Starvation in 15 geese (mean initial body mass, m = 6.3 kg) fasting for about 40 days (mean decrease in m = 2.5 kg) was characterized by three periods. Period I (3-8 days), an adaptation period, was marked by a considerable decrease in the daily rate of change in m (dm) as well as in resting metabolic rate (RMR), and by high fat mobilization. In period II (a period of economy) the decreases in dm, RMR, and daily rate of nitrogen excretion (dne) were reduced: when expressed per unit of body mass these rates were either constant or decreased slightly. Period III, a critical period, was characterized by a rapid increase in both dm and dne that appeared when body mass had dropped to 4.7-3.2 kg. In parallel there was a greater decrease in intracellular fluid volume below 5 kg. Throughout the fast, in contrast to fasting mammals, plasma glucose and alanine concentrations were maintained at high levels (8-10 and 0.4 mM, respectively), and there was no increase in acetoacetate. However, after 20 days of fasting, plasma beta-hydroxybutyrate concentration (beta-OHB) increased to about 20 mM, while blood pH remained constant and blood PCO2 decreased. Thus, compensation for metabolic acidosis was partly attributed to respiratory alkalosis. Throughout the fast, the variations in beta-OHB were a mirror image of those for daily changes in body mass and in nitrogen excretion. This presumably reflects a hormonal change, but might also suggest a key role of beta-OHB in the control of energy expenditure and/or in regulation of body mass as well as in protein sparing.

BackgroundThe hematopoietic syndrome of the acute radiation syndrome (HSARS) is a life-threatening condition in humans exposed to total body irradiation (TBI); no drugs are approved for treating this condition. Recombinant human interleukin-12 (rHuIL-12) is being developed for HSARS mitigation under the FDA Animal Rule, where efficacy is proven in an appropriate animal model and safety is demonstrated in humans.MethodsIn this blinded study, rhesus monkeys (9 animals/sex/dose group) were randomized to receive a single subcutaneous injection of placebo (group 1) or rHuIL-12 at doses of 50, 100, 250, or 500 ng/kg (groups 2–5, respectively), without antibiotics, fluids or blood transfusions, 24–25 hours after TBI (700 cGy).ResultsSurvival rates at Day 60 were 11%, 33%, 39%, 39%, and 50% for groups 1–5, respectively (log rank p < 0.05 for each dose vs. control). rHuIL-12 also significantly reduced the incidences of severe neutropenia, severe thrombocytopenia, and sepsis (positive hemoculture). Additionally, bone marrow regeneration following TBI was significantly greater in monkeys treated with rHuIL-12 than in controls.ConclusionsData from this study demonstrate that a single injection of rHuIL-12 delivered one day after TBI can significantly increase survival and reduce radiation-induced hematopoietic toxicity and infections. These data significantly advance development of rHuIL-12 toward approval under the Animal Rule as an effective stand-alone medical countermeasure against the lethal effects of radiation exposure.

Multiple treatment modalities are available for MF, but most result in inevitable relapse. Therefore, new treatment strategies that improve response rate and prolong response duration are greatly needed. TSEBT is a highly effective therapy in MF. LD-TSEBT (12 Gy) is much more tolerable than the conventional 36+ Gy dose, thereby allowing for re-treatment; however, LD-TSEBT has a less favorable complete response rate and response duration. Combining LD-TSEBT with immunostimulatory modalities in MF has a strong biological rationale, since radiation-induced exposure of cancer-specific antigens should be synergistic with concomitant stimulation of anti-cancer immune responses. Interleukin-12 is a robust candidate for radioimmunotherapy, as IL-12 has significant anti-MF activity as monotherapy, is very well tolerated without overlapping toxicity with TSEBT, and is a potent stimulator of innate and adaptive immunity. We report on a single-arm open-label phase 2a trial of combination of LD-TSEBT and NM-IL-12. Ten patients are planned for enrollment. Eligibility includes MF-type CTCL stages IB-IIIB and patients must be eligible for LD-TSEBT. TSEBT is started on study day 1 (fractionated 4 Gy/week, up to 12 Gy). NM-IL-12 is administered subcutaneously at 150 ng/kg on days 2 and 15, followed by 6 maintenance doses q4w at 100 ng/kg. The primary endpoint is safety with secondary endpoints being response rate and PFS. Currently, 6 patients are enrolled, 5 evaluable for response; 4 male; median age 55; three have stage IB, one IIB and one IIIB. Median number of previous therapies is 2 (0-6). The treatment was well-tolerated with only grade 1 or 2 AEs; most common AEs include grade 1 headache and chills. One patient achieved CR, 2 PR, and 2 SD. Median follow-up is so far 15 weeks and 5 patients remain on study. One patient has been withdrawn from the study due to development of a suspected PLC (pityriasis lichenoides chronica)-like skin reaction requiring topical steroid therapy. PK and PD analysis was completed in the first 4 patients. It demonstrated measurable drug levels in all patients studied, Cmax being 10.8-56.1 pg/ml achieved at 5-24 hours post injection. Interferon-γ and IP-10 (hallmark PD markers of IL-12 activity) were measurable after the first and the second injections in all patients. Levels of the inhibitory cytokine IL-10 were generally measurable after NM-IL-12 injections, but were very low (<1 pg/ml up to 12 pg/ml) - much lower than previously reported in IL-12 studies, thus unlikely to be associated with negative immunosuppressive feedback. There was no clear correlation between PK and PD measurements and either efficacy or toxicity, but the number of patients fully evaluated is small. Total RNA was extracted from patient blood samples and subjected to qRT-PCR. Gene specific primers were used to analyse intracellular expression of the IL-12 receptor and immune cell markers. Ongoing work investigates an activated cellular phenotype at week 15 of LD-TSEBT and NM-IL-12 treatment compared with the respective baseline samples. Overall these early results demonstrate that NM-IL-12 can be safely administered together with LD-TSEBT in CTCL patients. Encouraging clinical activity is observed including a CR. Enrollment is currently ongoing and planned PD and correlative biomarker studies are in progress. A phase 2B randomized trial of NM-IL-12 and low dose TSEBT compared against low-dose TSEBT alone in patients with MF is being developed based on the apparent benign AE profile as compared to approved systemic therapies. Disclosures Kim: Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tetralogic: Research Funding; Neumedicine: Research Funding; Merck: Research Funding; Kyowa Kirin Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Portola: Consultancy; miRagen: Research Funding; Innate: Research Funding; Horizon Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Soligenix: Research Funding. Ma:Neumedicines: Employment. Kha:Neumedicines: Employment. Lawrence:Neumedicines: Employment, Patents & Royalties. Vainstein:Neumedicines: Employment. Basile:Neumedicines: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Interleukin-12 (IL-12) is a heterodimeric cytokine with pleiotropic effects as a potent immunoregulatory molecule and a hematopoietic stimulatory factor. Studies in irradiated non-human primates (NHP) have shown the ameliorating effects of recombinant human IL-12 (HemaMaxTM, rHuIL-12) in a) regenerating hematopoietic tissue following radiation-induced bone marrow ablation, b) lowering the incidence of hemorrhage, and c) decreasing the risk of infections, collectively leading to an increase in NHP survival following lethal radiation exposure. A Phase 1b study was performed in healthy human subjects to evaluate the safety of rHuIL-12 following a single subcutaneously (sc) administered unit dose of 12μg to 32 healthy human subjects. Placebo was administered to 8 subjects. Peripheral blood samples were collected before and after rHuIL-12 administration and up to study day 14. Immunophenotyping of blood cell populations was conducted by Fluorescence Activated Cell Sorting (FACS). rHuIL-12 administration resulted in a transient decrease in peripheral blood mean CD45+ leukocytes, CD45+ lymphocytes, CD45+CD16+CD56+ NK cells, and CD45+CD34+ hematopoietic progenitor cells. Nadirs were reached on day 2, 24 hours after rHuIL-12 administration, for lymphocytes (54.2% reduction from baseline), CD16+CD56+ NK cells (80.4% reduction from baseline), and CD34+ hematopoietic progenitor cells (37.9% reduction from baseline). Cell levels returned to approximately baseline levels on day 5 for CD16+CD56+ NK cells, by day 7 for lymphocytes and CD34+ cells and by day 9 for CD45+leukocytes. Placebo was without effect. Plasma concentrations of IFNγ (Interferon γ) and the chemokine CXCL10 (Interferon γ-induced protein 10), as determined by ELISA, were increased on days 3 and 4 in rHuIL-12-treated subjects. Placebo was without effect. Cell surface expression of IL-12Rβ2, a subunit of the heterodimeric receptor for IL-12, was also studied by flow cytometry. IL-12Rβ2+ NK (CD16+CD56+) cells showed a decrease in levels on day 2, with a reduction of 80.4% from baseline. In addition, the mean fluorescence intensity (MFI) was determined for bright CD56, on NK cells. There was an increase in CD56 MFI for the rHuIL-12-treated subjects, while no effect was seen in the placebo treated subjects. The peak of expression occurred on day 3 (an increase of 55.5%) and had returned to baseline by day 11. Increased CD56 expression is found on a NK subset uniquely equipped to traffic to sites of innate and adaptive immune responses. IL-12, initially called NK cell stimulating factor, can regulate NK cell recruitment to tissues by inducing migration and interaction with endothelial cells. The transient change in IL-12Rβ2+ NK (CD16+CD56+) cells after rHuIL-12 administration, together with the increase in bright CD56+NK cells, suggests that NK cells may be leaving the peripheral blood and migrating into the tissues, fulfilling their role of immune surveillance. Using human peripheral blood mononuclear cells (PBMC) obtained from healthy donors rHuIL-12 was shown by qPCR to induce IFNγ and CXCL10 mRNA expression. Furthermore rHuIL-12 induced a transient upregulation of co-receptors IL-12Rβ2 and CXCR3 (the Gαiprotein-coupled receptor for CXCL10) on lymphocytes, as determined by FACS. Our data indicate that rHuIL-12 administration to healthy human subjects induces IL-12Rβ2+, CD16+CD56+ NK cell migration from the peripheral blood into the tissue compartment, through a mechanism facilitated by IFNγ-induced CXCL10 chemokine and its receptor CXCR3. Further it is likely that this mechanism is the basis for the transient decrease of CD45+ leukocytes, CD45+ lymphocytes, CD45+CD16+CD56+ NK cells, and CD45+CD34+hematopoietic progenitor cells, described above, following rHuIL-12 administration to healthy human subjects. This project has been funded in part with Federal funds from the Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, Office of the Secretary, Department of Health and Human Services, under Contract No. HHSO100201100037C. Disclosures: Thomas: Neumedicines: Employment. Lawrence:Neumedicines: Employment. Mar:Neumedicines Inc: Employment. Kha:Neumedicines: Employment. Vainstein:Neumedicines Inc.: Employment. Gokhale:Neumedicines Inc.: Employment. Basile:Neumedicines Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Interleukin-12 (IL-12) has potent immunoregulatory and hematopoietic properties, and exerts significant biological effects on natural killer (NK) cells, inducing IFNγ production and enhancing cytotoxicity. Two distinct NK cell populations correlate with their immunoregulatory functions. Mature CD56dimCD16bright NK cells represent 90% of the NK cells resident in the blood and can exert cytotoxic effects on transformed cells. Cytokine producing immature CD56brightCD16+/- NK cells exist in the blood (10% of total circulating NK cells) but are most prominently located in secondary lymphoid tissues. In the continued clinical development of recombinant human IL-12 (HemaMax™, rHuIL-12), to be used in combination with radiotherapy or chemotherapy for the treatment of cancer patients, we have performed a clinical safety study in healthy human subjects. A single subcutaneous (sc) dose of rHuIL-12 (12μg) was administered to 17 healthy human subjects. Placebo was administered to 5 healthy subjects. Peripheral blood samples were collected before rHuIL-12 administration, and up to Day 14 post administration. Immunophenotyping of blood cell populations was conducted by FACS. rHuIL-12 caused a transient decrease in peripheral blood CD56dimCD16bright NK cells, with a nadir (60% reduction from baseline) reached on Day 2 following rHuIL-12 administration. CD56dimCD16bright NK cell levels returned almost to baseline levels on Day 5. Placebo was without effect. Conversely rHuIL-12 caused an elevation in peripheral blood CD56brightCD16+/- NK cells, particularly between Days 2 and 3 after rHuIL-12 administration, which was sustained until a peak was reached on Day 5 (265% above baseline). Levels returned to baseline by Day 11, while placebo was without effect. rHuIL-12 did not impact the less functional CD56-CD16bright NK cell subset. CD56dimCD16bright NK cells expressing the IL-12 receptor β2 subunit (IL-12Rβ2+) showed a substantial, and transient, decrease in levels on Day 2. The plasma concentration of IFNγ was elevated to a peak over 35 fold above baseline level at 10hr. after rHuIL-12 administration. Human NK cells were negatively selected from highly enriched leukapheresis-derived blood and stimulated in vitro with 10 pM rHuIL-12. After 16hr. incubation these predominantly CD56dimCD16brightNK cells showed enhanced release of IFNγ and the increased killing of K562 cells, a human erythroleukemic cell line, when compared with vehicle controls. qPCR analysis of the human NK cell lysates showed rHuIL-12-induced elevation of CD56 (302%) and IL-12Rβ2 (587%) mRNA, when compared with vehicle controls. rHuIL-12 did not influence CD16 mRNA expression, but did increase the level of CD62L (L selectin, 206%) mRNA. The rapid 60% fall in circulating mature CD56dimCD16bright NK cells after rHuIL-12 administration to healthy human subjects suggests their immediate exit from peripheral blood into the tissue compartments. This could be mediated by the observed increase in NK cell CD62L mRNA expression seen in vitro. The sustained increase in immature CD56brightCD16+/- NK cell levels between Day 3 and 6 suggests their IL-12-induced development from CD34+ hematopoietic progenitor cells. In summary rHuIL-12 administration to healthy human subjects demonstrates differential effects on the two key NK cell populations in peripheral blood, increasing CD56brightCD16+/- NK cell numbers, potentially stimulating IFNγ release from and enhancing the cytotoxicity of the CD56dimCD16bright NK cells, and preparing this population for migration into tissues. rHuIL-12 thus shows excellent potential as an immunotherapeutic and hematopoietic agent for the treatment of cancer patients, by impacting the maturation, activation, immunoregulation, and cytolytic properties of NK cells. Disclosures Thomas: Neumedicines: Employment, Equity Ownership. Lawrence:Neumedicines: Employment, Equity Ownership. Mar:Neumedicines: Employment, Equity Ownership. Kha:Neumedicines: Employment, Equity Ownership. Basile:Neumedicines: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.