The complement C5 inhibitor crovalimab in paroxysmal nocturnal hemoglobinuria.

Complement C5 inhibition is the standard of care (SoC) for patients with paroxysmal nocturnal hemoglobinuria (PNH) with significant clinical symptoms. Constant and complete suppression of the terminal complement pathway and the high serum concentration of C5 pose challenges to drug development that result in IV-only treatment options. Crovalimab, a sequential monoclonal antibody recycling technology antibody was engineered for extended self-administered subcutaneous dosing of small volumes in diseases amenable for C5 inhibition. A 3-part open-label adaptive phase 1/2 trial was conducted to assess safety, pharmacokinetics, pharmacodynamics, and exploratory efficacy in healthy volunteers (part 1), as well as in complement blockade-naive (part 2) and C5 inhibitor-treated (part 3) PNH patients. Twenty-nine patients were included in part 2 (n = 10) and part 3 (n = 19). Crovalimab concentrations exceeded the prespecified 100-µg/mL level and resulted in complete and sustained terminal complement pathway inhibition in treatment-naive and C5 inhibitor-pretreated PNH patients. Hemolytic activity and free C5 levels were suppressed below clinically relevant thresholds (liposome assay <10 U/mL and <50 ng/mL, respectively). Safety was consistent with the known profile of C5 inhibition. As expected, formation of drug-target-drug complexes was observed in all 19 patients switching to crovalimab, manifesting as transient mild or moderate vasculitic skin reactions in 2 of 19 participants. Both events resolved under continued treatment with crovalimab. Subcutaneous crovalimab (680 mg; 4 mL), administered once every 4 weeks, provides complete and sustained terminal complement pathway inhibition in patients with PNH, warranting further clinical development (ClinicalTrials.gov identifier, NCT03157635).

Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice.

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.

Implications of Ocular Confounding Factors for Aqueous Humor Proteomic and Metabolomic Analyses in Retinal Diseases.

Purpose: To assess the impact of ocular confounding factors on aqueous humor (AH) proteomic and metabolomic analyses for retinal disease characterization. Methods: This study recruited 138 subjects (eyes): 102 with neovascular age-related macular degeneration (nAMD), 18 with diabetic macular edema (DME), and 18 with cataract (control group). AH samples underwent analysis using Olink Target 96 proteomics and Metabolon's metabolomics platform Data analysis included correlation, differential abundance, and gene-set analysis. Results: In total, 756 proteins and 408 metabolites were quantified in AH. Total AH protein concentration was notably higher in nAMD (3.2-fold) and DME (4.1-fold) compared to controls. Pseudophakic eyes showed higher total AH protein concentrations than phakic eyes (e.g., 1.6-fold in nAMD) and a specific protein signature indicative of matrix remodeling. Unexpectedly, pupil-dilating drugs containing phenylephrine/tropicamide increased several AH proteins, notably interleukin-6 (5.4-fold in nAMD). Correcting for these factors revealed functionally relevant protein correlation clusters and disease-relevant, differentially abundant proteins across the groups. Metabolomics analysis, for which the relevance of confounder adjustment was less apparent, suggested insufficiently controlled diabetes and chronic hyperglycemia in the DME group. Conclusions: AH protein concentration, pseudophakia, and pupil dilation with phenylephrine/tropicamide are important confounding factors for AH protein analyses. When these factors are considered, AH analyses can more clearly reveal disease-relevant factors. Translational Relevance: Considering AH protein concentration, lens status, and phenylephrine/tropicamide administration as confounders is crucial for accurate interpretation of AH protein data.

Correlation of Aqueous, Vitreous, and Serum Protein Levels in Patients With Retinal Diseases.

Purpose: To further establish aqueous humor (AH) as a clinically suitable source of protein biomarkers in retinal diseases by evaluating the correlation of a large panel of proteins between AH, vitreous humor (VH), and serum (SE). Methods: We enrolled 60 subjects (eyes) with various non-infectious retinal diseases. AH, VH, and SE proteins were analyzed using the Olink Target 96 platform (1196 protein assays in total). We compared these three matrices in terms of quantification overlap, principal component analysis, and correlation. Results: In the AH, VH, and SE samples, 841, 917, and 1133 proteins, respectively, were consistently quantified above the limit of detection in more than 30% of patients. AH and VH shared 812 of these proteins. AH and VH samples overlapped along principal component 1, but SE samples were distinct. We identified 490 proteins with significant (false discovery rate [FDR]-adjusted P < 0.05) and relevant correlations (correlation coefficient > 0.5) between AH and VH, compared to only 33 and 40 proteins for VH and SE and for AH and SE, respectively. Conclusions: Due to a close correlation between protein concentrations in the AH and VH and a clear difference from the SE, AH has the potential to serve as a substitute for VH and may hold significance in identifying protein biomarkers and novel targets related to retinal diseases. Translational Relevance: This study further supports AH as a clinically suitable source of protein biomarkers in retinal diseases. In addition, the identified AH and VH correlations can inform the selection of protein biomarker candidates in future translational research.

A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer Oligonucleotides.

The successful development of high-affinity gapmer antisense oligonucleotide (ASO) therapeutics containing locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions has been hampered by the risk of hepatotoxicity. Here, we present an in vitro approach using transfected mouse fibroblasts to predict the potential hepatic liabilities of LNA-modified ASOs (LNA-ASOs), validated by assessing 236 different LNA-ASOs with known hepatotoxic potential. This in vitro assay accurately reflects in vivo findings and relates hepatotoxicity to RNase H1 activity, off-target RNA downregulation, and LNA-ASO-binding affinity. We further demonstrate that the hybridization-dependent toxic potential of LNA-ASOs is also evident in different cell types from different species, which indicates probable translatability of the in vitro results to humans. Additionally, we show that the melting temperature (Tm) of LNA-ASOs maintained below a threshold level of about 55°C greatly diminished the hepatotoxic potential. In summary, we have established a sensitive in vitro screening approach for assessing the hybridization-dependent toxic potential of LNA-ASOs, enabling prioritization of candidate molecules in drug discovery and early development.

Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia.

Single-stranded oligonucleotides (ON) comprise a promising therapeutic platform that enables selective modulation of currently undruggable targets. The development of novel ON drug candidates has demonstrated excellent efficacy, but in certain cases also some safety liabilities were reported. Among them are events of thrombocytopenia, which have recently been evident in late stage trials with ON drugs. The underlying mechanisms are poorly understood and the risk for ON candidates causing such events cannot be sufficiently assessed pre-clinically. We investigated potential thrombocytopenia risk factors of ONs and implemented a set of in vitro assays to assess these risks. Our findings support previous observations that phosphorothioate (PS)-ONs can bind to platelet proteins such as platelet collagen receptor glycoprotein VI (GPVI) and activate human platelets in vitro to various extents. We also show that these PS-ONs can bind to platelet factor 4 (PF4). Binding to platelet proteins and subsequent activation correlates with ON length and connected to this, the number of PS in the backbone of the molecule. Moreover, we demonstrate that locked nucleic acid (LNA) ribosyl modifications in the wings of the PS-ONs strongly suppress binding to GPVI and PF4, paralleled by markedly reduced platelet activation. In addition, we provide evidence that PS-ONs do not directly affect hematopoietic cell differentiation in culture but at higher concentrations show a pro-inflammatory potential, which might contribute to platelet activation. Overall, our data confirm that certain molecular attributes of ONs are associated with a higher risk for thrombocytopenia. We propose that applying the in vitro assays discussed here during the lead optimization phase may aid in deprioritizing ONs with a potential to induce thrombocytopenia.

The dioxin/aryl hydrocarbon receptor mediates downregulation of osteopontin gene expression in a mouse model of gastric tumourigenesis.

The dioxin/aryl hydrocarbon receptor functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding primarily drug-metabolizing enzymes. Expression of a constitutively active mutant of the aryl hydrocarbon receptor (CA-AhR) in transgenic mice results in development of stomach tumours, correlating with increased mortality. We have used suppression subtractive hybridization techniques followed by macroarray analysis to elucidate which genes are differentially expressed during this process. In the glandular stomach of CA-AhR mice, we observed decreased mRNA expression of osteopontin (OPN), a noncollagenous protein of bone matrix that is also involved in several important functions including regulation of cytokine production, macrophage accumulation, cell motility and adhesion. Downregulated expression of OPN during tumour development was confirmed by RT-PCR and RNA blot analysis. Immunohistochemical analysis showed that this decrease was confined to the corpus region, correlating with the restricted localization of the tumours. Decreased OPN mRNA expression was also observed in other organs of CA-AhR mice. Taken together, these results show that OPN is negatively regulated by the dioxin receptor, and that downregulation of its expression correlates with development of stomach tumours in mice expressing a constitutively active mutant of dioxin receptor.

Parathyroid hormone inhibits c-Jun N-terminal kinase activity in rat osteoblastic cells by a protein kinase A-dependent pathway.

Treatment of osteoblastic cells with PTH initiates dual signaling cascades resulting in activation of both PKA and PKC. It has been shown that PTH either inhibits or stimulates ERKs depending on dose of the hormone; nevertheless, the ability of PTH to regulate other members of the MAPK family is unknown. Another member of this family, c-Jun-NH(2)-terminal kinase (JNK), is preferentially activated by cytokines and cellular stresses and plays a key role in regulating the activity of various transcription factors. We demonstrate that treatment of UMR 106-01 cells and rat calvarial osteoblasts with PTH (10(-8) M), N-terminal peptides of PTH that selectively activate PKA, or 8-bromo-cAMP (activates PKA) results in the inhibition of JNK activity from high basal levels. Examination of the upstream members of the JNK cascade revealed that both stress-activated protein kinase/extracellular signal-related kinase kinase 1/MAPK kinase 4 and MAPK/extracellular signal-related kinase kinase kinase 1 activities were also inhibited after treatment with PTH (10(-8) M). We conclude that treatment of osteoblastic cells with PTH is sufficient to inhibit high basal JNK activity by activation of the PKA signaling cascade.

Improved subtractive suppression hybridization combined with high density cDNA array screening identifies differentially expressed viral and cellular genes.

Suppression subtractive hybridization (SSH) combines normalization and suppression PCR effect step in a single cycle to isolate differentially expressed genes in two cDNA samples. The PCR suppression effect is mediated by long inverted terminal repeats. The efficiency of the restriction enzyme digestion and the adapter ligation are crucial in the success of the SSH. We modified the original SSH protocol in order to improve the efficiency of the subtraction. A magnetic bead based separation step has been included after the ligation step, to purify the successfully ligated fraction of the tester. EBV(NEO) infected Akata(-) Burkitt's lymphoma cell line was compared with the EBV(-) Akata(-) cell line to isolate differentially expressed genes with the improved SSH protocol. Some 44 cDNA clones that showed the greatest differences in expression have been sequenced. Of them, 20 showed more than 3-fold difference in expression. Seven of the 20 genes were EBV genes. To quantitate the expression levels, high density nylon cDNA array hybridization was optimized. Statistical analysis of the data revealed that the spotting of the arrayer is exceptionally reproducible, which makes the comparison of the hybridization of parallel filters possible.

Impact of the lymphoma idiotype on in vivo tumor protection in a vaccination model based on targeting antigens to antigen-presenting cells.

Trioma cell vaccination is a potent new immunologic approach for the therapy of malignant B-cell lymphoma. It is based on targeting tumor antigens to internalizing receptors on antigen-presenting cells (APCs). Tumor cells are fused to an APC-specific hybridoma, where they are converted to trioma cells that include potentially all lymphoma-derived antigens and that express the APC-binding arm. In this study, the mechanisms of trioma-mediated tumor immunity in immunocompetent mice were dissected, and it was shown in this model system that humoral anti-idiotypic immunity is indeed detectable after idiotype-specific immunization but that it does not reflect the degree of tumor protection obtained in vivo. Immunization against the idiotype alone was not sufficient for efficient tumor rejection in vivo. Targeting tumor antigens to APCs is only successful in terms of inducing tumor protection when designed as a polyvalent vaccination protocol.