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: Biomedicines

Article Title: Differential Protein-Coding Gene Expression Profile in Patients with Prostate Cancer

doi: 10.3390/biomedicines12112509

Figure Lengend Snippet: Genes classified as drug targets in the DrugBank database. Protein-coding genes as targets for drugs related to the treatment of prostate cancer in all states (investigation, experimental, approved). In the case of target genes for many drugs, only the first 10 are reported.

Article Snippet: DB05933 , MK-4721 , A fully human monoclonal antibody directed to Agensys’ proprietary target Prostate Stem Cell Antigen (PSCA), an antigen expressed at significant levels on tumor cells from the majority of patients with all stages of prostate, pancreatic, and bladder cancers [investigation]. , PSCA [O43653].

Techniques: Diagnostic Assay, Positron Emission Tomography, Membrane, Imaging, Biomarker Discovery, Expressing, Derivative Assay

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles from seminal plasma interact with T cells in vitro and drive their differentiation into regulatory T‐cells

doi: 10.1002/jev2.12457

Figure Lengend Snippet: Isolation and characterization of spEVs. spEVs were collected from pooled seminal plasma samples from vasectomized men by ultracentrifugation on top of an iohexol cushion, and then loaded at the bottom of an iohexol density gradient and floated upward into the gradient by ultracentrifugation. Gradient fractions were collected from the top and analysed by SDS‐PAGE, followed by total protein staining (a), or immunoblotting for the presence of Annexin A1, CD9, HSP70, PSCA, Galectin‐3, and CD47 (b). Molecular weight markers are indicated on the left in kDa. Density gradient fractions containing spEVs (4‐7) were pooled and spEVs isolated further by SEC. SEC fractions 6‐17 were analysed by SDS‐PAGE followed by total protein staining (c) or immunoblotting for the presence of Annexin A1, CD9, HSP70, PSCA, Galectin‐3 and CD47 (d). Molecular weight markers are indicated on the left in kDa. SEC fractions containing isolated spEVs (9‐13) were pooled and analysed by transmission electron microscopy (e) and NTA (f).

Article Snippet: Primary antibodies include mouse anti‐human CD9 (HI9a; 312102; Biolegend; 1:2000); mouse anti‐human PSCA (clone 7F5; sc‐80654; Santa Cruz Biotechnology; 1:1000); mouse anti‐human HSP70 (N27F3‐4; ADI‐SPA‐820‐D; Enzo; 1:1000); mouse anti‐human Annexin A1 (clone 29; 610066; BD Biosciences; 1:1000); rat anti‐human Galectin‐3 (M3/38; CL8942B; CEDARLANE; 1:500) and mouse anti‐human CD47 (B6H12; sc‐12730; Santa Cruz Biotechnology; 1:500).

Techniques: Isolation, Clinical Proteomics, SDS Page, Staining, Western Blot, Molecular Weight, Transmission Assay, Electron Microscopy