WO2022246646A1 - 一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒及其在肿瘤治疗中的应用 - Google Patents

一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒及其在肿瘤治疗中的应用 Download PDF

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WO2022246646A1
WO2022246646A1 PCT/CN2021/095796 CN2021095796W WO2022246646A1 WO 2022246646 A1 WO2022246646 A1 WO 2022246646A1 CN 2021095796 W CN2021095796 W CN 2021095796W WO 2022246646 A1 WO2022246646 A1 WO 2022246646A1
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cancer
oncolytic adenovirus
synergistic
tumor
adenovirus
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French (fr)
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黎江
苏英晗
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江苏万戎生物医药科技有限公司
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Priority to EP21942233.4A priority Critical patent/EP4349975A1/en
Priority to PCT/CN2021/095796 priority patent/WO2022246646A1/zh
Publication of WO2022246646A1 publication Critical patent/WO2022246646A1/zh
Priority to US18/520,513 priority patent/US20240165176A1/en

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Definitions

  • the invention belongs to the fields of life science and biomedicine, and specifically relates to a broad-spectrum chimeric oncolytic adenovirus for multi-mechanism synergistic and synergistic immunotherapy applied to tumor treatment and its application in tumor treatment.
  • Oncolytic virus-mediated gene therapy has high tumor cell killing efficiency, good specificity, high safety, few side effects, and low cost, making it a new option after the three conventional treatment methods (surgery, radiotherapy, and chemotherapy) and immunotherapy. Another important emerging tumor treatment model.
  • the biggest challenge of oncolytic virus therapy is how to improve the safety and effectiveness of the product. Through some safety regulation mechanisms, it can ensure that the oncolytic virus replicates in tumor cells to a large extent, but does not replicate in normal cells, the targeting specificity of tumors is improved, and its safety is guaranteed.
  • the mechanisms for the proliferation and killing of adenovirus-targeted tumor cells are rich and diverse.
  • the first generation of oncolytic adenovirus achieved specific replication of tumor cells with P53 gene deficiency by deleting the E1b-55kD protein gene.
  • the second-generation OAV uses various tumor-specific promoters to regulate the expression of adenovirus proliferation gene E1a or/and E1b, and the development of the third-generation OAV is the combined application of various regulatory elements. After several generations and various transformations, the specificity and effectiveness of OAV against tumors have been improved. Theoretically speaking, as a carrier, OAV carries an anti-cancer gene.
  • the purpose of the present invention is to solve the problem that the existing tumor immunotherapy has poor effect on solid tumors, and to provide a multi-mechanism synergistic anti-cancer agent that can be used as an immune checkpoint inhibitor and CAR-T/CAR-NK cell therapy synergist Chimeric broad-spectrum oncolytic adenovirus and its application in tumor therapy.
  • the present invention provides a chimeric broad-spectrum oncolytic adenovirus for synergistic and synergistic immunotherapy with multiple mechanisms.
  • the oncolytic adenovirus simultaneously expresses IL-12, IFN- ⁇ and CCL5;
  • the coat protein Hexon of the oncolytic adenovirus is chimerized from the Hexon sequences of the two serotype viruses Ad5 and Ad48, and the ciliary protein Fiber is chimerized from the Fiber sequences of the two serotype viruses Ad5 and Ad11;
  • the oncolytic adenovirus simultaneously adopts a strong tumor-specific promoter and an oxygen-dependent element switch, and deletes the Elb-55kD gene; the oncolytic adenovirus simultaneously knocks out partial protein coding sequences in E1a-CR2, E1b-19kD, and E3 regions.
  • the IFN- ⁇ and CCL5 gene cDNA sequences share an expression frame driven by a human cytomegalovirus (hCMV) promoter, and the IL-12 gene
  • hCMV human cytomegalovirus
  • mCMV mouse cytomegalovirus
  • the overall sequence of the two expression cassettes and the promoter for simultaneously expressing the three factors of IL-12, IFN- ⁇ and CCL5 is shown in the 29422-33108 bp of SEQ ID NO:3.
  • the coding sequence of the coat protein Hexon of the oncolytic adenovirus is shown in bp 18327-21170 of SEQ ID NO:3.
  • the coding sequence of the ciliary protein Fiber of the oncolytic adenovirus is shown in bp 33373-34356 of SEQ ID NO:3.
  • the whole genome sequence of the oncolytic adenovirus is shown in SEQ ID NO:3.
  • the present invention provides an oncolytic adenovirus that expresses IL-12, IFN- ⁇ and CCL5 at the same time.
  • the IFN- ⁇ and CCL5 gene cDNA sequences share one
  • the expression cassette is driven by the human cytomegalovirus (hCMV) promoter
  • the IL-12 gene cDNA sequence expression cassette is driven by the mouse cytomegalovirus (mCMV) promoter, and the two expression cassettes are arranged in a "foot-to-foot" arrangement.
  • the overall sequence of the two expression cassettes and promoters that simultaneously express IL-12, IFN- ⁇ and CCL5 three factors is shown in the 29422-33108bp of SEQ ID NO:3 .
  • the present invention provides a chimeric broad-spectrum oncolytic adenovirus for synergistic and synergistic immunotherapy with multiple mechanisms as described above or a combination of three factors expressing IL-12, IFN- ⁇ and CCL5 as described above Use of the oncolytic adenovirus in preparing medicines for treating and/or preventing and/or adjuvantly treating cancer or antitumor.
  • the cancer or tumor is breast cancer, liver cancer, gallbladder cancer, gastric cancer, colon cancer, lung cancer, prostate cancer, lymphoma, colorectal cancer, ovarian cancer, cervical cancer, cholangiocarcinoma, esophageal cancer cancer, kidney cancer, glioma, melanoma, pancreatic cancer, bladder cancer, or head and neck cancer.
  • the present invention provides the fourth-generation OAV design scheme based on 4T technology (4 triple-construction techniques), and improves the anti-cancer efficacy and biological safety of OAV to a new level as much as possible.
  • the main features are as follows:
  • Triple Transgene a chimeric broad-spectrum oncolytic adenovirus with multiple mechanisms for synergistic and synergistic immunotherapy provided by the present invention, simultaneously loads three anti-cancer genes in the E3 region of the adenovirus, including Two immune-enhancing cytokine genes (IL-12 and IFN- ⁇ ) and one immune cell chemokine gene (CCL5) established a direct and active molecular mechanism for the synergistic effect of OAV combined with immunotherapy.
  • IL-12 and IFN- ⁇ immune cell chemokine gene
  • CCL5 immune cell chemokine gene
  • cytokines and tumor Antigens not only have immune activation, but also can change the tumor microenvironment, which can not only enhance the effect of radiotherapy and chemotherapy, but also improve the effect of immune checkpoint molecule antibody therapy and immune cell therapy (Oncoimmunology 2015,4:e988098).
  • OAV provided by the present invention expresses the three factors of IL-12, IFN- ⁇ and CCL5, and has a synergistic mechanism of active and direct potentiation of immune checkpoint inhibitors and CAR-T/CAR-NK cell therapeutic efficacy.
  • OAV is loaded with two immune-enhancing cytokines IL-12 and IFN- ⁇ . Since PD-1 antibody and PD-L1 antibody treatment need to activate T cells to exert anti-cancer effects, this process is first initiated by dendritic cells ( DC) synthesize and secrete IFN- ⁇ and IL-12. These two cytokines communicate with T cells to activate the killing activity of T cells (Immunity.2018, 49:1148-1161.e7). However, DCs are often lacking in the tumor microenvironment, and the secretion of IFN- ⁇ and IL-12 is insufficient.
  • DCs are often lacking in the tumor microenvironment, and the secretion of IFN- ⁇ and IL-12 is insufficient.
  • IL-12 and IFN- ⁇ When OAV is loaded with immune-enhancing cytokines IL-12 and IFN- ⁇ , DCs are not required when OAV is combined with PD-1 antibody, and the IL-12 and IFN- ⁇ expressed by the virus can directly activate microenvironmental killer T cells and rapidly Play anticancer effect.
  • IL-12 expressed by OAV When combined with CAR-T/CAR-NK, IL-12 expressed by OAV can prolong the survival time of CAR-T/CAR-NK cells, and IFN- ⁇ can enhance the killing activity of CAR-T/CAR-NK.
  • IFN- ⁇ themselves also have strong anti-cancer activity.
  • IFN- ⁇ is an old-fashioned anti-cancer immune factor that can induce the expression of various cytokines such as Interlukins and TNF.
  • IL-12 is a new darling of tumor immunotherapy, which can improve the level of the body's protective immune response, stimulate the proliferation and activation of T cells and NK cells, and induce the secretion of various cytokines such as IFN- ⁇ .
  • OAV is loaded with the immune cell chemokine CCL5, which can attract NK, T cells (including CAR-T/CAR-NK), DC and other immune cells to migrate and accumulate in the tumor (Cell 2018,172:1022-1037) .
  • CCL5 can increase the number of NK, T cells, DC and other cells in the tumor microenvironment, and synergistically enhance the effect of killing cancer cells.
  • CCL5 When used in combination with CAR-T/CAR-NK, CCL5 can not only chemoattract more CAR-T/CAR-NK cells into the tumor area, but also promote chemotactic NK, T cells, DC and other cells to synergistically enhance CAR- T/CAR-NK function.
  • the arrangement and connection sequence of the three factors in the oncolytic virus genome has a significant impact on its anti-tumor activity. By comparing the anti-tumor activity of the oncolytic virus with multiple combinations of the three factors in the sequence, the anti-cancer activity is finally determined.
  • the optimal sequence of the three factors in VirRon namely: the IFN- ⁇ and CCL5 gene cDNA sequences loaded by VirRon share an expression frame driven by the human cytomegalovirus (hCMV) promoter, and the IFN- ⁇ and CCL5 gene cDNA sequences are separated by T2A connection.
  • the IL-12 gene cDNA sequence expression cassette loaded in VirRon is driven by the mouse cytomegalovirus (mCMV) promoter.
  • mCMV mouse cytomegalovirus
  • the two expression boxes are arranged "foot to foot”.
  • the two expression cassettes are respectively driven by two different cytomegalovirus (CMV) promoters of human and mouse, which can avoid the unforeseen high-level structure caused by the same sequence repetition from affecting the success rate of virus packaging and anticancer activity.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon for multiple mechanisms synergistic and synergistic immunotherapy described in the present invention the reverse complementary sequence of the IFN- ⁇ gene cDNA coding sequence (not including the stop codon) loaded by it is as shown in SEQ The 32263-32760 bp of ID NO:3 is shown.
  • the chimeric broad-spectrum oncolytic adenovirus VirRon which is a multi-mechanism synergistic and synergistic immunotherapy described in the present invention, contains the IL-12 gene cDNA coding sequence as shown in the 29973-31583 bp of SEQ ID NO:3.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon with multiple mechanisms for synergistic and synergistic immunotherapy described in the present invention the reverse complementary sequence of the cDNA coding sequence of the CCL5 gene loaded in it, such as the 31924-32199 bp of SEQ ID NO:3 shown.
  • the IFN- ⁇ and CCL5 gene cDNA sequences loaded in it share an expression frame, and the IFN- ⁇ and CCL5 gene cDNA sequences Connected by T2A, the reverse complementary sequence of its T2A sequence is shown in the 32200-32262bp of SEQ ID NO:3.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon with multiple mechanisms for synergistic and synergistic immunotherapy described in the present invention its loaded IFN- ⁇ and CCL5 gene cDNA sequences share an expression frame, which is represented by human cytomegalovirus (hCMV) Promoter drive.
  • hCMV human cytomegalovirus Promoter drive.
  • the reverse complementary sequence of its hCMV promoter coding sequence is shown in the 32767-33108bp of SEQ ID NO:3.
  • the chimeric broad-spectrum oncolytic adenovirus VirRon described in the present invention is a multi-mechanism synergistic and synergistic immunotherapy.
  • the IL-12 gene cDNA sequence expression cassette loaded in it is driven by the mouse cytomegalovirus (mCMV) promoter. Its mCMV promoter sequence is shown in the 29422-29944bp of SEQ ID NO:3.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon which is a multi-mechanism synergistic and synergistic immunotherapy described in the present invention
  • the anti-cancer genes loaded in it are not limited to IL-12, IFN- ⁇ and CCL5, but also include other anti-cancer genes.
  • the multi-gene connection method is not limited to T2A sequence, but also includes F2A, P2A, IRES, etc. sequences and their mutants ;
  • the promoters used are not limited to mCMV and hCMV, but also include other promoters, enhancers and mutant sequences with gene drive functions.
  • the present invention provides a chimeric broad-spectrum oncolytic adenovirus VirRon with multiple mechanisms for synergistic and synergistic immunotherapy, and simultaneously uses the Ad11 serotype adenovirus Fiber coding sequence of subgenus B
  • the Hexon coding sequence of the adenovirus Hexon of the D subgenus Ad48 serotype is chimeric with the corresponding sequence of the C subgenus Ad5 adenovirus backbone to construct three serotype chimeric OAVs, which realizes the simultaneous improvement of virus resistance in one treatment system.
  • the three major advantages are the efficiency of tumor cell infection, avoiding the interception of the virus by the body's pre-stored neutralizing antibodies, and reducing the adhesion and uptake of the virus by the body's liver cells.
  • the human adenovirus family has 52 serotypes divided into 6 subgenera (A to F). Except for group B, each group of adenovirus uses the Coxsackievirus-adenovirus receptor (CAR) as its main adsorption receptor, and is effective against bone marrow hematopoietic cells, hematopoietic stem cells, and dendritic cells lacking CAR.
  • CAR Coxsackievirus-adenovirus receptor
  • Group B adenoviruses (Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34-35, Ad50, Ad55, etc.) mainly recognize a widely expressed complement regulatory protein CD46.
  • group B adenoviruses Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34-35, Ad50, Ad55, etc.
  • Ad5 exists widely in nature, and most people have been infected and have produced neutralizing antibodies, which can block the same serotype of the virus.
  • Ad5 is hepatotropic and can be absorbed by liver cells.
  • the hypervariable region (HVR) of the adenovirus coat protein Hexon is exposed on the surface of the adenovirus due to its location, and is the key site that causes the differences in liver infection ability and immunogenicity between different serotypes of adenoviruses.
  • the seven HVRs (L1: HVR1-6; L2: HVR7) in the Hexon molecule of Ad5 are selectively combined with rare serotypes such as D subgroups (Ad8-10, Ad25-30, Ad36-39, Ad42-49, Ad51, etc. )
  • the chimerism of the Hexon corresponding region of the virus is an effective method to help Ad5 evade pre-existing immunity and liver uptake.
  • Hexon is the main structural protein of adenovirus
  • the modification of it often leads to the instability of the structure of the adenovirus vector, so that the virus cannot be packaged effectively, so this is a very challenging research.
  • the backbone of the adenovirus vector can effectively package the target virus particles.
  • the chimerism of Ad48 adenovirus Hexon can ensure that OAV escapes the interception of pre-existing neutralizing antibodies in the body and the adhesion and uptake of hepatocytes to adenovirus.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon with multiple mechanisms for synergistic and synergistic immunotherapy described in the present invention its B subgenus Ad11 serotype adenovirus ciliary protein (Fiber) coding sequence and C subgenus Ad5 type adenovirus
  • Ad5F11b The sequence of the chimeric ciliary protein (Ad5F11b) corresponding to the chimeric sequence of Fiber is shown in the 33373-34356 bp of SEQ ID NO:3.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon described in the present invention has multiple mechanism synergistic and synergistic immunotherapy, its D subgenus Ad48 serotype adenovirus hexon (Hexon) coding sequence and C subgenus Ad5 type adenovirus
  • Ad5H48 D subgenus Ad48 serotype adenovirus hexon
  • Ad5H48 Ad5H48
  • the chimeric broad-spectrum oncolytic adenovirus for multi-mechanism synergistic and synergistic immunotherapy described in the present invention is not limited to the chimerism of C subgenus Ad5, B subgenus Ad11, D subgenus Ad48, and also Including reciprocal chimeric transformations of other serotypes in other subgenuses.
  • VirRon a chimeric broad-spectrum oncolytic adenovirus with multiple mechanisms for synergistic and synergistic immunotherapy provided by the present invention, uses a strong tumor-specific promoter and an oxygen-dependent element switch at the same time, and Delete the Elb-55kD gene to construct OAV products with dual safety regulation mechanisms at the transcription and translation levels.
  • One of the mechanisms to realize the specific proliferation and replication of tumor cells by OAV is to use the tumor-specific promoter as a cis-element to control the expression of genes necessary for the proliferation of adenovirus.
  • the combination of regulatory modules in the promoter and enhancer regulatory elements is optimized, and the tumor-specific broad-spectrum promoter controls the adenovirus E1a gene at the transcriptional level only in tumors.
  • E1a Intracellular expression; the end of E1a is fused with an oxygen-dependent element switch, the E1a protein is protected in the hypoxic environment of the tumor, and degraded by the proteasome in the normoxic state of the normal tissue, effectively preventing the expression of the E1a protein in normal cells at the translational level Leakage;
  • Elb transcription unit encodes Elb-55kD and Elb-19kD, Elb-55kD is an essential protein in normal cell proliferation and replication of adenovirus but not in tumor cells, selective deletion of Elb-55kD encoding gene can make Adenovirus maintains the ability to proliferate and replicate in tumor cells, but loses the ability to replicate in normal cells.
  • Elb-55kD protein can inactivate and degrade P53 protein, Elb-55kD deletion is beneficial for cells to maintain the anti-tumor activity of P53, while improving the targeting of viral vectors. Therefore, under the triple control of the above-mentioned regulatory mechanism, the safety and effectiveness of VirRon are improved.
  • the core sequence of the tumor-specific broad-spectrum promoter is intercepted at the transcription initiation site of the 5'-UTR region of the BIRC5 gene
  • the "-857 ⁇ +4bp" fragment, the full-length sequence is shown in the 467-1388bp of SEQ ID NO:3.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon and its application for multi-mechanism synergistic and synergistic immunotherapy described in the present invention, its oxygen-dependent element ON/OFF switch sequence is shown in the 2376-2423bp of SEQ ID NO:3 Show.
  • the multi-mechanism synergistic and synergistic immunotherapy described in the present invention is a chimeric broad-spectrum oncolytic gland Viruses, whose regulatory elements include the promoters, enhancers and mutant sequences of Ki67, hTERT, CEA, AFP, EGFR, DF3/MUC1, VEGFR, E2F, GFAP, Survivin and other factors, as well as the HIF-1 hypoxia response element Regulatory sequences such as (HRE), Egr-1 radiation sensitive element (CArG), hTERT internal E-box, oxygen-dependent switch ODD, and the combined application of the above sequences.
  • HRE Egr-1 radiation sensitive element
  • CArG Egr-1 radiation sensitive element
  • ODD oxygen-dependent switch
  • a chimeric broad-spectrum oncolytic adenovirus VirRon with multiple mechanisms for synergistic and synergistic immunotherapy described in the present invention deletes the entire coding sequence of Elb-19kD and the first codon of the coding sequence of Elb-55kD in its Elb transcription unit to 437 codons, the remaining coding sequence of Elb-55kD is shown in the 2818-2997bp of SEQ ID NO:3.
  • VirRon a chimeric broad-spectrum oncolytic adenovirus VirRon with multiple mechanisms for synergistic and synergistic immunotherapy provided by the present invention, simultaneously knocks out partial proteins of E1a-CR2, E1b-19kD, and E3 regions coding sequence.
  • the construction of OAV is based on serotype 5 (Ad5) of the weakest virulence of human adenovirus subgenus C, retains the expression of E1a protein and makes it regulated by a tumor-specific promoter to achieve specific replication for tumors.
  • E1a has three functional regions, namely CR1, CR2, and CR3.
  • the CR1 region can inhibit the expression of the Her-2/neu gene by binding to the transcriptional regulator P300/CBP, the CR2 region binds to the Rb protein family, and the CR3 region is a transcriptional activation region. . Therefore, E1a protein itself has anti-tumor effect, which can not only inhibit the transcription of Her-2/neu gene, block the activity of NF- ⁇ B, increase the expression of p53, but also inhibit type IV collagenase and plasminogen activator. E1a can also cause non-specific immune response, increase the killing effect of CTL cells, NK cells, macrophages and other ways, induce tumor cell apoptosis, inhibit tumor invasion and metastasis, and improve tumor cell resistance to chemotherapy and radiotherapy. sensitivity.
  • the Elb-19kDa gene is homologous to the apoptosis inhibitor gene Bcl-2, and the Elb-19kD protein can bind Bax or/and Bak to initiate the downstream apoptosis inhibition program and protect infected cells from TNF- ⁇ mediated killing effect.
  • Elb-19kD is deleted, so the specificity of the virus mutant to proliferate in tumor cells is improved, while the proliferative activity in normal cells is weakened. Elb-19kD deletion can promote the recovery of cancer cell apoptosis pathway, and is conducive to the rapid clearance of the virus in normal cells and the rapid release and dissemination in tumor cells, making OAV more specific and more efficient; E3 transcription The unit has 9 open reading frames, encoding protein to protect infected cells from host immune response, E3-gpl9k can weaken the killing effect of infected cells mediated by CTL; RID can block "death" ligands including TNF and Fas ligands and TRAIL-mediated apoptosis; RID can also inhibit the activation of NF- ⁇ B necessary for cell survival mediated by IL-1 and TNF; E3-6.7k can also independently inhibit the external and Apoptosis induced by internal signaling pathway; E3-14.7k is a broad inhibitor of TNF-mediated apoptos
  • Adenovirus death protein can promote cell lysis and virus release, but the molecular mechanism is unknown. It can be seen that deleting the E3 region while retaining the ADP gene during the construction of OAV can not only expand the capacity of the vector, but also promote the apoptosis of infected cancer cells, and facilitate the dissolution of cancer cells by OAV and the release of progeny viruses.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon with multiple mechanisms for synergistic and synergistic immunotherapy described in the present invention knocks out 12 bases "cacgaggctggc" in its E1a-CR2, and the mutant E1a (mE1a) sequence terminates
  • An oxygen-dependent element switch is inserted before the codon, and its sequence is shown in the 1405-2426bp of SEQ ID NO:3.
  • a chimeric broad-spectrum oncolytic adenovirus with multi-mechanism synergistic and synergistic immunotherapy described in the present invention the transformation of its viral structural protein is not limited to the knockout of partial protein coding sequences in E1a-CR2, E1b-19kD, and E3 regions , also including deletions, insertions, mutations and other transformations of E1a, E1b, E3 and other proteins.
  • the chimeric broad-spectrum oncolytic adenovirus for multi-mechanism synergistic and synergistic immunotherapy described in the present invention is a new generation of OAV product, which realizes the real purpose of multi-mechanism synergistic effect, specificity Well, it has strong anticancer activity and has obvious therapeutic effects on a variety of solid tumors. It overcomes the problems of poor curative effect and narrow antitumor spectrum of oncolytic adenovirus used alone in the past. It not only has good anticancer effect when used alone, but also can As an immune checkpoint inhibitor and CAR-T/CAR-NK cell therapy synergist, synergistic immunotherapy can greatly improve the efficacy of solid tumors. Its innovative significance is as follows:
  • adenovirus proliferation gene E1a is controlled by a tumor-specific broad-spectrum promoter, and the specific replication activity of OAV against tumor cells is enhanced at the transcriptional level; within the E1a protein Fusion of an oxygen-dependent element switch ensures that E1a is degraded under normal oxygen conditions in normal tissues, and restricts virus replication in normal cells at the translational level; selective deletion of Elb-55kD can maintain the ability of adenovirus to proliferate and replicate in tumor cells, while Lose the ability to replicate in normal cells; Elb-19kD deletion can promote the recovery of cancer cell apoptosis pathway, which is conducive to the rapid clearance of the virus in normal cells and the rapid release in tumor cells; the partial coding protein knockout of E3 region and The ADP gene is retained to protect the long-term survival of the adenovirus and mediate the high-efficiency expression of anti-cancer genes. Multiple mechanisms work together to improve the safety and
  • adenovirus E1a protein itself has anti-tumor effect, which can inhibit the transcription of Her-2/neu gene and block the activity of NF- ⁇ B , increase the expression of p53, and can inhibit the expression of protease genes such as type IV collagenase and plasminogen activator; E1a can also cause non-specific immune responses, improve the killing effect of CTL, NK, macrophages and other ways, Induce tumor cell apoptosis, inhibit tumor invasion and metastasis, and improve tumor cell sensitivity to chemotherapy and radiotherapy.
  • a deletion mutation is introduced into the CR2 region of E1a so that it cannot bind to the Rb protein, ensuring that the dephosphorylated Rb protein forms a complex with the transcription factor E2F, blocks the transcriptional activity of E2F, and enhances the anticancer activity.
  • OAV-infected cancer cells release a large number of cytokines, lyse cancer cells and release a large number of tumor-associated antigens, which play a further role in immune activation.
  • Viral proteins that are restricted to the expression of cancer cells similar to tumor-specific antigens, can also function as anti-cancer vaccines.
  • the OAV formed by the chimerism of three serotypes of adenovirus improves its infectivity and can effectively evade immune interception and liver uptake: the chimerism of the fiber knob of group B adenovirus and Ad5 fiber knob is beneficial to improve the virus's resistance to infection. Infection efficiency of cancer cells, especially cancer stem cells.
  • the HVR region of the Hexon molecule of Ad5 is selectively chimerized with the corresponding region of the Hexon of the D subgroup virus, which helps the virus to evade the interception of human neutralizing antibodies and the uptake by liver cells.
  • Figure 1 Structural diagram of the right arm backbone plasmid pPE3F11bH48-RC(+) of adenovirus.
  • Figure 2 Structural diagram of the adenovirus left-arm shuttle plasmid pAdSVP-mE1aODD.
  • Fig. 3 Structural diagram of exogenous gene shuttle plasmid pENTR12-C3.
  • Figure 4 Genome structure diagram of oncolytic adenovirus VirRon.
  • Oncolytic virus VirRon has high proliferative activity in liver cancer, breast cancer, gallbladder cancer, and lung cancer cell lines.
  • the multiplication factor is between 10 4 and 10 6 at 96 hours after infection, and its proliferative ability is very weak in normal cell lines.
  • Figure 6 The graph of the killing effect of oncolytic virus VirRon on cells, showing that VirRon has obvious killing and inhibiting effect on various solid tumor cells at a low MOI value, but has no killing activity on normal fibroblast BJ.
  • Figure 7 Comparison of the IC50 values of the oncolytic virus VirRon on cell killing, showing that the IC50 value of VirRon on the killing inhibition of various solid tumor cells is low, while the IC50 value on the killing of normal fibroblast BJ is as high as 1090.
  • Oncolytic virus VirRon expresses three anti-cancer genes "IL-12+IFN- ⁇ +CCL5", and the expression level gradually increases with time.
  • Figure 9 Effects of the arrangement and connection sequence of three anti-cancer genes in oncolytic adenovirus VirRon on their packaging efficiency and anti-cancer activity.
  • Figure 10 Effects of the combination of chimeric Hexon (Ad5H48) fragments in oncolytic adenovirus VirRon on its packaging and amplification efficiency.
  • Figure 14 Humanized mouse model experiment of VirRon combined with PD-1 antibody in the treatment of colon cancer xenografts.
  • Figure 15 NCG mouse model experiment of VirRon combined with CAR-T cells in the treatment of prostate cancer xenografts.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon a multi-mechanism synergistic and synergistic immunotherapy described in the present invention, is recombined from an adenovirus right-arm plasmid, an adenovirus left-arm plasmid and an exogenous gene shuttle plasmid.
  • the three plasmids and the recombination process are now introduced, as follows:
  • Adenovirus right arm backbone plasmid pPE3F11bH48-RC(+) was reconstructed from pBHGlox(delta)E13Cre plasmid (Catalog no.PD-01-40) and pBHGE3 plasmid (Catalog no.PD-01-12) provided by Microbix Biosystems, Canada. Come. Insert the SpeI+NotI fragment of pBHGE3 containing part of the E3 region sequence and the synthetic ADP gene sequence (as shown in the 30901-31182bp of SEQ ID NO:3) into the SpeI+PacI site of pBHGlox(delta)E13Cre , construct pPE3 plasmid.
  • pPE3F11bH48-RC(+) The full length of pPE3F11bH48-RC(+) is 36591bp, and its ciliary protein (Ad5F11b) is chimerized from the corresponding sequences of Ad5 and Ad11 serotype adenovirus (as shown in 33373-34356bp of SEQ ID NO:3), and its hexon
  • the protein (Ad5H48) is chimerized from the corresponding sequences of Ad5 and Ad48 serotype adenoviruses (as shown in the 18327-21170bp of SEQ ID NO: 3).
  • the adenovirus left-arm shuttle plasmid pAdSVP-mE1aODD was rebuilt from the pXC1 plasmid (Catalog no.PD-01-03) provided by Microbix Biosystems, Canada.
  • a BglII restriction site was introduced at 12 bp upstream of the E1a initiation codon of pXC1 to construct pXC2.
  • the shuttle plasmid pENTR12 was rebuilt from the pENTR11 plasmid (Catalog no. 11819-018) provided by Invitrogen, USA. Introduce the "mCMV promoter+SpeI/EcoRI/HindIII/BglII/NheI/BamHI/SalI+Poly(A)" sequence (SEQ ID NO: 2) between the NcoI and EcoRV restriction sites of pENTR11 to replace the original pENTR11 plasmid The multiple cloning site and ccdB gene sequence in the new plasmid pENTR12 were constructed.
  • Insert the IL-12 expression cassette and the IFN- ⁇ +CCL5 expression cassette between EcoRI and EcoRV, the multiple cloning site of pENTR12, and the double expression cassettes are arranged in a "foot-to-foot” arrangement to construct the foreign gene shuttle plasmid pENTR12-C3, full-length 5985bp ( Figure 3).
  • the complete sequence (including the site sequence) between the NcoI and EcoRV sites containing the IL-12 expression cassette and the IFN- ⁇ +CCL5 expression cassette in the VirRon genome transferred from pENTR12-C3 is as shown in SEQ ID NO: 29401 of 3 -33114bp shown.
  • the recombination packaging of VirRon virus uses the adenovirus right-arm plasmid pPE3F11bH48-RC(+) as the backbone, and is packaged after two rounds of specific recombination.
  • the exogenous gene expression cassette in the gene was transferred to the E3 region of pPE3F11bH48-RC(+); then the second round of sequence-specific recombination occurred in 293T cells with the adenovirus left-arm shuttle plasmid pAdSVP-mE1aODD and the product of the first round of recombination , the mE1aODD expression cassette controlled by the tumor-specific promoter in pAdSVP-mE1aODD was transferred to the E1 region of the adenoviral right-arm backbone plasmid. After two rounds of specific recombination, the ideal oncolytic adenovirus VirRon was accurately and rapidly packaged.
  • the structural diagram of the oncolytic adenovirus VirRon is shown in Figure 4.
  • a chimeric broad-spectrum oncolytic adenovirus VirRon a multi-mechanism synergistic and synergistic immunotherapy described in the present invention, can specifically proliferate and replicate in tumor cells, mediate the high-efficiency expression of anti-cancer genes, and finally destroy or inhibit tumor cells.
  • VirRon showed high-copy proliferation in most solid tumor cell lines including liver cancer, breast cancer, gallbladder cancer, and lung cancer, and the multiplication factor was as high as 316978.64 times; while the proliferation activity in normal cell lines was low, and in GES-1 The cells did not proliferate at all, and the hepatic cells L02 had weak proliferative activity, and the proliferative multiple was only 399.05 times (Fig. 5).
  • the specific killing inhibition of tumor cells and normal cells by oncolytic adenovirus VirRon was detected by CCK8 assay. Collect tumor cell lines and normal cell lines in the logarithmic growth phase, spread 96-well plates, 1 ⁇ 10 4 /well, after the cells adhere to the wall, replace with serum-free medium; add VirRon at gradient MOI for cell infection, corresponding to each Set 8 multiple wells for each MOI value, and culture in the incubator for 2 hours; replace the serum culture solution with 100 ⁇ l/well, after 48 hours of cultivation, discard the culture solution, add CCK8 solution, put it in the incubator for 4 hours, and measure the light absorption value at 490nm wavelength with a microplate reader , calculate the cell survival rate, and draw the cell survival curve.
  • VirRon had obvious killing and inhibitory effects on various solid tumor cells, and was closely related to the intensity of virus infection; different cell lines of the same type of tumor had different sensitivities to VirRon, and the killing IC50 of VirRon on lung cancer H460 cells The value is only 6.656pfu/cell. VirRon has no obvious killing activity on normal fibroblast BJ, and its IC50 value is as high as 1091pfu/cell ( Figure 6-7, Table 1).
  • Oncolytic adenovirus VirRon mediates the expression of anti-cancer genes
  • Oncolytic adenovirus VirRon is loaded with three anti-cancer immune factors IL-12, IFN- ⁇ , and CCL5, and the order of arrangement and connection of the three factors has a significant impact on the successful rate of virus packaging and anti-cancer activity.
  • Oncolytic adenoviruses were packaged in HEK293 cells by designing and constructing shuttle plasmids (V1-V4) with different sequences of three factors, and recombining with adenovirus backbone plasmids (Fig. 9A).
  • Ad5H48 The coat protein Hexon (Ad5H48) of the oncolytic adenovirus VirRon is chimerized from the Hexon sequences of the two serotypes of Ad5 and Ad48, in order to avoid the interception of the pre-existing Ad5 virus neutralizing antibody in the body and the adsorption of the virus by the liver.
  • the full length of Ad5H48 encoding cDNA is 2844bp.
  • Ad5H48-1 namely VirRon
  • Example 3 Animal experiments of a chimeric broad-spectrum oncolytic adenovirus VirRon, a multi-mechanism synergistic and synergistic immunotherapy of the present invention
  • liver cancer HCCLM3 cell suspension in the logarithmic growth phase was injected under the skin of the right axilla of nude mice, 5 ⁇ 10 6 cells/100 ⁇ l/mouse. Twelve days after inoculation, the tumor formation rate was 100%, and the maximum and minimum diameters of transplanted tumors were about 5.0 mm on average. They were randomly divided into 3 groups (VirRon group, AdSVP-H48-DsRed group, blank control group), 10 rats in each group.
  • each group one mouse with the largest tumor and one mouse with the smallest tumor were removed first, and the remaining 8 mice entered the experimental therapeutic observation.
  • the 2 virus treatment groups were given multi-point injections of the corresponding adenovirus intratumorally, with a dose of 3 ⁇ 10 8 pfu/100 ⁇ l per mouse, once every other day, a total of 5 times; the blank control group was simultaneously given virus preservation solution, 100 ⁇ l per mouse .
  • the tumor size was measured regularly, the tumor volume was calculated with the formula of "maximum diameter ⁇ minimum diameter 2 ⁇ 0.5", and the growth curve was drawn.
  • the 3 with the largest tumor and the 2 with the smallest tumor were excluded, and the remaining 25 were randomly divided into 5 groups (VirRon low-dose group, VirRon medium-dose group, VirRon high-dose group, AdSVP-H48 group, and blank control group). Group of 5.
  • the virus treatment group was given intratumoral multi-point injection of corresponding adenovirus.
  • the high-dose group had a dose of 3 ⁇ 10 8 pfu/100 ⁇ l each time
  • the middle-dose group had a dose of 2 ⁇ 10 8 pfu/100 ⁇ l each time
  • the low-dose group had a dose of 2 ⁇ 10 8 pfu/100 ⁇ l each time.
  • the dose of each mouse was 1 ⁇ 10 8 pfu/100 ⁇ l, once every other day, 5 times in total; the AdSVP-H48 group was injected with a medium dose, and the blank control group was simultaneously given virus preservation solution, 100 ⁇ l each time. After treatment, the tumor size was measured regularly, and the tumor volume was calculated with the formula of "maximum diameter ⁇ minimum diameter 2 ⁇ 0.5", and the growth curve was drawn. During the experiment, when the tumor volume of any group exceeds the upper limit of tumor volume of 2000mm3 allowed by the animal experiment ethics committee, the experimental observation will be terminated.
  • Gallbladder cancer has a high degree of malignancy and rapid progression, and there is no clinical treatment except surgery.
  • a fast-growing xenograft tumor model of SGC-996 By establishing a fast-growing xenograft tumor model of SGC-996, observe the efficacy and synergistic mechanism of VirRon treatment.
  • the gallbladder cancer SGC-996 cell suspension in logarithmic growth phase was injected subcutaneously in the right axilla of nude mice, 5 ⁇ 10 6 cells/100 ⁇ l/mouse.
  • the tumor formation rate was 100%, and the maximum and minimum diameters of transplanted tumors were about 5.0 mm on average. They were randomly divided into 4 groups (VirRon group, AdSVP-H48 empty virus group, Ad5-C3 non-proliferating virus group, blank control group), 10 rats in each group. In each group, one mouse with the largest tumor was removed first, and the remaining 9 mice entered the experimental therapeutic observation.
  • VirRon group, AdSVP-H48 group and Ad5-C3 group were injected with corresponding virus at multiple points in the tumor, with a dose of 2 ⁇ 10 8 pfu/100 ⁇ l per mouse, once every other day, a total of 5 times; the blank control group was given virus preservation solution simultaneously , 100 ⁇ l each time. After treatment, the tumor size was measured regularly, and the tumor volume was calculated with the formula of "maximum diameter ⁇ minimum diameter 2 ⁇ 0.5", and the growth curve was drawn.
  • Healthy C57-hPD1 humanized mice female, 5-6 weeks old, 46, purchased from Beijing Biocytogen Biotechnology Co., Ltd., subcutaneously inoculated mouse colon cancer cell line MC38 cells 1 ⁇ 10 6 on the right back
  • the number of cells per mouse when the average tumor size reaches 50mm 3 -80mm 3 , the 3 with the largest tumor and the 3 with the smallest tumor were excluded, and the remaining 40 were randomly divided into 4 groups (blank control, VirRon single drug, Keytruda single drug, Combined drug group), start drug treatment.
  • Multi-point intratumoral injection of VirRon 3 ⁇ 10 8 pfu/100 ⁇ l per mouse, once every other day, 5 times in total; intraperitoneal injection of Keytruda, 2.5mg/kg per mouse, 2 times a week, 6 times in total Second-rate.
  • the blank control group was given virus preservation solution synchronously, 100 ⁇ l each time.
  • the tumor size was measured regularly, and the tumor volume was calculated with the formula of "maximum diameter ⁇ minimum diameter 2 ⁇ 0.5", and the growth curve was drawn.
  • the tumor inhibition rate was 18.71% in the VirRon single drug group, 69.05% in the Keytruda single drug group, and 88.86% in the combined drug group (Figure 14).
  • CAR-T cells were prepared by the Tumor Research Institute of Xuzhou Medical University, targeting B7-H3. Healthy NCG mice, male, 4-5 weeks old, 19 mice, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Prostate cancer cell line Du145 cells were inoculated subcutaneously on the right back at 3 ⁇ 10 6 cells/mouse, and the tumor average When they grow to about 50mm3 , they are randomly divided into 4 groups (blank control, VirRon single-drug, CAR-T single-drug, combined medication group), with 5 rats in each group (4 in the CAR-T single-drug group), and start drug treatment.
  • the total dose of VirRon per mouse was 1 ⁇ 10 9 pfu, which was divided into 3 intratumoral injections, once every other day, for a total of 5 times; on the second day after virus injection, CAR-T was injected into the tail vein, 2 ⁇ 10 6 per mouse number of cells.
  • the blank control group was given PBS buffer synchronously. After treatment, the tumor size was measured regularly, and the tumor volume was calculated with the formula of "maximum diameter ⁇ minimum diameter 2 ⁇ 0.5", and the growth curve was drawn.
  • the tumor inhibition rate was 80.19% in the VirRon single drug group, 48.83% in the CAR-T single drug group, and 97.56% in the combined drug group.

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Abstract

提供了一种同时表达IL-12、IFN-γ和CCL5三个因子的嵌合型溶瘤腺病毒及其在肿瘤治疗中的应用，所述溶瘤腺病毒的外壳蛋白Hexon由Ad5和Ad48两个血清型病毒的Hexon序列嵌合而成，纤毛蛋白Fiber由Ad5和Ad11两个血清型病毒的Fiber序列嵌合而成。所述嵌合型溶瘤腺病毒能激活多种病毒结构蛋白的内在抗癌活性，提高感染肿瘤细胞能力的同时又能保证病毒本身逃避机体预存中和抗体的拦截以及肝细胞的粘附和摄取，提高增强杀伤癌细胞的作用。

Description

一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒及其在肿瘤治疗中的应用 技术领域
本发明属生命科学、生物医学领域，具体涉及应用于肿瘤治疗的一款多重机制协同和增效免疫治疗的广谱嵌合型溶瘤腺病毒及其在肿瘤治疗中的应用。
背景技术
溶瘤病毒介导的基因治疗对肿瘤细胞杀伤效率高、特异性好、安全性高、副作用小、成本低廉，使其成为继三大常规治疗方法(手术、放疗和化疗)以及免疫治疗之后的又一重要新兴肿瘤治疗模式。溶瘤病毒疗法最大的挑战，在于如何提高产品的安全性和有效性。通过一些安全的调控机制，能够保障溶瘤病毒很大程度上在肿瘤细胞内复制，而在正常细胞内不复制，肿瘤的靶向特异性提高，其安全性也就得到了保障。实现腺病毒靶向肿瘤细胞增殖杀伤的机制是丰富多样的，如早期第一代的溶瘤腺病毒(OAV)通过删除E1b-55kD蛋白基因实现靶向P53基因缺陷肿瘤细胞的特异性复制，之后第二代OAV采用各种肿瘤特异性启动子调控腺病毒增殖基因E1a或/和E1b的表达，发展至第三代OAV则是多种调控元件的联合应用。经过数代、多样化的改造，OAV针对肿瘤的特异性和有效性有所提高。从理论上讲，OAV作为载体携带抗癌基因，只要有肿瘤细胞存在，就能启动病毒增殖，抗癌基因随之高拷贝复制且高效率表达，继而产生病毒增殖溶瘤和基因表达抑癌的双重肿瘤杀伤效应，而且对正常细胞不产生影响。但在实际应用中，包括OAV在内的溶瘤病毒，其肿瘤靶向特异性以及破坏肿瘤细胞的效应，还没有达到人们的期望值，临床上单独应用溶瘤病毒治疗肿瘤的效果依然不理想。此外，由于受调控元件差异和抗癌基因选择的限制，OAV个性化程度较高，难以在异质性大的不同肿瘤中均产生较好的治疗效果。
发明内容
本发明的目的是针对现有肿瘤免疫治疗对实体瘤效果不佳的难题，提供一款具有多重机制协同抗癌、能作为免疫检查点抑制剂和CAR-T/CAR-NK细胞治疗增效剂的嵌合型广谱溶瘤腺病毒及其在肿瘤治疗中的应用。
第一方面，本发明提供了一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，所述溶瘤腺病毒同时表达IL-12、IFN-γ和CCL5三个因子；所述溶瘤腺病毒其外壳蛋白Hexon由Ad5和Ad48两个血清型病毒的Hexon序列嵌合而成，其纤毛蛋白Fiber由Ad5和Ad11两个血清型病毒的Fiber序列嵌合而成；所述溶瘤腺病毒同时采用肿瘤特异性强势启动子和氧依赖元件开关，并删除Elb-55kD基因；所述溶瘤腺病毒同时敲除E1a-CR2、E1b-19kD、E3区部分蛋白编码序列。
作为本发明的一种优选实施方式，在所述溶瘤腺病毒的基因组中，IFN-γ和CCL5基因cDNA序列共有一个表达框，以人巨细胞病毒(hCMV)启动子驱动，IL-12基因cDNA序列表达框，以鼠巨 细胞病毒(mCMV)启动子驱动，两个表达框呈“脚对脚”排列。
更优选地，同时表达IL-12、IFN-γ和CCL5三个因子的所述两个表达框和启动子的整体序列如SEQ ID NO:3的第29422-33108bp所示。
作为本发明的另一种优选实施方式，所述溶瘤腺病毒其外壳蛋白Hexon的编码序列如SEQ ID NO:3的第18327-21170bp所示。
作为本发明的另一种优选实施方式，所述溶瘤腺病毒其纤毛蛋白Fiber的编码序列如SEQ ID NO:3的第33373-34356bp所示。
作为本发明的另一种优选实施方式，所述溶瘤腺病毒的全基因组序列如SEQ ID NO:3所示。
第二方面，本发明提供了一种同时表达IL-12、IFN-γ和CCL5三个因子的溶瘤腺病毒，所述溶瘤腺病毒的基因组中，IFN-γ和CCL5基因cDNA序列共有一个表达框，以人巨细胞病毒(hCMV)启动子驱动，IL-12基因cDNA序列表达框，以鼠巨细胞病毒(mCMV)启动子驱动，两个表达框呈“脚对脚”排列。
作为本发明的一种优选实施方式，同时表达IL-12、IFN-γ和CCL5三个因子的所述两个表达框和启动子的整体序列如SEQ ID NO:3的第29422-33108bp所示。
第三方面，本发明提供了如上所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒或如上所述的同时表达IL-12、IFN-γ和CCL5三个因子的溶瘤腺病毒在制备治疗和/或预防和/或辅助治疗癌症或者抗肿瘤的药物中的用途。
作为本发明的一种优选实施方式，所述癌症或者肿瘤为乳腺癌、肝癌、胆囊癌、胃癌、结肠癌、肺癌、前列腺癌、淋巴瘤、大肠癌、卵巢癌、宫颈癌、胆管癌、食管癌、肾癌、神经胶质瘤、黑色素瘤、胰腺癌、膀胱癌或头颈癌。
本发明提供第四代基于4T技术(4triple-construction techniques)的OAV设计方案，尽最大可能将OAV抗癌疗效和生物安全性提高到一个新高度。主要特点如下：
1)三重装载(Triple Transgene)：本发明提供的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，同时在腺病毒E3区装载3个抗癌基因，其中包括2个增强免疫的细胞因子基因(IL-12和IFN-γ)和1个免疫细胞趋化因子基因(CCL5)，建立OAV联合免疫治疗协同增效的直接的、主动的分子机制。以OAV为载体装载抗癌基因，有助于抗癌基因在癌细胞内特异性高拷贝复制和高效率表达。但选择装载的抗癌基因，不仅仅满足于抗癌疗效叠加的要求，更优的方案是建立能与OAV本身、肿瘤微环境、机体免疫系统互作的协同机制。肿瘤微环境的抑制状态是实体瘤免疫治疗的主要障碍，有研究证实，溶瘤病毒感染的肿瘤细胞能释放大量细胞因子，裂解的肿瘤细胞还能释放多种肿瘤相关抗原，这些细胞因子和肿瘤抗原不但具有免疫激活作用，也能改变肿瘤微环境，不仅能增强放化疗作用，同样能提高免疫检查点分子的抗体治疗和免疫细胞治疗的效果(Oncoimmunology  2015,4:e988098)。但现有溶瘤病毒提高免疫治疗的作用是有限的、缺乏直接的协同增效机制。本发明所提供的OAV，表达IL-12、IFN-γ和CCL5三个因子，具有主动的、直接的增效免疫检查点抑制剂和CAR-T/CAR-NK细胞治疗疗效的协同机制。首先，OAV装载2个增强免疫的细胞因子IL-12和IFN-γ，由于PD-1抗体、PD-L1抗体治疗时需要激活T细胞才能发挥抗癌作用，这个过程先由树突状细胞(DC)合成分泌IFN-γ和IL-12，这两个细胞因子与T细胞进行信息交流，才能激活T细胞的杀伤活性(Immunity.2018,49:1148-1161.e7)。但肿瘤微环境中常常缺乏DC，IFN-γ和IL-12分泌量不足。当OAV装载增强免疫的细胞因子IL-12和IFN-γ，在OAV与PD-1抗体联用时无需DC参与，病毒表达的IL-12和IFN-γ可直接激活微环境杀伤性T细胞而快速发挥抗癌疗效。在与CAR-T/CAR-NK连用时，OAV表达的IL-12能延长CAR-T/CAR-NK细胞存活时间，IFN-γ能增强CAR-T/CAR-NK杀伤活性。当然，IL-12和IFN-γ本身也具有很强的抗癌活性，IFN-γ是老牌的抗癌免疫因子，能诱导Interlukins、TNF等多种细胞因子表达。IL-12是肿瘤免疫治疗的新宠，能提高机体保护性免疫应答水平，促进刺激T细胞及NK细胞增殖和激活，诱导分泌IFN-γ等多种细胞因子。其次，OAV装载免疫细胞趋化因子CCL5，可趋化NK、T细胞(包括CAR-T/CAR-NK)、DC等多种免疫细胞迁移到肿瘤内积聚(Cell 2018,172:1022-1037)。在OAV与PD-1抗体联用时，CCL5可增加肿瘤微环境中NK、T细胞、DC等细胞数量，协同增强杀伤癌细胞的作用。在与CAR-T/CAR-NK连用时，CCL5既可趋化更多CAR-T/CAR-NK细胞进入肿瘤区，也能促进趋化来的NK、T细胞、DC等细胞协同增强CAR-T/CAR-NK功能。实验证实，三个因子在溶瘤病毒基因组中的排列连接顺序对其抗肿瘤活性有明显的影响，通过比较三个因子多重组合排列顺序的溶瘤病毒抑制肿瘤细胞的活性，最终确定抗癌活性最佳的VirRon中三因子排列顺序，即：VirRon装载的IFN-γ和CCL5基因cDNA序列共有一个表达框，以人巨细胞病毒(hCMV)启动子驱动，IFN-γ和CCL5基因cDNA序列间由T2A连接。VirRon装载的IL-12基因cDNA序列表达框，以鼠巨细胞病毒(mCMV)启动子驱动。两个表达框呈“脚对脚”排列。两个表达框分别选择人和鼠两个不同的巨细胞病毒(CMV)启动子驱动，可避免相同序列重复造成的不可预见的高级结构影响病毒的包装成功率和抗癌活性。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其装载的IFN-γ基因cDNA编码序列(不包含终止密码子)的反向互补序列如SEQ ID NO:3的第32263-32760bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其装载的IL-12基因cDNA编码序列如SEQ ID NO:3的第29973-31583bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其装载的CCL5基因cDNA编码序列的反向互补序列如SEQ ID NO:3的第31924-32199bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其装载的IFN-γ和CCL5基因cDNA序列共有一个表达框，IFN-γ和CCL5基因cDNA序列间由T2A连接，其T2A 序列的反向互补序列如SEQ ID NO:3的第32200-32262bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其装载的IFN-γ和CCL5基因cDNA序列共有一个表达框，以人巨细胞病毒(hCMV)启动子驱动。其hCMV启动子编码序列的反向互补序列如SEQ ID NO:3的第32767-33108bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其装载的IL-12基因cDNA序列表达框，以鼠巨细胞病毒(mCMV)启动子驱动。其mCMV启动子序列如SEQ ID NO:3的第29422-29944bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其装载的抗癌基因并不限于IL-12、IFN-γ和CCL5，还包括其他具有抗癌活性的细胞因子、免疫因子、抑癌基因、自杀基因、抗体基因等及其突变体改构序列；其多基因连接方式不限于T2A序列，还包括F2A、P2A、IRES等序列及其突变体；其采用的启动子不限于mCMV和hCMV，还包括其他具有基因驱动功能的启动子、增强子及其突变体序列。
2)三重嵌合(Triple Chimerism)：本发明提供的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，同时应用B亚属Ad11血清型的腺病毒Fiber编码序列和D亚属Ad48血清型的腺病毒Hexon编码序列，与C亚属Ad5型腺病毒骨架相应序列嵌合，构建出三个血清型嵌合的OAV，实现了在一个治疗系统上同时提高病毒对肿瘤细胞感染效率、避免病毒被机体预存中和抗体拦截、降低机体肝细胞对病毒的粘附摄取三大优势。人类腺病毒家族有52个血清型，分6个亚属(A至F)。除了B组以外，各组腺病毒都用柯萨奇病毒腺病毒共用受体(Coxsackievirus-adenovirus receptor，CAR)作为其主要吸附受体，对缺乏CAR的骨髓造血细胞、造血干细胞、树突状细胞、部分肿瘤细胞尤其是肿瘤干细胞等感染效率很低。B组腺病毒(Ad3、Ad7、Ad11、Ad14、Ad16、Ad21、Ad34-35、Ad50、Ad55等)则主要识别一种广泛表达的补体调节蛋白CD46。采用B组腺病毒的纤毛蛋白(fiber knob)替代Ad5纤毛蛋白，构建嵌合病毒，使之增加识别CD46受体的能力，则有利于提高病毒对肿瘤细胞感染效率，特别是感染肿瘤干细胞的能力，有可能更彻底地根除肿瘤复发的根源；Ad5在自然界存在较广，大多数人已被感染并产生了中和抗体，能够拦截相同血清型的病毒。况且Ad5具有嗜肝细胞性，能够被肝细胞吸附。腺病毒外壳蛋白Hexon的高变区(HVR)因位置暴露于腺病毒表面，是导致不同血清型腺病毒之间对肝脏感染能力和免疫原性差异的关键部位。将Ad5的Hexon分子内7个HVR(L1：HVR1-6；L2：HVR7)选择性地与稀有血清型如D亚群(Ad8-10、Ad25-30、Ad36-39、Ad42-49、Ad51等)病毒的Hexon相应区域进行嵌合，是帮助Ad5逃避预存免疫和肝脏摄取的有效方法。然而，Hexon作为腺病毒的主要结构蛋白，对其进行改造往往会导致腺病毒载体结构不稳定，从而无法有效地包装病毒，因此这是一项很具有挑战性的研究。我们前期通过深入研究，设计多个Ad5和Ad48血清型病毒的Hexon编码序列片段进行组合，进行病毒重组包装和扩增测试，终于攻克了这项技术难关，构建 成功多个Hexon替代的嵌合型腺病毒载体骨架，能有效包装目的病毒颗粒。Ad48腺病毒Hexon的嵌合则能保证OAV逃避机体预存中和抗体的拦截以及肝细胞对腺病毒的粘附和摄取。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其B亚属Ad11血清型腺病毒纤毛蛋白(Fiber)编码序列与C亚属Ad5型腺病毒Fiber相应序列嵌合的嵌合型纤毛蛋白(Ad5F11b)的序列如SEQ ID NO:3的第33373-34356bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其D亚属Ad48血清型腺病毒六邻体(Hexon)编码序列与C亚属Ad5型腺病毒Hexon相应序列嵌合的嵌合型Hexon(Ad5H48)的序列如SEQ ID NO:3的第18327-21170bp所示。
本发明所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其血清型嵌合并不限于C亚属Ad5与B亚属Ad11、D亚属Ad48的嵌合，还包括其他亚属其他血清型的相互嵌合改造。
3)三重调控(Triple Regulation)：本发明提供的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，同时采用肿瘤特异性强势启动子和氧依赖元件开关，并删除Elb-55kD基因，构建在转录和翻译水平上具有双重安全调控机制的OAV产品。实现OAV对肿瘤细胞特异性增殖复制的机制之一，是以肿瘤特异性启动子作为顺式元件控制腺病毒增殖必需基因的表达。通过研究腺病毒E1a、E1b与癌细胞分子互作的机制，在启动子、增强子调控元件中优选调控模块组合，由肿瘤特异性广谱启动子在转录水平上控制腺病毒E1a基因只在肿瘤细胞内表达；E1a末端融合氧依赖元件开关，E1a蛋白在肿瘤缺氧环境中被保护，而在正常组织常氧状态下则被蛋白酶体降解，在翻译水平上有效防止E1a蛋白在正常细胞中表达的渗漏；Elb转录单元编码Elb-55kD和Elb-19kD，Elb-55kD是腺病毒在正常细胞增殖复制必需的而在肿瘤细胞中非必需的蛋白，Elb-55kD编码基因的选择性缺失可以使腺病毒在肿瘤细胞内保持增殖复制的能力，而在正常细胞中失去复制能力。Elb-55kD蛋白能够灭活并降解P53蛋白，Elb-55kD缺失有利于细胞保持P53的抗肿瘤活性，同时提高病毒载体的靶向性。因此在上述调控机制的三重控制下，VirRon的安全性和有效性得到提高。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其肿瘤特异性广谱启动子核心序列截取于BIRC5基因5’-UTR区域转录起始位点“-857～+4bp”片段，全长序列如SEQ ID NO:3的第467-1388bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon及其应用，其氧依赖元件ON/OFF开关序列如SEQ ID NO:3的第2376-2423bp所示。
我们前期已克隆、研究、保存了20多种肿瘤特异性的基因启动子、增强子及其突变体序列，本发明所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其调控元件包括Ki67、hTERT、CEA、AFP、EGFR、DF3/MUC1、VEGFR、E2F、GFAP、Survivin等因子的启动子、增强子及其突变体序列，还有HIF-1缺氧反应元件(HRE)、Egr-l辐射敏感元件(CArG)、hTERT内部E-box、氧 依赖开关ODD等调控序列，以及上述序列的组合应用。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，删除其Elb转录单元中Elb-19kD全部编码序列以及Elb-55kD编码序列第1个密码子到437个密码子，Elb-55kD剩余编码序列如SEQ ID NO:3的第2818-2997bp所示。
4)三重改造(Triple Modification)：本发明提供的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，同时敲除E1a-CR2、E1b-19kD、E3区部分蛋白编码序列。OAV构建以致病力最弱的人类腺病毒C亚属中的5型血清型(Ad5)为基础，保留E1a蛋白表达并使其受肿瘤特异性启动子调控，实现针对肿瘤的特异性复制。E1a有3个功能区，即CR1、CR2、CR3，CR1区通过与转录调节因子P300/CBP结合能抑制Her-2/neu基因的表达，CR2区与Rb蛋白家族结合，CR3区是转录活化区。因此E1a蛋白本身就具有抗肿瘤作用，既可以通过抑制Her-2/neu基因的转录、阻断NF-κB的活性、提高p53的表达，又可以抑制Ⅳ型胶原酶、纤溶酶原激活剂等蛋白酶基因表达；E1a还能引起非特异性免疫反应，提高CTL细胞、NK细胞、巨噬细胞的杀伤效应等多种途径，诱导肿瘤细胞凋亡，抑制肿瘤侵袭转移，提高肿瘤细胞对化疗、放疗的敏感性。对E1a的CR2区引入12个碱基的缺失突变，使其不能结合Rb蛋白，保证去磷酸化的Rb蛋白与转录因子E2F形成复合物，阻断E2F的转录活性，可以释放E1a内在抗癌机制，增强产品抗癌活性；Elb-19kDa基因与凋亡抑制基因Bcl-2同源，Elb-19kD蛋白能够结合Bax或/和Bak启动下游的凋亡抑制程序，保护感染的细胞免受TNF-α介导的杀伤作用。Elb-19kD缺失，因而该病毒突变体在肿瘤细胞内增殖的特异性得以提高，而在正常细胞内的增殖活性被削弱。Elb-19kD缺失能够促进癌细胞凋亡通路的恢复，且有利于病毒在正常细胞内的快速清除以及在肿瘤细胞内的快速释放和播散，使OAV特异性更好，效能更强；E3转录单位有9个开放读框，编码蛋白保护感染细胞免受宿主免疫反应的杀伤，E3-gpl9k可减弱CTL介导的感染细胞杀伤效应；RID可阻断“死亡”配体包括TNF、Fas配体和TRAIL介导的细胞凋亡；RID还可抑制IL-1和TNF介导的细胞存活所必须的NF-κB的激活；E3-6.7k除了使TRAIL受体下调之外还可单独抑制外部和内部信号通路诱导的细胞凋亡；E3-14.7k是TNF介导细胞凋亡的广泛抑制剂，也可结合并抑制Caspases-8从而阻止Fas信号通路启动的细胞凋亡；E3-14.7k与受感染的细胞内FIP蛋白(for 14.7K-interacting protein，FIP-1、-2、-3)作用，可使E3-14.7k蛋白在细胞凋亡和存活、炎症反应、维持膜稳定、核浆转运等信号转导通路中处于重要地位，这一多功能的E3蛋白的分子机制仍需深入研究。腺病毒死亡蛋白(ADP)可促进细胞溶解以及病毒释放，然而分子机制不明。由此可见，在OAV构建过程中删除E3区同时保留ADP基因，既可以扩大载体容量，又能够促进受感染的癌细胞的凋亡，并有利于促进OAV溶解癌细胞以及子代病毒释放。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，敲除其E1a-CR2中12个碱基“cacgaggctggc”，并且突变型E1a(mE1a)序列终止密码子前插入氧依赖元件开关，其 序列如SEQ ID NO:3的第1405-2426bp所示。
本发明所述一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其病毒结构蛋白的改造不限于E1a-CR2、E1b-19kD、E3区部分蛋白编码序列的敲除，还包括E1a、E1b、E3以及其他蛋白的缺失、插入、突变等改造。
综上所述，本发明所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，是全新一代OAV产品，实现了真正意义的多重机制协同增效的目的，特异性好，抗癌活性强，对多种实体瘤具有明显的治疗效果，克服了以往溶瘤腺病毒单独使用疗效不佳和抗瘤谱窄的问题，既具有单独应用的良好抗癌效果，也能作为免疫检查点抑制剂和CAR-T/CAR-NK细胞治疗增效剂，协同免疫治疗大幅提高实体瘤的疗效。其创新性意义如下：
1、多重机制精准调控OAV增殖提高其安全性和有效性：以肿瘤特异性广谱启动子控制腺病毒增殖基因E1a表达，在转录水平增强OAV针对肿瘤细胞的特异性复制活力；在E1a蛋白内部融合氧依赖元件开关，保证E1a在正常组织常氧状态下被降解，在翻译水平限制病毒在正常细胞内复制；选择性缺失Elb-55kD可使腺病毒在肿瘤细胞内保持增殖复制的能力，而在正常细胞中失去复制能力；Elb-19kD缺失能够促进癌细胞凋亡通路的恢复，有利于病毒在正常细胞内的快速清除，在肿瘤细胞内的快速释放；E3区的部分编码蛋白敲除和ADP基因保留，达到保护腺病毒长期生存并介导抗癌基因高效表达的目的。多重机制协同，共同提高OAV安全性和有效性。
2、激活病毒内在抗肿瘤机制协同提高OAV溶瘤作用和抗癌疗效：腺病毒E1a蛋白本身即具有抗肿瘤作用，既可以通过抑制Her-2/neu基因的转录、阻断NF-κB的活性、提高p53的表达，又可以抑制Ⅳ型胶原酶、纤溶酶原激活剂等蛋白酶基因表达；E1a还能引起非特异性免疫反应，提高CTL、NK、巨噬细胞的杀伤效应等多种途径，诱导肿瘤细胞凋亡，抑制肿瘤侵袭转移，提高肿瘤细胞对化疗、放疗的敏感性。在此基础上，对E1a的CR2区引入缺失突变，使其不能结合Rb蛋白，保证去磷酸化的Rb蛋白与转录因子E2F形成复合物，阻断E2F的转录活性，可以增强抗癌活性。特别是OAV感染癌细胞释放大量细胞因子，裂解癌细胞释放大量肿瘤相关抗原，发挥进一步的免疫激活作用。局限于癌细胞表达的病毒蛋白，类似于肿瘤特异抗原，还能发挥抗癌疫苗的功能。
3、三个血清型腺病毒嵌合形成的OAV提高其感染力并能有效逃避免疫拦截和肝脏摄取：采用B组腺病毒纤毛蛋白(fiber knob)与Ad5纤毛蛋白嵌合，有利于提高病毒对癌细胞特别是对肿瘤干细胞的感染效率。将Ad5的Hexon分子内HVR区选择性地与D亚群病毒的Hexon相应区域嵌合，有助于病毒逃避人体中和抗体拦截和肝细胞摄取。
4、建立免疫调节肿瘤微环境的多机制协同增效的治疗策略并在研究中获得明确疗效：以OAV为载体装载抗癌基因，有助于抗癌基因随病毒增殖在癌细胞内特异性高拷贝复制和高效率表达。通过表达能激活肿瘤微环境免疫的抗癌基因或免疫调节基因，研制多机制协同抗癌的高效OAV新产品， 能够与免疫检查点抑制剂治疗和免疫细胞治疗等多种治疗手段具有协同增效，在提高病毒安全性和有效性方面实现多重重大突破，达到广谱、特异、安全、高效的抗癌效果。
附图说明
图1.腺病毒右臂骨架质粒pPE3F11bH48-RC(+)结构图。
图2.腺病毒左臂穿梭质粒pAdSVP-mE1aODD结构图。
图3.外源基因穿梭质粒pENTR12-C3结构图。
图4.溶瘤腺病毒VirRon基因组结构图。
图5.溶瘤病毒VirRon在肝癌、乳腺癌、胆囊癌、肺癌细胞系增殖活性高，增殖倍数在感染后96h介于10 4和10 6之间，在正常细胞系中增殖力很弱。
图6.溶瘤病毒VirRon对细胞杀伤作用的曲线图，显示VirRon在较低MOI值时即对多种实体瘤细胞均有明显的杀伤抑制作用，而对正常成纤维细胞BJ无杀伤活性。
图7.溶瘤病毒VirRon对细胞杀伤作用的IC50值比较，显示VirRon对多种实体瘤细胞杀伤抑制作用的IC50值较低，而对正常成纤维细胞BJ杀伤的IC50值高达1090。
图8.溶瘤病毒VirRon表达“IL-12+IFN-γ+CCL5”三个抗癌基因，随时间延长表达量逐渐增高。
图9.溶瘤腺病毒VirRon中三个抗癌基因排列连接顺序对其包装效率和抗癌活性的影响。
图10.溶瘤腺病毒VirRon中嵌合型Hexon(Ad5H48)片段组合对其包装和扩增效率的影响。
图11.溶瘤病毒VirRon抗肝癌HCCLM3裸鼠移植瘤的实验。
图12.溶瘤病毒VirRon抗乳腺癌MDA-MB-231裸鼠移植瘤的实验。
图13.溶瘤病毒VirRon抗胆囊癌SGC996裸鼠移植瘤的实验。
图14.VirRon联合PD-1抗体治疗结肠癌移植瘤的人源化小鼠模型实验。
图15.VirRon联合CAR-T细胞治疗前列腺癌移植瘤的NCG小鼠模型实验。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1 本发明的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon的研制
本发明所述的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，由腺病毒右臂质粒、腺病毒左臂质粒和外源基因穿梭质粒重组而成。现介绍这3个质粒及重组过程，具体如下：
(1)腺病毒右臂骨架质粒
腺病毒右臂骨架质粒pPE3F11bH48-RC(+)由加拿大Microbix Biosystems公司提供的pBHGlox(delta)E13Cre质粒(Catalog no.PD-01-40)和pBHGE3质粒(Catalog no.PD-01-12)改建而来。将pBHGE3含有部分E3区序列的SpeI+NotI酶切片段以及合成的ADP基因序列(如SEQ ID NO:3的第30901-31182bp所示)，插入到pBHGlox(delta)E13Cre的SpeI+PacI位点间，构建pPE3质粒。将 合成的attR1+ccdB+attR2序列(949bp，SEQ ID NO:1)插入到pPE3的PacI位点，将合成的Ad5F11b片段替换Ad5-Fiber序列，将合成的Ad5H48片段替换Ad5-Hexon序列，最终构建pPE3F11bH48-RC(+)质粒(图1)。pPE3F11bH48-RC(+)全长36591bp，其纤毛蛋白(Ad5F11b)由Ad5和Ad11血清型腺病毒相应序列嵌合而成(如SEQ ID NO:3的第33373-34356bp所示)，其六邻体蛋白(Ad5H48)由Ad5和Ad48血清型腺病毒相应序列嵌合而成(如SEQ ID NO:3的第18327-21170bp所示)。
(2)腺病毒左臂穿梭质粒
腺病毒左臂穿梭质粒pAdSVP-mE1aODD由加拿大Microbix Biosystems公司提供的pXC1质粒(Catalog no.PD-01-03)改建而来。在pXC1的E1a起始密码子上游12bp处引入BglII酶切位点，构建pXC2。合成BIRC5基因5’-UTR区域转录起始位点“-857～+4bp”片段、突变型E1a及氧依赖元件开关序列(mE1a-ODD)，共2355bp(如SEQ ID NO:3的第467-2821bp所示)，插入到pXC2的2个BglII位点之间，替换pXC2中的E1a和E1b序列，构建成pAdSVP-mE1aODD，全长9402bp(图2)。
(3)外源基因穿梭质粒
穿梭质粒pENTR12由美国Invitrogen公司提供的pENTR11质粒(Catalog no.11819-018)改建而来。在pENTR11的NcoI和EcoRV酶切位点之间引入“mCMV启动子+SpeI/EcoRI/HindIII/BglII/NheI/BamHI/SalI+Poly(A)”序列(SEQ ID NO:2)，替换原pENTR11质粒中的多克隆位点和ccdB基因序列，构建成新的质粒pENTR12。在pENTR12多克隆位点EcoRI和EcoRV之间插入IL-12表达框和IFN-γ+CCL5表达框，双表达框呈“脚对脚”排列，构建成外源基因穿梭质粒pENTR12-C3，全长5985bp(图3)。最终由pENTR12-C3转移到VirRon基因组中包含IL-12表达框和IFN-γ+CCL5表达框的NcoI和EcoRV位点之间的完整序列(含位点序列)如SEQ ID NO:3的第29401-33114bp所示。
(4)VirRon病毒的重组包装程序
VirRon病毒的重组包装以腺病毒右臂质粒pPE3F11bH48-RC(+)为骨架，经两轮特异性重组包装而成。首先用外源基因穿梭质粒pENTR12-C3与腺病毒右臂骨架质粒pPE3F11bH48-RC(+)在DB3.1大肠杆菌感受态细菌内发生第一轮attL/attR位点特异性重组，将pENTR12-C3中的外源基因表达框转移到pPE3F11bH48-RC(+)的E3区；然后再用腺病毒左臂穿梭质粒pAdSVP-mE1aODD与第一轮重组的产物在293T细胞内发生第二轮序列特异性重组，将pAdSVP-mE1aODD中的肿瘤特异性启动子控制的mE1aODD表达框转移到腺病毒右臂骨架质粒的E1区。经过两轮特异性重组，准确快速地包装出理想的溶瘤腺病毒VirRon。溶瘤腺病毒VirRon结构图如图4所示。本发明所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，其完整序列如SEQ ID NO:3所示。
实施例2 本发明的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon的细胞学实验
本发明所述的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon，能够特异性地 在肿瘤细胞内增殖复制，介导抗癌基因高效表达，最后破坏或抑制肿瘤细胞。
(1)溶瘤腺病毒VirRon的特异性增殖复制
收集对数生长期的实体瘤细胞系以及正常细胞系，铺96孔板，1×10 4/孔，细胞贴壁后，换用无血清培养液；以MOI＝5pfu/cell加入VirRon进行细胞感染。病毒感染2h后换5％血清培养液，继续培养0h、48h、96h后，收集0h、48h、96h时间点的细胞及上清液，TCID50方法检测病毒滴度，以0h为基准，计算病毒增殖倍数。结果发现，VirRon在包括肝癌、乳腺癌、胆囊癌、肺癌等多数实体瘤细胞系中呈高拷贝增殖，增殖倍数最高可达316978.64倍；而在正常细胞系中增殖活性较低，在GES-1细胞中完全不增殖，在肝细胞L02中有较弱增殖活性，增殖倍数仅为399.05倍(图5)。
(2)溶瘤腺病毒VirRon对肿瘤细胞的特异性杀伤抑制
通过CCK8实验检测溶瘤腺病毒VirRon对肿瘤细胞和正常细胞的特异性杀伤抑制。收集对数生长期的肿瘤细胞系以及正常细胞系，铺96孔板，1×10 4/孔，细胞贴壁后，换用无血清培养液；以梯度MOI加入VirRon进行细胞感染，对应于每个MOI值设8个复孔，孵箱内培养2h；换血清培养液100μl/孔，培养48h后，弃培养液，加入CCK8溶液，置孵箱内4h，酶标仪测定490nm波长光吸收值，计算细胞存活率，绘制细胞生存曲线。结果发现，VirRon对多种实体瘤细胞均有明显的杀伤抑制作用，并且与病毒感染强度密切相关；同一类型肿瘤的不同细胞系对VirRon的敏感性有所不同，VirRon对肺癌H460细胞的杀伤IC50值仅有6.656pfu/cell。VirRon对正常成纤维细胞BJ无明显杀伤活性，其IC50值高达1091pfu/cell(图6-7，表1)。
表1.溶瘤腺病毒VirRon对培养细胞的杀伤作用(IC50对比)
  细胞系 IC50(pfu/cell)
乳腺癌 MDA-MB-231 82.04
  MDA-MB-453 10.52
  MCF-7 16.72
肝癌 Hep3B 28.94
  MHCC97L 46.43
  MHCC97H 82.09
  HCCLM3 9.85
胆囊癌 NOZ 16.01
  GBC-SD 79.41
胃癌 MKN45 189.6
  BGC823 11.40
  SNU-1 47.23
结肠癌 SW480 21.22
  SW620 113.4
  Caco2 104.7
肺癌 H1299 12.65
  H460 6.66
  A549 16.66
正常细胞 BJ 1091
(3)溶瘤腺病毒VirRon介导抗癌基因的表达
收集对数生长期的实体瘤细胞系以及正常细胞系，铺6孔板，1×10 6/孔，24h细胞贴壁后，换用无血清培养液；以MOI＝5pfu/cell的感染强度加入VirRon进行细胞感染。继续培养24h、48h、72h后，收集上清，通过ELISA方法检测IL-12、IFN-γ、CCL5基因蛋白表达情况。结果发现，VirRon能够介导IFN-γ、IL-12、CCL5蛋白高效表达，并且随时间延长表达量逐渐增高(图8)。
(4)溶瘤腺病毒中抗癌基因排列连接顺序对其包装效率和抗癌活性的影响
溶瘤腺病毒VirRon装载三个抗癌免疫因子IL-12、IFN-γ、CCL5，三个因子的排列连接顺序对病毒包装成功率和抗癌活性都有明显影响。通过设计构建三个因子不同排列顺序的穿梭质粒(V1～V4)，与腺病毒骨架质粒重组，在HEK293细胞中包装溶瘤腺病毒(图9A)。结果发现，V1观察至第13天，细胞未出现噬斑病变，病毒包装不成功；V2和V3在第11天、V4在第7天分别出现噬斑，病毒重组成功(图9B)。在噬斑出现后48h收集细胞上清，ELISA检测三个因子的表达，V2、V3、V4表达IL-12和CCL5水平较高，但V2表达IFN-γ表达量极低，显著低于V3和V4(图9C)。培养肺癌A549细胞，以梯度(MOI＝0.01～1000pfu/cell)感染强度感染V2、V3、V4病毒，采用CCK-8试剂盒检测72h病毒对细胞增殖活性的影响，证实V4对A549细胞的抑制效应最明显(图9D)。最终确定V4(即VirRon)中三个因子的排列连接顺序既有利于病毒的重组包装和基因表达，也有益于提高病毒的抗癌活性。
(5)溶瘤腺病毒中嵌合型Hexon(Ad5H48)片段组合对其包装和扩增效率的影响
溶瘤腺病毒VirRon的外壳蛋白Hexon(Ad5H48)由Ad5和Ad48两个血清型病毒的Hexon序列嵌合而成，目的是避免体内预存Ad5型病毒中和抗体的拦截和肝脏对病毒的吸附。Ad5H48编码cDNA全长2844bp，我们设计了3种不同片段组合的嵌合方式，在HEK293细胞中进行病毒重组包装和扩增(图10A)。实验发现，Ad5H48-1(即VirRon)在第7天出现细胞噬斑，Ad5H48-2观察至第14天未出现噬斑病变，Ad5H48-3在第12天出现噬斑，证实Ad5H48-1(即VirRon)出毒最快(图10B)。Ad5H48-1和Ad5H48-3以MOI＝10pfu/cell感染HEK293细胞，96h后分别收集细胞和上清，TCID50法检测病毒滴度，无论是细胞内还是上清中Ad5H48-1滴度都高于Ad5H48-3(图10C)。最终证实Ad5H48-1(即VirRon)包装和扩增效率最佳。
实施例3 本发明的一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒VirRon的动物学实验
(1)VirRon抗肝癌的动物模型实验
健康纯种BALB/C裸鼠30只，4周龄，雄性，上海诚勤生物科技有限公司提供，合格证号SCXK(沪)2012-0002。取对数生长期肝癌HCCLM3细胞悬液注射于裸鼠右腋皮下，5×10 6细胞数/100μl/ 只。接种后12天，成瘤率100％，移植瘤最大最小直径平均约5.0mm。随机分为3组(VirRon组、AdSVP-H48-DsRed组、空白对照组)，每组10只。每组首先剔除瘤体最大和最小的小鼠各一只，其余8只进入实验治疗性观察。2个病毒治疗组给予相应腺病毒瘤内多点注射，每次每只剂量3×10 8pfu/100μl，隔天一次，共5次；空白对照组同步给予病毒保存液，每次每只100μl。治疗后，定期测量瘤体大小，以“最大径×最小径 2×0.5”公式计算瘤体体积，绘制生长曲线。实验过程中，任意一组瘤体体积超过动物实验伦理委员会允许的肿瘤体积上限2000mm 3时，即终止实验观察。结果发现，实验观察至治疗后28天，VirRon治疗组抑瘤率为65.51％，与对照组有明显差异(P＝0.0001)；对照病毒为未携带治疗基因但插入红色荧光报告基因的AdSVP-H48-DsRed，其也产生明显的溶瘤作用，抑瘤率45.91％，与对照组相比差异有统计学意义(P＝0.01)(图11)。
(2)VirRon抗乳腺癌的动物模型实验
健康纯种BALB/C裸鼠30只，4周龄，雌性，上海诚勤生物科技有限公司提供，合格证号SCXK(沪)2012-0002。取对数生长期乳腺癌MDA-MB-231细胞悬液注射于裸鼠右腋皮下，5×10 6细胞数/100μl/只。接种后10天，成瘤率100％，移植瘤最大最小直径平均约3.0mm。剔除瘤体最大的3只、瘤体最小的2只，其余25只随机分为5组(VirRon低剂量组、VirRon中剂量组、VirRon高剂量组、AdSVP-H48组、空白对照组)，每组5只。病毒治疗组给予相应腺病毒瘤内多点注射，高剂量组每次每只剂量3×10 8pfu/100μl，中剂量组每次每只剂量2×10 8pfu/100μl，低剂量组每次每只剂量1×10 8pfu/100μl，隔天一次，共5次；AdSVP-H48组为中剂量注射，空白对照组同步给予病毒保存液，每次每只100μl。治疗后，定期测量瘤体大小，以“最大径×最小径 2×0.5”公式计算瘤体体积，绘制生长曲线。实验过程中，任意一组瘤体体积超过动物实验伦理委员会允许的肿瘤体积上限2000mm 3时，即终止实验观察。结果发现，实验观察至治疗后30天，VirRon高、中、低剂量组组抑瘤率分别为74.15％、52.80％、40.66％，未携带治疗基因的对照病毒AdSVP-H48抑瘤率仅33.38％(图12)。
(3)VirRon抗胆囊癌的动物模型实验
胆囊癌恶性度高、进展速度快，除手术外临床无治疗措施。通过建立SGC-996快速增长的移植瘤模型，观察VirRon治疗的疗效及协同机制。健康纯种BALB/C裸鼠40只，4周龄，雄性，上海诚勤生物科技有限公司提供，合格证号SCXK(沪)2012-0002。取对数生长期胆囊癌SGC-996细胞悬液注射于裸鼠右腋皮下，5×10 6细胞数/100μl/只。接种后14天，成瘤率100％，移植瘤最大最小直径平均约5.0mm。随机分为4组(VirRon组、AdSVP-H48空载病毒组、Ad5-C3非增殖病毒组、空白对照组)，每组10只。每组首先剔除瘤体最大的小鼠各一只，其余9只进入实验治疗性观察。VirRon组、AdSVP-H48组和Ad5-C3组给予相应病毒瘤内多点注射，每次每只剂量2×10 8pfu/100μl，隔天一次，共5次；空白对照组同步给予病毒保存液，每次每只100μl。治疗后，定期测量瘤体大小，以“最大径×最小径 2×0.5”公式计算瘤体体积，绘制生长曲线。结果发现，实验观察至治疗后44天，VirRon 治疗组抑瘤率为49.47％，与对照组比较有明显差异(P＝0.0241)；未携带治疗基因的空载对照溶瘤腺病毒AdSVP-H48和表达三个细胞因子IL-12、IFN-γ、CCL5的非增殖型对照腺病毒Ad5-C3的抑瘤率仅为24.03％和15.26％，统计显示药物协同作用的Q值为1.39，证实VirRon发挥出了AdSVP-H48病毒的溶瘤作用和细胞因子的抗癌作用的协同增效作用(图13)。
(4)VirRon联合PD-1抗体治疗结肠癌移植瘤的人源化小鼠模型实验
健康C57-hPD1人源化小鼠，雌性，5-6周龄，46只，购自北京百奥赛图基因生物技术有限公司，右侧背部皮下接种小鼠结肠癌细胞系MC38细胞1×10 6细胞数/只，肿瘤平均长到50mm 3-80mm 3时，剔除瘤体最大的3只，瘤体最小的3只，其余40只随机分成4组(空白对照、VirRon单药、Keytruda单药，联合用药组)，开始给药治疗。VirRon瘤内多点注射，每次每只剂量3×10 8pfu/100μl，隔天一次，共5次；Keytruda腹腔注射给药，每次每只2.5mg/kg，每周2次，共6次。空白对照组同步给予病毒保存液，每次每只100μl。治疗后，定期测量瘤体大小，以“最大径×最小径 2×0.5”公式计算瘤体体积，绘制生长曲线。至首次治疗后18天，VirRon单药组抑瘤率18.71％、Keytruda单药组抑瘤率69.05％，联合用药组抑瘤率88.86％(图14)。VirRon+Keytruda联合用药组与VirRon单药组、Keytruda单药组、对照组相比均有显著差异(P＝0.0013、P＝0.0110、P＝0.0004)，统计显示药物协同作用的Q值为1.19，证实VirRon与Keytruda联合用药具有协同增效的作用。
(5)VirRon联合CAR-T细胞治疗前列腺癌移植瘤的NCG小鼠模型实验
CAR-T细胞由徐州医科大学肿瘤研究所制备，靶点为B7-H3。健康NCG小鼠，雄性，4-5周龄，19只，购自江苏集萃药康生物科技股份有限公司，右侧背部皮下接种前列腺癌细胞系Du145细胞3×10 6细胞数/只，肿瘤平均长到50mm 3左右时，随机分成4组(空白对照、VirRon单药、CAR-T单药，联合用药组)，每组5只(CAR-T单药组4只)，开始给药治疗。VirRon每只小鼠总剂量1×10 9pfu，分3次瘤内注射，隔天一次，共5次；完成病毒注射后第二天，给予CAR-T尾静脉注射，每只2×10 6细胞数。空白对照组同步给予PBS缓冲液。治疗后，定期测量瘤体大小，以“最大径×最小径 2×0.5”公式计算瘤体体积，绘制生长曲线。至首次治疗后43天，VirRon单药组抑瘤率80.19％、CAR-T单药组抑瘤率48.83％，联合用药组抑瘤率高达97.56％。VirRon+CAR-T联合用药组与VirRon单药组、CAR-T单药组、对照组相比均有显著差异(P＝0.0008、P＝0.0000、P＝0.0000)(图15左)，证实VirRon与CAR-T联合用药能提高疗效。观察期结束，抽取小鼠血液，流式细胞术检测CAR-T细胞含量，结果发现，VirRon与CAR-T联合时，VirRon能明显延长CAR-T存活时间，提高血液中CAR-T细胞数量(图15右)。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (10)

  1. 一款多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其特征在于，所述溶瘤腺病毒同时表达IL-12、IFN-γ和CCL5三个因子；所述溶瘤腺病毒其外壳蛋白Hexon由Ad5和Ad48两个血清型病毒的Hexon序列嵌合而成，其纤毛蛋白Fiber由Ad5和Ad11两个血清型病毒的Fiber序列嵌合而成；所述溶瘤腺病毒同时采用肿瘤特异性强势启动子和氧依赖元件开关，并删除Elb-55kD基因；所述溶瘤腺病毒同时敲除E1a-CR2、E1b-19kD、E3区部分蛋白编码序列。
  2. 根据权利要求1所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其特征在于，在所述溶瘤腺病毒的基因组中，IFN-γ和CCL5基因cDNA序列共有一个表达框，以人巨细胞病毒(hCMV)启动子驱动，IL-12基因cDNA序列表达框，以鼠巨细胞病毒(mCMV)启动子驱动，两个表达框呈“脚对脚”排列。
  3. 根据权利要求2所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其特征在于，同时表达IL-12、IFN-γ和CCL5三个因子的所述两个表达框和启动子的整体序列如SEQ ID NO:3的第29422-33108bp所示。
  4. 根据权利要求1所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其特征在于，所述溶瘤腺病毒其外壳蛋白Hexon的编码序列如SEQ ID NO:3的第18327-21170bp所示。
  5. 根据权利要求1所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其特征在于，所述溶瘤腺病毒其纤毛蛋白Fiber的编码序列如SEQ ID NO:3的第33373-34356bp所示。
  6. 根据权利要求1所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒，其特征在于，所述溶瘤腺病毒的全基因组序列如SEQ ID NO:3所示。
  7. 一款同时表达IL-12、IFN-γ和CCL5三个因子的溶瘤腺病毒，其特征在于，所述溶瘤腺病毒的基因组中，IFN-γ和CCL5基因cDNA序列共有一个表达框，以人巨细胞病毒(hCMV)启动子驱动，IL-12基因cDNA序列表达框，以鼠巨细胞病毒(mCMV)启动子驱动，两个表达框呈“脚对脚”排列。
  8. 根据权利要求7所述的同时表达IL-12、IFN-γ和CCL5三个因子的溶瘤腺病毒，其特征在于，同时表达IL-12、IFN-γ和CCL5三个因子的所述两个表达框和启动子的整体序列如SEQ ID NO:3的第29422-33108bp所示。
  9. 权利要求1所述的多重机制协同和增效免疫治疗的嵌合型广谱溶瘤腺病毒或权利要求7所述的同时表达IL-12、IFN-γ和CCL5三个因子的溶瘤腺病毒在制备治疗和/或预防和/或辅助治疗癌症或者抗肿瘤的药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述癌症或者肿瘤为乳腺癌、肝癌、胆囊癌、胃癌、结肠癌、肺癌、前列腺癌、淋巴瘤、大肠癌、卵巢癌、宫颈癌、胆管癌、食管癌、肾癌、神经胶 质瘤、黑色素瘤、胰腺癌、膀胱癌或头颈癌。
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