RESUMO
High quality biological reagents are a prerequisite for pharmacological research. Herein a protein production screening approach, including quality assessment methods, for protein-based discovery research is presented. Trends from 2895 expression constructs representing 253 proteins screened in mammalian and bacterial hosts-91% of which are successfully expressed and purified-are discussed. Mammalian expression combined with the use of solubility-promoting fusion proteins is deemed suitable for most targets. Furthermore, cases utilizing stable cell line generation and choice of fusion protein for higher yield and quality of difficult-to-produce proteins (Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) and Neurturin) are presented and discussed. In the case of Neurturin, choice of fusion protein impacted the target binding 80-fold. These results highlight the need for exploration of construct designs and careful Quality Control (QC) of difficult-to-produce protein reagents.
Assuntos
Mamíferos , Neurturina , Animais , Linhagem Celular , Proteínas Recombinantes de Fusão/genéticaRESUMO
Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.
RESUMO
OBJECTIVE: Reelin (RELN) is a large glycoprotein involved in synapse maturation and neuronal organization throughout development. Deficits in RELN signaling contribute to multiple psychological disorders, such as autism spectrum disorder, schizophrenia, and bipolar disorder. Nutritional stress alters RELN expression in brain regions associated with these disorders; however, the involvement of RELN in the neural circuits involved in energy metabolism is unknown. The RELN receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR) are involved in lipid metabolism and expressed in the hypothalamus. Here we explored the involvement of RELN in hypothalamic signaling and the impact of diet-induced obesity (DIO) on this system. METHODS: Adult male mice were fed a chow diet or maintained on a high-fat diet (HFD) for 12-16 weeks. HFD-fed DIO mice exhibited decreased ApoER2 and VLDLR expression and increased RELN protein in the hypothalamus. Electrophysiology was used to determine the mechanism by which the central fragment of RELN (CF-RELN) acts on arcuate nucleus (ARH) satiety-promoting proopiomelanocortin (POMC) neurons and the impact of DIO on this circuitry. RESULTS: CF-RELN exhibited heterogeneous presynaptic actions on inhibitory inputs onto ARH-POMC-EGFP neurons and consistent postsynaptic actions. Additionally, central administration of CF-RELN caused a significant increase in ARH c-Fos expression and an acute decrease in food intake and body weight. CONCLUSIONS: We conclude that RELN signaling is modulated by diet, that RELN is involved in synaptic signaling onto ARH-POMC neurons, and that altering central CF-RELN levels can impact food intake and body weight.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Obesidade/metabolismo , Pró-Opiomelanocortina/metabolismo , Serina Endopeptidases/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/induzido quimicamente , Proteína ReelinaAssuntos
Diabetes Mellitus Tipo 1 , Animais , Antígenos CD40 , Ligante de CD40 , Camundongos , Camundongos Endogâmicos NOD , PeptídeosRESUMO
Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope "bins" based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Interleucinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Autoimunidade , Linfócitos B/imunologia , Células CHO , Linhagem Celular , Cricetinae , Epitopos/imunologia , Humanos , Interleucinas/administração & dosagem , Interleucinas/química , Interleucinas/genética , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/imunologia , Coelhos , Ratos , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/imunologia , Linfócitos T/imunologiaRESUMO
Members of the CD28 family play important roles in regulating T-cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and Tregs. The nectin-family proteins CD155 and CD112 serve as counter-structures for VSTM3, and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well-characterized CD28-CTLA-4-CD80-CD86 network. In the same way that soluble CTLA-4 can be used to block T-cell responses, we show that soluble Vstm3 attenuates T-cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T-cell responses.
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Antígenos CD28/imunologia , Receptores Imunológicos/imunologia , Linfócitos T/imunologia , Animais , Doenças Autoimunes/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Humanos , Camundongos , Ratos , Receptores Imunológicos/deficiência , Linfócitos T/químicaRESUMO
Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRbeta and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Biespecíficos/farmacologia , Imunoterapia , Neoplasias Experimentais/tratamento farmacológico , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos , Inibidores da Angiogênese/administração & dosagem , Animais , Anticorpos Biespecíficos/administração & dosagem , Linhagem Celular Tumoral , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Humanos , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Neoplasias Experimentais/imunologia , Neovascularização Fisiológica/efeitos dos fármacos , Ligação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Receptor beta de Fator de Crescimento Derivado de Plaquetas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.
Assuntos
Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Interleucina-17/imunologia , Interleucina-23/imunologia , Engenharia de Proteínas , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacocinética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Bases de Dados de Proteínas , Escherichia coli/genética , Feminino , Meia-Vida , Humanos , Cinética , Camundongos , Estabilidade Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
Corticotroph-derived glycoprotein hormone (CGH), also referred to as thyrostimulin, is a noncovalent heterodimer of glycoprotein hormone alpha 2 (GPHA2) and glycoprotein hormone beta 5 (GPHB5). Here, we demonstrate that both subunits of CGH are expressed in the corticotroph cells of the human anterior pituitary, as well as in skin, retina, and testis. CGH activates the TSH receptor (TSHR); (125)I-CGH binding to cells expressing TSHR is saturable, specific, and of high affinity. In competition studies, unlabeled CGH is a potent competitor for (125)I-TSH binding, whereas unlabeled TSH does not compete for (125)I-CGH binding. Binding and competition analyses are consistent with the presence of two binding sites on the TSHR transfected baby hamster kidney cells, one that can interact with either TSH or CGH, and another that binds CGH alone. Transgenic overexpression of GPHB5 in mice produces elevations in serum T(4) levels, reductions in body weight, and proptosis. However, neither transgenic overexpression of GPHA2 nor deletion of GPHB5 produces an overt phenotype in mice. In vivo administration of CGH to mice produces a dose-dependent hyperthyroid phenotype including elevation of T(4) and hypertrophy of cells within the inner adrenal cortex. However, the distinctive expression patterns and binding characteristics of CGH suggest that it has endogenous biological roles that are discrete from those of TSH.