HemaMax™, a recombinant human interleukin-12, is a potent mitigator of acute radiation injury in mice and non-human primates
Lena A Basile, Dolph Ellefson, Zoya Gluzman-Poltorak, Katiana Junes-Gill, Vernon Mar, Sarita Mendonca, Joseph D Miller, Jamie Tom, Alice Trinh, Timothy K Gallaher, Lena A Basile, Dolph Ellefson, Zoya Gluzman-Poltorak, Katiana Junes-Gill, Vernon Mar, Sarita Mendonca, Joseph D Miller, Jamie Tom, Alice Trinh, Timothy K Gallaher
Abstract
HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8-9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit-expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD(50/30)) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12-14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin.
Conflict of interest statement
Competing Interests: Lena A. Basile, Dolph Ellefson, Zoya Gluzman-Poltorak, Katiana Junes-Gill, Vernon Mar, Sarita Mendonca, Jamie Tom, Alice Trinh, and Timothy K. Gallaher are employees of Neumedicines Inc. Joseph D. Miller is a consultant to Neumedicines Inc. HemaMax is a Neumedicines' product; there are numerous relevant patents, details of which are available on request. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
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References
- Drouet M, Herodin F. Radiation victim management and the haematologist in the future: time to revisit therapeutic guidelines? Int J Radiat Biol. 2010;86:636–648.
- Donnelly EH, Nemhauser JB, Smith JM, Kazzi ZN, Farfan EB, et al. Acute radiation syndrome: assessment and management. South Med J. 2010;103:541–546.
- . Effects of radiation levels on human body. . Accessed June 19, 2011.
- Williams JP, Brown SL, Georges GE, Hauer-Jensen M, Hill RP, et al. Animal models for medical countermeasures to radiation exposure. Radiat Res. 2010;173:557–578.
- Johnson SM, Torrice CD, Bell JF, Monahan KB, Jiang Q, et al. Mitigation of hematologic radiation toxicity in mice through pharmacological quiescence induced by CDK4/6 inhibition. J Clin Invest. 2010;120:2528–36.
- Burdelya LG, Krivokrysenko VI, Tallant TC, Strom E, Gleiberman AS, et al. An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models. Science. 2008;320:226–30.
- Vijay-Kumar M, Aitken JD, Sanders CJ, Frias A, Sloane VM, et al. Flagellin treatment protects against chemicals, bacteria, viruses, and radiation. J Immunol. 2008;180:8280–5.
- Chen BJ, Deoliveira D, Spasojevic I, Sempowski GD, Jiang C, et al. Growth hormone mitigates against lethal irradiation and enhances hematologic and immune recovery in mice and nonhuman primates. PLoS One. 2010;5:e11056.
- Singh VK, Yadav VS. Role of cytokines and growth factors in radioprotection. Exp Mol Pathol. 2005;78:156–69.
- Hérodin F, Drouet M. Cytokine-based treatment of accidentally irradiated victims and new approaches. Exp Hematol. 2005;33:1071–80.
- Weiss JF, Landauer MR. History and development of radiation-protective agents. Int J Radiat Biol. 2009;85:539–73.
- Dumont F, Le Roux A, Bischoff P. Radiation countermeasure agents: an update. Expert Opin Ther Pat. 2010;20:73–101.
- Basile LA, Gallaher TK, Shibata D, Miller JD, Douer D. Multilineage hematopoietic recovery with concomitant antitumor effects using low dose Interleukin-12 in myelosuppressed tumor-bearing mice. J Transl Med. 2008;6:26.
- Chen T, Burke KA, Zhan Y, Wang X, Shibata D, et al. IL-12 facilitates both the recovery of endogenous hematopoiesis and the engraftment of stem cells after ionizing radiation. Exp Hematol. 2007;35:203–213.
- Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev. 2002;13:155–168.
- Jacobsen SE, Veiby OP, Smeland EB. Cytotoxic lymphocyte maturation factor (interleukin 12) is a synergistic growth factor for hematopoietic stem cells. J Exp Med. 1993;178:413–418.
- Bellone G, Trinchieri G. Dual stimulatory and inhibitory effect of NK cell stimulatory factor/IL-12 on human hematopoiesis. J Immunol. 1994;153:930–937.
- Ploemacher RE, van Soest PL, Boudewijn A, Neben S. Interleukin-12 enhances interleukin-3 dependent multilineage hematopoietic colony formation stimulated by interleukin-11 or steel factor. Leukemia. 1993;7:1374–1380.
- Ploemacher RE, van Soest PL, Voorwinden H, Boudewijn A. Interleukin-12 synergizes with interleukin-3 and steel factor to enhance recovery of murine hemopoietic stem cells in liquid culture. Leukemia. 1993;7:1381–1388.
- Hirayama F, Katayama N, Neben S, Donaldson D, Nickbarg EB, et al. Synergistic interaction between interleukin-12 and steel factor in support of proliferation of murine lymphohematopoietic progenitors in culture. Blood. 1994;83:92–98.
- Broxmeyer HE, Lu L, Platzer E, Feit C, Juliano L, et al. Comparative analysis of the influences of human gamma, alpha and beta interferons on human multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. J Immunol. 1983;131:1300–1305.
- Gimble JM, Medina K, Hudson J, Robinson M, Kincade PW. Modulation of lymphohematopoiesis in long-term cultures by gamma interferon: direct and indirect action on lymphoid and stromal cells. Exp Hematol. 1993;21:224–230.
- Means RT, Jr, Krantz SB. Inhibition of human erythroid colony-forming units by gamma interferon can be corrected by recombinant human erythropoietin. Blood. 1991;78:2564–2567.
- Terrell TG, Green JD. Comparative pathology of recombinant murine interferon-gamma in mice and recombinant human interferon-gamma in cynomolgus monkeys. Int Rev Exp Pathol. 1993;34 Pt B:73–101.
- Zoumbos NC, Djeu JY, Young NS. Interferon is the suppressor of hematopoiesis generated by stimulated lymphocytes in vitro. J Immunol. 1984;133:769–774.
- Kurz K, Gluhcheva Y, Zvetkova E, Konwalinka G, Fuchs D. Interferon-gamma-mediated pathways are induced in human CD34(+) haematopoietic stem cells. Immunobiology. 2010;215:452–457.
- Zhao X, Ren G, Liang L, Ai PZ, Zheng B, et al. Brief report: interferon-gamma induces expansion of Lin(−)Sca-1(+)C-Kit(+) Cells. Stem Cells. 2010;28:122–126.
- U.S. Food and Drug Administration. Center for Drug Evaluation and Research. FDA guidance estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. 2005. . Accessed June 13, 2011.
- Yuan R, Maeda Y, Li W, Lu W, Cook S, et al. Erythropoietin: a potent inducer of peripheral immuno/inflammatory modulation in autoimmune EAE. PLoS One. 2008;3:e1924.
- Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449:1003–1007.
- Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, et al. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell. 2010;6:25–36.
- Garcia MI, Ghiani M, Lefort A, Libert F, Strollo S, et al. LGR5 deficiency deregulates Wnt signaling and leads to precocious Paneth cell differentiation in the fetal intestine. Dev Biol. 2009;331:58–67.
- Zou JJ, Schoenhaut DS, Carvajal DM, Warrier RR, Presky DH, et al. Structure-function analysis of the p35 subunit of mouse interleukin 12. J Biol Chem. 1995;270:5864–5871.
- Bekaii-Saab TS, Roda JM, Guenterberg KD, Ramaswamy B, Young DC, et al. A phase I trial of paclitaxel and trastuzumab in combination with interleukin-12 in patients with HER2/neu-expressing malignancies. Mol Cancer Ther. 2009;8:2983–2991.
- Lenzi R, Edwards R, June C, Seiden MV, Garcia ME, et al. Phase II study of intraperitoneal recombinant interleukin-12 (rhIL-12) in patients with peritoneal carcinomatosis (residual disease <1 cm) associated with ovarian cancer or primary peritoneal carcinoma. J Transl Med. 2007;5:66.
- Little RF, Aleman K, Kumar P, Wyvill KM, Pluda JM, et al. Phase 2 study of pegylated liposomal doxorubicin in combination with interleukin-12 for AIDS-related Kaposi sarcoma. Blood. 2007;110:4165–4171.
- Melichar B, Lenzi R, Rosenblum M, Kudelka AP, Kavanagh JJ, et al. Intraperitoneal fluid neopterin, nitrate, and tryptophan after regional administration of interleukin-12. J Immunother. 2003;26:270–276.
- Neta R, Stiefel SM, Finkelman F, Herrmann S, Ali N. IL-12 protects bone marrow from and sensitizes intestinal tract to ionizing radiation. J Immunol. 1994;153:4230–4237.
- Trinchieri G. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv Immunol. 1998;70:83–243.
- Langrish CL, McKenzie BS, Wilson NJ, de Waal MR, Kastelein RA, et al. IL-12 and IL-23: master regulators of innate and adaptive immunity. Immunol Rev. 2004;202:96–105.
- Gattoni A, Parlato A, Vangieri B, Bresciani M, Derna R. Interferon-gamma: biologic functions and HCV therapy (type I/II) (1 of 2 parts). Clin Ter. 2006;157:377–386.
- Macdougall IC, Cooper AC. Erythropoietin resistance: the role of inflammation and pro-inflammatory cytokines. Nephrol Dial Transplant. 2002;17(Suppl 11):39–43.
- Morceau F, Dicato M, Diederich M. Pro-inflammatory cytokine-mediated anemia: regarding molecular mechanisms of erythropoiesis. Mediators Inflamm. 2009;2009:405016.
- Dinarello CA, Fantuzzi G. Interleukin-18 and host defense against infection. J Infect Dis. 2003;187(Suppl 2):S370–S384.
- Gracie JA, Robertson SE, McInnes IB. Interleukin-18. J Leukoc Biol. 2003;73:213–224.
- Stonier SW, Schluns KS. Trans-presentation: a novel mechanism regulating IL-15 delivery and responses. Immunol Lett. 2010;127:85–92.
- Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine Growth Factor Rev. 2001;12:53–72.
- Werner ER, Werner-Felmayer G, Fuchs D, Hausen A, Reibnegger G, et al. Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present. J Biol Chem. 1990;265:3189–3192.
- Fuchs D, Hausen A, Reibnegger G, Werner ER, Dierich MP, et al. Neopterin as a marker for activated cell-mediated immunity: application in HIV infection. Immunol Today. 1988;9:150–155.
- Anagnostou A, Liu Z, Steiner M, Chin K, Lee ES, et al. Erythropoietin receptor mRNA expression in human endothelial cells. Proc Natl Acad Sci U S A. 1994;91:3974–3978.
- Brines M, Cerami A. Emerging biological roles for erythropoietin in the nervous system. Nat Rev Neurosci. 2005;6:484–494.
- Buemi M, Cavallaro E, Floccari F, Sturiale A, Aloisi C, et al. The pleiotropic effects of erythropoietin in the central nervous system. J Neuropathol Exp Neurol. 2003;62:228–236.
- Fraser JK, Tan AS, Lin FK, Berridge MV. Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes. Exp Hematol. 1989;17:10–16.
- Jaquet K, Krause K, Tawakol-Khodai M, Geidel S, Kuck KH. Erythropoietin and VEGF exhibit equal angiogenic potential. Microvasc Res. 2002;64:326–333.
- Sela S, Shurtz-Swirski R, Sharon R, Manaster J, Chezar J, et al. The polymorphonuclear leukocyte–a new target for erythropoietin. Nephron. 2001;88:205–210.
- Lifshitz L, Prutchi-Sagiv S, Avneon M, Gassmann M, Mittelman M, et al. Non-erythroid activities of erythropoietin: Functional effects on murine dendritic cells. Mol Immunol. 2009;46:713–721.
- Prutchi SS, Lifshitz L, Orkin R, Mittelman M, Neumann D. Erythropoietin effects on dendritic cells: potential mediators in its function as an immunomodulator? Exp Hematol. 2008;36:1682–1690.
- Cetin H, Olgar S, Oktem F, Ciris M, Uz E, et al. Novel evidence suggesting an anti-oxidant property for erythropoietin on vancomycin-induced nephrotoxicity in a rat model. Clin Exp Pharmacol Physiol. 2007;34:1181–1185.
- Genc S, Akhisaroglu M, Kuralay F, Genc K. Erythropoietin restores glutathione peroxidase activity in 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine-induced neurotoxicity in C57BL mice and stimulates murine astroglial glutathione peroxidase production in vitro. Neurosci Lett. 2002;321:73–76.
- Kumral A, Gonenc S, Acikgoz O, Sonmez A, Genc K, et al. Erythropoietin increases glutathione peroxidase enzyme activity and decreases lipid peroxidation levels in hypoxic-ischemic brain injury in neonatal rats. Biol Neonate. 2005;87:15–18.
- Liu J, Narasimhan P, Song YS, Nishi T, Yu F, et al. Epo protects SOD2-deficient mouse astrocytes from damage by oxidative stress. Glia. 2006;53:360–365.
- Wang ZY, Shen LJ, Tu L, Hu DN, Liu GY, et al. Erythropoietin protects retinal pigment epithelial cells from oxidative damage. Free Radic Biol Med. 2009;46:1032–1041.
- Akimoto T, Kusano E, Inaba T, Iimura O, Takahashi H, et al. Erythropoietin regulates vascular smooth muscle cell apoptosis by a phosphatidylinositol 3 kinase-dependent pathway. Kidney Int. 2000;58:269–282.
- Chong ZZ, Kang JQ, Maiese K. Erythropoietin is a novel vascular protectant through activation of Akt1 and mitochondrial modulation of cysteine proteases. Circulation. 2002;106:2973–2979.
- Chong ZZ, Kang JQ, Maiese K. Hematopoietic factor erythropoietin fosters neuroprotection through novel signal transduction cascades. J Cereb Blood Flow Metab. 2002;22:503–514.
- Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, et al. A novel protective effect of erythropoietin in the infarcted heart. J Clin Invest. 2003;112:999–1007.
- Gerosa F, Baldani-Guerra B, Nisii C, Marchesini V, Carra G, et al. Reciprocal activating interaction between natural killer cells and dendritic cells. J Exp Med. 2002;195:327–333.
- Lodoen MB, Lanier LL. Natural killer cells as an initial defense against pathogens. Curr Opin Immunol. 2006;18:391–398.
- Varma TK, Lin CY, Toliver-Kinsky TE, Sherwood ER. Endotoxin-induced gamma interferon production: contributing cell types and key regulatory factors. Clin Diagn Lab Immunol. 2002;9:530–543.
- de Barros AP, Takiya CM, Garzoni LR, Leal-Ferreira ML, Dutra HS, et al. Osteoblasts and bone marrow mesenchymal stromal cells control hematopoietic stem cell migration and proliferation in 3D in vitro model. PLoS One. 2010;5:e9093.
- Ahmed N, Khokher MA, Hassan HT. Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts. Stem Cells. 1999;17:92–99.
- Hamada T, Mohle R, Hesselgesser J, Hoxie J, Nachman RL, et al. Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation. J Exp Med. 1998;188:539–548.
- Hodohara K, Fujii N, Yamamoto N, Kaushansky K. Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK). Blood. 2000;95:769–775.
- Kiel MJ, Morrison SJ. Maintaining hematopoietic stem cells in the vascular niche. Immunity. 2006;25:862–864.
- Wang JF, Liu ZY, Groopman JE. The alpha-chemokine receptor CXCR4 is expressed on the megakaryocytic lineage from progenitor to platelets and modulates migration and adhesion. Blood. 1998;92:756–764.
- Dominici M, Rasini V, Bussolari R, Chen X, Hofmann TJ, et al. Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation. Blood. 2009;114:2333–2343.
- Savino R, Ciliberto G. A paradigm shift for erythropoietin: no longer a specialized growth factor, but rather an all-purpose tissue-protective agent. Cell Death Differ. 2004;11(Suppl 1):S2–S4.
- Zhang S, Wang Q. Factors determining the formation and release of bioactive IL-12: regulatory mechanisms for IL-12p70 synthesis and inhibition. Biochem Biophys Res Commun. 2008;372:509–512.
- Lacy MQ, Jacobus S, Blood EA, Kay NE, Rajkumar SV, et al. Phase II study of interleukin-12 for treatment of plateau phase multiple myeloma (E1A96): a trial of the Eastern Cooperative Oncology Group. Leuk Res. 2009;33:1485–1489.
- Lenzi R, Rosenblum M, Verschraegen C, Kudelka AP, Kavanagh JJ, et al. Phase I study of intraperitoneal recombinant human interleukin 12 in patients with Mullerian carcinoma, gastrointestinal primary malignancies, and mesothelioma. Clin Cancer Res. 2002;8:3686–3695.
- Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood. 1997;90:2541–2548.
- Chao NJ. Accidental or intentional exposure to ionizing radiation: biodosimetry and treatment options. Exp Hematol. 2007;35:24–27.
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