RESUMEN
Human tissue kallikreins (KLKs) constitute a family of 15 serine proteases that are distributed in various tissues and implicated in several pathological disorders. KLK7 is an unusual serine protease that presents both trypsin-like and chymotrypsin-like specificity and appears to be upregulated in pathologies that are related to skin desquamation processes, such as atopic dermatitis, psoriasis and Netherton syndrome. In recent years, various groups have worked to develop specific inhibitors for this enzyme, as KLK7 represents a potential target for new therapeutic procedures for diseases related to skin desquamation processes. In this work, we selected nine different single-chain variable fragment antibodies (scFv) from a human naïve phage display library and characterized their inhibitory activities against KLK7. The scFv with the lowest IC50 against KLK7 was affinity maturated, which resulted in the generation of four new scFv-specific antibodies for the target protease. These new antibodies were expressed in the scFv-Fc format in HEK293-6E cells, and the characterization of their inhibitory activities against KLK7 showed that three of them presented IC50 values lower than that of the original antibody. The cytotoxicity analysis of these recombinant antibodies demonstrated that they can be safely used in a cellular model. In conclusion, our research showed that in our case, a phage-display methodology in combination with enzymology assays can be a very suitable tool for the development of inhibitors for KLKs, suggesting a new strategy to identify therapeutic protease inhibitors for diseases related to uncontrolled kallikrein activity.
Asunto(s)
Calicreínas/antagonistas & inhibidores , Proteínas Recombinantes/inmunología , Inhibidores de Serina Proteinasa/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Chlorocebus aethiops , Células HEK293 , Humanos , Calicreínas/inmunología , Proteínas Recombinantes/toxicidad , Inhibidores de Serina Proteinasa/toxicidad , Anticuerpos de Cadena Única/toxicidad , Enfermedades de la Piel/terapia , Células VeroRESUMEN
Antibody-based near-infrared photoimmunotherapy (NIR-PIT) is an attractive strategy for cancer treatment. Tumor cells can be selectively and efficiently killed by the targeted delivery of an antibody-photoabsorber complex followed by exposure to NIR light. Glycoprotein A33 antigen (GPA33) is highly expressed in most human colorectal cancers (CRCs) and is an ideal diagnostic and therapeutic target. We previously produced a single-chain fragment of a variable antibody against GPA33 (A33scFv antibody). Here, we investigate the efficacy of NIR-PIT by combining A33scFv with the NIR photoabsorber IR700 (A33scFv-IR700). In vitro, recombinant A33scFv displayed specific binding and delivery of an NIR dye to GPA33-positive tumor cells. Furthermore, A33scFv-IR700-mediated NIR-PIT was successful in rapidly and specifically killing GPA33-positive colorectal tumor cells. NIR-PIT treatment induced the release of lactate dehydrogenase from tumor cells, followed by cell necrosis, rather than apoptosis, through the promotion of reactive oxygen species accumulation in tumor cells. In mice bearing LS174T tumor grafts, A33scFv selectively accumulated in GPA33-positive tumors. Following only a single injection of the conjugate and subsequent illumination, A33scFv-IR700-mediated NIR-PIT induced a significant increase in therapeutic response in LS174T-tumor mice compared with that in the non-NIR-PIT groups (p < 0.001). Because the GPA33 antigen is specifically expressed in CRC tumors, A33scFv-IR700 might be a promising antibody fragment-photoabsorber conjugate for NIR-PIT of CRC.
Asunto(s)
Muerte Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/radioterapia , Inmunoconjugados/uso terapéutico , Inmunoterapia/métodos , Glicoproteínas de Membrana/inmunología , Fototerapia/métodos , Anticuerpos de Cadena Única/inmunología , Animales , Muerte Celular/inmunología , Neoplasias Colorrectales/inmunología , Células HT29 , Humanos , L-Lactato Deshidrogenasa/metabolismo , Espectrometría de Masas , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Necrosis/metabolismo , Fármacos Fotosensibilizantes/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Anticuerpos de Cadena Única/efectos de la radiación , Anticuerpos de Cadena Única/toxicidad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: von Willebrand factor (VWF) is crucial to hemostasis, but also plays a role in inflammatory processes. Unfortunately, no proper monoclonal antibodies to study VWF function in mice are currently available. We therefore aimed to generate single-domain antibodies (sdAbs) recognizing murine VWF and blocking its function in vivo. APPROACH AND RESULTS: Llama-derived sdAbs recognizing both human and murine VWF were isolated via phage display technology. One of them (designated KB-VWF-006) recognized the VWF A1 domain with picomolar affinity. This sdAb avidity was strongly enhanced via dimerization using a triple Ala linker (KB-VWF-006bi). When administered in vivo to wild-type mice, KB-VWF-006bi dose dependently induced bleeding in a tail clip model. In 2 distinct models of inflammation, KB-VWF-006bi efficiently interfered with leukocyte recruitment and vascular leakage. CONCLUSIONS: KB-VWF-006bi is an sdAb recognizing the A1 domain of human VWF and murine VWF that interferes with VWF-platelet interactions in vivo. By using this sdAb, we now also show that the A1 domain is pertinent to the participation of VWF in the inflammatory response.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Inflamación/tratamiento farmacológico , Leucocitos/efectos de los fármacos , Anticuerpos de Cadena Única/farmacología , Factor de von Willebrand/antagonistas & inhibidores , Animales , Especificidad de Anticuerpos , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Reacciones Cruzadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hemorragia/inducido químicamente , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Dominios Proteicos , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/toxicidad , Factor de von Willebrand/genética , Factor de von Willebrand/inmunología , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS: Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined. RESULTS: Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death. CONCLUSIONS: These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.
Asunto(s)
Proteína ADAMTS13/antagonistas & inhibidores , Autoanticuerpos , Púrpura Trombocitopénica Trombótica/inmunología , Púrpura Trombocitopénica Trombótica/terapia , Proteína ADAMTS13/inmunología , Animales , Autoanticuerpos/genética , Clonación Molecular , ADN Complementario/administración & dosificación , Humanos , Ratones , Modelos Animales , Terapia Molecular Dirigida/métodos , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/toxicidad , Factor de von Willebrand/metabolismoRESUMEN
D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel immunotoxin that reacts with wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFRvIII proteins overexpressed in glioblastomas. This study assessed the toxicity of intracerebral administration of D2C7-IT to support an initial Food and Drug Administration Investigational New Drug application. After the optimization of the formulation and administration, two cohorts (an acute and chronic cohort necropsied on study days 5 and 34) of Sprague-Dawley (SD) rats (four groups of 5 males and 5 females) were infused with the D2C7-IT formulation at total doses of 0, 0.05, 0.1, 0.4 µg (the acute cohort) and 0, 0.05, 0.1, 0.35 µg (the chronic cohort) for approximately 72 h by intracerebral convection-enhanced delivery using osmotic pumps. Mortality was observed in the 0.40 µg (5/10 rats) and 0.35 µg (4/10 rats) high-dose groups of each cohort. Body weight loss and abnormal behavior were only revealed in the rats treated with high doses of D2C7-IT. No dose-related effects were observed in clinical laboratory tests in either cohort. A gross pathologic examination of systemic tissues from the high-dose and control groups in both cohorts exhibited no dose-related or drug-related pathologic findings. Brain histopathology revealed the frequent occurrence of dose-related encephalomalacia, edema, and demyelination in the high-dose groups of both cohorts. In this study, the maximum tolerated dose of D2C7-IT was determined to be between 0.10 and 0.35 µg, and the no-observed-adverse-effect-level was 0.05 µg in SD rats. Both parameters were utilized to design the Phase I/II D2C7-IT clinical trial.
Asunto(s)
Convección , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/toxicidad , Inmunotoxinas/administración & dosificación , Inmunotoxinas/toxicidad , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/toxicidad , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Femenino , Concentración 50 Inhibidora , Inyecciones Intraventriculares , Masculino , Ratas Sprague-DawleyRESUMEN
Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.
Asunto(s)
Inmunotoxinas/farmacología , Proteínas Luminiscentes/farmacología , Anticuerpos de Cadena Única/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Células CHO , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromatografía de Afinidad , Cricetulus , Escherichia coli , Fluorescencia , Vectores Genéticos , Humanos , Inmunotoxinas/aislamiento & purificación , Inmunotoxinas/toxicidad , Proteínas Luminiscentes/síntesis química , Proteínas Luminiscentes/aislamiento & purificación , Proteínas Luminiscentes/toxicidad , Microscopía Fluorescente , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/toxicidad , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos de Cadena Única/toxicidadRESUMEN
PURPOSE: Many genetic disorders, including chondrodysplasias, and acquired disorders impair growth plate function, resulting in short and sometimes malformed bones. There are multiple endocrine and paracrine factors that promote chondrogenesis at the growth plate, which could potentially be used to treat these disorders. Targeting these growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing undesirable effects on non-target tissues. METHODS: Using yeast display technology, we selected single-chain variable antibody fragments that bound to human and mouse matrilin-3, an extracellular matrix protein specifically expressed in cartilage tissue. The ability of the selected antibody fragments to bind matrilin-3 and to bind cartilage tissue in vitro and in vivo was assessed by ELISA and immunohistochemistry. RESULTS: We identified antibody fragments that bound matrilin-3 with high affinity and also bound with high tissue specificity to cartilage homogenates and to cartilage structures in mouse embryo sections. When injected intravenously in mice, the antibody fragments specifically homed to cartilage. CONCLUSIONS: Yeast display successfully selected antibody fragments that are able to target cartilage tissue in vivo. Coupling these antibodies to chondrogenic endocrine and paracrine signaling molecules has the potential to open up new pharmacological approaches to treat childhood skeletal growth disorders.
Asunto(s)
Placa de Crecimiento/efectos de los fármacos , Fragmentos de Inmunoglobulinas/farmacología , Proteínas Matrilinas/metabolismo , Anticuerpos de Cadena Única/farmacología , Animales , Especificidad de Anticuerpos , Clonación Molecular , Placa de Crecimiento/embriología , Placa de Crecimiento/metabolismo , Células HEK293 , Humanos , Fragmentos de Inmunoglobulinas/administración & dosificación , Fragmentos de Inmunoglobulinas/toxicidad , Inmunohistoquímica , Proteínas Matrilinas/genética , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/toxicidad , Levaduras/genéticaRESUMEN
Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/toxicidad , Docilidad , Priones/química , Priones/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/toxicidad , Sitios de Unión de Anticuerpos , Calpaína/metabolismo , Cerebelo , Síndrome de Creutzfeldt-Jakob/metabolismo , Reactivos de Enlaces Cruzados , Mapeo Epitopo , Femenino , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/toxicidad , Técnicas In Vitro , Ligandos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Estrés Oxidativo , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/inmunología , Priones/genética , Especies Reactivas de Oxígeno/metabolismo , Eliminación de Secuencia/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/toxicidadRESUMEN
Two novel human antitumor immunoconjugates, made up of a human anti-ErbB2 scFv, Erbicin, fused with either a human RNase or the Fc region of a human IgG1, are selectively cytotoxic for ErbB2-positive cancer cells in vitro and in vivo. The Erbicin-derived immunoagents (EDIA) target an epitope different from that of trastuzumab, the only humanized antibody currently prescribed for treatment of ErbB2-positive breast cancer (BC). As Trastuzumab has shown cardiotoxic effects, in this study, we evaluated if any side effects were exerted also by EDIA, used as single agents or in combination with anthracyclines. Furthermore, we compared the in vitro and in vivo cardiotoxic effects of EDIA with those of the other available anti-ErbB2 drugs: Trastuzumab, 2C4 (Pertuzumab), and Lapatinib. In this article, we show that EDIA, in contrast with Trastuzumab, 2C4, and Lapatinib, have no toxic effects on human fetal cardiomyocytes in vitro, and do not induce additive toxicity when combined with doxorubicin. Furthermore, EDIA do not impair cardiac function in vivo in mice, as evaluated by Color Doppler echocardiography, whereas Trastuzumab significantly reduces radial strain (RS) at day 2 and fractional shortening (FS) at day 7 of treatment in a fashion similar to doxorubicin. Also 2C4 and Lapatinib significantly reduce RS after only 2 days of treatment, even though they showed cardiotoxic effects less pronounced than those of Trastuzumab. These results strongly indicate that RS could become a reliable marker to detect early cardiac dysfunction and that EDIA could fulfill the therapeutic need of patients ineligible to Trastuzumab treatment because of cardiac dysfunction.
Asunto(s)
Anticuerpos Monoclonales Humanizados/toxicidad , Antineoplásicos/toxicidad , Corazón/efectos de los fármacos , Receptor ErbB-2/antagonistas & inhibidores , Anticuerpos de Cadena Única/toxicidad , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos/administración & dosificación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Doxorrubicina/toxicidad , Sinergismo Farmacológico , Humanos , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Anticuerpos de Cadena Única/administración & dosificación , TrastuzumabRESUMEN
HA22-LR is a recombinant immunotoxin for the treatment of B-cell malignancies that contains the Fv portion of an anti-CD22 antibody fused to a functional portion of Pseudomonas exotoxin A. In the present study, we attempted to improve this molecule. First, we produced a single-chain version of HA22-LR (scdsFv-HA22-LR) in which a peptide linker was introduced between the disulfide-linked light and heavy chains to enable production via single fermentation. No difference in cytotoxic activity was observed between scdsFv-HA22-LR and prototype HA22-LR. Next, we attempted to increase the affinity of scdsFv-HA22-LR by using alanine scanning mutagenesis of complementarity determining regions (CDRs) to assess the specific contribution of each CDR residue to the antigen binding. We found that mutation of asparagine 34 in VLCDR1, which is located at the VL/VH interface, to alanine (N34A) caused a substantial increase in affinity and activity. Estimated KD values measured by fluorescence-activated cell sorting were lowered by 10-fold: 0.056 nM in the N34A mutant compared to 0.58 nM in wild type (WT). Cell viability assays of CD22-positive B-cell lymphoma and leukemia cell lines showed that the N34A mutant had increased cytotoxicity ranging from ~2 (HAL-1, IC 50(WT): 2.37 ± 0.62 ng/ml, IC 50(N34A): 1.32 ± 0.41 ng/ml) to 10 (SUDHL-6, IC 50(WT): 0.47 ± 0.090 ng/ml, IC 50(N34A): 0.048 ± 0.018 ng/ml)-fold compared to WT immunotoxin. The present study suggests that the N34A mutant of scdsFv-HA22-LR could have important consequences in a clinical setting.