RESUMEN
Certain combinations of non-competitive anti-EGFR antibodies have been reported to produce new effects on cells compared to either antibody used separately. New and enhanced combination-activity includes increased inhibition of signaling, increased receptor internalization and degradation, reduced proliferation of tumor cell lines and induction of complement-dependent cytotoxicity (CDC) effector function. To test requirements and mechanisms to elicit enhanced combination-activity with different EGFR binding domains, we created an anti-EGFR biparatopic antibody. A biparatopic antibody interacts through two different antigen-binding sites to a single antigen. A heterodimeric antibody with one binding domain derived from the C225 antibody and one binding domain derived from the humanized 425 (hu425) antibody was built on the strand-exchange engineered domain (SEED) scaffold. This anti-EGFR biparatopic-SEED antibody was compared to parental antibodies used alone and in combination, and to the corresponding monovalent anti-EGFR-SEED antibodies used alone or in combination. We found that the anti-EGFR biparatopic-SEED had enhanced activity, similar to the combination of the two parental antibodies. Combinations of monovalent anti-EGFR-SEED antibodies did not produce enhanced effectiveness in cellular assays. Our results show that the anti-EGFR biparatopic antibody created using the SEED scaffold has enhanced combination-activity in a single molecule. Furthermore, these data suggest that the potential to cross-link the two different epitopes is an important requirement in the mechanism of enhanced combination-activity.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales/inmunología , Receptores ErbB/inmunología , Anticuerpos Biespecíficos/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados/química , Sitios de Unión de Anticuerpos , Línea Celular Tumoral , Proliferación Celular , Cetuximab , Epítopos/inmunología , Humanos , Estructura Terciaria de ProteínaRESUMEN
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
A novel high-affinity inhibitor of tumor necrosis factor (TNF) is described, which is created by the fusion of the extracellular domains of TNF-binding protein 1 (TBP-1) to both the alpha and beta chains of an inactive version of the heterodimeric protein hormone, human chorionic gonadotropin. The resulting molecule, termed TNF-soluble high-affinity receptor complex (SHARC), self-assembles into a heterodimeric protein containing two functional TBP-1 moieties. The TNF-SHARC is a potent inhibitor of TNF-alpha bioactivity in vitro and has a prolonged pharmacokinetic profile compared with monomeric TBP-1 in vivo. Consistent with the long half-life, the duration of action in an lipopolysaccharide-mediated proinflammatory mouse model is prolonged similarly. In a collagen-induced arthritis mouse model, this molecule demonstrates improved efficacy over monomeric TBP-1. Based on these results, we demonstrated that inactivated heterodimeric protein hormones are flexible and efficient scaffolds for the creation of soluble high-affinity receptor complexes.
Asunto(s)
Gonadotropina Coriónica/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Proteínas Recombinantes de Fusión/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacocinética , Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Gonadotropina Coriónica Humana de Subunidad beta/genética , Electroforesis en Gel de Poliacrilamida , Femenino , Hormonas Glicoproteicas de Subunidad alfa/genética , Humanos , Interleucina-6/sangre , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C3H , Peso Molecular , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
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.