Journal: Frontiers in Immunology

Article Title: IL-12-armed oncolytic HSV-2 enhances CAR T cell efficacy against pancreatic cancer in xenografted models

doi: 10.3389/fimmu.2025.1664289

Figure Lengend Snippet: Mesothelin chimeric antigen receptor (CAR)-T cells SS1-ICOSBBZ-CAR-T showed specific and modest anti-tumor capacity in vitro and in vivo . (A) Schematic diagram of the CAR structure. We constructed a third-generation MSLN-targeted CAR-T. This CAR incorporates the single-chain variable fragment (scFv) derived from the SS1 antibody for MSLN recognition, an ICOS-derived transmembrane domain, the intracellular domains contained two co-stimulatory molecules ICOS and 4-1BB, and the T cell receptor ζ (TCR-ζ) signaling domain. (B) Percentage of CAR positive T cells at day 12 (left) and CD4+ and CD8+ subsets (right) composition of the final CAR-T cell product. The CAR was stained by human mesothelin protein conjugated to PE. (C) Specific cytolytic activity of SS1-ICOSBBZ-CAR-T cells in vitro using luciferase assay. CAR-T cells were incubated with tumor cell lines for 30 hours at different E:T ratio. Panc-1-luc (mesothelin negative, left), Capan-2-luc (mesothelin positive, middle), Panc-1-luc-MSLN (mesothelin positive, right). (D) Schematic timeline of the antitumor efficacy experiment design. 2 × 10 6 Capan-2-luc cells were subcutaneously inoculated (s.c.) into the right flank of six-week-old B-NDG mice, tumors were established for 10 days prior to treatment initiation. Then, SS1-ICOSBBz-CAR-T cells or PBS (200 μL control) were administered via tail vein injection, (n = 5). (E) Bioluminescence imaging of mice at indicated days after treatment. (F) Quantification of tumor burden dynamics following CAR-T therapy. Tumor volume was monitored weekly during 35 days, and tumor volume changes were calculated relative to baseline (Day 0 set as 0% change). The tumor size at day 7 served as the reference for weekly measurements. Statistical significance of volume changes was assessed using t-test (n = 5 per group). (G) Body weight of mice treated with SS1-ICOSBBZ-CAR-T cells were measured. All data are presented as means ± standard deviation. *P < 0.05, **P < 0.01.

Article Snippet: Vero, SKOV3 (human ovarian), Panc-1, and Capan-2 cell lines were obtained from ATCC.

Techniques: In Vitro, In Vivo, Construct, Derivative Assay, Staining, Activity Assay, Luciferase, Incubation, Control, Injection, Imaging, Standard Deviation

Journal: Frontiers in Immunology

Article Title: IL-12-armed oncolytic HSV-2 enhances CAR T cell efficacy against pancreatic cancer in xenografted models

doi: 10.3389/fimmu.2025.1664289

Figure Lengend Snippet: In vitro and in vivo cytotoxicity effects of oHSV-2-IL-12 on pancreatic cancer cells. (A) Capan-2-luc cells were infected with oHSV-2-IL-12 at MOIs of 0.1 and 0.5. Cell morphology was monitored at 24 and 48 hours post-infection using light microscopy. Representative images depict virus-induced cytopathic effects, including cell rounding and detachment, indicative of oncolytic activity. (B) Real-time cytolysis assessed by RTCA and determination of KT80 values. Representative RTCA graphs to illustrate cytolytic effects of oHSV-2-IL-12 on Capan-2-luc cells. Capan-2-luc cancer cells were plated in duplicates in RTCA plates and incubated overnight. Thereafter oHSV-2-IL-12 was added at MOI 0.05, 0.5 and 5. The impedance was recorded every 10 min for 96 h. Cell index (CI) values were monitored every 10 minutes for 96 hours using the xCELL igence RTCA system. CI values were normalized to the time point of virus addition. Percent cytolysis was calculated, and the time required to reach 80% cytolysis (KT80) was determined for each MOI (right). (C) IL-12 secretion by Capan-2-luc cells infected with oHSV-2-IL-12 in vitro and in vivo . Capan-2-luc cells were infected with oHSV-2-IL-12 at MOIs of 0.5. After 24 hours and 48 hours, supernatants were collected, and IL-12 levels were measured by ELISA (left). Six-week-old B-NDG mice bearing subcutaneous Capan-2-luc tumors received intratumoral injections of oHSV-2-IL-12 on days 0, 3, and 6. On day 9 post-initial injection, tumors were harvested, homogenized, and IL-12 levels were measured by ELISA (n=3). Statistical significance between the two MOI groups was assessed using an unpaired two-tailed Student’s t-test (n=3). (D) Schematic representation of the in vivo treatment protocol using oHSV-2-IL-12 in six-week-old B-NDG mice bearing subcutaneous Capan-2-luc tumors. Mice received three injections of oHSV-2-IL-12 (i.t.) at two-day intervals,. Weekly assessments included BLI, body weight measurement, and tumor volume evaluation. (E) In vivo cancer cells BLI results obtained weekly following oHSV-2-IL-12 (i.t.) inoculation. Mice were divided into three dose groups: high (1×10 6 CCID 50 ), medium (1×10 5 CCID 50 ), and low (1×10 4 CCID 50 ). (F) Weekly tumor volume measurements of different groups. Tumor response to oHSV-2-IL-12 therapy was analyzed by two-way ANOVA with Tukey’s multiple comparisons (n=5/group). (G) Weekly body weight changes during treatment. Statistical significance: **** p <0.0001. ns, not significant.

Article Snippet: Vero, SKOV3 (human ovarian), Panc-1, and Capan-2 cell lines were obtained from ATCC.

Techniques: In Vitro, In Vivo, Infection, Light Microscopy, Virus, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Injection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: IL-12-armed oncolytic HSV-2 enhances CAR T cell efficacy against pancreatic cancer in xenografted models

doi: 10.3389/fimmu.2025.1664289

Figure Lengend Snippet: Combination oHSV-2-IL-12 and CAR-T achieved complete tumor clearance by promoting expansion of SS1-ICOSBBZ-CAR-T cells. (A) Schematic diagram of the combined oHSV-2-IL-12 and SS1-ICOSBBz-CAR-T treatment regimen in 6-week-old B-NDG mice bearing subcutaneous Capan-2-luc tumors established for 14 days. Xenografted pancreatic tumors were injected i.t. with 100 μL PBS, 1×10 5 CCID 50 oHSV-2-GFP, oHSV-2-IL-12 on treatment days 0, 3, and 6. 200 μL PBS or 1×10 7 SS1-ICOSBBz-CAR-T cells were i.v. in B-NDG mice on treatment day 2. On treatment day 7, retro-orbital venous blood of B-NDG mice were sampled every 3 days. (B) Representative tumor BLI measurement. (C) Weekly tumor volume measurements were performed. (D) Mice were weighed weekly. (E) CAR-T cells in mouse retro-orbital venous blood detected by flow cytometry every 3 days starting from treatment day 7. Significance was assessed using two way ANOVA combined with Tukey’s multiple comparisons test (each group n=5). (F) CAR-T cells frequency in splenic single-cell suspensions in mice were detected by flow cytometry after sacrificing mice on treatment day 8 and day 11. ns:p>0.05;**p<0.01;***p<0.001;**** p<0.0001.

Article Snippet: Vero, SKOV3 (human ovarian), Panc-1, and Capan-2 cell lines were obtained from ATCC.

Techniques: Injection, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: IL-12-armed oncolytic HSV-2 enhances CAR T cell efficacy against pancreatic cancer in xenografted models

doi: 10.3389/fimmu.2025.1664289

Figure Lengend Snippet: In vivo combination oHSV-2-IL-12 and SS1-ICOSBBz-CAR-T prevents antigen-specific tumor rechallenge. (A) Schematic of tumor rechallenge in mice receiving combination therapy with oHSV-2-IL-12 and MSLN-ICOSBBz-CAR-T. Following complete tumor clearance within two weeks post-treatment (1×10 5 oHSV-2-IL-12 + 1×10 5 SS1-ICOSBBz-CAR-T), mice were rechallenged via bilateral subcutaneous flank injections of 2×10 6 panc-1-luc cells (mesothelin-negative) and 2×10 6 Capan-2-luc cells. Tumor growth was monitored by weekly BLI and tumor volume measurements. (B) Panc-1-luc subcutaneously implanted into the left flank of B-NDG mice after 2 weeks elimination of first Capan-2-luc, tumor growth was monitored by weekly BLI. (C) Similarly, a re-challenge was performed by Capan-2-luc cells (S.c.) into the ipsilateral flank of B-NDG mice. Tumor growth was monitored weekly via in vivo BLI (right). (D) Time-dependent second tumor burden was measured and quantified for indicated experimental mouse groups. Experimental mice were sacrificed when the primary tumor size reaches 1000 mm 3 (n = 5).

Article Snippet: Vero, SKOV3 (human ovarian), Panc-1, and Capan-2 cell lines were obtained from ATCC.

Techniques: In Vivo

Journal: Frontiers in Immunology

Article Title: IL-12-armed oncolytic HSV-2 enhances CAR T cell efficacy against pancreatic cancer in xenografted models

doi: 10.3389/fimmu.2025.1664289

Figure Lengend Snippet: OHSV-2-IL-12 enables complete tumor eradication with reduced CAR-T cell dosage by promoting proliferation of SS1-ICOSBBz-CAR-T cells in peripheral blood of B-NDG mice. (A) Schematic of combination therapy with reduced CAR-T dosage and sampling regimen. SS1-ICOSBBz-CAR-T cell dose was reduced to 1/20 (5×10 5 cells) from 1×10 7 . B-NDG mice (6-week-old) bearing subcutaneous Capan-2-luc tumors (2×10 6 cells implanted 14 days prior) received: intratumoral injections (100 μL) of PBS, oHSV-2-GFP (1×10 5 CCID 50 ), or oHSV-2-IL-12 (1×10 5 CCID 50 ) on days 0, 3, and 6. PBS or SS1-ICOSBBz-CAR-T cells (5×10 5 ) were administrated via tail vein injections on day 2. Orbital venous plexus blood collection on day 9. Weekly assessments included bioluminescence imaging (BLI), body weight measurement, and tumor volume caliper measurement. (B) Representative weekly in vivo BLI images. (C) Tumor volume measurements (mean ± SEM; n=5/group). Statistical significance determined by unpaired *t*-test. (D) Body weight tracking (mean ± SEM; n=5/group). (E) Flow cytometric analysis of CAR-T cells in peripheral blood on day 7 post-CAR-T infusion (day 9 post-first OV injection; mean ± SEM; n=5/group). Significance analyzed by one-way ANOVA with Tukey’s multiple comparisons test. ns: not significant (p>0.05); * p<0.05, **p<0.01.

Article Snippet: Vero, SKOV3 (human ovarian), Panc-1, and Capan-2 cell lines were obtained from ATCC.

Techniques: Sampling, Imaging, In Vivo, Injection

Journal: Frontiers in Immunology

Article Title: IL-12-armed oncolytic HSV-2 enhances CAR T cell efficacy against pancreatic cancer in xenografted models

doi: 10.3389/fimmu.2025.1664289

Figure Lengend Snippet: (A) Cytokine/chemokine profiles of polyfunctionality of SS1-ICOSBBZ-CAR-T after cocultured with Capan-2-luc. (A) polyfunctionality of SS1-ICOSBBZ-CAR-T cells after co-cultured with target cells Capan-2-luc. (B) single-cell PSI computed for mesothelin-targeted CAR-T cells co-cultured with Canpan-2 cells for 48 hours at the single-cell level. (C) Polyfunctional heat map displaying major functional cytokines/chemokines secreted by CAR-T cells after target cell stimulation. Each column corresponds to a specific cytokine or combination of cytokines, and the red squares represent the frequency at which the group was secreted by the corresponding subsets of CAR-T.

Article Snippet: Vero, SKOV3 (human ovarian), Panc-1, and Capan-2 cell lines were obtained from ATCC.

Techniques: Cell Culture, Functional Assay, Cell Stimulation