Journal: The Journal of Clinical Investigation

Article Title: Off-the-shelf invariant NKT cells expressing anti-PSCA CAR and IL-15 promote pancreatic cancer regression in mice

doi: 10.1172/JCI179014

Figure Lengend Snippet: ( A ) Schematic diagrams of the clinical grade vectors. tEGFR was included as both a detection marker and a safety switch, allowing for in vivo iNKT cell depletion by administering an anti-EGFR antibody. ( B ) Representative flow cytometric analysis of PSCA CAR_sIL-15 iNKT cells and sIL-15 iNKT cells shows the proportion of CD3 and iNKT (TCR Vα24-Jα18) expression 2 days after transduction. sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells show high NKT purity of approximately 97%. The experiment was conducted with 3 donors with similar results. ( C ) The transduction ratio of PSCA CAR_sIL-15/sIL-15 iNKT cells was detected by measuring tEGFR expression 2 days after transduction and analyzed by flow cytometry. The transduction efficiencies, approximately 42%, were similar in both sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells. The experiment was conducted with 3 donors with similar results. SSC, side scatter. ( D ) The level of apoptosis of sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells was measured by the coexpression of annexin V and 7-AAD 2 days after transduction by flow cytometry. Both sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells exhibited very low levels of apoptosis. ( E ) Quantification of PSCA CAR_sIL-15 and sIL-15 iNKT cell fold expansion following 12 days of secondary expansion (mean ± SD, n = 3). Not significant (Student’s t test). Both sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells can be expanded more than 5,000-fold. ( F ) Surface expression of exhaustion markers LAG-3, PD-1, and TIM-3 on sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells, measured by flow cytometry. The results are displayed as mean ± SD ( n = 3). Not significant (2-way ANOVA).

Article Snippet: The PDAC cell lines were stained with PE-conjugated mouse anti-human PSCA antibody (clone: 7F5; Santa Cruz Biotechnology) and isotype antibody (BioLegend) to determine the PSCA antigen expression levels.

Techniques: Marker, In Vivo, Expressing, Transduction, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Off-the-shelf invariant NKT cells expressing anti-PSCA CAR and IL-15 promote pancreatic cancer regression in mice

doi: 10.1172/JCI179014

Figure Lengend Snippet: ( A ) Surface density expression of PSCA on human PDAC cell lines was measured by mean fluorescent intensity (MFI) using flow cytometry. Capan-1, MIA PaCa-2, and Aspc-1 cells highly expressed PSCA, while Panc-1 and BxPC-3 cells had low PSCA expression. ( B ) Summary of percentages of sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells positive for CD69 and CD25 following a 24-hour coincubation with Capan-1, MIA PaCa-2, or BxPC-3 (gated on iNKT cells). Data are presented as mean ± SD ( n = 3). ( C ) Representative flow cytometric analysis shows the expression of CD69 and CD25 on sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells after coincubation with target cells. ( D ) Representative flow cytometric analysis (left) and summary graph (right) show CD107a expression on sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells after coincubation with target cells. ( E ) Representative flow cytometric analysis (left) and summary graph (right) show TNF-α expression in sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells after coincubation with target cells. ( F ) Representative flow cytometric analysis (left) and summary graph (right) show IFN-γ expression of sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells after coincubation with target cells. All experiments were repeated using ≥ 3 donors and presented as mean ± SD ( B , D , E , and F ). Statistical analyses were performed using 1-way ANOVA, with P values corrected for multiple comparisons using the Holm-Šídák method.* P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The PDAC cell lines were stained with PE-conjugated mouse anti-human PSCA antibody (clone: 7F5; Santa Cruz Biotechnology) and isotype antibody (BioLegend) to determine the PSCA antigen expression levels.

Techniques: Expressing, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Off-the-shelf invariant NKT cells expressing anti-PSCA CAR and IL-15 promote pancreatic cancer regression in mice

doi: 10.1172/JCI179014

Figure Lengend Snippet: ( A ) RTCA results measuring cytotoxicity of sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells against PSCA + Capan-1, PSCA + MIA Paca-2, and PSCA + Aspc-1 or PSCA – Panc-1 and PSCA – BxPC-3 tumor cells at an E:T ratio of 1:1. Experiments were repeated with 3 donors. ( B ) Representative microscopic images show the killing as noted in A after 90 hours of coincubation. Experiments were repeated with 3 donors. ( C ) Freshly isolated human primary NK cells were cultured in the presence of supernatants from nontransduced iNKT (NT supernatant), sIL-15 iNKT, PSCA CAR iNKT, or PSCA CAR_sIL-15 cells for 2 days. Capan-1 cells were labeled with 51 Cr and served as target cells. The labeled target cells were added to the cultured NK cells in the presence of respective supernatants for an additional 12 hours. The cytotoxicity levels were measured by 51 Cr release assay. n = 4 donors. NT versus PSCA, P = 0.2218; NT versus sIL-15, P < 0.0001; PSCA versus PSCA CAR sIL-15, P < 0.0001; PSCA versus sIL-15, P < 0.0001; sIL-15 versus PSCA s15, P = 0.1157. Statistical analyses were performed by 1-way ANOVA with P values corrected for multiple comparisons by Bonferroni’s method.

Article Snippet: The PDAC cell lines were stained with PE-conjugated mouse anti-human PSCA antibody (clone: 7F5; Santa Cruz Biotechnology) and isotype antibody (BioLegend) to determine the PSCA antigen expression levels.

Techniques: Isolation, Cell Culture, Labeling, Release Assay

Journal: The Journal of Clinical Investigation

Article Title: Off-the-shelf invariant NKT cells expressing anti-PSCA CAR and IL-15 promote pancreatic cancer regression in mice

doi: 10.1172/JCI179014

Figure Lengend Snippet: ( A ) Treatment schema for i.p. plus i.v. injection of PSCA CAR_sIL-15 iNKT cells in a human metastatic PDAC model established by i.p. injection of PSCA + Capan-1_luc cells into NSG mice. The image was created in BioRender. ( B ) Tumor growth, directly correlated with color intensity, was monitored by BLI until week 9. ( C ) Graphical depiction of BLI from B up to day 32. The results are displayed as mean ± SD ( n = 4). * P < 0.05; ** P < 0.01 (2-way ANOVA). ( D ) Overall Kaplan–Meier survival curve. ** P < 0.01 (log-rank test, n = 5). Compared with the 2 control groups, PSCA CAR_sIL-15 iNKT cells significantly inhibited the progression of metastatic PDAC and prolonged the survival of the tumor-bearing mice. ( E ) Schematic diagram as in A but with MIA PaCa-2_luc PDAC tumor cells. ( F ) Representative images of the pancreas and liver from each treatment group at the endpoint of the in vivo experiment. Red arrows mark metastatic tumors in the liver. PSCA CAR_sIL-15 iNKT cells demonstrated strong therapeutic effects, as evidenced by their ability to kill MIA PaCa-2 cells in the pancreas and the liver. ( G ) Summary of relative fold change in BLI over 15 days as shown in H . The results are displayed as mean ± SD ( n = 5). ** P < 0.01; *** P < 0.001 (2-way ANOVA). ( H ) The growth of the tumor was monitored by BLI imaging until week 6. ( I ) Overall Kaplan-Meier survival curve. *** P < 0.001 (log-rank test, n = 5). PSCA CAR_sIL-15 iNKT cells completely eradicated PDAC in vivo. sIL-15 iNKT cells were inferior to PSCA CAR_sIL-15 iNKT cells but also exhibited some degree of efficacy in delaying tumor progression in mice bearing MIA PaCa-2 cells. ( J ) Assessment of blood cells and HGB on day 15 after PDAC cell transplantation (12 days after treatment with PBS, sIL-15 iNKT cells, or PSCA CAR_sIL-15 iNKT cells). Values represent mean ± SD ( n = 5). Not significant (2-way ANOVA).

Article Snippet: The PDAC cell lines were stained with PE-conjugated mouse anti-human PSCA antibody (clone: 7F5; Santa Cruz Biotechnology) and isotype antibody (BioLegend) to determine the PSCA antigen expression levels.

Techniques: Injection, Control, In Vivo, Imaging, Transplantation Assay

Journal: The Journal of Clinical Investigation

Article Title: Off-the-shelf invariant NKT cells expressing anti-PSCA CAR and IL-15 promote pancreatic cancer regression in mice

doi: 10.1172/JCI179014

Figure Lengend Snippet: ( A ) Schematic diagram of treatment with PSCA CAR_sIL-15 iNKT cells in a human orthotopic PDAC model established by i.p. injection of MIA PaCa-2_luc cells into NSG mice. The image was created in BioRender. ( B ) Representative images of the pancreas and the liver of each group in the MIA PaCa-2-transplanting PDAC mouse model at the endpoint of the in vivo experiments. Red arrows mark metastatic tumors in the liver. In this orthotopic PDAC model, PSCA CAR_sIL-15 iNKT cells efficiently eliminated carcinoma in situ within the pancreas and decreased metastatic lesion formation in the liver. ( C ) Summary statistical data of mouse tumor burden changes of each treatment group. The results are displayed as mean ± SD. ** P < 0.01; **** P < 0.0001 (2-way ANOVA). n = 7 for the untreated and PSCA CAR_sIL-15 groups. n = 6 for the sIL-15 group. ( D ) The growth of the tumor was monitored by BLI imaging until week 8. ( E ) Overall Kaplan-Meier survival curve. *** P < 0.001 (log-rank test). n = 7 for the untreated and PSCA CAR_sIL-15 groups. n = 6 for the sIL-15 group. Treatment with PSCA CAR_sIL-15 iNKT cells resulted in complete clearance of orthotopic tumors and reached 100% survival. ( F ) Assessment of blood cell populations on day 15 after PDAC cell transplantation (12 days after iNKT cell treatment). Peripheral blood counts and HGB in the PSCA CAR_sIL-15 iNKT group were not changed compared with the untreated group and sIL-15 iNKT cell treatment group. Values represent mean ± SD ( n = 7 for the untreated and PSCA CAR_sIL-15 groups. n = 4 for the sIL-15 group.). 2-way ANOVA.

Article Snippet: The PDAC cell lines were stained with PE-conjugated mouse anti-human PSCA antibody (clone: 7F5; Santa Cruz Biotechnology) and isotype antibody (BioLegend) to determine the PSCA antigen expression levels.

Techniques: Injection, In Vivo, In Situ, Imaging, Transplantation Assay

Journal: The Journal of Clinical Investigation

Article Title: Off-the-shelf invariant NKT cells expressing anti-PSCA CAR and IL-15 promote pancreatic cancer regression in mice

doi: 10.1172/JCI179014

Figure Lengend Snippet: ( A ) MFI of PSCA (blue solid histograms) on GR cell lines (Capan-1 GR and MIA Paca-2 GR) compared with parental cell lines (red solid histograms) as measured flow cytometry. ( B ) Cytotoxicity of gemcitabine measured by RTCA in the presence of different concentrations of gemcitabine on GR and parental PDAC cell lines. Capan-1 GR and MIA Paca-2 GR were not while their parental cell lines were killed by gemcitabine at the concentrations of 1.6 μM and 3.2 μM. ( C ) Cytotoxicity of sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells against GR PDAC cell lines (Capan-1 GR and MIA Paca-2 GR) and the parental PDAC cell lines at an E:T ratio of 1:2, measured by RTCA assay. PSCA CAR_sIL-15 iNKT cells maintained their potent killing ability against Capan-1 GR and MIA PaCa-2 GR cells compared with the parental cells. ( A – C ) Experiments were repeated 3 times or with 3 different donors. Expression of T cell activation markers CD69 ( D ) and CD25 ( E ) on sIL-15 iNKT cells and PSCA CAR_sIL-15 iNKT cells following a 24-hour coincubation with Capan-1 GR, Capan-1, MIA PaCa-2 GR, or MIA PaCa-2 (gated on EGFR + cells). Data are represented as mean ± SD ( n = 3). There were comparable levels of CD69 and CD25 expression in PSCA CAR_sIL-15 iNKT cells when they were coincubated with the GR cells and the parental cells. ( F ) The parent cell lines Capan-1_luc and Capan-1 GR_luc were injected i.p. (2 × 10 5 cells/mouse). Three days later, tumor engraftment was confirmed by BLI and visually displayed in vivo weekly up to 8 weeks. The fold changes of BLI for each treatment group were measured. n = 4 for the untreated group. n = 5 for the sIL-15 group. n = 6 for the PSCA CAR_sIL-15 group. ( G ) Overall Kaplan-Meier survival curve. log-rank test ( n = 4 for the untreated group. n = 5 for the sIL-15 group. n = 6 for the PSCA CAR_sIL-15 group.). ( H ) The growth of the tumor was monitored by BLI imaging until week 8. A single dose of PSCA CAR_sIL-15 iNKT cells still significantly suppressed Capan-1 GR tumor progression, and the antitumor effect of CAR_sIL-15 iNKT cells was not different when compared with the Capan-1 mouse tumor.

Article Snippet: The PDAC cell lines were stained with PE-conjugated mouse anti-human PSCA antibody (clone: 7F5; Santa Cruz Biotechnology) and isotype antibody (BioLegend) to determine the PSCA antigen expression levels.

Techniques: Flow Cytometry, Expressing, Activation Assay, Injection, In Vivo, Imaging

Journal: The Journal of Clinical Investigation

Article Title: Off-the-shelf invariant NKT cells expressing anti-PSCA CAR and IL-15 promote pancreatic cancer regression in mice

doi: 10.1172/JCI179014

Figure Lengend Snippet: ( A ) In vitro cytotoxicity of off-the-shelf sIL-15 iNKT cells and off-the-shelf PSCA CAR_sIL-15 iNKT cells against PSCA + PDAC cell lines, measured by RTCA at an E:T ratio 1:1. Experiments were repeated with 3 donors. ( B ) In vivo assessment of Capan-1 tumor burden was monitored by BMI during treatment with off-the-shelf cryopreserved sIL-15 iNKT cells and off-the-shelf cryopreserved PSCA CAR_sIL-15 iNKT cells until week 7. ( C ) Graphical summary of mouse tumor burden during treatment shown in B . Data are displayed as mean ± SD until day 30. *** P < 0.001 (2-way ANOVA). n = 6 for the untreated and sIL-15 groups. n = 7 for the PSCA CAR_sIL-15 group. ( D ) Overall Kaplan-Meier survival curve as the result of treatment shown in B . *** P < 0.001 (log-rank test). n = 6 for the untreated and the sIL-15 groups. n = 7 for the PSCA CAR_sIL-15 group. ( E ) NSG mice were injected with 5 × 10 5 Capan-1-luc cells on day 1. On day 7, mice received fresh PSCA CAR_sIL-15 iNKT cells or frozen PSCA CAR_sIL-15 iNKT cells (i.p. 4 × 10 6 and i.v. 2 × 10 6 /mouse, respectively). Tumor burden was monitored by BLI until day 49. ( F ) Overall Kaplan-Meier survival curve resulting from E . n = 6 per group. One mouse receiving fresh PSCA CAR_sIL-15 iNKT cells died accidentally during imaging. ( G ) NSG mice were injected with 5 × 10 5 Capan-1-luc cells on day 1. On day 7, mice were injected with off-the-shelf cryopreserved PSCA CAR-sIL-15 iNKT cells (i.p. 4 × 10 6 plus i.v. 2 × 10 6 /mouse). Blood, bone marrow, lung, liver, pancreas, kidney, and spleen were harvested on days 1, 7, 14, and 21 after PSCA CAR_sIL-15 iNKT cell injection. PSCA CAR_sIL-15 iNKT cell persistence was detected by flow cytometry using hCD45 + ( n = 3 or 4 mice per time point).

Article Snippet: The PDAC cell lines were stained with PE-conjugated mouse anti-human PSCA antibody (clone: 7F5; Santa Cruz Biotechnology) and isotype antibody (BioLegend) to determine the PSCA antigen expression levels.

Techniques: In Vitro, In Vivo, Injection, Imaging, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Off-the-shelf invariant NKT cells expressing anti-PSCA CAR and IL-15 promote pancreatic cancer regression in mice

doi: 10.1172/JCI179014

Figure Lengend Snippet: ( A ) Schematic diagram of treatment with PSCA CAR_sIL-15 iNKT cells or PSCA CAR_sIL-15 T cells in a Capan-1–transplanted metastatic PDAC humanized mouse model to investigate the risk of GvHD. ( B ) Fourteen days after transplantation, the percentage of repopulated human CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD19 + B cells, and CD56 + NK cells were assessed in the peripheral blood of the mice. ( C ) The burden of tumor was monitored by BLI on days 24 and day 31. ( D ) Clinical GvHD scores were observed on day 42. *** P < 0.001. n = 3 per group. ( E ) Splenic dimensions of tumor-bearing mice treated with PSCA CAR_sIL-15 T cells (left) or PSCA CAR_sIL-15 iNKT cells (right) measured on day 42. ( F ) SGM3 mice engrafted with PBMCs were injected with 2 × 10 6 Capan-1-luc cells. Seven days later, mice were injected either PBS ( n = 3) or off-the-shelf cryopreserved iNKT cells (4 × 10 6 cells/mouse i.p. plus 2 × 10 6 cells/mouse i.v., n = 6). Sera were harvested 1 (day 1) and 3 days (day 3) after the iNKT cell injection to assess for CRS-associated cytokines. The sera from the PBS group were collected 1 day after injection (Day 1). Data are displayed as the mean ± SD. Statistical analyses were conducted using a 2-sided t test.

Article Snippet: The PDAC cell lines were stained with PE-conjugated mouse anti-human PSCA antibody (clone: 7F5; Santa Cruz Biotechnology) and isotype antibody (BioLegend) to determine the PSCA antigen expression levels.

Techniques: Transplantation Assay, Injection

Journal: Journal for immunotherapy of cancer

Article Title: Impact of tumor localization on antitumor immunity with conditionally activated CTLA-4 blockade.

doi: 10.1136/jitc-2024-010566

Figure Lengend Snippet: Figure 1 DVD anti-CTLA-4 shows specific, safe antitumor efficacy in murine subcutaneous PSCA+ tumors. (A) Antibodies used in subcutaneous tumor experiments. In DVD antibodies (untargeted: ttDVD; uncleavable: unDVD; symmetric: sDVD; asymmetric: aDVD), tissue specificity is conferred by murine PSCA targeting in the outer variable domain. On tumor-induced upregulation of matriptase (MT-SP1), the linkers between both variable domains are cleaved and the CTLA-4-targeting inner variable domains are exposed and functional. (B) Uncleaved and matriptase-cleaved sDVD and aDVD resolved in 4–12% gradient SDS- PAGE gel. Briefly, 20 µg of each antibody were incubated overnight in the presence of 5 µL (2.5 µg) recombinant matriptase at room temperature. 3 µg of lysate were loaded onto SDS-PAGE gels and protein content was detected by SafeBlue staining. (C) Experiment design for the assessment of the antitumor efficacy of the DVD anti-CTLA-4 antibodies. 1 million mouse prostate TRAMP-C2 tumor cells were implanted on the right flanks of C57BL/6J mice (n=5–8). When tumors reached 150 mm3 (day 0), mice were enrolled into treatment groups, and injected intraperitoneally with antibodies on days 3, 6 and 9. Tumor growth was monitored twice weekly. (D) Tumor volume curves for mice (n=7–8 per group) from n=1 of three experiments in (C). *p<0.05 sDVD versus: isotype, unDVD; #, p<0.05 aDVD versus: isotype, unDVD; Φ, p<0.05 sDVD, aCTLA-4 versus ttDVD. One-way analysis of variance applying Kruskal-Wallis test was used for all shown p values. Error bars represent SEM. (E) Kaplan-Meier survival curves for mice (n=7–8 per group) from n=1 of three experiments in (C). *p<0.05 aCTLA-4, sDVD and aDVD versus rest by log-rank Mantel-Cox test. (F) Percentage of body weight variation for mice (n=7–8 per group) from n=1 of three experiments in (C). Error bars represent SEM. *p<0.05 aCTLA-4 versus isotype, aDVD; # p<0.05 aCTLA-4 versus isotype, unDVD, sDVD and aDVD. (G) Total gated CD45+ CD3+ CD8+ T-cell counts from lymph nodes of mice (n=4 from n=1 of three experiments) involved in (C) by flow cytometry. aCTLA-4, anti-cytotoxic T-lymphocyte associated protein 4; DVD, dual variable domain; HC, heavy chain; LC, light chain; MW, molecular weight; PSCA, prostate stem cell antigen; SDS-PAGE: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis; VH: variable domain of the heavy chain; VL, variable domain of the light chain; ttDVD, tetanus-toxin targeted DVD.

Article Snippet: Membranes were blocked and then incubated overnight at 1:1,000 with antimouse PSCA (17171- 1- AP, Proteintech) or 1:10,000 antivinculin (AB129002, Abcam) as loading control in TBS- T (170–6435, Bio- Rad).

Techniques: Functional Assay, SDS Page, Incubation, Recombinant, Staining, Injection, Flow Cytometry, Molecular Weight, Polyacrylamide Gel Electrophoresis

Journal: Journal for immunotherapy of cancer

Article Title: Impact of tumor localization on antitumor immunity with conditionally activated CTLA-4 blockade.

doi: 10.1136/jitc-2024-010566

Figure Lengend Snippet: Figure 2 PSCA-targeted anti-CTLA-4 shows specific antitumor efficacy in systemic PSCA+ tumors. (A) Experiment summary for characterization of PSCA+ MC38 cells. Briefly, luciferase-expressing empty vector (EV) PSCA− or PSCA+ MC38 cells were injected intravenously in C57BL/6J mice (n=5 per group). Treatment was administered intraperitoneally on days 7, 10 and 13 after implantation. Tumor growth was monitored by in vivo luminescence quantification twice a week. (B) Murine PSCA (mPSCA) expression on Luc-MC38 cell lines by western blot. (C) In vivo bioluminescence quantification of luciferase-expressing MC38 cells in mice (n=4) from n=1 of two experiments described in (A) on day 21 after tumor cell injection, shown as average photon radiance. (D) Representative individual whole-body images of mice from n=2 experiments involved in (A) on days 7, 14, 21 and 30 after systemic tumor implantation, colored by tumor luminescence intensity. Red circles are regions of interest (ROIs) considered for bioluminescence quantification with Living Image Software. Total photon counts are shown above ROIs. Mouse drawings with crossed eyes represent the percentage of deceased mice for each group (20% each). (E) Kaplan-Meier survival curves for mice (n=10) involved in (A) (n=2 experiments). *p<0.05 versus EV isotype, EV sDVD, EV unDVD, mPSCA isotype, mPSCA unDVD by log-rank Mantel-Cox test. (F) Percentage of body weight variation for mice (n=10) involved in (A) (n=2 experiments). Error bars represent SEM. *p<0.05 EV aCTLA-4, PSCA aCTLA-4 versus rest; # p<0.05 EV aCTLA-4 versus EV sDVD, PSCA isotype by one-way analysis of variance. (G) GI tracts from animals (n=3) harboring PSCA+tumors from n=1 experiment were stained for CD4. For each experimental group, a representative ROI is shown on top, and DAB+ (CD4+) counts below. (H) Mean fluorescence intensity offset histograms of TNF-α in GI-derived CD4+T cells from PSCA+challenged mice by flow cytometry. aCTLA-4, anti-cytotoxic T-lymphocyte associated protein 4; DVD, dual variable domain; GI, gastrointestinal; mPSCA, murine prostate stem cell antigen; PE, phycoerythrin; sDVD, symmetric DVD; TNF, tumor necrosis factor; unDVD, uncleavable DVD.

Article Snippet: Membranes were blocked and then incubated overnight at 1:1,000 with antimouse PSCA (17171- 1- AP, Proteintech) or 1:10,000 antivinculin (AB129002, Abcam) as loading control in TBS- T (170–6435, Bio- Rad).

Techniques: Luciferase, Expressing, Plasmid Preparation, Injection, In Vivo, Western Blot, Tumor Implantation, Software, Staining, Fluorescence, Derivative Assay, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Impact of tumor localization on antitumor immunity with conditionally activated CTLA-4 blockade.

doi: 10.1136/jitc-2024-010566

Figure Lengend Snippet: Figure 3 PSCA-targeted DVD anti-CTLA-4 induces antigen-specific responses and improved T-cell fitness in systemically implanted PSCA+ tumors. (A) Percentage of CD8+ T cells within the immune CD45+ compartment from flow cytometry data from lung tumors of mice (n=3) harvested on day 15 after tumor injection. As in the rest of the figure, representative data from n=1 of two experiments is shown. (B) Percentage of intratumoral FoxP3+ cells within gated CD4+ T cells. (C) Percentage of intratumoral CD44+ CD62L− cells within gated CD8+ T cells. (D) Percentage of intratumoral MC38 antigen-specific ADPGK- reactive CD8+ T cells. (E) Representative flow cytometry pseudocolor dot plots showing MC38 antigen ADPGK reactivity in gated CD8+ T cells from mouse lung tumors. Shown values are percentages within all CD8+. (F) MFI of effector molecule perforin (PRF1) in intratumoral ADPGK+ CD8+ T cells. (G) MFI of proliferation marker Ki67 in gated intratumoral ADPGK+ CD8+ T cells. (H) Percentage of PD-1+ TCF1+ cells in gated intratumoral ADPGK+ T cells. (I) Part-of-whole pie charts showing mean percentages of cells expressing 0, any given 1, any combination of 2, or 3 exhaustion markers PD-1, LAG-3, TIM3 in intratumoral ADPGK+ CD8+ T cells, as assessed by Boolean gating from flow cytometry data. Right, percentage of intratumoral PD-1+ TIM3+ LAG-3+ in ADPGK+ CD8+ T cells. aCTLA-4, anti-cytotoxic T-lymphocyte associated protein 4; DVD, dual variable domain; EV, empty vector; LAG-3, lymphocyte-activation gene 3; MFI, mean fluorescence intensity; PD-1, programmed cell death protein 1; PSCA, prostate stem cell antigen; sDVD, symmetric DVD; unDVD, uncleavable DVD; TIM3, T-cell immunoglobulin and mucin-domain containing-3.

Article Snippet: Membranes were blocked and then incubated overnight at 1:1,000 with antimouse PSCA (17171- 1- AP, Proteintech) or 1:10,000 antivinculin (AB129002, Abcam) as loading control in TBS- T (170–6435, Bio- Rad).

Techniques: Flow Cytometry, Injection, Marker, Expressing, Plasmid Preparation, Activation Assay, Fluorescence

Journal: Journal for immunotherapy of cancer

Article Title: Impact of tumor localization on antitumor immunity with conditionally activated CTLA-4 blockade.

doi: 10.1136/jitc-2024-010566

Figure Lengend Snippet: Figure 4 Symmetric DVD induces systemic immune responses. (A) Percentage of CD4+ T cells within the immune CD45+ compartment from flow cytometry data from spleens of mice (n=3) harvested on day 15 after tumor injection. As in the rest of the figure, representative data from n=1 of two experiments is shown. (B) Percentage of FoxP3+ cells within gated CD4+ T cells. (C) Percentage of CD44+ CD62L− cells within gated CD4+ T cells. (D) Percentage of splenic CD8+ T cells within the immune CD45+ compartment. (E) Percentage of splenic MC38 antigen-specific ADPGK-reactive CD8+ T cells. Shown p values obtained by one-way analysis of variance adjusted for multiple comparisons; *p<0.05 versus rest. (F) Representative flow cytometry pseudocolor dot plots showing MC38 antigen ADPGK reactivity in gated CD8+ T cells from mouse spleens. Shown values are percentages within all CD8+. (G) MFI of effector molecule perforin (PRF1) in gated splenic ADPGK+ CD8+ T cells. *p<0.05 versus rest. (H) MFI of proliferation marker Ki67 in gated splenic ADPGK+ CD8+ T cells. *p<0.05 versus rest. (I) Percentage of PD-1+ TCF1+ cells in gated splenic ADPGK+ CD8+ T cells. *p<0.05 versus rest. (J) Part-of-whole pie charts showing mean percentages of cells expressing 0, any given 1, any combination of 2, or 3 exhaustion markers PD-1, LAG-3, TIM3 in splenic ADPGK+ CD8+ T cells, as assessed by Boolean gating from flow cytometry data. Right, percentage of splenic PD-1− TIM3− LAG-3− in ADPGK+ CD8+ T cells. (K) Percentage of CD8+ T cells within the immune CD45+ compartment in mouse tumor-draining lymph nodes (TDLNs). (L) Percentage of MC38 antigen-specific ADPGK-reactive CD8+ T cells in TDLNs. (M) Representative flow cytometry pseudocolor dotplots showing MC38 antigen ADPGK reactivity in gated CD8+ T cells from mouse TDLNs. Shown values are percentages within all CD8+. aCTLA-4, anti-cytotoxic T-lymphocyte associated protein 4; DVD, dual variable domain; EV, empty vector; LAG-3, lymphocyte-activation gene 3; MFI, mean fluorescence intensity; PD-1, programmed cell death protein 1; PSCA, prostate stem cell antigen; sDVD, symmetric DVD; unDVD, uncleavable DVD; TIM3, T-cell immunoglobulin and mucin-domain containing-3.

Article Snippet: Membranes were blocked and then incubated overnight at 1:1,000 with antimouse PSCA (17171- 1- AP, Proteintech) or 1:10,000 antivinculin (AB129002, Abcam) as loading control in TBS- T (170–6435, Bio- Rad).

Techniques: Flow Cytometry, Injection, Marker, Expressing, Plasmid Preparation, Activation Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: The application of emerging immunotherapy in the treatment of prostate cancer: progress, dilemma and promise

doi: 10.3389/fimmu.2025.1544882

Figure Lengend Snippet: Different CAR-T cells clinical trials.

Article Snippet: PSCA-Targeted CAR-T Cells (BPX-60 , mCRPC , PSCA , Phase 1Phase 2 , 151 , NCT02744287 , Suspended , 2016-04-20 , Bellicum Pharmaceuticals.

Techniques:

Journal: Journal for Immunotherapy of Cancer

Article Title: Impact of tumor localization on antitumor immunity with conditionally activated CTLA-4 blockade

doi: 10.1136/jitc-2024-010566

Figure Lengend Snippet: DVD anti-CTLA-4 shows specific, safe antitumor efficacy in murine subcutaneous PSCA + tumors. ( A ) Antibodies used in subcutaneous tumor experiments. In DVD antibodies (untargeted: ttDVD; uncleavable: unDVD; symmetric: sDVD; asymmetric: aDVD), tissue specificity is conferred by murine PSCA targeting in the outer variable domain. On tumor-induced upregulation of matriptase (MT-SP1), the linkers between both variable domains are cleaved and the CTLA-4-targeting inner variable domains are exposed and functional. ( B ) Uncleaved and matriptase-cleaved sDVD and aDVD resolved in 4–12% gradient SDS-PAGE gel. Briefly, 20 µg of each antibody were incubated overnight in the presence of 5 µL (2.5 µg) recombinant matriptase at room temperature. 3 µg of lysate were loaded onto SDS-PAGE gels and protein content was detected by SafeBlue staining. ( C ) Experiment design for the assessment of the antitumor efficacy of the DVD anti-CTLA-4 antibodies. 1 million mouse prostate TRAMP-C2 tumor cells were implanted on the right flanks of C57BL/6J mice (n=5–8). When tumors reached 150 mm 3 (day 0), mice were enrolled into treatment groups, and injected intraperitoneally with antibodies on days 3, 6 and 9. Tumor growth was monitored twice weekly. ( D ) Tumor volume curves for mice (n=7–8 per group) from n=1 of three experiments in ( C ). *p<0.05 sDVD versus: isotype, unDVD; #, p<0.05 aDVD versus: isotype, unDVD; Φ, p<0.05 sDVD, aCTLA-4 versus ttDVD. One-way analysis of variance applying Kruskal-Wallis test was used for all shown p values. Error bars represent SEM. ( E ) Kaplan-Meier survival curves for mice (n=7–8 per group) from n=1 of three experiments in ( C ). *p<0.05 aCTLA-4, sDVD and aDVD versus rest by log-rank Mantel-Cox test. ( F ) Percentage of body weight variation for mice (n=7–8 per group) from n=1 of three experiments in ( C ). Error bars represent SEM. *p<0.05 aCTLA-4 versus isotype, aDVD; # p<0.05 aCTLA-4 versus isotype, unDVD, sDVD and aDVD. ( G ) Total gated CD45 + CD3 + CD8 + T-cell counts from lymph nodes of mice (n=4 from n=1 of three experiments) involved in ( C ) by flow cytometry. aCTLA-4, anti-cytotoxic T-lymphocyte associated protein 4; DVD, dual variable domain; HC, heavy chain; LC, light chain; MW, molecular weight; PSCA, prostate stem cell antigen; SDS-PAGE: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis; VH: variable domain of the heavy chain; VL, variable domain of the light chain; ttDVD, tetanus-toxin targeted DVD.

Article Snippet: Membranes were blocked and then incubated overnight at 1:1,000 with anti-mouse PSCA (17171-1-AP, Proteintech) or 1:10,000 anti-vinculin (AB129002, Abcam) as loading control in TBS-T (170–6435, Bio-Rad).

Techniques: Functional Assay, SDS Page, Incubation, Recombinant, Staining, Injection, Flow Cytometry, Molecular Weight, Polyacrylamide Gel Electrophoresis

Journal: Journal for Immunotherapy of Cancer

Article Title: Impact of tumor localization on antitumor immunity with conditionally activated CTLA-4 blockade

doi: 10.1136/jitc-2024-010566

Figure Lengend Snippet: PSCA-targeted anti-CTLA-4 shows specific antitumor efficacy in systemic PSCA + tumors. ( A ) Experiment summary for characterization of PSCA + MC38 cells. Briefly, luciferase-expressing empty vector (EV) PSCA− or PSCA + MC38 cells were injected intravenously in C57BL/6J mice (n=5 per group). Treatment was administered intraperitoneally on days 7, 10 and 13 after implantation. Tumor growth was monitored by in vivo luminescence quantification twice a week. ( B ) Murine PSCA (mPSCA) expression on Luc-MC38 cell lines by western blot. ( C ) In vivo bioluminescence quantification of luciferase-expressing MC38 cells in mice (n=4) from n=1 of two experiments described in ( A ) on day 21 after tumor cell injection, shown as average photon radiance. ( D ) Representative individual whole-body images of mice from n=2 experiments involved in ( A ) on days 7, 14, 21 and 30 after systemic tumor implantation, colored by tumor luminescence intensity. Red circles are regions of interest (ROIs) considered for bioluminescence quantification with Living Image Software. Total photon counts are shown above ROIs. Mouse drawings with crossed eyes represent the percentage of deceased mice for each group (20% each). ( E ) Kaplan-Meier survival curves for mice (n=10) involved in ( A ) (n=2 experiments). *p<0.05 versus EV isotype, EV sDVD, EV unDVD, mPSCA isotype, mPSCA unDVD by log-rank Mantel-Cox test. ( F ) Percentage of body weight variation for mice (n=10) involved in ( A ) (n=2 experiments). Error bars represent SEM. *p<0.05 EV aCTLA-4, PSCA aCTLA-4 versus rest; # p<0.05 EV aCTLA-4 versus EV sDVD, PSCA isotype by one-way analysis of variance. ( G ) GI tracts from animals (n=3) harboring PSCA+tumors from n=1 experiment were stained for CD4. For each experimental group, a representative ROI is shown on top, and DAB+ (CD4+) counts below. ( H ) Mean fluorescence intensity offset histograms of TNF-α in GI-derived CD4+T cells from PSCA+challenged mice by flow cytometry. aCTLA-4, anti-cytotoxic T-lymphocyte associated protein 4; DVD, dual variable domain; GI, gastrointestinal; mPSCA, murine prostate stem cell antigen; PE, phycoerythrin; sDVD, symmetric DVD; TNF, tumor necrosis factor; unDVD, uncleavable DVD.

Article Snippet: Membranes were blocked and then incubated overnight at 1:1,000 with anti-mouse PSCA (17171-1-AP, Proteintech) or 1:10,000 anti-vinculin (AB129002, Abcam) as loading control in TBS-T (170–6435, Bio-Rad).

Techniques: Luciferase, Expressing, Plasmid Preparation, Injection, In Vivo, Western Blot, Tumor Implantation, Software, Staining, Fluorescence, Derivative Assay, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Impact of tumor localization on antitumor immunity with conditionally activated CTLA-4 blockade

doi: 10.1136/jitc-2024-010566

Figure Lengend Snippet: PSCA-targeted DVD anti-CTLA-4 induces antigen-specific responses and improved T-cell fitness in systemically implanted PSCA + tumors. ( A ) Percentage of CD8 + T cells within the immune CD45 + compartment from flow cytometry data from lung tumors of mice (n=3) harvested on day 15 after tumor injection. As in the rest of the figure, representative data from n=1 of two experiments is shown. ( B ) Percentage of intratumoral FoxP3 + cells within gated CD4 + T cells. ( C ) Percentage of intratumoral CD44 + CD62L− cells within gated CD8 + T cells. ( D ) Percentage of intratumoral MC38 antigen-specific ADPGK-reactive CD8 + T cells. ( E ) Representative flow cytometry pseudocolor dot plots showing MC38 antigen ADPGK reactivity in gated CD8 + T cells from mouse lung tumors. Shown values are percentages within all CD8 + . ( F ) MFI of effector molecule perforin (PRF1) in intratumoral ADPGK + CD8 + T cells. ( G ) MFI of proliferation marker Ki67 in gated intratumoral ADPGK + CD8 + T cells. ( H ) Percentage of PD-1 + TCF1 + cells in gated intratumoral ADPGK + T cells. ( I ) Part-of-whole pie charts showing mean percentages of cells expressing 0, any given 1, any combination of 2, or 3 exhaustion markers PD-1, LAG-3, TIM3 in intratumoral ADPGK + CD8 + T cells, as assessed by Boolean gating from flow cytometry data. Right, percentage of intratumoral PD-1 + TIM3 + LAG-3 + in ADPGK + CD8 + T cells. aCTLA-4, anti-cytotoxic T-lymphocyte associated protein 4; DVD, dual variable domain; EV, empty vector; LAG-3, lymphocyte-activation gene 3; MFI, mean fluorescence intensity; PD-1, programmed cell death protein 1; PSCA, prostate stem cell antigen; sDVD, symmetric DVD; unDVD, uncleavable DVD; TIM3, T-cell immunoglobulin and mucin-domain containing-3.

Article Snippet: Membranes were blocked and then incubated overnight at 1:1,000 with anti-mouse PSCA (17171-1-AP, Proteintech) or 1:10,000 anti-vinculin (AB129002, Abcam) as loading control in TBS-T (170–6435, Bio-Rad).

Techniques: Flow Cytometry, Injection, Marker, Expressing, Plasmid Preparation, Activation Assay, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Impact of tumor localization on antitumor immunity with conditionally activated CTLA-4 blockade

doi: 10.1136/jitc-2024-010566

Figure Lengend Snippet: Symmetric DVD induces systemic immune responses. ( A ) Percentage of CD4 + T cells within the immune CD45 + compartment from flow cytometry data from spleens of mice (n=3) harvested on day 15 after tumor injection. As in the rest of the figure, representative data from n=1 of two experiments is shown. ( B ) Percentage of FoxP3 + cells within gated CD4 + T cells. ( C ) Percentage of CD44 + CD62L− cells within gated CD4 + T cells. ( D ) Percentage of splenic CD8 + T cells within the immune CD45 + compartment. ( E ) Percentage of splenic MC38 antigen-specific ADPGK-reactive CD8 + T cells. Shown p values obtained by one-way analysis of variance adjusted for multiple comparisons; *p<0.05 versus rest. ( F ) Representative flow cytometry pseudocolor dot plots showing MC38 antigen ADPGK reactivity in gated CD8 + T cells from mouse spleens. Shown values are percentages within all CD8 + . ( G ) MFI of effector molecule perforin (PRF1) in gated splenic ADPGK + CD8 + T cells. *p<0.05 versus rest. ( H ) MFI of proliferation marker Ki67 in gated splenic ADPGK + CD8 + T cells. *p<0.05 versus rest. ( I ) Percentage of PD-1 + TCF1 + cells in gated splenic ADPGK + CD8 + T cells. *p<0.05 versus rest. ( J ) Part-of-whole pie charts showing mean percentages of cells expressing 0, any given 1, any combination of 2, or 3 exhaustion markers PD-1, LAG-3, TIM3 in splenic ADPGK + CD8 + T cells, as assessed by Boolean gating from flow cytometry data. Right, percentage of splenic PD-1 − TIM3 − LAG-3 − in ADPGK + CD8 + T cells. ( K ) Percentage of CD8 + T cells within the immune CD45 + compartment in mouse tumor-draining lymph nodes (TDLNs). ( L ) Percentage of MC38 antigen-specific ADPGK-reactive CD8 + T cells in TDLNs. ( M ) Representative flow cytometry pseudocolor dotplots showing MC38 antigen ADPGK reactivity in gated CD8 + T cells from mouse TDLNs. Shown values are percentages within all CD8 + . aCTLA-4, anti-cytotoxic T-lymphocyte associated protein 4; DVD, dual variable domain; EV, empty vector; LAG-3, lymphocyte-activation gene 3; MFI, mean fluorescence intensity; PD-1, programmed cell death protein 1; PSCA, prostate stem cell antigen; sDVD, symmetric DVD; unDVD, uncleavable DVD; TIM3, T-cell immunoglobulin and mucin-domain containing-3.

Article Snippet: Membranes were blocked and then incubated overnight at 1:1,000 with anti-mouse PSCA (17171-1-AP, Proteintech) or 1:10,000 anti-vinculin (AB129002, Abcam) as loading control in TBS-T (170–6435, Bio-Rad).

Techniques: Flow Cytometry, Injection, Marker, Expressing, Plasmid Preparation, Activation Assay, Fluorescence