Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Expression of CD70 and B7-H3 on human tumor tissues. (A) Representative images of IHC staining of CD70 and B7-H3 on human tumor tissue microarrays were shown. (Scale bar, 20 μm) (B) IHC result of CD70 and B7-H3 staining in normal tissues including brain, esophagus, stomach, intestine, pancreas, appendix. The representative images were shown. (Scale bar, 50 μm) (C) Differential expression profile analysis of B7-H3 and CD70 in tumor and normal tissues based on the TCGA database.

Article Snippet: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

Techniques: Expressing, Immunohistochemistry, Staining, Quantitative Proteomics

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Expression of CD70 and B7-H3 in human solid tumor cell lines. (A, B) Representative images showed the immunofluorescence staining of B7-H3 and CD70 together with DAPI in NCI-H460, A375, MDA-MB-435 and 786-O tumor cells. (Scale bar: 20 μm) (C, D) Flow cytometry result indicated high expression of CD70 and B7-H3 on the four solid tumor cell lines.

Article Snippet: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Construct of CAR (A) Schematic diagrams showing the composition of the four CARs used in this study: CD70 CAR 1 , CD70 CAR 2 , B7-H3 CAR and TanCAR. (B) The lentiviral backbone plasmid encodes the TanCAR. (C) Cartoon depicted of TanCAR targeting respective tumor antigens.

Article Snippet: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

Techniques: Construct, Plasmid Preparation

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Functional analysis of CD70 CAR 1 and CD70 CAR 2 . (A) To assess the affinity of two CD70 binding fragments, immunofluorescence was performed using human trCD27.mFc and CD70 scFv.hFc chimeric protein as the primary antibody. Images showed the immunofluorescence staining of CD70 by NCI-H460 and A375 tumor cells. (Scale bar: 20 μm) (B) 4-hour 51 Cr cytotoxicity assays indicated a higher tumor killing of CD70 CAR 2 -T cells against target cells. (C) Microscopy images were captured 8 hours after A375 or H460 cells cocultured with CD70 CAR 1 , CD70 CAR 2 and NT T cells at a ratio of 2 effector cell to 1 target cells. (Scale bar: 50 μm) (D) ELISA results showed the IFN-γ and IL-2 secretion levels by CD70 CAR 1 , CD70 CAR2 and NT T cells encountering A375 or H460 cells.

Article Snippet: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

Techniques: Functional Assay, Binding Assay, Immunofluorescence, Staining, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Activity of TanCAR-T cells against tumor cells expressing CD70 and/or B7-H3. (A) Four-hour 51 Cr cytotoxicity assays of TanCAR-T cells against tumor cells expressing CD70 and/or B7-H3, compared with unispecific CAR and NT T cells. (B) Analysis of IFNγ and IL2 secretion level from supernatants of co-cultures of TanCAR, B7-H3 CAR, CD70 CAR 2 and NT T cells with multiple tumor cells, as detected by ELISA. Shown are pooled data from 3 independent experiments done in triplicates.

Article Snippet: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

doi: 10.7150/thno.43991

Figure Lengend Snippet: Antitumor response of TanCAR-T cells against CD70 and B7-H3 positive tumors in vivo . (A) The treatment scheme showed the timing of subcutaneous injection of tumor cells, vein-tail injection of CAR-T cells T cells and in vivo optical imaging. (B) Antitumor response of TanCAR-T cells in human subcutaneous xenograft models. NCI-H460.ffLuc or A375.ffLuc tumor bearing (confirmed by imaging 6 days after tumor implantation, 8/group) mice were adoptively transferred through tail vein injection with NT, CD70 CAR 2 , B7-H3 CAR or high/low doses (5×10 6 or 1×10 6 /mouse) of TanCAR T cells on 7 days and 10 days post tumor inoculation. (C) Tumor growth was measured weekly by using Living Image software, and mean values per treated group were shown. (D) Kaplan-Meier survival curve were performed 75 days after T cells injection. Mice treated with TanCAR-T cells had a significantly longer survival probability in comparison with mice with NT, CD70 CAR 2 or B7-H3 CAR-T cells.

Article Snippet: For the primary antibody, the murine anti-CD70 antibody (San Cruz Biotechnology Catalog no. sc-365539) and the rabbit anti-B7-H3 antibody (Cell Signaling Technology Catalog no.14058T) was used.

Techniques: In Vivo, Injection, Optical Imaging, Imaging, Tumor Implantation, Software, Comparison

Journal: Cancer research

Article Title: Distinctive Features of the Differentiated Phenotype and Infiltration of Tumor-reactive Lymphocytes in Clear Cell Renal Cell Carcinoma

doi: 10.1158/0008-5472.CAN-12-0588

Figure Lengend Snippet: CD70 expression on tumors and the effect of purified CD70 on naïve CD8+ or CD4+ T cells. A. CD70 expression on non-immune cells in renal fresh tumor digests comparing to melanoma. Fresh tumor digests were labeled with fluorescence-conjugated Lineage 1 cocktail antibodies (Lin 1) and CD70, and then analyzed on FACS Canto II. Lin 1− cells represent non-immune cells in the fresh tumor digests. FACS plots represented CD70 expression in RCC or melanoma. B. CD70 expression on renal tumor lines and melanoma lines. Tumor lines established from 10 RCC patients and 10 melanoma patients were thawed and grown till their confluency. The tumor cells were then harvested and labeled either with PE-conjugated anti-CD70 Abs (filled histogram) or isotype control (open histogram), and analyzed on FACS Canto II. C. The effect of purified CD70 on naïve CD8+ or CD4+ T cells. Peripheral blood mononuclear cells (PBMC) were thawed and cultured in RPMI with 10% HS overnight, and sorted for CD8+CD62L+CD45RO− or CD4+CD62L+CD45RO− naïve T cell populations on FACS Aria. Sorted naïve CD8 or CD4 T cells (2×105/well) were added to the 96-well plate that were pre-coated with anti-human CD3 (10μg/ml) and/or purified human CD70 protein (10μg/ml) and cultured in RPMI + 10%HS + IL-2 (50cu/ml) for 5 days. 5 days later, T cells were labeled with fluorescence-conjugated Abs, CD3, CD8, CD4, CD62L and CD45RO, and analyzed on FACS Canto II. D. The dose-dependent effect of purified CD70 on naïve CD8+ or CD4+ T cells. Sorted naïve CD8+ or CD4+ T cells were cultured with different doses of purified human CD70 protein (0–10μg/ml) in the presence or absence of anti-CD3 Ab (10μg/ml). The experiments in C and D were performed multiple times and gave similar results.

Article Snippet: Purified human CD70 fused with murine CD8 alpha (CD70-muCD8, Ancell Corporation; 0.03–10μg/ml) or controls were plated onto a 96-well flat-bottom plate that was pre-coated with anti-mouse CD8 alpha or PBS alone (Ancell; 10μg/ml) overnight at 4°C.

Techniques: Expressing, Purification, Labeling, Fluorescence, Cell Culture

Journal: Journal of Clinical Investigation

Article Title: Neonatal Fc receptor antagonist efgartigimod safely and sustainably reduces IgGs in humans

doi: 10.1172/jci97911

Figure Lengend Snippet: Figure 3. Effect of efgartigimod and IVIg on tracer IgG levels in cynomol- gus monkeys. Animals (n = 2/group) were injected with 1 mg/kg of the tracer Ab FR70-hIgG1, followed 2 days later with i.v. infusion of 70 mg/ kg efgartigimod or 2 g/kg IVIg. Tracer levels in serum, detected by murine CD70-binding ELISA, were plotted relative to predose levels. Values are shown as mean ± SEM. %T0, percentage change from baseline.

Article Snippet: Tracer Ab levels in blood serum were determined by murine CD70-binding ELISA using MaxiSorp plates (Thermo Fisher Scientific, catalog 31000) and murine CD70 (R&D Systems, catalog 783-CL/CF).

Techniques: Injection, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: Neonatal Fc receptor antagonist efgartigimod safely and sustainably reduces IgGs in humans

doi: 10.1172/jci97911

Figure Lengend Snippet: Figure 4. Efgartigimod dose-escalation study in cynomolgus monkeys. Animals (n = 2/group) were injected with 1 mg/kg of the tracer Ab FR70- hIgG1, followed 2 days later with i.v. infusion of 0.2 to 200 mg/kg efgar- tigimod or vehicle. Tracer levels in serum (A) are detected by murine CD70- binding ELISA. Endogenous IgG levels in serum (B) were plotted relative to predose levels. Cyno, cynomolgus. Values are shown as mean ± SEM.

Article Snippet: Tracer Ab levels in blood serum were determined by murine CD70-binding ELISA using MaxiSorp plates (Thermo Fisher Scientific, catalog 31000) and murine CD70 (R&D Systems, catalog 783-CL/CF).

Techniques: Injection, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75

doi: 10.1038/sj.bjc.6605816

Figure Lengend Snippet: Characterisation of anti-CD70 1C1 and 5D12. ( A ) Western blots containing FLAG-tagged CD70 ECD (lanes 1 and 5) and membrane extracts from HEK 293 cells (lanes 2 and 6), HEK 293:CD70 transfectants (lanes 3 and 7) and 786-O cells (lanes 4 and 8) were reacted with mAb 1C1 (left panel) or mAb 5D12 (right panel). The mAb 1C1 and 5D12 specifically bound recombinant CD70 (lane 1 and 4) and three distinct bands ( * ) from CD70+ membrane extracts (lanes 3, 4, 7, and 8). Extracts from parental HEK 293 cells were negative as expected (lane 2 and 6). The β -actin control shows comparable quantities of protein were loaded for each membrane extract (lower panels). ( B ) IHC analysis on FFPE normal human tissues showing limited expression of CD70 in lymphoid tissues. Red stain indicates presence of CD70+ cells. 1C1 mAb was used for these set of images. × 400 magnification, scale bar=50 μ m.

Article Snippet: Cells were incubated for 30 min on ice with PE-conjugated murine anti-CD70 antibodies (BD Pharmingen, San Diego, CA, USA), washed with cold staining medium, and evaluated with a Becton Dickinson FACScan flow cytometer.

Techniques: Western Blot, Membrane, Recombinant, Control, Expressing, Staining

Journal: British Journal of Cancer

Article Title: Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75

doi: 10.1038/sj.bjc.6605816

Figure Lengend Snippet: Comparative CD70 expression in pancreatic and ovarian cancer cell lines by immunohistochemistry and quantitative FACS

Article Snippet: Cells were incubated for 30 min on ice with PE-conjugated murine anti-CD70 antibodies (BD Pharmingen, San Diego, CA, USA), washed with cold staining medium, and evaluated with a Becton Dickinson FACScan flow cytometer.

Techniques: Expressing, Immunohistochemistry, Transfection

Journal: British Journal of Cancer

Article Title: Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75

doi: 10.1038/sj.bjc.6605816

Figure Lengend Snippet: Correlation of CD70 expression in cell lines by qFACS, IHC, and reverse trancriptase–PCR: ( A ) Quantitative flow cytometric analyses were carried out on ovarian (SK-OV-3, TOV-21G), and pancreatic (Panc-1) cell lines using a PE-conjugate murine anti-CD70 ab (BD Pharmingen). CD70 copy number was determined using standard labelled bead controls (QiFiKit, DAKO). ( B ) Corresponding cell pellets were harvested and processed as FFPE samples and immunohistochemistry using 1C1 (as shown) or 5D12 was carried out using alkaline phosphatase-labelled secondary antibody and Fast Red as the chromogen. Images were taken using a Zeiss microscope at 400 × magnification. Scale bar=50 μ m. ( C ) Detection of human CD70 expression by reverse trancriptase–PCR: expression of CD70 in various CD70+ cell lines was determined using cDNA preparations obtained from the cells (lanes 1: AN3CA ovarian; 2: SKOV-3 ovarian; 3: TOV-21G ovarian; 4: NCI-H716 colorectal; 5: 786-O renal; 6: PANC-1 pancreatic; 7: IGROV-1 ovarian (CD70 negative); 8: H 2 O). Primers that partially spanned the coding regions were used for CD70. PCR products were sequenced to confirm their identity. hCD70 ( TNFSF7 ) fragment=852 bp, including exons 1,2, and 3. Human GAPDH ( hGAPDH) (580 bp) was used as housekeeping gene PCR control.

Article Snippet: Cells were incubated for 30 min on ice with PE-conjugated murine anti-CD70 antibodies (BD Pharmingen, San Diego, CA, USA), washed with cold staining medium, and evaluated with a Becton Dickinson FACScan flow cytometer.

Techniques: Expressing, Immunohistochemistry, Microscopy, Control

Journal: British Journal of Cancer

Article Title: Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75

doi: 10.1038/sj.bjc.6605816

Figure Lengend Snippet: Summary of CD70 expression from tissue microarray analysis of carcinomas using 1C1

Article Snippet: Cells were incubated for 30 min on ice with PE-conjugated murine anti-CD70 antibodies (BD Pharmingen, San Diego, CA, USA), washed with cold staining medium, and evaluated with a Becton Dickinson FACScan flow cytometer.

Techniques: Expressing, Microarray

Journal: British Journal of Cancer

Article Title: Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75

doi: 10.1038/sj.bjc.6605816

Figure Lengend Snippet: Immunohistochemistry analysis using anti-CD70 mAb (1C1) in tumour microarrays. CD70 expression in pancreatic ( A ) and ovarian ( B ) tumours is shown. Of the 140 and 241 individual cases of pancreatic and ovarian carcinoma, respectively, 35 cases (25%) and 37 cases (15%) were found to be CD70+. In this analysis, the area of transformed cells within each biopsy stained positively for CD70 expression is categorised into <25%, 25–50%, 50–75%, and >75%. The intensity of CD70 expression is expressed as: 1=weak, 2=mild, 3=moderate, 4=strong ( × 400 magnification). To illustrate the heterogeneity in CD70 expression, the percent area positive is plotted against staining intensity. In these plots, the number in each in the graphs denotes the number of CD70+ cases. Representative images for low, heterogeneous (25–50% positive, 2+) and high, homogeneous (>75% positive, 4+) CD70 for pancreatic and ovarian carcinoma are shown.

Article Snippet: Cells were incubated for 30 min on ice with PE-conjugated murine anti-CD70 antibodies (BD Pharmingen, San Diego, CA, USA), washed with cold staining medium, and evaluated with a Becton Dickinson FACScan flow cytometer.

Techniques: Immunohistochemistry, Expressing, Transformation Assay, Staining

Journal: British Journal of Cancer

Article Title: Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75

doi: 10.1038/sj.bjc.6605816

Figure Lengend Snippet: In vitro and In vivo Anti-tumour Activity of SGN-75: ( A and B ) In vitro cytotoxicity assay showing potency of SGN-75 (triangles) on SK-OV-3 ovarian and CD70-transfected MiaPaCa-2 pancreatic carcinoma cell lines. The controls, unconjugated h1F6 control ( ) and non-binding ADC ( A , squares) have no effect. IC 50 for SGN-75 and non-binding ADC are noted. Similarly, no cytotoxicity is observed in the untransfected MiaPaCa-2 control ( B , squares). ( C and D ) In vivo anti-tumour activity of SGN-75 in0-transfected MiaPaCa-2 pancreatic carcinoma tumours in nude mice. Mice treated with SGN-75 (squares) showed a significantly reduced median tumour volume ( C ) and a higher percent survival ( D ) when compared with untreated (circles) and non-binding ADC (triangles).

Article Snippet: Cells were incubated for 30 min on ice with PE-conjugated murine anti-CD70 antibodies (BD Pharmingen, San Diego, CA, USA), washed with cold staining medium, and evaluated with a Becton Dickinson FACScan flow cytometer.

Techniques: In Vitro, In Vivo, Activity Assay, Cytotoxicity Assay, Transfection, Control, Binding Assay