RESUMO
Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.
Assuntos
Adesinas de Escherichia coli/metabolismo , Anticorpos Monoclonais/imunologia , Aderência Bacteriana , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Escherichia coli Uropatogênica/metabolismo , Animais , Feminino , Masculino , Camundongos Endogâmicos C57BL , Modelos MolecularesRESUMO
The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Amplamente Neutralizantes , Cristalografia por Raios X , Anticorpos Anti-HIV/química , Infecções por HIV/virologia , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies.
Assuntos
Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Receptores de Inositol 1,4,5-Trifosfato/imunologia , Animais , Anticorpos Monoclonais/genética , Autoanticorpos/genética , Autoantígenos/genética , Anticorpos Amplamente Neutralizantes , Cardiolipinas/genética , Cardiolipinas/imunologia , Regiões Determinantes de Complementaridade/genética , Epitopos/genética , Anticorpos Anti-HIV/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Camundongos , Camundongos Transgênicos , Proteoma/genética , Proteoma/imunologiaRESUMO
Mesothelin (MSLN) is a cell-surface glycoprotein expressed at low levels on normal mesothelium but overexpressed in many cancers. Mesothelin has been implicated to play role/s in cell adhesion and multiple signaling pathways. Mucin-16/CA125 is an enormous cell-surface glycoprotein, also normally expressed on mesothelium and implicated in the progression and metastasis of several cancers, and directly binds mesothelin. However, the precise biological function/s of mesothelin and mucin-16/CA125 remain mysterious. We report protein engineering and recombinant production, qualitative and quantitative binding studies, and a crystal structure determination elucidating the molecular-level details governing recognition of mesothelin by mucin-16/CA125. The interface is small, consistent with the â¼micromolar binding constant and is free of glycan-mediated interactions. Sequence comparisons and modeling suggest that multiple mucin-16/CA125 modules can interact with mesothelin through comparable interactions, potentially generating a high degree of avidity at the cell surface to overcome the weak affinity, with implications for functioning and therapeutic interventions.
Assuntos
Antígeno Ca-125 , Proteínas Ligadas por GPI , Mesotelina , Modelos Moleculares , Ligação Proteica , Mesotelina/metabolismo , Humanos , Antígeno Ca-125/metabolismo , Antígeno Ca-125/química , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Cristalografia por Raios X , Sítios de Ligação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Engenharia de Proteínas , Proteínas de MembranaRESUMO
MHC class I "single-chain trimer" molecules, coupling MHC heavy chain, ß2-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation.
Assuntos
Antígenos de Histocompatibilidade Classe I , Peptídeos , Humanos , Camundongos , Animais , Antígenos de Histocompatibilidade Classe I/genética , Peptídeos/metabolismo , Epitopos/químicaRESUMO
PG9 and PG16 are antibodies isolated from a subject infected with HIV-1 and display broad anti-HIV neutralizing activities. They recognize overlapping epitopes, which are preferentially expressed on the membrane-anchored trimeric form of the HIV envelope glycoprotein (Env). PG9 and PG16 were reported not to bind to soluble mimetics of Env. The engineering of soluble Env proteins on which the PG9 and PG16 epitopes are optimally exposed will support efforts to elicit broad anti-HIV neutralizing antibodies by immunization. Here, we identified several soluble gp140 Env proteins that are recognized by PG9 and PG16, and we investigated the molecular details of those binding interactions. The IgG versions of PG9 and PG16 recognize the soluble trimeric gp140 form less efficiently than the corresponding monomeric gp140 form. In contrast, the Fab versions of PG9 and PG16 recognized the monomeric and trimeric gp140 forms with identical binding kinetics and with binding affinities similar to the high binding affinity of the anti-V3 antibody 447D to its epitope. Our data also indicate that, depending on the Env backbone, the interactions of PG9 and PG16 with gp140 may be facilitated by the presence of the gp41 ectodomain and are independent of the proper enzymatic cleavage of gp140 into gp120 and gp41. The identification of soluble Env proteins that express the PG9 and PG16 epitopes and the detailed characterization of the molecular interactions between these two antibodies and their ligands provide important and novel information that will assist in improving the engineering of future Env immunogens.
Assuntos
Anticorpos Monoclonais/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína , Solubilidade , Ressonância de Plasmônio de Superfície , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Cystine-dense peptides (CDPs) are a miniprotein class that can drug difficult targets with high affinity and low immunogenicity. Tools for their design, however, are not as developed as those for small-molecule and antibody drugs. CDPs have diverse taxonomic origins, but structural characterization is lacking. Here, we adapted Iterative Threading ASSEmbly Refinement (I-TASSER) and Rosetta protein modeling software for structural prediction of 4298 CDP scaffolds and performed in silico prescreening for CDP binders to targets of interest. Mammalian display screening of a library of docking-enriched, methionine and tyrosine scanned (DEMYS) CDPs against PD-L1 yielded binders from four distinct CDP scaffolds. One was affinity-matured, and cocrystallography yielded a high-affinity (KD = 202 pM) PD-L1-binding CDP that competes with PD-1 for PD-L1 binding. Its subsequent incorporation into a CD3-binding bispecific T cell engager produced a molecule with pM-range in vitro T cell killing potency and which substantially extends survival in two different xenograft tumor-bearing mouse models. Both in vitro and in vivo, the CDP-incorporating bispecific molecule outperformed a comparator antibody-based molecule. This CDP modeling and DEMYS technique can accelerate CDP therapeutic development.
Assuntos
Anticorpos Biespecíficos , Linfócitos T , Animais , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antígeno B7-H1 , Complexo CD3 , Cistina , Modelos Animais de Doenças , Mamíferos , PeptídeosRESUMO
Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , SARS-CoV-2/metabolismo , Sequências de Repetição em Tandem , Sequência de Aminoácidos , COVID-19/virologia , Simulação por Computador , Cristalização , Células HEK293 , Humanos , Modelos Moleculares , Testes de Neutralização , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Protein engineering has enabled the design of molecular scaffolds that display a wide variety of sizes, shapes, symmetries and subunit compositions. Symmetric protein-based nanoparticles that display multiple protein domains can exhibit enhanced functional properties due to increased avidity and improved solution behavior and stability. Here we describe the creation and characterization of a computationally designed circular tandem repeat protein (cTRP) composed of 24 identical repeated motifs, which can display a variety of functional protein domains (cargo) at defined positions around its periphery. We demonstrate that cTRP nanoparticles can self-assemble from smaller individual subunits, can be produced from prokaryotic and human expression platforms, can employ a variety of cargo attachment strategies and can be used for applications (such as T-cell culture and expansion) requiring high-avidity molecular interactions on the cell surface.
Assuntos
Nanopartículas/química , Engenharia de Proteínas , Proteínas/química , Sequências de Repetição em Tandem/genética , Motivos de Aminoácidos/genética , Técnicas de Cultura de Células , Humanos , Modelos Moleculares , Domínios Proteicos/genética , Estabilidade Proteica , Proteínas/genética , Linfócitos T/químicaRESUMO
The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (KD as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Doenças do Sistema Nervoso Central/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Peptídeos/farmacologia , Animais , Antígenos CD/química , Antígenos CD/efeitos dos fármacos , Antígenos CD/genética , Antígenos CD/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Cistina/química , Cistina/genética , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Camundongos , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Neurotensina/química , Neurotensina/farmacologia , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Receptores da Transferrina/química , Receptores da Transferrina/efeitos dos fármacos , Receptores da Transferrina/genéticaRESUMO
In the original version of this Article the colour key for the amino acid enrichment score was inadvertently omitted from the lower panel of Figure 5b during the production process. This has now been corrected in the PDF and HTML versions of the Article.
RESUMO
Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology. Cystine-dense peptides have the potential to disrupt such interactions, and are used in drug-like roles by every clade of life, but their study has been hampered by a reputation for being difficult to produce, owing to their complex disulfide connectivity. Here we describe a platform for identifying target-binding cystine-dense peptides using mammalian surface display, capable of interrogating high quality and diverse scaffold libraries with verifiable folding and stability. We demonstrate the platform's capabilities by identifying a cystine-dense peptide capable of inhibiting the YAP:TEAD interaction at the heart of the oncogenic Hippo pathway, and possessing the potency and stability necessary for consideration as a drug development candidate. This platform provides the opportunity to screen cystine-dense peptides with drug-like qualities against targets that are implicated for the treatment of diseases, but are poorly suited for conventional approaches.
Assuntos
Cistina/análise , Peptídeos/química , Peptídeos/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Descoberta de Drogas , Proteínas de Escherichia coli/química , Glicosilação , Humanos , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
BACKGROUND: CD28 signal blockade following T cell receptor activation is under intense investigation as a tolerance-inducing therapy for transplantation. Our goal is to produce a CD28-specific reagent as a therapy for the prevention of graft rejection and graft-versus-host disease in the canine model of allogeneic hematopoietic cell transplantation (HCT). METHODS: We infused a monoclonal mouse anti-canine CD28 antibody (1C6 mAb) into four dogs and a fragment of antigen-binding (1C6 Fab) into two dogs. Pharmacokinetics, pathology, cytokine release, and the crystal structure of 1C6 Fv were evaluated. RESULTS: Within an hour of an IV injection of the 1C6 mAb, the dogs became leukopenic and developed a steroid-refractory cytokine storm. Two of the dogs developed high fevers, one experienced diffuse alveolar hemorrhage, and another developed gastrointestinal hemorrhage. The cytokine storm was characterized by elevated plasma levels of MCP-1, IP-10, IL-10, IL-6, and TNF-α. In addition, one dog showed elevated levels of IL-2, IL-8, and IL-18. In contrast, infusion of 1C6 Fab was well tolerated without any side effects. Dry-coating 1C6 mAb onto tissue culture plates induced CD3-independent proliferation and TNF-alpha production. Crystal structure analysis revealed that 1C6 binds to canine CD28 in a manner different than previously reported for conventional agonistic or superagonistic antibodies. CONCLUSIONS: These results indicate that dogs and humans develop a similar cytokine storm following infusion ofanti-CD28 mAb, providing an appropriate large animal for further study. 1C6 Fab warrants evaluation as a tolerance-inducing reagent in the canine model of allogeneic HCT.
RESUMO
Computational protein design has promise for vaccine design and other applications. We previously transplanted the HIV 4E10 epitope onto non-HIV protein scaffolds for structural stabilization and immune presentation. Here, we developed two methods to optimize the structure of an antigen, flexible backbone remodeling and resurfacing, and we applied these methods to a 4E10 scaffold. In flexible-backbone remodeling, an existing backbone segment is replaced by a de novo designed segment of prespecified length and secondary structure. With remodeling, we replaced a potentially immunodominant domain on the scaffold with a helix-loop segment that made intimate contact to the protein core. All three domain trim designs tested experimentally had improved thermal stability and similar binding affinity for the 4E10 antibody compared to the parent scaffold. A crystal structure of one design had a 0.8 Å backbone RMSD to the computational model in the rebuilt region. Comparison of parent and trimmed scaffold reactivity to anti-parent sera confirmed the deletion of an immunodominant domain. In resurfacing, the surface of an antigen outside a target epitope is redesigned to obtain variants that maintain only the target epitope. Resurfaced variants of two scaffolds were designed in which 50 positions amounting to 40% of the protein sequences were mutated. Surface-patch analyses indicated that most potential antibody footprints outside the 4E10 epitope were altered. The resurfaced variants maintained thermal stability and binding affinity. These results indicate that flexible-backbone remodeling and resurfacing are useful tools for antigen optimization and protein engineering generally.
Assuntos
Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Antígenos/química , Antígenos/imunologia , Drogas Desenhadas , Vacinas contra a AIDS/genética , Substituição de Aminoácidos/genética , Antígenos/genética , Cristalografia por Raios X , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Modelos Moleculares , Estabilidade Proteica , Estrutura Terciária de Proteína , Temperatura , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.
Assuntos
Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Técnicas Biossensoriais/métodos , Antígeno Prostático Específico/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/normas , Humanos , Cinética , Ligantes , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/imunologia , Ligação Proteica , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos , Ressonância de Plasmônio de Superfície/normas , Fatores de TempoRESUMO
Soluble type II interleukin (IL)-1 receptor (sIL1R-II) binds human IL-1beta with high affinity and neutralizes its activity. Recombinant sIL1R-II is considered a potentially useful anti-IL-1 therapeutic, and preclinical studies have been undertaken with this molecule in primates. To better understand the cytokine-receptor interactions occurring in this nonhuman context, monkey IL-1 and IL1R-II were cloned, and their binding abilities were examined in vitro. IL-1beta from cynomolgus monkey was capable of binding and activating the human type I IL-1 receptor. However, unlike human IL-1beta, it was unable to effectively bind and become neutralized by sIL1R-II. Human and cynomolgus IL-1beta proteins are 96% identical, differing by only six amino acids. Structural and mutational analysis revealed that the unique sIL1R-II binding ability of human IL-1beta is due to a single amino acid difference compared with monkey IL-1beta.