Coupling CRISPR interference with FACS enrichment: New approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production.

Difficulties in obtaining and maintaining the desired level of the critical quality attributes (CQAs) of therapeutic proteins as well as the pace of the development are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, potency, plasma half-life, immunogenicity, and effector functions of the therapeutic. Hence, glycosylation is widely recognized as a process-dependent CQA of therapeutic glycoproteins. Due to the typically high heterogeneity of glycoforms attached to the proteins, control of glycosylation represents one of the most challenging aspects of biopharmaceutical development. Here, we explored a new glycoengineering approach in therapeutic glycoproteins development, which enabled us to achieve the targeted glycoprofile of the Fc-fusion protein in a fast manner. Coupling CRISPRi technology with lectin-FACS sorting enabled downregulation of the endogenous gene involved in fucosylation and further enrichment of CHO cells producing Fc-fusion proteins with reduced fucosylation levels. Enrichment of cells with targeted glycoprofile can lead to time-optimized clone screening and speed up cell line development. Moreover, the presented approach allows isolation of clones with varying levels of fucosylation, which makes it applicable to a broad range of glycoproteins differing in target fucosylation level.

Molecular Dynamics Simulations Reveal Interactions of an IgG1 Antibody With Selected Fc Receptors.

In a survey of novel interactions between an IgG1 antibody and different Fcγ receptors (FcγR), molecular dynamics simulations were performed of interactions of monoclonal antibody involved complexes with FcγRs. Free energy simulations were also performed of isolated wild-type and substituted Fc regions bound to FcγRs with the aim of assessing their relative binding affinities. Two different free energy calculation methods, Molecular Mechanical/Generalized Born Molecular Volume (MM/GBMV) and Bennett Acceptance Ratio (BAR), were used to evaluate the known effector substitution G236A that is known to selectively increase antibody dependent cellular phagocytosis. The obtained results for the MM/GBMV binding affinity between different FcγRs are in good agreement with previous experiments, and those obtained using the BAR method for the complete antibody and the Fc-FcγR simulations show increased affinity across all FcγRs when binding to the substituted antibody. The FcγRIIa, a key determinant of antibody agonistic efficacy, shows a 10-fold increase in binding affinity, which is also consistent with the published experimental results. Novel interactions between the Fab region of the antibody and the FcγRs were discovered with this in silico approach, and provide insights into the antibody-FcγR binding mechanism and show promise for future improvements of therapeutic antibodies for preclinical studies of biological drugs.

Loop Grafting between Similar Local Environments for Fc-Silent Antibodies.

Reduction of the affinity of the fragment crystallizable (Fc) region with immune receptors by substitution of one or a few amino acids, known as Fc-silencing, is an established approach to reduce the immune effector functions of monoclonal antibody therapeutics. This approach to Fc-silencing, however, is problematic as it can lead to instability and immunogenicity of the developed antibodies. We evaluated loop grafting as a novel approach to Fc-silencing in which the Fc loops responsible for immune receptor binding were replaced by loops of up to 20 amino acids from similar local environments in other human and mouse antibodies. Molecular dynamics simulations of the designed variants of an Fc region in a complex with the immune receptor FcγIIIa confirmed that loop grafting potentially leads to a significant reduction in the binding of the antibody variants to the receptor, while retaining their stability. In comparison, standard variants with less than eight substituted amino acids showed possible instability and a lower degree of Fc-silencing due to the occurrence of compensatory interactions. The presented approach to Fc-silencing is general and could be used to modulate undesirable side effects of other antibody therapeutics without affecting their stability or increasing their immunogenicity.

New assay for quantification of PEGylated proteins during in vitro permeability studies.

One of the major challenges when analyzing very low amounts of PEGylated proteins is finding a sensitive analytical method. Immunoassays are most frequently used, however, conjugation can partially or completely mask protein epitopes, which can substantially lower the response and influence the quantitation range. Here we describe a novel assay that allows quantification of low amounts of PEGylated or differently conjugated proteins. The basic principle is similar to the classic sandwich ELISA but there are no antibodies used neither for capture nor for detection. Instead, Ni(2+) chelation is exploited for capture and affinity between streptavidin and biotin for the detection step. The usefulness of the assay was proven in permeation studies (Caco-2 cell model) using diversely conjugated TNF-a protein. This approach could be extended to numerous other proteins eliminating the need to develop a separate assay for each protein/project.

Virtual screening yields inhibitors of novel antifungal drug target, benzoate 4-monooxygenase.

Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)benzoic acid (compound 2) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process.

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification.

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

The Characterization and Potential use of G-CSF Dimers and their Pegylated Conjugates.

G-CSF successfully prevents chemotherapy-induced neutropenia. Two second-generation drugs with improved therapeutic properties are already available and the development of new forms is still ongoing. For an efficient receptor dimerization two G-CSF molecules have to bind. Development of G-CSF dimers acting as receptor dimerizers was explored and their potential use evaluated. The in vitro biological activities of the prepared dimers were lower than G-CSF monomer activity, presumably due to non-optimal spatial orientation of the molecules. Most likely two dimers had to bind to trigger receptor dimerization instead of one dimer acting as a dimerizer. Although significantly lower in the residual in vitro biological activity, the diPEG-Fdim conjugate exhibited pharmacokinetical (PK) and pharmacodynamical (PD) properties comparable to pegfilgrastim or even better. An interesting PD profile with the second maximum in absolute neutrophil count (ANC) and a balanced elevated ANC profile over the longer time interval was namely observed.

CYP53A15 of Cochliobolus lunatus, a target for natural antifungal compounds.

A novel cytochrome P450, CYP53A15, was identified in the pathogenic filamentous ascomycete Cochliobolus lunatus. The protein, classified into the CYP53 family, was capable of para hydroxylation of benzoate. Benzoate is a key intermediate in the metabolism of aromatic compounds in fungi and yet basically toxic to the organism. To guide functional analyses, protein structure was predicted by homology modeling. Since many naturally occurring antifungal phenolic compounds are structurally similar to CYP53A15 substrates, we tested their putative binding into the active site of CYP53A15. Some of these compounds inhibited CYP53A15. Increased antifungal activity was observed when tested in the presence of benzoate. Some results suggest that CYP53A15 O-demethylation activity is important in detoxification of other antifungal substances. With the design of potent inhibitors, CYP53 enzymes could serve as alternative antifungal drug targets.

Obstacles and pitfalls in the PEGylation of therapeutic proteins.

In recent years, PEGylation has become widely used as a post-production modification methodology for improving the biomedical efficacy and physicochemical properties of therapeutic proteins. Several marketed drugs that have already been in use for more than a decade have proved the applicability and safety of this technology and, with the successes already achieved, it is expected that PEGylation will be applied to other potential therapeutic proteins. The non-biodegradable nature of PEG, however, may become a limiting factor for the next generation of protein pharmaceuticals, for which use in high concentrations and in the long term is the aim, especially considering the trend for the use of branched and high molecular mass PEGs. This review addresses various obstacles and pitfalls in the production of PEGylated biopharmaceuticals, in particular, the specificity of PEGylation reactions, separation and purification issues, the analysis of inherently polydisperse PEG reagents and PEGylated products, the consistency of products and processes, and the accurate determination of pharmacokinetic properties.

Production of nonclassical inclusion bodies from which correctly folded protein can be extracted.

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.