Generation of sequence-specific, high affinity anti-DNA antibodies.

By taking advantage of the extreme stability of a protein-DNA complex, we have obtained two highly specific monoclonal antibodies against a predetermined palindromic DNA sequence corresponding to the binding site of the E2 transcriptional regulator of the human papillomavirus (HPV-16). The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constants in the low and subnanomolar range. This affinity matches that of the natural DNA binding domain and is severalfold higher than the affinity of a homologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These antibodies discriminate effectively among a number of double- and single-stranded synthetic DNAs with factors ranging from 125- to 20,000-fold the dissociation constant of the specific DNA sequence used in the immunogenic protein-DNA complex. Moreover, they are capable of fine specificity tuning, since they both bind less tightly to another HPV-16 E2 binding site, differing in only 1 base pair in a noncontact flexible region. Beyond the relevance of obtaining a specific anti-DNA response, these results provide a first glance at how DNA as an antigen is recognized specifically by an antibody. The accuracy of the spectroscopic method used for the binding analysis suggests that a detailed mechanistic analysis is attainable.

A synthetic, nitrogenated antimetastatic and anti-angiogenic compound with low toxicity in vivo.

The design of more effective therapies for metastatic disease involves development of new compounds able to specifically block the malignant process. We demonstrated previously that a new synthetic nitrogenated compound 3'-1-chloroethyl-2,3-dihydro-1H-imidazo-(2, 1-i)-purine-4-ium-7-yl-3'-deoxy-1',5', 6'-tri-O-(methylsulfonyl)-muco-inositol chloride (DIC) had an anti-proliferative activity on tumor cells in vitro. In the present work we demonstrate that DIC induces apoptosis on the LM3 murine mammary adenocarcinoma cell line in vitro and has anti-angiogenic activity in vivo. We also evaluated toxicity, biodistribution and anti-neoplastic properties of DIC in vivo. Toxicity studies allowed us to establish the LD50 (750 mg/kg body weight). Administration of 250 mg/kg/day (LD10) for 6 days did not cause overt toxic effects. Biodistribution assays revealed that DIC was rapidly eliminated (60% at t=10 min), although it accumulated in tumor tissue at higher concentrations than in other tissues. Daily s.c. treatment with DIC (LD10) for 24 days significantly reduced the number of spontaneous lung metastases. These results suggest that DIC has the ability of impairing the metastatic development by inhibiting angiogenesis and inducing apoptosis on tumor cells.