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.

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).

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.

BGC823 Cell Line with the Stable Expression of iRFP720 Retains Its Primary Properties with Promising Fluorescence Imaging Ability.

Reliable animal models are required for understanding the molecular events of gastric tumor growth and metastasis. Tracing techniques based on iRFP720 may optimize the noninvasive monitoring of tumors in vivo. The present study established a human gastric adenocarcinoma cell line BGC823-iRFP720-GFP (abbreviated as BGC823-iRFP) that stably expressed iRFP720 and green fluorescent protein (GFP) by piggyBac transposon system. The monoclonal cell line BGC823-iRFP was isolated under puromycin selection. The cell morphology and proliferation ability of BGC823-iRFP cells in vitro were similar to that of the BGC823 cells. The iRFP720 and GFP expressions were confirmed by laser confocal microscopy and Cytation™ 5. Hematoxylin and eosin staining, immunohistochemical analysis, and animal experiments also revealed that BGC823-iRFP exhibited no significant changes in morphology, growth kinetics, and tumorigenicity in vivo. IVIS Lumina III imaging indicated that the iRFP720 signals of the BGC823-iRFP cells could be used to evaluate the antitumor efficacy of oncolytic viruses and chemotherapy drugs. Therefore, the BGC823-iRFP cells would be a useful tool for gastric cancer research and antitumor drug evaluation.

PiggyBac-modified CD19-expressing 4T1 cell line for the evaluation of CAR construct.

Reliable and stable target cell lines are required for evaluating the efficiency and studying the mechanism of chimeric antigen receptor T (CAR-T) immunotherapy both in vitro and in vivo. Jurkat cells can be used as an alternative for human primary lymphocytes to evaluate the constructs and function of the "CAR". This study established a murine 4T1-CD19 cell line that stably expressed a cd19 gene. The 4T1-CD19 cells had similar growth kinetics to its parent cell 4T1. The protein CD19 expression of the 4T1-CD19 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The second-generation CAR was constructed and transfected into Jurkat cells. The expression of CAR protein was analyzed by flow cytometry and western blot. Finally, the interaction between the CAR and CD19 was confirmed by the upregulation of the IL-2 mRNA level of Jurkat-CAR stimulated by 4T1-CD19. Therefore, the 4T1-CD19 cell line and Jurkat-CAR have been successfully established, and may be used to access the function of various CAR constructs both in vitro and in vivo.