RESUMEN
Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/fisiología , Receptores de Esteroides/genética , Secuencias Reguladoras de Ácidos Nucleicos , Células Cultivadas , Citocromo P-450 CYP3A/genética , Genoma Humano , Células Hep G2/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/fisiología , Histonas/metabolismo , Humanos , Polimorfismo de Nucleótido Simple , Receptor X de Pregnano , Regiones Promotoras Genéticas , Receptores de Esteroides/metabolismo , Reproducibilidad de los Resultados , Rifampin/farmacología , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The discovery of expression quantitative trait loci ("eQTLs") can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (nâ=â206) and Illumina (nâ=â60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (nâ=â266). We found that â¼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3'UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits.
Asunto(s)
Genoma Humano , Hígado/metabolismo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Regiones no Traducidas 3' , Factores de Edad , Población Negra , Mapeo Cromosómico , Clonación Molecular , Femenino , Perfilación de la Expresión Génica , Vectores Genéticos , Estudio de Asociación del Genoma Completo , Genotipo , Células Hep G2 , Hispánicos o Latinos , Humanos , Mediciones Luminiscentes , Masculino , Análisis de Componente Principal , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Factores Sexuales , Transfección , Población BlancaRESUMEN
Promoters are important regulatory elements that contain the necessary sequence features for cells to initiate transcription. To functionally characterize a large set of human promoters, we measured the transcriptional activities of 4575 putative promoters across eight cell lines using transient transfection reporter assays. In parallel, we measured gene expression in the same cell lines and observed a significant correlation between promoter activity and endogenous gene expression (r = 0.43). As transient transfection assays directly measure the promoting effect of a defined fragment of DNA sequence, decoupled from epigenetic, chromatin, or long-range regulatory effects, we sought to predict whether a promoter was active using sequence features alone. CG dinucleotide content was highly predictive of ubiquitous promoter activity, necessitating the separation of promoters into two groups: high CG promoters, mostly ubiquitously active, and low CG promoters, mostly cell line-specific. Computational models trained on the binding potential of transcriptional factor (TF) binding motifs could predict promoter activities in both high and low CG groups: average area under the receiver operating characteristic curve (AUC) of the models was 91% and exceeded the AUC of CG content by an average of 23%. Known relationships, for example, between HNF4A and hepatocytes, were recapitulated in the corresponding cell lines, in this case the liver-derived cell line HepG2. Half of the associations between tissue-specific TFs and cell line-specific promoters were new. Our study underscores the importance of collecting functional information from complementary assays and conditions to understand biology in a systematic framework.
Asunto(s)
Secuencia de Bases/fisiología , Especificidad de Órganos/genética , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Composición de Base/fisiología , Sitios de Unión/genética , Línea Celular , Biología Computacional/métodos , Epigénesis Genética/fisiología , Expresión Génica/genética , Expresión Génica/fisiología , Células Hep G2 , Factor Nuclear 4 del Hepatocito/genética , Humanos , Unión Proteica , Transcripción Genética , TransfecciónRESUMEN
MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Genes Relacionados con las Neoplasias , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Proteínas de Neoplasias/biosíntesis , ARN Neoplásico/metabolismo , Carcinoma Hepatocelular/genética , Línea Celular , Humanos , Neoplasias Hepáticas/genética , MicroARNs/genética , Proteínas de Neoplasias/genética , ARN Neoplásico/genéticaRESUMEN
The use of T-cell engagers (TCEs) to treat solid tumors is challenging, and several have been limited by narrow therapeutic windows due to substantial on-target, off-tumor toxicities due to the expression of low levels of target antigens on healthy tissues. Here, we describe TNB-928B, a fully human TCE that has a bivalent binding arm for folate receptor alpha (FRα) to selectively target FRα overexpressing tumor cells while avoiding the lysis of cells with low levels of FRα expression. The bivalent design of the FRα binding arm confers tumor selectivity due to low-affinity but high-avidity binding to high FRα antigen density cells. TNB-928B induces preferential effector T-cell activation, proliferation, and selective cytotoxic activity on high FRα expressing cells while sparing low FRα expressing cells. In addition, TNB-928B induces minimal cytokine release compared to a positive control TCE containing OKT3. Moreover, TNB-928B exhibits substantial ex vivo tumor cell lysis using endogenous T-cells and robust tumor clearance in vivo, promoting T-cell infiltration and antitumor activity in mouse models of ovarian cancer. TNB-928B exhibits pharmacokinetics similar to conventional antibodies, which are projected to enable favorable administration in humans. TNB-928B is a novel TCE with enhanced safety and specificity for the treatment of ovarian cancer.
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Anticuerpos Biespecíficos , Neoplasias Ováricas , Animales , Anticuerpos Biespecíficos/uso terapéutico , Carcinoma Epitelial de Ovario , Femenino , Receptor 1 de Folato/metabolismo , Receptor 1 de Folato/uso terapéutico , Humanos , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Linfocitos TRESUMEN
The therapeutic potential of targeting CD19 in B cell malignancies has garnered attention in the past decade, resulting in the introduction of novel immunotherapy agents. Encouraging clinical data have been reported for T cell-based targeting agents, such as anti-CD19/CD3 bispecific T-cell engager blinatumomab and chimeric antigen receptor (CAR)-T therapies, for acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma (B-NHL). However, clinical use of both blinatumomab and CAR-T therapies has been limited due to unfavorable pharmacokinetics (PK), significant toxicity associated with cytokine release syndrome and neurotoxicity, and manufacturing challenges. We present here a fully human CD19xCD3 bispecific antibody (TNB-486) for the treatment of B-NHL that could address the limitations of the current approved treatments. In the presence of CD19+ target cells and T cells, TNB-486 induces tumor cell lysis with minimal cytokine release, when compared to a positive control. In vivo, TNB-486 clears CD19+ tumor cells in immunocompromised mice in the presence of human peripheral blood mononuclear cells in multiple models. Additionally, the PK of TNB-486 in mice or cynomolgus monkeys is similar to conventional antibodies. This new T cell engaging bispecific antibody targeting CD19 represents a novel therapeutic that induces potent T cell-mediated tumor-cell cytotoxicity uncoupled from high levels of cytokine release, making it an attractive candidate for B-NHL therapy.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Citocinas/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Linfoma no Hodgkin/tratamiento farmacológico , Animales , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales Humanizados/farmacocinética , Antígenos CD19/inmunología , Antineoplásicos Inmunológicos/farmacocinética , Complejo CD3/antagonistas & inhibidores , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Técnicas de Cocultivo , Humanos , Células K562 , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/metabolismo , Macaca fascicularis , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Therapeutic options currently available for metastatic castration-resistant prostate cancer (mCRPC) do not extend median overall survival >6 months. Therefore, the development of novel and effective therapies for mCRPC represents an urgent medical need. T cell engagers (TCEs) have emerged as a promising approach for the treatment of mCRPC due to their targeted mechanism of action. However, challenges remain in the clinic due to the limited efficacy of TCEs observed thus far in solid tumors as well as the toxicities associated with cytokine release syndrome (CRS) due to the usage of high-affinity anti-CD3 moieties such as OKT3. METHODS: Using genetically engineered transgenic rats (UniRat and OmniFlic) that express fully human IgG antibodies together with an NGS-based antibody discovery pipeline, we developed TNB-585, an anti-CD3xPSMA TCE for the treatment of mCRPC. TNB-585 pairs a tumor-targeting anti-PSMA arm together with a unique, low-affinity anti-CD3 arm in bispecific format. We tested TNB-585 in T cell-redirected cytotoxicity assays against PSMA+ tumor cells in both two-dimensional (2D) cultures and three-dimensional (3D) spheroids as well as against patient-derived prostate tumor cells. Cytokines were measured in culture supernatants to assess the ability of TNB-585 to induce tumor killing with low cytokine release. TNB-585-mediated T cell activation, proliferation, and cytotoxic granule formation were measured to investigate the mechanism of action. Additionally, TNB-585 efficacy was evaluated in vivo against C4-2 tumor-bearing NCG mice. RESULTS: In vitro, TNB-585 induced activation and proliferation of human T cells resulting in the killing of PSMA+ prostate tumor cells in both 2D cultures and 3D spheroids with minimal cytokine release and reduced regulatory T cell activation compared with a positive control antibody that contains the same anti-PSMA arm but a higher affinity anti-CD3 arm (comparable with OKT3). In addition, TNB-585 demonstrated potent efficacy against patient-derived prostate tumors ex vivo and induced immune cell infiltration and dose-dependent tumor regression in vivo. CONCLUSIONS: Our data suggest that TNB-585, with its low-affinity anti-CD3, may be efficacious while inducing a lower incidence and severity of CRS in patients with prostate cancer compared with TCEs that incorporate high-affinity anti-CD3 domains.
Asunto(s)
Anticuerpos Biespecíficos/administración & dosificación , Antígenos de Superficie/inmunología , Complejo CD3/inmunología , Glutamato Carboxipeptidasa II/inmunología , Inmunoglobulina G/genética , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Animales , Anticuerpos Biespecíficos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Macaca fascicularis , Masculino , Ratones , Células PC-3 , Neoplasias de la Próstata Resistentes a la Castración/inmunología , Ratas , Ratas Transgénicas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαßγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rßγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule's in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/farmacología , Subunidad gamma Común de Receptores de Interleucina/agonistas , Subunidad beta del Receptor de Interleucina-2/agonistas , Linfocitos/efectos de los fármacos , Animales , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Subunidad gamma Común de Receptores de Interleucina/inmunología , Subunidad beta del Receptor de Interleucina-2/inmunología , Macaca fascicularis , Masculino , Ratones Endogámicos BALB CRESUMEN
Approximately 10% of genes in the human genome are distributed such that their transcription start sites are located less than 1 kb apart on opposite strands. These divergent gene pairs have a single intergenic segment of DNA, which in some cases appears to share regulatory elements, but it is unclear whether these regions represent functional bidirectional promoters or two overlapping promoters. A recent study showed that divergent promoters are enriched for consensus binding sequences of a small group of transcription factors, including the ubiquitous ets-family transcription factor GA-binding protein (GABP). Here we show that GABP binds to more than 80% of divergent promoters in at least one cell type. Furthermore, we demonstrate that GABP binding is correlated and associated with bidirectional transcriptional activity in a luciferase transfection assay. In addition, we find that the addition of a strict consensus GABP site into a set of promoters that normally function in only one direction significantly increases activity in the opposite direction in 67% of cases. Our findings demonstrate that GABP regulates the majority of divergent promoters and suggest that bidirectional transcriptional activity is mediated through GABP binding and transactivation at both divergent and nondivergent promoters.
Asunto(s)
Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Transcripción Genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Humanos , Luciferasas/metabolismo , Regiones Promotoras Genéticas/genética , Unión ProteicaRESUMEN
This perspective highlights the history and challenges of developing CD3-based bispecific T-cell engagers (TCEs) as cancer therapeutics as well as considerations and potential strategies for designing the next generation TCE molecules. The goal of this article is to raise awareness of natural T-cell biology and how to best harness the tumor cell killing capacity of cytotoxic T-cells with TCEs. In light of 30 years of concerted efforts to advance TCEs in early clinical development, many of the first-generation bispecific antibodies have exhibited lackluster safety, efficacy, and manufacturability profiles. As of January 2020, blinatumomab remains the only approved TCE. Many of the current set-backs in early clinical trials implicate the high-affinity CD3 binding domains employed and the respective bispecific platforms as potential culprits. The underlying conviction of the authors is that by taking corrective measures, TCEs can transform cancer therapy. Through openness, transparency, and much needed feedback from ongoing clinical studies, the field can continuously improve the design and effectiveness of next generation T-cell redirecting therapeutics.
RESUMEN
Chimeric antigen receptor (CAR)-expressing T cells targeting B-cell maturation antigen (BCMA) have activity against multiple myeloma, but improvements in anti-BCMA CARs are needed. We demonstrated recipient anti-CAR T-cell responses against a murine single-chain variable fragment (scFv) used clinically in anti-BCMA CARs. To bypass potential anti-CAR immunogenicity and to reduce CAR binding domain size, here we designed CARs with antigen-recognition domains consisting of only a fully human heavy-chain variable domain without a light-chain domain. A CAR designated FHVH33-CD8BBZ contains a fully human heavy-chain variable domain (FHVH) plus 4-1BB and CD3ζ domains. T cells expressing FHVH33-CD8BBZ exhibit similar cytokine release, degranulation, and mouse tumor eradication as a CAR that is identical except for substitution of a scFv for FHVH33. Inclusion of 4-1BB is critical for reducing activation-induced cell death and promoting survival of T cells expressing FHVH33-containing CARs. Our results indicate that heavy-chain-only anti-BCMA CARs are suitable for evaluation in a clinical trial.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Animales , Antígeno de Maduración de Linfocitos B/inmunología , Línea Celular Tumoral , Supervivencia Celular , Células Cultivadas , Humanos , Leucocitos Mononucleares/inmunología , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Dominios Proteicos/genética , Dominios Proteicos/inmunología , Ingeniería de Proteínas , Receptores Quiméricos de Antígenos/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
BACKGROUND: Regulation of gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific DNA-protein interactions. In this study we finely map the genome-wide targets of the CREB protein across all known and predicted human promoters, and characterize the functional consequences of a subset of these binding events using high-throughput reporter assays. To measure CREB binding, we used HaloCHIP, an antibody-free alternative to the ChIP method that utilizes the HaloTag fusion protein, and also high-throughput promoter-luciferase reporter assays, which provide rapid and quantitative screening of promoters for transcriptional activation or repression in living cells. RESULTS: In analysis of CREB genome-wide binding events using a comprehensive DNA microarray of human promoters, we observe for the first time that CREB has a strong preference for binding at bidirectional promoters and unlike unidirectional promoters, these binding events often occur downstream of transcription start sites. Comparison between HaloCHIP-chip and ChIP-chip data reveal this to be true for both methodologies, indicating it is not a bias of the technology chosen. Transcriptional data obtained from promoter-luciferase reporter arrays also show an unprecedented, high level of activation of CREB-bound promoters in the presence of the co-activator protein TORC1. CONCLUSION: These data suggest for the first time that TORC1 provides directional information when CREB is bound at bidirectional promoters and possible pausing of the CREB protein after initial transcriptional activation. Also, this combined approach demonstrates the ability to more broadly characterize CREB protein-DNA interactions wherein not only DNA binding sites are discovered, but also the potential of the promoter sequence to respond to CREB is evaluated.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Genes Reporteros/genética , Ensayos Analíticos de Alto Rendimiento , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Sitios de Unión , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , ADN/metabolismo , Genómica , Células HeLa , Humanos , Luciferasas/genética , Regiones Promotoras Genéticas/genética , Activación TranscripcionalRESUMEN
T-cell-recruiting bispecific antibodies (T-BsAbs) have shown potent tumor killing activity in humans, but cytokine release-related toxicities have affected their clinical utility. The use of novel anti-CD3 binding domains with more favorable properties could aid in the creation of T-BsAbs with improved therapeutic windows. Using a sequence-based discovery platform, we identified new anti-CD3 antibodies from humanized rats that bind to multiple epitopes and elicit varying levels of T-cell activation. In T-BsAb format, 12 different anti-CD3 arms induce equivalent levels of tumor cell lysis by primary T-cells, but potency varies by a thousand-fold. Our lead CD3-targeting arm stimulates very low levels of cytokine release, but drives robust tumor antigen-specific killing in vitro and in a mouse xenograft model. This new CD3-targeting antibody underpins a next-generation T-BsAb platform in which potent cytotoxicity is uncoupled from high levels of cytokine release, which may lead to a wider therapeutic window in the clinic.
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Anticuerpos Biespecíficos/metabolismo , Anticuerpos Monoclonales/metabolismo , Complejo CD3/inmunología , Neoplasias/terapia , Linfocitos T/inmunología , Animales , Animales Endogámicos , Antígenos de Neoplasias/inmunología , Citocinas/metabolismo , Citotoxicidad Inmunológica , Femenino , Humanos , Células Jurkat , Activación de Linfocitos , Ratones , Neoplasias/inmunología , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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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
Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.
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Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Hibridomas/inmunología , Animales , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos/genética , Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Genes de las Cadenas Pesadas de las Inmunoglobulinas/inmunología , Genes de las Cadenas Ligeras de las Inmunoglobulinas/genética , Genes de las Cadenas Ligeras de las Inmunoglobulinas/inmunología , HumanosRESUMEN
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
We used cDNA microarrays in a systematic study of the gene expression responses of HeLa cells and primary human lung fibroblasts to heat shock, endoplasmic reticulum stress, oxidative stress, and crowding. Hierarchical clustering of the data revealed groups of genes with coherent biological themes, including genes that responded to specific stresses and others that responded to multiple types of stress. Fewer genes increased in expression after multiple stresses than in free-living yeasts, which have a large general stress response program. Most of the genes induced by multiple diverse stresses are involved in cell-cell communication and other processes specific to higher organisms. We found substantial differences between the stress responses of HeLa cells and primary fibroblasts. For example, many genes were induced by oxidative stress and dithiothreitol in fibroblasts but not HeLa cells; conversely, a group of transcription factors, including c-fos and c-jun, were induced by heat shock in HeLa cells but not in fibroblasts. The dataset is freely available for search and download at http://microarray-pubs.stanford.edu/human_stress/Home.shtml.
Asunto(s)
Ditiotreitol/análogos & derivados , Regulación de la Expresión Génica/genética , Respuesta al Choque Térmico/genética , Estrés Oxidativo/genética , Adenocarcinoma/genética , Ditiotreitol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Familia de Multigenes/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Presión Osmótica , Tunicamicina/farmacología , Levaduras/genética , Levaduras/metabolismoRESUMEN
Previous work has implicated heat shock transcription factor 1 (HSF1) as the primary transcription factor responsible for the transcriptional response to heat stress in mammalian cells. We characterized the heat shock response of mammalian cells by measuring changes in transcript levels and assaying binding of HSF1 to promoter regions for candidate heat shock genes chosen by a combination of genome-wide computational and experimental methods. We found that many heat-inducible genes have HSF1 binding sites (heat shock elements, HSEs) in their promoters that are bound by HSF1. Surprisingly, for 24 heat-inducible genes, we detected no HSEs and no HSF1 binding. Furthermore, of 182 promoters with likely HSE sequences, we detected HSF1 binding at only 94 of these promoters. Also unexpectedly, we found 48 genes with HSEs in their promoters that are bound by HSF1 but that nevertheless did not show induction after heat shock in the cell types we examined. We also studied the transcriptional response to heat shock in fibroblasts from mice lacking the HSF1 gene. We found 36 genes in these cells that are induced by heat as well as they are in wild-type cells. These results provide evidence that HSF1 does not regulate the induction of every transcript that accumulates after heat shock, and our results suggest that an independent posttranscriptional mechanism regulates the accumulation of a significant number of transcripts.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Animales , Células Cultivadas , Biología Computacional , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Humanos , Células K562 , Ratones , Regiones Promotoras Genéticas/genética , Factores de Transcripción , Activación TranscripcionalRESUMEN
Although licensed human papillomavirus (HPV) vaccines are most efficacious in persons never infected with HPV, they also reduce infection and disease in previously infected subjects, indicating natural immunity is not entirely protective against HPV re-infection. The aim of this exploratory study was to examine the B cell memory elicited by HPV infection and evaluate whether vaccination merely boosts antibody (Ab) levels in previously infected subjects or also improves the quality of B cell memory. Toward this end, the memory B cells (Bmem) of five unvaccinated, HPV-seropositive subjects were isolated and characterized, and subject recall responses to a single HPV vaccine dose were analyzed. Vaccination boosted Ab levels 24- to 930-fold (median 77-fold) and Bmem numbers 3- to 27-fold (median 6-fold). In addition, Abs cloned from naturally elicited Bmem were generally non-neutralizing, whereas all those isolated following vaccination were neutralizing. Moreover, Ab and plasmablast responses indicative of memory recall responses were only observed in two subjects. These results suggest HPV vaccination augments both the magnitude and quality of natural immunity and demonstrate that sexually active persons could also benefit from HPV vaccination. This study may have important public policy implications, especially for the older 'catch-up' group within the vaccine's target population.