RESUMEN
The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This new protein engineered platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. Alternating sequences from human IgA and IgG in the SEED CH3 domains generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of AG and GA SEED CH3 domains. Using a clinically validated antibody (C225), we tested whether Fab derivatives constructed on the SEED platform retain desirable therapeutic antibody features such as in vitro and in vivo stability, favorable pharmacokinetics, ligand binding and effector functions including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In addition, we tested SEED with combinations of binder domains (scFv, VHH, Fab). Mono- and bivalent Fab-SEED fusions retain full binding affinity, have excellent biochemical and biophysical stability, and retain desirable antibody-like characteristics conferred by Fc domains. Furthermore, SEED is compatible with different combinations of Fab, scFv and VHH domains. Our assessment shows that the new SEED platform expands therapeutic applications of natural antibodies by generating heterodimeric Fc-analog proteins.
Asunto(s)
Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Especificidad de Anticuerpos , Ingeniería de Proteínas/métodos , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Afinidad de Anticuerpos , Línea Celular Tumoral , Proteínas del Sistema Complemento/inmunología , Receptores ErbB/inmunología , Semivida , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Masculino , Ratones , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.
Asunto(s)
Empalme Alternativo/genética , Proteínas de la Membrana/genética , Receptores Acoplados a Proteínas G/genética , Secuencia de Bases , Humanos , Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos , Relaxina/metabolismoRESUMEN
The ARS Component B gene (EMBL ID: HSARS81S, AC: X99977) encodes a 9 kD non-glycosylated polypeptide (also known as SLURP-1, SwissProt/TrEMBL: P55000), a soluble member of the human Ly6/uPAR superfamily. ARS Component B gene mutations have been implicated in Mal de Meleda. In this study we show by immunohistochemistry that SLURP-1 (secreted Ly-6/uPAR related protein, the protein product of the ARS Component B gene) is localized to human skin, exocervix, gums, stomach and esophagus. In the epidermis, keratinocytes underlying the stratum corneum are highly positive for SLURP1 immunostaining and cultured keratinocytes secrete the expected 9 kD protein. Circulating SLURP1 is detected in human plasma and urine. In the mouse, expression is evident in skin, eye, whole lung, trachea, esophagus and stomach. Human ARS Component B mRNA expression is regulated by retinoic acid, epidermal growth factor and interferon-gamma. The tissue localization and the association with Mal de Meleda suggest that ARS Component B and its protein product SLURP1 are implicated in maintaining the physiological and structural integrity of the keratinocyte layers of the skin.
Asunto(s)
Antígenos Ly/genética , Queratinocitos/metabolismo , Queratodermia Palmoplantar/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Inmunohistoquímica , Queratodermia Palmoplantar/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Estructura Molecular , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vertebrate reproduction is tightly regulated by conserved glycoprotein hormones produced by the pituitary gland. Follicle-stimulating hormone (FSH) in tetrapods and gonadotropic hormone I (GTH-I) in fishes are orthologous glycoprotein hormones that control the timing of egg production and the number of eggs produced. Zebrafish, a well-established genetic model for developmental biology, also offers potential advantages for studies of reproductive toxicology, especially for modeling the impact of pollutants on fish reproductive processes. To facilitate these studies we have identified, expressed, and characterized the zebrafish GTH-I receptor. This receptor (zfGTHR-I)exhibits strong sequence similarity to the tetrapod FSH receptors and to GTHR-I from salmon and catfish. Human 293 cells transfected with zfGTHR-I exhibit increased cAMP levels after treatment with carp pituitary extracts or human FSH, but not when treated with a ligand to a related receptor (human chorionic gonadotropin). Northern blotting and RT-PCR analyses indicate that zfGTHR is expressed in ovaries from sexually mature fish, but not in immature fish. Several alternative splice variants of the receptor affecting putative exons 2-4 that encode dramatically shortened receptor fragments lacking the transmembrane domain as well as regions previously implicated in ligand binding were identified by RT-PCR. The zfGTHR-I sequence opens the way to study effects of genetic mutations or chemicals on ovarian zfGTHR-I expression and function in zebrafish.