Development and Validation of a Cell-Based Binding Neutralizing Antibody Assay for an Antibody-Drug Conjugate.

The utilization of antibody-drug conjugates (ADCs) has gained considerable attention in the field of targeted cancer therapy due to their ability to synergistically combine the specificity of monoclonal antibodies (mAbs) and the potency of small molecular drugs. However, the immunogenic nature of the antibody component within ADCs warrants the need for robust immunogenicity testing, including a neutralizing antibody (NAb) assay. Since the mechanism of action (MOA) of the ADC is to first bind to the target cells and then release the payload intracellularly to kill the cells, the most relevant NAb assay format would be a cell-based killing assay. However, in this paper, we present a case where a cell-based killing assay could not be developed after multiple cell lines and NAb-positive controls (PC) had been tested. Surprisingly, contrary to our expectations, all NAb PCs tested exhibited an increased killing effect on the target cells, instead of the expected protective response. This unexpected phenomenon most likely is due to the non-specific internalization of drug/NAb complexes via FcγRs, as an excessive amount of human IgG1 and mouse IgG2a, but not mouse IgG1, greatly inhibited drug or drug/NAb complexes induced cell death. To overcome this obstacle, we implemented a novel cell-based binding assay utilizing the Meso Scale Discovery (MSD) platform. We also propose that an in vitro cell killing NAb assay is limited to at best monitoring the target binding and internalization induced cell death, but not by-stander killing induced by prematurely released or dead-cell released payload, hence cannot really mimic the in vivo MOA of ADC.

Considerations in the Immunogenicity Assessment Strategy for Oligonucleotide Therapeutics (ONTs).

Oligonucleotide therapeutics (ONTs) are a diverse group of short synthetic nucleic acid-based molecules that exploit innovative intracellular molecular strategies to create novel treatments for a variety of medical conditions. ONT molecules (~7-15 kDa) reside between traditional large and small molecules, and there has been debate regarding their immunogenicity risk. To date, 13 ON drugs have been approved, and as the field is relatively new, there are currently no specific regulatory guidelines to indicate how to develop, validate, and interpret the immunogenicity assays of ONTs. Some investigators do not test for immune responses to ONs while others test for antibodies (Abs) to components within the formulation, which may or may not include aspects of characterization such as domain mapping of ONT conjugates. Similar to other biopharmaceuticals, the immunogenic properties of ONTs could be influenced by sequence, route, dosage, target population, co-medications, etc. The current anti-drug antibody (ADA) data for different approved ONTs suggest that their administration poses a low immunogenicity risk without any significant impact on pharmacokinetics (PK), pharmacodynamics (PD), and safety; nevertheless, until the field matures with data from many more ON drugs, it remains prudent to assess immunogenicity. The emphasis of this article is to highlight how current ADA methodologies might be applied to the development of ONTs, discuss factors that may pose immunogenicity risks, and provide the authors' current position on immunogenicity assessment strategies for ONTs. We also discuss assay parameters that may be appropriate for the detection and characterization of ADAs, including the evaluation of neutralizing ADAs, ADA isotyping, Abs to dsDNA, and pre-existing ADA. Immunogenicity risk assessments (IRAs) and early interactions with regulators will inform how to proceed in late stage/pivotal studies.

Analysis of regulatory guidance on antidrug antibody testing for therapeutic protein products.

Therapeutic proteins have the potential to induce unwanted immune responses. The potential impact of immunogenicity on pharmacokinetics, pharmacodynamics, safety and efficacy are well established. Here, we analyze key aspects of current US FDA and EMA guidelines on the development and validation of antidrug antibody assays. Although FDA and EMA guidance documents are in harmony on most points, EMA allows greater leeway for scientific judgement, while FDA recommends specific approaches that may not be appropriate in some situations. Many white papers suggest approaches different from the guidance documents, however, these can conflict with each other and are themselves only scientifically valid in certain situations. Here, we indicate when alternatives to guidance may be needed and what those approaches might be.

Genetic diversity of near genome-wide hepatitis C virus sequences during chronic infection: evidence for protein structural conservation over time.

Infection with hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and end-stage liver disease worldwide. The genetics of HCV infection in humans and the disease course of chronic hepatitis C are both remarkably variable. Although the response to interferon treatment is largely dependent on HCV genotypes, whether or not a relationship exists between HCV genome variability and clinical course of hepatitis C disease still remains unknown. To more thoroughly understand HCV genome evolution over time in association with disease course, near genome-wide HCV genomes present in 9 chronically infected participants over 83 total study years were sequenced. Overall, within HCV genomes, the number of synonymous substitutions per synonymous site (d(S)) significantly exceeded the number of non-synonymous substitutions per site (d(N)). Although both d(S) and d(N) significantly increased with duration of chronic infection, there was a highly significant decrease in d(N)/d(S) ratio in HCV genomes over time. These results indicate that purifying selection acted to conserve viral protein structure despite persistence of high level of nucleotide mutagenesis inherent to HCV replication. Based on liver biopsy fibrosis scores, HCV genomes from participants with advanced fibrosis had significantly greater d(S) values and lower d(N)/d(S) ratios compared to participants with mild liver disease. Over time, viral genomes from participants with mild disease had significantly greater annual changes in d(N), along with higher d(N)/d(S) ratios, compared to participants with advanced fibrosis. Yearly amino acid variations in the HCV p7, NS2, NS3 and NS5B genes were all significantly lower in participants with severe versus mild disease, suggesting possible pathogenic importance of protein structural conservation for these viral gene products.

Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver.

Our previous morphological studies illustrated the association of sterols with Plasmodium infecting hepatocytes. Because malaria parasites cannot synthesize sterols, they must scavenge these lipids from the host. In this paper, we have examined the source/s of sterols for intrahepatic Plasmodium and evaluated the importance of sterols for liver stage development. We show that Plasmodium continuously diverts cholesterol from hepatocytes until release of merozoites. Removal of plasma lipoproteins from the medium results in a 70% reduction of cholesterol content in hepatic merozoites but these parasites remain infectious in animals. Plasmodium salvages cholesterol that has been internalized by low-density lipoprotein but reduced expression of host low-density lipoprotein receptors by 70% does not influence liver stage burden. Plasmodium is also able to intercept cholesterol synthesized by hepatocytes. Pharmacological blockade of host squalene synthase or downregulation of the expression of this enzyme by 80% decreases by twofold the cholesterol content of merozoites without further impacting parasite development. These data enlighten that, on one hand, malaria parasites have moderate need of sterols for optimal development in hepatocytes and, on the other hand, they can adapt to survive in cholesterol-restrictive conditions by exploitation of accessible sterols derived from alternative sources in hepatocytes to maintain proper infectivity.

Metamorphosis of the malaria parasite in the liver is associated with organelle clearance.

Malaria parasites encounter diverse conditions as they cycle between their vertebrate host and mosquito vector. Within these distinct environments, the parasite undergoes drastic transformations, changing both its morphology and metabolism. Plasmodium species that infect mammals must first take up residence in the liver before initiating red blood cell infection. Following penetration into hepatocytes, the parasite converts from an invasion-competent, motile, elongated sporozoite to a metabolically active, round trophozoite. Relatively little is known about the cellular events involved in sporozoite metamorphosis. Our data uncover the early cellular events associated with these transformations. We illustrate that the beginning of metamorphosis is marked by the disruption of the membrane cytoskeleton beneath the plasma membrane, which results in a protruding area around the nucleus. As this bulbous region expands, the two distal ends of the sporozoite gradually retract and disappear, leading to cell sphericalization. This shape change is associated with major interior renovations and clearance of superfluous organelles, e.g. micronemes involved in invasion. The membrane cytoskeleton is reorganized into dense lamellar arrays within the cytoplasm and is partially expulsed by converting parasites. Simultaneously, micronemes are compartmentalized into large exocytic vesicles and are then discharged into the environment. At the completion of metamorphosis, the parasites only retain organelles necessary for replication. These observations lay the groundwork for further investigations on the developmental pathways implicated in the metamorphosis of the malaria parasite.

Hepatitis C virus induces oxidative stress, DNA damage and modulates the DNA repair enzyme NEIL1.

BACKGROUND AND AIMS: Hepatitis C virus (HCV)-induced chronic inflammation may induce oxidative stress which could compromise the repair of damaged DNA, rendering cells more susceptible to spontaneous or mutagen-induced alterations, the underlying cause of liver cirrhosis and hepatocellular carcinoma. In the current study we examined the induction of reactive oxygen species (ROS) resulting from HCV infection and evaluated its effect on the host DNA damage and repair machinery. METHODS: HCV infected human hepatoma cells were analyzed to determine (i) ROS, (ii) 8-oxoG and (iii) DNA glycosylases NEIL1, NEIL2, OGG1. Liver biopsies were analyzed for NEIL1. RESULTS: Human hepatoma cells infected with HCV JFH-1 showed 30-60-fold increases in ROS levels compared to uninfected cells. Levels of the oxidatively modified guanosine base 8-oxoguanine (8-oxoG) were significantly increased sixfold in the HCV-infected cells. Because DNA glycosylases are the enzymes that remove oxidized nucleotides, their expression in HCV-infected cells was analyzed. NEIL1 but not OGG1 or NEIL2 gene expression was impaired in HCV-infected cells. In accordance, we found reduced glycosylase (NEIL1-specific) activity in HCV-infected cells. The antioxidant N-acetyl cystein (NAC) efficiently reversed the NEIL1 repression by inhibiting ROS induction by HCV. NEIL1 expression was also partly restored when virus-infected cells were treated with interferon (IFN). HCV core and to a lesser extent NS3-4a and NS5A induced ROS, and downregulated NEIL1 expression. Liver biopsy specimens showed significant impairment of NEIL1 levels in HCV-infected patients with advanced liver disease compared to patients with no disease. CONCLUSION: Collectively, the data indicate that HCV induction of ROS and perturbation of NEIL1 expression may be mechanistically involved in progression of liver disease and suggest that antioxidant and antiviral therapies can reverse these deleterious effects of HCV in part by restoring function of the DNA repair enzyme/s.

Investigation of putative multisubtype hepatitis C virus infections in vivo by heteroduplex mobility analysis of core/envelope subgenomes.

The frequency that multiple different subtypes of hepatitis C virus (HCV) simultaneously infect a given individual is controversial. To address this question, heteroduplex mobility analysis (HMA) of portions of the HCV core and envelope 1 region was optimized for sensitive and specific detection of mixtures of HCV genomes of different genotype or subtype. Using the standard HCV genotyping approach of 5'-untranslated region (UTR) analysis, 28 of 374 (7.5%) chronic hepatitis C research subjects were classified as having either multiple-subtype HCV infections (n = 21) or switching HCV subtypes over time (n = 7), the latter pattern implying viral superinfection. Upon retesting of specimens by HMA, 25 of 28 multiple-subtype results could not be reproduced. All three patients with positive results were injection drug users with potential multiple HCV exposures. To address the hypothesis of tissue sequestration of multiple-subtype HCV infections, liver (n = 22), peripheral blood mononuclear cell (n = 13), perihepatic lymph node (n = 16), and serum (n = 19) specimens from 23 subjects with end-stage hepatitis C were collected and analyzed by the HMA technique. Whereas 5'-UTR results implicated mixed-subtype HCV infections in 2 subjects, HMA testing revealed no evidence of a second HCV subtype in any tissue compartment (0 of 70 compartments [0%]) or within any given subject (0 of 23 subjects [0%]). In summary, a large proportion of mixed-genotype and switching-genotype patterns generated by 5'-UTR analysis were not reproducible using the HMA approach, emphasizing the need for additional study.

New host nuclear functions are not required for the modifications of the parasitophorous vacuole of Toxoplasma.

The obligate intracellular parasite Toxoplasma develops within a parasitophorous vacuole (PV) uniquely adapted for its survival in mammalian cells. Post-invasion events extensively modify the PV, resulting in interactions with host cell structures. Recent studies emphasized that Toxoplasma is able to co-opt host gene expression, suggesting that host transcriptional activities are required for parasite infection. By using an experimental enucleation model, we investigated the potential need for Toxoplasma to modify its PV by modulating gene expression in the cell wherein it resides. Unexpectedly, cytoplasts can be actively invaded by Toxoplasma and sustain its replication inside a vacuole until egress and transmission to neighbouring cells. Although randomly distributed in the cytoplast, the PV associates with host centrosomes and the Golgi, is surrounded by host microtubules, and recruits host endoplasmic reticulum and mitochondria. Parasites are proficient in diverting exogenous nutrients from the endocytic network of cytoplasts. In enucleated cells invaded by an avirulent strain of T. gondii, the PV can normally transform into cysts. These observations suggest that new host nuclear functions are not proximately required for the post-invasion events underlying the remodelling of the host cell in which the parasites are confined, and therefore for the generation of infectious parasites in vitro.

Cellular interactions of Plasmodium liver stage with its host mammalian cell.

The Plasmodium liver forms are bridgehead stages between the mosquito sporozoite stages and mammalian blood stages that instigate the malaria disease. In hepatocytes, Plasmodium achieves one of the fastest growth rates among eukaryotic cells. However, nothing is known about host hepatic cell interactions, e.g. nutrient scavenging and/or subversion of cellular functions necessary for Plasmodium development and replication. Plasmodium usually invades hepatocytes by establishing a parasitophorous vacuole wherein it undergoes multiple nuclear division cycles. We show that Plasmodium preferentially develops in the host juxtanuclear region. By comparison with the parasitophorous vacuole of other apicomplexan parasites which associate with diverse host organelles, the Plasmodium parasitophorous vacuole only forms an association with the host endoplasmic reticulum. Intrahepatic Plasmodium actively modifies the permeability of its vacuole to allow the transfer of a large variety of molecules from the host cytosol to the vacuolar space through open channels. In contrast with malaria blood stages, the pores within the parasitophorous vacuole membrane of the liver stage display a smaller size as they restrict the passage of solutes to less than 855Da. These pores are stably maintained during parasite karyokinesis until complete cellularisation. Host-derived cholesterol accumulated at the parasitophorous vacuole membrane may modulate the channel activity. These observations define the parasitophorous vacuole of the Plasmodium liver stage as a dynamic and highly permeable compartment that can ensure the sustained supply of host molecules to support parasite growth in the nutrient-rich environment of liver cells.

Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent.

Phylogenetic characterization of soil isolate NJ-15, based on sequence homology of a partial 746-bp fragment of 16SrDNA amplicon, with the ribosomal database sequences (http://www.msu.edu/RDP/cgis/phylip.cgi), validated the strain as Pseudomonas aeruginosa. The strain NJ-15 produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. Besides, the strain also exhibited significant production of both the siderophore and hydrogen cyanide (HCN) on chrome azurol S and King's B media, respectively. The data revealed lower HCN production under iron-limiting conditions vis-à-vis higher HCN release with iron stimulation. Significant growth inhibition of phytopathogenic fungi occurred in the order as Fusarium oxysporum > Trichoderma herizum > Alternaria alternata > Macrophomina phasiolina upon incubation with strain NJ-15 cells. Thus, the secondary metabolites producing new Pseudomonas aeruginosa strain NJ-15 exhibited innate potential of plant growth promotion and biocontrol activities in vitro.