WO2022100746A1 - 双特异性单链抗体、负载抗体的重组溶瘤病毒及病毒组合 - Google Patents

双特异性单链抗体、负载抗体的重组溶瘤病毒及病毒组合 Download PDF

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WO2022100746A1
WO2022100746A1 PCT/CN2021/130773 CN2021130773W WO2022100746A1 WO 2022100746 A1 WO2022100746 A1 WO 2022100746A1 CN 2021130773 W CN2021130773 W CN 2021130773W WO 2022100746 A1 WO2022100746 A1 WO 2022100746A1
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ohsv2
virus
hgm
csf
cells
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刘滨磊
蔡林康
张思琪
胡翰
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武汉滨会生物科技股份有限公司
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Priority to CN202180076298.2A priority Critical patent/CN116802281A/zh
Priority to US17/568,738 priority patent/US11505607B2/en
Publication of WO2022100746A1 publication Critical patent/WO2022100746A1/zh
Priority to US18/047,285 priority patent/US20230192852A1/en
Priority to PCT/CN2022/126017 priority patent/WO2023082958A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
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    • A—HUMAN NECESSITIES
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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
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16621—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • 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 field of biotechnology, in particular to a bispecific single-chain antibody, an antibody-loaded recombinant oncolytic virus and a virus combination.
  • BiTEs (bispecific T cell engagers) is a bispecific single-chain antibody that uses T cells as effector cells. It has two antigen-binding arms, which can simultaneously bind to T cells and target cells, and activate cytotoxic T cells to kill lesions. cell. Compared with other bispecific antibodies, BiTEs are more molecularly flexible, can better facilitate the connection of the CD3 complex and tumor targets, and are not bound by T cell receptors and MHC class I molecules on target cells, It does not require the participation of costimulatory molecules, and it is a form of antibody with great application potential.
  • Herps simplex virus is a double-stranded DNA virus of about 154 kb that can replicate in the nucleus of infected host cells.
  • HSV vectors have the following advantages: 1) extensive host cells; 2) high virus titers; 3) large capacity of foreign genes.
  • the disadvantage of the HSV vector is its toxicity.
  • the prior art provides a variety of recombinant oncolytic viruses that use type II herpes simplex virus as a vector and are loaded with different functional genes.
  • the purpose of the present invention is to provide a bispecific single-chain antibody with better effect, an antibody-loaded recombinant oncolytic virus and a virus combination.
  • the bispecific single-chain antibody designed in the present invention is obtained by coupling CD3 antibody and PD-L1 antibody, and is referred to as BiTEs-PD-L1 for short.
  • the BiTEs-PD-L1 provided by the invention is a bispecific antibody that can simultaneously bind to CD3 and PD-L1 on the surface of tumor cells.
  • BiTEs-PD-L1 enters the body and binds to T cells, it can effectively activate T cells and guide T cells kill tumor cells.
  • BiTEs-PD-L1 binds to tumor cells, it can expose the tumor cells and attract T cells to kill them. At the same time, it can relieve the immunosuppression of PD-1/PD-L1 and delay the exhaustion of T cells.
  • the present invention also provides a recombinant oncolytic virus carrying the gene of the above-mentioned bispecific single-chain antibody, the recombinant oncolytic virus comprises type II herpes simplex virus as a vector, and the bispecific single-chain antibody is integrated into the vector
  • the gene encoding the single-chain antibody named oHSV2-Bite.
  • the recombinant oncolytic virus is type II herpes simplex virus HG52dICP47d34.5-BiTEs-PDL-1, and its deposit number is CCTCC NO: V202053. It has been deposited in the Chinese Type Culture Collection Center of Wuhan University on September 8, 2020.
  • the viral backbone used is a recombinant herpes simplex virus type II knocking out the ICP34.5 and ICP47 regions
  • the BiTEs-PD-L1 gene uses the pHG52d34.5-CMV-BiTEs-PD-L1 vector (specifically know the sequence of BiTEs-PD-L1).
  • those skilled in the art can easily construct the vector plasmid) and integrate it into the knocked out ICP34.5 region by means of homologous recombination.
  • the bispecific single chain antibody gene By integrating the bispecific single chain antibody gene into the oncolytic virus, the bispecific single chain antibody can be expressed in vivo for a long time, up to 7 days.
  • the present invention also provides a virus combination comprising two or more recombinant herpes simplex viruses loaded with functional genes, one of which is oHSV2-Bite.
  • the virus combination also includes one or more of oHSV2-hGM-CSF, oHSV2-OX40L, oHSV2-PD1v, oHSV2-neo, and oHSV2-IL12.
  • the virus combination is oHSV2-Bite and oHSV2-hGM-CSF, specifically, oHSV2-hGM-CSF has been deposited, and its deposit number is CGMCC No.3600; more preferably, the virus combination is oHSV2-Bite, oHSV2-hGM-CSF and oHSV2-OX40L, oHSV2-OX40L uses oHSV2-hGM-CSF as the backbone, and the inserted foreign gene number is GenBank: BC153120.1.
  • the present invention also provides another virus combination comprising oHSV2-hGM-CSF and oHSV2-OX40L.
  • the virus combination also includes one or more of oHSV2-PD1v, oHSV2-neo, and oHSV2-IL12.
  • the present invention also provides another virus combination comprising oHSV2-PD1v, oHSV2-neo, oHSV2-IL12.
  • oHSV2-PD1v takes oHSV2-hGM-CSF as the backbone, and the sequence of the inserted exogenous gene is shown in SEQ ID No. 4
  • oHSV2-neo takes oHSV2-hGM-CSF as the backbone, and the inserted exogenous gene
  • the sequence of the gene is shown in SEQ ID No.3
  • oHSV2-IL12 is based on oHSV2-hGM-CSF as the backbone, and the sequence of the inserted foreign gene is shown in SEQ ID No.2;
  • the bispecific single-chain antibody BiTEs-PD-L1 can effectively activate T cells and guide T cells to kill tumor cells.
  • oHSV2-Bite can specifically proliferate inside tumor cells, has the characteristics of high safety and simple preparation process, improves the half-life of BiTEs-PD-L1 in the body, and shortens the number of administrations and dosage.
  • FIG. 1 shows the electrophoretic purification of BiTEs-PD-L1.
  • Figure 2 is a flow cytometry image in the BiTEs-PD-L1 cell binding experiment.
  • FIG. 3 is a flow cytometry diagram in a tumor cell PD-L1 expression detection experiment.
  • Figure 4 is a flow cytometry image in the BiTEs-PD-L1-mediated PBMC activation experiment.
  • Figure 5 shows the results of IFN- ⁇ detection in the BiTEs-PD-L1-mediated PBMC activation experiment.
  • Figure 6 is a graph showing the comparison of the results of the BiTEs-PD-L1-mediated killing of BGC823 cells by PBMC.
  • Figure 7 is a graph showing the comparison of the results of BiTEs-PD-L1-mediated killing of Huh-7 cells by PBMC.
  • Figure 8 is a graph showing the comparison of the results of the BiTEs-PD-L1-mediated killing of A375 cells by PBMC.
  • Figure 9 shows the high-throughput real-time imaging observation in the oHSV2-BiTEs-PD-L1 combined PBMC killing experiment.
  • Figure 10 is a data comparison diagram of oHSV2-BiTEs-PD-L1 combined with PBMC killing.
  • Figure 11 is a comparison diagram of the overall effect of single virus treatment of mouse colon cancer CT26-iRFP.
  • Fig. 12 is a graph showing the comparison of the effect of single virus treatment on mouse colon cancer CT26-iRFP on the 24th day.
  • Figure 13 is a graph showing the comparison of the overall effect of multiple viruses in the treatment of murine colon cancer CT26-iRFP.
  • Figure 14 is a graph showing the comparison of the 24th day effect of multiple viruses in the treatment of murine colon cancer CT26-iRFP.
  • Figure 15 is a graph comparing cell killing results.
  • Figure 16 is a graph showing the comparison of the overall effect of various viruses on the treatment of murine colon cancer CT26-hPDL1.
  • Figure 17 is a graph showing the comparison of the overall effect of multiple viruses on the treatment of murine colon cancer CT26-hPDL1.
  • Figure 18 is a graph comparing the mean tumor size on day 28 of murine colon cancer CT26-hPDL1 treated with various viruses.
  • the pHG52d34.5-CMV-BiTEs-PD-L1 plasmid was transfected into HEK-293T using calcium phosphate transfection reagent, the supernatant was collected after 48h and 72h, and the supernatant was analyzed by Ni-NTA affinity chromatography medium (AKTA).
  • the obtained product was dialyzed using a 20 kDa dialysis card, replaced with a PBS buffer, and frozen at -70°C.
  • Blocking use blocking solution (5g nonfat dry milk + 100mL PBST), block at 37°C for 2h. After blocking, the samples were placed on a shaker and washed three times with PBST for 10 min each time.
  • blocking solution 5g nonfat dry milk + 100mL PBST
  • BGC823-GFP cells were plated in 96-well plates and cultured overnight in a cell incubator at 37 °C, 5% CO 2 .
  • H2 refers to herpes simplex virus type II HG52 strain (oHSV2)
  • d3 refers to the deletion of ICP34.5
  • d4 refers to the deletion of ICP47
  • Phage colony stimulating factor (hGM-CSF) expression cassette Disclosed in Chinese invention patent CN201010116275.3 "Recombinant type II herpes simplex virus vector and its preparation method, recombinant virus, pharmaceutical composition and application").
  • the oHSV2-hGM-CSF virus involved in the following experiments is the same as in Example 6, and can also be written as oHSV2-GMCSF. Then, based on the oHSV2-hGM-CSF skeleton, exogenous therapeutic genes were inserted into the ICP34.5 site respectively to construct a A series of oncolytic viruses are shown in the table below.
  • oHSV2-hGM-CSF hGM-CSF for short
  • oHSV2-IL15 IL15 for short
  • oHSV2-IL12 IL12 for short
  • oHSV2-PD1v referred to as PD1v
  • antiPD1 antiPD1
  • antiPD1 antiPD1
  • neo oncolytic virus anti-tumor effect
  • Tumor formation was induced by subcutaneous injection of murine colon cancer cells (CT26) into the right flank of BALB/c mice and randomized into groups containing a negative control group (Control).
  • CT26 murine colon cancer cells
  • Control a negative control group
  • the treatment was started when the tumor volume reached about 120 mm, and the first day of treatment was the first day.
  • the mice in the experimental group were injected with the corresponding virus in the tumor, and the mice in the control group were injected with inositol sorbitol buffer (IS) to resuspend the virus.
  • IS inositol sorbitol buffer
  • Buffer is a commonly used reagent, which can be sold on the market or prepared by yourself, and its composition difference will not affect the experimental results). After the first dose, the test mice were observed.
  • CT26 tumor cell culture SOP "In vivo induction of CT26 tumor SOP"
  • Viruses All viruses are produced according to the same standard protocol. All viruses were resuspended in formulation buffer and frozen in aliquots at -70°C, with a quick thaw prior to use.
  • mice Female BALB/c normal mice, purchased from Hubei Provincial Center for Food and Drug Safety Evaluation, were raised in a pathogen-free animal laboratory. Animals were 5-7 weeks old (16-20 g body weight) at the time of dosing. Distinguish by marking the mouse tail.
  • Murine colon cancer cells purchased from the National Experimental Cell Resource Sharing Platform, CT26-iRFP was modified on the basis of parental cells to continuously express near-infrared fluorescent protein) were cultured according to standard protocols.
  • the medium was DMEM/F12 with 10% fetal bovine serum. Prior to tumor induction, cells were harvested, centrifuged at 820 g for 5 min, and remixed with serum-free DMEM/F12.
  • tumor induction female BALB/c normal mice (7-9 mice per group) were subcutaneously injected into the right flank with 100 ⁇ L of a suspension containing 2 ⁇ 10 6 CT26 mouse colon cancer cells. Tumor long diameter (a) and short diameter (b) were then measured daily.
  • Treatment was started when the average tumor volume reached about 120 mm 3 .
  • the first day of treatment was the first day.
  • the tumor-bearing mice in the treatment group were injected with the corresponding virus (2 ⁇ 10 5 CCID 50 /100 ⁇ l/mice); two groups of control tumor-bearing mice were injected with an equal volume of IS Buffer (100 ⁇ L).
  • E6 (Effect evaluation of single virus treatment of murine colon cancer CT26-iRFP): Dilute the virus with IS Buffer (inositol sorbitol buffer) to a titer of 1x10 7 CCID 50 /ml and give 100 ⁇ l per mouse, that is, each small Mice were dosed with 1x106 CCID50 .
  • IS Buffer inositol sorbitol buffer
  • E5 (cocktail virus comparison): Viruses were diluted with IS Buffer (inositol sorbitol buffer) to a titer of 1 ⁇ 10 6 CCID 50 /ml to give 100 ⁇ l per mouse, ie 1 ⁇ 10 5 CCID 50 per mouse.
  • IS Buffer inositol sorbitol buffer
  • Cocktail virus Dilute several viruses to the target titer first, and then mix several required viruses one-to-one to obtain a mixed virus called cocktail virus.
  • the hGM-CSF+PD1v+neo group in Figure 13 and Figure 14 is to dilute the three viruses oHSV2-hGM-CSF, oHSV2-PD1v and oHSV2-neo to 1x10 6 CCID 50 /ml Then, 1 ml of each virus was mixed together to obtain a final 3 ml of virus cocktail.
  • the BALB/c tumor-bearing mouse model of mouse colon cancer cell CT26-hPDL1 was used to compare various virus combinations.
  • Tumor cell line CT26-hPDL1 (GFP expression 99.8% and hPDL1 expression 73.4%, purchased from the national experimental cell resource sharing platform, CT26-hPDL1 was modified on the basis of parental cells to continuously express human PDL1 protein)
  • mice 130 female BablC mice (6-8 weeks old)
  • oHSV2 virus Dilute the virus with IS Buffer (Inositol Sorbitol Buffer) to a titer of 1 ⁇ 10 7 CCID 50 /ml and give 100 ⁇ l per mouse, that is, 1 ⁇ 10 6 CCID 50 per mouse.
  • IS Buffer Inositol Sorbitol Buffer
  • Administration time Injection intratumoral injection, administration on the 1st, 4th, and 7th days, and other unspecified steps are the same as those in Example 7.
  • oHSV2-BiTEs-PD-L1 is abbreviated as BiTEs
  • oHSV2-OX40L is abbreviated as OX40L
  • group 12 is abbreviated as (6MIX)
  • other viruses are abbreviated as in Example 7.
  • the virus combination therapy is generally more effective than the single virus group, especially the OX40L+BiTEs+hGM-CSF group has a higher survival rate, is more effective in inhibiting tumor growth, and its comprehensive effect is even stronger than the 6mix virus. group, other groups such as hGM-CSF+BiTEs also had better effects.

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Abstract

本发明提供了一种能够同时结合CD3和肿瘤细胞表面PD-L1的双特异性单链抗体BiTEs-PD-L1、负载抗体的重组溶瘤病毒及病毒组合，当BiTEs-PD-L1进入体内与T细胞结合后，可以有效激活T细胞，并引导T细胞对肿瘤细胞进行杀伤。

Description

双特异性单链抗体、负载抗体的重组溶瘤病毒及病毒组合 技术领域
本发明涉及生物技术领域，具体地指一种双特异性单链抗体、负载抗体的重组溶瘤病毒及病毒组合。
背景技术
BiTEs(bispecific T cell engagers)是一种以T细胞作为效应细胞的双特异性单链抗体，它具有两个抗原结合臂，可以同时和T细胞及靶细胞结合，并激活细胞毒性T细胞杀伤病变细胞。和其它双特异性抗体相比，BiTEs的分子柔韧性更好，能更好地促进CD3复合体和肿瘤靶标的连接，并且它不受T细胞受体和靶细胞上MHC I类分子的约束，不需要共刺激分子的参与，是一种极具应用潜力的抗体形式。
单纯疱疹病毒(Herps simplex virus，HSV)是一种长约154kb的双链DNA病毒，可在受染宿主细胞核中复制。HSV载体具有以下优点：1)宿主细胞广泛；2)病毒滴度高；3)外源基因容量大。HSV载体的缺点在于它的毒性。
现有技术中提供了多种以II型单纯疱疹病毒为载体的，负载有不同功能性基因的重组溶瘤病毒。
发明内容
本发明的目的在于提供一种效果较好的双特异性单链抗体、负载抗体的重组溶瘤病毒及病毒组合。
为实现上述目的，首先，本发明所设计的双特异性单链抗体，它是由CD3抗体与PD-L1抗体偶联得到，简称为BiTEs-PD-L1。
上述方案中，双特异性单链抗体BiTEs-PD-L1的氨基酸序列如SEQ ID No.1所示。
其次，本发明提供了上述双特异性单链抗体在制备抗肿瘤药物中的应用。发明提供的BiTEs-PD-L1是一种能够同时结合CD3和肿瘤细胞表面PD-L1的双特异性抗体，当BiTEs-PD-L1进入体内与T细胞结合后，可以有效激活T细胞，并引导T细胞对肿瘤细胞进行杀伤。当BiTEs-PD-L1与肿瘤细胞结合后，可以将肿瘤细胞暴露出来，吸引T细胞对其进行杀伤，同时可以解除PD-1/PD-L1的免疫抑制，延缓T细胞的耗竭。
再次，本发明还提供了一种携带上述双特异性单链抗体的基因的重组溶瘤病毒，所述重组溶瘤病毒包含作为载体的II型单纯疱疹病毒，载体内整合有所述双特异性单链抗体的编码基因，名为oHSV2-Bite。
上述方案中，所述重组溶瘤病毒为II型单纯疱疹病毒HG52dICP47d34.5-BiTEs-PDL-1，其保藏编号为CCTCC NO：V202053。已于2020年9月8日在武汉大学的中国典型培养物保藏中心保藏。
所使用病毒骨架为敲除ICP34.5和ICP47区域的重组Ⅱ型单纯疱疹病毒，BiTEs-PD-L1基因利用pHG52d34.5-CMV-BiTEs-PD-L1载体(在具体知晓BiTEs-PD-L1序列的前提下，本领域技术人员很容易构建该载体质粒)通过同源重组的方式整合到已敲除的ICP34.5区域。通过将双特异性单链抗体基因整合至溶瘤病毒内，使双特异性单链抗体在体内的能长期表达，最长可达7天。
本发明还提供了一种病毒组合，包括两种及以上的负载有功能基因的重组单纯疱疹病毒，其中一种为oHSV2-Bite。
上述方案中，病毒组合还包含oHSV2-hGM-CSF、oHSV2-OX40L、oHSV2-PD1v、 oHSV2-neo、oHSV2-IL12中一种或几种。优选地，所述病毒组合为oHSV2-Bite与oHSV2-hGM-CSF，具体来说，oHSV2-hGM-CSF是已保藏的，其保藏编号为CGMCC No.3600；更优选地，所述病毒组合为oHSV2-Bite、oHSV2-hGM-CSF和oHSV2-OX40L，oHSV2-OX40L是以oHSV2-hGM-CSF为骨架，插入的外源基因编号为GenBank:BC153120.1。
本发明还提供了另一种病毒组合，包含oHSV2-hGM-CSF和oHSV2-OX40L。
上述方案中，病毒组合还包含oHSV2-PD1v、oHSV2-neo、oHSV2-IL12中一种或几种。
本发明还提供了另一种病毒组合，包含oHSV2-PD1v、oHSV2-neo、oHSV2-IL12。具体来说，oHSV2-PD1v是以oHSV2-hGM-CSF为骨架，插入的外源基因的序列如SEQ ID No.4所示；oHSV2-neo是以oHSV2-hGM-CSF为骨架，插入的外源基因的序列如SEQ ID No.3所示；oHSV2-IL12是以oHSV2-hGM-CSF为骨架，插入的外源基因的序列如SEQ ID No.2所示；。
本发明的有益效果：
1、双特异性单链抗体BiTEs-PD-L1可以有效激活T细胞，并引导T细胞对肿瘤细胞进行杀伤。
2、oHSV2-Bite能够特异性的在肿瘤细胞内部增殖，具有安全性高，制备工艺简单的特点，提高了BiTEs-PD-L1在机体内存留的半衰期，缩短了给药次数和给药剂量。
3、验证了多种负载外源基因病毒组合的功效，筛选出了数种抗肿瘤效果优异的病毒组合。
附图说明
图1为BiTEs-PD-L1的电泳纯化图。
图2为BiTEs-PD-L1细胞结合实验中的流式检测图。
图3为肿瘤细胞PD-L1表达检测实验中的流式检测图。
图4为BiTEs-PD-L1介导的PBMC激活实验中的流式检测图。
图5为BiTEs-PD-L1介导的PBMC激活实验中IFN-γ检测结果。
图6为BiTEs-PD-L1介导的PBMC杀伤BGC823细胞实验结果的效果比较图。
图7为BiTEs-PD-L1介导的PBMC杀伤Huh-7细胞实验结果的效果比较图。
图8为BiTEs-PD-L1介导的PBMC杀伤A375细胞实验结果的效果比较图。
图9为oHSV2-BiTEs-PD-L1联合PBMC杀伤实验中高通量实时成像观测图。
图10为oHSV2-BiTEs-PD-L1联合PBMC杀伤的数据比较图。
图11为单种病毒治疗鼠结肠癌CT26-iRFP的总体效果比较图。
图12为单种病毒治疗鼠结肠癌CT26-iRFP的第24天效果比较图。
图13为多种病毒治疗鼠结肠癌CT26-iRFP的总体效果比较图。
图14为多种病毒治疗鼠结肠癌CT26-iRFP的第24天效果比较图。
图15为细胞杀伤结果比较图。
图16为多种病毒治疗鼠结肠癌CT26-hPDL1的总体效果比较图。
图17为多种病毒治疗鼠结肠癌CT26-hPDL1的总体效果比较图。
图18为多种病毒治疗鼠结肠癌CT26-hPDL1的第28天肿瘤平均大小比较图。
具体实施方式
以下结合附图和具体实施例对本发明作进一步的详细描述。以下实施例是在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
第一部分 双特异性单链抗体及负载该抗体的重组溶瘤病毒的应用
实施例1
BiTEs-PD-L1的制备过程
使用磷酸钙转染试剂将pHG52d34.5-CMV-BiTEs-PD-L1质粒转染进HEK-293T内，48h和72h后收集上清，使用Ni-NTA亲和层析介质(AKTA)对上清中的BiTEs-PD-L1进行吸附。利用洗涤缓冲液(50mM Na 2HPO 4，0.3M NaCl，10mM imidazole pH＝8.0)和洗脱缓冲液(50mM Na 2HPO 4，0.3M NaCl，250mM imidazole pH＝8.0)将BiTEs-PD-L1从介质上洗脱。将得到的产物使用20kDa的透析卡进行透析，置换于PBS缓冲液，冻存于-70℃。
实施例2
BiTEs-PD-L1 Western鉴定
1)将收集的蛋白样品与4×SDS-PAGE蛋白上样缓冲液混匀，煮沸10min。
2)配置SDS-PAGE凝胶，12％的分离胶，分别在点样孔中加入5μL的蛋白Marker和6μL的样品，开始电泳，上层胶电压为80V，下层胶电压为120V，当溴酚蓝跑至底部时停止电泳。结果如图1所示，1泳道为阴性对照样品，2泳道为BiTEs-PD-L1样品(55kDa)。
3)电泳结束进行切胶，切除上层胶以及多余的胶，同时切掉底端溴酚蓝的部分。
4)剪裁滤纸和NC膜，NC膜的大小与凝胶的大小相等，小滤纸稍稍小于胶，将NC膜，蛋白胶和滤纸浸没于转膜缓冲液中。在转膜仪上依次放上大滤纸，NC膜，蛋白胶和小滤纸(滤纸不能相互接触，不要有气泡)，转膜15min，15V。
5)当转膜完成后，使用PBST(PBS+0.05％Tween 20)洗涤3次，每次10min。
6)封闭：使用封闭液(5g脱脂奶粉+100mL PBST)，37℃封闭2h。封闭结束后，将样品置于摇床上，使用PBST清洗3次，每次10min。
7)加入mouse 6×His抗体孵育：稀释比例为1:2000，取5μL一抗到10mL PBST中，室温置于摇床上孵育2h(或4℃过夜)。孵育完毕后，将样品置于摇床上，使用PBST清洗3次，每次10min。
8)加入rabbit anti-mouse-HPR抗体孵育：稀释比例为1:2000，取5μL的二抗到10mL的PBST中，室温置于摇床上孵育1h。孵育完毕后，将样品置于摇床上，使用PBST清洗3次，每次10min。
9)最后一次洗涤完成后进行显色，ECL显色液A，B等体积混合，一共1mL。使用1mL的移液枪吸取染色液滴加于NC膜上，避光显色5min，使用纯水终止显色，将NC膜晾干避光保存。
实施例3
BiTEs-PD-L1细胞结合实验
将1×10 6个Jurkat细胞和A375细胞分别重悬于100μL PBS缓冲液中，将A375细胞分别与1μg人源的PD-L1抗体和500μL BiTEs-PD-L1上清孵育，将Jurkat细胞分别与1μg人源的CD3抗体和500μL BiTEs-PD-L1上清孵育，4℃孵育25min，加入PBS清洗两次，将BiTEs-PD-L1孵育组加入1μg 6×His-FITC抗体，4℃孵育25min，使用PBS清洗两次，上流式检测，结果如图2所示。
实施例4
1)肿瘤细胞PD-L1表达检测
将2×10 4个Huh-7，Panc-1，BGC823，A549，A375细胞铺于96孔板中，37℃，5％CO 2的细胞培养箱中过夜培养。隔天按2:1的效靶比加入PBMC，37℃，5％CO 2的细胞培养箱培养48h。去上清，加入PBS清洗两次。使用胰酶将肿瘤细胞消化下来，使用PBS将肿瘤细胞清洗两次。与1μg人源的PD-L1抗体，4℃孵育25min，使用PBS清洗两次，上流式检测，结果如图3所示。
2)BiTEs-PD-L1介导的PBMC激活
将2×10 4个Panc-1，BGC823，A549细胞铺于96孔板中，37℃，5％CO 2的细胞培养箱中过夜培养。隔天按2:1的效靶比分别加入Mock PBMC和BiTEs-PD-L1孵育的PBMC，37℃，5％CO 2的细胞培养箱培养48h。取上清中的PBMC，使用PBS将清洗两次，与1μg人源的CD4，CD8，CD25抗体4℃孵育25min，使用PBS清洗两次，上流式检测，结果如图4所示。
将2×10 4个Huh-7，Panc-1，BGC823，A549，A375细胞铺于96孔板中，37℃，5％CO 2的细胞培养箱中过夜培养。隔天按2:1的效靶比分别加入Mock PBMC和BiTEs-PD-L1孵育的PBMC，37℃，5％CO 2的细胞培养箱培养48h。取上清，离心去除细胞。使用Elisa试剂盒对上清中的IFN-γ进行检测，结果如图5所示。
3)BiTEs-PD-L1介导的PBMC杀伤
将2×10 4个Huh-7，BGC823，A375细胞铺于96孔板中，37℃，5％CO 2的细胞培养箱中过夜培养。隔天按2:1的效靶比分别加入Mock PBMC和BiTEs-PD-L1孵育的PBMC，37℃，5％CO 2的细胞培养箱培养48h，显微镜下观察杀伤情况，结果如图6～8所示。
实施例5
oHSV2-BiTEs-PD-L1的详细制备过程
1)将4×10 5个ICP4细胞接种于6孔板中，在37℃，5％CO 2环境中培养。隔天使用磷酸钙转染试剂将oHSV2-GFP的基因组和pHG52d34.5-CMV-BiTEs-PD-L1质粒一同转染进ICP4细胞内，24～48h后观察是否形成噬毒斑。
2)将生成噬毒斑的细胞孔进行冻融，5min，2000g离心收集上清，100倍，1000倍，10000倍稀释后感染ICP4细胞。在荧光显微镜下挑选出不带绿色荧光的噬毒斑，进一步感染ICP4细胞，重复挑选出不带绿色荧光的噬毒斑，直至孔板的噬毒斑均不带绿色荧光，即得到oHSV2-BiTEs-PD-L1。进一步提取病毒基因组，使用PCR对病毒进行鉴定。
实施例6
oHSV2-BiTEs-PD-L1联合PBMC杀伤
将2×10 4个BGC823-GFP细胞铺于96孔板中，37℃，5％CO 2的细胞培养箱中过夜培养。隔天按2:1的效靶比分别加入PBMC，同时按MOI＝0.1加入oHSV2-hGM-CSF和oHSV2-BiTEs-PD-L1，组别如下表1，使用高通量实时成像系统对细胞进行观察，结果如图9～图10所示。
表1.oHSV2-BiTEs-PD-L1联合PBMC杀伤实验分组
组别 杀伤率
PBMC 0％
oHSV2-hGM-CSF 23.33％
oHSV2-BiTEs-PD-L1 7.756％
oHSV2-hGM-CSF+PBMC 44.20％
oHSV2-BiTEs-PD-L1+PBMC 93.77％
实验中涉及的oHSV2-hGM-CSF病毒，于2010年2月03日保藏至位于北京市朝阳区北辰西路1号院3号的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.3600。被保藏的生物材料H2d3d4-hGF株，其株号的含义为：H2指II型单纯疱疹病毒HG52株(oHSV2)；d3指剔除ICP34.5；d4指剔除ICP47；hGF指插入人粒细胞-巨噬细胞集落刺激因子(hGM-CSF)表达盒。(在中国发明专利CN201010116275.3“重组Ⅱ型单纯疱疹病毒载体及其制备方法、重组病毒、药物组合物及应用”中公开)。
第二部分 病毒组合应用实验
以下实验中涉及的oHSV2-hGM-CSF病毒同实施例6，也可写为oHSV2-GMCSF，随后以oHSV2-hGM-CSF骨架为基础在ICP34.5位点分别插入了外源治疗基因，构建了系列溶瘤病毒，见下表。
Figure PCTCN2021130773-appb-000001
实施例7
1.实验目的
采用鼠结肠癌细胞CT26-iRFP植瘤的BALB/c荷瘤鼠模型比较oHSV2-hGM-CSF(简称hGM-CSF)、oHSV2-IL15(简称IL15)、oHSV2-IL12(简称IL12)、oHSV2-PD1v(简称PD1v)、oHSV2-antiPD1(简称antiPD1)、oHSV2-neo(简称neo)溶瘤病毒的抗肿瘤作用。
通过在BALB/c鼠右侧肋腹皮下注射鼠结肠癌细胞(CT26)诱导肿瘤形成，并随机分组，含阴性对照组(Control)。待肿瘤体积达到约120mm 3时开始治疗，首次治疗当日记为第1天，并在实验组小鼠瘤内分别注射对应病毒，对照组瘤内注射重悬病毒的肌醇山梨醇缓冲液(IS Buffer为常用试剂，可市售或自行配制，其成分差异不对实验结果产生影响)，治疗共进行3次(第1/4/7天给药)。在首次给药后，对受试小鼠进行观察。
2.实验原理
实验原理参照《CT26肿瘤细胞培养SOP》、《体内诱导CT26肿瘤SOP》
3.实验设计与实验方法
3.1实验材料、动物试剂、耗材
病毒：所有病毒按照同一标准方案进行生产。所有病毒都是重悬于制剂缓冲液并分装冻存在-70℃冰箱中，在使用前进行快速解冻。
动物：雌性BALB/c正常小鼠，购自湖北省食品药品安全评价中心，在无致病原的动物实验室中进行饲养。动物给药时其大小为5-7周(体重16-20g)。通过标记小鼠尾巴加以区分。
3.2实验方法
3.2.1 CT26细胞的制备
按照标准方案对来源于鼠结肠癌细胞(CT26-iRFP，购买自国家实验细胞资源共享平台，CT26-iRFP为在亲本细胞的基础进行改造，使其持续表达近红外荧光蛋白)进行培养。培养基为含10％胎牛血清的DMEM/F12。在肿瘤诱导之前，收集细胞，820g离心5min，然后用无血清DMEM/F12重混。
3.2.2肿瘤模型
肿瘤诱导，雌性BALB/c正常小鼠(每组7-9只小鼠)于右侧肋腹皮下注射100μL含有2×10 6CT26鼠结肠癌细胞悬液。随后每天测量肿瘤长径(a)、短径(b)。肿瘤体积(tumor volume,TV)的计算公式为：V＝1/2×a×b 2。其中a和b分别表示长和宽。
3.2.3病毒治疗
在肿瘤平均体积达到约120mm 3时，开始治疗，第一次治疗当日记为第1天，于第1、4、7天，治疗组荷瘤小鼠分别瘤内注射对应的病毒(2×10 5CCID 50/100μl/只)；两组对照荷瘤小鼠瘤内注射等体积的IS Buffer(100μL)。
E6(单一病毒治疗鼠结肠癌CT26-iRFP效果评估)：将病毒用IS Buffer(肌醇山梨醇缓冲液)稀释到滴度为1x10 7CCID 50/ml每只小鼠给100μl，即每只小鼠给药1x10 6CCID 50。
E5(鸡尾酒病毒比较)：将病毒用IS Buffer(肌醇山梨醇缓冲液)稀释到滴度为1x10 6CCID 50/ml每只小鼠给100μl，即每只小鼠给药1x10 5CCID 50。
鸡尾酒病毒：将几种病毒先稀释到目的滴度，随后将几种需要的病毒一比一混合得到的混合病毒称鸡尾酒病毒。为了简化图示中分组标识，如图13、图14中hGM-CSF+PD1v+neo组就是将oHSV2-hGM-CSF、oHSV2-PD1v、oHSV2-neo三种病毒均稀释到1x10 6CCID 50/ml后，每种病毒取1ml混合在一起，得到最终3毫升鸡尾酒病毒。
4.实验结果有效性的评价标准
用统计软件处理数据，用Mean±SEM表示。组间比较应用单因素方差分析。P＜0.05具有统计学意义。
分组实验结果如图11至图15所示(单一病毒治疗结果如图11、图12，鸡尾酒病毒治疗结果如图13、图14)，IL12和IL15分别激活T和NK细胞，PD1v能与PD-L1高亲和力结合，阻断PD-1/PD-L1负调控。随后，对系列溶瘤病毒进行了体内外抗肿瘤疗效评估。用表达近红外红色荧光蛋白鼠结肠癌(CT26-iRFP)荷瘤鼠模型通过瘤内注射给药比较各携带外源治疗基因oHSV2的疗效，结果表明，相较于其他病毒，oHSV2-hGM-CSF和oHSV2-neo的抑瘤效果最为明显。
从图15中可以观察到，在对A549细胞和CT26细胞两种细胞采用MOI为0.1和1感染携带不同外源因子的溶瘤病毒进行体外杀伤实验。观察第24h和48h可以发现，和对照组相比，实验组的肿瘤细胞均出现了不同程度的形态改变，甚至死亡。提示溶瘤病毒在体外实验中具有肿瘤细胞杀伤效应。
实施例8 鸡尾酒病毒治疗实验
1.实验目的
采用鼠结肠癌细胞CT26-hPDL1植瘤的BALB/c荷瘤鼠模型比较多种病毒组合。
2.实验材料
肿瘤细胞系：CT26-hPDL1(GFP表达量99.8％和hPDL1表达量73.4％，购买自国家实验细胞资源共享平台，CT26-hPDL1为在亲本细胞的基础进行改造，使其持续表达人源PDL1蛋白)
小鼠：(6-8周龄)的雌性BablC小鼠130只
oHSV2病毒：将病毒用IS Buffer(肌醇山梨醇缓冲液)稀释到滴度为1x10 7CCID 50/ml每只小鼠给100μl，即每只小鼠给药1x10 6CCID 50。
3.实验步骤
给药时间注射：瘤内注射，第1、4、7天给药，其他未注明的步骤同实施例7。
分组：
Figure PCTCN2021130773-appb-000002
上述分组中除了oHSV2-BiTEs-PD-L1简称BiTEs，oHSV2-OX40L简称OX40L，第12组简称(6MIX)，其他病毒简称同实施例7。
4.实验数据收集：将第一次治疗时间记为第一天，随后在治疗的第1、4、7、10、14、21、28天测量肿瘤的长径和短径。
结果如下表和附图16～18所示。
表1 病毒治疗组的肿瘤生长数据表
Figure PCTCN2021130773-appb-000003
Figure PCTCN2021130773-appb-000004
结果显示，总体来说病毒组合疗法要比单一病毒组效果要好，特别是OX40L+BiTEs+hGM-CSF组的生存率较高，对抑制瘤体生长较为有效，其综合效果甚至强于6mix的病毒组，其他组如hGM-CSF+BiTEs也同样有较好的效果。这些数据表明，负载外源基因的多种病毒联合应用是具有很强的抗肿瘤功效，但并非外源基因种类越多就越好。

Claims (17)

  1. 一种双特异性单链抗体，它是由CD3抗体与PD-L1抗体偶联得到。
  2. 根据权利要求1所述的双特异性单链抗体，其特征在于，其氨基酸序列如SEQ ID No.1所示。
  3. 权利要求1所述双特异性单链抗体在制备抗肿瘤药物中的应用。
  4. 一种携带权利要求1所述双特异性单链抗体的基因的重组溶瘤病毒，所述重组溶瘤病毒包含作为载体的II型单纯疱疹病毒，载体内整合有所述双特异性单链抗体的编码基因。
  5. 根据权利要求4所述的重组溶瘤病毒，所述重组溶瘤病毒的保藏编号为CCTCC NO：V202053。
  6. 一种病毒组合，包括两种及以上的负载有功能基因的重组单纯疱疹病毒，其中一种如权利要求4所述，名为oHSV2-Bite。
  7. 根据权利要求6述的病毒组合，其特征在于，所述病毒组合还包含oHSV2-hGM-CSF、oHSV2-OX40L、oHSV2-PD1v、oHSV2-neo、oHSV2-IL12中一种或几种。
  8. 根据权利要求6述的病毒组合，其特征在于，所述病毒组合还包含oHSV2-hGM-CSF，其保藏编号为CGMCC No.3600。
  9. 根据权利要求8述的病毒组合，其特征在于，所述病毒组合还包含oHSV2-OX40L，所述oHSV2-OX40L是以oHSV2-hGM-CSF为骨架，插入的外源基因编号为GenBank:BC153120.1。
  10. 一种病毒组合，包含oHSV2-hGM-CSF和oHSV2-OX40L。
  11. 根据权利要求10所述的病毒组合，其特征在于，所述病毒组合还包含oHSV2-PD1v、oHSV2-neo、oHSV2-IL12中一种或几种。
  12. 根据权利要求8所述的病毒组合，其特征在于，所述oHSV2-hGM-CSF的保藏编号为CGMCC No.3600。
  13. 根据权利要求10所述的病毒组合，其特征在于，所述oHSV2-OX40L是以oHSV2-hGM-CSF为骨架，插入的外源基因编号为GenBank:BC153120.1。
  14. 一种病毒组合，包含oHSV2-PD1v、oHSV2-neo、oHSV2-IL12。
  15. 根据权利要求14所述的病毒组合，其特征在于，所述oHSV2-PD1v是以oHSV2-hGM-CSF为骨架，插入的外源基因的序列如SEQ ID No.4所示。
  16. 根据权利要求14所述的病毒组合，其特征在于，所述oHSV2-neo是以oHSV2-hGM-CSF为骨架，插入的外源基因的序列如SEQ ID No.3所示。
  17. 根据权利要求14所述的病毒组合，其特征在于，所述oHSV2-IL12是以oHSV2-hGM-CSF为骨架，插入的外源基因的序列如SEQ ID No.2所示。
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