RESUMEN
Generation of bispecific antibodies (BsAbs) having two unique Fab domains requires heterodimerization of the two heavy chains and pairing of each heavy chain with its cognate light chain. An alternative bispecific scaffold (Bipod) comprising an scFv and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. However, unpredictable levels of chain expression and scFv-induced aggregation can complicate purification and reduce the yield of desired Bipod. Here, we describe a high-throughput method for generation of Bipods based on protein A and CH1 domain affinity capture. This method exploits over-expression of the scFv chain to maximize heterodimer yield. Bipods purified by this method have purity suitable for cell-based functional assays and in vivo studies.
Asunto(s)
Anticuerpos Biespecíficos/química , Fragmentos Fab de Inmunoglobulinas/química , Ingeniería de Proteínas/métodos , Anticuerpos de Cadena Única/química , Animales , Productos Biológicos/uso terapéutico , Células CHO , Cricetulus , ADN/química , Dimerización , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Epítopos/química , Humanos , Inmunoglobulina G/genética , Inmunosupresores/uso terapéutico , Mutación , Neoplasias/terapia , Plásmidos , Dominios ProteicosRESUMEN
AIM: To investigate the protein kinase B (Akt) signalling proteins phosphatase and tensin homolog (PTEN), phosphorylated-Akt (p-Akt) and cyclin-dependent kinase inhibitor 1B (p27(Kip1)) in non-seminomatous germ cell tumors (NST) with a view to future investigative approaches. MATERIALS AND METHODS: The expressions of PTEN, p-Akt and p27(Kip1) were immunohistochemically assessed in 17 teratomas, 27 embryonal cell carcinomas, 6 yolk sac tumors and 24 benign testicular parenchymas. The cytoplasmic and corresponding nuclear expressions were compared and correlated to tumor entity. RESULTS: PTEN was dramatically reduced in all the NST subgroups. Concentrated nuclear p27(Kip1) and loss of the cytoplasmic form was found in teratomas and embryonal cell carcinomas. Neither altered expression nor negative Akt regulation was found. The yolk sac tumors showed late cytoplasmic shift of PTEN and p27(Kip1). CONCLUSION: Both, the absence of overexpression of p-Akt and of negative correlations to PTEN and p27(Kip1) suggest that signalling of these parameters in NST might include additional mechanisms such as crosstalk to other pathways rather than classical Akt activation.
