Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

Robust and low-cost ELISA based on IgG-Fc tagged recombinant proteins to screen for anti-SARS-CoV-2 antibodies.

The development of new diagnostic assays become a priority for managing COVID-19. To this aim, we presented here an in-house ELISA based on the production of two major recombinant and high-quality antigens from SARS-CoV-2. Full-length N and S-RBD fragment proteins fused to mouse IgG2a-Fc were produced in the CHO cell line. Secreted recombinant proteins were easily purified with standard Protein A chromatography and were used in an in-house ELISA to detect anti-N and anti-RBD IgGs in the plasma of COVID-19 RTPCR-positive patients. High reactivity against recombinant antigens was readily detected in all positive plasma samples, whereas no recognition was observed with control healthy subject's plasmas. Remarkably, unpurified recombinant N protein obtained from cell culture supernatant was also suitable for the monitoring by ELISA of IgG levels in positive patients. This work provides an early prospection for low price but high-quality serological kit development.

Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.

Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.

Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins.

Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells. Many alternative production systems and improved constructs are also being actively investigated. IgGs glycans represent only an average of around 3% of the total mass of the molecule. Despite this low percentage, particular glycoforms are involved in essential immune effector functions. On the other hand, glycoforms that are not commonly biosynthesized in human may be allergenic, immunogenic and accelerate the plasmatic clearance of the linked antibody. These glyco-variants have to be identified, controlled and limited for therapeutic uses. Glycosylation depends on multiple factors like production system, selected clonal population, manufacturing process and may be genetically or chemically engineered. The present account reviews the glycosylation patterns observed for the current approved therapeutic antibodies produced in mammalian cell lines, details classical and state-of-the-art analytical methods used for the characterization of glycoforms and discusses the expected benefits of manipulating the carbohydrate components of antibodies by bio- or chemical engineering as well as the expected advantages of alternative biotechnological production systems developed for new generation of therapeutic antibodies and Fc-fusion proteins.

[Construction of BPI23-Fc gamma 1 recombinant protein prokaryotic expression vector and the expression and biological assessment of BPI23-Fc gamma 1 recombinant protein].

OBJECTIVE: To construct pBV-BPI600-Fc gamma 1(700) recombinant expression vector, to transform into Escherichia coli DH5 alpha, and to induce the expression of BPI23-Fc gamma 1 anti-bacterial recombinant protein. METHODS: Genes which encode BPI23 and Fc gamma 1 were amplified by RT-PCR from mRNA that was extracted from HL-60 cell and normal human leukocytes; Recombinant cloning vector and recombinant expression vector were constructed. pBV-BPI600-Fc gamma 1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5 alpha and BPI23-Fc gamma 1 recombinant protein was expressed by temperature induced method. RESULTS: (1) Expected amplified products BPI600 bp and Fc gamma 1(700) bp were obtained by RT-PCR. (2) pUC18-BPI180, pUC18-BPI420, and pUC18-Fc gamma 1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. (3) pBV-BPI600-Fc gamma 1(700) recombinant expression vector was successfully constructed, and results of the enzyme digestion analysis were identical with expected ones. (4) pBV-BPI600-Fc gamma 1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5 alpha and BPI23-Fc gamma 1 recombinant protein was expressed by temperature-induced method, and its expression level was accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fc gamma 1 recombinant protein had anti-bacterial activity and biological functions of complement fixation, opsonization. CONCLUSION: pBV-BPI600-Fc gamma 1(700) recombinant expression vector was successfully constructed, and BPI23-Fc gamma 1 recombinant protein with BPI and IgGFc double biological activity was expressed in Escherichia coli.