Walter N. Hittelman

Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. We designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy. We assessed the construct’s antigen-binding specificity and GrB enzymatic activity, as well as in vitro cytotoxicity and internalization into target and control cells. We also assessed pharmacokinetic and toxicology parameters in vivo. GrB-Fc-4D5 was highly cytotoxic to Her2 positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM, respectively, and against a panel of HER2+ cell lines regardless of endogenous expression levels of the PI-9 inhibitor. Contemporaneous studies with Kadcyla demonstrated similar levels of in vitro activity against virtually all cells tested. GrB-Fc-4D5 internalized rapidly into target SKOV3 cells within 1 h of exposure rapidly delivering GrB to the cytoplasmic compartment. In keeping with its relatively high molecular weight (160 kDa), the construct demonstrated a terminal-phase serum half-life in mice of 39.2 h. Toxicity studies conducted on BALB/c mice demonstrated no statistically significant changes in SGPT, SGOT or serum LDH. Histopathologic analysis of tissues from treated mice demonstrated no drug-related changes in any tissues examined. GrB-Fc-4D5 shows excellent, specific cytotoxicity and demonstrates no significant toxicity in normal, antigen-negative murine models. This construct constitutes a novel approach against HER2-expressing tumors and is an excellent candidate for further development.

The fusion protein VEGF121/rGel composed of the growth factor VEGF121 and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF121/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.

Based on findings that cancer cell clonogens exhibit stem cell features, it has been suggested that cancer stem-like cells are relatively radioresistant owing to different intrinsic and extrinsic factors, including quiescence, activated radiation response mechanisms (e.g., enhanced DNA repair, upregulated cell cycle control mechanisms and increased free-radical scavengers) and a surrounding microenvironment that enhances cell survival mechanisms (e.g., hypoxia and interaction with stromal elements). However, these radiosensitivity features are probably dynamic in nature and come into play at different times during the course of chemo/radiotherapy. Therefore, different molecularly targeted radiosensitization strategies may be needed at different stages of therapy. This article describes potential sensitization approaches based on the dynamics and changing properties of cancer stem-like cells during therapy.

Retinoids have shown antiproliferative and chemopreventive activity. We analyzed data from a randomized, placebo-controlled chemoprevention trial to determine whether a 3-month treatment with either 9-cis-retinoic acid (RA) or 13-cis-RA and alpha-tocopherol reduced Ki-67, a proliferation biomarker, in the bronchial epithelium. Former smokers (n = 225) were randomly assigned to receive 3 months of daily oral 9-cis-RA (100 mg), 13-cis-RA (1 mg/kg) and alpha-tocopherol (1200 IU), or placebo. Bronchoscopic biopsy specimens obtained before and after treatment were immunohistochemically assessed for changes in the Ki-67 proliferative index (i.e., percentage of cells with Ki-67-positive nuclear staining) in the basal and parabasal layers of the bronchial epithelium. Per-subject and per-biopsy site analyses were conducted. Multicovariable analyses, including a mixed-effects model and a generalized estimating equations model, were used to investigate the treatment effect (Ki-67 labeling index and percentage of bronchial epithelial biopsy sites with a Ki-67 index > or = 5%) with adjustment for multiple covariates, such as smoking history and metaplasia. Coefficient estimates and 95% confidence intervals (CIs) were obtained from the models. All statistical tests were two-sided. In per-subject analyses, Ki-67 labeling in the basal layer was not changed by any treatment; the percentage of subjects with a high Ki-67 labeling in the parabasal layer dropped statistically significantly after treatment with 13-cis-RA and alpha-tocopherol treatment (P = .04) compared with placebo, but the drop was not statistically significant after 9-cis-RA treatment (P = .17). A similar effect was observed in the parabasal layer in a per-site analysis; the percentage of sites with high Ki-67 labeling dropped statistically significantly after 9-cis-RA treatment (coefficient estimate = -0.72, 95% CI = -1.24 to -0.20; P = .007) compared with placebo, and after 13-cis-RA and alpha-tocopherol treatment (coefficient estimate = -0.66, 95% CI = -1.15 to -0.17; P = .008). In per-subject analyses, treatment with 13-cis-RA and alpha-tocopherol, compared with placebo, was statistically significantly associated with reduced bronchial epithelial cell proliferation; treatment with 9-cis-RA was not. In per-site analyses, statistically significant associations were obtained with both treatments.

Epithelial tumors develop through a multistep process driven by genomic instability frequently associated with etiologic agents such as prolonged tobacco smoke exposure or human papilloma virus (HPV) infection. The purpose of the studies reported here was to examine the nature of genomic instability in epithelial tissues at cancer risk in order to identify tissue genetic biomarkers that might be used to assess an individual's cancer risk and response to chemopreventive intervention. As part of several chemoprevention trials, biopsies were obtained from risk tissues (i.e., bronchial biopsies from chronic smokers, oral or laryngeal biopsies from individuals with premalignancy) and examined for chromosome instability using in situ hybridization. Nearly all biopsy specimens show evidence for chromosome instability throughout the exposed tissue. Increased chromosome instability was observed with histologic progression in the normal to tumor transition of head and neck squamous cell carcinomas. Chromosome instability was also seen in premalignant head and neck lesions, and high levels were associated with subsequent tumor development. In bronchial biopsies of current smokers, the level of ongoing chromosome instability correlated with smoking intensity (e.g., packs/day), whereas the chromosome index (average number of chromosome copies per cell) correlated with cumulative tobacco exposure (i.e., pack‐years). Spatial chromosome analyses of the epithelium demonstrated multifocal clonal outgrowths. In former smokers, random chromosome instability was reduced; however, clonal populations appeared to persist for many years, perhaps accounting for continued lung cancer risk following smoking cessation.