RESUMEN
Multispecific antibodies have gained significant importance in a broad indication space due to their ability to engage multiple epitopes simultaneously and to thereby overcome therapeutic barriers. With growing therapeutic potential, however, the molecular complexity increases, thus intensifying the demand for innovative protein engineering and analytical strategies. A major challenge for multispecific antibodies is the correct assembly of light and heavy chains. Engineering strategies exist to stabilize the correct pairing, but typically individual engineering campaigns are required to arrive at the anticipated format. Mass spectrometry has proven to be a versatile tool to identify mispaired species. However, due to manual data analysis procedures, mass spectrometry is limited to lower throughputs. To keep pace with increasing sample numbers, we developed a high-throughput-capable mispairing workflow based on intact mass spectrometry with automated data analysis, peak detection, and relative quantification using Genedata Expressionist. This workflow is capable of detecting mispaired species of â¼1000 multispecific antibodies in three weeks and thus is applicable to complex screening campaigns. As a proof of concept, the assay was applied to engineering a trispecific antibody. Strikingly, the new setup has not only proved successful in mispairing analysis but has also revealed its potential to automatically annotate other product-related impurities. Furthermore, we could confirm the assay to be format-agnostic, as shown by analyzing several different multispecific formats in one run. With these comprehensive capabilities, the new automated intact mass workflow can be applied as a universal tool to detect and annotate peaks in a format-agnostic approach and in high-throughput, thus enabling complex discovery campaigns.
Asunto(s)
Anticuerpos , Espectrometría de Masas , EpítoposRESUMEN
We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro --> Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.
Asunto(s)
Osteoporosis/prevención & control , Ovariectomía , Tirotropina/administración & dosificación , Animales , Densidad Ósea , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Inyecciones , Ratones , Osteoclastos/metabolismo , Osteoporosis/metabolismo , Ratas , Receptores de Tirotropina/metabolismo , Proteínas Recombinantes/administración & dosificación , Estrés Mecánico , Tiroxina/sangreRESUMEN
Deamidation evaluation and mitigation is an important aspect of therapeutic antibody developability assessment. We investigated the structure and function of the Asn-Gly deamidation in a human anti-CD52 IgG1 antibody light chain complementarity-determining region 1, and risk mitigation through protein engineering. Antigen binding affinity was found to decrease about 400-fold when Asn33 was replaced with an Asp residue to mimic the deamidation product, suggesting significant impacts on antibody function. Other variants made at Asn33 (N33H, N33Q, N33H, N33R) were also found to result in significant loss of antigen binding affinity. The co-crystal structure of the antigen-binding fragment bound to a CD52 peptide mimetic was solved at 2.2Å (PDB code 6OBD), which revealed that Asn33 directly interacts with the CD52 phosphate group via a hydrogen bond. Gly34, but sits away from the binding interface, rendering it more amendable to mutagenesis without affecting affinity. Saturation mutants at Gly34 were prepared and subjected to forced deamidation by incubation at elevated pH and temperature. Three mutants (G34R, G34K and G34Q) showed increased resistance to deamidation by LC-MS peptide mapping, while maintaining high binding affinity to CD52 antigen measured by Biacore. A complement -dependent cytotoxicity assay indicated that these mutants function by triggering antibody effector function. This study illustrates the importance of structure-based design and extensive mutagenesis to mitigate antibody developability issues.
Asunto(s)
Anticuerpos Monoclonales/química , Antígeno CD52/química , Regiones Determinantes de Complementariedad/química , Inmunoglobulina G/química , Cadenas Ligeras de Inmunoglobulina/química , Amidas/química , Anticuerpos Monoclonales/genética , Citotoxicidad Celular Dependiente de Anticuerpos , Asparagina/genética , Bioingeniería , Antígeno CD52/genética , Antígeno CD52/inmunología , Regiones Determinantes de Complementariedad/genética , Cristalografía por Rayos X , Humanos , Inmunoglobulina G/genética , Cadenas Ligeras de Inmunoglobulina/genética , Mutagénesis Sitio-Dirigida , Mapeo Peptídico , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
UNLABELLED: We show the systemic administration of low levels of TSH increases bone volume and improves bone microarchitecture and strength in aged OVX rats. TSH's actions are mediated by its inhibitory effects on RANKL-induced osteoclast formation and bone resorption coupled with stimulatory effects on osteoblast differentiation and bone formation, suggesting TSH directly affects bone remodeling in vivo. INTRODUCTION: Thyroid-stimulating hormone (TSH) receptor haploinsufficient mice with normal circulating thyroid hormone levels have reduced bone mass, suggesting that TSH directly affects bone remodeling. We examined whether systemic TSH administration restored bone volume in aged ovariectomized (OVX) rats and influenced osteoclast formation and osteoblast differentiation in vitro. MATERIALS AND METHODS: Sprague-Dawley rats were OVX at 6 months, and TSH therapy was started immediately after surgery (prevention mode; n = 80) or 7 mo later (restoration mode; n = 152). Hind limbs and lumbar spine BMD was measured at 2- or 4-wk intervals in vivo and ex vivo on termination at 8-16 wk. Long bones were subjected to microCT, histomorphometric, and biomechanical analyses. The direct effect of TSH was examined in osteoclast and osteoblast progenitor cultures and established rat osteosarcoma-derived osteoblastic cells. Data were analyzed by ANOVA Dunnett test. RESULTS: In the prevention mode, low doses (0.1 and 0.3 microg) of native rat TSH prevented the progressive bone loss, and importantly, did not increase serum triiodothyroxine (T3) and thyroxine (T4) levels in aged OVX rats. In restoration mode, animals receiving 0.1 and 0.3 microg TSH had increased BMD (10-11%), trabecular bone volume (100-130%), trabecular number (25-40%), trabecular thickness (45-60%), cortical thickness (5-16%), mineral apposition and bone formation rate (200-300%), and enhanced mechanical strength of the femur (51-60%) compared with control OVX rats. In vitro studies suggest that TSH's action is mediated by its inhibitory effects on RANKL-induced osteoclast formation, as shown in hematopoietic stem cells cultivated from TSH-treated OVX rats. TSH also stimulates osteoblast differentiation, as shown by effects on alkaline phosphatase activity, osteocalcin expression, and mineralization rate. CONCLUSIONS: These results show for the first time that systemically administered TSH prevents bone loss and restores bone mass in aged OVX rats through both antiresorptive and anabolic effects on bone remodeling.
Asunto(s)
Huesos/efectos de los fármacos , Ovariectomía , Tirotropina/farmacología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Fenómenos Biomecánicos , Densidad Ósea/efectos de los fármacos , Huesos/anatomía & histología , Huesos/química , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Femenino , Fémur/anatomía & histología , Fémur/química , Fémur/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Miembro Posterior/química , Miembro Posterior/efectos de los fármacos , Vértebras Lumbares/química , Vértebras Lumbares/efectos de los fármacos , Vértebras Lumbares/fisiología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteoclastos/química , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoporosis/fisiopatología , Osteoporosis/prevención & control , Ratas , Ratas Sprague-Dawley , Tirotropina/uso terapéutico , Tiroxina/sangre , Tibia/química , Tibia/efectos de los fármacos , Tibia/fisiología , Triyodotironina/sangreRESUMEN
The development of an effective AIDS vaccine has been challenging because of viral genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs). We engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to those of human bnAbs, and conferred complete immunity against a mixture of simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and they may be applicable for treatment of diverse diseases, including infections, cancer, and autoimmunity.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/farmacocinética , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Antígenos CD4/inmunología , Cristalografía por Rayos X , Anticuerpos Anti-VIH/administración & dosificación , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Humanos , Macaca mulatta , Ingeniería de Proteínas , Síndrome de Inmunodeficiencia Adquirida del Simio/sangreRESUMEN
A bioassay was developed to assess the potency of TGFbeta antagonists by measuring IL-11 production in TGFbeta-1 treated human lung epithelial cells (A549). The production of IL-11 by A549 cells, measured by ELISA, was shown to be proportional to the TGFbeta-1 concentration. The A549 cells were responsive to all three isoforms of TGFbeta in the range of 3.0 ng/mL to 1.4 pg/mL, with an 18 to 24 h exposure time found to be within the linear portion of the bioassay response range. The Effective Dose at 80% of the maximal response (ED80) of TGFbeta-1 determined for the assay was 0.3 ng/mL. With this level of TGFbeta-1, a human anti-TGFbeta-1 antibody (CAT-192) yielded an approximate median Inhibitory Concentration (IC50) value of 3 microg/mL. To investigate assay specificity, alternate members of the TGFbeta superfamily were evaluated. Recombinant human activin B, inhibin A and BMP-2 (Bone Morphogenetic Protein-2) did not elicit a significant IL-11 response from the A549 cell line. Bioassay qualification was performed to obtain estimates of precision and accuracy, as well as to establish plate validity criteria. Assay precision was estimated at 30%, while the accuracy was +/-13%. Additionally, the ability of the A549 cell potency assay to detect potency differences in structurally modified samples was investigated.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Bioensayo/métodos , Interleucina-11/análisis , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Interleucina-11/biosíntesis , Isoformas de Proteínas , Sensibilidad y Especificidad , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of beta-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitro activity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. beta-Subunit C-terminal truncation up to residue 113 did not effect the in vitro activity of the hormone. The relationship between the heterogeneity of oligosaccharide structures on rhTSH and specific activity of the glycoprotein hormone was also examined. Oligosaccharide profiles were generated for preparations of rhTSH containing similar sialic acid levels. A weak correlation was observed between relative levels of monosialylated biantennary, bisialylated biantennary, and trisialylated triantennary oligosaccharide species and in vitro activity of the recombinant hormone (p < 0.05). To examine the effect of chemically induced methionine oxidation on the activity of rhTSH, the hormone was treated with tert-butyl hydroperoxide and then characterized. Using peptide mapping and mass spectrometry, the degree of oxidation of the five methionine residues within rhTSH was measured. Met-71 in the alpha-subunit was the most susceptible to oxidation whereas Met-9 in the beta-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the beta-subunit, and Met-29 and Met-47 in the alpha-subunit were less than half of that observed for Met-71. The in vitro activity of rhTSH initially declined with increasing oxidation; however, the loss in activity plateaued at approximately 50% of the control sample activity. In summary, despite the possible effects that posttranslational modifications may have on the bioactivity of a protein, a limited degree of variation in bioactivity was observed for the rhTSH preparations described in this study.
Asunto(s)
Procesamiento Proteico-Postraduccional , Tirotropina/metabolismo , Cromatografía Liquida , Dicroismo Circular , Fluorescencia , Humanos , Espectrometría de Masas , Oligosacáridos/análisis , Oxidación-Reducción , Mapeo Peptídico , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Tirotropina/química , Tirotropina/efectos de los fármacos , terc-Butilhidroperóxido/farmacologíaRESUMEN
Thyroid-stimulating hormone (TSH) and follicle-stimulating hormone (FSH) have both been recently implicated in bone remodelling. Clinical evidence, as well as data from TSH receptor and thyroid hormone receptor knockout mice, suggest that TSH has a direct effect on skeletal homeostasis, although some data are conflicting. Recently, the exogenous administration of TSH has been shown to positively impact bone in oophrectomised rats. These data, along with their potential implications for the treatment of severe osteoporosis, are discussed.
Asunto(s)
Remodelación Ósea/fisiología , Hormona Folículo Estimulante/fisiología , Tirotropina/fisiología , Animales , Densidad Ósea/fisiología , Modelos Animales de Enfermedad , Ratones , Osteoporosis/fisiopatología , RatasRESUMEN
Thyroid stimulating hormone (TSH), a pituitary glycoprotein hormone, is a potent inducer of intracellular cAMP production. Two methods for measuring TSH bioactivity were evaluated and compared. One assay is based on using a radioimmunoassay (RIA) to measure the recombinant human TSH-induced increase in cAMP using a bovine thyroid membrane isolate. The other is based on a Chinese hamster ovary (CHO) cell line that has been transfected with the TSH receptor and a cAMP-responsive luciferase reporter. The within-assay coefficient of variation for the membrane-based assay was determined to be approximately 35% compared with approximately 25% for the cell-based assay. Twenty-one preparations of recombinant human TSH (rhTSH) were tested using both methods. No significant difference was detected between the data sets and no assay bias was present. Both assay systems provide a suitable means for measuring the activity of rhTSH. The advantage of the membrane-based assay is the relatively small quantity of TSH needed for analysis. However, the average time required to analyse a sample using the membrane-based method was more than twice as long as that needed to test a sample in the cell-based assay. Other advantages of the cell-based method include the use of a 96-well format, which facilitates the analysis of several concentrations of rhTSH within one assay plate, and the use of a non-radioactive endpoint.