Anti-tumor properties of CD40 ligand when delivered as a transgene by the conditional replicative oncolytic adenovirus AdEH to breast cancer cells.

Includes bibliographical references (p. 116-137).Cancer-selective biotherapy and gene therapy have been considered to be the next horizon towards developing a cure for breast cancer. CD40 ligand (CD40L), a member of the tumor necrosis factor superfamily, relays critical growth signals in various hematological malignancies and carcinomas. We previously demonstrated that recombinant CD40L can directly inhibit breast cancer cell growth. However a potential limitation of CD40L therapy is systemic toxicity. To improve efficacy of gene delivery and limit CD40L expression to the tumor micro-environment, we have generated a conditionally replicative virus (AdEHCD40L) that delivers CD40L selectively to breast carcinomas. Tumor/tissue-specific promoters (hypoxic/HIF-1α response and estrogen response elements) were incorporated to limit CD40L expression to the tumor microenvironment. Viral E1A and CD40L transgene expression was examined in breast cancer lines with low constitutive (T47D) or no (BT-20) HIF-1α expression. Both cell lines displayed significantly increased CD40L expression under viral- permissive conditions (T47D: 65.5 ± 3.9% with increased HIF-1α vs. 38.5 ± 2.8% under uninduced condition, p = 0.01; BT20: 43.2 ± 14.9% vs. 10.6 ± 0.2%, p = 0.03). AdEHCD40L produced markedly stronger inhibition compared to the parental construct (T47D: 53.7 ± 15.2% vs. 32.1 ± 11.7%, p = 0.02; BT20: 25.8 ± 10.2% vs. 15.2 ± 6.8%, p = 0.03), suggesting that growth inhibition encompassed CD40L-mediated and viral oncolytic events. Replicative activity of AdEHCD40L was comparable to the wild type adenovirus in breast cancer cells and attenuated in normal lung fibroblast cells, with reduced growth inhibitory impact. Preliminary findings on mechanisms of AdEHCD40L cytotoxicity indicated increased apoptotic (Annexin V+) and necrotic (propidium iodide incorporation) activities that were accompanied by reduced IBα, phosphorylation, G2M/S cell cycle arrest, culminating in an increased sub-G0 fraction, and altered chemokine/cytokine expression. AdEHCD40L biodistribution and its maximum tolerated dose (2x108 pfu) evaluated in mice were comparable to that of other conditional-replicative adenoviruses. Anti-tumor efficacy of AdEHCD40L showed a reduction in mean tumor diameter of MDA-MB-231 (44-58%) and T47D (49%) human breast cancer xenografts in SCID mice. These findings illustrate the applicability of CD40L gene transfer approach for experimental treatment of human breast cancer.by Erica Manuela Gomes.Ph.D