Journal: Journal for Immunotherapy of Cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: B7H3 expression in cell lines and the binding of delivered B7H3 BiTE to T and target cell. ( A ) B7H3 expression on MGC-803, SH-SY5Y, U87, SGC-7901 and Raji cells were detected using flow cytometry. ( B ) Binding of delivered B7H3 BiTE to target cells. Several cell lines were incubated with supernatants of the OAd-B7H3-BiTE-infected HEK293 cells. B7H3 BiTE binding was detected via flow cytometry with anti–anti-CD3-scFv antibody. ( C ) CD3 expression on Jurkat cells was detected using flow cytometry. ( D ) Binding of delivered B7H3 BiTE to T cells. PBMCs were incubated with supernatants of the OAd-B7H3-BiTE-infected HEK293 cells. B7H3 BiTE binding was detected via flow cytometry with anti–anti-CD3-scFv antibody. ( E ) Competition between delivered B7H3 BiTE and OKT3. PBMCs were incubated with supernatants of OAd or OAd-B7H3-BiTE-infected HEK293 cells. Then, cells were stained with fluorescein-conjugated OKT3 and examined throughflow cytometry. ( F ) Cell-bridging assay. A mixture of MGC-803/U87 and PBMCs was incubated with the supernatants of the OAd or OAd-B7H3-BiTE-infected HEK293 cells. The percentage of T/target cell pairs was quantified using flow cytometry. Statistical analysis was performed by unpaired Student’s t-test (n=3). NS: not significant; **p<0.01 and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; OAd, oncolytic adenovirus; PBMC, peripheral blood mononuclear cell; scFv, single-chain variable fragment.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy adults using a PBMC Isolation Solution Kit (P8680; Solarbio).

Techniques: Expressing, Binding Assay, Flow Cytometry, Incubation, Infection, Staining, Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: T-cell activation mediated by oncolytic viruses-delivered B7H3 BiTE. ( A – D ) PBMCs were cultured alone or co-cultured with the indicated cell lines in the absence (Mock) or presence of the supernatants of OAd or OAd-B7H3-BiTE-infected cells for 48 hours. ( A ) Representative brightfield images are shown. ( B ) Representative flow cytometry histograms showing FSC-A of CD4 + and CD8 + T cells. ( C ) Cumulative data of flow cytometry showing CD69 or CD107a expression in CD4 + and CD8 + T cells. ( D ) The levels of IFN-γ and IL-2 in supernatants at 48 hours measured by ELISA. ( E ) T-cell proliferation assay. CFSE-stained PBMCs were co-cultured with MGC-803 cells and incubated with or without the supernatants of OAd or OAd-B7H3-BiTE-infected cells. Proliferation was determined after incubation for 4 days. Statistical analysis was performed by unpaired Student’s t-test (n=3). NS: not significant; **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester; IFN, interferon; IL, interleukin; OAd, oncolytic adenovirus; PBMC, peripheral blood mononuclear cell.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy adults using a PBMC Isolation Solution Kit (P8680; Solarbio).

Techniques: Activation Assay, Cell Culture, Infection, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Staining, Incubation

Journal: Journal for Immunotherapy of Cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: T-cell cytotoxicity mediated by OAd-B7H3-BiTE. ( A – C ) After labeling with CFSE, MGC-803 ( A ), U87 ( B ) or Raji ( C ) cells were infected with OAd or OAd-B7H3-BiTE; uninfected cells (Mock) were used as a control. After 24 hours, peripheral blood mononuclear cells were added and co-cultured for 48 hours. Representative flow cytometry plots (left) and cumulative data (right) are shown. Dead target cells are defined as CFSE + Zombie Dye + . The percentage of dead target cells is calculated as follows: CFSE + Zombie-Dye + /CFSE + . Statistical analysis was performed by unpaired Student’s t-test (n=3). **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester; OAd, oncolytic adenovirus.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy adults using a PBMC Isolation Solution Kit (P8680; Solarbio).

Techniques: Labeling, Infection, Control, Cell Culture, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: OAd-B7H3-BiTE treatment elicits potent therapeutic effects in vivo. ( A ) Schematic diagram of treatment schedule for MGC-803 bearing mice ( B – G ). ( B ) Representative flow cytometry plots showing human CD45 + CD3 + T cells in tumor and spleen of untreated mice. Flow cytometry plots gated on live-cell population. ( C ) Summary data for average tumor growth (left) and body weight changes (right) for all treatment groups. ( D ) Tumor pictures and weights for all treatment groups. ( E – F ) Primary tumors were collected and analyzed with reverse transcription-quantitative PCR to examine the expression of E1a ( E ) and B7H3 BiTE ( F ). ( G ) Representative H&E staining of the major organs for all treatment groups. Statistical analysis was performed by unpaired two-way analysis of variance ( C ) or unpaired Student’s t-test ( D ) (n=5). ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; mRNA, messenger RNA; OAd, oncolytic adenovirus; PBS, phosphate-buffered saline; PBMC, peripheral blood mononuclear cell; s.c., subcutaneous injection.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy adults using a PBMC Isolation Solution Kit (P8680; Solarbio).

Techniques: In Vivo, Flow Cytometry, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Staining, Saline, Injection