RESUMEN

Immunoglobulin G (IgG) is the main isotype of antibody in human blood. IgG consists of four subclasses (IgG1 to IgG4), encoded by separate constant region genes within the Ig heavy chain locus (IGH). Here, we report a genome-wide association study on blood IgG subclass levels. Across 4334 adults and 4571 individuals under 18 years, we discover ten new and identify four known variants at five loci influencing IgG subclass levels. These variants also affect the risk of asthma, autoimmune diseases, and blood traits. Seven variants map to the IGH locus, three to the Fcγ receptor (FCGR) locus, and two to the human leukocyte antigen (HLA) region, affecting the levels of all IgG subclasses. The most significant associations are observed between the G1m (f), G2m(n) and G3m(b*) allotypes, and IgG1, IgG2 and IgG3, respectively. Additionally, we describe selective associations with IgG4 at 16p11.2 (ITGAX) and 17q21.1 (IKZF3, ZPBP2, GSDMB, ORMDL3). Interestingly, the latter coincides with a highly pleiotropic signal where the allele associated with lower IgG4 levels protects against childhood asthma but predisposes to inflammatory bowel disease. Our results provide insight into the regulation of antibody-mediated immunity that can potentially be useful in the development of antibody based therapeutics.

Asunto(s)

Asma , Estudio de Asociación del Genoma Completo , Inmunoglobulina G , Polimorfismo de Nucleótido Simple , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/genética , Adulto , Femenino , Masculino , Asma/genética , Asma/inmunología , Asma/sangre , Niño , Adolescente , Receptores de IgG/genética , Persona de Mediana Edad , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/sangre , Alelos , Adulto Joven , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/sangre , Cromosomas Humanos Par 17/genética , Predisposición Genética a la Enfermedad , Antígenos HLA/genética , Antígenos HLA/inmunología , Proteínas de la Membrana

RESUMEN

Variable number tandem repeat (VNTR) polymorphisms of the human neonatal IgG Fc receptor α-chain gene (FCGRT) are known to influence the expression levels of FCGRT and IgG in serum. Monkeys are considered to be a relevant biological model for studying the effects of immunobiological drugs. The study determined the functional VNTR polymorphisms of the FCGRT gene in 109 male rhesus macaques from the nursery of the Kurchatov Complex of Medical Primatology. PCR amplification of samples was carried out followed by electrophoretic separation of DNA fragments in a 2% agarose gel. Individual DNA amplification products were sequenced (according to Sanger system) in forward and reverse directions to confirm the specificity. The genotyping showed that the VNTR polymorphism of the FCGRT gene in the studied population of rhesus macaques is presented by 9 variants. The frequency of the VNTR5 allele associated with lower IgG levels was 14.2%, and the most common one was the VNTR7 allele (25.2%). We also identified alleles that have not been previously reported: VNTR3, VNTR4, VNTR6, VNTR8, and VNTR9. The study allows to consider rhesus macaques as a potential model for studying the immunological response depending on the genetic VNTR variant of FCGRT.

Asunto(s)

Alelos , Macaca mulatta , Repeticiones de Minisatélite , Polimorfismo Genético , Animales , Macaca mulatta/genética , Repeticiones de Minisatélite/genética , Polimorfismo Genético/genética , Masculino , Frecuencia de los Genes/genética , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Receptores Fc/genética , Genotipo , Antígenos de Histocompatibilidad Clase I

RESUMEN

We previously described an in vitro single-chain fragment (scFv) library platform originally designed to generate antibodies with excellent developability properties. The platform design was based on the use of clinical antibodies as scaffolds into which replicated natural complementarity-determining regions purged of sequence liabilities were inserted, and the use of phage and yeast display to carry out antibody selection. In addition to being developable, antibodies generated using our platform were extremely diverse, with most campaigns yielding sub-nanomolar binders. Here, we describe a platform advancement that incorporates Fab phage display followed by single-chain antibody-binding fragment Fab (scFab) yeast display. The scFab single-gene format provides balanced expression of light and heavy chains, with enhanced conversion to IgG, thereby combining the advantages of scFvs and Fabs. A meticulously engineered, quality-controlled Fab phage library was created using design principles similar to those used to create the scFv library. A diverse panel of binding scFabs, with high conversion efficiency to IgG, was isolated against two targets. This study highlights the compatibility of phage and yeast display with a Fab semi-synthetic library design, offering an efficient approach to generate drug-like antibodies directly, facilitating their conversion to potential therapeutic candidates.

Asunto(s)

Afinidad de Anticuerpos , Fragmentos Fab de Inmunoglobulinas , Biblioteca de Péptidos , Anticuerpos de Cadena Única , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Humanos , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/química , Afinidad de Anticuerpos/inmunología , Técnicas de Visualización de Superficie Celular/métodos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/química

RESUMEN

Mutational changes that affect the binding of the C2 fragment of Streptococcal protein G (GB1) to the Fc domain of human IgG (IgG-Fc) have been extensively studied using deep mutational scanning (DMS), and the binding affinity of all single mutations has been measured experimentally in the literature. To investigate the underlying molecular basis, we perform in silico mutational scanning for all possible single mutations, along with 2 µs-long molecular dynamics (WT-MD) of the wild-type (WT) GB1 in both unbound and IgG-Fc bound forms. We compute the hydrogen bonds between GB1 and IgG-Fc in WT-MD to identify the dominant hydrogen bonds for binding, which we then assess in conformations produced by Mutation and Minimization (MuMi) to explain the fitness landscape of GB1 and IgG-Fc binding. Furthermore, we analyze MuMi and WT-MD to investigate the dynamics of binding, focusing on the relative solvent accessibility of residues and the probability of residues being located at the binding interface. With these analyses, we explain the interactions between GB1 and IgG-Fc and display the structural features of binding. In sum, our findings highlight the potential of MuMi as a reliable and computationally efficient tool for predicting protein fitness landscapes, offering significant advantages over traditional methods. The methodologies and results presented in this study pave the way for improved predictive accuracy in protein stability and interaction studies, which are crucial for advancements in drug design and synthetic biology.

Asunto(s)

Proteínas Bacterianas , Enlace de Hidrógeno , Inmunoglobulina G , Simulación de Dinámica Molecular , Mutación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Inmunoglobulina G/genética , Unión Proteica , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo

RESUMEN

Autoimmune diseases are commonly associated with a polygenic inheritance pattern. In rare instances, causal monogenic variants have been identified. The study by Liu et al. in this issue of the JCI provides an example of monogenic variants occurring in patients with IgG4-related disease (IgG4-RD). The authors investigated a familial cluster of IgG4-RD that consisted of an affected father and two daughters; the mother was unaffected. Genome sequencing of this quad identified a variant in IKZF1 (encoding IKAROS) and another variant in UBR4 (encoding E3 ubiquitin ligase). Both variants were present in the father and both daughters but absent in the unaffected mother. Using multidimensional profiling of immune cells and functional experiments in primary cells, the authors determined a molecular pathway contributing to T cell activation in IgG4-RD. Importantly, the characterization of these variants provides insights into pathogenic mechanisms in IgG4-RD and, potentially, other autoimmune diseases.

Asunto(s)

Factor de Transcripción Ikaros , Enfermedad Relacionada con Inmunoglobulina G4 , Ubiquitina-Proteína Ligasas , Humanos , Enfermedad Relacionada con Inmunoglobulina G4/genética , Enfermedad Relacionada con Inmunoglobulina G4/inmunología , Enfermedad Relacionada con Inmunoglobulina G4/patología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/inmunología , Femenino , Masculino , Inmunoglobulina G/inmunología , Inmunoglobulina G/genética , Linfocitos T/inmunología , Linfocitos T/patología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Variación Genética

RESUMEN

Antibody discovery processes are continually advancing, with an ever-increasing number of potential binding sequences being identified out of in vivo, in vitro, and in silico sources. In this work we describe a rapid system for high yield recombinant antibody (IgG and Fab) expression using Gibson assembled linear DNA fragments (GLFs). The purified recombinant antibody yields from 1 ml expression for this process are approximately five to ten-fold higher than previous methods, largely due to novel usage of protecting flanking sequences on the 5' and 3' ends of the GLF. This method is adaptable for small scale (1 ml) expression and purification for rapid evaluation of binding and activity, in addition to larger scales (30 ml) for more sensitive assays requiring milligram quantities of antibody purified over two columns (Protein A and size exclusion chromatography). When compared to plasmid-based expression, these methods provide nearly equivalent yield of high-quality material across multiple applications, allowing for reduced costs and turnaround times to enhance the antibody discovery process.

Asunto(s)

Fragmentos Fab de Inmunoglobulinas , Inmunoglobulina G , Proteínas Recombinantes , Inmunoglobulina G/genética , Inmunoglobulina G/química , Inmunoglobulina G/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/química , Expresión Génica , Humanos , Anticuerpos/genética , Anticuerpos/química

RESUMEN

The immunoglobulin heavy constant gamma (IGHG) gene cluster encoding immunoglobulin G (IgG) subclasses is highly polymorphic, resulting in amino acid variation along the antibody constant heavy chain referred to as allotypes. IGHG1 and IGHG3 are the two most polymorphic IgG subclasses in humans, with 4 classical IgG1 allotypes and 13 allotypes described for IgG3, though recent studies suggest greater allelic diversity, especially in underrepresented ethnic populations. Polymerase chain reaction (PCR) and Sanger sequencing of IGHG amplicons allow for the identification of the single nucleotide polymorphisms (SNPs) responsible for the observed amino acid substitutions. Here, we provide a detailed protocol for the amplification of IGHG1 and IGHG3 segments by PCR, sample preparation for Sanger sequencing, and analysis of sequencing data to identify SNPs associated with different IgG1 and IgG3 allotypes.

Asunto(s)

Inmunoglobulina G , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Humanos , Inmunoglobulina G/genética , Reacción en Cadena de la Polimerasa/métodos , Alotipos de Inmunoglobulinas/genética , Análisis de Secuencia de ADN/métodos

RESUMEN

In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer-dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.

Asunto(s)

Dependovirus , Edición Génica , Animales , Femenino , Dependovirus/genética , Dependovirus/inmunología , Ratones , Embarazo , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/sangre , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/inmunología , Vectores Genéticos/inmunología , Intercambio Materno-Fetal/inmunología , Intercambio Materno-Fetal/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Sistemas CRISPR-Cas , Feto/inmunología , Inmunidad Materno-Adquirida/inmunología

RESUMEN

The hybridoma method for production of monoclonal antibodies has been a cornerstone of biomedical research for several decades. Here we convert the monoclonal antibody sequence from mouse-derived hybridomas into a "devilized" recombinant antibody with devil IgG heavy chain and IgK light chain. The chimeric recombinant antibody can be used in functional assays, immunotherapy, and to improve understanding of antibodies and Fc receptors in Tasmanian devils. The process can be readily modified for other species.

Asunto(s)

Hibridomas , Inmunoglobulina G , Marsupiales , Animales , Ratones , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Hibridomas/inmunología , Marsupiales/inmunología , Marsupiales/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

The self-association of therapeutic antibodies can result in elevated viscosity and create problems in manufacturing and formulation, as well as limit delivery by subcutaneous injection. The high concentration viscosity of some antibodies has been reduced by variable domain mutations or by the addition of formulation excipients. In contrast, the impact of Fc mutations on antibody viscosity has been minimally explored. Here, we studied the effect of a panel of common and clinically validated Fc mutations on the viscosity of two closely related humanized IgG1, κ antibodies, omalizumab (anti-IgE) and trastuzumab (anti-HER2). Data presented here suggest that both Fab-Fab and Fab-Fc interactions contribute to the high viscosity of omalizumab, in a four-contact model of self-association. Most strikingly, the high viscosity of omalizumab (176 cP) was reduced 10.7- and 2.2-fold by Fc modifications for half-life extension (M252Y:S254T:T256E) and aglycosylation (N297G), respectively. Related single mutations (S254T and T256E) each reduced the viscosity of omalizumab by ~6-fold. An alternative half-life extension Fc mutant (M428L:N434S) had the opposite effect in increasing the viscosity of omalizumab by 1.5-fold. The low viscosity of trastuzumab (8.6 cP) was unchanged or increased by ≤2-fold by the different Fc variants. Molecular dynamics simulations provided mechanistic insight into the impact of Fc mutations in modulating electrostatic and hydrophobic surface properties as well as conformational stability of the Fc. This study demonstrates that high viscosity of some IgG1 antibodies can be mitigated by Fc mutations, and thereby offers an additional tool to help design future antibody therapeutics potentially suitable for subcutaneous delivery.

Asunto(s)

Fragmentos Fc de Inmunoglobulinas , Inmunoglobulina G , Mutación , Omalizumab , Trastuzumab , Humanos , Trastuzumab/química , Viscosidad , Omalizumab/química , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Inmunoglobulina G/química , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética

RESUMEN

A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.

Asunto(s)

Inmunoglobulina G , Pepsina A , Espectrometría de Masas en Tándem , Animales , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Espectrometría de Masas en Tándem/métodos , Ratones , Cromatografía Liquida/métodos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/genética , Ratas , Mutación , Cromatografía de Afinidad/métodos

RESUMEN

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

Asunto(s)

Anticuerpos Biespecíficos , Inmunoglobulina G , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/genética , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/genética , Medición de Riesgo , Trastuzumab/inmunología , Trastuzumab/genética , Animales , Bevacizumab/inmunología , Bevacizumab/genética , Mutación

RESUMEN

Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6ß and WFS1, are rational engineering targets. Although both ATF6ß and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6ß decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6ß and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6ß improved the UPR specifically later in fed-batch leading to increased overall productivity.

Asunto(s)

Factor de Transcripción Activador 6 , Cricetulus , Inmunoglobulina G , Respuesta de Proteína Desplegada , Animales , Células CHO , Factor de Transcripción Activador 6/metabolismo , Factor de Transcripción Activador 6/genética , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Respuesta de Proteína Desplegada/genética , Estrés del Retículo Endoplásmico/genética , Técnicas de Silenciamiento del Gen , Ingeniería Celular/métodos , Técnicas de Cultivo Celular por Lotes/métodos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética

RESUMEN

Observational studies revealed changes in Immunoglobulin G (IgG) N-glycosylation during the aging process. However, it lacks causal insights and remains unclear in which direction causal relationships exist. The two-sample bidirectional Mendelian randomization (MR) design was adopted to explore causal associations between IgG N-glycans and the senescence-associated secretory phenotype (SASP). Inverse variance weighted (IVW) and Wald ratio methods were used as the main analyses, supplemented by sensitivity analyses. Forward MR analyses revealed causal associations between the glycan peak (GP) and SASP, including GP6 (odds ratio [OR] = 0.428, 95% confidence interval [CI] = 0.189-0.969) and GP17 (OR = 0.709, 95%CI = 0.504-0.995) with growth/differentiation factor 15 (GDF15), GP19 with an advanced glycosylation end-product-specific receptor (RAGE) (OR = 2.142, 95% CI = 1.384-3.316), and GP15 with matrix metalloproteinase 2 (MMP2) (OR = 1.136, 95% CI =1.008-1.282). The reverse MR indicated that genetic liability to RAGE was associated with increased levels of GP17 (OR = 1.125, 95% CI = 1.003-1.261) and GP24 (OR = 1.222, 95% CI = 1.046-1.428), while pulmonary and activation-regulated chemokines (PARC) exhibited causal associations with GP10 (OR = 1.269, 95% CI = 1.048-1.537) and GP15 (OR = 1.297, 95% CI = 1.072-1.570). The findings provided suggested evidence on the bidirectional causality between IgG N-glycans and SASP, which might reveal potential regulatory mechanisms.

Asunto(s)

Inmunoglobulina G , Análisis de la Aleatorización Mendeliana , Fenotipo , Humanos , Glicosilación , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Polisacáridos/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Polimorfismo de Nucleótido Simple , Glicoproteínas

RESUMEN

Improving current cell line development workflows can either focus on increasing the specific productivity of the cell lines or shortening timelines to reach the clinic as fast as possible. In this work, using the Beacon platform, we have combined two distinct protocols - early cloning with low-viability pools, and IgG membrane staining-, to concomitantly reach both objectives, and generate highly productive CHO clones in shorter timelines. Fast-sorting approaches using low-viability pools in combination with the Beacon platform have recently been reported to shorten CLD timelines. However, the low recovery led to a drastic reduction in the clone number obtained postcloning. Here, we report a combined approach of fast-sorting and fluorescent membrane staining. With this new protocol, the cells reach a correct recovery, allowing to fully exploit the Beacon screening capacities. In addition, by using a fluorescent staining recognizing the secreted IgG, we were able to enrich the fraction of highly secreting cells prior to cloning and we obtained significant increases in the cell's specific productivity. The combination of these two protocols has a synergistic effect, and as they help discarding the dead and nonproducing populations prior to cloning, they increase the throughput power of the Beacon platform and the detection of super productive clones.

Asunto(s)

Cricetulus , Células CHO , Animales , Inmunoglobulina G/genética , Dispositivos Laboratorio en un Chip , Citometría de Flujo

RESUMEN

Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3's Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.

Asunto(s)

COVID-19 , Fragmentos Fc de Inmunoglobulinas , Inmunoglobulina G , SARS-CoV-2 , Streptococcus pyogenes , Animales , Humanos , Ratones , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , COVID-19/virología , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones Endogámicos BALB C , Fagocitosis , Ingeniería de Proteínas/métodos , SARS-CoV-2/inmunología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/inmunología

RESUMEN

The CRISPR-Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease-protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9-RNP. We opted for protein trans-splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine-less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG-Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double-strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N-terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG-Cas9 provides the first step towards a novel CRISPR-Cas9 targeting approach involving the preformed RNP complex.

Asunto(s)

Sistemas CRISPR-Cas , Inmunoglobulina G , Inteínas , Inmunoglobulina G/química , Inmunoglobulina G/genética , Humanos , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/química

RESUMEN

Production of therapeutic monoclonal antibody (mAb) in transgenic plants has several advantages such as large-scale production and the absence of pathogenic animal contaminants. However, mAb with high mannose (HM) type glycans has shown a faster clearance compared to antibodies produced in animal cells. The neonatal Fc receptor (FcRn) regulates the persistence of immunoglobulin G (IgG) by the FcRn-mediated recycling pathway, which salvages IgG from lysosomal degradation within cells. In this study, Fc-engineering of antirabies virus therapeutic mAb SO57 with the endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) (mAbpK SO57) in plant cell was conducted to enhance its binding activity to human neonatal Fc receptor (hFcRn), consequently improve its serum half-life. Enzyme-linked immunosorbent assay (ELISA) and Surface plasmon resonance assay showed altered binding affinity of the Fc region of three different mAbpK SO57 variants [M252Y/S254T/T256E (MST), M428L/N434S (MN), H433K/N434F (HN)] to hFcRn compared to wild type (WT) of mAbpK SO57. Molecular modeling data visualized the structural alterations in these mAbpK SO57. All of the mAbpK SO57 variants had HM type glycan structures similar to the WT mAbpK SO57. In addition, the neutralizing activity of the three variants against the rabies virus CVS-11 was effective as the WT mAbpK SO57. These results indicate that the binding affinity of mAbpK SO57 variants to hFcRn can be modified without alteration of N-glycan structure and neutralization activity. Taken together, this study suggests that Fc-engineering of antirabies virus mAb can be applied to enhance the efficacy of therapeutic mAbs in plant expression systems.

Asunto(s)

Antígenos de Histocompatibilidad Clase I , Inmunoglobulina G , Receptores Fc , Humanos , Anticuerpos Monoclonales/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Polisacáridos , Receptores Fc/genética , Ingeniería de Proteínas/métodos , Plantas/genética , Plantas/metabolismo

RESUMEN

BACKGROUND: Immunoglobulin G (IgG) N-glycosylation is considered a potential biomarker for aging and various pathological conditions. However, whether these changes in IgG N-glycosylation are a consequence or a contributor to the aging process remains unclear. This study aims to investigate the causality between IgG N-glycosylation and aging using Mendelian randomization (MR) analysis. METHODS: We utilized genetic variants associated with IgG N-glycosylation traits, the frailty index (FI), and leukocyte telomere length (LTL) from a previous genome-wide association study (GWAS) on individuals of European ancestry. Two-sample and multivariable MR analyses were conducted, employing the inverse-variance weighted (IVW) method. Sensitivity analyses were performed to assess potential confounding factors. RESULTS: Using the IVW method, we found suggestive evidence of a causal association between GP14 and FI (ß 0.026, 95% CI 0.003 to 0.050, p = 0.027) and LTL (ß -0.020, 95% CI -0.037 to -0.002, p = 0.029) in the two-sample MR analysis. In the multivariable MR analysis, suggestive evidence was found for GP23 and FI (ß -0.119, 95% CI -0.219 to -0.019, p = 0.019) and GP2 and LTL (ß 0.140, 95% CI 0.020 to 0.260, p = 0.023). CONCLUSIONS: In conclusion, our results supported a potentially causal effect of lower GP23 levels on an advanced aging state. Additional verification is required to further substantiate the causal relationship between glycosylation and aging.

Asunto(s)

Estudio de Asociación del Genoma Completo , Análisis de la Aleatorización Mendeliana , Humanos , Glicosilación , Inmunoglobulina G/genética , Envejecimiento/genética

RESUMEN

Despite the large number of existing bispecific antibody (bsAb) formats, the generation of novel bsAbs is still associated with development and bioprocessing challenges. Here, we present RUBY, a novel bispecific antibody format that allows rapid generation of bsAbs that fulfill key development criteria. The RUBYTM format has a 2 + 2 geometry, where two Fab fragments are linked via their light chains to the C-termini of an IgG, and carries mutations for optimal chain pairing. The unique design enables generation of bsAbs with mAb-like attributes. Our data demonstrate that RUBY bsAbs are compatible with small-scale production systems for screening purposes and can be produced at high yields (>3 g/L) from stable cell lines. The bsAbs produced are shown to, in general, contain low amounts of aggregates and display favorable solubility and stress endurance profiles. Further, compatibility with various IgG isotypes is shown and tailored Fc gamma receptor binding confirmed. Also, retained interaction with FcRn is demonstrated to translate into a pharmacokinetic profile in mice and non-human primates that is comparable to mAb controls. Functionality of conditional active RUBY bsAbs is confirmed in vitro. Anti-tumor effects in vivo have previously been demonstrated, and shown to be superior to a comparable mAb, and here it is further shown that RUBY bsAbs penetrate and localize to tumor tissue in vivo. In all, the RUBY format has attractive mAb-like attributes and offers the possibility to mitigate many of the development challenges linked to other bsAb formats, facilitating both high functionality and developability.

Asunto(s)

Anticuerpos Biespecíficos , Neoplasias , Animales , Ratones , Línea Celular , Inmunoglobulina G/genética