Raymond M. Reilly

Problems of Delivery of Monoclonal Antibodies Pharmaceutical and Pharmacokinetic Solutions

SummaryMonoclonal antibodies to tumour-associated antigens have great theoretical potential for the specific targeting of radioactivity and anti-neoplastic agents to tumours. The clinical success of monoclonal antibody—based cancer diagnosis and therapy depends, however, on solving a number of pharmacokinetic delivery problems. These include: (i) slow elimination of monoclonal antibodies from the blood and poor vascular permeability; (ii) low and heterogeneous tumour uptake; (iii) cross-reactivity with normal tissues; (iv) metabolism of monoclonal antibody conjugates; and (v) immunogenicity of murine forms in humans.As a result of extensive pharmaceutical and pharmacokinetic research conducted over the past 10 to 15 years, several potential solutions to these delivery problems have been identified. Blood concentrations of antibody conjugates may be reduced through regional administration, the use of antibody fragments, interventional strategies and various pre-targeting techniques. Tumour uptake may be increased through administration of higher doses, or the use of agents to increase tumour vascular permeability. Tumour retention of antibody conjugates may be improved by inhibition of metabolism, by using more stable linkage chemistry. Alternatively, normal tissue retention may be decreased through the use of metabolisable chemical linkages inserted between the antibody and conjugated moiety.Very small antigen-binding fragments and peptides that exhibit improved tumour penetration and more rapid elimination from the blood and normal tissues have been prepared by genetic engineering techniques. Chimeric (mouse/human) and human monoclonal antibodies have been developed to circumvent the problem of immunogenicity. Future research will continue to be focused on improvements in the design of monoclonal antibodies for tumour targeting, with the ultimate goal of finally uncovering the ‘magic bullet’ envisioned by Paul Ehrlich almost a century ago.

111In-Labeled Trastuzumab (Herceptin) Modified with Nuclear Localization Sequences (NLS): An Auger Electron-Emitting Radiotherapeutic Agent for HER2/neu-Amplified Breast Cancer

The cytotoxicity and tumor-targeting properties of the anti-HER2/neu monoclonal antibody trastuzumab modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localization sequence ([NLS] italicized) of simian virus 40 large T-antigen and radiolabeled with 111In were evaluated. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) for reaction with NLS-peptides and labeled with 111In using diethylenetriaminepentaacetic acid (DTPA). The immunoreactivity of 111In-NLS-trastuzumab was determined by its ability to displace the binding of trastuzumab to SK-BR-3 human breast cancer (BC) cells. Cellular uptake and nuclear localization were evaluated in SK-BR-3, MDA-MB-361, and MDA-MB-231 BC cells, expressing high, intermediate, or very low levels of HER2/neu, respectively, by cell fractionation and confocal microscopy. Biodistribution and nuclear uptake were compared in athymic mice bearing MDA-MB-361 xenografts. The cytotoxicity of 111In-trastuzumab and 111In-NLS-trastuzumab was studied by clonogenic assays, and DNA damage was assessed by probing for phosphorylated histone H2AX (γH2AX) foci. Results: The dissociation constant for binding of 111In-NLS-trastuzumab to SK-BR-3 cells was reduced <3-fold compared with that of 111In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The receptor-mediated internalization of 111In-trastuzumab in SK-BR-3, MDA-MB-361, and MDA-MB-231 cells increased significantly from 7.2% ± 0.9%, 1.3% ± 0.1%, and 0.2% ± 0.05% to 14.4% ± 1.8%, 6.3% ± 0.2%, and 0.9% ± 0.2% for 111In-NLS-trastuzumab harboring 6 NLS-peptides, respectively. NLS-trastuzumab localized in the nuclei of BC cells, whereas unmodified trastuzumab remained surface-bound. Conjugation of 111In-trastuzumab to NLS-peptides did not affect its tissue biodistribution but promoted specific nuclear uptake in MDA-MB-361 xenografts (2.4–2.9 %ID/g [percentage injected dose per gram] for 111In-NLS-trastuzumab and 1.1 %ID/g for 111In-trastuzumab). 111In-NLS-trastuzumab was 5- and 2-fold more potent at killing SK-BR-3 and MDA-MB-361 cells than 111In-trastuzumab, respectively, whereas toxicity toward MDA-MB-231 cells was minimal. 111In-NLS-trastuzumab was 6-fold more effective at killing SK-BR-3 cells than unlabeled trastuzumab. Formation of γH2AX foci occurred in a greater proportion of BC cells after incubation with 111In-NLS-trastuzumab compared with 111In-trastuzumab or unlabeled trastuzumab. Conclusion: NLS-peptides routed 111In-trastuzumab to the nucleus of HER2/neu-positive human BC cells, rendering the radiopharmaceutical lethal to the cells through the emission of nanometer−micrometer range Auger electrons. The greater cytotoxic potency of 111In-NLS-trastuzumab compared with unlabeled trastuzumab in vitro and its favorable tumor-targeting properties in vivo suggest that it could be an effective targeted radiotherapeutic agent for HER2/neu-amplified BC in humans.

Implications on clinical scenario of gold nanoparticle radiosensitization in regards to photon energy, nanoparticle size, concentration and location

Gold nanoparticle (AuNP) radiosensitization represents a novel approach to enhance the effectiveness of ionizing radiation. Its efficiency varies widely with photon source energy and AuNP size, concentration, and intracellular localization. In this Monte Carlo study we explored the effects of those parameters to define the optimal clinical use of AuNPs. Photon sources included (103)Pd and (125)I brachytherapy seeds; (169)Yb, (192)Ir high dose rate sources, and external beam sources 300 kVp and 6 MV. AuNP sizes were 1.9, 5, 30, and 100 nm. We observed a 10(3) increase in the rate of photoelectric absorption using (125)I compared to 6 MV. For a (125)I source, to double the dose requires concentrations of 5.33-6.26 mg g(-1) of Au or 7.10 × 10(4) 30 nm AuNPs per tumor cell. For 6 MV, concentrations of 1560-1760 mg g(-1) or 2.17 × 10(7) 30 nm AuNPs per cell are needed, which is not clinically achievable. Examining the proportion of energy transferred to escaping particles or internally absorbed in the nanoparticle suggests two clinical strategies: the first uses photon energies below the k-edge and takes advantage of the extremely localized Auger cascade. It requires small AuNPs conjugated to tumor targeted moieties and nuclear localizing sequences. The second, using photon sources above the k-edge, requires a higher gold concentration in the tumor region. In this approach, energy deposited by photoelectrons is the main contribution to radiosensitization; AuNP size and cellular localization are less relevant.

Oral delivery of antibodies : Future pharmacokinetic trends

SummaryAntibodies have been investigated as specific targeting agents for cancer diagnosis and therapy, to inactivate toxic substances including drugs and also as passive immunotherapy for neoplastic or infectious diseases. In most cases the antibodies were administered systemically by the intravenous route. More recently, however, there has been increasing interest in the oral administration of antibodies for localised treatment of infections or other conditions in the gastrointestinal tract.The normal physiological handling of ingested proteins is degradation by proteases in the stomach and intestine into small peptides or amino acids which are subsequently absorbed. Proteolytic enzymes involved in the degradation of orally administered immunoglobulins include pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase. These enzymes initially degrade the antibodies to F(ab′)2, Fab and Fc fragments. The F(ab′)2 and Fab fragments, however, retain some of their neutralising activity locally in the gastrointestinal tract. Various approaches are possible to increase the stability of orally administered antibodies against proteolysis, including formulation in liposomes, coating with polymers and genetic engineering of resistant forms.The clinical application of orally administered antibodies includes the treatment and prevention of gastrointestinal infections caused by enteric pathogens such as rotavirus, Escherichia coli or Vibrio cholerae in susceptible individuals including those with immunodeficiency diseases and patients with bone marrow transplants. There is also a suggestion that such agents may be useful in preventing chemotherapy-induced gastrointestinal mucositis.Future opportunities for research include the design of oral dosage forms of antibodies which resist proteolysis and can deliver a greater fraction of immunoreactive antibody locally in the gastrointestinal tract for the treatment of infections or perhaps even to allow the absorption of antibodies for the treatment or prevention of systemic conditions.

Associations between the uptake of 111In-DTPA-trastuzumab, HER2 density and response to trastuzumab (Herceptin) in athymic mice bearing subcutaneous human tumour xenografts

PurposeThe purpose of the study was to investigate the associations between uptake of 111In-DTPA-trastuzumab, tumour HER2 density and response to trastuzumab (Herceptin) of human breast cancer (BC) xenografts in athymic mice.Materials and methodsThe tumour uptake of 111In-DTPA-trastuzumab in athymic mice bearing BC xenografts with increasing HER2 density (0 to 3+) was evaluated. Specific uptake ratios were established in biodistribution (SUR) and imaging studies (ROI-SUR) using 111In-labeled mouse IgG (111In-DTPA-mIgG). Further corrections were made for circulating radioactivity using tumour-to-blood ratios defined as a localization index (LI) and region-of-interest localization index (ROI-LI), respectively. Mice were treated with trastuzumab (Herceptin). A tumour growth inhibition index (TGI) was calculated and relative TGIs calculated by dividing the TGI of control by that of trastuzumab-treated mice.ResultsStrong, nonlinear associations with HER2 density were obtained if the uptake of 111In-DTPA-trastuzumab was corrected for nonspecific IgG localization (i.e., SUR; r2 = 0.99) and circulating radioactivity (i.e., LI; r2 = 0.87), but without these corrections, the association between HER2 density and tumour uptake was poor (r2 = 0.22). There was a strong association between ROI-SUR and ROI-LI values and HER2 expression (r2 = 0.90 and r2 = 0.95, respectively. All tumours were imaged. Relative TGI values were associated with increasing uncorrected tumour uptake of 111In-DTPA-trastuzumab but not always with HER2 density (i.e., MCF-HER2-18 cells with trastuzumab-resistance).ConclusionHER2 expression (0 to 3+) can be differentiated using 111In-DTPA-trastuzumab, but requires correction of tumour uptake for nonspecific IgG localization and circulating radioactivity. The uncorrected uptake of 111In-DTPA-trastuzumab was associated with tumour response to trastuzumab.

Early Radiation-Induced Endothelial Cell Loss and Blood–Spinal Cord Barrier Breakdown in the Rat Spinal Cord

Abstract Li, Y. Q., Chen, P., Jain, V., Reilly, R. M. and Wong, C. S. Early Radiation-Induced Endothelial Cell Loss and Blood–Spinal Cord Barrier Breakdown in the Rat Spinal Cord. Radiat. Res. 161, 143–152 (2004). Using a rat spinal cord model, this study was designed to characterize radiation-induced vascular endothelial cell loss and its relationship to early blood–brain barrier disruption in the central nervous system. Adult rats were given a single dose of 0, 2, 8, 19.5, 22, 30 or 50 Gy to the cervical spinal cord. At various times up to 2 weeks after irradiation, the spinal cord was processed for histological and immunohistochemical analysis. Radiation-induced apoptosis was assessed by morphology and TdT-mediated dUTP nick end labeling combined with immunohistochemical markers for endothelial and glial cells. Image analysis was performed to determine endothelial cell and microvessel density using immunohistochemistry with endothelial markers, namely endothelial barrier antigen, glucose transporter isoform 1, laminin and zonula occludens 1. Blood–spinal cord barrier permeability was assessed using immunohistochemistry for albumin and 99mTc-diethylenetriamine pentaacetic acid as a vascular tracer. Endothelial cell proliferation was assessed using in vivo BrdU labeling. During the first 24 h after irradiation, apoptotic endothelial cells were observed in the rat spinal cord. The decrease in endothelial cell density at 24 h after irradiation was associated with an increase in albumin immunostaining around microvessels. The decrease in the number of endothelial cells persisted for 7 days and recovery of endothelial density was apparent by day 14. A similar pattern of blood–spinal cord barrier disruption and recovery of permeability was observed over the 2 weeks, and an increase in BrdU-labeled endothelial cells was seen at day 3. These results are consistent with an association between endothelial cell death and acute blood–spinal cord barrier disruption in the rat spinal cord after irradiation.

In Vivo Distribution of Polymeric Nanoparticles at the Whole-Body, Tumor, and Cellular Levels

PurposeBlock copolymer micelles (BCMs) were functionalized with indium-111 and/or epidermal growth factor (EGF), which enabled investigation of the in vivo transport of passively and actively targeted BCMs. The integration of conventional and image-based techniques afforded novel quantitative means to achieve an in-depth insight into the fate of polymeric nanoparticles in vivo.MethodsPharmacokinetics and biodistribution studies were performed in athymic mice bearing human breast xenografts to evaluate the whole-body transport of NT-BCMs (non-targeted, EGF-) and T-BCMs (targeted, EGF+). The intratumoral distribution of BCMs was investigated using MicroSPECT/CT and autoradiographic imaging, complemented with quantitative MATLAB® analyses. Tumors were fractionated for quantifying intracellular uptake of BCMs via γ-counting.ResultsThe intratumoral distribution of NT-BCMs and T-BCMs were found to be heterogeneous, and positively correlated with tumor vascularization (r > 0.68 ± 0.04). The enhanced in vivo cell uptake and cell membrane binding of T-BCMs were found to delay their clearance from tumors overexpressing EGFR, and therefore resulted in enhanced tumor accumulation for the T-BCMs in comparison to the NT-BCMs.ConclusionsAdequate passive targeting is required in order to achieve effective active targeting. Tumor physiology has a significant impact on the transvascular and intratumoral transport of passively and actively targeted BCMs.

Design and characterization of HER-2-targeted gold nanoparticles for enhanced X-radiation treatment of locally advanced breast cancer.

Our purpose was to develop a human epidermal growth factor receptor-2 (HER-2) targeted nanotechnology-based radiosensitizer. HER-2 is overexpressed in 20-30% of all breast cancers and up to 2-fold higher in locally advanced disease (LABC). Trastuzumab was derivatized with a polyethylene glycol (OPSS-PEG-SVA) cross-linker to produce trastuzumab-PEG-OPSS. These immunoconjugates were analyzed by SDS-PAGE, and their immunoreactivity was assessed by flow cytometry using HER-2 overexpressing SK-BR-3 breast cancer cells. Reacting trastuzumab with increasing ratios of PEG resulted in an increase in molecular weight from approximately 148 kDa to 243 kDa, associated with increasing PEG substitution (0.6 to 18.9 PEG chains per trastuzumab). Attachment of approximately 7 PEG chains per trastuzumab resulted in 56% retention in immunoreactivity assessed by flow cytometry. The conjugates were then linked to 30 nm AuNPs. Using a novel (123)iodine-radiotracer based assay that overcomes the current limitations of spectrophotometric quantification of biological molecules on AuNPs we estimate 14.3 ± 2.7 antibodies were attached to each AuNP when 2 × 10(11) AuNPs were reacted with 20 μg of trastuzumab-PEG-OPSS. Specificity of trastuzumab-PEG-AuNPs for HER-2 and internalization in SK-BR-3 cells was demonstrated by comparing the uptake of trastuzumab-PEG-AuNPs or PEG-AuNPs by darkfield microscopy. The ability of trastuzumab-PEG-AuNPs in combination with 300 kVp X-rays to enhance DNA double strand breaks (DSBs) in SK-BR-3 cells was assessed by immunofluorescence using the γ-H2AX assay. γ-H2AX assay results revealed 5.1-fold higher DNA-DSBs with trastuzumab-PEG-AuNPs and X-radiation as compared to treatment with X-radiation alone. The trastuzumab-PEG-AuNPs are a promising targeted nanotechnology-based radiosensitizer for improving LABC therapy. The design and systematic approaches taken to surface modify and characterize trastuzumab-PEG-AuNPs described in this study would have application to other molecularly targeted AuNPs for cancer treatment.

Noninvasive monitoring of the fate of 111In-labeled block copolymer micelles by high resolution and high sensitivity microSPECT/CT imaging.

The validation of high sensitivity and high resolution microSPECT/CT imaging for tracking the in vivo pathway and fate of an 111Indium-labeled (111In) amphiphilic diblock copolymer micelle formulation is presented. Heterobifunctional poly(ethylene glycol) was used to initiate cationic ring opening polymerization of epsilon-caprolactone, which was then conjugated to 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA) for chelation with 111In. The micelles were characterized in terms of their physicochemical properties including size, size distribution, zeta-potential, and radiochemical purity. Elimination kinetics and tissue deposition were evaluated in healthy mice following administration of 111In-micelles, 111In-DTPA-b-PCL unimers (i.e., administered under the critical micelle concentration) or 111In-Bn-DTPA. Healthy and MDA-MB-231 tumor-bearing mice were imaged using microSPECT/CT following iv administration of 111In-micelles or 111In-Bn-DTPA. Overall, incorporation of 111In onto the surface of thermodynamically stable micelles results in long plasma residence times for the radionuclide and preferential localization within the spleen (22 +/- 5% i.d/g), liver (13 +/- 3% i.d./g), and tumor (9 +/- 2% i.d./g). MicroSPECT/CT imaging provided noninvasive longitudinal visualization of circulation dynamics and tissue deposition. A strong correlation between image-based region of interest (ROI) analysis and biodistribution data was found, implying that nuclear imaging can be used as a noninvasive tool to accurately quantify tissue distribution. As well, the image-based assessment provided unique insight into the intratumoral distribution of the micelles in vivo.

Carbon Nanotubes: Potential Benefits and Risks of Nanotechnology in Nuclear Medicine

Targeting of tumors with radionuclides for radiotherapeutic purposes is often limited by inadequate delivery to lesions using currently available targeting vehicles (e.g., monoclonal antibodies and peptides), relatively low and heterogeneous epitope/receptor expression on cancer cells, as well as dose-limiting toxicities to normal tissues (1). Nevertheless, there have been successes, particularly for radioimmunotherapy of non-Hodgkin’s B-cell lymphoma with 90Y-ibritumomab tiuxetan (Zevalin; Biogen Idec) or 131I-tositumomab (Bexxar; GlaxoSmithKline) (1). Notwithstanding these