Gary L. Griffiths

Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells

BackgroundEffective transvascular delivery of nanoparticle-based chemotherapeutics across the blood-brain tumor barrier of malignant gliomas remains a challenge. This is due to our limited understanding of nanoparticle properties in relation to the physiologic size of pores within the blood-brain tumor barrier. Polyamidoamine dendrimers are particularly small multigenerational nanoparticles with uniform sizes within each generation. Dendrimer sizes increase by only 1 to 2 nm with each successive generation. Using functionalized polyamidoamine dendrimer generations 1 through 8, we investigated how nanoparticle size influences particle accumulation within malignant glioma cells.MethodsMagnetic resonance and fluorescence imaging probes were conjugated to the dendrimer terminal amines. Functionalized dendrimers were administered intravenously to rodents with orthotopically grown malignant gliomas. Transvascular transport and accumulation of the nanoparticles in brain tumor tissue was measured in vivo with dynamic contrast-enhanced magnetic resonance imaging. Localization of the nanoparticles within glioma cells was confirmed ex vivo with fluorescence imaging.ResultsWe found that the intravenously administered functionalized dendrimers less than approximately 11.7 to 11.9 nm in diameter were able to traverse pores of the blood-brain tumor barrier of RG-2 malignant gliomas, while larger ones could not. Of the permeable functionalized dendrimer generations, those that possessed long blood half-lives could accumulate within glioma cells.ConclusionThe therapeutically relevant upper limit of blood-brain tumor barrier pore size is approximately 11.7 to 11.9 nm. Therefore, effective transvascular drug delivery into malignant glioma cells can be accomplished by using nanoparticles that are smaller than 11.7 to 11.9 nm in diameter and possess long blood half-lives.

Anti-CD74 Antibody-Doxorubicin Conjugate, IMMU-110, in a Human Multiple Myeloma Xenograft and in Monkeys

Purpose: IMMU-110 is a drug immunoconjugate composed of doxorubicin conjugated to the humanized anti-CD74 monoclonal antibody, hLL1, at a doxorubicin/monoclonal antibody ratio of ∼8:1 (mol/mol). CD74 is a rapidly internalizing molecule associated with HLA-DR, which has high expression by several tumor types. Here, we describe safety evaluations of IMMU-110 in mice and monkeys as well as efficacy studies in a xenograft model of the human multiple myeloma cell line, MC/CAR. Experimental Design:In vitro binding of IMMU-110 was determined by a cell-based ELISA and cytotoxicity of IMMU-110 assayed with a tetrazolium assay. Pharmacokinetics and biodistribution of radiolabeled IMMU-110 were examined in tumor-free BALB/c mice, and the therapeutic effectiveness was evaluated in severe combined immunodeficient mice bearing MC/CAR cells. Acute toxicity of IMMU-110 was studied in CD74-positive cynomolgus monkeys (Macaca fascicularis). Results:In vitro, IMMU-110 specifically binds to CD74 and is cytotoxic against MC/CAR cells. In vivo, IMMU-110 displayed a pharmacokinetic and biodistribution profile identical to that of unconjugated hLL1 monoclonal antibody, except for higher kidney uptake. Treatment with a single dose of IMMU-110 as low as 50 μg antibody/mouse (or 1.4 μg doxorubicin/mouse), 5 days postinjection of the multiple myeloma cells, resulted in cure of most mice. In mice, no host toxicity of IMMU-110 was observed at the highest protein dose tested (125 mg/kg). In cynomolgus monkeys, bone marrow toxicity was observed at 30 and 90 mg/kg doses. Conclusions: The excellent safety and efficacy profile of IMMU-110 supports clinical testing of this immunoconjugate in the treatment of CD74-positive B-cell malignancies.

Physiologic upper limit of pore size in the blood-tumor barrier of malignant solid tumors

BackgroundThe existence of large pores in the blood-tumor barrier (BTB) of malignant solid tumor microvasculature makes the blood-tumor barrier more permeable to macromolecules than the endothelial barrier of most normal tissue microvasculature. The BTB of malignant solid tumors growing outside the brain, in peripheral tissues, is more permeable than that of similar tumors growing inside the brain. This has been previously attributed to the larger anatomic sizes of the pores within the BTB of peripheral tumors. Since in the physiological state in vivo a fibrous glycocalyx layer coats the pores of the BTB, it is possible that the effective physiologic pore size in the BTB of brain tumors and peripheral tumors is similar. If this were the case, then the higher permeability of the BTB of peripheral tumor would be attributable to the presence of a greater number of pores in the BTB of peripheral tumors. In this study, we probed in vivo the upper limit of pore size in the BTB of rodent malignant gliomas grown inside the brain, the orthotopic site, as well as outside the brain in temporalis skeletal muscle, the ectopic site.MethodsGeneration 5 (G5) through generation 8 (G8) polyamidoamine dendrimers were labeled with gadolinium (Gd)-diethyltriaminepentaacetic acid, an anionic MRI contrast agent. The respective Gd-dendrimer generations were visualized in vitro by scanning transmission electron microscopy. Following intravenous infusion of the respective Gd-dendrimer generations (Gd-G5, N = 6; Gd-G6, N = 6; Gd-G7, N = 5; Gd-G8, N = 5) the blood and tumor tissue pharmacokinetics of the Gd-dendrimer generations were visualized in vivo over 600 to 700 minutes by dynamic contrast-enhanced MRI. One additional animal was imaged in each Gd-dendrimer generation group for 175 minutes under continuous anesthesia for the creation of voxel-by-voxel Gd concentration maps.ResultsThe estimated diameters of Gd-G7 dendrimers were 11 ± 1 nm and those of Gd-G8 dendrimers were 13 ± 1 nm. The BTB of ectopic RG-2 gliomas was more permeable than the BTB of orthotopic RG-2 gliomas to all Gd-dendrimer generations except for Gd-G8. The BTB of both ectopic RG-2 gliomas and orthotopic RG-2 gliomas was not permeable to Gd-G8 dendrimers.ConclusionThe physiologic upper limit of pore size in the BTB of malignant solid tumor microvasculature is approximately 12 nanometers. In the physiologic state in vivo the luminal fibrous glycocalyx of the BTB of malignant brain tumor and peripheral tumors is the primary impediment to the effective transvascular transport of particles across the BTB of malignant solid tumor microvasculature independent of tumor host site. The higher permeability of malignant peripheral tumor microvasculature to macromolecules smaller than approximately 12 nm in diameter is attributable to the presence of a greater number of pores underlying the glycocalyx of the BTB of malignant peripheral tumor microvasculature.

Processing of antibody-radioisotope conjugates after binding to the surface of tumor cells

Background. Previous experiments indicated that most antibodies binding to cell surface antigens are internalized gradually and degraded within lysosomes, with a half‐life of degradation of approximately 1 day, for most antibodies. The research discussed in this article extended our studies to eight additional antibodies reacting with six different antigens, including three antigens anchored in the membrane by glycosyl‐phosphatidylinositol. The authors also tested antibodies labeled with 111indium, as well as 125iodine, to determine whether different radiolabels would be processed differently.

Cytotoxicity with Auger electron‐emitting radionuclides delivered by antibodies

We investigated the in vitro cytotoxic potential of Auger electron‐emitting radionuclides delivered to the cytoplasm or, more specifically, to lysosomes, via antibodies. The antibody (Ab) used was LL1, which is specific for CD74, an epitope of the major histocompatibility complex (MHC) class II antigen invariant chain, Ii, present on the cell surface. It is taken up in large amounts, approximately 107 Ab molecules per cell per day, and delivered to lysosomes. The radioisotopes tested included 111In, 99mTc and 125I. With sufficient specific activity, approximately 10 mCi/mg Ab, all of these isotopes were potent cytotoxic agents. 125I was active only if a “residualizing” form was used, meaning a form that is trapped within cells after catabolism of the Ab to which it was conjugated (conventional oxidative iodination produces a non‐residualizing label). The conjugates of 111In and 99mTc used are known to be residualizing. One hundred percent cell kill in vitro was obtained with 111In and 125I, under conditions in which a non‐reactive control Ab, conjugated in the same way, produced no significant toxicity. 99mTc was also potent and specific, but appeared somewhat less active than the other isotopes under the conditions evaluated. Although few Abs are accreted by cells at the same rate as LL1, it may be possible to use other Abs to deliver similar amounts of radioactivity, if Abs with higher specific activity can be produced. Such conjugated radioisotopes may be useful for attacking tumor cells in vivo, particularly for single cells or micrometastases. Int. J. Cancer 81:985–992, 1999.

Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22

Abstract LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin’s lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (111In). The biodistribution of a mixture of 125I (non-residualizing chloramine-T compared to residualizing DLT), 111In-labeled LL2 murine IgG2a or its fragments [F(ab′)2, Fab′], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., 111In and DLT-125I) than with the non-residualizing iodine label. For example, tumor/blood ratios of 111In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing 111In-labeled LL2 for diagnosis, and residualizing 131I and 90Y labels for therapy.

Overcoming the nephrotoxicity of radiometal-labeled immunoconjugates†

Elevated renal uptake and extended retention of radiolabeled antibody fragments and peptides is a problem in the therapeutic application of such agents. However, cationic amino acids have been shown to reduce renal accretion. The aims of the current study were to evaluate whether this methodology would benefit therapy with yttrium 90 (90Y)‐labeled antibody fragments (Fab, F(ab)2), to establish the relationship between radiation dosimetry and observed biologic effects, and to compare the antitumor efficacy of antibody fragments with that of whole immunoglobulin (Ig)G.

Brain redox imaging using blood–brain barrier-permeable nitroxide MRI contrast agent

Reactive oxygen species (ROS) and compromised antioxidant defense may contribute to brain disorders such as stroke, amyotrophic lateral sclerosis, etc. Nitroxides are redox-sensitive paramagnetic contrast agents and antioxidants. The ability of a blood—brain barrier (BBB)-permeable nitroxide, methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (MC-P), as a magnetic resonance-imaging (MRI) contrast agent for brain tissue redox imaging was tested. MC-P relaxation in rodent brain was quantified by MRI using a fast Look-Locker T1-mapping sequence. In the cerebral cortex and thalamus, the MRI signal intensity increased up to 50% after MC-P injection, but increased only by 2.7% when a BBB-impermeable nitroxide, 3CxP (3-carboxy-2,2,5,5,5-tetramethylpyrrolidine-1-oxyl) was used. The maximum concentrations in the thalamus and cerebral cortex after MC-P injection were calculated to be 1.9±0.35 and 3.0±0.50 mmol/L, respectively. These values were consistent with the ex vivo data of brain tissue and blood concentration obtained by electron paramagnetic resonance (EPR) spectroscopy. Also, reduction rates of MC-P were significantly decreased after reperfusion following transient MCAO (middle cerebral artery occlusion), a condition associated with changes in redox status resulting from oxidative damage. These results show the use of BBB-permeable nitroxides as MRI contrast agents and antioxidants to evaluate the role of ROS in neurologic diseases.

Mitochondrial NADH Fluorescence Is Enhanced by Complex I Binding

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10-fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.

Selection of radioimmunoconjugates for the therapy of well-established or micrometastatic colon carcinoma

In order to optimize radioimmunotherapy (RAIT) as a cancer‐treatment modality, it is necessary to select the appropriate radionuclide and antibody carrier. We evaluated the therapeutic potential of a single cycle of Mu‐9 anti‐CSAp monoclonal antibody (MAb) labeled with 3 different radionuclides, 131I, 90Y and 188Re. Intact antibodies and bivalent fragments with different blood clearance kinetics, normal organ distribution and varying tumor accretion and retention are also evaluated. Efficacy of treatment for large and small tumor burden was assessed in nude mice bearing s.c. GW‐39 human colonic‐carcinoma xenografts or intrapulmonary micrometastatic GW‐39 colonies at the maximal tolerated dose of each agent. The magnitude and duration of myelosuppression associated with each radioantibody was considered by monitoring peripheral blood counts, marrow colony‐forming unit activity and hematopoietic tissue weight. Radiation‐dose estimates were calculated based on the kinetics of antibody accretion and elimination from tumor and normal tissues, and the results were correlated with tumoricidal activity and dose‐limiting toxicity results. These studies, therefore, represent a detailed analysis, in a well‐defined experimental tumor system, of several parameters (antibody form, radioisotope, tumor size) influencing the overall outcome of RAIT using equitoxic doses. It was found that myelosuppression is the primary dose‐limiting toxicity for all radioantibodies except 90Y‐F(ab′)2, even though the different agents showed varied organ distribution. In a single‐cycle treatment schedule of Mu‐9 MAb, the 131I‐labeled IgG is the radioimmunoconjugate of choice for the treatment of s.c. and intrapulmonary growth of the GW‐39 human colonic‐carcinoma xenograft in nude mice. Int. J. Cancer 72:477–485, 1997.