Development of TaqMan-based real-time PCR based on ψ gene for quantitative detection of CAR-T cells.

Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = -3.3841x + 41.402 (R2 = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/µL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.

oHSV2-mGM repolarizes TAMs and cooperates with αPD1 to reprogram the immune microenvironment of residual cancer after radiofrequency ablation.

BACKGROUND: Due to the size and location of the tumor, incomplete radiofrequency ablation (iRFA) of the target tumor inhibits tumor immunity. In this study, a murine herpes simplex virus (oHSV2-mGM) armed with granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed to explore its effect on innate and adaptive immunity during iRFA, and the inhibitory effect of programmed cell death-1 (PD1) on tumor. METHODS: We verified the polarization and activation of RAW264.7 cells mediated by oHSV2-mGM in vitro. Subsequently, we evaluated the efficacy of oHSV2-mGM alone and in combination with αPD1 in the treatment of residual tumors after iRFA in two mouse models. RNA-seq was used to characterize the changes of tumor microenvironment. RESULTS: oHSV2-mGM lysate effectively stimulated RAW264.7 cells to polarize into M1 cells and activated M1 phenotypic function. In the macrophage clearance experiment, oHSV2-mGM activated the immune response of tumor in mice. The results in vivo showed that oHSV2-mGM showed better anti-tumor effect in several mouse tumor models. Finally, oHSV2-mGM combined with PD1 antibody can further enhance the anti-tumor effect of oHSV2-mGM and improve the complete remission rate of tumor in mice. CONCLUSION: The application of oHSV2-mGM leads to the profound remodeling of the immune microenvironment of residual tumors. oHSV2-mGM also works in synergy with PD1 antibody to achieve complete remission of tumors that do not respond well to monotherapy at immune checkpoints. Our results support the feasibility of recombinant oncolytic virus in the treatment of residual tumors after iRFA, and propose a new strategy for oncolytic virus treatment of tumors.

VP5 protein of oncolytic herpes simplex virus type 2 induces apoptosis in A549 cells through TP53I3 protein.

Oncolytic virotherapy stands out as a burgeoning and promising therapeutic paradigm, harnessing the intrinsic cytotoxicity of oncolytic viruses for selective replication and dissemination within tumors. The primary mode of action revolves around the direct eradication of tumor cells. In our previous investigations, we formulated an oncolytic herpes simplex virus type 2 (OH2) and substantiated its anti-tumor efficacy both in vivo and in vitro. Subsequently, we embarked on a phase I/II clinical trial in China (NMPA, 2018L02743) and the USA (FDA, IND 27137) to assess OH2's safety, biodistribution, and anti-tumor activity as a standalone agent in patients with advanced solid tumors. In this investigation, our primary focus was to comprehend the influence of the major capsid protein VP5 of OH2 on its efficacy as an antitumor agent. Our findings underscore that the VP5 protein significantly amplifies OH2's oncolytic impact on A549 cells. Additionally, we observed that VP5 actively promotes the induction of apoptosis in A549 cells, both in vivo and in vitro. Through comprehensive transcriptional sequencing, we further authenticated that the VP5 protein triggers apoptosis-related signaling pathways and Gene Ontology (GO) terms in A549 cells. Moreover, we scrutinized differentially expressed genes in the p53-dependent apoptosis pathway and conducted meticulous in vitro validation of these genes. Subsequently, we delved deeper into unraveling the functional significance of the TP53I3 gene and conclusively affirmed that the VP5 protein induces apoptosis in A549 cells through the TP53I3 gene. These revelations illuminate the underlying mechanisms of OH2's antitumor activity and underscore the pivotal role played by the VP5 protein. The outcomes of our study harbor promising implications for the formulation of effective oncolytic virotherapy strategies in cancer treatment.

Exploring immune evasion of SARS-CoV-2 variants using a pseudotyped system.

In the United States, coronavirus disease 2019 (COVID-19) cases have consistently been linked to the prevailing variant XBB.1.5 of SARS-CoV-2 since late 2022. A system has been developed for producing and infecting cells with a pseudovirus (PsV) of SARS-CoV-2 to investigate the infection in a Biosafety Level 2 (BSL-2) laboratory. This system utilizes a lentiviral vector carrying ZsGreen1 and Firefly luciferase (Fluc) dual reporter genes, facilitating the analysis of experimental results. In addition, we have created a panel of PsV variants that depict both previous and presently circulating mutations found in circulating SARS-CoV-2 strains. A series of PsVs includes the prototype SARS-CoV-2, Delta B.1.617.2, BA.5, XBB.1, and XBB.1.5. To facilitate the study of infections caused by different variants of SARS-CoV-2 PsV, we have developed a HEK-293T cell line expressing mCherry and human angiotensin converting enzyme 2 (ACE2). To validate whether different SARS-CoV-2 PsV variants can be used for neutralization assays, we employed serum from rats immunized with the PF-D-Trimer protein vaccine to investigate its inhibitory effect on the infectivity of various SARS-CoV-2 PsV variants. According to our observations, the XBB variant, particularly XBB.1.5, exhibits stronger immune evasion capabilities than the prototype SARS-CoV-2, Delta B.1.617.2, and BA.5 PsV variants. Hence, utilizing the neutralization test, this study has the capability to forecast the effectiveness in preventing future SARS-CoV-2 variants infections.

OH2 oncolytic virus: A novel approach to glioblastoma intervention through direct targeting of tumor cells and augmentation of anti-tumor immune responses.

Glioblastoma (GBM), the deadliest central nervous system cancer, presents a poor prognosis and scant therapeutic options. Our research spotlights OH2, an oncolytic viral therapy derived from herpes simplex virus 2 (HSV-2), which demonstrates substantial antitumor activity and favorable tolerance in GBM. The extraordinary efficacy of OH2 emanates from its unique mechanisms: it selectively targets tumor cells replication, powerfully induces cytotoxic DNA damage stress, and kindles anti-tumor immune responses. Through single-cell RNA sequencing analysis, we discovered that OH2 not only curtails the proliferation of cancer cells and tumor-associated macrophages (TAM)-M2 but also bolsters the infiltration of macrophages, CD4+ and CD8+ T cells. Further investigation into molecular characteristics affecting OH2 sensitivity revealed potential influencers such as TTN, HMCN2 or IRS4 mutations, CDKN2A/B deletion and IDO1 amplification. This study marks the first demonstration of an HSV-2 derived OV's effectiveness against GBM. Significantly, these discoveries have driven the initiation of a phase I/II clinical trial (ClinicalTrials.gov: NCT05235074). This trial is designed to explore the potential of OH2 as a therapeutic option for patients with recurrent central nervous system tumors following surgical intervention.

[Construction and identification of a stable CT26 cell line expressing CD19-FLUC-GFP].

Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.

[Replication and expression of oncolytic herpes simplex virus type â ¡ in tumors].

This study aimed to measure the duration and replication level of oncolytic herpes simplex virus type 2 (oHSV2) at the tumor injection site in BALB/c mice. Additionally, the expression level of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HSV-2 antibody in the serum was also measured. High and low doses of oHSV2-Fluc (firefly luciferin, Fluc) were injected into the mice's tumors to track the change and duration of fluorescence expression. The copy number of oHSV2 gene in tumor tissues was determined using quantitative real-time polymerase chain reaction (qPCR). Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of hGM-CSF and HSV-2 antibody in the serum. The tumor volume in the high-dose group was significantly lower than that in the control group (P < 0.01). Intratumor injection of oHSV2-Fluc showed that the carried Fluc could continue to express in the tumor, with fluorescence still detectable at day 11 and declining to undetectable level by day 18. The mRNA expression of oHSV2 was detected in tumor tissues of both high and low dose groups on day 9 using qPCR. ELISA results showed that the levels of HSV2 antibody and hGM-CSF in both high and low dose groups were significantly increased compared to the control group (P < 0.05) after collecting orbital blood. These findings suggest that oHSV2 can replicate in the tumor and sustainably express exogenous factors, thus effectively targeting and killing the tumor. Furthermore, intratumoral injection of oHSV2 resulted in higher levels of hGM-CSF and HSV-2 antibodies found in the mice's serum.

Preclinical safety assessment of an oncolytic herpes simplex virus type 2 expressed PD-L1/CD3 bispecific antibody.

Oncolytic virotherapy is an emerging and safe therapeutic approach based on the inherent cytotoxicity of oncolytic viruses and their ability to replicate and spread within tumors in a selective manner. We constructed a new type of oncolytic herpes simplex virus armed with Bispecific Antibody (BsAb) molecules targeting PD-L1/CD3 (oHSV2-PD-L1/CD3-BsAb) to treat human malignancies. We demonstrated the anti-tumor efficacy of oHSV2-PD-L1/CD3-BsAb. To move forward with clinical trials of oHSV2-PD-L1/CD3-BsAb, we conducted a comprehensive preclinical safety evaluation, including hemolysis test, anaphylaxis test, repeated dose toxicity test in cynomolgus monkeys, biodistribution in cynomolgus monkeys and tissue cross-reactivity of PD-L1/CD3-BsAb with human and cynomolgus monkey tissues in vitro. Our preclinical safety evaluation indicated that oHSV2-PD-L1/CD3-BsAb is safe and suitable for clinical trials. After undergoing a thorough evaluation by the United States Food and Drug Administration (FDA), oHSV2-PD-L1/CD3-BsAb has successfully obtained approval to initiate Phase I clinical trials in the United States (FDA IND: 28717).

Immune-check blocking combination multiple cytokines shown curative potential in mice tumor model.

OBJECTIVE: In order to ensure the stable transcription of target genes, we constructed a eukaryotic high expression vector carrying an immune-check inhibitor PD-1v and a variety of cytokines, and studied their effects on activating immune response to inhibit tumor growth. METHODS: A novel eukaryotic expression plasmid vector named pT7AMPCE containing T7RNA polymerase, T7 promoter, internal ribosome entry site (IRES), and poly A tailing signal was constructed by T4 DNA ligase, on which homologous recombination was used to clone and construct the vector carrying PD-1v, IL-2/15, IL-12, GM-CSF, and GFP. In vitro transfection of CT26 cells was performed, and the protein expression of PD-1v, IL-12 and GM-CSF was detected by Western blot and ELISA after 48 h. Mice were subcutaneously inoculated with CT26-IRFP tumor cells in the rib abdomen, and the tumor tissues were injected with PD-1v, IL-2/15, IL-12, and GM-CSF recombinant plasmids for treatment during the experimental period. The efficacy of the treatment was evaluated by assay tumor size and survival time of tumor-bearing mice during the experiment. Expression levels of IFN-γ, TNF, IL-4, IL-2, and IL-5 in mouse blood were measured using the CBA method. Tumor tissues were extracted and immune cell infiltration in tumor tissues was detected by HE staining and the IHC method. RESULTS: The recombinant plasmids carrying PD-1v, IL-2/15, IL-12, and GM-CSF were successfully constructed, and the Western blot and ELISA results showed that PD-1v, IL-12, and GM-CSF were expressed in the supernatant of CT26 cells 48 h after in vitro cell transfection. The combined application of PD-1v, IL-2/15, IL-12, and GM-CSF recombinant plasmids significantly inhibited tumor growth in mice, and the tumor growth rate was significantly lower than that in the blank control group and GFP plasmid control group (p < 0.05). Cytometric bead array data suggested that the combination of PD-1v and various cytokines can effectively activate immune cells. HE and IHC analysis revealed plenty of immune cell infiltrates in the tumor tissue, and a large proportion of tumor cells showed the necrotic phenotype in the combination treatment group. CONCLUSION: The combination of immune check blockade and multiple cytokine therapy can significantly activate the body's immune response and inhibit tumor growth.

PF-D-Trimer, a protective SARS-CoV-2 subunit vaccine: immunogenicity and application.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has had and continues to have a significant impact on global public health. One of the characteristics of SARS-CoV-2 is a surface homotrimeric spike protein, which is primarily responsible for the host immune response upon infection. Here we present the preclinical studies of a broadly protective SARS-CoV-2 subunit vaccine developed from our trimer domain platform using the Delta spike protein, from antigen design through purification, vaccine evaluation and manufacturability. The pre-fusion trimerized Delta spike protein, PF-D-Trimer, was highly expressed in Chinese hamster ovary (CHO) cells, purified by a rapid one-step anti-Trimer Domain monoclonal antibody immunoaffinity process and prepared as a vaccine formulation with an adjuvant. Immunogenicity studies have shown that this vaccine candidate induces robust immune responses in mouse, rat and Syrian hamster models. It also protects K18-hACE2 transgenic mice in a homologous viral challenge. Neutralizing antibodies induced by this vaccine show cross-reactivity against the ancestral WA1, Delta and several Omicrons, including BA.5.2. The formulated PF-D Trimer is stable for up to six months without refrigeration. The Trimer Domain platform was proven to be a key technology in the rapid production of PF-D-Trimer vaccine and may be crucial to accelerate the development and accessibility of updated versions of SARS-CoV-2 vaccines.

Transarterial viroembolization improves the therapeutic efficacy of immune-excluded liver cancer: Three birds with one stone.

OBJECTIVE: To investigate the mechanism and efficacy of transarterial viroembolization (TAVE) with an oncolytic virus (OH2) for the treatment of liver cancer in rabbit VX2 tumor models. MATERIALS AND METHODS: Subcutaneous tumor and liver cancer models were established to determine the optimal viral titer and administration modality of OH2. Different liver cancer models were established to evaluate the locoregional tumor response, synergistic and standby effects, survival benefit, and specific antitumor immune memory after TAVE treatment. The immune cell densities in tumor tissues were measured. RESULTS: The optimal viral titer of OH2 was 1 × 107 CCID50. TAVE was the most effective modality with greater homogeneous OH2 distribution and therapeutic efficacy compared to other administration routes of transarterial virus infusion (TAVI), commonly adopted intratumor injection (TI), and intravenous injection (IV). Additionally, TAVE treatment significantly improved the locoregional tumor response, standby effect, and survival benefit compared to the TAVI, transarterial embolization (TAE), and control groups. TAVE modified the immune cell densities for immune-excluded liver cancer, partially destroyed vessel metastases, and established antitumor immune memory. The synergistic treatment efficacy of TAVE was superior to the simple addition of two independent monotherapies. CONCLUSION: TAVE was the optimal and a safe modality for treating immune-excluded liver cancer, and its synergistic effect achieved a remarkable tumor response, standby effect, survival benefit, and antitumor immune memory, which providing an innovative therapeutic modality for clinical practice. DATA AVAILABILITY: Data is available from the corresponding author upon requirement.

Single-cell transcriptomics of peripheral blood reveals anti-tumor systemic immunity induced by oncolytic virotherapy.

Rationale: Oncolytic virus (OV) therapy as a cancer therapy that improves immune status makes it a favorable candidate for optimizing immunotherapy strategies. Existing studies have focused on characterizing the disturbance of the tumor microenvironment (TME) by OV therapy. However, the changes in systemic immunity induced by OV were largely ignored, which would prevent the further understanding and optimization of oncolytic viruses. Methods: The HSV-2-based oncolytic virus OH2 was used to treat tumor-bearing mouse models. The peripheral blood samples were then collected for single-cell RNA sequencing (scRNA-seq). The scRNA-seq data were analyzed using Cell Ranger, Seurat, and other bioinformatics tools. Key findings were further validated by ELISA, immunohistochemistry, flow cytometry, in vivo experiments, and clinical samples. Results: Our data showed that OH2 therapy effectively activated systemic immunity and induced a sustained anti-tumor immune response. One major impact of OH2 on systemic immunity was to boost Ccl5 production, which correlated with clinical response. Besides, the cytotoxic ability of peripheral cytotoxic Cd8+ T cells and mature NK cells was elevated by OH2. Further analysis revealed that the interaction of monocytes with T cells and NK cells was critical for systemic immune remodeling and activation. We also found that systemic immune responses induced by OH2 could effectively reshape the microenvironment of distant tumor lesions and inhibit their progression. Conclusions: This study is the first to comprehensively characterize the effects of OV therapy on systemic immunity, which not only sheds new light on the anti-tumor mechanisms of OH2, but also contributes to the establishment of companion diagnostics for OH2 treatment and the improvement of oncolytic therapy strategies.

SIRPα antibody combined with oncolytic virus OH2 protects against tumours by activating innate immunity and reprogramming the tumour immune microenvironment.

BACKGROUND: The combination of oncolytic viruses (OVs) with immune checkpoint blockades is a research hotspot and has shown good efficacy. Here, we present the first attempt to combine oncolytic herpes simplex virus 2 (OH2) with an anti-SIRPα antibody as an antitumour treatment. Our results provide unique insight into the combination of innate immunity with OV. METHODS: We verified the polarization and activation of OH2 in RAW264.7 cells in vitro. Subsequently, we evaluated the antitumour ability of OH2 and anti-SIRPα combined therapy in a tumour-bearing mouse model. RNA-seq and Single-cell RNA-seq were used to characterize the changes in the tumour microenvironment. RESULTS: The OH2 lysates effectively stimulated RAW264.7 cells to polarize towards the M1 but not the M2 phenotype and activated the function of the M1 phenotype in vitro. In the macrophage clearance experiment, OH2 therapy induced polarization of M1 macrophages and participated in the antitumour immune response in a tumour-bearing mouse model. Treatment with a combination of OH2 and anti-SIRPα effectively inhibited tumour growth and significantly prolonged the survival time of the mice, and this result was more obvious in the mouse model with a larger tumour volume at the beginning of the treatment. These results suggest that combination therapy can more profoundly reshape the TME and activate stronger innate and adaptive immune responses. CONCLUSIONS: Our data support the feasibility of oncolytic virus therapy in combination with anti-SIRPα antibodies and suggest a new strategy for oncolytic virus therapy.

A novel oncolytic virus induces a regional cytokine storm and safely eliminates malignant ascites of colon cancer.

BACKGROUND: Given malignant ascites with a terrible prognosis and a unique immune microenvironment, our purpose is to evaluate whether oncolytic herpes simplex virus type 2(OH2) is able to safely eliminate ascites of colon cancer and through which specific mechanism it exerts antitumor immunity. METHODS: We established an ascites mice model through intraperitoneal injection of CT26 cells and obtained an appropriate dose range for in vivo tests. Efficacy and safety of OH2 were detected by weight of ascites, blood routine analysis, histopathological examination, and the survival time of mice. The specific mechanism underlying antitumor immunity was analyzed by cytometric bead array, flow cytometry, and single-cell RNA sequencing. Furthermore, anti-interleukin (IL)-6R antibody tocilizumab was synchronously or sequentially delivered with OH2 to explore the role of the regional cytokine storm, mainly IL-6 hypersecretion. RESULTS: OH2 was able to eliminate ascites and significantly prolong the survival of mice-bearing CT26 tumor cells by intraperitoneal injection, without obvious systemic damage to the main organs even though a regional cytokine storm. Hypersecretion of pro-inflammatory cytokines, mainly IL-6, and increased infiltration of CD4+ and CD8+ T cells were observed in ascites mice treated by OH2, compared with those treated by 5-fluorouracil or nonresponders. Furthermore, the initial-stage blocking of the IL-6 pathway was able to considerably suppress antitumor immune responses driven by OH2. Surprisingly, we discovered upregulations of the immune checkpoint genes such as Cd274 and Pdcd1 by single-cell RNA sequencing. CONCLUSIONS: OH2 could safely eliminate malignant ascites of colon cancer and convert the cold immune microenvironment by inducing a remarkably regional cytokine storm in ascites, mainly IL-6, in the early stage of antitumor immune responses beyond directed oncolytic virotherapy.

A novel cocktail therapy based on quintuplet combination of oncolytic herpes simplex virus-2 vectors armed with interleukin-12, interleukin-15, GM-CSF, PD1v, and IL-7 × CCL19 results in enhanced antitumor efficacy.

BACKGROUND: Selectively replicating herpes simplex virus-2 (HSV-2) vector is a promising treatment for cancer therapy. The insertion of multiple transgenes into the viral genome has been performed to improve its oncolytic activity. METHODS: Herein, we simultaneously constructed five "armed" oncolytic viruses (OVs), designated oHSV2-IL12, -IL15, GM-CSF, -PD1v, and IL7 × CCL19. These OVs delete the ICP34.5 and ICP47 genes with the insertion of transgenes into the deleted ICP34.5 locus. The anti-tumor efficacy in vivo was tested in the syngeneic 4T1 and CT26 tumor-bearing mice model. RESULTS: The OVs showed comparable oncolytic capability in vitro. The combination therapy of oHSV2-IL12, -IL15, GM-CSF, -PD1v, and IL7 × CCL19 exhibited the highest tumor inhibition efficacy compared with the treatment of single OV or two OVs combination. CONCLUSIONS: The OVs armed with different transgenes combination therapy also named 5-valent oHSV2 (also called cocktail therapy) might be an effective therapeutic strategy for solid tumors.

Bispecific Antibody Expressed by an Oncolytic Herpes Simplex Virus Type 2 Can Transform Heterologous T Cells Into Uniform Tumor Killer Cells.

BsAb (bispecific antibody)-armed oncolytic viruses (OVs) are effective in regulating tumor microenvironment. However, oHSV2 (oncolytic herpes simplex virus type 2) expressing immune checkpoints targeting BsAb molecules are not reported. Here, we generated oHSV2-armed PD-L1/CD3 BsAb and established pharmacodynamic evaluation models, which suggested that our oHSV2-BsAb molecules have an improved oncolytic potency in vitro and in vivo. The oHSV2 viruses armed with BsAb molecules targeting programmed cell death ligand 1 (PD-L1)/CD3 or CD19/CD3 (oHSV2-PD-L1/CD3-BsAb or oHSV2-CD19/mCD3-BsAb) were constructed; besides inducing oncolysis in virus-infected tumor cells, the modified oncolytic virus oHSV2-PD-L1/CD3-BsAb can also activate peripheral blood mononuclear cells (PBMCs) by releasing PD-L1/CD3 BsAb and thereby induce PBMC-mediated killing of PD-L1-positive tumor cells, regardless of PD-L1 expression level. The expressed PD-L1/CD3 BsAb can upregulate the activation markers of T cells in PBMCs and induce different cytokine secretion. The activation of T cells and the enrichment of related immune regulatory pathways are further confirmed by proteomics. It also demonstrated that the OVs or PBMCs could upregulate PD-L1 expression on the surface of tumor cells through transforming "cold tumors" with low PD-L1 expression into "hot tumors" with high PD-L1 expression, which can facilitate the targeting of BsAb molecules and enhance the effect of oncolysis. oHSV2-PD-L1/CD3-BsAb or oHSV2-CD19/mCD3-BsAb showed an enhanced oncolytic effect in vitro and in vivo compared to backbone virus oHSV2-GFP. Our results showed that the newly designed oHSV2-BsAb had enhanced therapeutic effects against solid tumors and provided a new option of immunotherapy.

NK cell tumor therapy modulated by UV-inactivated oncolytic herpes simplex virus type 2 and checkpoint inhibitors.

Oncolytic virotherapy is a new and safe therapeutic strategy for cancer treatment. In our previous study, a new type of oncolytic herpes simplex virus type 2 (oHSV2) was constructed. Following the completion of a preclinical study, oHSV2 has now entered into clinical trials for the treatment of melanoma and other solid tumors (NCT03866525). Oncolytic viruses (OVs) are generally able to directly destroy tumor cells and stimulate the immune system to fight tumors. Natural killer (NK) cells are important components of the innate immune system and critical players against tumor cells. But the detailed interactions between oncolytic viruses and NK cells and these interaction effects on the antitumor immune response remain to be elucidated. In particular, the functions of activating surface receptors and checkpoint inhibitors on oHSV2-treated NK cells and tumor cells are still unknown. In this study, we found that UV-oHSV2 potently activates human peripheral blood mononuclear cells, leading to increased antitumor activity in vitro and in vivo. Further investigation indicated that UV-oHSV2-stimulated NK cells release IFN-γ via Toll-like receptor 2 (TLR2)/NF-κB signaling pathway and exert antitumor activity via TLR2. We found for the first time that the expression of a pair of checkpoint molecules, NKG2A (on NK cells) and HLA-E (on tumor cells), is upregulated by UV-oHSV2 stimulation. Anti-NKG2A and anti-HLA-E treatment could further enhance the antitumor effects of UV-oHSV2-stimulated NK92 cells in vitro and in vivo. As our oHSV2 clinical trial is ongoing, we expect that the combination therapy of oncolytic virus oHSV2 and anti-NKG2A/anti-HLA-E antibodies may have synergistic antitumor effects in our future clinical trials.

Telomerase-positive circulating tumor cells are associated with poor prognosis via a neutrophil-mediated inflammatory immune environment in glioma.

BACKGROUND: Gliomas are the most common aggressive cancer in the central nervous system. Considering the difficulty in monitoring glioma response and progression, an approach is needed to evaluate the progression or survival of patients with glioma. We propose an application to facilitate clinical detection and treatment monitoring in glioma patients by using telomerase-positive circulating tumor cells (CTCs) and to further evaluate the relationship between the immune microenvironment and CTCs in glioma patients. METHODS: From October 2014 to June 2017, 106 patients newly diagnosed with glioma were enrolled. We used the telomerase reverse transcriptase CTC detection method to detect and analyze the CTC statuses of glioma patients before and after surgery. FlowSight and FISH confirmed the CTCs detected by the telomerase-based method. To verify the correlation between CTCs and the immune response, peripheral white blood cell RNA sequencing was performed. RESULTS: CTCs were common in the peripheral blood of glioma patients and were not correlated with the pathological classification or grade of patients. The results showed that the presence of postoperative CTCs but not preoperative CTCs in glioma patients was a poor prognostic factor. The level of postoperative CTCs, which predicts a poor prognosis after surgery, may be associated with neutrophils. RNA sequencing suggested that postoperative CTCs were positively correlated with innate immune responses, especially the activation of neutrophils and the generation of neutrophil extracellular traps, but negatively correlated with the cytotoxic response. CONCLUSIONS: Our results showed that telomerase-positive CTCs can predict a poor prognosis of patients with glioma. Our results also showed a correlation between CTCs and the immune macroenvironment, which provides a new perspective for the treatment of glioma.

Investigation of morphological changes of HPS membrane caused by cecropin B through scanning electron microscopy and atomic force microscopy.

BACKGROUND: Antimicrobial peptides (AMPs) have been identified as promising compounds for consideration as novel antimicrobial agents. OBJECTIVES: This study analyzed the efficacy of cecropin B against Haemophilus parasuis isolates through scanning electron microscopy (SEM) and atomic force microscopy (AFM) experiments. RESULTS: Cecropin B exhibited broad inhibition activity against 15 standard Haemophilus parasuis (HPS) strains and 5 of the clinical isolates had minimum inhibition concentrations (MICs) ranging from 2 to 16 µg/mL. Microelectrophoresis and hexadecane adsorption assays indicated that the more hydrophobic and the higher the isoelectric point (IEP) of the strain, the more sensitive it was to cecropin B. Through SEM, multiple blisters of various shapes and dents on the cell surface were observed. Protrusions and leakage were detected by AFM. CONCLUSIONS: Based on the results, cecropin B could inhibit HPS via a pore-forming mechanism by interacting with the cytoplasmic membrane of bacteria. Moreover, as cecropin B concentration increased, the bacteria membrane was more seriously damaged. Thus, cecropin B could be developed as an effective anti-HPS agent for use in clinical applications.

Intratumoral OH2, an oncolytic herpes simplex virus 2, in patients with advanced solid tumors: a multicenter, phase I/II clinical trial.

BACKGROUND: OH2 is a genetically engineered oncolytic herpes simplex virus type 2 designed to selectively amplify in tumor cells and express granulocyte-macrophage colony-stimulating factor to enhance antitumor immune responses. We investigated the safety, tolerability and antitumor activity of OH2 as single agent or in combination with HX008, an anti-programmed cell death protein 1 antibody, in patients with advanced solid tumors. METHODS: In this multicenter, phase I/II trial, we enrolled patients with standard treatment-refractory advanced solid tumors who have injectable lesions. In phase I, patients received intratumoral injection of OH2 at escalating doses (106, 107 and 108CCID50/mL) as single agent or with fixed-dose HX008. The recommended doses were then expanded in phase II. Primary endpoints were safety and tolerability defined by the maximum-tolerated dose and dose-limiting toxicities (DLTs) in phase I, and antitumor activity assessed per Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) and immune-RECIST in phase II. RESULTS: Between April 17, 2019 and September 22, 2020, 54 patients with metastatic cancers were enrolled. Forty patients were treated with single agent OH2, and 14 with OH2 plus HX008. No DLTs were reported with single agent OH2 in phase I. Four patients, having metastatic mismatch repair-proficient rectal cancer or metastatic esophageal cancer, achieved immune-partial response, with two from the single agent cohort and two from the combination cohort. The duration of response were 11.25+ and 14.03+ months for the two responders treated with single agent OH2, and 1.38+ and 2.56+ months for the two responders in the combination cohort. The most common treatment-related adverse event (TRAE) with single agent OH2 was fever (n=18, 45.0%). All TRAEs were of grade 1-2, except one case of grade 3 fever in the 108CCID50/mL group. No treatment-related serious AEs occurred. Single agent OH2 induced alterations in the tumor microenvironment, with clear increases in CD3+ and CD8+ cell density and programmed death-ligand 1 expression in the patients' post-treatment biopsies relative to baseline. CONCLUSIONS: Intratumoral injection of OH2 was well-tolerated, and demonstrated durable antitumor activity in patients with metastatic esophageal and rectal cancer. Further clinical development of OH2 as single agent or with immune checkpoint inhibitors in selected tumor types is warranted.