RESUMEN
Prominent inclusion bodies can develop in the endoplasmic reticulum (ER) when overexpressed antibodies possess intrinsically high condensation propensities. These observations suggest that antibodies deemed to show notable solubility problems may reveal such characteristics preemptively in the form of ER-associated inclusion bodies during antibody overexpression. To define the relationships between solubility problems and inclusion body phenotypes, we investigated the biosynthesis of a model human IgG2λ that shows severe opalescence in an acidic formulation buffer yet retains high solubility at physiological pH. Consistent with the pH-dependent solubility characteristics, the model antibody did not induce notable inclusion body in the physiological pH environment of the ER lumen. However, when individual subunit chains of the antibody were expressed separately, the light chain (LC) spontaneously induced notable crystal-like inclusion bodies in the ER. The LC crystallization event was readily reproducible in vitro by simply concentrating the purified LC protein at physiological pH. Two independent structural determinants for the LC crystallization were identified through rational mutagenesis approach by monitoring the effect of amino acid substitutions on intracellular LC crystallogenesis. The effect of mutations on crystallization was also recapitulated in vitro using purified LC proteins. Importantly, when introduced directly into the model antibody, a mutation that prevents the LC crystallization remediated the antibody's solubility problem without compromising the secretory output or antigen binding. These results illustrate that the ER can serve as a "physiological test tube" that not only reports secretory cargo's high condensation propensity at physiological pH, but also provides an orthogonal method that guides antibody engineering strategy.
Asunto(s)
Cadenas lambda de Inmunoglobulina/química , Células Cultivadas , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Cadenas lambda de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/inmunología , Modelos Moleculares , Conformación Proteica , SolubilidadRESUMEN
Antibody-dependent cell-mediated cytotoxicity (ADCC) has been identified as one of the potentially critical effector functions underlying the clinical efficacy of some therapeutic immunoglobin G1 (IgG1) antibodies. It has been well established that higher levels of afucosylated N-linked glycan structures on the Fc region enhance the IgG binding affinity to the FcγIIIa receptor and lead to increased ADCC activity. However, whether terminal galactosylation of an IgG1 impacts its ADCC activity is less understood. Here, we used a new strategy for glycan enrichment and remodeling to study the impact of terminal galactose on ADCC activity for therapeutic IgG1s. Our results indicate that the degree of influence of terminal galactose on in vitro ADCC activity depends on the presence or absence of the core fucose, which is typically linked to the first N-acetyl glucosamine residue of an N-linked glycosylation core structure. Specifically, terminal galactose on afucosylated IgG1 mAbs enhanced ADCC activity with impact coefficients (ADCC%/Gal%) more than 20, but had minimal influence on ADCC activity on fucosylated structures with impact coefficient in the range of 0.1-0.2. Knowledge gained here can be used to guide product and process development activities for biotherapeutic antibodies that require effector function for efficacy, and also highlight the complexity in modulating the immune response through N-linked glycosylation of antibodies.
Asunto(s)
Anticuerpos Monoclonales/química , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Fragmentos Fc de Inmunoglobulinas/química , Receptores de IgG/genética , Anticuerpos Monoclonales/genética , Fucosa/química , Fucosa/genética , Galactosa/química , Galactosa/genética , Glicosilación/efectos de los fármacos , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/química , Inmunoglobulina G/genética , Polisacáridos/química , Polisacáridos/genética , Receptores de IgG/químicaRESUMEN
Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Reacción en el Punto de Inyección/inmunología , Agregado de Proteínas/inmunología , Tejido Subcutáneo/efectos de los fármacos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Femenino , Centro Germinal/efectos de los fármacos , Centro Germinal/inmunología , Humanos , Reacción en el Punto de Inyección/sangre , Inyecciones Subcutáneas , Masculino , Ratones , Solubilidad , Tejido Subcutáneo/inmunología , Distribución TisularRESUMEN
An increasing number of therapeutic proteins are being developed for delivery through the subcutaneous (SC) route of administration. Relative to intravenous (IV) administration, the SC route offers more convenience to patients, flexibility in dosing, and potential to reduce health care costs. There is a perception that SC administration can pose a higher immunogenicity risk than IV administration for a given protein. To evaluate whether there is a difference in therapeutic protein immunogenicity associated with administration routes, a more detailed understanding of the interactions with the immune system by each route is needed. Few approved therapeutic proteins have available clinical immunogenicity data sets in the public domain that represent both IV and SC administration routes. This has prevented a direct comparison of the 2 routes of administration across a large sample size. Of the 6 marketed products where SC and IV route-related incidences of anti-drug antibody (ADA) were available, 4 were associated with higher immunogenicity incidence with SC. In other cases, there was no apparent difference between the SC and IV routes. Overall, the ADA incidence was low (<15%) with no impact on safety or efficacy. The challenges associated with identifying specific risk factors unique to SC administration are discussed.
Asunto(s)
Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Proteínas/inmunología , Administración Intravenosa/métodos , Animales , Humanos , Inyecciones Intravenosas/métodos , Inyecciones Subcutáneas/métodos , Factores de RiesgoRESUMEN
The underlying reasons for why some mAb (monoclonal antibody) clones are much more inclined to induce a Russell body (RB) phenotype during immunoglobulin biosynthesis remain elusive. Although RBs are morphologically understood as enlarged globular aggregates of immunoglobulins deposited in the endoplasmic reticulum (ER), little is known about the properties of the RB-inducing mAb clones as secretory cargo and their physical behaviors in the extracellular space. To elucidate how RB-inducing propensities, secretion outputs, and the intrinsic physicochemical properties of individual mAb clones are interrelated, we used HEK293 cells to study the biosynthesis of 5 human IgG mAbs for which prominent solution behavior problems were known a priori. All 5 model mAbs with inherently high condensation propensities induced RB phenotypes both at steady state and under ER-to-Golgi transport block, and resulted in low secretion titer. By contrast, one reference mAb that readily crystallized at neutral pH in vitro produced rod-shaped crystalline bodies in the ER without inducing RBs. Another reference mAb without notable solution behavior issues did not induce RBs and was secreted abundantly. Intrinsic physicochemical properties of individual IgG clones thus directly affected the biosynthetic steps in the ER, and thereby produced distinctive cellular phenotypes and influenced IgG secretion output. The findings implicated that RB formation represents a phase separation event or a loss of colloidal stability in the secretory pathway organelles. The process of RB induction allows the cell to preemptively reduce the extracellular concentration of potentially pathogenic, highly aggregation-prone IgG clones by selectively storing them in the ER.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Retículo Endoplásmico/metabolismo , Inmunoglobulina G/biosíntesis , Cuerpos de Inclusión/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Western Blotting , Células CHO , Cricetinae , Cricetulus , Cristalización , Retículo Endoplásmico/inmunología , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/química , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cuerpos de Inclusión/inmunología , Microscopía Fluorescente , Estrés Mecánico , Temperatura , TransfecciónRESUMEN
Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.
Asunto(s)
Retículo Endoplásmico/metabolismo , Expresión Génica , Inmunoglobulina G/biosíntesis , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/genética , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/genética , Proteínas Recombinantes/genéticaRESUMEN
A-kinase anchoring proteins (AKAPs) regulate cyclic AMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP 2 (D-AKAP2) binds to the dimerization/docking (D/D) domain of both RI and RII regulatory subunits of PKA with high affinity. Here we have determined the structures of the RIalpha D/D domain alone and in complex with D-AKAP2. The D/D domain presents an extensive surface for binding through a well-formed N-terminal helix, and this surface restricts the diversity of AKAPs that can interact. The structures also underscore the importance of a redox-sensitive disulfide in affecting AKAP binding. An unexpected shift in the helical register of D-AKAP2 compared to the RIIalpha:D-AKAP2 complex structure makes the mode of binding to RIalpha novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/metabolismo , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/química , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/genética , Disulfuros/química , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Metionina/metabolismo , Receptores Fc/metabolismo , Proteína Estafilocócica A/metabolismo , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Células CHO , Cricetinae , Cricetulus , Concentración de Iones de Hidrógeno , Proteínas Inmovilizadas/metabolismo , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/química , Inmunoglobulina G/genética , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Unión Proteica , Alineación de Secuencia , terc-Butilhidroperóxido/químicaRESUMEN
A kinase-anchoring proteins (AKAPs) target PKA to specific microdomains by using an amphipathic helix that docks to N-terminal dimerization and docking (D/D) domains of PKA regulatory (R) subunits. To understand specificity, we solved the crystal structure of the helical motif from D-AKAP2, a dual-specific AKAP, bound to the RIIalpha D/D domain. The 1.6 Angstrom structure reveals how this dynamic, hydrophobic docking site is assembled. A stable, hydrophobic docking groove is formed by the helical interface of two RIIalpha protomers. The flexible N terminus of one protomer is then recruited to the site, anchored to the peptide through two essential isoleucines. The other N terminus is disordered. This asymmetry provides greater possibilities for AKAP docking. Although there is strong discrimination against RIalpha in the N terminus of the AKAP helix, the hydrophobic groove discriminates against RIIalpha. RIalpha, with a cavity in the groove, can accept a bulky tryptophan, whereas RIIalpha requires valine.