WO2022135357A1 - A hIL7/hCCL19 DOUBLE GENE RECOMBINANT ONCOLYTIC VIRUS AND ITS PREPARATION METHOD AND USE - Google Patents

A hIL7/hCCL19 DOUBLE GENE RECOMBINANT ONCOLYTIC VIRUS AND ITS PREPARATION METHOD AND USE Download PDF

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WO2022135357A1
WO2022135357A1 PCT/CN2021/139843 CN2021139843W WO2022135357A1 WO 2022135357 A1 WO2022135357 A1 WO 2022135357A1 CN 2021139843 W CN2021139843 W CN 2021139843W WO 2022135357 A1 WO2022135357 A1 WO 2022135357A1
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oncolytic virus
virus
recombinant oncolytic
cancer
recombinant
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PCT/CN2021/139843
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English (en)
French (fr)
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Xuhui Zhang
Xianlin Luo
Xiaofeng Chen
Wenjia LI
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Sunshine Lake Pharma Co., Ltd.
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Definitions

  • the present invention relates to the field of genetic engineering, in particular, the present invention relates to a hIL7/hCCL19 double gene recombinant oncolytic virus and its preparation method and use.
  • the biggest problem faced by current oncolytic viruses is curative effect.
  • the anti-tumor effect of oncolytic viruses is significant, but the effect is not ideal.
  • the overall remission rate of the oncolytic treatment group of the T-vec product approved by the FDA is only 26%.
  • How to modify the oncolytic virus or use a combination to completely eliminate the tumor is the main work of most researchers. Due to the molecular nature of CCL19, repeated high-concentration administrations are required. It has major side effects and cannot be used well.
  • the viral vector carries the CCL19 factor for continuous local expression to avoid rapid elimination of CCL19.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • the present invention provides a recombinant oncolytic virus, the genome of which integrates two genes IL7 and CCL19 with a strong synergistic effect, thereby enhancing the effect of the oncolytic virus.
  • the double gene carrier type virus constructed by the present invention maintains the sensitivity to tumor cells, while the virus replication ability does not decrease.
  • the first aspect of the present invention provides a recombinant oncolytic virus.
  • the recombinant oncolytic virus comprises:
  • the present invention integrates two immunotherapeutic genes IL7 and CCL19 with strong synergistic effects into the genome of the attenuated oncolytic virus.
  • the expression of CCL19 can effectively enhance the infiltration capacity of the body's immune cells and improve the tumor immune microenvironment.
  • IL7 stabilizes the chemotactic immune cells at the tumor site and synergistically enhances the therapeutic effect of oncolytic viruses.
  • CCL19 can chemoattract immune cells in the body, and IL7 is a key factor for the maturation of immune cells.
  • IL7 plays an important role in the development and survival of T cells and the generation of memory T cells. It is especially critical to restore the number of T cells by promoting steady-state proliferation in the case of lymphopenia. At present, there is no relevant report on oncolytic viruses loaded with these two genes. At the same time, the inventors creatively discovered that compared with previous oncolytic viruses whose sensitivity is usually reduced after modification, the double gene carrier type virus constructed by the present invention maintains the sensitivity to tumor cells, while the replication ability does not decrease.
  • the recombinant oncolytic virus according to the embodiment of the present invention may also have at least one of the following additional technical features:
  • the IL7 is a mammal-derived IL7 protein
  • the CCL19 is a mammal-derived CCL19 protein.
  • the IL7 protein is hIL7 protein
  • the CCL19 protein is hCCL19 protein
  • the nucleic acid sequence encoding hIL7 is selected from the nucleic acid sequence shown in SEQ ID NO: 1, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%homology with the nucleic acid sequence shown in SEQ ID NO: 1;
  • the nucleic acid sequence encoding hCCL19 is selected from the nucleic acid sequence shown in SEQ ID NO: 2, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%homology with the nucleic acid sequence shown in SEQ ID NO: 2.
  • the first nucleic acid sequence and the second nucleic acid sequence are inserted into the ICP34.5 gene position in the genome of the recombinant oncolytic virus.
  • the recombinant oncolytic virus is a recombinant oncolytic virus with optimization and modification.
  • the optimization and modification includes knocking out the ICP47 gene.
  • the recombinant oncolytic virus is derived from an attenuated oncolytic virus.
  • the oncolytic virus is an oncolytic RNA virus or an oncolytic DNA virus
  • the oncolytic virus is selected from oncolytic adenovirus, oncolytic herpes virus, oncolytic alpha virus, oncolytic reovirus, oncolytic coxsackie virus, and oncolytic vaccinia virus.
  • the type of the oncolytic virus is selected from HSV-1 and HSV-2.
  • the HSV-1 includes F strain, HF strain, KOS strain, HrR3 strain and 17 strain.
  • the HSV-2 includes the HG52 strain.
  • the second aspect of the present invention provides a kit.
  • the kit comprises the nucleic acid molecule of the recombinant oncolytic virus described in the first aspect.
  • the nucleic acid molecule refers to the genome of the recombinant oncolytic virus that integrates the genes IL7 and CCL19.
  • the third aspect of the present invention provides a vector.
  • the vector includes expression cassettes of IL7 and CCL19 genes.
  • the expression cassette of the IL7 gene and the expression cassette of the CCL19 gene are in opposite directions, and the gene expression is initiated by different promoters.
  • the IL7 is a mammal-derived IL7 protein
  • the CCL19 is a mammal-derived CCL19 protein.
  • the IL7 is hIL7
  • the CCL19 is hCCL19.
  • the fourth aspect of the present invention provides a method of preparing the recombinant oncolytic virus described in the first aspect. According to an embodiment of the present invention, the method comprises:
  • targeting the predetermined gene position of the oncolytic virus genome and realizing shearing is realized by the Crisper-Cas9 gene editing system.
  • the gene targeted to the predetermined gene position of the oncolytic virus genome is the ICP34.5 gene.
  • the IL7 is a mammal-derived IL7 protein
  • the CCL19 is a mammal-derived CCL19 protein.
  • the IL7 is hIL7
  • the CCL19 is hCCL19.
  • the fifth aspect of the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the recombinant oncolytic virus according to the first aspect or the vector according to the third aspect or the recombinant oncolytic virus prepared by the method according to the fourth aspect.
  • each unit dose of the pharmaceutical composition contains 10 ⁇ 2-10 ⁇ 11 pfu of recombinant oncolytic virus.
  • the sixth aspect of the present invention provides use of the recombinant oncolytic virus of the first aspect or the vector of the third aspect or the recombinant oncolytic virus prepared by the method of the fourth aspect in the preparation of a medicament for treating or preventing tumors.
  • the tumor is selected from lung cancer, liver cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer and pancreatic cancer.
  • the sixth aspect of the present invention provides a method of treating or preventing tumors in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant oncolytic virus of the first aspect or the vector of the third aspect or the recombinant oncolytic virus prepared by the method of the fourth aspect.
  • the tumor is selected from lung cancer, liver cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer and pancreatic cancer.
  • the sixth aspect of the present invention provides the recombinant oncolytic virus of the first aspect or the vector of the third aspect or the recombinant oncolytic virus prepared by the method of the fourth aspect for use in treating or preventing tumors in a subject.
  • the tumor is selected from lung cancer, liver cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer and pancreatic cancer.
  • the seventh aspect of the present invention provides a method of recruiting immune cells to tumors.
  • the method includes contacting the tumor with the recombinant oncolytic virus according to the first aspect or the recombinant oncolytic virus prepared by the method according to the fourth aspect.
  • the eighth aspect of the present invention provides a method of inhibiting the growth of tumor cells or promoting the death of tumor cells.
  • the method includes contacting tumor cells with the recombinant oncolytic virus according to the first aspect or the recombinant oncolytic virus prepared by the method according to the fourth aspect.
  • the tumor cell is selected from lung cancer cell, liver cancer cell, breast cancer cell, osteosarcoma cell, ovarian cancer cell, cervical cancer cell, prostate cancer cell, glioma cell, melanoma cell, colorectal cancer cell, esophageal cancer cell and pancreatic cancer cell.
  • the recombinant oncolytic virus is provided in a dose sufficient to cause death of the tumor cells.
  • Figure 1 shows the pMD18T-HOM-hIL7-hCCL19 donor plasmid map
  • Figure 2 shows the identification results of recombinant oncolytic virus monoclonal ⁇ 47-hIL7-hCCL19 after recombination.
  • M represents the DL5000 marker
  • lanes 2-5 are the identification results of recombinant oncolytic virus monoclonal ⁇ 47-hIL7-hCCL19-1
  • lanes 7-10 are the identification results of recombinant oncolytic virus monoclonal ⁇ 47-hIL7-hCCL19-2;
  • Figures 3A-3B show the results of sequencing to verify whether the double gene expression cassette sequences of ⁇ 47-hIL7-hCCL19-1 and ⁇ 47-hIL7-hCCL19-2 contain mutations, respectively;
  • Figure 4 shows the expression results of hCCL19 in Vero cells
  • Figure 5 shows the expression results of hIL7 in Vero cells
  • Figures 6A-6C show the traces of IC50 fitted according to the inhibition rate
  • Figure 7 shows the titer measurement results of the experimental group ( ⁇ 47-hIL7-hCCL19) and the control group ( ⁇ 47) .
  • the present invention provides a recombinant oncolytic virus, which comprises a first nucleic acid sequence encoding IL7 or a functional analogue thereof; and a second nucleic acid sequence encoding CCL19 or a functional analogue thereof.
  • Functional analogue refers to an analogue that maintains the functional activity of IL7 or CCL19. Its amino acid sequence may be slightly different from the amino acid sequence of IL7 and CCL19 (for example, it has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%homology with the amino acid sequence of IL7 or CCL19) , but the activity is similar.
  • the IL7 is a mammal-derived IL7 protein
  • the CCL19 is a mammal-derived CCL19 protein.
  • Mammals are, for example, humans, monkeys, rabbits, dogs, cows and the like.
  • the IL7 and CCL19 are derived from humans, that is, hIL7 and hCCL19, respectively.
  • the nucleic acid sequence encoding hIL7 is selected from the nucleic acid sequence shown in SEQ ID NO: 1, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%homology with the nucleic acid sequence shown in SEQ ID NO: 1.
  • sequence of SEQ ID NO: 1 is specifically:
  • the nucleic acid sequence encoding hCCL19 is selected from the nucleic acid sequence shown in SEQ ID NO: 2, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%homology with the nucleic acid sequence shown in SEQ ID NO: 2.
  • sequence of SEQ ID NO: 2 is specifically:
  • amino acid sequence of hIL7 protein is shown in SEQ ID NO: 3:
  • amino acid sequence of hCCL19 protein is shown in SEQ ID NO: 4:
  • the hIL7 and hCCL19 are inserted into the coding region of the ICP34.5 gene in the genome of the recombinant oncolytic virus.
  • CCL19 can chemoattract naive T cells and mature DC cells.
  • IL-7 is an essential cytokine for T cell development and can maintain a stable number of T cells.
  • ICP34.5 is a neurotoxic factor in HSV-1 and HSV-2 (ICP34.5 gene has high sequence homology in HSV-1 and HSV-2, the sequence is relatively conservative, and its function is similar in HSV-1 and HSV-2) , which can improve the safety of the virus after being knocked out. At the same time, the virus lacking ICP34.5 can selectively kill tumor cells.
  • Two immunotherapeutic genes IL7 and CCL19 with a strong synergistic effect are integrated into the genome of the attenuated oncolytic virus, which can enhance the therapeutic effect of the oncolytic virus.
  • the double gene carrier type virus constructed by the present invention maintains the sensitivity to tumor cells, while the replication ability does not decrease.
  • the recombinant oncolytic virus is a recombinant oncolytic virus with optimization and modification.
  • Optimization and modification refers to further modification of the oncolytic virus, such as knocking out certain factors that hinder or inhibit the triggering of the immune response, or knocking out genes that affect the safety of the virus.
  • the optimization and modification can occur before the recombination of IL7 and CCL19 into the viral genome in the present invention, or after the recombination.
  • the modification may be to knock out the ICP47 gene in the genome of the oncolytic virus.
  • the ICP47 gene can prevent cell antigen presentation in HSV-infected cells. Knockout of this gene in the oncolytic virus can increase the expression of MHC I on the surface of tumor cells infected by the virus, and improve the ability of antigen presentation, thereby promoting the oncolytic virus to kill tumor cells.
  • the oncolytic virus is an oncolytic RNA virus or an oncolytic DNA virus.
  • the oncolytic virus is selected from oncolytic adenovirus, oncolytic herpes virus, oncolytic alpha virus, oncolytic reovirus, oncolytic coxsackie virus and oncolytic vaccinia virus.
  • the type of the oncolytic virus is selected from HSV-1, HSV-2, including any laboratory strain or clinical isolate;
  • the HSV-1 can be any existing HSV-1 strain, such as F strain, HF strain, KOS strain, 17 strain, HrR3 strain;
  • the HSV-2 may be any existing HSV-2 strain, for example, it may be the HG52 strain.
  • a method of preparing a recombinant oncolytic virus comprising:
  • the IL7 is hIL7
  • the CCL19 is hCCL19.
  • the coding frame is operably linked to a promoter, and the promoter includes at least one selected from the group consisting of CMV, CAG, EF1 ⁇ , Rous sarcoma virus long terminal repeat (RSV LTR) , and metallothionein I (MTI) .
  • the promoter includes at least one selected from the group consisting of CMV, CAG, EF1 ⁇ , Rous sarcoma virus long terminal repeat (RSV LTR) , and metallothionein I (MTI) .
  • the hIL7 and hCCL19 genes are cloned into a vector with RSV and CMV promoters, and then the two expression cassettes are amplified by PCR technology, and then connected to the T vector with the homology arms of the viral genome at both ends using the homologous recombination technology.
  • the transcription direction of the two expression cassettes is opposite, and the donor plasmid is obtained after verification and sequencing.
  • the designed CRISP/Cas9 primer sgRNA sequence is ligated to the cas9 protein expression plasmid to construct a targeting plasmid.
  • the liposome kit is used to transfect the viral genome and two plasmids into 293FT cells, and the CRISPR/Cas9 principle is used for the modification of the virus. After screening and verification, the modified double gene recombinant virus is subjected to protein expression identification and tumor cell killing and replication evaluation.
  • SEQ ID NO: 5 The partial sequence of the construct is shown in SEQ ID NO: 5 (the sequence shown in SEQ ID NO: 5 includes the expression cassettes of hIL7 and hCCL19 genes as shown in Figure 1 and the HOM1 + HOM2 sequences flanking the two expression cassettes) , specifically for:
  • the HSV-1 oncolytic virus used in the following examples of the present invention is a KOS strain, and the “ ⁇ 47 virus genome” refers to the HSV-1 oncolytic virus genome with the ICP47 gene knocked out.
  • ⁇ 47 refers to the control HSV-1 oncolytic virus with the ICP47 gene knocked out
  • ⁇ 47-hIL7-hCCL19 refers to the double gene (hIL7 and hCCL19 genes) recombinant virus with the ICP47 gene knocked out (wherein the hIL7 and hCCL19 double genes are inserted at the ICP34.5 gene position)
  • ⁇ 47-hIL7-hCCL19-1” and “ ⁇ 47-hIL7-hCCL19-2” indicate two clones of the double gene recombinant virus with the ICP47 gene knocked out, respectively.
  • CRISPR-Cas9-sgRNA-34.5 refers to a plasmid construct that uses the Crisper-Cas9 gene editing system to cut the ICP34.5 gene of the oncolytic virus genome.
  • PMD18-T-HOM-hIL7-hCCL19 refers to a donor plasmid to which the hIL7 and hCCL19 genes are linked, which inserts hIL7 and hCCL19 into the ICP34.5 gene position in the HSV-1 oncolytic virus genome through genetic recombination.
  • the coding hIL7 and hCCL19 gene sequences were inserted between the homologous arm sequences on the left and right sides of the ICP34.5 gene, the pMD18T-HOM-hIL7-hCCL19 donor plasmid was obtained.
  • the ICP34.5 gene of the HSV-1/ICP47-knockout virus strain was replaced by the therapeutic genes hIL7 (human IL7) and hCCL19 (human CCL19) through the Crisper-Cas9 system, the recombinant virus was constructed, and the successfully constructed virus was screened.
  • the first nucleotide of the start codon of the ICP34.5 gene was taken as the first position for sequential coding, and the gene insertion position was between the 134th nucleotide of the start codon and the 160th nucleotide downstream of the stop codon, corresponding to the wild-type HSV-1 genome (GeneBank: JQ780693.1) between nt522-nt1271 and nt124324-nt125073.
  • the Cas9-sgRNA-puro vector (Shanghai Shenggong) was linearized with BbsI enzyme, and the vector fragments were recovered by gel.
  • the designed and synthesized sgRNA primers (SgRNA-F target sequence: 5’-tcggtctaacgttacacccgAGG-3’, as shown in SEQ ID NO: 6; sgRNA-R target sequence: 5’-gagccgcgcatatatacgctTGG-3’, as shown in SEQ ID NO: 7) obtained sgRNA fragments through primer annealing complementary reaction, and T4 DNA ligase was used to connect the vector fragments with the same sticky ends and sgRNA fragments to obtain the targeting plasmid.
  • the 293FT cell suspension was inoculated in a 12-well plate, with 1ml per well.
  • the inoculation density was 5*10 ⁇ 4 and 10 ⁇ 5 respectively. It was cultured in a 37 ⁇ , 5%CO2 incubator. During this period, the cell growth density was observed. When the cell confluence reached 70% ⁇ 90%, transfection was performed.
  • the virus (recombinant virus obtained in Example 1) was infected with VERO cells spreading over a six-well plate, the size of the virus plaque was observed, and DMEM medium containing 0.01%neutral red was added for staining. Only the monoclonal virus plaques in each well were picked and amplified, and then the genome was extracted for PCR verification.
  • HOM1-F 5’-CGCACAGTCCCAGGTAACCTC-3’, as shown in SEQ ID NO: 8;
  • PCR program 98°C, 3min; (98°C, 10s; 55°C, 15s; 68°C, 3min) x 35 cycles; 72°C, 5min; 12°C, forever.
  • the experimental group ⁇ 47-hIL7-hCCL19 referred to the ⁇ 47-hIL7-hCCL19-1 recombinant oncolytic virus, and the negative control was the ⁇ 47 oncolytic virus.
  • Vero African green monkey kidney cells
  • MOI the ratio of virus to cell number
  • Table 3 showed the toxicity of ⁇ 47-hIL7-hCCL19 and ⁇ 47 to different tumor cells.
  • Figures 6A-6C were the half-maximal inhibitory concentration (IC50) traces fitted according to the inhibition rate by Graphpad, indicating that the double gene carrier type virus ⁇ 47-hIL7-hCCL19 maintained sensitivity to tumor cells in a variety of tumor cells.

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