RESUMO
Carriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology. scFv and hTM-scFv targeted to band 3/GPA increased membrane rigidity and sensitized RBCs to hemolysis induced by mechanical stress, while reducing sensitivity to hypo-osmotic hemolysis. Similar properties were seen for other ligands bound to GPA and band 3 on human and murine RBCs. In contrast, binding of scFv or hTM-scFv to RhCE did not alter deformability or sensitivity to mechanical and osmotic stress at similar copy numbers bound per RBCs. Contrasting responses were also seen for immunoglobulin G antibodies against band 3, GPA, and RhCE. RBC-bound hTM-scFv generated activated protein C (APC) in the presence of thrombin, but RhCE-targeted hTM-scFv demonstrated greater APC generation per bound copy. Both Wrb- and RhCE-targeted fusion proteins inhibited fibrin deposition induced by tumor necrosis factor-α in an endothelialized microfluidic model using human whole blood. RhCE-bound hTM-scFv more effectively reduced platelet and leukocyte adhesion, whereas anti-Wrb scFv appeared to promote platelet adhesion. These data provide a translational framework for the development of engineered affinity ligands to safely couple therapeutics to human RBCs.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Eritrócitos/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Trombose/tratamento farmacológico , Animais , Proteína 1 de Troca de Ânion do Eritrócito/imunologia , Humanos , Inflamação/tratamento farmacológico , Macaca , Camundongos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Trombomodulina/administração & dosagem , Trombomodulina/genética , Trombose/patologiaRESUMO
Diverse human illnesses are characterized by loss or inactivation of endothelial thrombomodulin (TM), predisposing to microvascular inflammation, activation of coagulation, and tissue ischemia. Single-chain antibody fragment (scFv)/TM) fusion proteins, previously protective against end-organ injury in murine models of inflammation, are attractive candidates to treat inflammatory thrombosis. However, animal models have inherent differences in TM and coagulation biology, are limited in their ability to resolve and control endothelial biology, and do not allow in-depth testing of "humanized" scFv/TM fusion proteins, which are necessary for translation to the clinical domain. To address these challenges, we developed a human whole-blood, microfluidic model of inflammatory, tissue factor (TF)-driven coagulation that features a multichannel format for head-to-head comparison of therapeutic approaches. In this model, fibrin deposition, leukocyte adhesion, and platelet adhesion and aggregation showed a dose-dependent response to tumor necrosis factor-α activation and could be quantified via real-time microscopy. We used this model to compare hTM/R6.5, a humanized, intracellular adhesion molecule 1 (ICAM-1)-targeted scFv/TM biotherapeutic, to untargeted antithrombotic agents, including soluble human TM (shTM), anti-TF antibodies, and hirudin. The targeted hTM/R6.5 more effectively inhibited TF-driven coagulation in a protein C (PC)-dependent manner and demonstrated synergy with supplemental PC. These results support the translational prospects of ICAM-targeted scFv/TM and illustrate the utility of the microfluidic system as a platform to study humanized therapeutics at the interface of endothelium and whole blood under flow.
RESUMO
Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such "vascular immunotargeting" to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans.
Assuntos
Sistemas de Liberação de Medicamentos , Endotélio/imunologia , Lipossomos/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Linhagem Celular , Células Endoteliais/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/farmacocinética , Ratos , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacocinéticaRESUMO
Nanocarriers (NCs) coated with antibodies (Abs) to extracellular epitopes of the transmembrane glycoprotein PECAM (platelet endothelial cell adhesion molecule-1/CD31) enable targeted drug delivery to vascular endothelial cells. Recent studies revealed that paired Abs directed to adjacent, yet distinct epitopes of PECAM stimulate each other's binding to endothelial cells in vitro and in vivo ("collaborative enhancement"). This phenomenon improves targeting of therapeutic fusion proteins, yet its potential role in targeting multivalent NCs has not been addressed. Herein, we studied the effects of Ab-mediated collaborative enhancement on multivalent NC spheres coated with PECAM Abs (Ab/NC, â¼180 nm diameter). We found that PECAM Abs do mutually enhance endothelial cell binding of Ab/NC coated by paired, but not "self" Ab. In vitro, collaborative enhancement of endothelial binding of Ab/NC by paired Abs is modulated by Ab/NC avidity, epitope selection, and flow. Cell fixation, but not blocking of endocytosis, obliterated collaborative enhancement of Ab/NC binding, indicating that the effect is mediated by molecular reorganization of PECAM molecules in the endothelial plasmalemma. The collaborative enhancement of Ab/NC binding was affirmed in vivo. Intravascular injection of paired Abs enhanced targeting of Ab/NC to pulmonary vasculature in mice by an order of magnitude. This stimulatory effect greatly exceeded enhancement of Ab targeting by paired Abs, indicating that '"collaborative enhancement"' effect is even more pronounced for relatively large multivalent carriers versus free Abs, likely due to more profound consequences of positive alteration of epitope accessibility. This phenomenon provides a potential paradigm for optimizing the endothelial-targeted nanocarrier delivery of therapeutic agents.
Assuntos
Plaquetas/metabolismo , Epitopos/imunologia , Nanosferas/química , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Animais , Membrana Celular/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Camundongos , Camundongos Endogâmicos C57BL , Nanosferas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/química , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Ligação ProteicaRESUMO
A microfluidic platform is presented which fully automates all incubation steps of a three-stage, multiplexed magnetic bead immunoassay, such as the Luminex® xMAP technology. Magnetic actuation is used to transfer the microbeads between co-infused adjacent laminar flow streams to transport the beads into and out of incubation and wash solutions, with extended incubation channels to allow sufficient bead incubation times (1-30 min, commonly 5 min per stage) to enable high-sensitivity. The serial incubation steps of the immunoassay are completed in succession within the device with no operator interaction, and the continuous flow operation with magnetic bead transfer defines the incubation sequencing requiring no external fluidic controls beyond syringe pump infusion. The binding kinetics of the assay is empirically characterized to determine the required incubation times for specific assay sensitivities in the range 1 pg/ml to 100 ng/ml. By using a Luminex® xMAP duplex assay, concurrent detection of IL-6 and TNF-α was demonstrated on-chip with a detection range 10 pg/ml to 1 ng/ml. This technology enables rapid automation of magnetic microbead assays, and has the potential to perform continuous concentration monitoring.