Ravibhushan Singh

Publications of Ravibhushan Singh

• GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials.

  Authors: Jung Hee Woo, Jen-Sing Liu, Soo Hyun Kang, Ravibhushan Singh, Seong Kyu Park, Yunpeng Su, Janelle Ortiz, David M Neville, Mark C Willingham, Arthur E Frankel

  Protein expression and purification. 04/2008; 58(1):1-11.

  The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical gradeThe bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical grade bivalent anti-T cell immunotoxin for phase I/II clinical trials, a single batch of 120 L bioreactor culture was performed using the Pichia pastoris mutEF2JC307-8(2) strain expressing the bivalent anti-T cell immunotoxin. After 162 h induction of the culture by methanol, the culture medium was harvested by a 0.1 microm hollow-fiber microfiltration step. The recombinant protein was purified by a 3-step purification procedure (Butyl 650 M capturing step, borate anion exchange step and final Poros anion exchange step). The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Expression level was 207 mg/L of culture supernatant and the final production yield was 69.6% or 144.2mg/L of culture supernatant. The final product was characterized by multiple assays. Vialed product was sterile. The drug concentration was 0.8 mg/mL in 150 mM NaCl, 5% glycerol, 1mM EDTA, and 5mM Tris (pH 8.0). Purity by SDS-PAGE was 98%. Aggregates by Superdex 200 HPLC were <1%. Potency revealed a 20 h IC(50) of 17f M on Jurkat cells. Endotoxin level was 0.02 U/mg. Chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The drug did not react with tested frozen human tissue sections except for T cells. LD(10) in mice was between 500 and 75 0microg/kg. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 1.5 year at -80 degrees C. The scalable synthesis of this protein drug should be useful for production for phase I/II clinical trials and can be applicable for other diphtheria toxin fusion drugs for clinical development.

• Recombinant prostate-specific antigen proaerolysin shows selective protease sensitivity and cell cytotoxicity.

  Authors: Ravibhushan Singh, Jeff L Browning, Ralph Abi-Habib, Kevin Wong, Simon A Williams, Rosemina Merchant, Samuel R Denmeade, Thomas J Buckley, Arthur E Frankel

  Anti-cancer drugs. 09/2007; 18(7):809-16.

  Native proaerolysin is a channel-forming bacterial protoxin that binds to cell-surface receptors and then is activated by furin or furin-like proteases. We genetically engineered proaerolysin byNative proaerolysin is a channel-forming bacterial protoxin that binds to cell-surface receptors and then is activated by furin or furin-like proteases. We genetically engineered proaerolysin by replacing the furin-cleavage sequence with a prostate-specific antigen-selective sequence. The recombinant modified proaerolysin was expressed and purified from Aeromonas salmonicida in good yields and purity. Recombinant modified proaerolysin had no furin sensitivity and markedly increased prostate-specific antigen sensitivity relative to wild-type proaerolysin. Human prostate cancer cells were significantly more sensitive to recombinant modified proaerolysin in the presence of active prostate-specific antigen when compared with the absence of prostate-specific antigen or the presence of potent prostate-specific antigen inhibitors. Most normal human cells with the exception of prostate and renal epithelial cells showed very low sensitivity to recombinant modified proaerolysin. Our results suggest that recombinant modified proaerolysin is a potent prostate-specific antigen-sensitive protoxin that deserves further development for regional therapy of benign and malignant prostate growths.

• Systematic urokinase-activated anthrax toxin therapy produces regressions of subcutaneous human non-small cell lung tumor in athymic nude mice.

  Authors: Yunpeng Su, Janelle Ortiz, Shihui Liu, Thomas H Bugge, Ravibhushan Singh, Stephen H Leppla, Arthur E Frankel

  Cancer research. 05/2007; 67(7):3329-36.

  The novel recombinant anthrax toxin, PrAgU2/FP59, composed of the urokinase-activated protective antigen and a fusion protein of Pseudomonas exotoxin and lethal factor was tested for anti-lung cancerThe novel recombinant anthrax toxin, PrAgU2/FP59, composed of the urokinase-activated protective antigen and a fusion protein of Pseudomonas exotoxin and lethal factor was tested for anti-lung cancer efficacy in an in vivo human tumor model. Male athymic nude mice (age 4-6 weeks) were inoculated s.c. with 10 million H1299 non-small cell lung cancer (NSCLC) cells in the left flank. When tumor volumes reached 200 mm(3) (6-8 days), i.p. injection of 100 muL saline or different ratios and doses of PrAgU2/FP59 in 100 muL saline were given every 3 days for four doses and an additional dose at day 29. Animals were monitored twice daily and tumor measurements were made by calipers. The maximum tolerated doses of PrAgU2/FP59 differed dependent on the ratios of PrAgU2 to FP59 over the range of 3:1 to 25:1, respectively. At tolerated doses, tumor regressions were seen in all animals. Complete histologic remission lasting 60 days occurred in 30% of animals. PrAgU2/FP59 showed dramatic anti-NSCLC efficacy and warrants further clinical development for therapy of patients with advanced NSCLC.

• MUC1: a target molecule for cancer therapy.

  Authors: Ravibhushan Singh, Dilip Bandyopadhyay

  Cancer biology & therapy. 05/2007; 6(4):481-6.

  MUC1 is a mucin family protein, overexpressed in more than 90% of breast cancers in an underglycosylated form, exposing the core peptides of the extracellular domain that act as a potential targetMUC1 is a mucin family protein, overexpressed in more than 90% of breast cancers in an underglycosylated form, exposing the core peptides of the extracellular domain that act as a potential target for antibody-mediated therapy. The highly conserved repeats of 20 amino acids VNTR varies between 20 and 125 depending on the allele. Each tandem repeat contains five potential O-glycosylation sites that have been exploited in designing cancer therapeutics. The core peptides in the tandem repeated VNTR domain are masked in normal cells and become exposed in the cancer cells associated mucins. The characteristics of the MUCI epitopes constituted with the tandem repeats and the carbohydrates present on MUC1 induce immune responses that favor targeted immunotherapy. In addition, aberrant glycosylation also plays an important role in enhancing the internalization of MUC1 into the cytoplasm making MUC1 a very attractive cytoplasmic delivery system of drugs and other therapeutic agents. MUC1 being present on most of the cancers of glandular epithelial origin, appears as a potential target for therapeutic interventions in these cancers.

• Target-specific cytotoxic activity of recombinant immunotoxin scFv(MUC1)-ETA on breast carcinoma cells and primary breast tumors.

  Authors: Ravibhushan Singh, Urmila Samant, Stephen Hyland, Pradip R Chaudhari, Winfried S Wels, Dilip Bandyopadhyay

  Molecular cancer therapeutics. 03/2007; 6(2):562-9.

  MUC1 is a mucin family protein, overexpressed in more than 90% of breast cancers in an underglycosylated form, exposing the core peptides of the extracellular domain that act as a potential targetMUC1 is a mucin family protein, overexpressed in more than 90% of breast cancers in an underglycosylated form, exposing the core peptides of the extracellular domain that act as a potential target for antibody-mediated therapy. We have developed an anti-MUC1 scFv antibody from a phage library of mice immunized with synthetic peptide MUC1-variable number of tandem repeats. MUC1 binding phages were affinity selected through biopanning using a biotin-streptavidin pull-down method. The selected phage clones showed target-specific binding to MUC1-expressing cells. Fusion of truncated Pseudomonas aeruginosa exotoxin A (ETA) to a high binder, phage-derived scFv clone and bacterial expression and purification of recombinant scFv(MUC1)-ETA immunotoxin were done with good yield and purity. In vitro target-specific cytotoxic activity and target-specific binding of immunotoxin were shown on MUC1-expressing cells and primary breast tumor samples. A truncated ETA fusion protein expressed from the same vector but lacking scFv did not show cytotoxic effects, confirming target specificity. Our results suggest that the scFv(MUC1)-ETA immunotoxin has therapeutic potential and deserves further development and characterization for MUC1-specific breast cancers treatment.

• Systemic anthrax lethal toxin therapy produces regressions of subcutaneous human melanoma tumors in athymic nude mice.

  Authors: Ralph J Abi-Habib, Ravibhushan Singh, Stephen H Leppla, John J Greene, Yan Ding, Bree Berghuis, Nicholas S Duesbery, Arthur E Frankel

  Clinical cancer research : an official journal of the American Association for Cancer Research. 01/2007; 12(24):7437-43.

  PURPOSE: Anthrax Lethal Toxin (LeTx), composed of protective antigen and lethal factor, catalytically cleaves mitogen-activated protein kinase (MAPK) kinases and inhibits the MAPK signaling pathways.PURPOSE: Anthrax Lethal Toxin (LeTx), composed of protective antigen and lethal factor, catalytically cleaves mitogen-activated protein kinase (MAPK) kinases and inhibits the MAPK signaling pathways. The majority of metastatic melanomas possess the V599E BRAF mutation, which constitutively activates MAPK1/2 signaling. LeTx is cytotoxic to BRAF mutant melanoma cell lines in vitro, whereas most normal cells are resistant to this toxin. In this study, we determine the in vivo potency and safety of systemically administered LeTx. EXPERIMENTAL DESIGN: A s.c. xenograft melanoma model in athymic nude mice was treated with different i.p. doses of LeTx. RESULTS: In this study, we show that in vivo systemic LeTx treatment of s.c. xenograft melanoma tumors in athymic nude mice yields partial and complete tumor regressions with minor toxicity to mice. When animal toxicity was observed, we did not find any histologic evidence of tissue damage. CONCLUSIONS: LeTx is one of the rare targeted agents to produce complete remissions of human melanomas in an animal model and thus warrants further preclinical development.

• A urokinase-activated recombinant anthrax toxin is selectively cytotoxic to many human tumor cell types.

  Authors: Ralph J Abi-Habib, Ravibhushan Singh, Shihui Liu, Thomas H Bugge, Stephen H Leppla, Arthur E Frankel

  Molecular cancer therapeutics. 11/2006; 5(10):2556-62.

  Urokinase plasminogen activator (uPA) is a tumor-specific protease highly expressed in several types of solid tumors and rarely present on normal cells under physiologic conditions. Due to its highUrokinase plasminogen activator (uPA) is a tumor-specific protease highly expressed in several types of solid tumors and rarely present on normal cells under physiologic conditions. Due to its high expression on metastatic tumors, several different strategies have been used to target the urokinase system. These have mostly led to tumor growth inhibition rather than tumor regression. A different approach was adopted by replacing the furin activation site on a recombinant anthrax toxin with a urokinase activation site. The resulting toxin, PrAgU2/FP59, was highly potent against tumors both in vitro and in vivo. In this study, we show that PrAgU2/FP59 is toxic to a wide range of tumor cell lines, including non-small cell lung cancer, pancreatic cancer, and basal-like breast cancer cell lines. Of the few cell lines found to be resistant to PrAgU2/FP59, most became sensitive upon addition of exogenous pro-uPA. PrAgU2/FP59 was much less toxic to normal human cells. The potency of PrAgU2/FP59 was dependent on anthrax toxin receptor, uPA receptor, and uPA levels but not on total plasminogen activator inhibitor-1 levels. In this study, we show that PrAgU2/FP59 is a wide-range, highly potent, and highly selective toxin that is capable of specifically targeting uPA-expressing tumor cells, independently of the tissue of origin of these cells. Furthermore, we identify three molecular markers, anthrax toxin receptor, uPA, and uPA receptor, which can be used as predictors of tumor cell sensitivity to PrAgU2/FP59.

• GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials

  Authors: Jung Hee Woo, Jen-Sing Liu, Soo Hyun Kang, Ravibhushan Singh, Seong Kyu Park, Yunpeng Su, Janelle Ortiz, David M. Neville, Mark C. Willingham, Arthur E. Frankel

  Protein Expression and Purification.

  The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical gradeThe bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical grade bivalent anti-T cell immunotoxin for phase I/II clinical trials, a single batch of 120 L bioreactor culture was performed using the Pichia pastoris mutEF2JC307-8(2) strain expressing the bivalent anti-T cell immunotoxin. After 162 h induction of the culture by methanol, the culture medium was harvested by a 0.1 μm hollow-fiber microfiltration step. The recombinant protein was purified by a 3-step purification procedure (Butyl 650 M capturing step, borate anion exchange step and final Poros anion exchange step). The final material was filter sterilized, aseptically vialed, and stored at −80 °C. Expression level was 207 mg/L of culture supernatant and the final production yield was 69.6% or 144.2 mg/L of culture supernatant. The final product was characterized by multiple assays. Vialed product was sterile. The drug concentration was 0.8 mg/mL in 150 mM NaCl, 5% glycerol, 1 mM EDTA, and 5 mM Tris (pH 8.0). Purity by SDS–PAGE was 98%. Aggregates by Superdex 200 HPLC were <1%. Potency revealed a 20 h IC50 of 17 fM on Jurkat cells. Endotoxin level was 0.02 U/mg. Chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The drug did not react with tested frozen human tissue sections except for T cells. LD10 in mice was between 500 and 750 μg/kg. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 1.5 year at −80 °C. The scalable synthesis of this protein drug should be useful for production for phase I/II clinical trials and can be applicable for other diphtheria toxin fusion drugs for clinical development.