Postchemotherapy and Tumor-Selective Targeting with the La-Specific DAB4 Monoclonal Antibody Relates to Apoptotic Cell Clearance

DAB4 binds preferentially to dead malignant cells in presence of dead normal cells in vitro. Fixed number of dead CFSE-labeled thymocytes was mixed with increasing number of dead EL4 cells, and DAB4 or antibody isotype control (Sal5) binding to either cell type was analyzed by flow cytometry. (A) MFI of DAB4 binding (after subtraction of Sal5 MFI) to dead thymocytes (gated as CFSE⁺ cells) was plotted as function of percentage of dead EL4 cells in total number of dead cells analyzed. (B) MFI of DAB4 binding to EL4 cells (gated as CFSE⁻ cells) as function of percentage of EL4 cells in total number of dead cells analyzed (n = 3). CFSE = carboxyfluorescein succinimidyl ester.

Clearance of apoptotic cells in vivo modulates tumor accumulation of DAB4. (A) C57BL/6 mice and syngeneic C1qa^−/− mice were untreated or administered apoptotic Jurkat cells intraperitoneally. Plasma was collected 24 h later, and level of dsDNA present was measured using Quant-iT PicoGreen dsDNA kit (n = 5). ***P < 0.001. (B) EL4 tumor–bearing C57BL/6 and C1qa^−/− mice were untreated (Control) or treated with cyclophosphamide and etoposide (Chemo). ¹⁴C-DAB4 (100 μg at specific activity of 130 dpm/μg) was administered 24 h later, organs were collected 48 h later, and %ID/g was determined (n = 5). *P < 0.05, compared with chemotherapy-treated C57BL/6 mice. ***P < 0.001, compared with control mice.

Biodistribution of radiolabeled DAB4 in mice bearing human carcinoma xenografts. PC-3 tumor–bearing BALB/c nude mice were untreated (Control) or treated with etoposide (50 mg/kg) (Chemo) intraperitoneally, and tumors were collected 72 h after treatment for TUNEL staining (A) or flow cytometric analysis (B) of specific binding of DAB4 to dead (7-AAD⁺) cells, n = 3. (C) ¹¹¹In-DOTA-DAB4 or ¹¹¹In-DOTA-Sal5 (100 μg at specific activity of 77.7 MBq/mg) was administered after chemotherapy, mice were euthanized 72 h later, and accumulation of radiolabeled antibody in organs (%ID/g) was measured (n = 5). ***P < 0.001. (D, left) Untreated PC-3 tumor–bearing mice were administered 14.8 MBq of ¹⁸F-FDG and imaged 90 min later before injection of ¹⁷⁷Lu-DOTA-DAB4 (top, anterior view; bottom, lateral view; tumor arrowed). (D, right) Tumors were collected 48 h later for high-resolution β autoradiography and hematoxylin and eosin staining (n = 2). LI = large intestine; N = necrotic; SI = small intestine; V = viable.

Specific binding of DAB4 to human ALL cells after in vitro treatment with DNA-damaging cytotoxic drugs. Leukemic blasts from ALL patient were untreated (Control) or treated with etoposide (Etop; 40 μg/mL), cisplatin (Cis; 20 μg/mL), gemcitabine (Gem; 200 μg/mL), etoposide (40 μg/mL) with cisplatin (20 μg/mL) (Etop + cis), etoposide (40 μg/mL) with gemcitabine (200 μg/mL) (Etop + gem), or gemcitabine (200 μg/mL) with cisplatin (20 μg/mL) (Gem + cis). Cells were analyzed by flow cytometry for DAB4 binding after gating based on 7-AAD⁺ events. MFI of DAB4 binding was analyzed at 24 h (white bars), 48 h (black bars), and 72 h (blue bars) after treatment (n = 3). ***P < 0.001.