HemaMax™, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

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/LD50/30) 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

Rationale for combination therapy of chronic myelogenous leukaemia with imatinib and irradiation or alkylating agents: implications for pretransplant conditioning

The tyrosine kinase activity of the BCR–ABL oncoprotein results in reduced apoptosis and thus prolongs survival of chronic myelogenous leukaemia cells. The tyrosine kinase inhibitor imatinib (formerly STI571) was reported to selectively suppress the proliferation of BCR–ABL-positive cells. Assuming that imatinib could be included in pretransplantation conditioning therapies, we tested whether combinations of imatinib and γ-irradiation or alkylating agents such as busulfan or treosulfan would display synergistic activity in BCR–ABL-positive chronic myelogenous leukaemia BV173 and EM-3 cell lines. Further, primary cells of untreated chronic myelogenous leukaemia patients were assayed for colony forming ability under combination therapy with imatinib. Additionally, the cytotoxic effect of these combinations on BCR–ABL-negative cells was investigated. In the cell lines a tetrazolium based MTT assay was used to quantify growth inhibition after exposure to cytotoxic drugs alone or to combinations with imatinib. Irradiation was applied prior to exposure to imatinib. Interaction of drugs was analysed using the median-effect method of Chou and Talalay. The combination index was calculated according to the classic isobologram equation. The combination imatinib + γ-irradiation proved to be significantly synergistic over a broad range of cell growth inhibition levels in both BCR–ABL-positive cell lines and produced the strongest reduction in primary chronic myelogenous leukaemia colony-forming progenitor cells. Combinations of imatinib + busulfan and imatinib + treosulfan showed merely additive to antagonistic effects. Imatinib did not potentiate the effects of irradiation or cytotoxic agents in BCR–ABL-negative cells. Our data provide the basis to further develop imatinib-containing conditioning therapies for stem cell transplantation in chronic myelogenous leukaemia

Serum after Autologous Transplantation Stimulates Proliferation and Expansion of Human Hematopoietic Progenitor Cells

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34+ cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34+, CD133+, CD45−) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool

Multilineage hematopoietic recovery with concomitant antitumor effects using low dose Interleukin-12 in myelosuppressed tumor-bearing mice

<p>Abstract</p> <p>Background</p> <p>Interleukin-12 (IL-12) is a cytokine well known for its role in immunity. A lesser known function of IL-12 is its role in hematopoiesis. The promising data obtained in the preclinical models of antitumor immunotherapy raised hope that IL-12 could be a powerful therapeutic agent against cancer. However, excessive clinical toxicity, largely due to repeat dose regimens, and modest clinical response observed in the clinical trials have pointed to the necessity to design protocols that minimize toxicity without affecting the anti-tumor effect of IL-12. We have focused on the lesser known role of IL-12 in hematopoiesis and hypothesized that an important clinical role for IL-12 in cancer may be as an adjuvant hematological cancer therapy. In this putative clinical function, IL-12 is utilized for the prevention of cancer therapy-related cytopenias, while providing concomitant anti-tumor responses over and above responses observed with the primary therapy alone. This putative clinical function of IL-12 focuses on the dual role of IL-12 in hematopoiesis and immunity.</p> <p>Methods</p> <p>We assessed the ability of IL-12 to facilitate hematopoietic recovery from radiation (625 rad) and chemotherapy (cyclophosphamide) in two tumor-bearing murine models, namely the EL4 lymphoma and the Lewis lung cancer models. Antitumor effects and changes in bone marrow cellularity were also assessed.</p> <p>Results</p> <p>We show herein that carefully designed protocols, in mice, utilizing IL-12 as an adjuvant to radiation or chemotherapy yield facile and consistent, multilineage hematopoietic recovery from cancer therapy-induced cytopenias, as compared to vehicle and the clinically-utilized cytokine granulocyte colony-stimulating factor (G-CSF) (positive control), while still providing concomitant antitumor responses over and above the effects of the primary therapy alone. Moreover, our protocol design utilizes single, low doses of IL-12 that did not yield any apparent toxicity.</p> <p>Conclusion</p> <p>Our results portend that despite its past failure, IL-12 appears to have significant clinical potential as a hematological adjuvant cancer therapy.</p

TRANSIENT AND PERMANENT ENGRAFTMENT POTENTIAL OF MURINE HEMATOPOIETIC STEM-CELL SUBSETS - DIFFERENTIAL-EFFECTS OF HOST CONDITIONING WITH GAMMA-RADIATION AND CYTOTOXIC DRUGS

Transplant of sorted donor (BG-Gpi-1(a)) hematopoietic stem cell subsets and host (BG-Gpi-1(b)) treatment with total body irradiation (TBI) or cytotoxic drugs were compared for induction of short- and long-term engraftment in a murine chimera model of congenic bone marrow transplantation (BMT). Parallel studies on donor and host marrow were performed in vitro in long-term bone marrow cultures to determine early and late cobblestone area forming cell (CAFC) frequencies in the grafts or in the transplant recipients 1 day after conditioning. Bone marrow cells (BMC) sorted for high wheat germ agglutinin affinity (WGA(+++)) were enriched about 30-fold for early developing CAFC (colony-forming unit-spleen [CFU-S]) but not for primitive late CAFC (pre-CFU-S). This fraction showed only temporary engraftment when transplanted in irradiated recipients. In contrast, the low affinity (WGA(+)) fraction were preferentially enriched (200- to 300-fold) for late developing CAFC and were very effective for providing stable long-term engraftment following transplantation. Substituting radiation for chemotherapy in the host conditioning treatment also had diverse effects on the development of bone marrow engraftment. Pretreatment with 5-fluorouracil (5-FU, 200 mg/kg) allowed a discrete wave of donor engraftment that peaked at 2 to 4 weeks and then subsided to leave mostly host cells at 10 weeks and beyond. Busulfan preparation gave over 50% engraftment at 1 month after BMT but this continued to increase to reach stable donor chimerism of 80 to 90% beyond 10 weeks. The level of long-term engraftment given by 50 mg/kg busulfan appeared similar to that induced by doses of 6 to 8 Gy TBI. The changing patterns of erythroid chimerism for each preparative agent showed a remarkable correlation with depletion of defined hematopoietic stem cell subsets as quantified using the CAFC assay at 1 day after recipient treatment. These findings collectively show that the level of depletion of host CFU-S (CAFC-10) determines the extent of early and transient repopulation, whereas the degree of pre-CFU-S (CAFC-35) depletion determines the percentage of stable chimerism. Preliminary data on bone marrow CAFC content at 9 months after busulfan and BMT revealed a long-term reduction of the stem cell pool by about 50% but with relatively minor effects on supporting bone marrow stromas. The differential cytotoxic effects on stem cell subsets in relation to subsequent donor marrow engraftment offer a systematic and mechanistic approach toward identifying more effective chemotherapeutic compounds for use in clinical BMT conditioning regimens