RESUMEN
Antibody display technologies enable the successful isolation of antigen-specific antibodies with therapeutic potential. The key feature that facilitates the selection of an antibody with prescribed properties is the coupling of the protein variant to its genetic information and is referred to as genotype phenotype coupling. There are several different platform technologies based on prokaryotic organisms as well as strategies employing higher eukaryotes. Among those, phage display is the most established system with more than a dozen of therapeutic antibodies approved for therapy that have been discovered or engineered using this approach. In recent years several other technologies gained a certain level of maturity, most strikingly mammalian display. In this review, we delineate the most important selection systems with respect to antibody generation with an emphasis on recent developments.
Asunto(s)
Anticuerpos , Biblioteca de Péptidos , Animales , Anticuerpos/genética , Anticuerpos/uso terapéutico , Mamíferos/genéticaRESUMEN
Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.
Asunto(s)
Neoplasias Óseas/genética , Receptores de Hialuranos/metabolismo , Neoplasias Pulmonares/genética , Neurofibromina 2/genética , Osteosarcoma/genética , Animales , Neoplasias Óseas/patología , Huesos/patología , Adhesión Celular/genética , Línea Celular Tumoral/trasplante , Proliferación Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Receptores de Hialuranos/genética , Pulmón/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Noqueados , Osteosarcoma/secundarioRESUMEN
Research on the development of scientific reasoning has put the main focus on children's experimentation skills, in particular on the control-of-variables strategy. However, there are more scientific methods than just experimentation. Observation is defined as an independent scientific method that includes not only the description of what is observed, but also all phases of the scientific inquiry, such as questioning, hypothesizing, testing, and interpreting. Previous research has shown that the quality of observations depends on specific knowledge in the domain. We argue that observation competency shares the domain-general ability to differentiate hypotheses from evidence with other scientific methods. The present study investigates the relations of both domain-general scientific thinking and domain-specific knowledge in biology with observation competency in grade K children. We tested relations between observation competency, domain-general scientific reasoning, domain-specific knowledge, and language abilities of 75 children (age 4;9 to 6;7). Both scientific reasoning and domain-specific knowledge proved to be significant predictors of observation competency, explaining 35% of the variance. In a mediation analysis, we found a significant indirect effect of language via these two predictors. Thus, the present results indicate that observation skills require not only domain-specific knowledge but also domain-general scientific reasoning abilities.
RESUMEN
In addition to conventional hetero-tetrameric antibodies, the adaptive immune repertoire of camelids comprises the so-called heavy chain-only antibodies devoid of light chains. Consequently, antigen binding is mediated solely by the variable domain of the heavy chain, referred to as VHH. In recent years, these single-domain moieties emerged as promising tools for biotechnological and biomedical applications. In this chapter, we describe the generation of VHH antibody yeast surface display libraries from immunized Alpacas and Lamas as well as the facile isolation of antigen-specific molecules in a convenient fluorescence-activated cell sorting (FACS)-based selection process.