RESUMEN
The programmed death 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are negative regulators of T-cell immune function. Removal of these "brakes" in T cells results in increased activation of the immune system and controlling and eradicating tumor. The development of immune checkpoint inhibitors (ICIs) is a revolutionary milestone in tumor immunotherapy. Obtaining the atomic structure of the human immune checkpoint receptor/ICI therapeutic antibody complex is essential for understanding its inhibition mechanism and the rational design of improved biotherapeutics. In this chapter, we describe the methods for efficient production of extracellular domain of human immune checkpoint receptors and Fv fragments of ICI therapeutic antibodies in milligram quantities sufficient for structural studies, taking examples of the PD-1/pembrolizumab Fv and CTLA-4-ipilimumab Fv complexes.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Región Variable de Inmunoglobulina/farmacología , Neoplasias/tratamiento farmacológico , Difracción de Rayos X/métodos , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/uso terapéutico , Biosimilares Farmacéuticos/química , Biosimilares Farmacéuticos/farmacología , Biosimilares Farmacéuticos/uso terapéutico , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/aislamiento & purificación , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/uso terapéutico , Ipilimumab/química , Ipilimumab/farmacología , Ipilimumab/uso terapéutico , Modelos Moleculares , Neoplasias/inmunología , Neoplasias/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/aislamiento & purificación , Dominios Proteicos/genética , Dominios Proteicos/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Recent developments for therapeutic antibody have provided new options for cancer treatments. Modification of antibody molecules pursuing improvement of binding efficacy for Fcgamma receptors and enabling efficient recycling of antibody have been performed. Novel constructs of antibody possessing multivalent specificity and conjugated agents have also been developed. Based on exploration of new class of target molecules for therapeutic antibody, antibodies enhancing anti-tumor immunity such as anti-CTLA-4 antibody have appeared. These advancements would achieve more effective and safer therapy for various kinds of cancers.
Asunto(s)
Anticuerpos/uso terapéutico , Neoplasias/tratamiento farmacológico , Receptores de IgG/efectos de los fármacos , Anticuerpos/inmunología , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/aislamiento & purificación , Humanos , Inmunoconjugados/uso terapéutico , Neoplasias/inmunología , Receptores de IgG/inmunologíaRESUMEN
Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on P. pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from â¼2 to â¼8mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (K(D)=13nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model.
