pre transformed human universal cdna library  (TaKaRa)

Journal: Journal for immunotherapy of cancer

Article Title: Novel PAP-targeted CAR-T therapy enhances antitumor efficacy through CoupledCAR approach.

doi: 10.1136/jitc-2024-011238

Figure Lengend Snippet: Figure 2 PAP-specific CAR-T cells show antitumor activity both in vitro and in vivo. (A) The expression of tmPAP on PC3 and PC3-tmPAP cells. (B) The binding ability of antibodies to the tmPAP antigen on PC3-tmPAP cells. (C) Schematic diagram of the design of PAP CAR. (D) Killing ability of PAP CAR-T and non-transduced T (NT) cells. (E) The concentrations of IFN-γ, IL-2 and TNF-α in the cell co-culture supernatants. (F) Schematic illustration of the in vivo studies. (G) The expansion of CAR-T cells in the peripheral blood of mice in both the PAP CAR-T and NT groups on Day 14. (H) IFN-γ release, IL-2 release and TNF-α release in the peripheral blood in both the PAP CAR-T and NT groups on Day 14. (I) Tumor volumes in the mice over time (an unpaired t-test was used to compare between NT and PAP CAR-T on Day 21). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and p≥0.05; NS, no significance. CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; i.v., intravenous; PAP, prostatic acid phosphatase; s.c., subcutaneous; tmPAP, transmembrane PAP; TNF, tumor necrosis factor.

Article Snippet: METHODS Preparation of transmembrane PAP antigen and screening of anti-PAP specific single-chain variable fragment The transmembrane PAP (tmPAP) gene (Sino Biological, HG10959- M) was cloned into the recombinant antigen expression vector PTSE- His (Beijing Bettermab Biotechnology).

Techniques: Activity Assay, In Vitro, In Vivo, Expressing, Binding Assay, Co-Culture Assay

Journal: Journal for immunotherapy of cancer

Article Title: Novel PAP-targeted CAR-T therapy enhances antitumor efficacy through CoupledCAR approach.

doi: 10.1136/jitc-2024-011238

Figure Lengend Snippet: Figure 4 The CoupledCAR approach enhanced the expansion and functionality of PAP CAR-T cells in vitro. (A–D) After 96 hours of co-culture with different CD19 CAR-T cells and the B-cell ratio, the percentage of CD4+ T cell (A) and CD8+ T cell (B) expansion, and absolute cell numbers of expanded CD4+ T cells (C) and CD8+ T cells were determined by flow cytometry (D). (E, F) The expression levels of CD137 (E) and CD69 (F) in PAP CAR-T cells, NT cells, and CD19 CAR-T cells. (G–H) The expression of PD-1 (G) and LAG-3 (H) in PAP CAR-T cells, NT cells, and CD19 CAR-T cells. (I–L) The percentages of stem- like central memory T cells (CD45ROnegCD62Lpos) (I), central memory T cells (CD45ROposCD62Lpos) (J), effector memory T cells (CD45ROnegCD62Lneg) (K) and effector T cells (CD45ROposCD62Lneg) (L). (M) NT cells, PAP CAR-T cells, and CD19 CAR-T cells were grouped and either cultured alone or pre-mixed with B cells in different ratios, followed by co-culture with PC3- tmPAP cells. The specific lysis against PC3-tmPAP tumor cells was analyzed. Intergroup comparisons in (A–L) were performed using unpaired t-tests. Intergroup comparisons in (M) were analyzed using one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and p≥0.05; NS, no significance. CAR, chimeric antigen receptor; LAG-3, Lymphocyte Activation Gene-3; NT, non-transduced; PAP, prostatic acid phosphatase; tmPAP, transmembrane PAP; PD-1, Programmed Cell Death Protein 1.

Article Snippet: METHODS Preparation of transmembrane PAP antigen and screening of anti-PAP specific single-chain variable fragment The transmembrane PAP (tmPAP) gene (Sino Biological, HG10959- M) was cloned into the recombinant antigen expression vector PTSE- His (Beijing Bettermab Biotechnology).

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Expressing, Cell Culture, Lysis, Activation Assay

Journal: Journal for immunotherapy of cancer

Article Title: Novel PAP-targeted CAR-T therapy enhances antitumor efficacy through CoupledCAR approach.

doi: 10.1136/jitc-2024-011238

Figure Lengend Snippet: Figure 5 The CoupledCAR approach enhanced the efficient expansion and enhanced the functionality of PAP CAR-T cells in vivo. (A) Schematic illustration of the in vivo studies. The prostate cancer xenograft model was established by the subcutaneous injection of 2.0×106 PC3-tmPAP cells, followed by the administration of different T and B cell or MOCK infusion strategies 24 days later (D0). The mice were divided into the MOCK group (n=7), NT group (n=7), B cell group (n=7), PAP CAR-T group (n=7), PAP CAR-T cell+CD19 CAR-T cell group (n=7), CD19 CAR-T cell group (n=7), CD19 CAR-T cell+B cell group (n=7), PAP CAR-T cell+B cell group (n=7) and PAP CAR-T cell+CD19 CAR-T cell+B cell group (n=7). A total of 1.0×106 B cells were infused on Day 3, 6 and 9 in the groups that received B-cell infusion. (B–G) Peripheral blood was collected on Day 14 to analyze CAR-T cell expansion (B, C) and cytokine release (D, E). The tumor volumes were measured every 3 days. (An unpaired t-test was used to compare between PAP CAR-T cell+CD19 CAR-T cell+B cell group and PAP CAR-T group, PAP CAR-T cell+CD19 CAR-T cell+B cell group and PAP CAR-T cell+CD19 CAR-T cell group, PAP CAR-T cell+CD19 CAR-T cell+B cell group and PAP CAR-T cell+B cell group on Day 15) (F). (G) The infiltration of CD3+, CD4+, CD8+, and PAP-CAR-transduced T cells into mouse tumors was assessed by immunohistochemistry. Scale bars represent 200 µm. (H–K). The histograms show the infiltration of CD3, CD4, CD8, and PAP CAR-T cells in tumor tissues. Intergroup comparisons in (B) were analyzed using an unpaired t-test. Intergroup comparisons in (C–F) and (H–K) were analyzed using one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and p≥0.05; NS, no significance. CAR, chimeric antigen receptor; GFP, green fluorescence protein; IFN, interferon; i.v., intravenous; NT, non-transduced; PAP, prostatic acid phosphatase; PB, peripheral blood; tmPAP, transmembrane PAP; s.c., subcutaneous.

Article Snippet: METHODS Preparation of transmembrane PAP antigen and screening of anti-PAP specific single-chain variable fragment The transmembrane PAP (tmPAP) gene (Sino Biological, HG10959- M) was cloned into the recombinant antigen expression vector PTSE- His (Beijing Bettermab Biotechnology).

Techniques: In Vivo, Injection, Immunohistochemistry, Fluorescence

Journal: PLoS ONE

Article Title: Rho GTPase Cdc42 Is a Direct Interacting Partner of Adenomatous Polyposis Coli Protein and Can Alter Its Cellular Localization

doi: 10.1371/journal.pone.0016603

Figure Lengend Snippet: (i) Cdc42-interacting APC clone from the brain cDNA library. The β-galactosidase activity of different bait and prey pairs in the Y2H screen. (a) Cdc42/N-WASP (positive control), (b) Cdc42/APC, (c) Cdc42/pACT2 (negative control) and (d) pAS2-1/N-WASP (negative control). (ii) Affinity pulldown assay. IRSp53 (positive control), GFP (negative control) and APC 222–653 were transcribed and translated using the TNT T7 coupled reticulocyte lysate systems kit in the presence of 35 S-methionine. The translated products were incubated with GST-tagged Cdc42V12 or Cdc42N17 or GST bound to glutathione-sepharose beads. The protein complexes were eluted by boiling and resolved by SDS-PAGE and detected by autoradiography. (a) Input for IRSp53, GFP and APC 222–653 respectively and (b) pulldowns using different affinity columns. First two lanes are Cdc42V12 pulldowns of 35 S-IRSp53 and 35 S-GFP respectively and the following three lanes are GST, Cdc42N17 and Cdc42V12 pulldowns of 35 S-APC 222–653 . (iii) APC 222–653 interaction with Cdc42V12 as shown by AP-FRET. mRFP-APC 222–653 and GFP-Cd42V12 were coexpressed in CHO cells and AP-FRET analysis was carried out in the ROI (box). ROI line graphs showing the FRET assay results are shown below the image. Scale bar = 5µm. (iv) AP-FRET controls. AP-FRET analysis was carried out in the ROI (box). Line graphs showing the FRET assay results are shown on the right of the respective panels. (a) cell expressing cytosolic GFP-mRFP, a tandem fusion serving as positive control, (b) cell coexpressing cytosolic GFP and mRFP serving as negative control and (c) cell coexpressing mRFP-APC 222–653 and GFP-Cdc42N17. Scale bar = 5 µm. (v) Table shows the %FE and CC values of various APC constructs in the presence of (Cdc42V12, Cdc42N17, Rac1 or RhoA) along with positive and negative controls. Data is shown as ± SD, with three experiments and n = 7–10 cells for each experiment.

Article Snippet: Matchmaker™ pre-transformed human cDNA libraries were screened using the Matchmaker™ GAL4 two-hybrid system 3 according to the manufacturer's protocol (Clontech).

Techniques: cDNA Library Assay, Activity Assay, Positive Control, Negative Control, Incubation, SDS Page, Autoradiography, Expressing, Construct