Anti-transferrin Receptor Monoclonal Antibody Research Articles

Towards a translational physiologically-based pharmacokinetic (PBPK) model for receptor-mediated transcytosis of anti-transferrin receptor monoclonal antibodies in the central nervous system.

In this manuscript, we present a translational physiologically-based pharmacokinetic (PBPK) model to characterize receptor-mediated transcytosis (RMT) of anti-transferrin receptor (TfR) monoclonal antibodies (mAbs) in the central nervous system (CNS). The model accounts for the state-of-the-art knowledge of the brain's anatomy and physiology, and physiological parameters were fixed according to different species. By estimating a few parameters associated with the TfR concentration, the TfR turnover, and the internalization rate, the model simultaneously characterizes plasma, whole brain, interstitial fluid (ISF), and cerebrospinal fluid (CSF) PK of unbound and bound anti-TfR mAbs with different binding affinities in mice, rats, and monkeys obtained from various literature sources within a threefold prediction error. The final PBPK model was validated using external anti-TfR mAb PK data in mice and monkeys with different affinities and doses. The simulation reasonably predicted plasma and brain PK of monovalent/bivalent anti-TfR mAbs within a threefold prediction error and characterized a bell-shaped relationship between the brain ISF/plasma AUC ratio and the KD value. Although further refinements of the PBPK model and clinical validation are required, this PBPK model may provide physiologically-based translation of CNS disposition of anti-TfR mAbs by accounting for the physiological difference of the endogenous RMT system among different species. The PBPK model may also guide selection of other endogenous receptors, lead optimization, and clinical development of novel CNS-targeted mAbs.

Selenium nanoparticles for targeted stroke therapy through modulation of inflammatory and metabolic signaling

Ischemic cerebral stroke is a major cause of death and morbidity. Currently, no neuroprotective agents have been shown to impact the clinical outcomes in cerebral stroke cases. Here, we report therapeutic effects of Se nanoparticles on ischemic stroke in a murine model. Anti-transferrin receptor monoclonal antibody (OX26)-PEGylated Se nanoparticles (OX26-PEG-Se NPs) were designed and synthesized and their neuroprotective effects were measured using in vitro and in vivo approaches. We demonstrate that administration of the biodegradable nanoparticles leads to resolution of brain edema, protection of axons in hippocampus region, and myelination of hippocampal area after cerebral ischemic stroke. Our nanoparticle design ensures efficient targeting and minimal side effects. Hematological and biochemical analyses revealed no undesired NP-induced changes. To gain mechanistic insights into the therapeutic effects of these particles, we characterized the changes to the relevant inflammatory and metabolic signaling pathways. We assessed metabolic regulator mTOR and related signaling pathways such as hippo, Ubiquitin-proteasome system (ERK5), Tsc1/Tsc2 complex, FoxO1, wnt/β-catenine signaling pathway. Moreover, we examined the activity of jak2/stat3 signaling pathways and Adamts1, which are critically involved in inflammation. Together, our study provides a promising treatment strategy for cerebral stroke based on Se NP induced suppression of excessive inflammation and oxidative metabolism.

Targeted Delivery of Auristatin-Modified Toxins to Pancreatic Cancer Using Aptamers.

Pancreatic cancer is one of the most lethal malignancies. Treatment with the first-line agent, gemcitabine, is often unsuccessful because it, like other traditional chemotherapeutic agents, is non-specific, resulting in off-target effects that necessitate administration of subcurative doses. Alternatively, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are highly toxic small molecules that require ligand-targeted delivery. MMAE has already received FDA approval as a component of an anti-CD30 antibody-drug conjugate, brentuximab vedotin. However, in contrast to antibodies, aptamers have distinct advantages. They are chemicals, which allows them to be produced synthetically and facilitates the rapid development of diagnostics and therapeutics with clinical applicability. In addition, their small size allows for enhanced tissue distribution and rapid systemic clearance. Here, we assayed the toxicity of MMAE and MMAF conjugated to an anti-transferrin receptor aptamer, Waz, and an anti-epidermal growth factor receptor aptamer, E07, on the pancreatic cancer cell lines Panc-1, MIA PaCa-2, and BxPC3. In vitro, our results indicate that these aptamers are a viable option for the targeted delivery of toxic payloads to pancreatic cancer cells.

Resveratrol and Grape Extract-loaded Solid Lipid Nanoparticles for the Treatment of Alzheimer's Disease.

The aggregation of amyloid-β peptide (Aβ) has been linked to the formation of neuritic plaques, which are pathological hallmarks of Alzheimer’s disease (AD). Various natural compounds have been suggested as therapeutics for AD. Among these compounds, resveratrol has aroused great interest due to its neuroprotective characteristics. Here, we provide evidence that grape skin and grape seed extracts increase the inhibition effect on Aβ aggregation. However, after intravenous injection, resveratrol is rapidly metabolized into both glucuronic acid and sulfate conjugations of the phenolic groups in the liver and intestinal epithelial cells (within less than 2 h), which are then eliminated. In the present study, we show that solid lipid nanoparticles (SLNs) functionalized with an antibody, the anti-transferrin receptor monoclonal antibody (OX26 mAb), can work as a possible carrier to transport the extract to target the brain. Experiments on human brain-like endothelial cells show that the cellular uptake of the OX26 SLNs is substantially more efficient than that of normal SLNs and SLNs functionalized with an unspecific antibody. As a consequence, the transcytosis ability of these different SLNs is higher when functionalized with OX-26.

Identification of Stages of Erythroid Differentiation in Bone Marrow and Erythrocyte Subpopulations in Blood Circulation that Are Preferentially Lost in Autoimmune Hemolytic Anemia in Mouse.

Repeated weekly injections of rat erythrocytes produced autoimmune hemolytic anemia (AIHA) in C57BL/6 mice after 5–6 weeks. Using the double in vivo biotinylation (DIB) technique, recently developed in our laboratory, turnover of erythrocyte cohorts of different age groups during AIHA was monitored. Results indicate a significant decline in the proportion of reticulocytes, young and intermediate age groups of erythrocytes, but a significant increase in the proportion of old erythrocytes in blood circulation. Binding of the autoantibody was relatively higher to the young erythrocytes and higher levels of intracellular reactive oxygen species (ROS) were also seen in these cells. Erythropoietic activity in the bone marrows and the spleen of AIHA induced mice was examined by monitoring the relative proportion of erythroid cells at various stages of differentiation in these organs. Cells at different stages of differentiation were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Erythroid cells in bone marrow declined significantly in AIHA induced mice, erythroblast C being most affected (50% decline). Erythroblast C also recorded high intracellular ROS level along with increased levels of membrane-bound autoantibody. No such decline was observed in spleen. A model of AIHA has been proposed indicating that binding of autoantibodies may not be a sufficient condition for destruction of erythroid cells in bone marrow and in blood circulation. Last stage of erythropoietic differentiation in bone marrow and early stages of erythrocytes in blood circulation are specifically susceptible to removal in AIHA.

Use of PEGylated Immunoliposomes to Deliver Dopamine Across the Blood-Brain Barrier in a Rat Model of Parkinson's Disease.

To treat neurodegenerative disorders such as Parkinson's disease (PD), drugs must be able to cross the blood-brain barrier (BBB). Patients with PD are deficient in dopamine (DA), a neurotransmitter that cannot pass through the BBB. Liposomes modified by adding polyethylene glycol (PEGylated liposomes (PLs)) can be conjugated with antibody to form DA-PEGylated immunoliposomes (DA-PILs), and we tested their use as carriers of DA for treating PD. PEGylated liposomes (PLs) were prepared by evaporation method, and [(3) H]dopamine was encapsulated within the dried lipid film using a freeze/thaw cycle to form DA-PL. Thiolated OX26 MAb, an antitransferrin receptor monoclonal antibody, was then conjugated to 46-nm PEGylated liposomes. Particle size, zeta potential, and stability were assessed, and in vivo effects were determined after the intravenous injection of DA, DA-PL, and DA-PIL by examining brain tissue in normal rats and rats that underwent transection of the medial forebrain bundle to induce PD. The uptake of DA-PIL in the brains of this PD rat model increased about 8-fold compared with that of DA alone and about 3-fold compared with that of encapsulated DA-PEGylated liposomes (DA-PL). The volume of distribution of DA-PIL in the brain by the perfusion method was 4-fold higher than that of DA-PL, indicating that conjugation of OX26 MAb to the transferrin receptor of brain capillary endothelium mediated the effective delivery of DA to brain tissue. Dopamine can be effectively delivered to the brain by means of a PIL-based drug delivery system in PD rats.

Cellular uptake of PLGA nanoparticles targeted with anti-amyloid and anti-transferrin receptor antibodies for Alzheimer’s disease treatment

During the last few decades, relevant efforts have been reported to design nanocarriers for drug transport through the blood brain barrier (BBB). New drugs, such as peptide iAβ5, capable to inhibit the aggregates associated with Alzheimeŕs disease (AD) are being tested but the most frequent drawback is to reach the brain in the desired concentrations due to the low BBB permeability-surface area. Our approach, as a proof of concept to improve drug transport through the BBB, is based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles with surface functionalized with anti-transferrin receptor monoclonal antibody (OX26) and anti-Aβ (DE2B4) to deliver encapsulated iAβ5 into the brain. Porcine brain capillary endothelial cells (PBCECs) were used as a BBB model to evaluate the system efficacy and toxicity. The uptake of immune nanoparticles with a controlled delivery of the peptide iAβ5 was substantially increased compared to the nanoparticles (NPs) without monoclonal antibody functionalization.

Preferential Elimination of Older Erythrocytes in Circulation and Depressed Bone Marrow Erythropoietic Activity Contribute to Cadmium Induced Anemia in Mice.

Feeding cadmium chloride (50 or 1000 ppm CdCl2 in drinking water, ad libitum) to C57BL/6 mice resulted in a significant and sustained fall in blood erythrocyte count and hemoglobin levels that started 4 and 3 weeks after the start of 50 and 1000 ppm cadmium doses respectively. A transient yet significant reticulocytosis occurred during the first 4 weeks of cadmium treatment. Using the recently developed double in vivo biotinylation (DIB) technique, turnover of erythrocyte cohorts of different age groups was simultaneously monitored in control and cadmium treated mice. A significant accumulation of younger erythrocytes and a concomitant decline in the relative proportions of older erythrocytes in circulation was observed in both 50 and 1000 ppm cadmium groups indicating that older erythrocytes were preferentially eliminated in cadmium induced anemia. A significant increase in the erythropoietin levels in plasma was seen in mice exposed to 1000 ppm cadmium. Levels of inflammatory cytokines (IL1A, IL6, TNFα, IFNγ) were however not significantly altered in cadmium treated mice. A significant increase in cellular levels of reactive oxygen species (ROS) was observed in older erythrocytes in circulation but not in younger erythrocytes. Erythropoietic activity in the bone marrows and spleens of cadmium treated mice was examined by monitoring the relative proportion of cells belonging to the erythroid line of differentiation in these organs. Erythroid cells in bone marrow declined markedly (about 30%) in mice in the 1000 ppm cadmium group but the decline was not significant in the 50 ppm cadmium group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Decline of erythroid cells was essentially confined to pro-erythroblast and erythroblast-A, along with a concurrent increase in the splenic erythroid population indicating a stress response. In short cadmium exposure causes preferential clearance of older erythrocytes from circulation along with a depressed erythropoietic activity at higher doses.

Dual ligand immunoliposomes for drug delivery to the brain

Drug delivery systems that can reach brain areas affected by amyloid deposits are still underdeveloped. We propose pegylated liposomes functionalized with two antibodies, the anti-transferrin receptor monoclonal antibody (OX26MAb) and the anti-amyloid beta peptide antibody (19B8MAb), as nanocarriers of drugs for Alzheimer’s disease therapy. Two distinct conjugation methods are investigated. In one formulation, the OX26MAb is conjugated to the tip of polyethylene glycol molecules through the maleimide group and the 19B8MAb is bound through the streptavidin–biotin complex. In the second system the conjugation reagents are swapped between the antibodies. Fluorescence spectroscopy experiments on porcine brain capillary endothelial cells show that the cellular uptake of the immunoliposomes is substantially more efficient if OX26MAb antibody is conjugated through the streptavidin–biotin complex instead of the maleimide group. The ability of the immunoliposomes to cross the blood brain barrier was established by in vivo studies in wild type rats. Our results demonstrate the importance of the conjugation method used to bind the antibody that targets the blood brain barrier to immunoliposomes for efficient drug delivery to the brain.

Evaluation of Efficacy of Radioimmunotherapy with 90Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models.

ObjectivePancreatic cancer is an aggressive tumor and the prognosis remains poor. Therefore, development of more effective therapy is needed. We previously reported that 89Zr-labeled TSP-A01, an antibody against transferrin receptor (TfR), is highly accumulated in a pancreatic cancer xenograft, but not in major normal organs. In the present study, we evaluated the efficacy of radioimmunotherapy (RIT) with 90Y-TSP-A01 in pancreatic cancer mouse models.MethodsTfR expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2) was evaluated by immunofluorescence staining. 111In-labeled anti-TfR antibodies (TSP-A01, TSP-A02) were evaluated in vitro by cell binding assay with the three cell lines and by competitive inhibition assay with MIAPaCa-2. In vivo biodistribution was evaluated in mice bearing BxPC-3 and MIAPaCa-2 xenografts. Tumor volumes of BxPC-3 and MIAPaCa-2 were sequentially measured after 90Y-TSP-A01 injection and histological analysis of tumors was conducted.ResultsMIAPaCa-2 cells showed the highest TfR expression, followed by AsPC-1 and BxPC-3 cells. 111In-TSP-A01 and 111In-TSP-A02 bound specifically to the three cell lines according to TfR expression. The dissociation constants for TSP-A01, DOTA-TSP-A01, TSP-A02, and DOTA-TSP-A02 were 0.22, 0.28, 0.17, and 0.22 nM, respectively. 111In-TSP-A01 was highly accumulated in tumors, especially in MIAPaCa-2, but this was not true of 111In-TSP-A02. The absorbed dose for 90Y-TSP-A01 was estimated to be 8.3 Gy/MBq to BxPC-3 and 12.4 Gy/MBq to MIAPaCa-2. MIAPaCa-2 tumors treated with 3.7 MBq of 90Y-TSP-A01 had almost completely disappeared around 3 weeks after injection and regrowth was not observed. Growth of BxPC-3 tumors was inhibited by 3.7 MBq of 90Y-TSP-A01, but the tumor size was not reduced.Conclusion 90Y-TSP-A01 treatment achieved an almost complete response in MIAPaCa-2 tumors, whereas it merely inhibited the growth of BxPC-3 tumors. 90Y-TSP-A01 is a promising RIT agent for pancreatic cancer, although further investigation is necessary to improve the efficacy for the radioresistant types like BxPC-3.

Selective loss of younger erythrocytes from blood circulation and changes in erythropoietic patterns in bone marrow and spleen in mouse anemia induced by poly-dispersed single-walled carbon nanotubes

Administration of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs, 10 µg intravenously on alternate days) induced a sustained anemia in mice. Using a modified double in vivo biotinylation (DIB) technique, blood counts of reticulocytes and erythrocytes of different age groups were simultaneously enumerated in control and AF-SWCNT treated mice. A sustained reticulocytosis was observed in AF-SWCNT treated mice. Young erythrocytes (up to 7 days old) that are normally resistant to elimination in control mice were eliminated at a significant rate in AF-SWCNT treated mice. Old erythrocytes, however, accumulated in circulation indicating that younger erythrocytes were selectively eliminated from the blood circulation of AF-SWCNT treated mice. Cells representing various stages of erythroid differentiation in bone marrow and spleen were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Proportion of erythroid cells was significantly reduced (up to 27%) in bone marrow (BM) indicating a fall in erythropoietic activity. A concomitant increase in the spleen erythroid population was however observed that could be a compensatory response. Changes in erythroid populations in bone marrow and spleen correlated with changes in erythroblast-A population in these organs that represent an early stage of erythroid differentiation. Uptake of intravenously administered fluorescence tagged AF-SWCNTs (FAF-SWCNTs) was relatively low (3–4%) in erythroid cells in bone marrow and spleen. A significantly higher proportion of pro-erythroblasts and erythroblast-A (early stages of erythroid differentiation) took-up FAF-SWCNTs. Uptake of AF-SWCNTs by early precursors of erythroid differentiation with toxic consequences may be a contributing factor in AF-SWCNT induced anemia.

Preclinical evaluation of ⁸⁹Zr-labeled human antitransferrin receptor monoclonal antibody as a PET probe using a pancreatic cancer mouse model.

Pancreatic cancer is aggressive and its prognosis remains poor; thus, effective therapy is urgently needed. Transferrin receptor (TfR) is highly expressed in pancreatic cancer and is considered to be a good candidate for molecular-targeted therapy. We radiolabeled and evaluated fully human anti-TfR monoclonal antibodies as a new PET probe for evaluating the biodistribution of the anti-TfR antibody in pancreatic cancer. TfR expression was evaluated in four human pancreatic cancer (MIAPaCa-2, PANC-1, BxPC-3, and AsPC-1) and murine A4 cell lines. The binding of 125I-labeled anti-TfR antibodies (TSP-A01, TSP-A02, TSP-A03, and TSP-A04) to MIAPaCa-2 cells was compared. 125I-labeled, 67Ga-labeled, and 89Zr-labeled TSP-A01 were evaluated by cell binding, competitive inhibition, and internalization assays. Biodistribution studies of 125I-labeled and 89Zr-labeled TSP-A01 were conducted in mice bearing MIAPaCa-2 and A4 tumors. PET imaging with [89Zr]TSP-A01 was carried out. MIAPaCa-2 cells showed the highest TfR expression in vitro and in vivo, whereas A4 cells showed no expression. Of the four antibodies, [125I]TSP-A01 showed the highest binding to MIAPaCa-2 cells, but not to A4 cells. The dissociation constant of TSP-A01 was 0.29 nmol/l. Uptake of radiolabeled TSP-A01, especially [89Zr]TSP-A01, was significantly higher in MIAPaCa-2 tumors than in A4 tumors. PET with [89Zr]TSP-A01 clearly visualized MIAPaCa-2 xenografts but not A4 xenografts. [89Zr]TSP-A01 is a promising PET probe for evaluating the accumulation of anti-TfR antibody in pancreatic cancer and has the potential to facilitate the selection of appropriate patients who would benefit from anti-TfR antibody therapy.

Recent advances in adult T-cell leukemia therapy: focus on a new anti-transferrin receptor monoclonal antibody

HTLV-I is an endemic retrovirus responsible for the adult T-cell leukemia/lymphoma (ATLL). This aggressive lymphoid proliferation is associated with a bad prognosis due to the resistance of HTLV-I-infected cells to most classical chemotherapeutic agents. Here we review recent advances in ATLL immunotherapy. We particularly focus on promising data from our group, characterizing a new mouse monoclonal antibody (mAb A24) against the human transferrin receptor (TfR-1). Monoclonal antibodies to target cell differentiation markers on ATLL cells have already been proposed as therapeutic agents. However, in clinical trials acute forms of ATLL were resistant to these immunotherapies. A24 binds TfR-1 (K(d) 2.7 nM) and competes with transferrin for receptor binding. It blocks the proliferation of malignant cells (TfR-1(high)), such as HTLV-I-infected T cells but not of resting cells. A24 induces TfR-1 endocytosis in lysosomal compartments where the receptor is degraded leading to intracellular iron deprivation. In HTLV-I-infected cells, A24 targets and induces apoptosis of both chronic and acute ATLL forms, independent of antibody aggregation, antibody-dependent cellular cytotoxicity and/or complement addition. The antibody efficacy was confirmed in animal models. We are currently developing strategies to use A24 in clinical trials.

Translocation of Activated Heterotrimeric G Protein Gαo to Ganglioside-enriched Detergent-resistant Membrane Rafts in Developing Cerebellum

The association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 immunoprecipitated phosphoproteins of 40, 53, 56, and 80 kDa from the rat cerebellum. Of these proteins, the 40-kDa protein was identified as the alpha-subunit of a heterotrimeric G protein, G(o) (Galpha(o)). Using sucrose density gradient analysis of cerebellar membranes, Galpha(o), but not Gbetagamma, was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5'-O-(thiotriphosphate) (GTPgammaS), which stabilizes G(o) in its active form. On the other hand, both Galpha(o) and Gbetagamma were excluded from the DRM raft fractions in the presence of guanyl-5'-yl thiophosphate, which stabilizes G(o) in its inactive form. Only Galpha(o) was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, Galpha(o) was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of Galpha(o) into the DRM rafts. Furthermore, Galpha(o) was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1alpha, a physiological ligand for the G protein-coupled receptor, stimulated [(35)S]GTPgammaS binding to Galpha(o) and caused Galpha(o) translocation to the DRM fractions and RhoA translocation to the membrane fraction, leading to the growth cone collapse of cerebellar granule neurons. The collapse was partly prevented by pretreatment with the cholesterol-sequestering and raft-disrupting agent methyl-beta-cyclodextrin. These results demonstrate the involvement of signal-dependent Galpha(o) translocation to the DRM in the growth cone behavior of cerebellar granule neurons.

Synthesis of pegylated immunonanoparticles.

This work describes the synthesis of pegylated immunonanoparticles by conjugation of an anti-transferrin receptor monoclonal antibody (MAb) to maleimide-grafted pegylated nanoparticles prepared from poly(lactic acid) (PLA) and a bi-functional polyethyleneglycol (PEG). Maleimide-PEG3500-PLA40000 and methoxyPEG2600-PLA40000 copolymers were synthesized by ring opening polymerization of L-lactide using stannous octoate as catalyst. Pegylated nanoparticles were prepared from these copolymers by a multiple emulsion/solvent evaporation method and thiolated OX26 MAb was conjugated through the maleimide function located at the distal end of the PEG spacer. The pegylated immunonanoparticles were characterized by quasi-elastic light scattering, gel permeation chromatography, turbidimetry assays, and transmission electron microscopy. NMR spectroscopy confirmed the synthesis of both copolymers and the preservation of the maleimide function. The pegylated immunonanoparticles had an average diameter of 121 +/- 5 nm and appeared spherical by transmission electron microscopy. The number of OX26 MAb molecules conjugated per individual pegylated nanoparticle was 67 +/- 4. The MAb conjugated to the surface of the pegylated immunonanoparticle was visualized directly by electron microscopy using a conjugate of 10 nm gold and an anti-mouse immunoglobulin secondary antibody. Pegylated immunonanoparticles can be synthesized with bifunctional PEG derivatives that bridge the nanoparticle and the targeting MAb. This novel formulation may enable the targeted delivery of small molecules, protein drugs, and gene medicines.

By-passing of P-glycoprotein Using Immunoliposomes

The over-expression of MDR1 P-glycoprotein has been associated with the development of multidrug-resistance in cancer cells. Methods used to overcome multidrug-resistance often involve the co-administration of inhibitors of P-glycoprotein. Here, we test the hypothesis that an immunoliposome-based drug delivery system may be used as an alternative approach to overcome multidrug-resistance since immunoliposomes penetrate target cells by receptor-mediated endocytosis which allows to by-pass membrane-associated P-glycoprotein. Targeting of immunoliposomes was achieved by the use of an anti-transferrin receptor monoclonal antibody (OX26 mAb). Incorporation of radiolabelled digoxin within OX26-immunoliposomes enhanced cellular uptake of digoxin by a factor of 25 in immortalised RBE4 rat brain capillary endothelial cells. Uptake of liposomal digoxin was insensitive to ritonavir, a P-glycoprotein inhibitor, and was reduced in presence of increasing free concentrations of OX26 mAb or nocodazole, a reversible inhibitor of endocytosis. In contrast, uptake of free digoxin was enhanced by a factor of 1.8 in presence of ritonavir and was insensitive to OX26 mAb or nocodazole. Cellular uptake and intracellular accumulation of liposomal digoxin (55% internalisation within 30 min) was demonstrated by acid wash of the cells and was confirmed by confocal microscopy studies. Endosomal release to the cytosol of propidium iodide loaded immunoliposomes was shown. These in vitro studies suggest that immunoliposome-based drug delivery systems can be used to by-pass P-glycoprotein and thus deliver drugs to the cytosol of a target cell.

Mechanisms underlying the immunosuppressive activity of antitransferrin receptor monoclonal antibody

Mechanisms underlying the immunosuppressive activity of antitransferrin receptor monoclonal antibody

Role of Ceruloplasmin in Cellular Iron Uptake: Addendum

We recently reported that ceruloplasmin (Cp) stimulates iron uptake by HepG2 cells by a transferrin-independent mechanism ([1][1]). We are grateful to Ian Trowbridge, at the Salk Institute, for a comment about our use of the anti-transferrin receptor monoclonal antibody H68.4 in this study. He

Recombinant immunocytokines targeting the mouse transferrin receptor: construction and biological activities.

Localized cytokine therapies with recombinant monoclonal antibody-cytokine fusion proteins, designated immunocytokines, have become of increasing interest for tumor immunotherapy, since they direct immunomodulatory cytokines into the tumor microenvironment. To investigate their mechanisms of action in a variety of syngeneic tumor models, recombinant mouse cytokines IL2 and GM-CSF were engineered as fusion proteins to the carboxyl terminus of a chimeric rat/mouse antitransferrin receptor antibody, ch17217 and expressed in stable-transfected Chinese hamster ovary cells. The recombinant immunocytokines were readily purified by affinity chromatography and their binding characteristics were identical to those shown for the ch17217 antibody. The IL2 immunocytokine had an activity similar to recombinant mouse IL2, whereas the GM-CSF immunocytokine had enhanced cytokine activity relative to recombinant mouse GM-CSF. The clearance rates of ch17217 and the GM-CSF and IL2 immunocytokines were relatively similar with elimination phases (t1/2alpha) of 1.8 h and distribution phases (t1/2beta) of 83, 88, and 91 h, respectively. Both immunocytokines demonstrated effective antitumor activity by suppressing the growth of hepatic metastases of mouse neuroblastoma and pulmonary metastases of mouse colon carcinoma in syngeneic A/J and BALB/c mice, respectively. These results indicate that biologically effective IL2 and GM-CSF immunocytokines combine the targeting ability of an antitransferrin receptor monoclonal antibody with the immunomodulatory functions of each cytokine. Because of the universal expression of the transferrin receptor on mouse tumor cell lines, these constructs should prove useful to determine their efficacy in a wide variety of syngeneic mouse tumor models and to perform detailed studies of their modes of action.

Examination of Blood‐Brain Barrier Transferrin Receptor by Confocal Fluorescent Microscopy of Unfixed Isolated Rat Brain Capillaries

Isolated rat brain capillaries were analyzed by confocal microscopy. Fluorescent immunoliposomes bearing the OX26 anti-transferrin receptor monoclonal antibody were synthesized and incubated with freshly isolated unfixed microvessels to visualize binding to luminal and abluminal membranes of the endothelium. Intactness of the endothelial structure was demonstrated by computer-aided reconstruction of a series of consecutive optical sections. These results indicate that analysis of unfixed brain capillaries by confocal microscopy offers the possibility of assigning the presence of membrane receptors to either the luminal or the basolateral plasma membrane domain.