沉默，这两种基因的表达量相比于未被敲除ICP47和ICP34.5的HSV-1，显著降低。
本申请所述的“UL23基因失活”是指插入失活，也就是说在UL23基因位置插入外源基因后，UL23基因的原有功能丧失。
本申请所述的“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如启动子序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。
以下实施例利用CRISPR/Cas9系统构建重组HSV-1病毒，具体包括重组构建，重组筛选，及病毒鉴定等。重组HSV-1病毒的生物学活性，具体包括scFv-PD1的表达情况，病毒在肿瘤细胞上感染杀伤能力等。
实施例1构建重组单纯疱疹病毒
将编码scFv-hPD1基因序列插入至含UL23基因左右两侧同源臂序列之间，获得pMD18T-HOM-scFv-hPD1质粒，如图1，通过Crisper-Cas9系统在HSV-1/ICP47-/ICP34.5-双敲除KOS病毒株的UL23基因上的LacZ位点替换为治疗性基因scFv-hPD1，构建重组病毒，接下来并用X-gal蓝白斑筛选方法筛选构建成功的病毒。
需要说明的是，由于在病毒的保存和使用过程中，病毒在一些碱基位点会发生变异，本实施例中用到的KOS病毒株相较于野生型KOS病毒株，具有99％的核苷酸序列相似性。
实施例2 HSV-1-UL23-scFv-hPD1重组病毒的筛选纯化
(1)实验材料：
P2代重组病毒2k-HSV-1-UL23-scFv-hPD1(其中，2k表示敲除ICP47和ICP34.5，P2表示病毒代数)。
(2)重组病毒的第三轮筛选纯化
1、Vero细胞铺板(6孔板)
2、病毒感染(6孔板，病毒原液稀释度为5×10 7)
3、中性红/x-gal染色
4、挑白斑，每孔有1-3个斑
5、vero细胞扩增病毒(24孔板)
6、收取病毒
病毒名称 HSV-1-UL23-scFv-hPD1-mAb-s11-1-1
挑取p3代病毒斑个数 5
实施例3 HSV-1-UL23-scFv-hPD1重组病毒的PCR验证
对三轮纯化后单一病毒斑HSV-1-UL23-scFv-hPD1进行PCR验证。
实验材料：
P3代重组病毒2k-HSV-1-UL23-scFv-hPD1(S11-1-1-1#、S11-1-1-3#)(其中，2k表示敲除ICP47和ICP34.5，S11表示打靶质粒的代号，1-1是病毒斑的代号，p3表示病毒代数)。(备注：1-1-1和1-1-3为每孔只有一个斑)
1、PCR同源臂外臂
引物为UL23HOM1 left-F2/B48431UL24R；
PCR产物大小：HSV-1-UL23-scFv-hPD1(4873bp)。
引物序列：
UL23HOM1 left-F2 ACCGGAGGGCTGTCGTGCATGGATATCA
B 48431 UL24 R ACGAGAACTGCGGTCGTTGTCCTAA
PCR体系：
组成 体积(μL)
5×PS GXL缓冲液 10.0
模板[HSV-1-UL23-scFv-hPD1] 1.0
正向引物(UL23HOM1 left-F2) 1.0
反向引物(B 48431 UL24 R) 1.0
primeSTAR GXL 1.0
dNTP混合物 4.0
ddH 2O 32.0
总体积 50.0
PCR程序：
98℃，3min；(98℃，10s；60℃，15s；68℃，3min)×35cycles；72℃，5min；12℃，forever。
结果与分析：PCR产物进行凝胶电泳检测，如图2所示，两个p3代重组病毒HSV-1-UL23-scFv-hPD1(S11-1-1-1#、S11-1-1-3#)作为最终筛选出的病毒，分别进行测序，如图3所示，测序验证UL23HOM1以左618bp到UL23HOM2以右500bp序列完整无突变，与p1代结果一致，表明scFv-hPD1已成功插入HSV-1-病毒基因组。
2、PCR打靶位点的LacZ基因
PCR体系：
Figure PCTCN2021074592-appb-000005
PCR程序：
98℃，3min；(98℃，10s；60℃，15s；68℃，1min)×35cycles；72℃，5min；12℃，forever。
测序结果与分析：PCR产物进行凝胶电泳检测，如图4所示，表明病毒较纯，未混有含有LacZ基因的病毒。
以下实施例4～5对多轮纯化后单一病毒斑的HSV-1-scFv-PD1进行蛋白表达 验证，并评价HSV1-scFv-PD1在不同肿瘤细胞的细胞毒性。
实施例4蛋白表达鉴定
设计时在scFv-hPD1 cDNA前插入了His-tag标签，因此可用抗His-tag抗体与scFv-hPD1结合，用于蛋白表达鉴定。
(1)实验材料：
实验组HSV1-scFv-hPD1指代2K-scFv-hPD1，阴性对照为HSV-1/2K。
(2)Western blot验证：
样品制备：以Vero(非洲绿猴肾细胞)为宿主细胞。
条件如下：HSV1-scFv-hPD1、HSV-1/2K感染MOI(病毒与细胞数量的比值)为0.01，分别在感染Vero细胞后，24、48、72小时收取培养病毒培养上清。胞内蛋白加入RIPA裂解液后，和上清液一起加入蛋白上样缓冲液，98℃加热10分钟后离心待用。
Western blot：将样品加载到12％的PAGE凝胶上进行电泳，待样品跑到凝胶底部后，80V电压60分钟将样品电转到PVDF膜上。转膜完成后，加入5％BSA封闭1-2小时。而后加入His-tag一抗与目的蛋白结合，再加入带HRP标签二抗孵育。最后加入ECL显影液，选择合适曝光时间进行显影。
(3)scFv-hPD1表达结果：
scFv-hPD1在Vero细胞中的表达及分泌结果如图5所示，在HSV1-scFv-hPD1感染Vero细胞24小时后，即开始在Vero细胞中表达scFv-hPD1，并成功分泌到上清中。48小时后上清液中累积的scFv-hPD1浓度相比24小时有所增加。
实施例5 HSV1-scFv-hPD1病毒细胞毒性实验
(1)实验材料：HSV1-scFv-hPD1，HSV1/2K，SKOV3(人卵巢癌细胞)；Fadu(人咽鳞癌细胞)；Hep3B2.1-7(人肝癌细胞)；Skmel-28(人黑色素瘤细胞)；Skmel-28(黑色素瘤细胞)。
(2)细胞杀伤验证：以合适的细胞密度接种于96孔培养板，培养过夜后，分别加入7个梯度浓度(MOI＝10、5、1、0.5、0.1、0.05、0.01)的两种病毒，再分别培养24或48小时，依照CCK8试剂盒说明书进行细胞活力的检测。
(3)细胞杀伤结果：
HSV1-scFv-hPD1，HSV-1/2K(简写为2K)对不同肿瘤细胞的毒性如表1 所示。
HSV1-scFv-hPD1病毒在较低剂量对Skmel-28黑色素瘤细胞、Fadu鼻咽癌细胞、Hep3B2.1-7肝癌细胞均具有良好的杀伤作用，24h时对Hep3B2.1-7肿瘤细胞杀伤力明显强于2K病毒，而24h时对HepG2肝癌细胞杀伤能力弱于2K病毒，可以得出本发明所构建的重组溶瘤病毒HSV1-scFv-hPD1选择性地在某些肿瘤细胞上的敏感性有所提高，如图6所示。
表1：
Figure PCTCN2021074592-appb-000006
实施例5 HSV1-scFv-hPD1病毒在肿瘤细胞系上的复制能力
(1)实验材料：HSV1-scFv-hPD1，HSV-1/2k(简写为2K)，细胞为Fadu鼻咽癌细胞、Hep3B2.1-7肝癌细胞和Skmel-28黑色素瘤细胞。
(2)细胞给药：取对数生长期上述细胞以合适的细胞密度接种于6孔培养板，培养过夜后，对6孔板细胞进行计数，根据每孔细胞数将病毒母液用含1％灭活FBS的高糖DMEM或RPMI-1640培养基稀释，配制成MOI＝0.1的病毒溶液。对应每孔依次加入300μl的病毒溶液，置于37℃，5％CO2条件下孵育，每15min摇动培养板使得病毒更好的吸附细胞，1.25h后添加1ml的培养基。2h后弃掉培养基，再补充2ml的培养基，放置CO 2培养箱中分别孵育24h、30h、42h、48h或72h。
(3)病毒滴度测定：培养结束后，收取病毒液。冻融三次(-80℃、37℃)后，梯度稀释收取的病毒液，取300ul感染Vero细胞(6孔板)，每15min摇动培养板使得病毒更好的吸附细胞，1.25h后添加2ml的培养基。2h后弃掉培养基，添加300ul的DMEM完全培养基、3ml 2％甲基纤维素固定病毒。培养3-4天后，吸弃覆盖培养基，加入10％HCHO溶液，1mL/孔，固定20min。之后吸掉甲醛溶液，加入1％结晶紫染色液，500μL/孔，染色30min。最后倒掉染色液， 自来水缓缓冲洗干净，吸水纸倒扣擦干，进行空斑计数，并计算病毒滴度。病毒滴度＝病毒稀释倍数*(1000/300)*空斑数。
(4)HSV1-scFv-hPD1病毒在肿瘤细胞的复制能力
HSV1-scFv-hPD1和HSV-1/2k病毒在不同肿瘤系细胞上的增殖结果如图7所示。实验结果显示HSV1-scFv-hPD1病毒在Hep3B2.1-7肝癌细胞上的复制能力略强于2K；在FaDu鼻咽癌细胞和Skmel-28黑色素瘤细胞上复制能力与2K相当，无显著性差异(P<0.05)。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (13)

  1. 一种构建体，其特征在于，所述构建体的载体来源于HSV,所述构建体携带免疫检查点调节因子基因编码框，所述免疫检查点调节因子基因编码框设置于UL23基因位置。
  2. 根据权利要求1所述的构建体，其特征在于，所述构建体的ICP47和双拷贝的ICP34.5基因被敲除。
  3. 根据权利要求1所述的构建体，其特征在于，免疫检查点调节因子基因编码框设置于UL23基因序列的第387-521位核苷酸之间位置。
  4. 根据权利要求1所述的构建体，其特征在于，所述免疫检查点调节因子基因编码框与启动子可操作地连接，所述启动子包括选自CMV、CAG、EF1α、RSV LTR、MTI的至少之一，所述免疫检查点调节因子选自细胞因子、趋化因子、细胞毒性肽、免疫调节多肽、天然受体的可溶结构域、RNAi、反义分子、抗体或蛋白质支架的至少之一。
  5. 根据权利要求4所述的构建体，其特征在于，所述细胞因子选自白介素(例如IL-2、IL-7、IL-12和IL-15)、干扰素(例如IFNα、IFNβ、IFNγ)、肿瘤坏死因子(例如TNFα、TNFβ)、集落刺激因子(例如GM-CSF)，及其任意组合；所述趋化因子选自CCL2、RANTES、CCL7、CCL9、CCL10、CCL12、CCL15、CCL19、CCL21、CCL20、XCL-1，及其任意组合；所述细胞毒性肽选自胸苷激酶TK(TK/GCV)、TRAIL、FasL，及其任意组合；所述免疫调节多肽选自CD40L、OX40L、可诱导共刺激分子(ICOS)、FTL3L、LIGHT、CD137L、CD70、4-1BB、GITR、CD28，及其任意组合；所述抗体选自抗PD-1抗体、抗PD-L1抗体、抗TIGIT抗体、抗BTLA抗体、抗CTLA-4抗体、抗Tim-3抗体、抗Lag-3抗体、抗CD137抗体、抗OX40抗体、抗GITR抗体、抗CD73抗体、抗KIR抗体、抗ICOS抗体、抗CSF1R抗体、抗EGFR抗体、抗VEGFR抗体、抗HER2抗体、抗PDGFR抗体，及其任意组合。
  6. 根据权利要求4所述的构建体，所述免疫检查点调节因子包含特异性结合PD1的抗体或其结合片段。
  7. 根据权利要求6所述的构建体，其特征在于，所述抗体为单链抗体scFv-PD1，所述scFv-PD1基因编码框具有如下所示的核苷酸序列，
    1)SEQ ID NO:1所示的核苷酸序列；
    2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同源性的核苷酸序列。
  8. 根据权利要求6所述的构建体，其特征在于，所述构建体具有如下所示的核苷酸序列，
    1)SEQ ID NO:2所示的核苷酸序列；
    2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、 至少99％同源性的核苷酸序列。
  9. 根据权利要求1所述的构建体，其特征在于，所述构建体还具有选自下列的一个或多个特征：
    (1)一个或多个非必需基因被缺失或突变(例如包含功能丧失性突变，或者被置换为外源核苷酸序列)；优选地，所述非必需基因选自UL3基因，UL4基因，UL14基因，UL16基因，UL21基因，UL24基因，UL31基因，UL32基因，US3基因，UL51基因，UL55基因，UL56基因，US2基因，LAT基因，及其任何组合；
    (2)必需基因未被缺失，且不包含功能丧失性突变；优选地，一个或多个必需基因(例如，ICP27基因、ICP4基因、VP5基因、gL基因、gH基因、gD基因、gK基因、gB基因、gN基因、UL5基因、UL6基因、UL8基因、UL9基因、UL12基因、UL25基因、UL26基因、UL28基因、UL29基因、UL30基因、UL33基因、UL36基因、UL38基因、UL42基因、UL48基因和/或UL52基因)的原生启动子被替换为肿瘤特异性启动子，例如hTERT、CMV、CAG、EF1α、劳斯肉瘤氏病毒长末端重复序列(RSV LTR)、金属硫蛋白I(MTI)、Egr的至少之一。
  10. 一种溶瘤病毒，其特征在于，携带权利要求1-9任一项所述的构建体。
  11. 根据权利要求10所述的溶瘤病毒，其特征在于，所述病毒表达特异性结合PD1的抗体或其结合片段。
  12. 一种药物组合物，其特征在于，包括权利要求1-9任一项所述的构建体或权利要求10-11任一项所述的溶瘤病毒。
  13. 权利要求1-9任一项所述的构建体、权利要求10-11任一项所述的溶瘤病毒或权利要求12所述的药物组合物在制备用于治疗或预防肿瘤的药物中的用途，任选地，所述药物用于选择性杀伤肿瘤细胞，所述肿瘤为肺癌，肝癌，鼻咽癌，乳腺癌，骨肉瘤，卵巢癌，前列腺癌，神经胶质瘤，黑色素瘤，结直肠癌，食管癌和胰腺癌。
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