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Oncolytic viruses: What are difficulties and challenges in tumor treatments?

Oncolytic viruses: What are difficulties and challenges in tumor treatments?


Oncolytic viruses: What are difficulties and challenges in tumor treatments? As a new type of tumor therapy, oncolytic virus has shown good application prospects in preclinical and clinical research.

However, oncolytic viruses still have certain limitations, including oncolytic efficacy, tumor targeting, biosafety, etc. The difficulties and challenges encountered in oncolytic virus therapy are described below.


Spread and infiltrate

In cancer, the intracellular connections of epithelial cells are barriers to the penetration of high-molecular-weight therapeutics, which can trigger drug resistance. In addition, in the process of metastasis, epithelial-mesenchymal transition and mesenchymal-epithelial transition, the phenotype changes, making the epithelial connection tight, which brings difficulties to treatment.

The epithelial junction is also a barrier to prevent intracellular penetration of OV, especially adenovirus. Some adenoviruses, including HAdV-B3, B14 and B14p, may release pentadiene dodecahedron (Pt-Dd) to overcome the connection in the early stages of infection and before cell division. Adenoviruses that do not produce Pt-Dd will be in the same The stage produces a lot of fibrin. However, HAdV-C5 is still the most commonly used serotype to construct oncolytic adenoviruses and does not release Pt-Dd. Yumul et al.’s modified Ad5Δ24 can produce epithelial junction opener (JO).

Compared with unmodified virus, oncolytic Ad expressing JO has stronger anti-tumor effect; joint application of JO and unmodified oncolytic adenovirus It can inhibit tumor growth better than injecting virus alone. JO is a HAdV-B3 fiber ball containing self-dimeric recombinant protein, in which the C-terminus of the fiber ball is modified to increase its affinity with desmoprotein 2 (DSG2).

DSG2 is a member of the cadherin family involved in cell-to-cell connections and is overexpressed in epithelial tumors. After JO binds to DSG2, the signal pathway activates the matrix metalloproteinase ADAM17, causing the division of the extracellular domain of DSG2 and the separation of epithelial cells.


Extracellular matrix (ECM) is composed of proteoglycans, which can hinder the spread of tumor factors in tumors. ECM degrading enzymes such as relaxin, matrix metalloproteinase (MMP)-1, -8, -9, chondroitinase and hyaluronidase are co-administered with OV, or induced in GLV-1h255 (VACV) and VCN-01 Their gene expression (OAdV) can increase the spread of OV to TME and improve the efficiency of OV treatment in cancer (Figure 1).

As mentioned above, the connection between ECM and cells is the main obstacle to the spread and spread of OV. In addition to proteases, cancer cell apoptosis also promotes the spread of viruses. Nagano et al. reported that cytotoxic drugs induced apoptosis and caspase-8 activation led to increased intratumoral penetration, thereby improving the anti-tumor efficacy of oncolytic HSV; they believed that the shrinkage or removal of apoptotic cancer cells produced channel-like structures and voids , To promote the spread of oncolytic HSV.


Oncolytic viruses: What are difficulties and challenges in treatments?

Figure 1 Strategies to avoid obstacles to OV clinical trials

(A) Promote the spread of the virus in the tumor. OV expressing hyaluronidase (HD) can decompose HA in ECM and enhance the spread of OV in tumors. (B) Sensitize tumor cells to OV therapy. OV secreting pro-apoptotic proteins can reverse tumor resistance to OV therapy. (C) Optimize OV transfer. Carrier cells protect OV from immune system damage and enhance the tumor targeting of OVs. (D) OV-mediated immunotherapy. OV-mediated oncolysis promotes the immune system’s response to tumor cells and improves the overall therapeutic response.


ImageTumor targeting

Although OV has tumor tropism based on some over-expressed receptors and adhesion molecules on tumor cells, the tumor tropism of wild OV is not enough. Genetically modified OV (GMOV) can express receptors with high affinity for tumor-associated antigen (TAA) (Figure 4). For example, inserting single-chain antibodies (scAb) against human epidermal growth factor receptor (HER)-2, epithelial cell adhesion molecule (EpCAM), and carcinoembryonic antigen (CEA) can increase the specificity of OV for tumors. VSV expressing HIV-derived glycoprotein (gp)-160 is a specific VSV for leukemia and T lymphoma (Figure 2).

Deficiency of IFN-I antiviral response, lack of tumor suppressor genes, such as retinoblastoma (Rb) and the increase of Ras signal in tumor cells can trigger the specific proliferation of OV in tumor cells. Inserting tumor-specific promoters that are highly expressed in tumor cells, such as prostate-specific antigen (PSA) and human telomerase reverse transcriptase (hTERT) promoters, enable specific expression of viral genes in tumor cells. Some microRNAs (miRNAs) are overexpressed in healthy cells, and their levels in tumor cells are negligible; therefore, targeting these miRNAs through miRNA targeting sequences (miRNA-TS) will destroy the viral RNA in normal cells, The low expression of miRNA-TS targets in tumor cells leads to the retention and replication of viral RNA in tumor cells (Figure 4).

The interaction of HAdV-C5 fibrin with many other adenoviruses and CARs on target cells leads to the interaction of the RGD (arginine-glycine-aspartic acid) motif on the viral penton base protein and the host cell integrin This interaction induces clathrin-mediated endocytosis and virus entry into the cell. Because CAR is down-regulated in many tumor cells, researchers have modified it to enhance the tumorigenicity of oncolytic Ad. One of the modifications to improve the efficiency of adenovirus infection is to insert it in the HI loop of the adenovirus fiber knob domain. RGD motif. Studies have shown that RGD-modified oncolytic adenovirus treatment of CAR-negative tumor models can significantly improve infection efficiency and anti-tumor activity. Another strategy for oncolytic Ads is to use different serotypes. Lenman et al. found that HAdV-G52 can bind to polysialic acid on target cells; due to the high level of polysialic acid expression on cancer cells including lung and brain, using HAdV-G52 as OV can preferentially infect corresponding cancers Type. In addition, antibody targeting through the fusion of antibody single-chain variable fragments (scfv) and capsid protein IX (pIX) or generation of fiber chimeras can also be used to redirect adenoviruses.

Another strategy for targeting viruses to tumor cells is to use a bispecific adapter, which consists of two arms, the virus binding arm and the tumor cell binding arm, which are connected by chemical or flexible connections. Because polySia is overexpressed on tumor cells including lung cancer, Kloos et al. designed a bispecific adapter containing polySia binding single-chain antibody domain and CAR outer domain to dissolve Neoplastic Ad was relocated to lung cancer, and it was found that pretreatment of adenovirus vector with adaptor can effectively infect tumor cells expressing polyribonucleic acid in tumor-bearing mice and improve survival rate. Another dual-specific adapter containing the fusion of the CAR outer domain and CXCL12, as a CXCR4 chemokine ligand, can enhance the infectivity of chemokine receptor-positive cancer cells while reducing liver toxicity. Nakano et al. also developed an adaptor protein consisting of an EGFR-binding single-chain antibody and the N-terminal domain of nectin1 to direct HSV-1 to EGFR.


Immune response

Another challenge of using OV therapy is the pre-existing immunity due to the short half-life after intravenous injection due to previous immunity or infection. Coating the oncolytic adenovirus with polymer can protect the adenovirus during delivery. The most highly utilized polymers are N-(2-hydroxypropyl)methacrylamide (HPMA), polyethyleneglycine and polyamidoamine. In addition to prolonging the half-life, the use of polymers can also target tumors by modifying oncolytic Ad/polymers.

Another strategy to protect OV from neutralizing antibodies is to use cell carriers as delivery vehicles (Figure 1). There are three main types of cells that can be used to deliver OV: immune cells, transformed cells, and progenitor cells. In addition to carrying OV, stem cells also have the ability to locate tumors, making them attractive vectors. In the tumor microenvironment that is essential for tumor growth, the expression of various growth factors, cytokines, chemokines and angiogenic factors attract stem cells to the tumor site. For example, in determining the homing process of stem cells and progenitor cells in the tumor environment, the upregulation of hypoxia and oncogenic factors is crucial; studies have shown that oncolytic adenovirus loaded neural stem cells (NSC) can increase the expression of VEGFR2 and CXCR4 , Thereby increasing the homing ability; in order to avoid the patient’s allograft reaction, it is recommended to use autologous stem cells. However, it is worth noting that the quality and quantity of cells isolated from patients who have received multiple rounds of treatment are variable.

Antiviral cytokines (including different types of IFNs) can hinder OV replication and reduce the anti-tumor response. To overcome this problem, some studies have used histone deacetylase (HDAC) inhibitors to induce epigenetic changes and Minimize the antiviral cytokine response in the tumor microenvironment. Pretreatment of cancer cells with sodium valproate (VPA, an HDAC inhibitor) can promote oncolytic HSV replication. However, studies have shown that VPA can inhibit the infiltration of NK cells and macrophages in the tumor microenvironment after virus infection, thereby Reduce the induction of anti-tumor immunity. In addition, HDAC inhibitors can also transform gene expression profiles to induce cancer cell arrest and apoptosis through epigenetic modification.

Genetic modification of OV can increase the expression of cytokines, chemokines, costimulatory molecules, tumor extracellular matrix (ECM) degrading enzymes and anti-angiogenic molecules, and enhance its anti-tumor effect (Figure 2, Figure 3). OVs carrying granulocyte macrophage colony stimulating factor (GM-CSF) gene, such as T-VEC, Pexa-Vec and CG0070, can recruit antigen presenting cells (APC) and CTL, thereby inducing more efficient antiviral responses with minimal Submitted by TAA. GMOV expressing pro-inflammatory cytokines has an enhanced anti-tumor effect. Inserting IL-12, IL-15, IL-18, TNF-α, IL-24 and IFN-γ genes into OV can also enhance the anti-tumor effect, and the toxicity is much lower than IL-2. At the same time, the application of non-secreted forms of these cytokines will only cause local effects, but will not cause systemic adverse reactions. Specific chemokines expressed by engineered OV (mainly VACV), such as CCL5, CCL19, CCL20, CCL21, can increase the infiltration of initial and memory T lymphocytes and DC into TME. At the same time, the use of one or more costimulatory ligands in OV, including CD40L, 4-1BBL, OX40L and B7-1, such as LOAd703 (a combination of CD40L and 4-1BBL) can increase antigen presentation and T cell activation. In addition, the insertion of TLR ligands (such as CpG-rich regions) into the OV genome can stimulate TLRs and further activate innate and acquired immunity.

Another way to enhance the immune response in TME is to eliminate immunosuppressive cells. GMOV expressing hydroxyprostaglandin dehydrogenase (HPGD) inactivates PGE2 and reduces MDSC in TME. The soluble CXCR4 expressed by GMOV acts as a decoy receptor to bind to CXCL12 secreted by tumor cells, inhibiting the effect of CXCL12 on the angiogenesis, metastasis and recruitment of MDSCs. Although OV can release TAA through various mechanisms, expression of TAA by GMOV or coating of TAA-derived peptide on the surface of OV will increase T cell response and improve OV treatment. So far, a large number of TAAs and peptides have been studied. Compared with peptide expression, peptide coating has the advantages of convenience, speed, lower cost, and the peptide coating method can be personalized for each patient. OV can be designed to express pro-apoptotic proteins, such as TNF-related apoptosis-inducing ligand (TRAIL) and apoptin that can induce tumor cell-specific apoptosis; inserting small interfering RNAs (siRNAs) into OV can also inhibit Oncogenes express and inhibit tumor growth.

The 2′,5′-oligoadenylate synthase (OAS)-RNase L system and RNA-dependent protein kinase (PKR) can also limit viral infections. After producing type I interferon in response to viral infection, double-stranded RNA activates the transcription of OAS-1, -2, and -3 genes, leading to activation of RNase L, and ultimately degrading virus and single-stranded RNA. In addition, infecting cells with double-stranded RNA and type I IFN systems activates PKR. PKR is a serine/threonine protein kinase that phosphorylates the alpha subunit of eukaryotic initiation factor 2 alpha (eIF2 alpha). The phosphorylation of eIF2α makes it in an inactive state and inhibits the synthesis of viral proteins. The study of Jha et al. showed that pretreatment with sunitinib (a PDGF-R and VEGF inhibitor) can enhance the efficacy of oncolytic virus therapy and inhibit the antiviral enzymes RNase L and PKR in vivo and in vitro. In addition, bevacizumab (an inhibitor of vascular endothelial growth factor) can also increase the spread of oncolytic adenovirus, possibly because bevacizumab reduces interstitial fluid pressure. In addition, the combination of anti-angiogenic drugs and OV has a synergistic effect. By inhibiting angiogenesis, it inhibits the supply of oxygen and nutrients necessary for tumor growth, and enhances the replication and spread of OV in tumors.


Hypoxia effect

Hypoxia is a characteristic of solid tumors. It occurs during the development and growth of tumors and can cause cell cycle arrest, which may affect the replication ability of Ad and any other viruses that depend on cell cycle progression. In order to overcome the inhibition of hypoxia on adenovirus replication and use hypoxic conditions for targeting, Clarke et al. designed an oncolytic adenovirus in which the expression of the E1A gene is controlled by a promoter containing hypoxia response elements, and an improved solution The tumor adenovirus has acquired the ability to replicate fully under hypoxic conditions.

Studies by two research groups (Aghi et al., Fasullo et al.) in 2009 showed that hypoxic environment enhanced virus replication of oncolytic HSV, which may be related to the reduction of oxygen cells or DNA damage induced by oxygen-derived free radicals by HSV. HSV replication) is related to the natural tropism. In addition, the transcription of several genes involved in HSV replication is activated by hypoxia-inducible factor-1α (HIF-1α). Infection with AF2240 (an oncolytic NDV) degrades HIF-1α under hypoxic conditions, leading to the down-regulation of HIF-1α target genes in different cancer cell lines. Other viruses (including vesicular stomatitis virus and vaccinia virus) can also enhance their replication under hypoxic conditions.



Biological safety of OV treatment

In addition to tumor cells, some OVs may also replicate in normal cells and cause damage. For example, T-VEC may still cause potential infection and cause long-term neurological adverse events (AE). The use of OV with low human pathogenicity, such as parvovirus and reovirus, can increase the safety of OV therapy by weakening OV or deleting virulence genes through repeated passages. Thymidine kinase (TK) and infected cell protein (ICP) 34.5 genes are in VACV and HSV-1 Replication plays a vital role.

The products of these genes are abundant in tumor cells. Therefore, GMOV lacking these genes can replicate in tumor cells, while the replication of viruses in healthy cells is due to the low level of such products. Impaired by expression. GL-ONC1 and Pexa-Vec (JX-594) are TK-free VACVs, and T-VEC, HSV-1716 and G207 are ICP34.5-free HSVs, which have shown good safety in clinical trials. Wild ZIKA virus has oncolytic potential in glioblastoma, but it can also infect normal nerves with serious complications.

Removing 10 nucleotides from the 3’of its genome can increase the oncolytic activity without reducing the oncolytic activity. Security. The mutation or deletion of the E1 gene in AdV, and the deletion of the TK, vaccinia growth factor (VGF), hemagglutinin, and B18R genes in the poxvirus can reduce the virulence of OV in normal cells. However, deleting virulence genes to increase safety can sometimes reduce the anti-tumor activity of OV.

Reorganizing a safe OV (such as NDV) with an efficient OV (such as VSV) is another way to improve the safety of OV. The recombinant VSV-NDV (rVSV-NDV) contains the envelope content from the NDV and the original skeleton of the VSV. The recombination of AdV with the less harmful Coxsackie virus or parvovirus constitutes an OV that is highly effective in tumor cell infection without damaging normal cells; the use of Ebola virus (EBOV) glycoprotein can also reduce rVSV -Neurotoxicity of VSV in EBOV. However, the natural homologous recombination of GMOV and wild-type OV may result in genetically modified and pathogenic viruses. In addition, the safety of OV therapy in immunocompromised individuals and pregnant women receiving radiotherapy and chemotherapy is still controversial.



Route of administration

One of the factors that affect the OV response is the mode of administration. Intratumoral injection can precisely control the OV concentration in TME, so as to obtain a better therapeutic effect. However, the complexity of intratumoral injection limits the reproducibility of administration. Intravenous injection is popular because of its convenience, reproducibility, and possibility of targeting metastases, but requires a tumor-specific delivery system and is more likely to cause systemic toxicity. Intraperitoneal, intrathecal/intracranial and intrapleural injections are suitable for targeting intra-abdominal organs, central nervous system (CNS) and lung tumors, respectively, but are limited to use in laboratory animals. The optimal route of administration is still a matter of debate, and there are no specific guidelines. It seems that oral/mucosal and nasal administration and other less aggressive routes of administration, at least for gastrointestinal and brain malignancies, can improve patient acceptability and should be considered in future studies.

Patient selection

Which patients are suitable for treatment? At present, there is no reliable predictive biomarker that can predict patients who will respond to oncolytic virus therapy. On the other hand, due to the experimental nature of most OVs, patients receiving oncolytic virus therapy usually have undergone multiple conventional cancer treatments, their immune system is destroyed, and the tumor has undergone a fundamental change from its initial form. Zloza et al. designed a study to measure the changes in peripheral blood mononuclear cell gene expression in patients with melanoma before and after oncolytic vaccinia virus treatment. Microarray data showed that after virus administration, 301 and 960 genes were up-regulated and down-regulated, respectively. Further analysis showed that immunoglobulin-like transcript 2 can be used as a therapeutic biomarker for patients treated with oncolytic vaccinia virus; and in another study, the high mobility group box 1 was recommended as an oncolytic adenovirus treatment Predictive and prognostic biomarkers.


Oncolytic viruses: What are difficulties and challenges in treatments?

Figure 2 New method of oncolytic virus treatment


Oncolytic viruses: What are difficulties and challenges in treatments?

Figure 3 Cytokine/chemokine east armed oncolytic virus


Oncolytic viruses: What are difficulties and challenges in treatments?

Figure 4 Genetic modification of OV


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