RESUMEN
Bispecific antibodies have become important formats for therapeutic discovery. They allow for potential synergy by simultaneously engaging two separate targets and enable new functions that are not possible to achieve by using a combination of two monospecific antibodies. Antagonistic antibodies dominate drug discovery today, but only a limited number of agonistic antibodies (i.e. those that activate receptor signaling) have been described. For receptors formed by two components, engaging both of these components simultaneously may be required for agonistic signaling. As such, bispecific antibodies may be particularly useful in activating multicomponent receptor complexes. Here, we describe a biparatopic (i.e. targeting two different epitopes on the same target) format that can activate the endocrine fibroblast growth factor (FGF) 21 receptor (FGFR) complex containing ß-Klotho and FGFR1c. This format was constructed by grafting two different antigen-specific VH domains onto the VH and VL positions of an IgG, yielding a tetravalent binder with two potential geometries, a close and a distant, between the two paratopes. Our results revealed that the biparatopic molecule provides activities that are not observed with each paratope alone. Our approach could help address the challenges with heterogeneity inherent in other bispecific formats and could provide the means to adjust intramolecular distances of the antibody domains to drive optimal activity in a bispecific format. In conclusion, this format is versatile, is easy to construct and produce, and opens a new avenue for agonistic antibody discovery and development.
Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Sitios de Unión de Anticuerpos , Línea Celular , Epítopos/metabolismo , Humanos , Proteínas Klotho , Ligandos , Ratas , Anticuerpos de Cadena Única/metabolismoRESUMEN
STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported. Leveraging a newly-identified monoclonal antibody that recognizes a conformation-sensitive epitope nested in the second extracellular loop of STEAP2, we demonstrate that the epitope formation was dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Monitoring the STEAP2-dependent internalization of this antibody uncovered STEAP2's rapid internalization from the cell surface and their subsequence trafficking to the Golgi region and endosome-like puncta. Acute inhibition of endocytosis also increased the detectable amount of STEAP2 at the plasma membrane. Collectively, these experiments demonstrate that an intricate balance of membrane flux between the secretory and endocytic pathways underlies the characteristic broad subcellular localization of STEAP2. By using a cell-based assay that detects the metalloreductase functions of cell surface-localizing STEAP4, STEAP2's metalloreductase activities were not detectable, suggesting that its enzymatic function is suppressed at the plasma membrane. The conformational modulation of STEAP2 by the local membrane cholesterol content can therefore serve as a potential mechanism to modulate STEAP2 function in a compartment-restricted manner, by coupling a pre-existing difference in cholesterol content among different cellular membranes to a dynamic trafficking process leading to broad subcellular distribution.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Oxidorreductasas/metabolismo , Animales , Transporte Biológico/fisiología , Movimiento Celular/fisiología , Endocitosis/fisiología , Endosomas/metabolismo , Humanos , Ratones , Conformación Molecular , Transporte de ProteínasRESUMEN
p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared with apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress induction. To understand the basis for such distinct temporal assembly of transcription complexes, we examined the role of core promoter structures in this process. We find that the p21 core promoter directs rapid, TATA box-dependent assembly of RNAP II preinitiation complexes (PICs), but permits few rounds of RNAP II reinitiation. In contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. We define a downstream element within the Fas/APO1 core promoter that is essential for its activation, and identify nuclear transcription factor Y (NF-Y) as its binding partner. NF-Y acts as a bifunctional transcription factor that regulates basal expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent reinitiation. These features are intrinsic, DNA-encoded properties of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes upon stress.
Asunto(s)
Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Factor de Unión a CCAAT/metabolismo , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión , Estrés Fisiológico/genética , TATA Box/genética , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
The tumor-associated antigen STEAP1 is a potential therapeutic target that is expressed in most prostate tumors and at increased levels in metastatic castration-resistant prostate cancer (mCRPC). We developed a STEAP1-targeted XmAb 2+1 T-cell engager (TCE) molecule, AMG 509 (also designated xaluritamig), that is designed to redirect T cells to kill prostate cancer cells that express STEAP1. AMG 509 mediates potent T cell-dependent cytotoxicity of prostate cancer cell lines in vitro and promotes tumor regression in xenograft and syngeneic mouse models of prostate cancer in vivo. The avidity-driven activity of AMG 509 enables selectivity for tumor cells with high STEAP1 expression compared with normal cells. AMG 509 is the first STEAP1 TCE to advance to clinical testing, and we report a case study of a patient with mCRPC who achieved an objective response on AMG 509 treatment. SIGNIFICANCE: Immunotherapy in prostate cancer has met with limited success due to the immunosuppressive microenvironment and lack of tumor-specific targets. AMG 509 provides a targeted immunotherapy approach to engage a patient's T cells to kill STEAP1-expressing tumor cells and represents a new treatment option for mCRPC and potentially more broadly for prostate cancer. See related commentary by Hage Chehade et al., p. 20. See related article by Kelly et al., p. 76. This article is featured in Selected Articles from This Issue, p. 5.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias de la Próstata Resistentes a la Castración , Masculino , Ratones , Animales , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/patología , Linfocitos T , Inmunoterapia , Anticuerpos Biespecíficos/uso terapéutico , Microambiente Tumoral , Antígenos de Neoplasias , Oxidorreductasas/uso terapéuticoRESUMEN
Cell division in animals must be regulated; during development, for example, proliferation often occurs in spatially and temporally restricted patterns, and loss of mitotic control underlies cancer. The epidermal growth factor receptor (EGFR) has been implicated extensively in the control of cell proliferation in metazoans; in addition, hyperactivity of the EGFR and its three relatives, ErbB2-ErbB4, are implicated in many cancers. But little is known about how these receptor tyrosine kinases regulate the cell cycle. In the developing Drosophila melanogaster imaginal eye disc, there is a single patterned mitosis that sweeps across the eye disc epithelium in the third larval instar. This 'second mitotic wave' is triggered by EGFR signalling and depends on expression of String, the Drosophila homologue of Cdc25 phosphatase, the ultimate regulator of mitosis in all eukaryotic cells. Here we show that two antagonistic transcriptional regulators, Pointed, an activator, and Tramtrack69, a repressor, directly regulate the transcription of string. The activity of at least one of these regulators, Pointed, is controlled by EGFR signalling. This establishes a molecular mechanism for how intercellular signalling can control string expression, and thereby cell proliferation.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes erbB-1 , Mitosis/genética , Fosfoproteínas Fosfatasas/genética , Proteínas Tirosina Fosfatasas , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Animales , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Drosophila melanogaster/embriología , Ojo/citología , Ojo/embriología , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso , Transducción de Señal/genética , Factores de Transcripción , Transcripción GenéticaRESUMEN
PURPOSE: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. EXPERIMENTAL DESIGN: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). RESULTS: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients. CONCLUSIONS: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Monoclonales , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares , Proteínas de la Membrana , Carcinoma Pulmonar de Células Pequeñas , Linfocitos T , Animales , Femenino , Humanos , Ratones , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas de la Membrana/antagonistas & inhibidores , Ratones Endogámicos NOD , Ratones SCID , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/inmunología , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Linfocitos B/inmunología , Inmunoglobulina G/inmunología , Animales , Linfocitos B/citología , Células CHO , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Humanos , RatonesRESUMEN
Therapeutic molecules derived from antibodies have become a dominant class of drugs used to treat human disease. Increasingly, therapeutic antibodies are discovered using transgenic animal systems that have been engineered to express human antibodies. While the engineering details differ, these platforms share the ability to raise an immune response that is comprised of antibodies with fully human idiotypes. Although the predominant transgenic host species has been mouse, the genomes of rats, rabbits, chickens, and cows have also been modified to express human antibodies. The creation of transgenic animal platforms expressing human antibody repertoires has revolutionized therapeutic antibody drug discovery. The observation that the immune systems of these animals are able to recognize and respond to a wide range of therapeutically relevant human targets has led to a surge in antibody-derived drugs in current development. While the clinical success of fully human monoclonal antibodies derived from transgenic animals is well established, recent trends have seen increasingly stringent functional design goals and a shift in difficulty as the industry attempts to tackle the next generation of disease-associated targets. These challenges have been met with a number of novel approaches focused on the generation of large, high-quality, and diverse antibody repertoires. In this perspective, we describe some of the strategies and considerations we use for manipulating the immune systems of transgenic animal platforms (such as XenoMouse®) with a focus on maximizing the diversity of the primary response and steering the ensuing antibody repertoire toward a desired outcome.
Asunto(s)
Animales Modificados Genéticamente/inmunología , Anticuerpos/inmunología , Formación de Anticuerpos , Linfocitos B/inmunología , Animales , HumanosRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic ß-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Obesidad/tratamiento farmacológico , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Adipocitos/metabolismo , Animales , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Dieta , Quimioterapia Combinada , Conducta Alimentaria , Polipéptido Inhibidor Gástrico/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos Similares al Glucagón/análogos & derivados , Péptidos Similares al Glucagón/farmacología , Péptidos Similares al Glucagón/uso terapéutico , Humanos , Fragmentos Fc de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Liraglutida/farmacología , Liraglutida/uso terapéutico , Ratones Obesos , Obesidad/patología , Primates , Receptores de la Hormona Gastrointestinal/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Respiración , Aumento de Peso/efectos de los fármacos , Pérdida de Peso/efectos de los fármacosRESUMEN
Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na V1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na V1.7 DII voltage sensor domain and block human Na V1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na V1.7 DII voltage sensor domain via ELISA, and does not bind Na V1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na V1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na V1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na V1.7 target sequences or specifically inhibit Na V1.7 current.
RESUMEN
The Siah proteins, mammalian homologues of the Drosophila Sina protein, function as ubiquitin-protein isopeptide ligase enzymes to target a wide range of cellular proteins for degradation. We report here a novel Drosophila protein that is homologous to Sina, named Sina-Homologue (SinaH). We show that it can direct the degradation of the transcriptional repressor Tramtrack (Ttk) using two different mechanisms. One is similar to Sina and requires the adaptor Phyllopod, and the other is a novel mechanism of recognition. This novel mode of targeting for degradation is specific for the 69-kDa Ttk isoform, Ttk69. Ttk69 contains a region that is required for binding of SinaH and for SinaH-directed degradation. This region contains an AXVXP motif, which is the consensus sequence found in Siah substrate proteins. These results suggest that degradation directed by SinaH differs from that directed by Sina and is more similar to that found in vertebrates. We speculate that SinaH may be involved in regulating the levels of developmentally important transcription factors.
Asunto(s)
Proteínas de Drosophila/farmacología , Drosophila/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Cartilla de ADN , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Datos de Secuencia Molecular , Proteínas Represoras/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacosRESUMEN
Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 in response to hormonal stimuli triggers cellular gene expression via the recruitment of the histone acetylase coactivator paralogs CREB binding protein (CBP) and p300 to the promoter. The NMR structure of the CREB:CBP complex, using relevant interaction domains called KID and KIX, respectively, reveals a shallow hydrophobic groove on the surface of KIX that accommodates an amphipathic helix in phospho (Ser-133) KID. Using an NMR-based screening approach on a preselected small-molecule library, we identified several compounds that bind to different surfaces on KIX. One of these, KG-501 (2-naphthol-AS-E-phosphate), targeted a surface distal to the CREB binding groove that includes Arg-600, a residue that is required for the CREB:CBP interaction. When added to live cells, KG-501 disrupted the CREB: CBP complex and attenuated target gene induction in response to cAMP agonist. These results demonstrate the ability of small molecules to interfere with second-messenger signaling cascades by inhibiting specific protein-protein interactions in the nucleus.