Michael G. Rosenblum

In vitro and in vivo studies of a VEGF121/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors

Vascular endothelial growth factor (VEGF) plays a key role in the growth and metastasis of solid tumors. We generated a fusion protein containing VEGF121 linked by a flexible G4S tether to the toxin gelonin (rGel) and expressed this as a soluble protein in bacteria. Purified VEGF121/rGel migrated as an 84-kDa homodimer under nonreducing conditions. VEGF121/rGel bound to purified, immobilized Flk-1, and the binding was competed by VEGF121. Both VEGF121/rGel and VEGF121 stimulated cellular kinase insert domain receptor (KDR) phosphorylation. The VEGF121/rGel fusion construct was highly cytotoxic to endothelial cells overexpressing the KDR/Flk-1 receptor. The IC50 of the construct on dividing endothelial cells expressing 105 or more KDR/Flk-1 receptors per cell was 0.5–1 nM, as compared with 300 nM for rGel itself. Dividing endothelial cells overexpressing KDR were approximately 60-fold more sensitive to VEGF121/rGel than were nondividing cells. Endothelial cells overexpressing FLT-1 were not sensitive to the fusion protein. Human melanoma (A-375) or human prostate (PC-3) xenografts treated with the fusion construct demonstrated a reduction in tumor volume to 16% of untreated controls. The fusion construct localized selectively to PC-3 tumor vessels and caused thrombotic damage to tumor vessels with extravasation of red blood cells into the tumor bed. These studies demonstrate the successful use of VEGF121/rGel fusion construct for the targeted destruction of tumor vasculature in vivo.

Phase I study of recombinant human interleukin-3 in patients with bone marrow failure.

Interleukin-3 (IL-3) is a T-cell-derived colony-stimulating factor (CSF) whose primary targets include relatively early, multipotential, hematopoietic progenitor cells. In this trial, we treated 24 patients with recombinant human IL-3 given by a daily 4-hour intravenous infusion for 28 days. The dose levels were 30, 60, 125, 250, 500, 750, and 1,000 micrograms/m2/d. At least three patients were entered at every dose level. Each participant suffered from bone marrow failure, with the underlying diagnosis being myelodysplastic syndrome (13 patients), aplastic anemia (eight patients), or aplasia after prolonged high-dose chemotherapy (three patients) for multiple myeloma, breast cancer, or acute myelogenous leukemia. Most patients tolerated therapy well, with the most frequent side effects being low-grade fever and headaches. Hematopoietic changes included modest increases in neutrophil counts (eight patients), eosinophil counts (six patients), platelet counts (three patients), and reticulocyte counts (two patients). An increase in blasts occurred in one patient who had refractory anemia with excess blasts in transformation and was reversible once IL-3 was discontinued. In addition, one patient with chronic myelomonocytic leukemia showed an increase in monocytes (and granulocytes). Progression to acute leukemia did not occur. Pharmacokinetic analyses showed a rapid clearance with a mean half-life of 18.8 minutes at the 60 micrograms/m2/d dose, and 52.9 minutes at the 250 micrograms/m2/d dose. Serum concentrations of 10 to 20 ng/mL of IL-3 were achievable at the 250 micrograms/m2/d dose. Our observations indicate that recombinant human IL-3 can be given safely at doses of 1,000 micrograms/m2/d or less. In addition, on the basis of preclinical data and the biologic activity observed in this study, further trials of this molecule, alone and in combination with other growth factors, are warranted in patients with pancytopenia.

Phase I trial of murine monoclonal antibody 14G2a administered by prolonged intravenous infusion in patients with neuroectodermal tumors.

PURPOSE The purpose of this phase I trial was to determine the toxicity and maximum-tolerated dose (MTD) of murine monoclonal antibody (Mab) 14G2a (anti-GD2) in cancer patients. PATIENTS AND METHODS Following tracer doses of iodine-131-labeled 14G2a to determine tumor uptake, 18 patients with refractory melanoma, neuroblastoma, or osteosarcoma received unlabeled 14G2a at total concentrations of 50, 100, and 200 mg/m2 administered as daily 24-hour infusions for 5 days. RESULTS The overall sensitivity of external immunoscintigraphy was 64 of 74 known metastases (86%). Toxicity from prolonged infusion of 14G2a consisted of severe generalized pain, hyponatremia, fever, rash, paresthesias, weakness, and chronic refractory postural hypotension (two patients). Toxicity was less severe in pediatric patients. The MTD of Mab was 100 mg/m2. Sixteen of 18 patients developed human antimouse antibodies (HAMA) to 14G2a. Terminal-phase half-life (T1/2) of unlabeled Mab was 6.6 +/- 1.8 hours for patients receiving 50 mg/m2 and 39.5 +/- 13.3 hours at the 100-mg/m2 level. Tumor biopsies from six melanoma patients were positive for GD2 antigen, but only two of six had trace amounts of 14G2a present. Three mixed responses (two melanoma, one osteosarcoma) and two partial responses (PRs; neuroblastoma) were observed. CONCLUSION Mab 14G2a has modest antitumor activity at the expense of significant toxicity. Dose-limiting neurologic sequelae may significantly limit phase II studies other than in pediatric patients with neuroblastoma.

Phase I trial of liposomal muramyl tripeptide phosphatidylethanolamine in cancer patients.

Twenty-eight evaluable patients with metastatic cancer refractory to standard therapy received escalating doses of muramyl tripeptide phosphatidylethanolamine (MTP-PE) (.05 to 12 mg/m2) in phosphatidylserine (PC):phosphatidylcholine (PS) liposomes (lipid:MTP-PE) ratio 250:1). Liposomal MTP-PE (L-MTP-PE) was infused over 1 hour twice weekly; doses were escalated within individual patients every 3 weeks as tolerated for a total treatment duration of 9 weeks. Routine clinical laboratory parameters, acute phase reactants and various immunologic tests were monitored at various time points during treatment. Toxicity was moderate (less than or equal to grade II) in 24 patients with chief side effects being chills (80% of patients), fever (70%), malaise (60%), and nausea (55%). In four patients L-MTP-PE treatment was deescalated due to severe malaise and recurrent fever higher than 38.8 degrees C. The maximum-tolerated dose (MTD) was 6 mg/m2. Significant (P less than .05) increases in WBC count, absolute granulocyte count, ceruloplasmin, beta 2-microglobulin, c-reactive protein, monocyte tumoricidal activity, and serum IL-1 beta were found. Significant decreases in serum cholesterol were also observed. Clearance of intravenously (iv)-infused technetium-99 (99mTc)-labeled liposomes containing MTP-PE in four patients was biphasic; gamma camera scans revealed uptake of radiolabel in liver, spleen, lung, nasopharynx, thyroid gland, and tumor (two patients). No objective tumor regression was seen. In view of its definite immunobiologic activity and lack of major toxicity, additional phase II and adjuvant trials of L-MTP-PE are warranted.

Imaging of VEGF receptor in a rat myocardial infarction model using PET.

Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factors receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject. Methods: In Sprague–Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3). Results: Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 ± 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 ± 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels. Conclusion: We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.

Tumor epidermal growth factor receptor studies in patients with non-small-cell lung cancer or head and neck cancer treated with monoclonal antibody RG 83852.

PURPOSE Tumor tyrosine kinase activity associated with the epidermal growth factor receptor (EGFR) and localization of anti-EGFR monoclonal antibody RG 83852 were studied in patients with non-small-cell lung cancer (NSCLC) and head and neck cancer. PATIENTS AND METHODS Fifteen patients were treated with escalating doses of RG 83852 by continuous intravenous infusion for 5 days. Fresh tumor specimens were obtained 24 hours after therapy in 10 patients (of whom five had a pretherapy sample taken). Tumor EGFR tyrosine kinase activity was determined in fresh tumor samples by autophosphorylation of EGFR isolated in immunocomplexes with RG 83852. Tumor EGFR saturation with RG 83852 was assessed semiquantitatively by comparing the EGFR tyrosine kinase activity in immunocomplexes of tumor specimens obtained after therapy with total EGFR tyrosine kinase activity assessed by exogenous addition of RG 83852 to tumor lysates. Modulation of EGFR tyrosine kinase activity after the administration of RG 83852 was assessed by comparing EGFR tyrosine kinase activity from the same malignant lesion obtained before and after therapy. Tumor localization of RG 83852 and EGFR saturation were also assessed by immunohistochemistry. RESULTS No significant side effects were observed up to a total dose of 600 mg/m2. Based on tyrosine kinase activity, a high degree of EGFR saturation (> or = 50%) was observed at doses > or = 200 mg/m2, and EGFR saturation was estimated to be 100% at a dose level of 600 mg/m2 both in tumor tissue and skin used as surrogate EGFR-positive tissue. Immunohistochemistry studies showed that RG 83852 localized in tumor tissue and skin, but not in stroma, at doses > or = 400 mg/m2, and high EGFR saturation was observed at 600 mg/m2. Tumor EGFR tyrosine kinase activity was studied in five patients (four with EGFR-positive tumors) before and 24 hours post-therapy; a threefold to fourfold upregulation of EGFR tyrosine kinase activity in posttherapy specimens was observed in two patients. Moderate upregulation of EGFR itself was suggested in both of these patients and in two additional patients by immunohistochemistry. CONCLUSION RG 83852 causes no toxic effects at doses that result in high tumor EGFR saturation. Treatment with RG 83852 may enhance EGFR tyrosine kinase activity and/or EGFR expression. Because high EGFR expression by tumors has been associated with increased sensitivity to cytotoxic therapy, the suggestion of antibody-mediated upregulation of EGFR by agents such as RG 83852 may prove useful in enhancing chemotherapeutic efficacy.

Influence of lipoproteins on renal cytotoxicity and antifungal activity of amphotericin B.

We examined the influence of high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs) on the toxicity of amphotericin B (AmpB) to fungal and renal cells. Candida albicans was incubated for 18 h at 37 degrees C with AmpB and deoxycholate (Fungizone) or liposomal AmpB (L-AmpB) (0.1 to 2.0 micrograms of AmpB per ml) in the presence or absence of HDLs or LDLs (0.5 mg of protein per ml). The MICs of AmpB and L-AmpB, whether or not HDLs or LDLs were present, were similar. LLC PK1 renal cells, derived from primary cultures of pig proximal tubular cells, were incubated for 18 h at 37 degrees C in serum-free medium that contained AmpB and deoxycholate or L-AmpB at 20 micrograms of AmpB per ml, HDLs or LDLs at 0.5 mg of protein per ml, mixtures of AmpB with HDLs or LDLs, and mixtures of L-AmpB with HDLs or LDLs. HDL-associated AmpB was less toxic than AmB to LLC PK1 cells (53.0% +/- 2.5% versus 81.3% +/- 3.6% cytotoxicity; P = 0.01), while LDL-associated AmpB was as toxic as AmpB. L-AmpB, HDL-associated L-AmpB, and LDL-associated L-AmpB were less toxic to LLC PK1 cells than was AmpB (48.3% +/- 1.5%, 25.5% +/- 2.2%, and 52.2% +/- 2.5% versus 81.3% +/- 3.6% cytotoxicity; P = 0.02). To further understand why HDL-associated AmpB reduced renal cytotoxic effects, the LLC PK1 cells were examined for the presence of HDL and LDL receptors. LLC PK1 cells expressed high-affinity (K(d) = 0.0538 nanograms/ml; 96,000 sites per cell) and low-affinity (K(d) = 222.22 nanograms/ml; 77 sites per cell) LDL receptors but only a low-affinity HDL receptor (K(d) = 71.43 nanograms/ml; 2 sites per cell). HDL-associated AmpB and LDL-associated AmpB were less toxic than AmpB to trypsinized LLC PK1 cells (46.6% +/- 10.9% and 16.8% +/- 15.98% versus 74.7% +/- 7.7% cytotoxicity; P = 0.02). HDL-associated AmB and LDL-associated L-AmpB were also less toxic than AmpB to the cells (20.4% +/- 6.2% and 13.5% +/- 8.6% versus 74.7% cytotoxicity; P = 0.01). The antifungal activities of AmpB and L-AmpB were not altered in the presence of HDLs or LDLs. We conclude that the reduced nephrotoxicity associated with the use of L-AmpB is related to a decreased uptake of AmpB by renal cells when AmpB is associated with HDLs because of the low level of expression of HDL receptors in these cells.

Noninvasive monitoring of orthotopic glioblastoma therapy response using RGD-conjugated iron oxide nanoparticles

Noninvasive imaging techniques have been considered important strategies in the clinic to monitor tumor early response to therapy. In the present study, we applied RGD peptides conjugated to iron oxide nanoparticles (IONP-RGD) as contrast agents in magnetic resonance imaging (MRI) to noninvasively monitor the response of a vascular disrupting agent VEGF(121)/rGel in an orthotopic glioblastoma model. RGD peptides were firstly coupled to IONPs coated with a crosslinked PEGylated amphiphilic triblock copolymer. In vitro binding assays confirmed that cellular uptake of particles was mainly dependent on the interaction between RGD and integrin α(v)β(3) of human umbilical vein endothelial cells (HUVEC). The tumor targeting of IONP-RGD was observed in an orthotopic U87 glioblastoma model. Finally, noninvasive monitoring of the tumor response to VEGF(121)/rGel therapy at early stages of treatment was successfully accomplished using IONP-RGD as a contrast agent for MRI, a superior method over common anatomical approaches which are based on tumor size measurements. This preclinical study can accelerate anticancer drug development and promote clinical translation of nanoprobes.

Detecting and Treating Cancer with Nanotechnology

Nanotechnology offers many opportunities for enhanced diagnostic and therapeutic medicine against cancer and other diseases. In this review, the special properties that result from the nanoscale size of quantum dots, metal colloids, superparamagnetic iron oxide, and carbon-based nanostructures are reviewed and interpreted against a background of the structural and electronic detail that gives rise to their nanotechnologic behavior. The detection and treatment of cancer is emphasized, with special attention paid to the biologic targeting of the disease. The future of nanotechnology in cancer research and clinical practice is projected to focus on ‘theranostic’ nanoparticles that are both diagnostic and therapeutic by design.

Development and characterization of a potent immunoconjugate targeting the Fn14 receptor on solid tumor cells.

TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor (FGF)-inducible 14 (Fn14) are a TNF superfamily ligand–receptor pair involved in many cellular processes including proliferation, migration, differentiation, inflammation, and angiogenesis. The Fn14 receptor is expressed at relatively low levels in normal tissues, but it is known to be dramatically elevated in a number of tumor types, including brain and breast tumors. Thus, it seems to be an excellent candidate for therapeutic intervention. We first analyzed Fn14 expression in human tumor cell lines. Fn14 was expressed in a variety of lines including breast, brain, bladder, skin, lung, ovarian, pancreatic, colon, prostate, and cervical cancer cell lines. We then developed an immunoconjugate containing a high-affinity anti-Fn14 monoclonal antibody (ITEM-4) conjugated to recombinant gelonin (rGel), a highly cytotoxic ribosome-inactivating N-glycosidase. Both ITEM-4 and the conjugate were found to bind to cells to an equivalent extent. Confocal microscopic analysis showed that ITEM4-rGel specifically and rapidly (within 2 hours) internalized into Fn14-positive T-24 bladder cancer cells but not into Fn14-deficient mouse embryonic fibroblasts. Cytotoxicity studies against 22 different tumor cell lines showed that ITEM4-rGel was highly cytotoxic to Fn14-expressing cells and was 8- to 8 × 104-fold more potent than free rGel. ITEM4-rGel was found to kill cells by inducing apoptosis with high-mobility group box 1 protein release. Finally, ITEM4-rGel immunoconjugate administration promoted long-term tumor growth suppression in nude mice bearing T-24 human bladder cancer cell xenografts. Our data support the use of an antibody–drug conjugate approach to selectively target and inhibit the growth of Fn14-expressing tumors. Mol Cancer Ther; 10(7); 1276–88. ©2011 AACR.