John J. Docherty

Cell electrophoresis research directed toward clinical cytodiagnosis.

The application of cell electrophoresis to cytodiagnosis requires that a scientifically established basis exists for identifying abnormal cells electrophoretically, that research to detect such differences in the cytodiagnostic setting is possible and that a rapid and simple method of cell electrophoresis is adaptable to the clinical setting. Data are presented indicating modifications of electrophoretic mobility due to herpes simplex virus type 1 infection and Rous sarcoma virus transformation of culture cells. A simple apparatus for electrophoretically separating cells on a density gradient and collecting them for subsequent analysis is described, and results of experiments with this apparatus are consistent with those obtained by microscopic electrophoresis. Laser-doppler spectroscopic electrophoresis is suggested as a rapid method adaptable to clinical application.

Abortive Herpes Simplex Virus Replication in Rous Sarcoma Virus Transformed Cells

Summary The XC cell line was developed from tumor cells induced in newborn Wistar rats by the Prague strain of Rous sarcoma virus (RSV). Attempts to propagate herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2) in these cells proved futile. However, both virus types were able to replicate to high levels in control nontransformed Wistar rat embryo (WRE) cells. Examination of this abortive infection revealed that both HSV-1 and HSV-2 were able to attach to the XC cells but were unable to induce virus protein, DNA, or specific thymidine kinase. Furthermore, while cell DNA synthesis was severely depressed in receptive WRE cells, no such effect was observed in resistant XC cells. These studies suggest that an early event, prior to viral DNA synthesis, is blocked in the replicative cycle of HSV. This study was conducted in part under support funds from the U.S. Public Health Service Biomedical Grant No. 5 S05 R RO7082-07 and the Pennsylvania Agriculture Experiment Station Project No. 1992. Authorized for publication as paper No. 4439 in the journal series of the Pennsylvania Agriculture Experiment Station.

The isolation of anti-gum arabic antibodies by affinity chromatography

Antibodies directed at gum arabic have been induced in rabbits immunized with gum arabic in Freunds complete adjuvant. These antibodies have been isolated in pure form by affinity chromatography on AH-Sepharose 4B containing gum arabic ligands. Oxidation of the susceptable carbohydrate residues of gum arabic with periodate or reduction of the glucuronic acid moieties with carbodiimide and borohydride converted the polysaccharide to products which no longer yielded precipitin reactions with the antibodies. The antibodies are therefore anti-carbohydrate antibodies with specificity for certain carbohydrate units of the gum arabic. Results of chemical modification and inhibition experiments indicate that 4-alpha-L-arabinofuranosyl-D-glucuronic acid units of the polysaccharide are the major immunodeterminant groups.

Detection of herpes simplex virus tumor-associated antigen in uterine cervical cancer tissue: five case studies.

Abstract Biopsies from five gynecologic cancer patients were examined for the presence of herpes simplex virus tumor-associated antigen (HSV-TAA) using the highly sensitive peroxidase-antiperoxidase technique. The results demonstrate that two of three patients with squamous cell carcinoma of the uterine cervix that were serologically positive for HSV-TAA, determined by complement fixation, contained HSV-TAA in the cytoplasm of the malignant cells of the biopsy. Tissue from one squamous cell carcinoma of the uterine cervix patient, as well as tissue from an adenoacanthoma patient, was negative for tissue HSV-TAA. However, both of these patients were also serologically negative for HSV-TAA. All patients, regardless of cancer type, had serum antibodies to herpes simplex virus types 1 and 2. These studies continue to suggest a role for HSV in certain gynecologic cancers.

Abstract 2851: Development of an alkalizing antibody-enzyme conjugate for NSCLC treatment that is in Phase I clinical testing.

Many solid human tumors generate an acidic and hypoxic microenvironment as a result of altered metabolic pathways and aberrant tumor vasculature. In certain tumors, the chronic exposure to acidic extracellular conditions has been reported to promote invasiveness and metastatic behaviour. In addition, the lower pH may promote resistance to weakly basic chemotherapeutic agents by altering their partitioning coefficient between the extracellular and intracellular compartments. L-DOS47, an immunoconjugate cancer therapeutic, has been developed to target this unique tumor microenvironment. L-DOS47 is a conjugate of a lung adenocarcinoma specific single domain antibody and a urease enzyme. The antibody serves as a targeting agent to deliver the enzyme to the affected site while the urease enzyme coverts urea, an abundant metabolite, into ammonia and generates a local pH increase. The combined effect of ammonia toxicity and pH increase is cytotoxic to cancer cells as shown both in culture and in xenograft models. In vitro experiments showed that L-DOS47 also dramatically potentiated the cytotoxicity of a number of chemotherapeutics. Imaging studies using A549 xenografts and labelled L-DOS47 applied intravenously showed that the drug molecule preferentially accumulated and persisted at the tumor site. L-DOS47 has been studied in a number of tissue-cross reactivity studies to identify suitable animal species for toxicological studies. Cryosections of normal human, cynomolgus monkey, Sprague-Dawley rat, and CD-1 mouse tissues were compared. L-DOS47 produced specific staining to control material (BxPC-3 cells) and those tissues that are known to express CEACAM6 - an antigen for L-DOS47. Both cytoplasmic and membrane bound staining were observed. Results of these studies suggest that the primate and both rodent species are suitable for animal toxicological studies. Pivotal GLP compliant animal toxicological studies in cynomologus monkey and rat together with several non-GLP pilot animal studies were done. In the primate GLP study, L-DOS47 (0, 17, 26, and 35 μg/kg) was administered by IV infusion on Days 1, 8, 15 and 22, followed by a 28-day recovery period. Main study animals were sacrificed on Day 25 and recovery animals on Day 50. Standard toxicology parameters were evaluated. In addition, toxicokinetics, cytokine stimulation and immunogenicity were also evaluated. A separate cardiovascular safety pharmacology study was also conducted. No treatment related clinical signs were observed for the animals treated with 17 or 26μg/kg of L-DOS47. Adverse signs were observed in some but not all of the high dose (35 μg/kg) treated animals. Based on adverse clinical signs observed at 35μg/kg, the NOAEL was determined to be 26 μg/kg/day in this study. Currently L-DOS47 is approved for phase I/II studies in Europe and the U.S.A. Patient enrollment has begun in Poland and first patient has been dosed. Citation Format: Heman Chao, Baomin Tian, Kim Gaspar, John Docherty, Wah Wong. Development of an alkalizing antibody-enzyme conjugate for NSCLC treatment that is in Phase I clinical testing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2851. doi:10.1158/1538-7445.AM2013-2851

DNA Binding of a 38,000-Dalton Herpes Simplex Virus 2-Specific Protein

Western transfer and immunoenzymatic staining with affinity-purified monospecific antiserum was used to detect a 38-Kd protein that bound to native and denatured DNA cellulose. This protein has previously been shown to be a delayed early herpes simplex virus type-2 specific protein.