Page 172 - 2014 Printable Abstract Book
P. 172
(PS2-63) Anti-ceramide antibody as mitigator of the acute radiation gastrointestinal syndrome. Vijay K.
1
1
2
2
3
Singh, PhD ; John Fuller ; Jimmy Rotolo, PhD ; and Richard Kolesnick, MD, AFRRI, USUHS, Bethesda, MD ;
3
Memorial Sloan-Kettering Cancer Center, New York, NY ; and Ceramide Therapeutics, Brooklyn, NY
2
Accumulating evidence indicates ceramide-mediated apoptosis within the gastrointestinal (GI)
microvascular endothelial compartment regulates radiation sensitivity of GI epithelial stem cells. Recent
studies describe a novel murine anti-ceramide IgM monoclonal antibody (Mab) that inhibits ceramide-
mediated endothelial apoptosis, providing radiation protection from the lethal radiation GI syndrome
(RGS) in mice. Further, prophylactic administration of humanized anti-ceramide IgG1 inhibits high-dose
radiation-induced microvascular endothelial apoptosis within the GI tract, and dose-dependently protects
crypt stem cells. Administering humanized Mab at 24 h post-irradiation similarly protects crypt stem cells,
indicating that anti-ceramide may represent a bonafide mitigator of the lethal RGS. Preliminary data also
show that anti-ceramide Fab fragments, generated by enzymatic digestion of humanized anti-ceramide
Mab, bind to and neutralize ceramide in vitro, and confer stem cell protection equal to full-length Mab.
These data suggest that a monovalent antibody fragment directed against ceramide may be sufficient for
therapeutic neutralization of ceramide. Recombinant antibody fragments often display favorable biologic
and commercial attributes compared to full-length immunoglobulins, including faster diffusion into the
bloodstream, higher tissue permeability due to smaller size and solubility, and reduced product
development costs as they can be expressed in bacteria rather than mammalian cells. Thus, we
engineered an anti-ceramide single-chain variable fragment (scFv) by fusing the variable heavy and light
chains of anti-ceramide IgG. Anti-ceramide scFv retains the in vitro ceramide-binding and neutralizing
activity of full length Mab. When tested against supralethal doses of gamma-radiation in mice, anti-
ceramide scFv demonstrates efficacy in protecting crypt survival against radiation doses as high as 14 Gy.
Anti-ceramide scFv was effective when administered 15 min prior to or as late as 24 h post radiation.
Furthermore, anti-ceramide scFv was as potent as full-length Mab at one tenth of the dose. In brief, anti-
ceramide scFv appears to be a very promising countermeasure for acute RGS among all candidate
therapeutics currently under development.
(PS2-64) Recombinant Human Interleukin-12, but not Filgrastim, Increases Survival after Radiation-
induced Myeloablation: Results from Randomized Blinded Placebo-Controlled Study in Rhesus
Macaques. Zoya Gluzman-Poltorak, PhD, MBA; Vladimir Vainstein, MD, MSc; and Lena A. Basile, PhD,
J.D., Neumedicines Inc., Pasadena, CA
rHuIL-12 (HemaMaxTM) is being developed for mitigation of HSARS under the FDA Animal Rule
using a NHP model of HSARS. Our previous randomized, blinded, placebo-controlled GLP study in the NHP
showed that a single subcutaneous injection of rHuIL-12 (50ng/kg-500 ng/kg) 24-25 hours after irradiation
significantly improved overall survival without the use of antibiotics, fluids or blood transfusions. rHuIL-
12 also reduced incidence of severe neutropenia and thrombocytopenia, systemic infection and internal
organ hemorrhage. Here we report the results from our second GLP randomized blinded placebo-
controlled NHP study that was designed to compare rHuIL-12 to filgrastim (rHuG-CSF) for treatment of
HSARS. Animals were irradiated (700 cGy) and treated with a single subcutaneous injection of vehicle or
rHuIL-12 175ng/kg 24-25 hours after irradiation, daily subcutaneous injections of rHuG-CSF at 10μg/kg/d
for 18 days starting 24-25 hours after irradiation, or combination of a single rHuIL-12 and 18 rHuG-CSF
injections (26-36 animals/group) without supportive care. Single injection of rHuIL-12 increased survival
170 | P a g e
1
1
2
2
3
Singh, PhD ; John Fuller ; Jimmy Rotolo, PhD ; and Richard Kolesnick, MD, AFRRI, USUHS, Bethesda, MD ;
3
Memorial Sloan-Kettering Cancer Center, New York, NY ; and Ceramide Therapeutics, Brooklyn, NY
2
Accumulating evidence indicates ceramide-mediated apoptosis within the gastrointestinal (GI)
microvascular endothelial compartment regulates radiation sensitivity of GI epithelial stem cells. Recent
studies describe a novel murine anti-ceramide IgM monoclonal antibody (Mab) that inhibits ceramide-
mediated endothelial apoptosis, providing radiation protection from the lethal radiation GI syndrome
(RGS) in mice. Further, prophylactic administration of humanized anti-ceramide IgG1 inhibits high-dose
radiation-induced microvascular endothelial apoptosis within the GI tract, and dose-dependently protects
crypt stem cells. Administering humanized Mab at 24 h post-irradiation similarly protects crypt stem cells,
indicating that anti-ceramide may represent a bonafide mitigator of the lethal RGS. Preliminary data also
show that anti-ceramide Fab fragments, generated by enzymatic digestion of humanized anti-ceramide
Mab, bind to and neutralize ceramide in vitro, and confer stem cell protection equal to full-length Mab.
These data suggest that a monovalent antibody fragment directed against ceramide may be sufficient for
therapeutic neutralization of ceramide. Recombinant antibody fragments often display favorable biologic
and commercial attributes compared to full-length immunoglobulins, including faster diffusion into the
bloodstream, higher tissue permeability due to smaller size and solubility, and reduced product
development costs as they can be expressed in bacteria rather than mammalian cells. Thus, we
engineered an anti-ceramide single-chain variable fragment (scFv) by fusing the variable heavy and light
chains of anti-ceramide IgG. Anti-ceramide scFv retains the in vitro ceramide-binding and neutralizing
activity of full length Mab. When tested against supralethal doses of gamma-radiation in mice, anti-
ceramide scFv demonstrates efficacy in protecting crypt survival against radiation doses as high as 14 Gy.
Anti-ceramide scFv was effective when administered 15 min prior to or as late as 24 h post radiation.
Furthermore, anti-ceramide scFv was as potent as full-length Mab at one tenth of the dose. In brief, anti-
ceramide scFv appears to be a very promising countermeasure for acute RGS among all candidate
therapeutics currently under development.
(PS2-64) Recombinant Human Interleukin-12, but not Filgrastim, Increases Survival after Radiation-
induced Myeloablation: Results from Randomized Blinded Placebo-Controlled Study in Rhesus
Macaques. Zoya Gluzman-Poltorak, PhD, MBA; Vladimir Vainstein, MD, MSc; and Lena A. Basile, PhD,
J.D., Neumedicines Inc., Pasadena, CA
rHuIL-12 (HemaMaxTM) is being developed for mitigation of HSARS under the FDA Animal Rule
using a NHP model of HSARS. Our previous randomized, blinded, placebo-controlled GLP study in the NHP
showed that a single subcutaneous injection of rHuIL-12 (50ng/kg-500 ng/kg) 24-25 hours after irradiation
significantly improved overall survival without the use of antibiotics, fluids or blood transfusions. rHuIL-
12 also reduced incidence of severe neutropenia and thrombocytopenia, systemic infection and internal
organ hemorrhage. Here we report the results from our second GLP randomized blinded placebo-
controlled NHP study that was designed to compare rHuIL-12 to filgrastim (rHuG-CSF) for treatment of
HSARS. Animals were irradiated (700 cGy) and treated with a single subcutaneous injection of vehicle or
rHuIL-12 175ng/kg 24-25 hours after irradiation, daily subcutaneous injections of rHuG-CSF at 10μg/kg/d
for 18 days starting 24-25 hours after irradiation, or combination of a single rHuIL-12 and 18 rHuG-CSF
injections (26-36 animals/group) without supportive care. Single injection of rHuIL-12 increased survival
170 | P a g e
