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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...
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... humanized mice have historically been tested in the context of infectious disease; in recent years, however, providers of HIS mice have focused their efforts on generating models for the burgeoning immuno-oncology field. Table 6 provides an overview of the three different HIS models and their potential for immunogenicity testing. ...Citations
... 16 A growing suite of potentially complementary in silico, in vitro and ex vivo experimental methods is being used to model and predict clinical immunogenicity risk of antibody and other protein therapeutics. [17][18][19] These risk assessment methods typically involve head-to-head comparison of therapeutic candidates with multiple benchmark proteins with known incidence of clinical ADAs. 20 Many of these methods focus on different aspects of T cell-dependent generation of ADAs, reflecting the key role of T cells in developing a mature antibody response, including variable domain somatic hypermutation and immunoglobulin classswitching. ...
... As mentioned previously, and extensively reviewed elsewhere, 17,19 each method used has inherent limitations that may lead to false-positive or negative predictions. For example, T cell epitopes detected by MAPPs include parental constant domain sequences that seem likely to be tolerized as self-peptides, 21 i.e., false positives. ...
Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.
... One of the challenges for the development and comparison of preclinical in vitro immunogenicity risk assays is the lack of availability of standard positive and negative control therapeutic proteins for use in assay qualification and as benchmarks for comparison of relative immunogenicity (2,3). The different CD4 T cell responses elicited by the two anti-IL21R ATR107 homologs used in this study are an illustration of the challenge. ...
T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.
... As some key steps of Td immune response against exogeneous can be recapitulated by cultured cells, many in vitro assay platforms were developed to monitor the T cell-mediated immunogenicity of biotherapeutics. 30,31 These methods involve the measurement of physical or functional binding of therapeutics-derived peptides with HLA II molecules as summarized in Table 1. Examples in the following paragraphs illustrate different methods for TCE characterization. ...
The surge in the clinical use of therapeutic antibodies has reshaped the landscape of pharmaceutical therapy for many diseases, including rare and challenging conditions. However, the administration of exogenous biologics could potentially trigger unwanted immune responses such as generation of anti-drug antibodies (ADAs). Real-world experiences have illuminated the clear correlation between the ADA occurrence and unsatisfactory therapeutic outcomes as well as immune-related adverse events. By retrospectively examining research involving immunogenicity analysis, we noticed the growing emphasis on elucidating the immunogenic epitope profiles of antibody-based therapeutics aiming for mechanistic understanding the immunogenicity generation and, ideally, mitigating the risks. As such, we have comprehensively summarized here the progress in both experimental and computational methodologies for the characterization of T and B cell epitopes of therapeutics. Furthermore, the successful practice of epitope-driven deimmunization of biotherapeutics is exceptionally highlighted in this article.
... The working group continues their work following the NCIRA publication on possible harmonization of antigenicity assays, in order to address regulatory requests and expectations regarding the validation of assays assessing innate and adaptive immune responses. 4 Their aim is to provide updated overviews on existing tools, outlines of potential future applications, generic protocols where possible, and recommendations for appropriate and inappropriate use, maximizing comparability between labs and increasing confidence by establishing good practices. The assays assessed include innate immune response modulating impurities (IIRMI) assays, dendritic cell maturation assays, in silico tools, MHC-associated peptide proteomics (MAPPs), T cell and B cell assays. ...
Biologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals and its one-day training course gives experts and newcomers across academia, industry, and regulatory agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we report the discussions that took place at the EIP’s 14th Symposium, held in April 2023. The topics covered included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assessment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated with new modalities, which were discussed in a dedicated session.
... Also, the moDCs used here are equivalent or similar in phenotype to those used in other immunogenicity risk assessment strategies, such as MAPPS and DC internalization assays 12,[39][40][41] . Keyhole limpet hemocyanin (KLH), a highly immunogenic protein, was used as the positive control which is common for in vitro immunogenicity risk assessment 42,43 . Twentyfour-hour exposure to increasing concentrations of KLH induced CXCR4 expression on moDC in a dose-dependent manner. ...
Background
The efficacy and safety of therapeutic proteins are undermined by immunogenicity driven by anti-drug antibodies. Immunogenicity risk assessment is critically necessary during drug development, but current methods lack predictive power and mechanistic insight into antigen uptake and processing leading to immune response. A key mechanistic step in T-cell-dependent immune responses is the migration of mature dendritic cells to T-cell areas of lymphoid compartments, and this phenomenon is most pronounced in the immune response toward subcutaneously delivered proteins.
Methods
The migratory potential of monocyte-derived dendritic cells is proposed to be a mechanistic marker for immunogenicity screening. Following exposure to therapeutic protein in vitro, dendritic cells are analyzed for changes in activation markers (CD40 and IL-12) in combination with levels of the chemokine receptor CXCR4 to represent migratory potential. Then a transwell assay captures the intensity of dendritic cell migration in the presence of a gradient of therapeutic protein and chemokine ligands.
Results
Here, we show that an increased ability of the therapeutic protein to induce dendritic cell migration along a gradient of chemokine CCL21 and CXCL12 predicts higher immunogenic potential. Expression of the chemokine receptor CXCR4 on human monocyte-derived dendritic cells, in combination with activation markers CD40 and IL-12, strongly correlates with clinical anti-drug antibody incidence.
Conclusions
Mechanistic understanding of processes driving immunogenicity led to the development of a predictive tool for immunogenicity risk assessment of therapeutic proteins. These predictive markers could be adapted for immunogenicity screening of other biological modalities.
... MAPPs assay enables the identification of peptides presented by MHC-II molecules on moDCs, thus validating the sequences identified by algorithms. DC maturation assays are rather used to evaluate the impact of product co-impurities on the development of immune responses [4,137]. Since a naive T-cell repertoire is mandatory for the induction of an antigen-specific T-cell response, the characterization of drug-responsive naive T cells is important for measuring the potential of immunogenicity [138][139][140]. ...
Internalization and processing by antigen‐presenting cells such as dendritic cells (DCs) are critical steps for initiating a T‐cell response to therapeutic antibodies. Consequences are the production of neutralizing anti‐drug antibodies altering the clinical response, the presence of immune complexes, and, in some rare cases, hypersensitivity reactions. In recent years, significant progress has been made in the knowledge of cellular uptake mechanisms of antibodies in DCs. Uptake of antibodies could be directly related to their immunogenicity by regulating the quantity of materials entering the DCs in relation to antibody structure. Here, we summarize the latest insights into cellular uptake mechanisms and pathways in DCs. We highlight the approaches to study endocytosis, the impact of endocytosis routes on T cell response, and discuss the link between how DCs internalize therapeutic antibodies and the potential mechanisms that could give rise to immunogenicity. Understanding these processes could help developing assays to evaluate the immunogenicity potential of biotherapeutics.
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... While MAPPS helps to vastly narrow down possibilities of culprit epitopes, this assay does not verify that these putative sequences are immunogenic (32). Therefore, each putative sequence region identified by MAPPS needed to be confirmed for their ability to stimulate a specific T cell response. ...
... To determine which of these suspect epitopes could confer T cell reactivity, a restimulated T cell line assay using healthy donor PBMCs was developed in-house, with modifications to what has been described previously (16,17,33). The restimulated T cell line assay is in essence, an extension of the traditional DC:T assay that evaluates sequence-based immunogenicity risk (32). A key differentiating factor is that the restimulated T cell line assay utilizes multiple rounds of stimulation instead of one. ...
... Peptide #4 and #5, despite being presented by 7 and 8 out of 20 donors respectively in the MAPPS assay ( Figure 5B), failed to confer T cell reactivity in the restimulated T cell line assay. MAPPS can be under-predictive if the appropriate sensitivity is not applied (12,32). However, this was not the case in our MAPPS assays, as we had sufficient consistency and sensitivity across both rounds of MAPPS assays (see Methods).The results from the restimulated T cell line assay align with our expectations that only a subset of sequences identified from the MAPPS assay, translate into T cell reactivity. ...
Introduction
In oncology, anti-drug antibody (ADA) development that significantly curtails response durability has not historically risen to a level of concern. The relevance and attention ascribed to ADAs in oncology clinical studies have therefore been limited, and the extant literature on this subject scarce. In recent years, T cell engagers have gained preeminence within the prolific field of cancer immunotherapy. These drugs whose mode of action is expected to potently stimulate anti-tumor immunity, may potentially induce ADAs as an unintended corollary due to an overall augmentation of the immune response. ADA formation is therefore emerging as an important determinant in the successful clinical development of such biologics.
Methods
Here we describe the immunogenicity and its impact observed to pasotuxizumab (AMG 212), a prostate-specific membrane antigen (PSMA)-targeting bispecific T cell engager (BiTE®) molecule in NCT01723475, a first-in-human (FIH), multicenter, dose-escalation study in patients with metastatic castration-resistant prostate cancer (mCRPC). To explain the disparity in ADA incidence observed between the SC and CIV arms of the study, we interrogated other patient and product-specific factors that may have explained the difference beyond the route of administration.
Results
Treatment-emergent ADAs (TE-ADA) developed in all subjects treated with at least 1 cycle of AMG 212 in the subcutaneous (SC) arm. These ADAs were neutralizing and resulted in profound exposure loss that was associated with contemporaneous reversal of initial Prostate Surface Antigen (PSA) responses, curtailing durability of PSA response in patients. Pivoting from SC to a continuous intravenous (CIV) administration route remarkably yielded no subjects developing ADA to AMG 212. Through a series of stepwise functional assays, our investigation revealed that alongside a more historically immunogenic route of administration, non-tolerant T cell epitopes within the AMG 212 amino acid sequence were likely driving the high-titer, sustained ADA response observed in the SC arm.
Discussion
These mechanistic insights into the AMG 212 ADA response underscore the importance of performing preclinical immunogenicity risk evaluation as well as advocate for continuous iteration to better our biologics.
... This work demonstrated the attractive predictability power of such an assay. In the last years, the Non-Clinical Immunogenicity Risk Assessment working group of the European Immunogenicity Platform (EIP) (Ducret et al, 2022) and ABIRISK (Rup et al, 2015) consortium have sought to harmonize currently accepted in vitro assays by giving standard accepted criteria systematically applied by industry or research groups. ...
... Therefore, balancing the number of preclinical candidates and the associated workload is crucial. The primary strategy to assess immunogenicity using MAPPs assay should involve a first step where the candidate molecules are screened on a set of HLA-genotyped PBMCs representing a good approximation of the general population (Ducret et al, 2022). ...
MHC-II-associated peptide proteomics (MAPPs) is a mass spectrometry-based (MS) method to identify naturally presented MHC-II-associated peptides that could elicit CD4+T cell activation. MAPPs assay is considered one of the assays that better characterize the safety of biotherapeutics by driving the selection of the best candidates concerning their immunogenicity risk. However, there is little knowledge about the impact of bead material on the recovery of MHC-II MS-eluted ligands in MAPPs assays. Here, we firstly describe a robust MAPPs protocol by implementing streptavidin magnetic beads for the isolation of these peptides instead of commonly used NHS-activated beads. Moreover, we assessed the impact of the cell medium used for cell cultures on the morphology and recovery of the in vitro-generated APCs, and its potential implications in the amount of MHC-II isolated peptides. We also described an example of a MAPPs assay application to investigate drug-induced immunogenicity of two bispecific antibodies and compared them with monospecific trastuzumab IgG1 control. This work highlighted the importance of MAPPs in the preclinical in vitro strategy to mitigate the immunogenicity risk of biotherapeutics.
... Studies have shown a difference in cell surface expression of the three main HLA II receptor families, in which there is a significantly higher (~5-10-fold) HLA-DR expression level in comparison to HLA-DP and HLA-DQ [15]. This correlates with the frequency distribution for HLA class II antigen-specific responses and number of epitopes in which 80%,~20%, and 5-10% of MHC-II-identified peptides are HLA-DR-, HLA-DP-, and HLA-DQ-specific, respectively [21,22]. Due to this, HLA-DR has been, and is still, the most intensively studied MHC-II isotype [19]. ...
... For example, the presence of the HLA-DQA1*05 allele, which is carried by approximately 40% of Europeans, has been shown to significantly increase the rate of immunogenicity in patients treated with adalimumab [23]. Overall, this indicates that an accurate preclinical drug immunogenicity assessment must integrate comprehensive in silicoand in vitro-based tools taking into account the HLA diversity encountered in the general population [21]. ...
... After maturation and challenge with the compound of interest, MHC-II peptide-receptor complexes are first immuneprecipitated using a suitable reagent, typically an antibody specific for HLA class II receptors. Bound MHC-II peptides are then acid-eluted from the receptor complexes and characterized by LC-MS/MS [21]. ...
Simple Summary
The major histocompatibility complex class II-associated peptide proteomics assay is widely used during preclinical immunogenicity risk assessments to identify biotherapeutic-derived peptides. These potential T cell epitopes are presented by dendritic cells and may trigger CD4⁺ T helper cell activation, which could lead to downstream anti-drug antibody secretion by plasma cells. Currently, the utility of this immunopeptidomics assay has often been restricted to studying human leukocyte antigen-DR receptors due to the lack of well-characterized human leukocyte antigen-DP, -DQ, and pan antibodies available. Here, we seek to accommodate major histocompatibility complex class II pan receptors by testing commonly commercially available antibody clones, and characterizing their specificity via the epitope prediction algorithm NetMHCIIpan. Although the application of these antibodies in the assay increased the identified compound-specific cluster profile, no individual antibody clone was able to recover the complete human leukocyte antigen II peptide repertoire. Our findings reveal that a mixed immunoprecipitation strategy utilizing a minimum of three antibody clones with differing specificities (human leukocyte antigen-DR-specific clone L243, pan-specific clone WR18, and -DQ-specific clone SPV-L3) leads to more robust compound-specific peptide detection in one single analysis. Ultimately, expanding the assay to leverage human leukocyte antigen pan receptors improves the predictability of additional potential T cell epitopes.
Abstract
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.
... There are many manuscripts for flow cytometry for other applications and suggestions for assay validation similar to those used in the presented case study above. Similar to clinical immunogenicity methods, the absence of assay harmonization and standardization is a challenge for in vitro flow cytometry assays used to predict immunogenicity [84]. Therefore, the recommendation was to build more experience, publications, working group and case studies are necessary to expand knowledge/ data on this application and Regulatory Agencies would be willing to discuss this together. ...
The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.
