Development of a semi-automated MHC-associated peptide proteomics (MAPPs) method using streptavidin bead-based immunoaffinity capture and nano LC-MS/MS to support immunogenicity risk assessment in drug development.

Major histocompatibility complex (MHC)-Associated Peptide Proteomics (MAPPs) is an ex vivo method used to assess the immunogenicity risk of biotherapeutics. MAPPs can identify potential T-cell epitopes within the biotherapeutic molecule. Using adalimumab treated human monocyte derived dendritic cells (DCs) and a pan anti-HLA-DR antibody (Ab), we systematically automated and optimized biotin/streptavidin (SA)-capture antibody coupling, lysate incubation with capture antibody, as well as the washing and elution steps of a MAPPs method using functionalized magnetic beads and a KingFisher Magnetic Particle processor. Automation of these steps, combined with capturing using biotinylated-Ab/SA magnetic beads rather than covalently bound antibody, improved reproducibility as measured by minimal inter-and intra-day variability, as well as minimal analyst-to-analyst variability. The semi-automated MAPPs workflow improved sensitivity, allowing for a lower number of cells per analysis. The method was assessed using five different biotherapeutics with varying immunogenicity rates ranging from 0.1 to 48% ADA incidence in the clinic. Biotherapeutics with ≥10%immunogenicity incidence consistently presented more peptides (1.8-28 fold) and clusters (10-21 fold) compared to those with <10% immunogenicity incidence. Our semi-automated MAPPs method provided two main advantages over a manual workflow- the robustness and reproducibility affords confidence in the epitopes identified from as few as 5 to 10 donors and the method workflow can be readily adapted to incorporate different capture Abs in addition to anti-HLA-DR. The incorporation of semi-automated MAPPs with biotinylated-Ab/SA bead-based capture in immunogenicity screening strategies allows the generation of more consistent and reliable data, helping to improve immunogenicity prediction capabilities in drug development. MHC associated peptide proteomics (MAPPs), Immunogenicity risk assessment, in vitro/ex vivo, biotherapeutics, Major Histocompatibility Complex Class II (MHC II), LC-MS, Immunoaffinity Capture, streptavidin magnetic beads.

Survey Outcome on Immunogenicity Risk Assessment Tools for Biotherapeutics: an Insight into Consensus on Methods, Application, and Utility in Drug Development.

A survey conducted by the Therapeutic Product Immunogenicity (TPI) community within the American Association of Pharmaceutical Scientists (AAPS) posed questions to the participants on their immunogenicity risk assessment strategies prior to clinical development. The survey was conducted in 2 phases spanning 5 years, and queried information about in silico algorithms and in vitro assay formats for immunogenicity risk assessments and how the data were used to inform early developability effort in discovery, chemistry, manufacturing and control (CMC), and non-clinical stages of development. The key findings representing the trends from a majority of the participants included the use of high throughput in silico algorithms, human immune cell-based assays, and proteomics based outputs, as well as specialized assays when therapeutic mechanism of action could impact risk assessment. Additional insights into the CMC-related risks could also be gathered with the same tools to inform future process development and de-risk critical quality attributes with uncertain and unknown risks. The use of the outputs beyond supporting early development activities was also noted with participants utilizing the risk assessments to drive their clinical strategy and streamline bioanalysis.

Introducing dendritic cell antibody internalization as an immunogenicity risk assessment tool.

Aim: Immunogenicity risk assessment assays are powerful tools that assess the relative immunogenicity of potential biotherapeutics. We detail here the development of a novel assay that measures the degree of antibody internalization by antigen-presenting cells as a predictor of immunogenicity. Results & methodology: The assay uses the fluorescence signal from the antibody bound to the outside of the cell as well as inside the cell to determine internalization. To calculate the amount of internalized antibody, the fluorescent signal from the outside was subtracted from the fluorescent signal from the inside, which is referred to as the internalization index. Conclusion: This assay format demonstrated that antibody-based biotherapeutics with higher clinical immunogenicity internalized to a higher degree than therapeutic antibodies with lower clinical immunogenicity.

A cell-based FcRn-dependent recycling assay for predictive pharmacokinetic assessment of therapeutic antibodies.

Aim: Evaluation of suitable pharmacokinetic properties is critical for successful development of IgG-based biotherapeutics. The prolonged half-lives of IgGs depend on the intracellular trafficking function of neonatal Fc receptor, which rescues internalized IgGs from lysosomal degradation and recycles them back to circulation. Results: Here, we developed a novel cell-based assay to quantify recycling of monoclonal antibodies in a transwell culture system that uses a cell line that stably expresses human neonatal Fc receptor. We tested seven therapeutic antibodies and showed that the recycling output of the assay strongly correlated with the clearance in humans. Conclusion: This recycling assay has potential application as a pharmacokinetic prescreening tool to facilitate development and selection of IgG-based candidate therapeutic monoclonal antibodies.

Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells.

Biotherapeutics, which are biologic medications that are natural or bioengineered products of living cells, have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop antigen-specific anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Here, we describe an in vitro method for assessing the immunogenic risk of a biotherapeutic. We found a correlation between clinical immunogenicity and the frequency with which a biotherapeutic stimulated an increase in CD134, CD137, or both cell surface markers on CD4+ T cells. Using high-throughput flow cytometry, we examined the effects of 14 biotherapeutics with diverse rates of clinical immunogenicity on peripheral blood mononuclear cells from 120 donors with diverse human leukocyte antigen class II-encoding alleles. Biotherapeutics with high rates of ADA development in the clinic had higher proportions of CD4+ T cells positive for CD134 or CD137 than biotherapeutics with low clinical immunogenicity. This method provides a rapid and simple preclinical test of the immunogenic potential of a new candidate biotherapeutic or biosimilar. Implementation of this approach during biotherapeutic research and development enables rapid elimination of candidates that are likely to cause ADA-related adverse events and detrimental consequences.

An in vitro FcRn- dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans.

A cell-based assay employing Madin-Darby canine kidney cells stably expressing human neonatal Fc receptor (FcRn) heavy chain and ß2-microglobulin genes was developed to measure transcytosis of monoclonal antibodies (mAbs) under conditions relevant to the FcRn-mediated immunoglobulin G (IgG) salvage pathway. The FcRn-dependent transcytosis assay is modeled to reflect combined effects of nonspecific interactions between mAbs and cells, cellular uptake via pinocytosis, pH-dependent interactions with FcRn, and dynamics of intracellular trafficking and sorting mechanisms. Evaluation of 53 mAbs, including 30 marketed mAb drugs, revealed a notable correlation between the transcytosis readouts and clearance in humans. FcRn was required to promote efficient transcytosis of mAbs and contributed directly to the observed correlation. Furthermore, the transcytosis assay correctly predicted rank order of clearance of glycosylation and Fv charge variants of Fc-containing proteins. These results strongly support the utility of this assay as a cost-effective and animal-sparing screening tool for evaluation of mAb-based drug candidates during lead selection, optimization, and process development for desired pharmacokinetic properties.

Comparative Assessment of Two Robot-Assisted Therapies for the Upper Extremity in People With Chronic Stroke.

OBJECTIVE: We investigated the effects on motor and daily function of robot-assisted therapies in people with chronic stroke using the Bi-Manu-Track (BMT) and InMotion 3.0 (IMT) compared with control treatment (CT). METHOD: In this comparative efficacy trial, 30 participants were randomized to receive BMT, IMT, or CT. Outcome measures included the Fugl-Meyer Assessment (FMA), Modified Ashworth Scale (MAS), Motor Activity Log (MAL), and Medical Research Council (MRC) scale. RESULTS: The IMT group improved more in FMA and proximal MAS scores than the BMT group (both ps < .01) and the CT group (p < .01 and p = .03, respectively). The IMT and BMT groups showed clinically relevant improvements after treatment on the MRC rather than the MAL. CONCLUSION: The results indicate that the IMT might improve motor function. The IMT and BMT groups showed similar benefits for muscle power but limited improvements in self-perceived use of the affected arm.

Ecological carrying capacity assessment of diving site: A case study of Mabul Island, Malaysia.

Despite considered a non-consumptive use of the marine environment, diving-related activities can cause damages to coral reefs. It is imminent to assess the maximum numbers of divers that can be accommodated by a diving site before it is subject to irreversible deterioration. This study aimed to assess the ecological carrying capacity of a diving site in Mabul Island, Malaysia. Photo-quadrat line transect method was used in the benthic survey. The ecological carrying capacity was assessed based on the relationship between the number of divers and the proportion of diver damaged hard corals in Mabul Island. The results indicated that the proportion of diver damaged hard corals occurred exponentially with increasing use. The ecological carrying capacity of Mabul Island is 15,600-16,800 divers per diving site per year at current levels of diver education and training with a quarterly threshold of 3900-4200 per site. Our calculation shows that management intervention (e.g. limiting diving) is justified at 8-14% of hard coral damage. In addition, the use of coral reef dominated diving sites should be managed according to their sensitivity to diver damage and the depth of the reefs.

Strategies to Determine Assay Format for the Assessment of Neutralizing Antibody Responses to Biotherapeutics.

Most biotherapeutics can elicit immune responses in dosed recipients generating anti-drug antibodies (ADAs). Neutralizing antibodies (NAbs) are a subpopulation of ADAs that can potentially impact patient safety and directly mediate loss of drug efficacy by blocking the biological activity of a therapeutic product. Therefore, NAb detection is an important aspect of immunogenicity assessment, requiring sensitive and reliable methods reflective of the therapeutic mechanism of action (MoA). Both cell-based and non cell-based assays are viable options for NAb assessment. However, the scientific approach for the selection of a suitable assay format (cell-based or non cell-based) for NAb assessment is not currently well defined. In this manuscript, the authors summarize the design and utility of cell-based and non cell-based NAb assays and recommend a NAb assay format selection approach that relies on a combination of three factors. These include (i) the therapeutic MoA, (ii) the evidence of desirable assay performance characteristics, and (iii) risk of immunogenicity. The utility of correlating NAb response with pharmacodynamic data is also discussed. The aim of this paper is to provide a consistent strategy that will guide the selection of scientifically justified assay formats capable of detecting clinically relevant NAbs for biotherapeutics with varying MoAs and diverse complexity.

Human health risk assessment based on trace metals in suspended air particulates, surface dust, and floor dust from e-waste recycling workshops in Hong Kong, China.

This study investigated health risks exerted on electronic waste (e-waste) recycling workers exposed to cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), nickel (Ni), mercury (Hg), and zinc (Zn) in Hong Kong. E-waste recycling workshops were classified into eight working areas: 1 = office, 2 = repair, 3 = dismantling, 4 = storage, 5 = desoldering, 6 = loading, 7 = cable shredding, and 8 = chemical waste. The aforementioned metal concentrations were analyzed in suspended air particulates, surface dust and floor dust collected from the above study areas in five workshops. Elevated Pb levels were measured in dismantling and desoldering areas (582 and 486 µg/100 cm(2) in surface and 3,610 and 19,172 mg/kg in floor dust, respectively). Blood lead levels of 10 and 39.5 µg/dl were estimated using United States Environmental Protection Agency's Adult Lead Model as a result of exposure to the floor dust from these two areas. Human health risk assessments were conducted to evaluate cancer and noncancer risks resulting from exposure to floor dust through the combined pathways of ingestion, dermal contact, and inhalation. Findings indicated that workers may be exposed to cancer risks above the acceptable range at 147 in a million at the 95th percentile in the dismantling area. Workers should be informed of associated risks to safeguard their health.

Projection of waste quantities: the case of e-waste of the People's Republic of China.

Although waste quantification and projection are important data for waste management, the reliability of their results is difficult to verify. The present study attempted to identify the best waste quantification methods using e-waste quantification studies of mainland China as case studies. Large discrepancies in the predicted amounts of e-waste generated were found no matter whether the same or different methods of estimation are used. Moreover, even when agreements between studies were found, the agreed figures were not necessarily the correct figures. However, since without hindsight it is not possible to tell whether a projection figure is accurate, the convergence rule and a prudent approach to counting on studies conducted with meticulous scientific procedures should be adopted. Two worrying trends are noted. First, the transparency of data collection and computation methods in these studies was not high; second, irresponsible citation practices were found to have already spread to academic studies. As a result, leading organizations in the academic community should consider establishing a platform devoted to the reporting of false or dubious citations.

Health risk assessment of organochlorine pesticides with emphasis on DDTs and HCHs in abandoned agricultural soils.

The aim of this study was to evaluate the consequence of changing land use from agriculture land to other use purposes with respect to OCPs non-cancer and cancer risk on human health, based on concentrations of DDTs and HCHs in soils collected from 55 locations representing 12 different land use types. There were no non-cancer risks of DDTs and γ-HCHs on adults and children, and there were very low cancer risks of DDD, o,p'-DDE, DDT, α-HCH, ß-HCH, γ-HCH based on their total concentrations in all samples. Nonetheless, there were significant correlations of DDT to its metabolites (DDE and DDD) (r = 0.506 and r = 0.648) and DDE to DDD (r = 0.438) both at p < 0.01. OCP levels should be routinely monitored in different environmental media and food in order to verify whether there is fresh input. Their potential risks on human health should also be assessed.

Advances in the assessment and control of the effector functions of therapeutic antibodies.

The Fc (crystallizable fragment) region of therapeutic antibodies can have an important role in their safety and efficacy. Although much is known about the structure-activity relationship of antibodies and the factors that influence Fc effector functions, a process has not yet been defined to clearly delineate how Fc functionality should be assessed and controlled during antibody development and manufacturing. In this article, we summarize the current knowledge of antibody Fc functionality, provide a strategy for assessing the effector functions of different classes of therapeutic antibodies (including Fc fusion proteins) and propose a path for routine testing and controls for manufacturers of antibody products.

Health risk assessment of abandoned agricultural soils based on heavy metal contents in Hong Kong, the world's most populated city.

The objective of this study was to evaluate the consequence of changing and using agricultural soils to other purposes in Hong Kong with respect to risk to human health. This study established concentrations of the following priority elements: As, Cu, Cd, Cr, Pb and Zn in terms of total burden (using mixed acid microwave digestion) and with respect to metal bioaccessibility (using an in vitro simulated gastric solution). 55 locations were sampled representing 12 different land use types, namely, agricultural (A), abandoned agricultural (Ab), organic farm (OF), container storage (CS), construction waste (CW), e-waste storage (EW (S)), e-waste dismantling workshop (EW (DW)), e-waste open burning site (EW (OBS)), open burning site (OBS), petrol station (PS), metal recycling workshop (MRW) and car dismantling workshop (CDW). The elemental concentrations were subsequently used to establish Hazard Indices (for adults and children). 95th percentile values of total elemental concentrations were used to derive a combined (ingestion, dermal and inhalation) Hazard Index (HI) only for adults where the EW (DW) land use type indicated the potential for increased harm (HI=1.16). On the other hand, where 5th percentile values of total elemental concentrations were used to derive a combined Hazard Index (HI) for children the HI values exceeded 1 for CS, MRW, PS, EW (DW), EW (OBS) and CDW land use types (respectively, 1.21, 1.19, 1.52, 1.21, 1.81 and 2.04).