RESUMEN
V-set immunoglobulin domain-containing 4 (VSIG4) is a B7 family protein with known roles as a C3 fragment complement receptor involved in pathogen clearance and a negative regulator of T cell activation by an undetermined mechanism. VSIG4 expression is specific for tumor-associated and select tissue-resident macrophages. Increased expression of VSIG4 has been associated with worse survival in multiple cancer indications. Based upon computational analysis of transcript data across thousands of tumor and normal tissue samples, we hypothesized that VSIG4 has an important role in promoting M2-like immune suppressive macrophages and that targeting VSIG4 could relieve VSIG4-mediated macrophage suppression by repolarizing tumor-associated macrophages (TAMs) to an inflammatory phenotype. We have also observed a cancer-specific pattern of VSIG4 isoform distribution, implying a change in the functional regulation in cancer. Through a series of in vitro, in vivo, and ex vivo assays we demonstrate that anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Anti-VSIG4 antibodies induce pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages. Across patient-derived tumor samples from multiple tumor types, anti-VSIG4 treatment resulted in the upregulation of cytokines associated with TAM repolarization and T cell activation and chemokines involved in immune cell recruitment. VSIG4 blockade is also efficacious in a syngeneic mouse model as monotherapy as it enhances efficacy in combination with anti-PD-1, and the effect is dependent on the systemic availability of CD8+ T cells. Thus, VSIG4 represents a promising new target capable of triggering an anti-cancer response via multiple key immune mechanisms.
Asunto(s)
Neoplasias , Macrófagos Asociados a Tumores , Animales , Humanos , Ratones , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Línea Celular Tumoral , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Citocinas/metabolismo , Femenino , Receptores de ComplementoRESUMEN
Clinical antibodies are an important class of drugs for the treatment of both chronic and acute diseases. Their manufacturability is subject to evaluation to ensure product quality and efficacy. One critical quality attribute is deamidation, a non-enzymatic process that is observed to occur during thermal stress, at low or high pH, or a combination thereof. Deamidation may induce antibody instability and lead to aggregation, which may pose immunogenicity concerns. The introduction of a negative charge via deamidation may impact the desired therapeutic function (i) within the complementarity-determining region, potentially causing loss of efficacy; or (ii) within the fragment crystallizable region, limiting the effector function involving antibody-dependent cellular cytotoxicity. Here we describe a transformative solution that allows for a comparative assessment of deamidation and its impact on stability and aggregation. The innovative streamlined method evaluates the intact protein in its formulation conditions. This breakthrough platform technology is comprised of a quantum cascade laser microscope, a slide cell array that allows for flexibility in the design of experiments, and dedicated software. The enhanced spectral resolution is achieved using two-dimensional correlation, co-distribution, and two-trace two-dimensional correlation spectroscopies that reveal the molecular impact of deamidation. Eight re-engineered immunoglobulin G4 scaffold clinical antibodies under control and forced degradation conditions were evaluated for deamidation and aggregation. We determined the site of deamidation, the overall extent of deamidation, and where applicable, whether the deamidation event led to self-association or aggregation of the clinical antibody and the molecular events that led to the instability. The results were confirmed using orthogonal techniques for four of the samples.
RESUMEN
Immunotherapy checkpoint inhibitors, such as antibodies targeting PD-1 and CTLA-4, have demonstrated the potential of harnessing the immune system to treat cancer. However, despite encouraging results particularly with respect to survival, only a minority of patients benefit from these therapies. In clinical studies aimed at understanding changes in the immune system following immunotherapy treatment, ICOS (Inducible T cell CO-Stimulator) was shown to be significantly up-regulated on CD4+ T cells and this was associated with clinical activity, indicating that ICOS stimulatory activity may be beneficial in the treatment of solid tumors. In this report, we describe the generation of specific, species cross-reactive, agonist antibodies to ICOS, including the humanized clinical candidate, JTX-2011 (vopratelimab). Preclinical studies suggest that the ICOS stimulating antibodies require Fc receptor cross-linking for optimal agonistic activity. Notably, the ICOS antibodies do not exhibit superagonist properties but rather require T cell receptor (TCR)-mediated upregulation of ICOS for agonist activity. Treatment with the ICOS antibodies results in robust anti-tumor benefit and long-term protection in preclinical syngeneic mouse tumor models. Additional benefit is observed when the ICOS antibodies are administered in combination with anti-PD-1 and anti-CTLA-4 therapies. Based on the preclinical data, JTX-2011 is currently being developed in the clinical setting for the treatment of solid tumors.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Reactividad Cruzada , Inmunoterapia/métodos , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Neoplasias Experimentales/terapia , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Femenino , Humanos , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Experimentales/inmunología , Receptores Fc/inmunologíaRESUMEN
The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)-K326I/A327Y/L328G (IYG) and N297D/S298A-IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT-IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT-IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.
Asunto(s)
Glicosilación , Inmunoglobulina G/metabolismo , Factores Inmunológicos/metabolismo , Ingeniería de Proteínas , Receptores de IgG/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Fenómenos Biofísicos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Semivida , Humanos , Inmunoglobulina G/genética , Factores Inmunológicos/genética , Factores Inmunológicos/farmacocinética , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Modelos Teóricos , Metástasis de la Neoplasia/prevención & control , Fagocitosis , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinéticaRESUMEN
We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between nuclear factor kappa B (NF-κB) essential modulator (NEMO) and IκB kinase subunit ß (IKKß), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NEMO binding domain (NBD) region of IKKß contains the highest concentration of hot-spot residues, the strongest of which are W739, W741, and L742 (ΔΔG = 4.3, 3.5, and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKß L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot-spot regions centered on IKKß residues L708/V709 and L719/I723. The computational approach successfully identified all three hot-spot regions on IKKß. Moreover, the method was able to accurately quantify the energetic importance of all hot-spot residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small-molecule inhibitors that target the NEMO/IKKß interaction. They additionally clarify the structural and energetic complementarity between "pocket-forming" and "pocket-occupying" hot-spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.
Asunto(s)
Quinasa I-kappa B/química , FN-kappa B/química , FN-kappa B/genética , Alanina/química , Algoritmos , Aminoácidos/química , Anisotropía , Biología Computacional , Vectores Genéticos , Quinasa I-kappa B/genética , Modelos Moleculares , Mutagénesis , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas Recombinantes de Fusión , Transducción de Señal , Difracción de Rayos XRESUMEN
Multicellular organisms rely on complex, fine-tuned protein networks to respond to environmental changes. We used in vitro evolution to explore the role of domain mutation and expansion in the evolution of network complexity. Using random mutagenesis to facilitate family expansion, we asked how versatile and robust the binding site must be to produce the rich functional diversity of the natural PDZ domain family. From a combinatorial protein library, we analyzed several hundred structured domain variants and found that one-quarter were functional for carboxyl-terminal ligand recognition and that our variant repertoire was as specific and diverse as the natural family. Our results show that ligand binding is hardwired in the PDZ fold and suggest that this flexibility may facilitate the rapid evolution of complex protein interaction networks.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Evolución Molecular Dirigida , Animales , Sitios de Unión/genética , Humanos , Ligandos , Mutagénesis , Estructura Terciaria de ProteínaRESUMEN
Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in Saccharomyces cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three- to tenfold improvements over wild-type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFalpha1pp) is screened for the enhanced secretion of a single-chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to 16-fold over wild type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a 180-fold improvement over previous reports in the secretion of full-length, functional, glycosylated human IgG(1). The production of full-length IgG(1) at milligram per liter titers in a simple, laboratory-scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs.
Asunto(s)
Anticuerpos/metabolismo , Biotecnología/métodos , Evolución Molecular Dirigida , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Anticuerpos/genética , Humanos , Datos de Secuencia Molecular , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Immunoglobulin G plays a vital role in adaptive immunity and antibody-based therapy through engagement of its Fc region by the Fc gamma receptors (Fc gammaRs) on immune cells. In addition to specific protein-protein contacts, N-linked glycosylation of the IgG Fc has been thought to be essential for the recognition of Fc by Fc gammaR. This requirement for the N-linked glycan has limited biomanufacture of therapeutic antibodies by restricting it to mammalian expression systems. We report here aglycosylated Fc domain variants that maintain engagement to Fc gammaRs, both in vitro and in vivo, demonstrating that Fc glycosylation is not strictly required for the activation of immune cells by IgG. These variants provide insight into how the N-linked glycan is used biologically in the recognition of Fc by Fc gammaRs, as well as represent a step toward the production in alternative expression systems of antibody-based therapeutics capable of eliciting immune effector functions.
Asunto(s)
Inmunoglobulina G/genética , Receptores Fc/inmunología , Variación Genética , Glicosilación , Inmunoglobulina G/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Receptores de IgG/inmunologíaRESUMEN
PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.
Asunto(s)
Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/genética , Dominios PDZ , Proteoma/análisis , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/clasificación , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Péptidos/análisis , Péptidos/genética , Filogenia , Estructura Secundaria de Proteína , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.
Asunto(s)
Serina Endopeptidasas/química , Secuencia de Aminoácidos , Chaperoninas/química , Escherichia coli/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Ligandos , Datos de Secuencia Molecular , Dominios PDZ , Péptidos/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.
Asunto(s)
Anticuerpos/genética , Anticuerpos/aislamiento & purificación , Ingeniería de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Anticuerpos/química , Anticuerpos/metabolismo , Citometría de Flujo/métodos , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/metabolismo , Magnetismo , Saccharomyces cerevisiae/genética , Coloración y EtiquetadoRESUMEN
Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the "binary code" strategy preclude explicit design of particular side chains at specified positions. Therefore, packing interactions cannot be specified a priori. To assess whether the binary code strategy can nonetheless produce well-folded de novo proteins, we constructed a second-generation library based upon a new structural scaffold designed to fold into 102-residue four-helix bundles. Characterization of five proteins chosen arbitrarily from this new library revealed that (1) all are alpha-helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy--if applied to an appropriately designed structural scaffold--can generate large collections of stably folded and/or native-like proteins.