In vivo characterization of fusion protein comprising of A1 subunit of Shiga toxin and human GM-CSF: Assessment of its immunogenicity and toxicity.

BACKGROUND: Most cancer cells become resistant to anti-cancer agents. In the last few years, a new approach for targeted therapy of human cancer has been developed using immunotoxins which comprise both the cell targeting and the cell killing moieties. METHODS: In the present study, the recombinant Shiga toxin A1 subunit fused to human granulocyte-macrophage colony stimulating factor (A1-GM-CSF), previously produced in E. coli, was further characterized. RESULTS: The recombinant protein could cause 50% cytotoxicity and induced apoptosis in cells bearing GM-CSF receptors. The non-specific toxicity of the fusion protein was assessed in C57BL/6 and BALB/c mice. No mortality was observed in either group of mice, with different concentration of fusion protein. CONCLUSION: The lymphocyte proliferation assay, induction of specific IgG response and a mixed (Th1/Th2) response were observed only in BALB/c mice. The mixed response in BALB/c mice (Th1/Th2) could be explained on the basis of the two components of the fusion protein i.e. A1 and GM-CSF.

Priming effects of lipopolysaccharide and inflammatory cytokines on canine granulocytes.

Granulocytes play a pivotal role in natural immunity. Under inflammatory conditions, granulocytes are universally primed by several agents, such as endotoxins and inflammatory cytokines. Primed granulocytes exert potent adhesiveness, chemotaxis, phagocytosis and reactive oxygen species (ROS) production, effectively eliminating invading agents. Reactivity against priming agents is known to vary with species; however, there have been few reports on the effects of priming agents on canine granulocytes. In the present study, we assayed the priming effects of lipopolysaccharide (LPS), recombinant canine tumor necrosis factor-alpha (rcTNF-alpha) and recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) on canine granulocyte function in vitro. Isolated recombinant canine were primed with various concentrations of LPS, rcTNF-alpha and rcGM-CSF, and CD11b expression was assayed. Furthermore, actin polymerization, phagocytosis and ROS production were then assayed at primer concentrations where enhancement of CD11b expression was observed. LPS did not enhance canine granulocyte function. Phagocytosis and actin polymerization were not enhanced by priming agents; however, rcTNF-alpha and rcGM-CSF enhanced CD11b expression and ROS production in canine granulocytes. These results suggest that priming effects are mainly reflected in CD11b expression and ROS production, with rcGM-CSF and rcTNF-alpha having a priming effect similar to that observed in humans.

Incorporating the use of GM-CSF in the treatment of chronic lymphocytic leukemia.

We evaluated the clinical activity of GM-CSF in combination with standard dose rituximab in patients with chronic lymphocytic leukemia (CLL). The rationale for exploring this combination is provided by the ability of GM-CSF to increase surface expression of CD20 in CLL cells and potentially render them a better target for rituximab. GM-CSF also enhances antibody-dependent cellular cytotoxicity against CLL cells. The combination of GM-CSF and rituximab was evaluated as initial treatment in elderly patients with indication for treatment and in patients at high risk for progression identified by elevated beta(2) microglobulin. This combination was also evaluated in patients with recurrent CLL. On the basis of the results of 118 patients, we observed an overall response rate of 65 and 9% complete remission and these results compare favourably with the results obtained with rituximab single agent. This combination was well tolerated with the most common toxicity consisting in mild GM-CSF injection site erythema. On the basis of this experience, we are currently evaluating the use of GM-CSF in combination with the chemoimmunotherapy regimen fludarabine, cyclophosphamide and rituximab.

Granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor exacerbate atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Recent studies have suggested a potential contribution of bone marrow-derived progenitor cells to vascular repair. Preliminary clinical studies have explored the possibility that mobilization of progenitor cells with granulocyte macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) can affect vascular repair. However, it is not known whether the short-term administration of G-CSF or GM-CSF exerts beneficial effects on atherosclerosis. METHODS AND RESULTS: Apolipoprotein E-deficient mice were treated with either GM-CSF or G-CSF at a dose of 10 microg x kg(-1) x d(-1) s.c. administered daily for 5 days per week on alternating weeks for a total of 20 doses over an 8-week treatment period. We found that in animals maintained on a high-fat diet, both G-CSF and GM-CSF actually demonstrated an increase in atherosclerotic lesion extent. The increase in atherosclerotic extent was not associated with an increase in either inflammatory cells or expression of proinflammatory genes. Interestingly, adventitial vascularity significantly increased, suggesting a mechanistic role for vasa vasorum neovascularization. CONCLUSIONS: These findings demonstrate that in this animal model of atherosclerosis, not only did administration of G-CSF or GM-CSF fail to demonstrate any beneficial therapeutic effect, but both resulted in a worsening of atherosclerosis.

Selective cytotoxicity of recombinant STXA1-GM-CSF protein in hematopoetic cancer cells.

Chimeric proteins are composed of a cell-targeting moiety and a cell-killing moiety. In this study, a chimeric protein, STXA1-GM-CSF, composed of catalytic domain of Shiga toxin (A1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed and expressed in E. coli. Cytotoxicity, receptor blocking, and neutralization experiments revealed that the chimeric protein induced cytotoxic effect on different cell lines. This effect was found to be specific, due to the presence of the killing moiety (A1), which exerts its effect through a specific GM-CSF-targeting domain, by binding to its receptor present on those cell lines. These initial investigations indicate that the chimeric protein was functional; further analyses are required for its application.

Reduced risk of acute GVHD following mobilization of HLA-identical sibling donors with GM-CSF alone.

We retrospectively reviewed the results of transplanting peripheral blood progenitor cell (PBPC) allografts from HLA-matched sibling donors mobilized using various hematopoietic cytokines. Patients had received allografts mobilized with Granulocyte colony-stimulating factor (G-CSF) (G, N = 65) alone, G plus Granulocyte-macrophage colony stimulating factor (GM-CSF) (G/GM, N = 70), or GM-CSF alone at 10 or 15 microg/kg/day (GM, N = 10 at 10 microg/kg/day and 21 at 15 microg/kg/day). The CD34+ and CD3+ cell content of grafts were significantly lower following GM alone compared to G alone (P < 0.001 and 0.04, respectively). Nonhematopoietic toxicity observed in donors precluded dose escalation of GM-CSF beyond 10 microg/kg/day. Hematopoietic recovery was similar among all three groups. Grades II-IV acute graft-versus-host disease (GVHD) was observed in only 13% of patients in the GM alone group compared to 49 and 69% in the G alone or G/GM groups, respectively (P < 0.001). In a multivariate analysis, receipt of PBPC mobilized with GM alone was associated with a lower risk of grades II-IV acute GVHD (hazard ratio 0.21; 95% CI 0.073, 0.58) compared to G alone or G/GM. There were no differences in relapse risk or overall survival among the groups. Donor PBPC grafts mobilized with GM-CSF alone result in prompt hematopoietic engraftment despite lower CD34+ cell doses and may reduce the risk of grades II-IV acute GVHD following HLA-matched PBPC transplantation.

A chimeric protein induces tumor cell apoptosis by delivering the human Bcl-2 family BH3-only protein Bad.

Deregulation of PI3K/Akt and Raf/Mek/Erk signal transduction cascades is one of the principal causes of neoplastic transformation. The inactivation of the proapoptotic protein Bad, upon phosphorylation by different kinases of these two pathways, may play an important role in different human malignancies. Therefore, we have expressed and purified a new chimeric protein, hGM-CSF-Bad, linking the human granulocyte-macrophage colony-stimulating factor to the N-terminus of the proapoptotic protein human Bad, to deliver Bad into tumor cells and induce apoptosis. Indeed, the human GM-CSF receptor is a good target because it is overexpressed on many leukemias and solid tumors and is not detectable on stem cells. We found that the chimeric protein binds the human GM-CSF receptor, is endocytosed, and appears to reach the cytosol via retrograde ER transport. After entering cells, the protein is able to induce apoptosis of human leukemia cells and human colon and gastric carcinoma cell lines (IC(50) values as low as 1 muM). We conclude that GM-CSF-Bad can overcome the inappropriate survival stimuli in transformed cells and restore the apoptotic pathway. The completely human sequence and the elevated selectivity for cancer cells could prevent immunogenicity and the nonspecific toxicity of targeted toxins in future clinical application of this fusion protein.

Randomized comparison of granulocyte colony-stimulating factor versus granulocyte-macrophage colony-stimulating factor plus intensive chemotherapy for peripheral blood stem cell mobilization and autologous transplantation in multiple myeloma.

Autologous peripheral blood stem cell transplantation for multiple myeloma offers higher response rates and improved survival compared with conventional chemotherapy. However, successful autografting requires effective cytoreduction and rapid hematologic reconstitution. We conducted a prospective randomized clinical trial to assess the efficacy of 2 cycles of priming chemotherapy with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for peripheral blood stem cell mobilization followed by autologous transplantation. The major study end points were the comparative utility of G-CSF versus GM-CSF, the percentage of patients achieving complete response after transplantation, and overall and progression-free survival. Priming chemotherapy included cyclophosphamide (4 g/m2), mitoxantrone (8 g/m2 every day for 2 days), and dexamethasone (20 mg/m2 every 12 hours for 2 days) followed by randomization to either G-CSF or GM-CSF daily until completion of leukapheresis. Conditioning for transplantation included cyclophosphamide (75 mg/kg every day for 2 days) plus total body irradiation (165 cGy twice daily for 3 days), and patients received maintenance immunotherapy with interferon alpha. Seventy-two patients were randomized, and 64 underwent autologous transplantation. The median age at transplantation was 52 years, and the median time from diagnosis to transplantation was 10 months; 58% of the patients had received >4 cycles of pretransplantation chemotherapy. The median number of CD34+ cells obtained after mobilization was 16.4 x 10(6)/kg in the G-CSF arm versus 12.8 x 10(6)/kg in the GM-CSF arm (P = .8). Neutrophil recovery was faster in the G-CSF group after both cycle 1 (median, 13 days with G-CSF and 16 days with GM-CSF; P < .01) and cycle 2 (median, 13 days versus 17 days in the 2 groups, respectively; P = .03). Although platelet recovery was similar after cycle 1, platelet recovery to >100000/microL was notably faster in the G-CSF group both after cycle 2 and after transplantation (P = .03). Response and overall and disease-free survival were similar in both cohorts. Overall, 23% of the patients achieved a complete response after priming chemotherapy, which improved to 33% after transplantation. An additional 47% attained a partial response after transplantation, for a total response rate of 80%. With a median follow-up of 2 years (range, 0.7-8 years), the overall survival was 88% (95% confidence interval [CI], 80%-96%) at 1 year and 65% (95% CI, 51%-79%) at 3 years. Progression-free survival was 73% (95% CI, 62%-84%) at 1 year and 40% (95% CI, 26%-54%) at 3 years. Relapse or progressive disease was the most common cause of death (25 [83%] of 30 deaths). We conclude that mobilization with chemotherapy plus G-CSF versus GM-CSF results in similar CD34+ progenitor collections, even in patients exposed to multiple cycles of alkylator-based chemotherapy. Earlier neutrophil and platelet recovery was seen with G-CSF priming. Two cycles of priming chemotherapy plus autologous transplantation yields survival rates similar to those in published reports, including those using tandem transplantation.

Diphtheria toxin-murine granulocyte-macrophage colony-stimulating factor-induced hepatotoxicity is mediated by Kupffer cells.

DT388GMCSF, a fusion toxin composed of the NH2-terminal region of diphtheria toxin (DT) fused to human granulocyte-macrophage colony-stimulating factor (GMCSF) has shown efficacy in the treatment of acute myeloid leukemia. However, the primary dose-limiting side effect is liver toxicity. We have reproduced liver toxicity in rats using the rodent cell-tropic DT-murine GMCSF (DT390mGMCSF). Serum aspartate aminotransferase and alanine aminotransferase were elevated 15- and 4-fold, respectively, in DT390mGMCSF-treated rats relative to controls. Histologic analysis revealed hepatocyte swelling; however, this did not lead to hepatic necrosis or overt histopathologic changes in the liver. Immunohistochemical staining showed apoptotic cells in the sinusoids, and depletion of cells expressing the monocyte/macrophage markers, ED1 and ED2, indicating that Kupffer cells (KC) are targets of DT390mGMCSF. In contrast, sinusoidal endothelial cells seemed intact. In vitro, DT390mGMCSF was directly cytotoxic to primary KC but not hepatocytes. Two related fusion toxins, DT388GMCSF, which targets the human GMCSF receptor, and DT390mIL-3, which targets the rodent IL-3 receptor, induced a less than 2-fold elevation in serum transaminases and did not deplete KC in vivo. In addition, DTU2mGMCSF, a modified form of DT390mGMCSF with enhanced tumor cell specificity, was not hepatotoxic and was significantly less toxic to KC in vivo and in vitro. These results show that DT390mGMCSF causes liver toxicity by targeting KC, and establish a model for studying how this leads to hepatocyte injury. Furthermore, alternative fusion toxins with potentially reduced hepatotoxicity are presented.

Collection of hematopoietic stem cells from patients with autoimmune diseases.

We reviewed data from 24 transplant centers in Asia, Australia, Europe, and North America to determine the outcomes of stem cell collection including methods used, cell yields, effects on disease activity, and complications in patients with autoimmune diseases. Twenty-one unprimed bone marrow harvests and 174 peripheral blood stem cell mobilizations were performed on 187 patients. Disease indications were multiple sclerosis (76 patients), rheumatoid arthritis (37 patients), scleroderma (26 patients), systemic lupus erythematosus (19 patients), juvenile chronic arthritis (13 patients), idiopathic autoimmune thrombocytopenia (8 patients), Behcet's disease (3 patients), undifferentiated vasculitis (3 patients), polychondritis (1 patient) and polymyositis (1 patient). Bone marrow harvests were used in the Peoples Republic of China and preferred worldwide for children. PBSC mobilization was the preferred technique for adult stem cell collection in America, Australia, and Europe. Methods of PBSC mobilization included G-CSF (5, 10, or 16 microg/kg/day) or cyclophosphamide (2 or 4 g/m2) with either G-CSF (5 or 10 microg/kg/day) or GM-CSF (5 microg/kg/day). Bone marrow harvests were without complications and did not affect disease activity. A combination of cyclophosphamide and G-CSF was more likely to ameliorate disease activity than G-CSF alone (P < 0.001). g-csf alone was more likely to cause disease exacerbation than the combination of cyclophosphamide and g-csf (P = 0.003). Three patients died as a result of cyclophosphamide-based stem cell collection (2.6% of patients mobilized with cyclophosphamide). When corrected for patient weight and apheresis volume, progenitor cell yields tended to vary by underlying disease, prior medication history and mobilization regimen. Trends in the approaches to, and results of, progenitor cell mobilization are suggested by this survey. While cytokine-based mobilization appears less toxic, it is more likely to result in disease reactivation. Optimization with regard to cell yields and safety are likely to be disease-specific and prospective disease-specific studies of mobilization procedures appear warranted.

Safety of a GM-CSF adjuvant-plasmid DNA malaria vaccine.

MuStDO 5 is a multivalent plasmid DNA vaccine for malaria comprised of five plasmid DNAs encoding five proteins from Plasmodium falciparum and one plasmid DNA encoding human GM-CSF. To evaluate the safety of MuStDO 5, a series of pre-clinical studies were conducted in mice and rabbits. In pharmacology studies in mice, GM-CSF could not be detected in the serum following either intramuscular or a combined intramuscular/intradermal administration of the vaccine, but was readily detected in the muscle following intramuscular administration. In a tissue distribution study in mice, MuStDO 5 plasmid DNA was detected by PCR initially in highly vascularized tissues, while at later time-points the plasmid DNA was detected primarily at the site(s) of injection. In GLP safety studies in mice and rabbits, repeated intramuscular/intradermal administration of the MuStDO 5 vaccine was found to be safe and well tolerated without any evidence of autoimmune pathology.

Effects of recombinant human granulocyte-macrophage colony-stimulating factor in an intensive treatment program for children with Ewing's sarcoma.

BACKGROUND AND OBJECTIVES: A treatment program including polychemotherapy at progressively escalating doses and sequential hemi-body irradiation (HBI) was adopted between 1987-1994 at our Pediatric Unit for high risk Ewing's sarcoma. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was added to the treatment program in a phase II study fashion to evaluate, in a pediatric setting, its tolerability, as well as its impact on drug dose escalation and on the need for supportive care. DESIGN AND METHODS: The study was open-label and sequential; GM-CSF administration (5 microg/Kg s.c./d x10) was planned after each chemotherapy cycle and after each HBI session in 18 consecutive patients (group A). Thirty-eight additional patients (group B) were treated by the same therapeutic program, without GM-CSF. In 12 patients (6 in each group) long-term bone marrow cultures (LTBMC) were performed to evaluate the myeloproliferative potential throughout the chemotherapeutic program. RESULTS: Seven of 18 (39%) patients experienced side effects from GM-CSF; 3/7 discontinued GM-CSF due to anaphylactic symptoms. The degree of neutropenia, as well as the frequency of infectious episodes and the need for supportive care were significantly lower in group A than in group B. Iatrogenic thrombocytopenia, and the possibility of performing drug-dose escalation were similar in the two groups. The 5-year event-free survival probabilities for group A and B were similar. LTBMC showed that the chemotherapy-related depletion of myeloid precursors could be more pronounced in patients receiving GM-CSF cyclically. INTERPRETATION AND CONCLUSIONS: In this series, GM-CSF was shown to be effective on iatrogenic neutropenia and related complications, with no impact on thrombopoiesis, drug dose escalation and outcome.

Exacerbation of acute inflammatory arthritis by the colony-stimulating factors CSF-1 and granulocyte macrophage (GM)-CSF: evidence of macrophage infiltration and local proliferation.

CSF-1 and GM-CSF have been implicated in the pathogenesis of rheumatoid arthritis. We report the effects of CSF-1 and GM-CSF in the development of an acute methylated bovine serum albumin (mBSA)-induced murine arthritis model. Examination of histopathological features revealed that the systemic administration of CSF-1 or GM-CSF following mBSA administration into the knee resulted in the exacerbation of arthritis. This included synovial hyperplasia and joint inflammation, most evident at 7 and 14 days post-mBSA administration, and the appearance of erosive pannus tissue. The exacerbation by CSF-1 and GM-CSF was not sustained but declined in incidence and severity by 21 days post-mBSA administration, similar to the effects of IL-1beta in this model, reported here and previously. Macrophages expressing Mac-2 and F4/80 were a prominent feature of the pathology observed, particularly the infiltration of Mac-2+ macrophages seen in all mice administered CSF-1, GM-CSF or IL-1beta. Present in inflamed knees was a locally dividing population of cells which included Mac-2+ and F4/80+ macrophages. These studies demonstrate that CSF-1 and GM-CSF can exacerbate and prolong the histopathology of acute inflammatory arthritis and lend support to monocytes/macrophages being a driving influence in the pathogenesis of inflammatory arthritis.

Prospective, randomized trial of sequential interleukin-3 and granulocyte- or granulocyte-macrophage colony-stimulating factor after standard-dose chemotherapy in cancer patients.

BACKGROUND AND OBJECTIVE: Several in vitro and animal studies have shown that IL-3 primes hematopoietic stem cells to become more sensitive to later acting growth factors. We wanted to compare the toxicity and the synergistic stimulatory effect of interleukin-3 (IL-3) followed by granulocyte colony-stimulating factor (G-CFS) or granulocyte-macrophage colony-stimulating factor (GM-CSF) on white blood cell (WBC) and platelet counts, after standard-dose chemotherapy (CT) in patients with solid tumors. DESIGN AND METHODS: Fifty consecutive cancer patients with thrombocytopenia and/or leukopenia registered during a previous course of CT were randomized to receive, after the following course, IL-3 (10 microg/kg/day, s.c., day 1-5) followed by G- or GM-CSF (5 microg/kg/day, day 6-8). RESULTS: The nadir of WBC in the cycles supported with the combination of IL-3 and G-CSF was significantly higher than that observed in the CT cycles not supported by growth factors (p < 0. 005). Furthermore, severe leukopenia was abrogated in all the cycles supported with IL-3+G-CSF, while in the cycles without cytokines, this event was registered in 62.5% of the cases (p < 0.0005). Finally, the recovery of WBC was achieved a mean of 4 days earlier in the cycles supported with IL-3+G-CSF. As for thrombocytoprotection, no significant differences were evidenced, but severe thrombocytopenia was abrogated in all the cycles supported by IL-3+G-CSF (p < 0.05). Furthermore, platelet recovery after CT was achieved on average 3.5 days earlier in the IL-3+G-CSF group than in the previous cycles. The nadir of WBC count in the cycles supported by the combination of IL-3 and GM-CSF was significantly higher than that observed in the CT cycles not supported by growth factors (p < 0.005). Furthermore, severe leukopenia was abrogated in 40% of the cycles supported by IL-3+GM-CSF, while in the cycles without cytokines, this event was registered in 80% of the cases (p < 0.005). Finally, the recovery of WBC was achieved a mean of 3.5 days earlier in the cycles supported by IL-3+GM-CSF. As far as thrombocytoprotection is concerned, there were no significant differences in the nadir between the cycles supported by the association IL-3+GM-CSF and the cycles not supported by cytokines. However, severe thrombocytopenia was registered in 20% of the cycles not supported by growth factors but in only 10% of the cycles supported by IL-3+GM-CSF (p < 0.05). Furthermore, platelet recovery after CT was achieved on average 3 days earlier in the IL-3+GM-CSF group. The combination of IL-3 and G-CSF would appear to be more effective than the combination of IL-3 and GM-CSF in the control of both severe thrombocytopenia and leukopenia. Indeed, severe leukopenia was abrogated in all the cycles in arm A, but only in 40% of the cycles in arm B (p < 0.0005). Furthermore, considering a platelet count below 49

Effects of CSFS and their combinations with chemotherapeutic agents (CH) on leukemic blasts (LB) in children (MTT-assay).

Thirty-five samples of bone marrow (BM) from 17 patients (pts) with ALL and 18 pts with AML (aged 9 m-20 yrs, median 7.7 yrs) were obtained. Using MTT-assay the sensitivity of LB to Ara-C, VP-16, DOX, G(GM)-CSF and their combinations was measured. LC50 was higher in pts with AML than with ALL: to Ara-C 1.94-fold (p < 0.05), to VP-16 1.62-fold (p = 0.2), to DOX 3.9-fold (p < 0.05). Incubation with G-CSF increased the viability of ALL and AML LB--104.3% and 104.1% respectively (the viability of leukemic cells without CSF accepted as 100%). Incubation with GM-CSF decreased the viability of ALL LB (96.5%) and increased the viability of AML LB (139.1%) (p = 0.08). Combining Ara-C with G- or GM-CSF resulted in equal or increased LC50 (compared with LC50 of Ara-C alone) in 100% cases of AML. For ALL: LC50 of "Ara-C+G-CSF" was equal or increased in 63.6% cases; LC50 of "Ara-C+GM-CSF"-in 62.5%. For VP-16 and DOX all pts (ALL, AML) except two had equal or increased LC50 of "CH+CSF" (compared with LC50 of CH alone). These data show: 1) AML LB were less sensitive to the investigated CH than ALL LB. 2) The LC50 of "CH+CSF" was equal or increased compared to the LC50 of CH for the absolute majority of cases with VP-16 and DOX. The same results were obtained with AML and in about 60% cases of ALL. The effect of the increasing of cytototoxity of CH in presence of CSF probably exists mostly at higher concentrations of CH than those that can be achieved in clinical practice.

Mobilization of blood-derived stem and progenitor cells in normal subjects by granulocyte-macrophage- and granulocyte-colony-stimulating factors.

BACKGROUND: It was previously reported that the combination of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) for 4 days mobilized more primitive CD34+ subsets than did either G-CSF or GM-CSF alone. STUDY DESIGN AND METHODS: The studies determine the optimal number of days of growth factor dosing for mobilization and collection of peripheral blood progenitor cells, by increasing the days of administration of GM-CSF and/or G-CSF or employing the sequential administration of GM-CSF followed by G-CSF. Sixty normal subjects were given injections of G-CSF or GM-CSF alone; GM-CSF and G-CSF concurrently for 4, 5, or 6 days; or a sequential regimen of GM-CSF for 3 or 4 days followed by G-CSF for 2 or 3 days. A 10-L apheresis was performed 24 hours after the last dose. RESULTS: The three most efficacious mobilization regimens consisted of sequential GM-CSF for 3 days followed by G-CSF for either 2 or 3 days and G-CSF alone for 5 days. Each of these regimens resulted in the collection of significantly greater numbers of CD34+ cells by apheresis than any of the 4-day dosing regimens with G-CSF and/or GM-CSF (sequential GM-CSF/G-CSF: 3 days/2 days = 3.58 +/- 0.53 x 106 CD34+ cells/kg; GM-CSF/G-CSF: 3 days/3 days = 4.45 +/- 1.08 x 10(6) CD34+ cells/kg; G-CSF: 5 days = 3.58 +/- 0.97 x 10(6) CD34+ cells/kg; all p<0.05 vs. G-CSF and/or GM-CSF for 4 days). Clonogenic assays generally paralleled the level of CD34+ cells. Regimens containing GM-CSF resulted in a higher percentage of the cells from primitive CD34+/CD38-/HLA-DR+ subset than G-CSF alone. CONCLUSION: Compared with 4-day dosing regimens with G-CSF and/or GM-CSF, mobilization of CD34+ cells in normal subjects using sequential GM-CSF for 3 days followed by G-CSF for 2 or 3 days or using G-CSF alone for 5 days increased the number CD34+ cells that can be collected by a single 10-L apheresis 24 hours after the last dose of cytokine.

Malignant progenitors from patients with acute myelogenous leukemia are sensitive to a diphtheria toxin-granulocyte-macrophage colony-stimulating factor fusion protein.

We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 x 10(-12) mol/L (range, 5 to >4,000 x 10(-12) mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing > or = 35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 x 10(-9) mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.

Toxicology and pharmacokinetics of DT388-GM-CSF, a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF) in C57BL/6 mice.

Because the majority of acute myeloid leukemia (AML) blasts express the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, we are developing a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human GM-CSF for multi-drug resistant AML. Our goal was to determine the toxicity and pharmacokinetics of DT388-GM-CSF in C57BL/6 mice. Because human GM-CSF does not cross-react with the mouse GM-CSF receptor, the toxicity observed should be nonspecific toxicity of DT388. We injected C57BL/6 mice i.p. with 0.1, 0.5, 1.0, 1.5, 1.75, 2.0, 3.5, 5.0, or 10 micrograms/day of DT388-GM-CSF for 5 consecutive days. For pharmacokinetics, blood samples were drawn at 20, 40, 60, 120, and 180 min after i.p. administration of 81 micrograms/kg of DT388-GM-CSF. In mice, the LD10 of DT388-GM-CSF is between 84.4 (1.5) and 104.4 (1.75) micrograms/kg (microgram) when administered for 5 consecutive days. All mice receiving > or = 201 micrograms/kg (3.5 micrograms) for 5 consecutive days died. Histopathologic examination of morbid animals showed only renal toxicity with acute proximal tubular necrosis. DT388-GM-CSF is stable in vivo based on nonreducing SDS-PAGE gel of plasma samples of 125I-labeled DT388-GM-CSF injected i.p.. The peak concentration of DT388-GM-CSF was 3.3 x 10(-8) M at 40 min and exhibited a t1/2 of 24 min. Based on its half-life, DT388-GM-CSF concentrations in the plasma are above the concentration inhibiting 50% protein synthesis and inducing apoptosis in 50% of HL-60 cells (AML cell line) for 5.2 h. Only four of 17 mice developed a weak immune response (0.9-160 ng/mL) 3 weeks after treatment. DT388-GM-CSF exhibits a short t1/2, but concentrations exceed those required in vitro to inhibit AML cell lines.

In vivo targeting of leukemic cells using diphtheria toxin fused to murine GM-CSF.

We have previously demonstrated that diphtheria toxin (DT) fused to human GM-CSF effectively eliminates human long-term leukemia initiating cells in SCID mice. However, because huGM-CSF does not react with the murine GM-CSF receptor possible side-effects to nonleukemic tissues could not be analyzed in the AML/SCID model. To overcome this problem, we used murine GM-CSF fused to DT and studied the therapeutic index in the rat leukemia model BNML/LT12. In DT-mGM-CSF dose escalation experiments, severe dose-dependent toxicity to organs such as liver, kidney and lung was observed. Therefore, the antileukemic effects were evaluated with the lower doses. Daily intraperitoneal bolus injections of 75 microg/kg/day for 7 days induced a 3 log leukemic cell kill. The dose of 75 microg/kg/day had no effect on the hemopoietic progenitor cell subsets. These in vivo studies show that the DT-GM-CSF fusion protein can be used for specifically targeting leukemic cells and thus has potential as a therapeutic agent in the treatment of AML.