RESUMO
Capping protein (CP) binds the fast growing barbed end of the actin filament and regulates actin assembly by blocking the addition and loss of actin subunits. Recent studies provide new insights into how CP and barbed-end capping are regulated. Filament elongation factors, such as formins and ENA/VASP (enabled/vasodilator-stimulated phosphoprotein), indirectly regulate CP by competing with CP for binding to the barbed end, whereas other molecules, including V-1 and phospholipids, directly bind to CP and sterically block its interaction with the filament. In addition, a diverse and unrelated group of proteins interact with CP through a conserved 'capping protein interaction' (CPI) motif. These proteins, including CARMIL (capping protein, ARP2/3 and myosin I linker), CD2AP (CD2-associated protein) and the WASH (WASP and SCAR homologue) complex subunit FAM21, recruit CP to specific subcellular locations and modulate its actin-capping activity via allosteric effects.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Capeamento de Actina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Modelos Moleculares , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Conformação ProteicaRESUMO
Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.
Assuntos
Actinas/química , Actinas/metabolismo , Microscopia Crioeletrônica/métodos , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Difosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Faloidina/química , Faloidina/metabolismo , Conformação ProteicaRESUMO
Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel "2Fc" mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.
Assuntos
Anticorpos Monoclonais/química , Antígenos de Histocompatibilidade Classe I/química , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Receptores Fc/química , Receptores de IgG/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Engenharia de Proteínas/métodos , Receptores Fc/genética , Receptores Fc/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vírus Sinciciais Respiratórios/química , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/metabolismoRESUMO
Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Förster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the CH3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reaction.
Assuntos
Anticorpos Biespecíficos/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Animais , Humanos , Cinética , Espectrometria de FluorescênciaRESUMO
The serum half-life and clearance of therapeutic monoclonal antibodies (mAbs) are critical factors that impact their efficacy and optimal dosing regimen. The pH-dependent binding of an mAb to the neonatal Fc receptor (FcRn) has long been recognized as an important determinant of its pharmacokinetics. However, FcRn affinity alone is not a reliable predictor of mAb half-life, suggesting that other biologic or biophysical mechanisms must be accounted for. mAb thermal stability, which reflects its unfolding and aggregation propensities, may also relate to its pharmacokinetic properties. However, no rigorous statistical regression methods have been used to identify combinations of physical parameters that best predict biologic properties. In this work, a panel of eight mAbs with published human pharmacokinetic data were selected for biophysical analyses of FcRn binding and thermal stability. Biolayer interferometry was used to characterize FcRn/mAb binding at acidic and neutral pH, while differential scanning calorimetry was used to determine thermodynamic unfolding parameters. Individual binding or stability parameters were generally weakly correlated with half-life and clearance values. Least absolute shrinkage and selection operator regression was used to identify the combination of two parameters with the best correlation to half-life and clearance as being the FcRn binding response at pH 7.0 and the change in heat capacity. Leave-one-out subsampling yielded a root mean square difference between observed and predicted half-life of just 2.7 days (16%). Thus, the incorporation of multiple biophysical parameters into a cohesive model may facilitate early-stage prediction of in vivo half-life and clearance based on simple in vitro experiments.
Assuntos
Anticorpos Monoclonais/sangue , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/sangue , Modelos Biológicos , Receptores Fc/metabolismo , Fenômenos Biofísicos , Meia-Vida , Humanos , Inativação Metabólica , Cinética , Aprendizado de Máquina , Valor Preditivo dos Testes , Ligação ProteicaRESUMO
Submicrometer aggregates are frequently present at low levels in antibody-based therapeutics. Although intuition suggests that the fraction of the aggregate or the size of the aggregate present might correlate with deleterious clinical properties or formulation difficulties, it has been challenging to demonstrate which aggregate states, if any, trigger specific biological effects. One source of uncertainty about the putative linkage between aggregation and safety or efficacy lies in the likelihood that noncovalent aggregation differs in ideal buffers versus in serum and biological tissues; self-association or association with other proteins may vary widely with environment. Therefore, methods for monitoring aggregation and aggregate behavior in biologically relevant matrices could provide a tool for better predicting aggregate-dependent clinical outcomes and provide a basis for antibody engineering prior to clinical studies. Here, we generate models for soluble aggregates of THIOMABs and a bispecific antibody (bsAb) of defined size and exploit fluorescence correlation spectroscopy to monitor their diffusion properties in serum and viscosity-matched buffers. The monomers, dimers, and trimers of both THIOMABs and a bsAb reveal a modest increase in diffusion time in serum greater than expected for an increase in viscosity alone. A mixture of larger aggregates containing mostly bsAb pentamers exhibits a marked increase in diffusion time in serum and much greater intrasample variability, consistent with significant aggregation or interactions with serum components. The results indicate that small aggregates of several IgG platforms are not likely to aggregate with serum components, but nanometer-scale aggregates larger than trimers can interact with the serum in an Ab-dependent manner.
Assuntos
Anticorpos Biespecíficos/química , Proteínas Sanguíneas/química , Imunoglobulina G/química , Agregados Proteicos , Trastuzumab/química , Algoritmos , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/análise , Anticorpos Biespecíficos/genética , Proteínas Sanguíneas/análise , Reagentes de Ligações Cruzadas/farmacologia , Difusão , Ditiotreitol/farmacologia , Composição de Medicamentos , Glutaral/farmacologia , Humanos , Hidrodinâmica , Imunoglobulina G/efeitos adversos , Imunoglobulina G/análise , Imunoglobulina G/genética , Peso Molecular , Tamanho da Partícula , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Reprodutibilidade dos Testes , Solubilidade , Reagentes de Sulfidrila/farmacologia , Trastuzumab/efeitos adversos , Trastuzumab/análise , ViscosidadeRESUMO
The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A (ref. 2). A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH3 (refs 3, 4). The structural basis of this specification is of particular interest. Yeast Scm3 and human HJURP are conserved non-histone proteins that interact physically with the (CenH3-H4)(2) heterotetramer and are required for the deposition of CenH3 at centromeres in vivo. Here we have elucidated the structural basis for recognition of budding yeast (Saccharomyces cerevisiae) CenH3 (called Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 in complex with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved amino terminus and a shorter α-helix at the carboxy terminus of Scm3(CBD) wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3(CBD) induces major conformational changes and sterically occludes DNA-binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome.
Assuntos
Centrômero/química , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Motivos de Aminoácidos , Sequência de Aminoácidos , Autoantígenos/química , Autoantígenos/metabolismo , Sítios de Ligação , Centrômero/metabolismo , Proteína Centromérica A , Sequência Conservada , DNA/química , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismoRESUMO
Myosins are molecular motors that power diverse cellular processes, such as rapid organelle transport, muscle contraction, and tension-sensitive anchoring. The structural adaptations in the motor that allow for this functional diversity are not known, due, in part, to the lack of high-resolution structures of highly tension-sensitive myosins. We determined a 2.3-Å resolution structure of apo-myosin-Ib (Myo1b), which is the most tension-sensitive myosin characterized. We identified a striking unique orientation of structural elements that position the motor's lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. Single-molecule and biochemical analyses show that the N terminus plays an important role in stabilizing the post power-stroke conformation of Myo1b and in tuning the rate of the force-sensitive transition. We propose that this region plays a general role in tuning the mechanochemical properties of myosins.
Assuntos
Miosina Tipo I/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , VertebradosRESUMO
Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein ("mAbtyrin") that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Humanos , Staphylococcus aureus , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/microbiologia , Anticorpos Monoclonais/uso terapêutico , Fagócitos/metabolismo , Leucocidinas/metabolismo , Leucocidinas/uso terapêuticoRESUMO
Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.
Assuntos
Anticorpos , Região Variável de Imunoglobulina , Região Variável de Imunoglobulina/química , Fragmentos de ImunoglobulinasRESUMO
TL1A (TNFSF15) is a TNF superfamily ligand which can bind the TNFRSF member death receptor 3 (DR3) on T cells and the soluble decoy receptor DcR3. Engagement of DR3 on CD4+ or CD8+ effector T cells by TL1A induces downstream signaling, leading to proliferation and an increase in secretion of inflammatory cytokines. We designed a stable recombinant TL1A molecule that (1) displays high monodispersity and stability, (2) displays the ability to activate T cells in vitro and in vivo, and (3) lacks binding to DcR3 while retaining functional activity via DR3. Together these results suggest the TL1A ligand can be amenable to therapeutic development on its own or paired with a tumor-targeting moiety.
Assuntos
Linfócitos T , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Contagem de Linfócitos , Transdução de SinaisRESUMO
Capping protein (CP) is a ubiquitously expressed, 62-kDa heterodimer that binds the barbed end of the actin filament with approximately 0.1 nm affinity to prevent further monomer addition. CARMIL is a multidomain protein, present from protozoa to mammals, that binds CP and is important for normal actin dynamics in vivo. The CARMIL CP binding site resides in its CAH3 domain (CARMIL homology domain 3) located at or near the protein's C terminus. CAH3 binds CP with approximately 1 nm affinity, resulting in a complex with weak capping activity (30-200 nm). Solution assays and single-molecule imaging show that CAH3 binds CP already present on the barbed end, causing a 300-fold increase in the dissociation rate of CP from the end (i.e. uncapping). Here we used nuclear magnetic resonance (NMR) to define the molecular interaction between the minimal CAH3 domain (CAH3a/b) of mouse CARMIL-1 and CP. Specifically, we show that the highly basic CAH3a subdomain is required for the high affinity interaction of CAH3 with a complementary "acidic groove" on CP opposite its actin-binding surface. This CAH3a-CP interaction orients the CAH3b subdomain, which we show is also required for potent anti-CP activity, directly adjacent to the basic patch of CP, shown previously to be required for CP association to and high affinity interaction with the barbed end. The importance of specific residue interactions between CP and CAH3a/b was confirmed by site-directed mutagenesis of both proteins. Together, these results offer a mechanistic explanation for the barbed end uncapping activity of CARMIL, and they identify the basic patch on CP as a crucial regulatory site.
Assuntos
Proteínas de Capeamento de Actina/química , Proteínas de Transporte/química , Proteínas de Capeamento de Actina/genética , Proteínas de Capeamento de Actina/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Camundongos , Proteínas dos Microfilamentos , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Propriedades de SuperfícieRESUMO
Capping protein (CP) is a ubiquitously expressed, heterodimeric 62-kDa protein that binds the barbed end of the actin filament with high affinity to block further filament elongation. Myotrophin (V-1) is a 13-kDa ankyrin repeat-containing protein that binds CP tightly, sequestering it in a totally inactive complex in vitro. Here, we elucidate the molecular interaction between CP and V-1 by NMR. Specifically, chemical shift mapping and intermolecular paramagnetic relaxation enhancement experiments reveal that the ankyrin loops of V-1, which are essential for V-1/CP interaction, bind the basic patch near the joint of the alpha tentacle of CP shown previously to drive most of the association of CP with and affinity for the barbed end. Consistently, site-directed mutagenesis of CP shows that V-1 and the strong electrostatic binding site for CP on the barbed end compete for this basic patch on CP. These results can explain how V-1 inactivates barbed end capping by CP and why V-1 is incapable of uncapping CP-capped actin filaments, the two signature biochemical activities of V-1.
Assuntos
Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Capeamento de Actina/genética , Animais , Galinhas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Espectroscopia de Ressonância Magnética , Camundongos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , CoelhosRESUMO
The global health crisis and economic tolls of COVID-19 necessitate a panoply of strategies to treat SARS-CoV-2 infection. To date, few treatment options exist, although neutralizing antibodies against the spike glycoprotein have proven to be effective. Because infection is initiated at the mucosa and propagates mainly at this site throughout the course of the disease, blocking the virus at the mucosal milieu should be effective. However, administration of biologics to the mucosa presents a substantial challenge. Here, we describe bifunctional molecules combining single-domain variable regions that bind to the polymeric Ig receptor (pIgR) and to the SARS-CoV-2 spike protein via addition of the ACE2 extracellular domain (ECD). The hypothesis behind this design is that pIgR will transport the molecule from the circulation to the mucosal surface where the ACE ECD would act as a decoy receptor for the nCoV2. The bifunctional molecules bind SARS-Cov-2 spike glycoprotein in vitro and efficiently transcytose across the lung epithelium in human tissue-based analyses. Designs featuring ACE2 tethered to the C-terminus of the Fc do not induce antibody-dependent cytotoxicity against pIgR-expressing cells. These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa.
Assuntos
Anticorpos Antivirais , Tratamento Farmacológico da COVID-19 , Receptores de Imunoglobulina Polimérica , SARS-CoV-2/imunologia , Anticorpos de Cadeia Única , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Células CHO , COVID-19/genética , COVID-19/imunologia , Cricetulus , Cães , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos , Mucosa Bucal/imunologia , Domínios Proteicos , Receptores de Imunoglobulina Polimérica/genética , Receptores de Imunoglobulina Polimérica/imunologia , Receptores de Imunoglobulina Polimérica/uso terapêutico , SARS-CoV-2/genética , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , SuínosRESUMO
Mucosal immunity is dominated by secretory IgA and IgM, although these are less favorable compared to IgG molecules for therapeutic development. Polymeric IgA and IgM are actively transported across the epithelial barrier via engagement of the polymeric Ig receptor (pIgR), but IgG molecules lack a lumen-targeted active transport mechanism, resulting in poor biodistribution of IgG therapeutics in mucosal tissues. In this work, we describe the discovery and characterization of single-domain antibodies (VHH) that engage pIgR and undergo transepithelial transport across the mucosal epithelium. The anti-pIgR VHH panel displayed a broad range of biophysical characteristics, epitope diversity, IgA competition profiles and transcytosis activity in cell and human primary lung tissue models. Making use of this diverse VHH panel, we studied the relationship between biophysical and functional properties of anti-pIgR binders targeting different domains and epitopes of pIgR. These VHH molecules will serve as excellent tools for studying pIgR-mediated transport of biologics and for delivering multispecific IgG antibodies into mucosal lumen, where they can target and neutralize mucosal antigens.
Assuntos
Produtos Biológicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Receptores de Imunoglobulina Polimérica , Anticorpos de Domínio Único , Transcitose/fisiologia , Animais , Descoberta de Drogas , Humanos , Imunoglobulina G , MucosaRESUMO
Generation of bispecific antibodies (BsAbs) having two unique Fab domains requires heterodimerization of the two heavy chains and pairing of each heavy chain with its cognate light chain. An alternative bispecific scaffold (Bipod) comprising an scFv and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. However, unpredictable levels of chain expression and scFv-induced aggregation can complicate purification and reduce the yield of desired Bipod. Here, we describe a high-throughput method for generation of Bipods based on protein A and CH1 domain affinity capture. This method exploits over-expression of the scFv chain to maximize heterodimer yield. Bipods purified by this method have purity suitable for cell-based functional assays and in vivo studies.
Assuntos
Anticorpos Biespecíficos/química , Fragmentos Fab das Imunoglobulinas/química , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/química , Animais , Produtos Biológicos/uso terapêutico , Células CHO , Cricetulus , DNA/química , Dimerização , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Epitopos/química , Humanos , Imunoglobulina G/genética , Imunossupressores/uso terapêutico , Mutação , Neoplasias/terapia , Plasmídeos , Domínios ProteicosRESUMO
The availability of the complete genome sequence of Bdellovibrio bacteriovorus provides an opportunity for investigating genes that play a significant role in predation. Using two independently derived facultatively predatory Bdellovibrio strains, we have designed a method to cultivate and screen transposon insertion mutants in 96-well microtiter dishes. Transposon insertion mutants were produced by introducing the plasposon pRL27, which carries a mini-Tn5. Mutants have been screened for predatory activity using 96-well plates. Seventeen independent nonpredatory mutants have been isolated, and DNA flanking the insertion has been sequenced. BLAST analysis revealed that most of these interrupted DNA sequences do not code for known proteins or functions. Two of the inactivated genes were analyzed further: one was found to code for a putative serine protease and the other a probable protein involved in secretion through the outer membrane. The methods described here are the first for the generation and isolation of predation-deficient mutants using random-transposon-insertion mutagenesis. As more mutants are isolated and their gene products analyzed, more light will be shed on how this predator carries out its exclusive life processes and perhaps how these products, or the organism itself, can be used for therapeutic, agricultural, and/or other purposes.
Assuntos
Bdellovibrio/genética , Bdellovibrio/isolamento & purificação , Elementos de DNA Transponíveis , Mutagênese Insercional/métodos , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Bacterianos , DNA Bacteriano/genética , Genes Bacterianos , Genes Essenciais , Vetores Genéticos , Dados de Sequência Molecular , Plasmídeos , Alinhamento de SequênciaRESUMO
Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or "TLQ") in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.
Assuntos
Anticorpos Biespecíficos/genética , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Mutação , Receptores de IgG/química , Proteína Estafilocócica A/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Sítios de Ligação , Cromatografia de Afinidade , Expressão Gênica , Células HEK293 , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteína Estafilocócica A/imunologia , Proteína Estafilocócica A/metabolismoRESUMO
The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.