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Is hydrophobic interaction chromatography the most suitable technique to characterize site-specific antibody-drug conjugates?
No full textAntibody drug conjugates (ADCs) belong to the fastest growing class of therapeutic agents for cancer therapy. In preclinical and clinical studies, there is a significant number of site-specific ADCs (also known as third generation ADCs), which are more homogeneous than their previous generations. These new ADC formats in which the inter-chain disulphide bridges (hinge cysteines) are not reduced also need to be deeply characterized. In particular, various quality attributes (QAs) have to be determined, such as free antibody level, average drug to antibody ratio (DAR) and drug distribution. In this contribution, we have demonstrated that these QAs can be determined using reversed phase liquid chromatography (RPLC) for the native ADCs, rather than the historical hydrophobic interaction chromatography (HIC) widely used for hinged cysteines ADC products. In addition, the RPLC approach can be directly coupled to mass spectrometry (MS) for identification of molecular species.
„The authors received feedback by the reviewers of Journal of Chromatography A and have addressed their minor comments. The changes are tracked in the change history of attached word document (i.e. the final manuscript for re-submission).
Regarding changes of IP relevant data, only the logD value of the linker-payload had to be added (due to a reviewer`s request). However, this is considered acceptable, since no other data on the antibody or the linker-payload (such as chemical structures or physical-chemical properties) are shown.
IRF2 is a master regulator of human keratinocyte stem cell fate
No full textSUMMARY
Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and provision of differentiated cells. Human epidermal keratinocytes retain stem cell potential in vitro but this is highly variable and lost over time suggesting extrinsic and intrinsic regulation.
As transcription factor regulatory circuits have been shown to govern cell identity and are sufficient to induce pluripotency or transdifferentiate cell types, we sought to define changes in transcriptional circuitry between two populations of keratinocytes with intrinsic high and low stem cell potential.
Using integrated chromatin and transcriptional profiling, we implicate the transcription factor IRF2 as antagonistic to stemness and show that its knockdown in keratinocytes with low stem cell potential is sufficient to increase self-renewal, migration and ability to generate 3D human skin constructs.
These data suggest that transcription factor regulatory circuits, in addition to maintaining cell identity, control cellular plasticity and could offer potential for therapeutic modulation of cell functio
Sustainability challenges in peptide synthesis and purification: from R&D to production
No full textIn recent years there has been a growing interest on therapeutic peptides within the pharmaceutical industry with more than 50 peptide drugs in the market, approximately 170 in clinical trials and >200 in preclinical development. However, the current state of the art in peptide synthesis involves primarily legacy technologies with usage of large amounts of highly hazardous reagents and solvents and little focus on green chemistry and engineering. In 2016 the ACS Pharmaceutical Roundtable identified the greener synthesis of peptides as a critical unmet need and a new Roundtable team formed to address this important area. The initial focus of this group is to highlight best practices in peptide synthesis and encourage much needed innovations. In this perspective, we aim to summarise the current challenges of peptide synthesis and purification in terms of sustainability, highlight possible solutions and encourage synergies between academia and industry and between the pharmaceutical industry and CROs/CMOs
Small-molecule factor B inhibitor for the treatment of complement-mediated diseases
No full textDysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development
Targeting Trypanosoma brucei FPPS by Fragment-based drug discovery
No full textTrypanosoma brucei (T. brucei) is the causative agent of the Human African Trypanosomiasis (HAT), which is a neglected disease with an endemic occurrence in 36 sub-Saharan African countries. The current standard of care suffers from low efficacy and severe side effects. Therefore, new drugs with better safety and efficacy profiles are urgently needed. Nitrogen-containing bisphosphonates, a current treatment for bone diseases, have been shown to block the growth of the T. brucei parasites by inhibiting farnesyl pyrophosphate synthase (FPPS); however, due to their particular pharmacokinetic properties they are not well suited for parasitic therapy. Recently, an additional allosteric site was discovered on the surface of human FPPS that, based on sequence analysis, is likely also present in T. brucei FPPS. The high unmet medical need combined with the discovery of a potential new target site prompted a fragment-based drug discovery approach to identify non-bisphosphonate binders on T. brucei FPPS, which is presented in this work.
Fragment screening was performed by NMR and X-ray crystallography. To this end, a robust T. brucei FPPS crystallization system was established enabling high-throughput determination of crystal structures up to 1.67 Å resolution. Structural superimpositions revealed that the allosteric site found on human FPPS is in fact present in T. brucei FPPS. This observation enabled subsequent protein-observed NMR and crystal soaking experiments with established human FPPS binders resulting in three protein-ligand complex structures with bound fragments in the previously unknown allosteric site. For most of the tested binders, Kd by SPR was outside of experimental range for T. brucei FPPS and only for one fragment the Kd on T. brucei FPPS was determined three orders of magnitude higher than the IC50 value on human FPPS. Crystal structural analysis revealed a different binding mode on human and T. brucei FPPS with reduced protein-ligand interactions on T. brucei FPPS, which explains the significantly different binding affinity.
Encouraged by the detection of first allosteric binders on T. brucei FPPS, fragment pools were screened by ligand-observed NMR and identified hits were followed-up by single compound ligand-observed NMR and protein-observed NMR resulting in 25 validated fragment hits for T. brucei FPPS. Validated hits were followed-up by crystal soaking and co-crystallization experiments and seven protein-ligand complex structures were solved using PanDDA. Out of the seven fragments, four fragments were bound in the active site, one fragment was detected in the allosteric site that was identified as part of this thesis, and two fragments were bound in surface-exposed binding sites. Notably, an active site bound fragment with a four atom long flexible linker adopted an orthogonal binding mode along αD when compared to the other three ligands. Sixteen fragment analogues of the elongated flexible active site fragment were tested by SAR by catalogue and archive, and one crystal structure with a fragment analogue was solved and was surprisingly found in the allosteric site.
In addition to the NMR fragment screen, an X-ray screen was performed at XChem (Diamond, UK) and at EMBL/ESRF (Grenoble, FR) resulting in seven protein-ligand structures. One fragment was positioned in the active site, three fragments in the allosteric site, two fragments in a cryptic site between helices αI and αH and one fragment at the opposite side of the allosteric site close to αG and αF. Fragment binding was further validated in protein-observed NMR.
As fragments identified by fragment screening typically exhibit low binding affinities up to the mM range, structure-based fragment optimisation based on a fragment merging and growing approach was performed. In total, ten compounds were synthesised and subjected to protein observed NMR and X-ray structural analysis. Strikingly, a fragment merger based on T. brucei and T. cruzi active site binders bound in a new binding site close to the SARM instead to the active site.
Taken together, this work presents high-resolution structures of T. brucei FPPS and identified 19 compounds binding to seven different sites thereby paving the way for future studies aiming to identify high-affinity non-bisphosphonate inhibitors for T. brucei FPPS with pharmacokinetic properties that are suitable for parasitic indications
The impact of assay recovery on the apparent permeability, a function of lysosomal trapping
No full text1. In vitro permeability assessment tools, like PAMPA, Caco-2, and MDCK, are frequently used to assess permeability and provide input in to various classification systems. Frequently, the measured recovery values in permeability assays are poor. Poor recovery may be a result of lysosomal trapping of compound. It was hypothesized that a relationship existed between diminished assay recovery of compound due to lysosomal trapping and underestimation of the Papp value.2. To examine this hypothesis, a series of experiments were conducted measuring cellular accumulation, percent recovery, and permeability in the absence or presence of an inhibitor of the V-type H+-ATPase, bafilomycin A1, to determine if a quantifiable relationship between lysosomal trapping, recovery, and permeability existed.3. Displacing compounds from lysosomes using bafilomycin A1 resulted in an improved compound recovery in the assay and a corresponding elevated permeability, where for each 10% loss in recovery, a Papp underestimate of ∼2.2 × 10-6 cm/s was observed. The findings highlight the potential for compound misclassification in various classification systems when assay recovery is not considered. Consideration of lysosomal trapping in the context of permeability assays may yield permeability values more reflective of the intrinsic permeability and the appropriate permeability classification
CRISPR/Cas13a‐powered electrochemical microfluidic biosensor for nucleic acid amplification‐free miRNA diagnostics
No full textNoncoding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated with many different diseases, such as cancer, dementia, and cardiovascular conditions. The key for effective treatment is an accurate initial diagnosis at an early stage, improving the patient's survival chances. In this work, the first clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a‐powered microfluidic, integrated electrochemical biosensor for the on‐site detection of microRNAs is introduced. Through this unique combination, the quantification of the potential tumor markers microRNA miR‐19b and miR‐20a is realized without any nucleic acid amplification. With a readout time of 9 min and an overall process time of less than 4 h, a limit of detection of 10 pm is achieved, using a measuring volume of less than 0.6 µL. Furthermore, the feasibility of the biosensor platform to detect miR‐19b in serum samples of children, suffering from brain cancer, is demonstrated. The validation of the obtained results with a standard quantitative real‐time polymerase chain reaction method shows the ability of the electrochemical CRISPR‐powered system to be a low‐cost, easily scalable, and target amplification‐free tool for nucleic acid based diagnostics
Pharmacological Inhibition of GPR4 remediates intestinal inflammation in a mouse colitis model
No full textInflammatory bowel disease (IBD) is characterized by chronic, recurring inflammation of the digestive tract. Current therapeutic approaches are limited and include biologics and steroids such as anti-TNFα monoclonal antibodies and corticosteroids, respectively. Significant adverse drug effects can occur for chronic usage and include increased risk of infection in some patients. GPR4, a pH-sensing G protein-coupled receptor, has recently emerged as a potential therapeutic target for intestinal inflammation. We have assessed the effects of a GPR4 antagonist, 2-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)phenyl)-5-(piperidin-4-yl)-1,3,4-oxadiazole (GPR4 antagonist 13, also known as NE 52-QQ57) in the dextran sulfate sodium (DSS)-induced acute colitis mouse model. The GPR4 antagonist 13 inhibited intestinal inflammation. The clinical parameters such as body weight loss and fecal score were reduced in the GPR4 antagonist 13 treatment group compared to vehicle control. Macroscopic disease indicators such as colon shortening, splenic expansion, and mesenteric lymph node enlargement were all reduced in severity in the GPR4 antagonist 13 treated mice. Histopathological features of active colitis were alleviated in GPR4 antagonist 13 treatment groups compared to vehicle control. Finally, inflammatory gene expression in the colon tissues and vascular adhesion molecule expression in the intestinal endothelia were attenuated by GPR4 antagonist 13. Our results indicate that GPR4 antagonist 13 provides a protective effect in the DSS-induced acute colitis mouse model, and inhibition of GPR4 can be explored as a novel anti-inflammatory approach
Secukinumab, a fully human anti-interleukin-17A monoclonal antibody, exhibits low immunogenicity in psoriasis patients treated up to 5 years
No full textIntroduction: Secukinumab is a fully human monoclonal antibody that selectively neutralizes IL-17A, a key cytokine involved in psoriasis (PsO) and psoriatic arthritis (PsA) development, and has shown long lasting efficacy and safety in the complete spectrum of psoriasis manifestations. Monoclonal antibody therapies may be associated with the production of treatment-emergent anti-drug antibodies (TE-ADA) that can affect drug pharmacokinetics, diminish clinical responses, or cause hypersensitivity reactions. Secukinumab exhibited minimal immunogenicity up to 52 weeks in patients with moderate-to-severe plaque psoriasis, as evidenced by TE-ADA in <1% patients.
Objective: To investigate the immunogenicity of secukinumab treatment up to 5 years in two phase 3 extension studies (NCT01640951 and NCT01365455) in patients with moderate-to-severe plaque psoriasis.
Material and Methods: Immunogenicity was evaluated up to Week 268 (5 years). TE-ADA were defined as positive anti-drug antibody (ADA) signals detected in post-treatment samples from patients with negative baseline signals. Confirmed positive samples were further analysed for their neutralizing potential.
Results: In total, 1821 patients entered the extension studies. Among patients receiving secukinumab and evaluated for ADAs (n = 1636), 32 developed TE-ADA, which resulted in an incidence of new TE-ADA cases below 1% per year. Neutralizing antibodies were detected in 9/32 (28%) patients with TE-ADA. Half of ADA-positive cases were transient. Among pharmacokinetic samples measured at the times of immunogenicity determination (n = 9992), 544 (5.4%) had secukinumab concentrations higher than the drug tolerance level of 53.8 µg/mL. There was no effect of TE-ADA, including neutralizing antibodies, on efficacy, safety, or pharmacokinetics of secukinumab.
Conclusions: The yearly secukinumab immunogenicity incidence over 5 years of treatment was consistently below 1% in patients with moderate-to-severe plaque psoriasis. Any TE-ADA, including neutralizing antibodies, were not associated with loss of secukinumab efficacy or with clinical concerns
MAA868, a novel FXI antibody with a unique binding mode, shows durable effects on markers of anticoagulation in humans
No full textA large unmet medical need exists for safer antithrombotic drugs because all currently approved anticoagulant agents interfere with hemostasis, leading to an increased risk of bleeding. Genetic and pharmacologic evidence in humans and animals suggests that reducing factor XI (FXI) levels has the potential to effectively prevent and treat thrombosis with a minimal risk of bleeding. We generated a fully human antibody (MAA868) that binds the catalytic domain of both FXI (zymogen) and activated FXI. Our structural studies show that MAA868 traps FXI and activated FXI in an inactive, zymogen-like conformation, explaining its equally high binding affinity for both forms of the enzyme. This binding mode allows the enzyme to be neutralized before entering the coagulation process, revealing a particularly attractive anticoagulant profile of the antibody. MAA868 exhibited favorable anticoagulant activity in mice with a dose-dependent protection from carotid occlusion in a ferric chloride-induced thrombosis model. MAA868 also caused robust and sustained anticoagulant activity in cynomolgus monkeys as assessed by activated partial thromboplastin time without any evidence of bleeding. Based on these preclinical findings, we conducted a first-in-human study in healthy subjects and showed that single subcutaneous doses of MAA868 were safe and well tolerated. MAA868 resulted in dose- and time-dependent robust and sustained prolongation of activated partial thromboplastin time and FXI suppression for up to 4 weeks or longer, supporting further clinical investigation as a potential once-monthly subcutaneous anticoagulant therapy