Journal: JCI Insight

Article Title: ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody

doi: 10.1172/jci.insight.145875

Figure Lengend Snippet: ( A ) Illustrated evolution of 3E10 into DX1. 3E10 was isolated from the MRL/lpr lupus mouse model. A 3E10 single chain variable fragment (scFv) with D31N mutation in the heavy chain variable domain complementarity determining region 1 (V H CDR1) was previously shown to have higher affinity for DNA and efficiency of cellular penetration compared with the original 3E10. 3E10 D31N di-scFv has greater impact on the DNA damage response and synthetic lethality to PTEN-deficient cancer cells due to its increased avidity for DNA compared with the scFv. The 3E10 D31N di-scFv was humanized, deimmunized, and CDR-optimized to yield DX1, which is now in development for testing in clinical trials ( , ). ( B ) DX1 penetrates into brain endothelial cells in an ENT2-dependent manner. hCMEC/D3 cells transfected with control or ENT2-targeting siRNA were treated with control buffer or DX1 alone, and then stained to detect DX1 penetration. Representative images are shown. Scale bar: 30 μm. ( C ) Control IgG shows minimal uptake into brain endothelial cells. hCMEC/D3 cells were treated with 0–10 μM of a control IgG (specifically an anti-PD1 antibody) and stained to detect uptake of IgG. Representative images are shown. Scale bar: 30 μm. ( D ) The ENT2 inhibitor DP interferes with DX1 penetration into brain endothelial cells. hCMEC/D3 cells were treated with control buffer, DX1, or DX1 + 50 μM DP and stained for DX1. Representative images are shown. Scale bar: 30 μm. These data demonstrate ENT2-dependent penetration by DX1 into hCMEC/D3 cells.

Article Snippet: Rat primary anti-DX1 antibody (clone D5) was provided by Patrys Ltd. and was used at 1:1000 in cell and tissue staining and immunoblots.

Techniques: Isolation, Mutagenesis, Clinical Proteomics, Transfection, Control, Staining

Journal: JCI Insight

Article Title: ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody

doi: 10.1172/jci.insight.145875

Figure Lengend Snippet: ( A ) Illustrated transwell model used to test BBB crossing by antibodies. hCMEC/D3 BECs and normal human astrocytes (NHA) were adhered to apical and basolateral surfaces of transwell inserts, respectively. The ability of control IgG or DX1 to cross this model was tested by measuring their appearance in the basolateral chamber after addition to the apical chamber. ( B and C ) DX1 crosses the transwell model of the BBB. The efficiency of IgG and DX1 transport across control blank inserts and inserts with BBB was compared by anti-IgG or anti-DX1 Western blot of basolateral chamber contents 1 hour after addition of IgG or DX1 to apical chambers. Representative Western blot showing DX1 at expected molecular weight (~54 kDa) in basolateral chambers of blank inserts (lanes 1–3) and BBB inserts (lanes 4–6) is shown in B . Each lane represents an independent experiment. Control IgG blot is shown in . Presence of the BBB reduced the relative control IgG content in basolateral chambers to 0.12 ± 0.01 relative to chambers lacking the BBB, while DX1 content was only reduced to 0.62 ± 0.07 ( P < 0.01 compared with control IgG, Student’s t test, n ≥ 3) as determined by ImageJ-based quantification of band intensities ( C ). ( D ) DP inhibits transport of DX1 across the hCMEC/D3 BBB. DX1 content in basolateral chambers was evaluated 15 and 30 minutes after addition of 5 μM DX1 ± 50 μM DP to apical chambers and quantified relative to DX1 content at the 30-minute time point in the absence of DP. * P < 0.01, Student’s t test, n = 3. These data demonstrate DP-sensitive DX1 transport across the hCMEC/D3 transwell BBB model, consistent with ENT2-dependent crossing of the BBB by DX1.

Article Snippet: Rat primary anti-DX1 antibody (clone D5) was provided by Patrys Ltd. and was used at 1:1000 in cell and tissue staining and immunoblots.

Techniques: Control, Western Blot, Molecular Weight

Journal: JCI Insight

Article Title: ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody

doi: 10.1172/jci.insight.145875

Figure Lengend Snippet: Human GBM model 1 GSCs engineered to express luciferase were inoculated into the brains of immunodeficient mice ( n = 10) to generate orthotopic PDX GBM tumors. ( A ) Representative IVIS images confirming presence of tumors (upper panel) and comparing DX1 localization to tumors (lower panel). DX1 was labeled with AF750 to facilitate its detection by IVIS. Mice were treated with i.v. and i.p. control buffer ( n = 2), i.v. DX1 AF750 (20 mg/kg) and i.p. control buffer ( n = 4), or i.v. DX1 AF750 (20 mg/kg) and i.p. DP (70 mg/kg) ( n = 4). IVIS measurements 24 hours after treatment demonstrated DX1 localization into the tumors, and DP significantly suppressed this uptake. ( B ) Quantification of radiance efficiencies. * P = 0.0142, ** P < 0.0001. Tukey’s multiple-comparison test–adjusted P values; numbers of mice evaluated at each point are indicated in the plots.

Article Snippet: Rat primary anti-DX1 antibody (clone D5) was provided by Patrys Ltd. and was used at 1:1000 in cell and tissue staining and immunoblots.

Techniques: Luciferase, Labeling, Control, Comparison

Journal: JCI Insight

Article Title: ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody

doi: 10.1172/jci.insight.145875

Figure Lengend Snippet: ( A – C ) DX1 suppresses GBM model 1 tumors. Tumor growth after inoculation of luciferase-expressing GBM model 1 GSCs into the brains of immunodeficient mice was followed by radiance efficiency in the brain by weekly IVIS. Two weeks after inoculation, mice began treatment with i.v. control buffer (group 1) or DX1 (20 mg/kg, group 2). ( A ) Radiance efficiencies are shown and demonstrate significant suppression of tumors by DX1 (note the y axis scale increases over the weeks). ** P < 0.04, Student’s t test, n = 7 per group. * P < 0.02, Student’s t test, n = 4 and 7 per group. ( B ) Representative IVIS images and H&E stains are shown. Scale bar: 1.25 mm. ( C ) Treatment with DX1 increased median survival (measured from initiation of treatment) to 24 days, compared with 17 days in control mice ( P < 0.01, log-rank test, n = 7). ( D – G ) DX1 suppresses GBM model 2 tumors. Two weeks after inoculation of GBM model 2 GSCs, mice began treatment with i.v. control buffer ( n = 10) or 20 mg/kg DX1 ( n = 10) 3 times per week. At 9 weeks after inoculation, brains from 3 mice per group were analyzed by H&E and Ki67 stains to facilitate measurement of mean tumor areas. ( D and E ) Representative H&E sections and mean tumor areas (* P = 0.04, Student’s t test, n = 3). Scale bar: 1.25 mm. ( F ) Sections of tumors from mice treated with control or DX1 stained to detect DX1 with protein L are shown and demonstrate that DX1 crossed the BBB to penetrate tumors and was associated with increased TUNEL staining. Scale bar: 50 μm. Results were confirmed by separately staining tumor sections with an anti-DX1 antibody . ( G ) DX1 prolonged median survival in GBM model 2 to 73 days, compared with 58 days in mice treated with control ( P = 0.02, log-rank test, n = 7).

Article Snippet: Rat primary anti-DX1 antibody (clone D5) was provided by Patrys Ltd. and was used at 1:1000 in cell and tissue staining and immunoblots.

Techniques: Luciferase, Expressing, Control, Staining, TUNEL Assay

Journal: JCI Insight

Article Title: ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody

doi: 10.1172/jci.insight.145875

Figure Lengend Snippet: Formation and growth of brain metastases in mice after intracardiac injection of luciferase-expressing 231-BR cells was followed by monitoring radiance efficiency in the brain. One week after injection, mice began treatment with i.v. control buffer (group 1) or DX1 (20 mg/kg, group 2). Weekly IVIS demonstrated significant suppression of tumor growth by DX1 delivered as 1 or 4 cycles. ( A – D ) Radiance efficiencies are plotted ( A – C ), and representative IVIS images from week 5 of the 4 cycle study are shown ( D ). * P < 0.04, ** P ≤ 0.01. Student’s t test; numbers of mice evaluated at each point are indicated in the plots. ( E ) One cycle of DX1 was associated with a nonsignificant increase in median survival to 35 days, compared with 30 days in control mice ( P = 0.42, log-rank test, n = 7). ( F ) Four cycles of DX1 had a greater effect, with median survival prolonged to 45 days, compared with 31 days in control mice ( P < 0.02, log-rank test, n = 7).

Article Snippet: Rat primary anti-DX1 antibody (clone D5) was provided by Patrys Ltd. and was used at 1:1000 in cell and tissue staining and immunoblots.

Techniques: Injection, Luciferase, Expressing, Control