RESUMEN
Recent advances are described for the isolation and affinity maturation of antibodies that couple in vitro somatic hypermutation (SHM) with mammalian cell display, replicating key aspects of the adaptive immune system. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID). AID-directed SHM in vitro in non-B cells, combined with mammalian display of a library of human antibodies, initially naïve to SHM, can be used to isolate and affinity mature antibodies via iterative cycles of fluorescence-activated cell sorting (FACS) under increasingly stringent sort conditions. SHM observed in vitro closely resembles SHM observed in human antibodies in vivo in both mutation type and positioning in the antibody variable region. In addition, existing antibodies originating from mouse immunization, in vivo based libraries, or alternative display technologies such as phage can also be affinity matured in a similar manner. The display system has been developed to enable simultaneous high-level cell surface expression and secretion of the same protein through alternate splicing, where the displayed protein phenotype remains linked to genotype, allowing soluble secreted antibody to be simultaneously characterized in biophysical and cell-based functional assays. This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.
Asunto(s)
Anticuerpos Monoclonales/genética , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Antígenos/inmunología , Secuencia de Bases , Separación Celular , Cartilla de ADN/genética , Evolución Molecular Dirigida , Descubrimiento de Drogas , Citometría de Flujo , Biblioteca de Genes , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Ingeniería de ProteínasRESUMEN
A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human regions was used to isolate low-affinity antibodies to human ß nerve growth factor (hßNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K(D). This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.
Asunto(s)
Anticuerpos/química , Membrana Celular/metabolismo , Mutación , Hipermutación Somática de Inmunoglobulina , Secuencia de Aminoácidos , Linfocitos B/inmunología , Citometría de Flujo/métodos , Glicosilación , Células HEK293 , Humanos , Inmunoglobulina G/química , Inmunoglobulina M/química , Cinética , Datos de Secuencia Molecular , Factor de Crecimiento Nervioso/química , Homología de Secuencia de AminoácidoRESUMEN
The T cell receptor (TCR) protein is a heterodimer composed of an α chain and a ß chain. TCR genes undergo somatic DNA rearrangements to generate the diversity of T cell binding specificities needed for effective immunity. Recently, high-throughput immunosequencing methods have been developed to profile the TCR α (TCRA) and TCR ß (TCRB) repertoires. However, these methods cannot determine which TCRA and TCRB chains combine to form a specific TCR, which is essential for many functional and therapeutic applications. We describe and validate a method called pairSEQ, which can leverage the diversity of TCR sequences to accurately pair hundreds of thousands of TCRA and TCRB sequences in a single experiment. Our TCR pairing method uses standard laboratory consumables and equipment without the need for single-cell technologies. We show that pairSEQ can be applied to T cells from both blood and solid tissues, such as tumors.
Asunto(s)
Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T CD8-positivos/metabolismo , Humanos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.