RESUMEN
Ang1 is a soluble ligand to receptor Tie2, and increasing the circulating Ang1 level may improve vascular stabilization under certain disease conditions. Here, we found that the circulating Ang1 level was significantly increased in cynomolgus monkeys treated with non-neutralizing anti-Ang1 antibodies. Improving the antibodies' pharmacokinetic properties by IgG Fc mutations further increased the circulating Ang1 level. However, the mutations decreased the thermal stability of the molecules, which may limit their use as therapeutic antibodies. Nevertheless, we showed that non-neutralizing antibodies may have therapeutic potential by increasing the level of a target molecule in the circulation.
Asunto(s)
Angiopoyetina 1/inmunología , Anticuerpos Monoclonales/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Angiopoyetina 1/sangre , Angiopoyetina 1/metabolismo , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Macaca fascicularis , Mutación , Unión Proteica , Receptor TIE-2/genética , Receptor TIE-2/inmunología , Receptor TIE-2/metabolismoRESUMEN
Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes.