RESUMEN
We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Descubrimiento de Drogas/métodos , Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Genes de las Cadenas Ligeras de las Inmunoglobulinas/genética , Cadenas kappa de Inmunoglobulina/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Antígenos/administración & dosificación , Antígenos/inmunología , Linfocitos B/inmunología , Centro Germinal/citología , Centro Germinal/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/genética , Modelos Animales , Ratas , Ratas Sprague-Dawley , Ratas TransgénicasRESUMEN
Heavy chain-only antibodies (HCAbs) do not associate with light chains and their VH regions are functional as single domains, forming the smallest active antibody fragment. These VH regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human VH regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains. Here, we present an innovative platform for the rapid development of diverse sets of human HCAbs that have been selected in vivo. Our unique approach combines antibody repertoire analysis with immunization of transgenic rats, called UniRats, that produce chimeric HCAbs with fully human VH domains in response to an antigen challenge. UniRats express HCAbs from large transgenic loci representing the entire productive human heavy chain V(D)J repertoire, mount robust immune responses to a wide array of antigens, exhibit diverse V gene usage and generate large panels of stable, high affinity, antigen-specific molecules.
Asunto(s)
Anticuerpos/química , Anticuerpos/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Ingeniería de Proteínas/métodos , Animales , Afinidad de Anticuerpos , Antígenos/inmunología , Linfocitos B/inmunología , Células CHO , Cricetulus , Cristalografía , Citometría de Flujo , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunización , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Estructura Secundaria de Proteína , Ratas , Ratas Transgénicas , Anticuerpos de Dominio Único/químicaRESUMEN
Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6-9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.