The MHC Associated Peptide Proteomics assay is a useful tool for the non-clinical assessment of immunogenicity.

The propensity of therapeutic proteins to elicit an immune response, poses a significant challenge in clinical development and safety of the patients. Assessment of immunogenicity is crucial to predict potential adverse events and design safer biologics. In this study, we employed MHC Associated Peptide Proteomics (MAPPS) to comprehensively evaluate the immunogenic potential of re-engineered variants of immunogenic FVIIa analog (Vatreptacog Alfa). Our finding revealed the correlation between the protein sequence affinity for MHCII and the number of peptides identified in a MAPPS assay and this further correlates with the reduced T-cell responses. Moreover, MAPPS enable the identification of "relevant" T cell epitopes and may contribute to the development of biologics with lower immunogenic potential.

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.

Expanding the MAPPs Assay to Accommodate MHC-II Pan Receptors for Improved Predictability of Potential T Cell Epitopes.

A critical step in the immunogenicity cascade is attributed to human leukocyte antigen (HLA) II presentation triggering T cell immune responses. The liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based major histocompatibility complex (MHC) II-associated peptide proteomics (MAPPs) assay is implemented during preclinical risk assessments to identify biotherapeutic-derived T cell epitopes. Although studies indicate that HLA-DP and HLA-DQ alleles are linked to immunogenicity, most MAPPs studies are restricted to using HLA-DR as the dominant HLA II genotype due to the lack of well-characterized immunoprecipitating antibodies. Here, we address this issue by testing various commercially available clones of MHC-II pan (CR3/43, WR18, and Tü39), HLA-DP (B7/21), and HLA-DQ (SPV-L3 and 1a3) antibodies in the MAPPs assay, and characterizing identified peptides according to binding specificity. Our results reveal that HLA II receptor-precipitating reagents with similar reported specificities differ based on clonality and that MHC-II pan antibodies do not entirely exhibit pan-specific tendencies. Since no individual antibody clone is able to recover the complete HLA II peptide repertoire, we recommend a mixed strategy of clones L243, WR18, and SPV-L3 in a single immunoprecipitation step for more robust compound-specific peptide detection. Ultimately, our optimized MAPPs strategy improves the predictability and additional identification of T cell epitopes in immunogenicity risk assessments.

Therapeutic Potential of d-MAPPS™ for Ocular Inflammatory Diseases and Regeneration of Injured Corneal and Retinal Tissue.

The invasion of microbial pathogens and/or sterile inflammation caused by physical/chemical injuries, increased ocular pressure, oxidative stress, and ischemia could lead to the generation of detrimental immune responses in the eyes, which result in excessive tissue injury and vision loss. The bioavailability of eye drops that are enriched with immunoregulatory and trophic factors which may concurrently suppress intraocular inflammation and promote tissue repair and regeneration is generally low. We recently developed "derived- Multiple Allogeneic Proteins Paracrine Signaling regenerative biologics platform technology d-MAPPS™", a bioengineered biological product which is enriched with immunomodulatory and trophic factors that can efficiently suppress detrimental immune responses in the eye and promote the repair and regeneration of injured corneal and retinal tissues. The results obtained in preclinical and clinical studies showed that d-MAPPS™ increased the viability of injured corneal cells, inhibited the production of inflammatory cytokines in immune cells, alleviated inflammation, and restored vision loss in patients suffering from meibomian gland dysfunction and dry eye disease. Herewith, we emphasized molecular mechanisms responsible for the therapeutic efficacy of d-MAPPS™ and we presented the main beneficial effects of d-MAPPS™ in clinical settings, indicating that the topical administration of d-MAPPS™ could be considered a new therapeutic approach for the treatment of ocular inflammatory diseases and for the repair and regeneration of injured corneal and retinal tissues.

Assay format diversity in pre-clinical immunogenicity risk assessment: Toward a possible harmonization of antigenicity assays.

A major impediment to successful use of therapeutic protein drugs is their ability to induce anti-drug antibodies (ADA) that can alter treatment efficacy and safety in a significant number of patients. To this aim, in silico, in vitro, and in vivo tools have been developed to assess sequence and other liabilities contributing to ADA development at different stages of the immune response. However, variability exists between similar assays developed by different investigators due to the complexity of assays, a degree of uncertainty about the underlying science, and their intended use. The impact of protocol variations on the outcome of the assays, i.e., on the immunogenicity risk assigned to a given drug candidate, cannot always be precisely assessed. Here, the Non-Clinical Immunogenicity Risk Assessment working group of the European Immunogenicity Platform (EIP) reviews currently used assays and protocols and discusses feasibility and next steps toward harmonization and standardization.

In vitro immunogenicity prediction: bridging between innate and adaptive immunity.

Development of antidrug antibodies (ADAs) is an undesirable potential outcome of administration of biotherapeutics and involves the innate and adaptive immune systems. ADAs can have detrimental clinical consequences: they can reduce biotherapeutic efficacy or produce adverse events. Because animal models are considered poor predictors of immunogenicity in humans, in vitro assays with human innate and adaptive immune cells are commonly used alternatives that can reveal cell-mediated unwanted immune responses. Multiple methods have been developed to assess the immune cell response following exposure to biotherapeutics and estimate the potential immunogenicity of biotherapeutics. This review highlights the role of innate and adaptive immune cells as the drivers of immunogenicity and summarizes the use of these cells in assays to predict clinical ADA.

Using Imaging Mass Cytometry to Define Cell Identities and Interactions in Human Tissues.

In the evolving landscape of highly multiplexed imaging techniques that can be applied to study complex cellular microenvironments, this review characterizes the use of imaging mass cytometry (IMC) to study the human kidney. We provide technical details for antibody validation, cell segmentation, and data analysis specifically tailored to human kidney samples, and elaborate on phenotyping of kidney cell types and novel insights that IMC can provide regarding pathophysiological processes in the injured or diseased kidney. This review will provide the reader with the necessary background to understand both the power and the limitations of IMC and thus support better perception of how IMC analysis can improve our understanding of human disease pathogenesis and can be integrated with other technologies such as single cell sequencing and proteomics to provide spatial context to cellular data.

The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein.

Precise elucidation of the antigen sequences for T cell immunosurveillance greatly enhances our ability to understand and modulate humoral responses to viral infection or active immunization. Mass spectrometry is used to identify 526 unique sequences from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein extracellular domain in a complex with human leukocyte antigen class II molecules on antigen-presenting cells from a panel of healthy donors selected to represent a majority of allele usage from this highly polymorphic molecule. The identified sequences span the entire spike protein, and several sequences are isolated from a majority of the sampled donors, indicating promiscuous binding. Importantly, many peptides derived from the receptor binding domain used for cell entry are identified. This work represents a precise and comprehensive immunopeptidomic investigation with the SARS-CoV-2 spike glycoprotein and allows detailed analysis of features that may aid vaccine development to end the current coronavirus disease 2019 (COVID-19) pandemic.

Enabling Routine MHC-II-Associated Peptide Proteomics for Risk Assessment of Drug-Induced Immunogenicity.

Major histocompatibility complex-II (MHC-II)-Associated Peptide Proteomics (MAPPs) is a mass spectrometry-based approach to identify and relatively quantitate naturally processed and presented MHC-II-associated peptides that can potentially activate T cells and contribute to the immunogenicity of a drug. Acceptance of the MAPPs technology as an appropriate preclinical (and potentially clinical) immunogenicity risk assessment tool depends not only on its technical stability and robustness but also on the ability to compare results across experiments and donors. To this end, we developed a specialized MAPPs data processing pipeline, dataMAPPs, which presents complex mass spectrometric data sets in the form of heat maps (heatMAPPs), enabling rapid and convenient comparison between conditions and donors. A customized normalization procedure based on identified endogenous peptides standardizes signal intensities within and between donors and enables cross-experimental comparison. We evaluated the technical reproducibility of the MAPPs platform using tool compounds with respect to the most prominent experimental factors and found that the systematic biological differences across donors by far outweighed any technical source of variation. We illustrate the capability of the MAPPs platform to generate data that may be used for preclinical risk assessment of drug-induced immunogenicity and discuss its applicability in the clinics.

Applying MAPPs Assays to Assess Drug Immunogenicity.

Immunogenicity against biotherapeutic proteins (BPs) and the potential outcome for the patient are difficult to predict. In vitro assays that can help to assess the immunogenic potential of BPs are not yet used routinely during drug development. MAPPs (MHC-associated peptide proteomics) is one of the assays best characterized regarding its value for immunogenicity potential assessment. This review is focusing on recent studies that have employed human HLA class II-MAPPs assays to rank biotherapeutic candidates, investigate clinical immunogenicity, and understand mechanistic root causes of immunogenicity. Advantages and challenges of the technology are discussed as well as the different areas of application.

T cell epitope mapping of secukinumab and ixekizumab in healthy donors.

Secukinumab, a human monoclonal antibody that selectively neutralizes IL-17A, has consistently shown low anti-drug antibody responses in patients with psoriasis, psoriatic arthritis, and ankylosing spondylitis. Secukinumab has also shown lower in vitro immunogenicity potential compared with other monoclonal antibodies used to treat psoriasis and psoriatic arthritis, and a significantly lower in vitro T cell precursor frequency compared with ixekizumab, which targets the same antigen. Here, secukinumab and ixekizumab were further examined regarding their specific T cell epitopes. Secukinumab- or ixekizumab-specific CD4 T cell lines were generated from 31 healthy, treatment-naïve donors via 28-day co-culture with mature monocyte-derived dendritic cells exposed to either antibody. Consistent with previous data, the frequency of preexisting T cells to secukinumab was significantly lower as compared with ixekizumab. Only two T cell lines from two different donors could be derived for secukinumab, but no specific T cell epitope was identified. In contrast, 32 T cell lines from eight donors were obtained for ixekizumab. For 11 of these T cell lines, the specific T cell epitopes could be identified and confirmed by major histocompatibility complex-associated peptide proteomics as being naturally presented peptides. All identified T cell epitopes cluster in four main regions that are overlapping with the complementarity-determining regions HCDR3, LCDR1, LCDR2 and LCDR3. Interestingly, ixekizumab CDRs contain amino acids that are not found in any of the germline family members. These amino acids may be associated with the higher number of T cell epitopes identified for ixekizumab light chain and may contribute to the increased in vitro immunogenicity potential observed for ixekizumab vs. secukinumab.

Reconstructing secondary test database from PHM08 challenge data set.

In this data article, a reconstructed database, which provides information from PHM08 challenge data set, is presented. The original turbofan engine data were from the Prognostic Center of Excellence (PCoE) of NASA Ames Research Center (Saxena and Goebel, 2008), and were simulated by the Commercial Modular Aero-Propulsion System Simulation (C-MAPSS) (Saxena et al., 2008). The data set is further divided into "training", "test" and "final test" subsets. It is expected from collaborators to train their models using "training" data subset, evaluate the Remaining Useful Life (RUL) prediction performance on "test" subset and finally, apply the models to the "final test" subset for competition. However, the "final test" results can only be submitted once by email to PCoE. Before the results are sent for performance evaluation, in order to pre-validate the dataset with true RUL values, this data article introduces reconstructed secondary datasets derived from the noisy degradation patterns of original trajectories. Reconstructed database refers to data that were collected from the training trajectories. Fundamentally, it is formed of individual partial trajectories in which the RUL is known as a ground truth. Its use provides a robust validation of the model developed for the PHM08 data challenge that would otherwise be ambiguous due to the high-risk of one-time submission. These data and analyses support the research data article "A Neural Network Filtering Approach for Similarity-Based Remaining Useful Life Estimations" (Bektas et al., 2018).

MAPPs for the identification of immunogenic hotspots of biotherapeutics; an overview of the technology and its application to the biopharmaceutical arena.

INTRODUCTION: Anti-drug antibody (ADA) responses are becoming an increasing concern as more highly engineered and sophisticated biotherapeutics enter the clinic. An arsenal of tools has been developed to identify potential T cell epitopes that may drive unwanted immunological responses to protein therapeutics; one such tool is termed 'Major Histocompatibility Complex-Associated Peptide Proteomics' (MAPPs). This review highlights the evolution of this MHC II profiling technology, its technological advantages and limitations, and its utility in helping to de-risk the immunogenicity of biotherapeutics. Areas covered: A comprehensive literature review was performed along with discussions with key leaders in the field of MAPPs to summarize the importance of monitoring potential immunogenicity of clinical molecules. Herein we also describe how MAPPs has been applied specifically for monitoring MHC II peptides derived from biotherapeutics. Expert commentary: Given the importance of this growing field we discuss the complementary tools used in conjunction with MAPPs and review case studies where this approach has informed clinical studies and in some cases allowed re-engineering of the biotherapeutic moiety to a less immunogenic format.

MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity.

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method's high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.

How Close Are We to Profiling Immunogenicity Risk Using In Silico Algorithms and In Vitro Methods?: an Industry Perspective.

In silico HLA-binding algorithms and in vitro T cell-based assays as predictive tools for human immunogenicity risk have made inroads in the biotherapeutic drug discovery and development process. Currently, these tools are being used only for candidate selection or characterization and not for making a go/no-go decision for further development. A clear limitation for a broader implementation is the lack of correlation between the predicted T cell epitope content/immune reactivity potential of a biotherapeutic and the subsequent ADA-related clinical immunogenicity outcome. The current state of technologies and their pros and cons were discussed as a part of the 2016 AAPS National Biotechnology Conference in a themed session. A review of the advances in the area and the session talks along with the ensuing discussions are summarized in this commentary.

A comparison of the ability of the human IgG1 allotypes G1m3 and G1m1,17 to stimulate T-cell responses from allotype matched and mismatched donors.

The immunogenicity of clinically administered antibodies has clinical implications for the patients receiving them, ranging from mild consequences, such as increased clearance of the drug from the circulation, to life-threatening effects. The emergence of methods to engineer variable regions resulting in the generation of humanised and fully human antibodies as therapeutics has reduced the potential for adverse immunogenicity. However, due to differences in sequence referred to as allotypic variation, antibody constant regions are not homogeneous within the human population, even within sub-classes of the same immunoglobulin isotype. For therapeutically administered antibodies, the potential exists for an immune response from the patient to the antibody if the allotype of patient and antibody do not match. Allotypic distribution in the human population varies within and across ethnic groups making the choice of allotype for a therapeutic antibody difficult. This study investigated the potential of human IgG1 allotypes to stimulate responses in human CD4(+) T cells from donors matched for homologous and heterologous IgG1 allotypes. Allotypic variants of the therapeutic monoclonal antibody trastuzumab were administered to genetically defined allotypic matched and mismatched donor T cells. No significant responses were observed in the mismatched T cells. To investigate the lack of T-cell responses in relation to mismatched allotypes, HLA-DR agretopes were identified via MHC associated peptide proteomics (MAPPs). As expected, many HLA-DR restricted peptides were presented. However, there were no peptides presented from the sequence regions containing the allotypic variations. Taken together, the results from the T-cell assay and MAPPs assay indicate that the allotypic differences in human IgG1 do not represent a significant risk for induction of immunogenicity.

Medicines Adaptive Pathways to Patients (MAPPs): A Story of International Collaboration Leading to Implementation.

After nearly a decade of discussion, analysis, and development, the Medicines Adaptive Pathways to Patients (MAPPs) initiative is beginning to see acceptance from regulators, industry, patients, and payers, with the first live pilot project initiated under the guidance of the European Medicines Agency in 2014. Although it is a significant achievement to see the first asset being placed into human trials under an adaptive pathway, there is much to be learned regarding the multinational and multi-stakeholder effort that has driven the growing acceptance of MAPPs as a methodology and concept, as well as the need for continued and increasing international collaboration to foster the wider adoption of MAPPs. Changes in available science and technology, as well as a number of challenges in the current system, outlined in this paper, are transforming approaches to medicines development and approval. It is these challenges that have led directly to the groundbreaking MAPPs collaboration between the Massachusetts Institute of Technology Center for Biomedical Innovation's New Drug Development Paradigms Initiative, the EMA, patient, payer and health technology assessment groups, the European Federation of Pharmaceutical Industries and Associations, and the Innovative Medicines Initiative-a European public-private partnership. This article examines the development of MAPPs, from inception of the concept, to the establishment of this trans-Atlantic initiative, and examines challenges for the future.

Promyelocytic leukemia bodies tether to early endosomes during mitosis.

During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

Medicine adaptive pathways to patients (MAPPs): using regulatory innovation to defeat Eroom's law.

Eroom's Law is, literally, Moore's law in reverse. The pharmaceutical sector invests $50 billion annually in research for new medicines but, "the number of new drugs approved per billion US dollars spent has halved roughly every 9 years since 1950, falling around 80-fold in inflation-adjusted terms". Pharmaceutical companies have invested enormous sums in new molecular entities (NME) in the areas of unmet medical need identified by the World Health Organization (WHO), but the approval rates from phase I are only 7% for cardiovascular disease, dropping to 4% for Alzheimer's disease. The increasing cost of research & development (R&D) is not only a factor of research management quality, but also indicative of an industry trying to address therapeutic areas that have incredibly complex biological mechanisms with budget-crushing failure rates. Medicine adaptive pathways to patients (MAPPs) build on the stratification breakthroughs of personalized medicine to facilitate new types of clinical trials that adapt to a given patient's response. At their core, MAPPs will have a limited commercial marketing authorization for a patient group who has access to new therapeutic agents while validating additional clinical endpoints at the same time. This gives MAPPs a theoretical ability to run trials that fulfil both the efficacy requirements for authorization and the effectiveness needs of national health technology assessments (HTA) simultaneously, providing patients with needed therapies in the most efficient timescale and trial size possible. In order to move science forward and meet these daunting medical challenges for patients, new collaborative approaches to testing the efficacy and effectiveness of new improved medicines such as MAPPs should be embraced by regulators in close partnership with patients, payers, and practitioners. To not do so puts the entire healthcare value chain, and the future health of patients, at risk.