A novel immune-related long non-coding RNAs risk model for prognosis assessment of lung adenocarcinoma.

BACKGROUND: The abundant immune-related long non-coding RNA (IRLNRs) in immune cells and immune microenvironment have the potential to forecast prognosis and evaluate the effect of immunotherapy. IRLNRs analysis will provide a new perspective for LUAC research. METHODS: We calculated the immune score of each sample according to the expression levels of immune-related genes (IRGs) and screened the survival-related IRLNRs (sIRLNRs) by Cox regression analysis. The expression levels of AC068338.3 and AL691432.2 in tissues and cell lines were confirmed by RT-qPCR. RESULTS: 36 IRLNRs were selected by Pearson correlation analysis. Ten sIRLNRs were significantly correlated with the clinical outcomes of LUAC patients. Five sIRLNRs were identified by multivariate COX regression analysis to establish the immune-related risk score model (IRRS). The overall survival (OS) in the high-risk group was shorter than that in the low-risk group. IRRS could be an independent prognostic factor with significant survival correlation The distributions of immune gene concentrations were different between high-risk group and low-risk group. Furthermore, we further verified that the expression levels of AC068338.3 and AL691432.2 in different LUAC cell lines and tumor tissues were lower than that in Human bronchial epithelial cell (HBE) and adjacent tissues respectively. The lower expression levels of AC068338.3 and AL691432.2 were detected with the more advance T-stages. CONCLUSIONS: Our results highlighted some sIRLNRs with significant clinical correlations and demonstrated their monitored and prognostic values for LUAC patients. The results of this study may provide a new perspective for immunological research and immunotherapy strategies.

Immunogenicity and toxicokinetics assessment of the mono-PEGylated recombinant human interleukin-11 in cynomolgus monkeys.

AIM: Protein therapeutics have potential to elicit immune responses resulting in undesirable anti-drug antibodies (ADA) that might affect product efficacy and patient safety, and should be assessed in animals before applying the treatment to humans. In this paper, we aim to assess the immunogenicity and toxicokinetics of the mono-PEGylated recombinant human interleukin-11 (rhIL-11), a novel protein therapeutic for the treatment of chemotherapy-induced thrombocytopenia, in repeated administration to cynomolgus monkeys. MAIN METHODS: Enzyme-linked immunosorbent assay (ELISA) methods were developed to measure ADA responses and plasma PEGylated IL-11 (PEG-IL11) concentration in monkeys. Assay parameters of immunogenicity and toxicokinetics methods were evaluated during validation in accordance with regulatory guidelines. We also employed cell-based assays to test the neutralizing activity of ADA provoked in monkeys. KEY FINDINGS: The results showed that weak immunogenicity occurred in some monkeys after receiving repeated dose of 0.1-0.3 mg/kg by subcutaneous administration and disappeared after the recovery period. More pronounced immunogenicity occurred at high dose of 0.9 mg/kg, with a higher positive rate and titer, and some ADAs had neutralizing activity, but it can be greatly reduced after recovery. Such ADAs generated in monkeys may be accounted for the plasma toxicokinetics changes of PEG-IL11 and a minor reduction in systemic exposure. SIGNIFICANCE: These methods have been successfully applied to immunogenicity and toxicokinetic studies of PEG-IL11 in repeated dose toxicity following subcutaneous administration to monkeys, and could be successfully used in clinical trials after some modifications.

Development of a FRET-Based Assay for Analysis of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immunogenicity Risk Assessment.

Treatment-emergent antidrug antibodies (TE-ADA) pose a major challenge to the development of biotherapeutics. The antidrug antibody responses are highly orchestrated and involve many types of immune cells and biological processes. Biological drug internalization and processing by antigen-presenting cells (APCs) are two initial and critical steps in the cascade of events leading to T cell-dependent ADA production. The assays thus far described in literature to evaluate immunogenicity potential/risk as a function of APC activity mainly focus on internalization of labeled drug candidates in vitro. Herein, we describe a high-throughput Förster Resonance Energy Transfer (FRET)-based assay for assessing both internalization and processing using CD14+ monocyte-derived dendritic cells (DCs) as APCs. Antigen-binding fragment F(ab')2 against IgG fragment crystallizable gamma (Fcγ) was labeled with the activatable FRET pair TAMRA-QSY7 as a universal probe for antibodies and proteins with a fragment crystallizable (Fc) domain. The assay was qualified using six mAbs of known clinical immunogenicity and one IgG1 isotype antibody using Design of Experiment (DoE). Correlation analysis of internalization and clinical immunogenicity data showed that this FRET-based internalization assay was able to detect clinically immunogenic antibodies. This method provides a tool for analyzing/screening the immunogenicity risk of biological candidates by assessing one of the critical components of the ADA formation process within the broader context of an immunogenicity risk assessment strategy.

Clinical Immunogenicity Risk Assessment for a Fusion Protein.

This document highlights some relevant factors in the assessment of immunogenicity risk of fusion protein therapeutics. Our aim is to highlight specific risks associated with this type of molecule, while also aligning with general risk assessment factors, through a hypothetical case study, where the therapeutic molecule of interest is a Receptor-Fc Fusion protein (RFF) expressed within a typical manufacturing process in Chinese Hamster Ovary Cells (CHO). Given that the components are comprised of endogenous sequences, the risk of developing an ADA response to this molecule is generally considered to be low. However, the consequences of such an immune response may be more severe, specifically, if there is cross reactivity with the endogenous receptor, inducing cell lysis, or if any ADA act as an agonist to trigger receptor signaling. The risk factors described below are not meant to provide a comprehensive list, but rather a framework for factors that should be considered. Immunogenicity risk is difficult to quantify and relies on a comprehensive analysis of both product and patient-related factors. The goal is not necessarily to quantify risk, but rather to demonstrate an understanding of factors that may pose a risk, implement a strategy to minimize risk factors and then align overall risk with a bioanalytical immunogenicity monitoring strategy. The consequences resulting from unexpected outcome will likely depend on severity and impact on patient safety. An immunogenicity risk assessment is an ongoing and continuous process throughout clinical development with the goal of maximizing the safety of patients.

Immunogenicity Risk Assessment for an Engineered Human Cytokine Analogue Expressed in Different Cell Substrates.

The purpose of this article is to illustrate how performance of an immunogenicity risk assessment at the earliest stage of product development can be instructive for critical early decision-making such as choice of host system for expression of a recombinant therapeutic protein and determining the extent of analytical characterization and control of heterogeneity in co- and post-translational modifications. Application of a risk-based approach for a hypothetical recombinant DNA analogue of a human endogenous cytokine with immunomodulatory functions is described. The manner in which both intrinsic and extrinsic factors could interact to influence the relative scale of risk associated with expression in alternative hosts, namely Chinese hamster ovary (CHO) cells, Pichia pastoris, Escherichia coli, or Nicotinia tabacum is considered in relation to the development of the investigational product to treat an autoimmune condition. The article discusses how particular product-related variants (primary amino acid sequence modifications and post-translational glycosylation or other modifications) and process-derived impurities (host cell proteins, endotoxins, beta-glucans) associated with the different expression systems might influence the impact of immunogenicity on overall clinical benefit versus risk for a therapeutic protein candidate that has intrinsic MHC Class II binding potential. The implications of the choice of expression system for relative risk are discussed in relation to specific actions for evaluation and measures for risk mitigation, including use of in silico and in vitro methods to understand intrinsic immunogenic potential relative to incremental risk associated with non-human glycan and protein impurities. Finally, practical guidance on presentation of this information in regulatory submissions to support clinical development is provided.

Clinical Immunogenicity Risk Assessment Strategy for a Low Risk Monoclonal Antibody.

This article provides a theoretical case-study risk assessment report for a low-risk monoclonal antibody (mAb) therapeutic. In terms of risk, there are considerations around risks to safety, but also risks regarding effects on pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Much of the discussion in this document is around the risk of immunogenicity incidence. A higher incidence of immunogenicity would necessitate a detailed review of the PK, efficacy and safety in anti-drug antibody (ADA) positive and ADA negative subjects, in order to evaluate potential effects. The publication is intended to provide a framework of some the current thought processes around assessing immunogenicity risk and for building strategies to mitigate those risks. For this example, we have created a hypothetical antibody, ABC-123, targeting a membrane protein on antigen presenting cells, for the treatment of rheumatoid arthritis (RA). This hypothetical antibody therapeutic is provided as an example for the purposes of risk assessment for a low risk molecule, although any application of similar approach would be case by case.

Pathophysiology, assessment and treatment of psoriatic dactylitis.

Dactylitis is diffuse swelling of the digits that is usually related to an underlying inflammatory or infiltrative disorder. Psoriatic arthritis (PsA) is the most common severe disease thought to cause dactylitis. Our understanding of the pathogenesis of PsA-related dactylitis comes from experimental animal models of PsA-like disease, as well as advances in imaging and other clinical studies. Clinical trials in PsA have increasingly included dactylitis as an important secondary outcome measure. These studies indicate that cytokines drive multi-locus microanatomical pan-digital pathology. Given the importance of pro-inflammatory cytokines, the pathogenesis of dactylitis is best understood as an initial aberrant innate immune response to biomechanical stress or injury, with subsequent adaptive immune mechanisms amplifying the dactylitis inflammatory response. Regarding the treatment of dactylitis, no studies have been conducted using dactylitis as the primary outcome measure, and the current knowledge comes from analysis of dactylitis as a secondary outcome measure.

Assessment of the immunogenicity of mechanically induced interferon aggregates in a transgenic mouse model.

Pump delivery of human interferon alpha-2B (IFNα2b) has the potential for inducing immunogenic drug aggregates. We therefore evaluated the immunogenicity of mechanically induced IFNα2b aggregates to assess this risk. Transgenic human-IFNα2b (TG) and wild-type (WT) FVB/N mice (n = 8 and n = 9/group, respectively) were administered mechanically agitated drug [45 Hz for 6 h (LLA) or 24 h (HLA)], chemically modified drug [low pH (pH 4.0) or metal oxidized (OXD)] or unstressed drug (native). Mice received IFNα2b (50 µg; 100 µg/mL; s.c.) formulations on days 0, 7, 14, and 21. Drug-binding and neutralizing antibody titers were determined after 28 d. Aggregate concentrations were highest in OXD and HLA formulations but OXD had more dimers/trimers. Geometric mean titers were 1:131, 1:728, 1:1573, 1:871, and 1:10,240 for WT mice (n = 9) and 1:207, 1:587, 1:1810, 1:571, and 1:2,153 for TG mice (n = 8) for native, LLA, HLA, pH4, and OXD groups, respectively. Mechanical agitation of IFNα2b induced equivalent titers of immunoglobulin to that of metal oxidation, both capable of binding to or neutralizing the drug in WT and TG mice. Thus, by limiting metal contamination and by inclusion of a stabilizing agent to mitigate drug aggregation, the risk of anti-drug immunoglobulin may be reduced in a pump delivery scenario.

2012 AAPS National Biotech Conference Open Forum: a perspective on the current state of immunogenicity prediction and risk management.

The immunogenicity profile of a biotherapeutic is determined by multiple product-, process- or manufacturing-, patient- and treatment-related factors and the bioanalytical methodology used to monitor for immunogenicity. This creates a complex situation that limits direct correlation of individual factors to observed immunogenicity rates. Therefore, mechanistic understanding of how these factors individually or in concert could influence the overall incidence and clinical risk of immunogenicity is crucial to provide the best benefit/risk profile for a given biotherapeutic in a given indication and to inform risk mitigation strategies. Advances in the field of immunogenicity have included development of best practices for monitoring anti-drug antibody development, categorization of risk factors contributing to immunogenicity, development of predictive tools, and development of effective strategies for risk management and mitigation. Thus, the opportunity to ask "where we are now and where we would like to go from here?" was the main driver for organizing an Open Forum on Improving Immunogenicity Risk Prediction and Management, conducted at the 2012 American Association of Pharmaceutical Scientists' (AAPS) National Biotechnology Conference in San Diego. The main objectives of the Forum include the following: to understand the nature of immunogenicity risk factors, to identify analytical tools used and animal models and management strategies needed to improve their predictive value, and finally to identify collaboration opportunities to improve the reliability of risk prediction, mitigation, and management. This meeting report provides the Forum participant's and author's perspectives on the barriers to advancing this field and recommendations for overcoming these barriers through collaborative efforts.

Statistical and bioanalytical considerations for establishing a depletion criterion for specificity testing during immunogenicity assessment of a biotherapeutic.

Immunogenicity assessment of fully human monoclonal antibody-based biotherapeutics requires sensitive and specific ligand binding assays. One of the components of specificity is the depletion of signal by a relevant biotherapeutic that is commonly based on an arbitrary depletion criterion of inhibition of the original response or reduction of the signal below the screening assay cut point (ACP). Hence, there is a need to develop a statistically derived physiologically relevant specificity criterion. We illustrate an optimization approach to determine the concentration of biotherapeutic required for the specificity evaluation. Naïve donor sample sets with and without circulating drug and antitherapeutic/drug antibody (ADA) were prepared. Next, a depletion cut point (DCP) using naïve and ADA-containing donor sets with the optimized biotherapeutic concentration was evaluated. A statistically derived design of experiment was used to establish a validated DCP. A reliable DCP requires naïve (no ADA) donors treated only with an optimized concentration of biotherapeutic. The additional DCPs generated using two distinct concentrations of ADA-spiked sample sets led to a physiologically irrelevant criterion that was not necessarily representative of real-time samples. This increased the risk of false positives or negatives. In this study, well-defined bioanalytical and statistical methods were employed to validate a DCP to confirm the presence of biotherapeutic specific ADA in human serum samples. A physiologically relevant and effective strategy to confirm specificity in immune reactive samples, especially those that are close to the ACP, is proposed through this study.

Novel data analysis methods to overcome cut point challenges and enable comprehensive assessment of antidrug binding activity in confirmatory assays.

Immunogenicity assessments in response to drug treatment are commonly performed using a tiered approach strategy. All samples are initially tested in a screening assay followed by the evaluation of the screened positive samples in a confirmatory assay. Percent inhibition of signal intensity by the competing unlabeled drug in a confirmatory assay is typically used to measure the specificity of antidrug binding activity in samples, and has been successfully applied to most immunogenicity assays. However, the percent inhibition approach may not be suitable in cases where broadly distributed and high percent inhibition values are observed in drug-naïve subjects or when persistent operator-dependent differences in assay performance are encountered. Herein, we present the case studies faced with such challenges and provide appropriate solutions by introducing two novel data analysis methods: (1) Reference Delta, and (2) Reference Percent Inhibition, - in which relative-to-baseline signal inhibition is calculated for each sample. These novel methods significantly improve the confirmatory assay's ability to detect the samples positive for antidrug antibodies (ADA), especially when challenges are encountered using the traditional percent inhibition approach. Furthermore, both methods can be implemented in parallel with the percent inhibition method, enabling not only confirmation of ADA specificity, but also providing additional insights about the relevance of this antidrug binding activity to drug treatment.

Challenges of developing and validating immunogenicity assays to support comparability studies for biosimilar drug development.

Imminent patent expiry for a number of biological products currently on the market (many of which are blockbusters) has created an increasing opportunity for the development of biosimilars in the biotechnology industry. The key for successful biosimilar development is to demonstrate biosimilarity to the originator drug. In addition to demonstrating the similarity of physical and chemical properties between biosimilar and originator compounds, regulatory agencies require that immunogenicity be evaluated in comparative studies between biosimilar and originator drugs. Immunogenicity assays are generally non-quantitative (qualitative) and proving similarity/comparability based on qualitative assays can be very challenging. This review will discuss the challenges of developing and validating immunogenicity assays to support preclinical and clinical comparative studies for biosimilar drug development as well as the challenges in association with the interpretation of the data.

[Future therapeutics in celiac disease].

Celiac disease is a common autoimmune disease affecting 1% of the Western populations. It is an inappropriate immune response, in genetically susceptible patients to dietary wheat, rye, barley and oats. Treatment involves a Lifelong gluten-free diet that predisposes to low compliance due to limited variety, high cost and low palatability, imposing social pressure and affecting quality of life. The result is an urgent need for alternative therapeutic strategies. Based on the growing actual knowledge on the intestinal inflammatory cascade, mucosal immunology and genetics of celiac disease, new attractive potential therapies are emerging. Possibilities include: searching for low immunogenic wheat variants or strains pretreated with enzymes or binders for lower toxicity. Other strategies involve decreasing transepithelial uptake or dampening of the adaptive immune response by transglutaminase inhibitors or blockage of the HLA groove and immune modulation to shift the TH1 to TH2 profile. Developing biological therapy aims to decrease intestinal homing, adhesion and activity of inflammatory cells, counteract the pro-inflammatory cytokines, clonal intestinal T cells or mesenchymal stem cell replacement or mitogenic intestinal repair safety, cost, affordability and clinical effectiveness are of prime concern. Most of the above strategies showed promising results ex-vivo. The future will highlight the in-vivo winner.

Temporal graphical models for cross-species gene regulatory network discovery.

Many genes and biological processes function in similar ways across different species. Cross-species gene expression analysis, as a powerful tool to characterize the dynamical properties of the cell, has found a number of applications, such as identifying a conserved core set of cell cycle genes. However, to the best of our knowledge, there is limited effort on developing appropriate techniques to capture the causality relations between genes from time-series microarray data across species. In this paper, we present hidden Markov random field regression with L(1) penalty to uncover the regulatory network structure for different species. The algorithm provides a framework for sharing information across species via hidden component graphs and is able to incorporate domain knowledge across species easily. We demonstrate our method on two synthetic datasets and apply it to discover causal graphs from innate immune response data.

Immunogenic males: a genome-wide analysis of reproduction and the cost of mating in Drosophila melanogaster females.

In Drosophila melanogaster, mating radically transforms female physiology and behaviour. Post-mating responses include an increase in the oviposition rate, a reduction in female receptivity and an activation of the immune system. The fitness consequences of mating are similarly dramatic--females must mate once in order to produce fertile eggs, but additional matings have a clear negative effect. Previously, microarrays have been used to examine gene expression of females differing in their reproductive status with the aim of identifying genes influenced by mating. However, as only virgin and single mated females were compared, transcriptional changes associated with reproduction (under natural selection) and male-induced effects (possibly under sexually antagonistic selection) cannot be disentangled. We partitioned these fundamentally different effects by instead examining the expression profiles of virgin, single mated and double mated females. We found substantial effects relating to reproduction and further effects that are only attributable to mating itself. Immune response genes dominate this male-induced effect indicating that the cost of mating may be due partly to this system's activation. We propose that both sexually antagonistic and natural selection have been important in the evolution of the innate immunity genes, thereby contributing to the sexual dimorphism and rapid evolution at these loci.