Bactericidal/permeability-increasing protein (rBPI21) and fluoroquinolone mitigate radiation-induced bone marrow aplasia and death.

Identification of safe, effective treatments to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the U.S. Food and Drug Administration has yet to approve any agent for a mass-casualty radiation disaster. Because patients undergoing hematopoietic stem cell transplantation undergo radiation treatment that produces toxicities similar to radiation-disaster exposure, we studied patients early after such treatment to identify new approaches to this problem. Patients rapidly developed endotoxemia and reduced plasma bactericidal/permeability-increasing protein (BPI), a potent endotoxin-neutralizing protein, in association with neutropenia. We hypothesized that a treatment supplying similar endotoxin-neutralizing activity might replace the BPI deficit and mitigate radiation toxicity and tested this idea in mice. A single 7-Gy radiation dose, which killed 95% of the mice by 30 days, was followed 24 hours later by twice-daily, subcutaneous injections of the recombinant BPI fragment rBPI21 or vehicle alone for 14 or 30 days, with or without an oral fluoroquinolone antibiotic with broad-spectrum antibacterial activity, including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle plus fluoroquinolone, the combined rBPI21 plus fluoroquinolone treatment improved survival, accelerated hematopoietic recovery, and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 plus fluoroquinolone initiated 24 hours after lethal irradiation, combined with their established favorable bioactivity and safety profiles in critically ill humans, suggests the potential clinical use of this radiation mitigation strategy and supports its further evaluation.

Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis.

Claudin-3 (CLDN3) is a tight junction protein that is overexpressed in 90% of ovarian tumors. Previous in vitro studies have indicated that CLDN3 overexpression promotes the migration, invasion, and survival of ovarian cancer cells. Here, we investigated the efficacy of lipidoid-formulated CLDN3 siRNA in 3 different ovarian cancer models. Intratumoral injection of lipidoid/CLDN3 siRNA into OVCAR-3 xenografts resulted in dramatic silencing of CLDN3, significant reduction in cell proliferation, reduction in tumor growth, and a significant increase in the number of apoptotic cells. Intraperitoneal injection of lipidoid-formulated CLDN3 siRNA resulted in a substantial reduction in tumor burden in MISIIR/TAg transgenic mice and mice bearing tumors derived from mouse ovarian surface epithelial cells. Ascites development was reduced in CLDN3 siRNA-treated mice, suggesting the treatment effectively suppressed metastasis. Toxicity was not observed after multiple i.p. injections. Importantly, treatment of mice with nonimmunostimulatory 2'-OMe modified CLDN3 siRNA was as effective in suppressing tumor growth as unmodifed siRNA. These results suggest that lipidoid-formulated CLDN3 siRNA has potential as a therapeutic for ovarian cancer.

Interferon-gamma ELISPOT assay for the quantitative measurement of antigen-specific murine CD8+ T-cells.

Effective screening of new vaccines and immunotherapeutics requires assay methods that can provide quantitative measurement of cellular immune responses. The enzyme-linked immunospot (ELISPOT) is a sensitive technique for the detection of cytokine-producing cells at the single cell level. This assay is rapid and reproducible and permits the direct enumeration of low-frequency antigen-specific T-cells. This protocol describes in detail an interferon (IFN)-gamma ELISPOT method for measuring antigen-specific murine CD8+ T-cells. Spleen cells from specific cytotoxic T-lymphocyte (CTL) peptide-primed mice are used source of CD8+ T-cells to demonstrate the utility of this technique. The assay procedure is facilitated by the use of a ready-to-use IFN-gamma ELISPOT assay kit and it can be adapted to other model systems in which CD8+ T-cell responses are monitored.

Repeated immunization with plasmid DNA formulated in poly(lactide-co-glycolide) microparticles is well tolerated and stimulates durable T cell responses to the tumor-associated antigen cytochrome P450 1B1.

Injection of microparticle-encapsulated DNA elicits immune responses to plasmid-encoded antigens in mice and humans. Cytochrome P450 CYP1B1 (CYP1B1) is a member of the CYP1 P450 enzyme family that is overexpressed in a variety of solid tumors. The work described herein was performed to study the kinetics of stimulating T cell responsiveness with an encapsulated DNA encoding CYP1B1 and provides support for the clinical development of this formulation. Immunization of HLA-A2/Kb transgenic mice with human CYP1B1 encoding plasmid DNA formulated in poly(lactide-co-glycolide) (PLG) microparticles elicits CD8+ T cells that respond to human CYP1B1-positive target cells. The duration of the immune response, the effect on the immune response of multiple injections, and the safety of repeated injections were studied. These results show that the PLG-encapsulated DNA therapeutic elicits durable immune responses to CYP1B1, the responses are dependent on repeat immunization, and that the formulation is well tolerated.

The shared tumor-associated antigen cytochrome P450 1B1 is recognized by specific cytotoxic T cells.

Cytochrome P450 1B1 (CYP1B1), a drug-metabolizing extrahepatic enzyme, was recently shown to be overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for anticancer therapy, particularly cancer immunotherapeutics. We identified HLA-A*0201-binding peptides and a naturally processed and presented T-cell epitope capable of inducing CYP1B1-specific cytotoxic T lymphocytes (CTLs) in HLA-A2 transgenic mice. Furthermore, the induction of CYP1B1-specific T cells was demonstrated in healthy donors and cancer patients. These T cells efficiently lysed target cells pulsed with the cognate peptide. More important, HLA-A2-matched tumor cell lines and primary malignant cells were also recognized by CYP1B1-specific CTLs. These findings form the basis of a phase 1 clinical trial exploring a DNA-based vector encoding CYP1B1 for widely applicable cancer immunotherapy conducted at the Dana-Farber Cancer Institute.

Immunotherapy of human cervical high-grade cervical intraepithelial neoplasia with microparticle-delivered human papillomavirus 16 E7 plasmid DNA.

OBJECTIVE: The purpose of this study was to evaluate the safety of the administration of a bacterial expression plasmid encoding a 13 amino acid sequence that is highly homologous with human papillomavirus E7 within poly (lactide-co-glycolide) microparticles (ZYC101) in women with HLA A2+ antigen and persistent cervical intraepithelial neoplasia grade 2/3 and human papillomavirus 16. STUDY DESIGN: Fifteen women entered an institutional review board-approved dose-escalating phase I study with the use of three levels of blood monitoring and urine studies, Papanicolaou tests, and colposcopy. Escalation required no serious adverse events. Immunologic responses were evaluated in peripheral blood with the use of human papillomavirus peptide-stimulated interferon gamma enzyme-linked immunosorbent assay for T-cell reactivity. In cervical secretions, immunoglobulin A anti-human papillomavirus 16 E2 concentrations were measured. Three doses every 3 weeks were followed 4 weeks later by surgical excision. RESULTS: No serious adverse events occurred. Five women had complete histologic responses; 11 women had human papillomavirus-specific T-cell responses. Four of five complete histologic responses developed immunoglobulin A anti-E2-specific antibody. CONCLUSION: ZYC101 warrants further investigation because of a 33% complete histologic responses, a 73% immunologic response, and no serious adverse events.