RESUMO

A critical attribute of therapeutic antibodies is their ability to engage with humoral or cellular effector mechanisms, and this depends on the ability of the Fc region to bind to complement (C1q) or Fc receptors. Investigators have sought to optimize these effects by engineering the Fc region to bind to a greater or lesser extent to individual receptors. Different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies representing a range of variants and compared their activity in cell-based assays for complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent phagocytosis using a range of individual Fc receptors. We have also compared the thermal stability of the variants by differential scanning fluorimetry (DSF). The results reveal a spectrum of activities which may be appropriate for different applications.

Assuntos

Citotoxicidade Celular Dependente de Anticorpos , Fragmentos Fc das Imunoglobulinas , Receptores de IgG , Humanos , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores de IgG/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/química , Mutação , Fagocitose , Ligação Proteica , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia

RESUMO

Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined. Therefore, we aimed to evaluate whether these recombinant mAbs can be used in enzyme-linked immunosorbent assay (ELISA). Recombinant HER2 mAbs from transgenic eggs were dissolved in phosphate-buffered saline and applied directly to 96-well microplates as immobilized antibodies without purification. The performance of ELISA using the unpurified recombinant HER2 mAbs from transgenic eggs was comparable to that of ELISA using commercially available purified recombinant HER2 mAbs. Moreover, ELISA using unpurified recombinant HER2 mAbs from transgenic eggs demonstrated high antigen specificity and was successfully applied to samples from cultured cell lysates derived from HER2-positive and HER2-negative cell lines. The unpurified recombinant HER2 mAbs from transgenic eggs were also efficiently used as immobilized antibodies in paper-based ELISA. In conclusion, our findings suggest that recombinant mAbs from transgenic eggs have the potential to be used to develop economic ELISA devices. To the best of our knowledge, this study is the first to use recombinant HER2 mAbs from transgenic eggs in ELISA.

Assuntos

Animais Geneticamente Modificados , Anticorpos Monoclonais , Reatores Biológicos , Galinhas , Ensaio de Imunoadsorção Enzimática , Receptor ErbB-2 , Proteínas Recombinantes , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Receptor ErbB-2/imunologia , Receptor ErbB-2/genética , Humanos , Linhagem Celular Tumoral

RESUMO

Prenatal administration of monoclonal antibodies (mAbs) is a strategy that could be exploited to prevent viral infections during pregnancy and early life. To reach protective levels in fetuses, mAbs must be transported across the placenta, a selective barrier that actively and specifically promotes the transfer of antibodies (Abs) into the fetus through the neonatal Fc receptor (FcRn). Because FcRn also regulates Ab half-life, Fc mutations like the M428L/N434S, commonly known as LS mutations, and others have been developed to enhance binding affinity to FcRn and improve drug pharmacokinetics. We hypothesized that these FcRn-enhancing mutations could similarly affect the delivery of therapeutic Abs to the fetus. To test this hypothesis, we measured the transplacental transfer of leronlimab, an anti-CCR5 mAb, in clinical development for preventing HIV infections, using pregnant rhesus macaques to model in utero mAb transfer. We also generated a stabilized and FcRn-enhanced form of leronlimab, termed leronlimab-PLS. Leronlimab-PLS maintained higher levels within the maternal compartment while also reaching higher mAb levels in the fetus and newborn circulation. Further, a single dose of leronlimab-PLS led to complete CCR5 receptor occupancy in mothers and newborns for almost a month after birth. These findings support the optimization of FcRn interactions in mAb therapies designed for administration during pregnancy.

Assuntos

Feto , Antígenos de Histocompatibilidade Classe I , Macaca mulatta , Receptores CCR5 , Receptores Fc , Animais , Gravidez , Receptores Fc/genética , Receptores Fc/imunologia , Receptores Fc/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Feto/imunologia , Receptores CCR5/genética , Receptores CCR5/imunologia , Animais Recém-Nascidos , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Infecções por HIV/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Troca Materno-Fetal/imunologia , Mutação , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/genética , Antagonistas dos Receptores CCR5/farmacologia , Anticorpos Monoclonais Humanizados/imunologia

RESUMO

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies with different Fc subclasses and variants and compared their activity for binding to C1q, Fc-gamma receptors and in cell-based assays. Most of the variants still have significant levels of activity in one or more of these assays and many of them have impaired temperature stability compared with the corresponding wild-type antibody.

Assuntos

Fragmentos Fc das Imunoglobulinas , Receptores de IgG , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores de IgG/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Mutação , Ligação Proteica , Antígenos CD20/imunologia , Antígenos CD20/genética , Antígenos CD20/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética

RESUMO

Epigenetic regulation plays a central role in the regulation of a number of cellular processes such as proliferation, differentiation, cell cycle, and apoptosis. In particular, small molecule epigenetic modulators are key elements that can effectively influence gene expression by precisely regulating the epigenetic state of cells. To identify useful small-molecule regulators that enhance the expression of recombinant proteins in Chinese hamster ovary (CHO) cells, we examined a novel dual-HDAC/LSD1 inhibitor I-4 as a supplement for recombinant CHO cells. Treatment with 2 µM I-4 was most effective in increasing monoclonal antibody production. Despite cell cycle arrest at the G1/G0 phase, which inhibits cell growth, the addition of the inhibitor at 2 µM to monoclonal antibody-expressing CHO cell cultures resulted in a 1.94-fold increase in the maximal monoclonal antibody titer and a 2.43-fold increase in specific monoclonal antibody production. In addition, I-4 significantly increased the messenger RNA levels of the monoclonal antibody and histone H3 acetylation and methylation levels. We also investigated the effect on HDAC-related isoforms and found that interference with the HDAC5 gene increased the monoclonal antibody titer by 1.64-fold. The results of this work provide an effective method of using epigenetic regulatory strategies to enhance the expression of recombinant proteins in CHO cells. KEY POINTS: • HDAC/LSD1 dual-target small molecule inhibitor can increase the expression level of recombinant monoclonal antibodies in CHO cells. • By affecting the acetylation and methylation levels of histones in CHO cells and downregulating HDAC5, the production of recombinant monoclonal antibodies increased. • It provides an effective pathway for applying epigenetic regulation strategies to enhance the expression of recombinant proteins.

Assuntos

Anticorpos Monoclonais , Cricetulus , Epigênese Genética , Proteínas Recombinantes , Células CHO , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Epigênese Genética/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Histonas/genética , Acetilação , Cricetinae , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Metilação

RESUMO

The engineering of pH-sensitive therapeutic antibodies, particularly for improving effectiveness and specificity in acidic solid-tumor microenvironments, has recently gained traction. While there is a justified need for pH-dependent immunotherapies, current engineering techniques are tedious and laborious, requiring repeated rounds of experiments under different pH conditions. Inexpensive computational techniques to predict the effectiveness of His pH-switches require antibody-antigen complex structures, but these are lacking in most cases. To circumvent these requirements, we introduce a sequence-based in silico method for predicting His mutations in the variable region of antibodies, which could lead to pH-biased antigen binding. This method, called Sequence-based Identification of pH-sensitive Antibody Binding (SIpHAB), was trained on 3D-structure-based calculations of 3,490 antibody-antigen complexes with solved experimental structures. SIpHAB was parametrized to enhance preferential binding either toward or against the acidic pH, for selective targeting of solid tumors or for antigen release in the endosome, respectively. Applications to nine antibody-antigen systems with previously reported binding preferences at different pHs demonstrated the utility and enrichment capabilities of this high-throughput computational tool. SIpHAB, which only requires knowledge of the antibody primary amino-acid sequence, could enable a more efficient triage of pH-sensitive antibody candidates than could be achieved conventionally. An online webserver for running SipHAB is available freely at https://mm.nrc-cnrc.gc.ca/software/siphab/runner/.

Assuntos

Endossomos , Neoplasias , Engenharia de Proteínas , Concentração de Íons de Hidrogênio , Humanos , Engenharia de Proteínas/métodos , Neoplasias/imunologia , Neoplasias/terapia , Endossomos/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Microambiente Tumoral/imunologia , Animais

RESUMO

Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.

Assuntos

Cricetulus , Proteínas Recombinantes , Células CHO , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Cricetinae , Humanos , Transposases/genética , Transposases/metabolismo

RESUMO

Monoclonal antibodies have widespread applications in disease treatment and antigen detection. They are traditionally produced using mammalian cell expression system, which is not able to satisfy the increasing demand of these proteins at large scale. Baculovirus expression vector system (BEVS) is an attractive alternative platform for the production of biologically active monoclonal antibodies. In this chapter, we demonstrate the production of an HIV-1 broadly neutralizing antibody b12 in BEVS. The processes including transfer vector construction, recombinant baculovirus generation, and antibody production and detection are described.

Assuntos

Baculoviridae , Vetores Genéticos , Baculoviridae/genética , Vetores Genéticos/genética , Animais , Humanos , Expressão Gênica , HIV-1/genética , HIV-1/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ensaio de Imunoadsorção Enzimática , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/genética , Células Sf9

RESUMO

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.

Assuntos

Hibridomas , Imunoglobulina G , Marsupiais , Animais , Camundongos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Hibridomas/imunologia , Marsupiais/imunologia , Marsupiais/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia

RESUMO

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.

Assuntos

Imunoglobulina G , Pepsina A , Espectrometria de Massas em Tandem , Animais , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Espectrometria de Massas em Tandem/métodos , Camundongos , Cromatografia Líquida/métodos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Ratos , Mutação , Cromatografia de Afinidade/métodos

RESUMO

Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (< 0.1% RMCE efficiency), which further depends on the expressed molecules, and underwent rapid population skewing towards dominant clones during routine cultivation. Clonal cell lines from one individual TI event demonstrated a significantly lower variance regarding production-relevant and phenotypic parameters as compared to cell lines from distinct TI events. This implies that the observed cellular diversity lies within pre-existing cell-intrinsic factors and that the majority of clonal variation did not develop during the CLD process, especially during single cell cloning. Using cellular barcodes as a proxy for cellular diversity, we improved our CLD screening workflow and enriched diversity of production-relevant parameters substantially. This work, by enabling clonal diversity monitoring and control, paves the way for an economically valuable and data-driven CLD process.

Assuntos

Células Clonais , Cricetulus , Código de Barras de DNA Taxonômico , Células CHO , Animais , Código de Barras de DNA Taxonômico/métodos , Genômica/métodos , Anticorpos Monoclonais/genética

RESUMO

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.

Assuntos

Cricetulus , Retículo Endoplasmático , Complexo de Golgi , Proteínas Recombinantes , Células CHO , Animais , Retículo Endoplasmático/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Complexo de Golgi/metabolismo , Glicosilação , Cricetinae , Resposta a Proteínas não Dobradas , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/genética , Transporte Proteico , Técnicas de Cultura Celular por Lotes/métodos

RESUMO

Monoclonal antibodies (mAbs) are a driving force in the biopharmaceutical industry. Therapeutic mAbs are usually produced in mammalian cells, but there has been a push towards the use of alternative production hosts, such as Escherichia coli. When the genes encoding for a mAb heavy and light chains are codon-optimized for E. coli expression, a truncated form of the heavy chain can form along with the full-length product. In this work, the role of codon optimization in the formation of a truncated product was investigated. This study used the amino acid sequences of several therapeutic mAbs and multiple optimization algorithms. It was found that several algorithms incorporate sequences that lead to a truncated product. Approaches to avoid this truncated form are discussed.

Assuntos

Anticorpos Monoclonais , Escherichia coli , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Escherichia coli/genética , Escherichia coli/metabolismo , Códon/genética , Algoritmos , Sequência de Aminoácidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Humanos , Expressão Gênica , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/química

RESUMO

Monoclonal antibodies (mAbs) are one of the most important classes of biologics with high therapeutic and diagnostic value, but traditional methods for mAbs generation, such as hybridoma screening and phage display, have limitations, including low efficiency and loss of natural chain pairing. To overcome these challenges, novel single B cell antibody technologies have emerged, but they also have limitations such as in vitro differentiation of memory B cells and expensive cell sorters. In this study, we present a rapid and efficient workflow for obtaining human recombinant monoclonal antibodies directly from single antigen-specific antibody secreting cells (ASCs) in the peripheral blood of convalescent COVID-19 patients using ferrofluid technology. This process allows the identification and expression of recombinant antigen-specific mAbs in less than 10 days, using RT-PCR to generate linear Ig heavy and light chain gene expression cassettes, called "minigenes", for rapid expression of recombinant antibodies without cloning procedures. This approach has several advantages. First, it saves time and resources by eliminating the need for in vitro differentiation. It also allows individual antigen-specific ASCs to be screened for effector function prior to recombinant antibody cloning, enabling the selection of mAbs with desired characteristics and functional activity. In addition, the method allows comprehensive analysis of variable region repertoires in combination with functional assays to evaluate the specificity and function of the generated antigen-specific antibodies. Our approach, which rapidly generates recombinant monoclonal antibodies from single antigen-specific ASCs, could help to identify functional antibodies and deepen our understanding of antibody dynamics in the immune response through combined antibody repertoire sequence analysis and functional reactivity testing.

Assuntos

Anticorpos Monoclonais , Células Produtoras de Anticorpos , COVID-19 , Proteínas Recombinantes , SARS-CoV-2 , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Células Produtoras de Anticorpos/imunologia , SARS-CoV-2/imunologia , COVID-19/imunologia , Anticorpos Antivirais/imunologia , Feminino

RESUMO

Immunoglobulin G-based monoclonal antibodies (mAbs) have been effective in treating various diseases, but their large molecular size can limit their penetration of tissue and efficacy in multifactorial diseases, necessitating the exploration of alternative forms. In this study, we constructed a phage display library comprising single-domain antibodies (sdAbs; or "VHHs"), known for their small size and remarkable stability, using a total of 1.6 × 109 lymphocytes collected from 20 different alpacas, resulting in approximately 7.16 × 1010 colonies. To assess the quality of the constructed library, next-generation sequencing-based high-throughput profiling was performed, analyzing approximately 5.65 × 106 full-length VHH sequences, revealing 92% uniqueness and confirming the library's diverse composition. Systematic characterization of the library revealed multiple sdAbs with high affinity for three therapeutically relevant antigens. In conclusion, our alpaca sdAb phage display library provides a versatile resource for diagnostics and therapeutics. Furthermore, the library's vast natural VHH antibody repertoire offers insights for generating humanized synthetic sdAb libraries, further advancing sdAb-based therapeutics.

Assuntos

Camelídeos Americanos , Biblioteca de Peptídeos , Anticorpos de Domínio Único , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Animais , Camelídeos Americanos/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Ensaios de Triagem em Larga Escala/métodos , Afinidade de Anticorpos , Técnicas de Visualização da Superfície Celular/métodos

RESUMO

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody's application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.

Assuntos

Anticorpos Monoclonais , Proteínas não Estruturais Virais , Animais , Feminino , Camundongos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Antivirais/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Hibridomas/imunologia , Camundongos Endogâmicos BALB C , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/genética

RESUMO

Monoclonal antibody biologics have significantly transformed the therapeutic landscape within the biopharmaceutical industry, partly due to the utilisation of discovery technologies such as the hybridoma method and phage display. While these established platforms have streamlined the development process to date, their reliance on cell transformation for antibody identification faces limitations related to library diversification and the constraints of host cell physiology. Cell-free systems like ribosome display offer a complementary approach, enabling antibody selection in a completely in vitro setting while harnessing enriched cellular molecular machinery. This review aims to provide an overview of the fundamental principles underlying the ribosome display method and its potential for advancing antibody discovery and development.

Assuntos

Anticorpos Monoclonais , Biblioteca de Peptídeos , Ribossomos , Ribossomos/imunologia , Humanos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Animais , Técnicas de Visualização da Superfície Celular , Descoberta de Drogas , Eucariotos/imunologia , Eucariotos/genética

RESUMO

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

Assuntos

Processamento Alternativo , Animais , Subunidades Proteicas/genética , Humanos , Galinhas , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/biossíntese , Células CHO , Éxons/genética , Cricetulus , Proteínas de Fluorescência Verde/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/biossíntese , Precursores de RNA/genética

RESUMO

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

Assuntos

Sistemas CRISPR-Cas , Cricetulus , Edição de Genes , Células CHO , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Anticorpos Monoclonais/genética , Proteínas Recombinantes/genética , Técnicas de Inativação de Genes/métodos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Cricetinae , Engenharia Genética/métodos

RESUMO

Ichnos has developed a multi-specific antibody platform based on the BEAT® (Bispecific engagement by antibodies based on the T-cell receptor) interface. The increased complexity of the bi- and multi-specific formats generated with this platform makes these molecules difficult-to-express proteins compared to standard monoclonal antibodies (mAbs). This report describes how expression limitations of a bi-specific bi-paratopic BEAT antibody were improved in a holistic approach. An initial investigation allowed identification of a misbalance in the subunits composing the BEAT antibody as the potential root cause. This misbalance was then addressed by a signal peptide optimization, and the overall expression level was increased by the combination of two vector design elements on a single gene vector. Further improvements were made in the selection of cell populations and an upstream (USP) platform process was applied in combination with a cell culture temperature shift. This allowed titer levels of up to 6 g/L to be reached with these difficult-to-express proteins. Furthermore, a high-density seeding process was developed that allowed titers of around 11 g/L for the BEAT antibody, increasing the initial titer by a factor of 10. The approach was successfully applied to a tri-specific antibody with titer levels reaching 10 g/L. In summary, a platform process for difficult-to-express proteins was developed using molecular biology tools, cell line development, upstream process optimization and process intensification.

Assuntos

Anticorpos Monoclonais , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/biossíntese , Células CHO , Cricetulus , Humanos