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.

Validation of a Dendritic Cell and CD4+ T Cell Restimulation Assay Contributing to the Immunogenicity Risk Evaluation of Biotherapeutics.

Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.

HLAII peptide presentation of infliximab increases when complexed with TNF.

CD4+ T-cell activation through recognition of Human Leukocyte Antigen II (HLAII)-presented peptides is a key step in the development of unwanted immune response against biotherapeutics, such as the generation of anti-drug antibodies (ADA). Therefore, the identification of HLAII-presented peptides derived from biotherapeutics is a crucial part of immunogenicity risk assessment and mitigation strategies during drug development. To date, numerous CD4+ T-cell epitopes have been identified by HLAII immunopeptidomics in antibody-based biotherapeutics using either their native or aggregated form. Antibody-target immune complexes have been detected in patients with ADA and are thought to play a role in ADA development by enhancing the presentation of CD4+ T-cell epitopes at the surface of antigen presenting cells (APCs). The aim of this study was to investigate the effect of biotherapeutic antibody-target immune complexes on the HLAII peptide presentation of biotherapeutics in human primary monocyte-derived dendritic cells (DCs). The trimeric tumor necrosis factor (TNF) and its biotherapeutic antagonists infliximab (INFL), adalimumab (ADAL), and a single armed Fab' were used as a model system. The HLAII immunopeptidome of DCs loaded with antagonists or their immune complexes with TNF was analyzed by trapped ion mobility time-of-flight mass spectrometry (timsTOF MS) leading to the identification of ~ 12,000 unique HLAII-associated peptides per preparation. Anti-TNF sequences were detected at a median of 0.3% of the total immunopeptidome, against a majority background of peptides from endogenous and media-derived proteins. TNF antagonist presentation spanned the variable and constant regions in a widespread manner in both light and heavy chains, consistent with previously discovered HLAII peptides. This investigation extends the collection of observed HLAII peptides from anti-TNF biotherapeutics to include sequences that at least partially span the complementary determining regions (CDRs), such as the LCDR1 for both INFL and ADAL. Although antagonist presentation varied significantly across donors, peptides from both bivalent antagonists INFL and ADAL were more highly presented relative to the Fab'. While TNF immune complexes did not alter overall HLAII presentation, a moderate increase in presentation of a subset of peptide clusters was observed in the case of INFL-TNF, which included HCDR2, HCDR3 and LCDR2 sequences.

Crystal structure of ultra-humanized anti-pTau Fab reveals how germline substitutions humanize CDRs without loss of binding'.

Administration of therapeutic antibodies can elicit adverse immune responses in patients through the generation of anti-drug antibodies that, in turn, reduce the efficacy of the therapeutic. Removal of foreign amino acid content by humanization can lower the immunogenic risk of the therapeutic mAb. We previously developed the ultra-humanization technology "Augmented Binary Substitution" (ABS) which enables single-step CDR germlining of antibodies. The application of ABS to a chicken anti-pTau antibody generated an ultra-humanized variant, anti-pTau C21-ABS, with increased human amino acid content in the CDRs and reduced in-silico predicted immunogenicity risk. Here, we report the high-resolution crystal structure of anti-pTau C21-ABS Fab in complex with the pTau peptide (7KQK). This study examines how ultra-humanization, via CDR germlining, is facilitated while maintaining near-identical antigen affinity (within 1.6-fold). The co-complex structure reveals that the ABS molecule targets the same antigenic epitope, accommodated by structurally-similar changes in the paratope. These findings confirm that ABS enables the germlining of amino acids within CDRs by exploiting CDR plasticity, to reduce non-human amino acid CDR content, with few alterations to the overall mechanism of binding.

Genome-wide pharmacogenetics of anti-drug antibody response to bococizumab highlights key residues in HLA DRB1 and DQB1.

In this largest to-date genetic analysis of anti-drug antibody (ADA) response to a therapeutic monoclonal antibody (MAb), genome-wide association was performed for five measures of ADA status among 8844 individuals randomized to bococizumab, which targets PCSK9 for LDL-C lowering and cardiovascular protection. Index associations prioritized specific amino acid substitutions at the DRB1 and DQB1 MHC class II genes rather than canonical haplotypes. Two clusters of missense variants at DRB1 were associated with general ADA measures (residues 9, 11, 13; and 96, 112, 120, 180) and a third cluster of missense variants in DQB1 was associated with ADA measures including neutralizing antibody (NAb) titers (residues 66, 67, 71, 74, 75). The structural disposition of the missense substitutions implicates peptide antigen binding and CD4 effector function, mechanisms that are potentially generalizable to other therapeutic mAbs.Clinicaltrials.gov: NCT01968954, NCT01968967, NCT01968980, NCT01975376, NCT01975389, NCT02100514.

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.

Correction to: Immunogenicity of Biosimilars for Rheumatic Diseases, Plaque Psoriasis, and Inflammatory Bowel Disease: A Review from Clinical Trials and Regulatory Documents.

The article Immunogenicity of Biosimilars for Rheumatic Diseases, Plaque Psoriasis, and Inflammatory Bowel Disease: A Review from Clinical Trials and Regulatory Documents, written by Vibeke Strand, Joao Gonçalves, Timothy P. Hickling, Heather E. Jones, Lisa Marshall and John D. Isaacs, was originally published Online First without Open Access.

Immunogenicity of Biosimilars for Rheumatic Diseases, Plaque Psoriasis, and Inflammatory Bowel Disease: A Review from Clinical Trials and Regulatory Documents.

The goal of this narrative review was to summarize immunogenicity data of biosimilars or biosimilar candidates for rheumatic diseases, plaque psoriasis, or inflammatory bowel disease (IBD), available in peer-reviewed publications or regulatory documents. PubMed records and regulatory documents were searched for immunogenicity data of TNFα or CD20 inhibitor biosimilars or biosimilar candidates. Data collected included the proportion of patients positive for anti-drug antibodies (ADAbs), proportion with neutralizing antibodies (nAbs) among ADAb-positive patients, ADAb/nAb assay characteristics, cross-reactivity, and the effects of ADAbs on pharmacokinetics, pharmacodynamics, efficacy, and safety. We identified eight biosimilars or biosimilar candidates for adalimumab (BI 695501, SB5, ABP 501, GP2017, PF-06410293, MSB-11022, FKB-327, ZRC-3197) four for etanercept (SB4, GP2015, CHS-0214, LBEC0101), and three each for infliximab (SB2, CT-P13, GP1111) and rituximab (CT-P10, GP2013, PF-05280586) with immunogenicity data. Randomized, head-to-head trials with reference products varied in design and methodology of ADAb/nAb detection. The lowest proportions of ADAb-positive (0-13%) and nAb-positive patients (0-3%) were observed in the trials of etanercept and its biosimilars, and the highest with adalimumab, infliximab, and their biosimilars (ADAbs: ≤ 64%; nAbs: ≤ 100%). The most common method of ADAb detection was electrochemiluminescence, and ADAb positivity was associated with nominally inferior efficacy and safety. Overall, there were no significant immunogenicity differences between biosimilars and reference products. However, there are many discrepancies in assessing and reporting clinical immunogenicity. In conclusion, immunogenicity data of biosimilars or biosimilar candidates for TNFα or CD20 inhibitors were collected in trials that varied in design and procedures for ADAb/nAb detection. In general, immunogenicity parameters of biosimilars are similar to those of their reference products.

Nonclinical immunogenicity risk assessment of therapeutic proteins.

Therapeutic protein drugs have significantly improved the management of many severe and chronic diseases. However, their development and optimal clinical application are complicated by the induction of unwanted immune responses. Therapeutic protein-induced antidrug antibodies can alter drug pharmacokinetics and pharmacodynamics leading to impaired efficacy and occasionally serious safety issues. There has been a growing interest over the past decade in developing methods to assess the risk of unwanted immunogenicity during preclinical drug development, with the aim to mitigate the risk during the molecular design phase, clinical development and when products reach the market. Here, we discuss approaches to therapeutic protein immunogenicity risk assessment, with attention to assays and in vivo models used to mitigate this risk.

Immunogenicity of immunomodulatory, antibody-based, oncology therapeutics.

The increasing use of multiple immunomodulatory (IMD) agents for cancer therapies (e.g. antibodies targeting immune checkpoints, bispecific antibodies, and chimeric antigen receptor [CAR]-T cells), is raising questions on their potential immunogenicity and effects on treatment. In this review, we outline the mechanisms of action (MOA) of approved, antibody-based IMD agents, potentially related to their immunogenicity, and discuss the reported incidence of anti-drug antibodies (ADA) as well as their clinical relevance in patients with cancer. In addition, we discuss the impact of the administration route and potential strategies to reduce the incidence of ADA and manage treated patients. Analysis of published reports indicated that the risk of immunogenicity did not appear to correlate with the MOA of anti-programmed death 1 (PD-1)/PD-ligand 1 monoclonal antibodies nor to substantially affect treatment with most of these agents in the majority of patients evaluated to date. Treatment with B-cell depleting agents appears associated with a low risk of immunogenicity. No significant difference in ADA incidence was found between the intravenous and subcutaneous administration routes for a panel of non-oncology IMD antibodies. Additionally, while the data suggest a higher likelihood of immunogenicity for antibodies with T-cell or antigen-presenting cell (APC) targets versus B-cell targets, it is possible to have targets expressed on APCs or T cells and still have a low incidence of immunogenicity.

Importance of Feedback and Feedforward Loops to Adaptive Immune Response Modeling.

The human adaptive immune system is a very complex network of different types of cells, cytokines, and signaling molecules. This complex network makes it difficult to understand the system level regulations. To properly explain the immune system, it is necessary to explicitly investigate the presence of different feedback and feedforward loops (FFLs) and their crosstalks. Considering that these loops increase the complexity of the system, the mathematical modeling has been proved to be an important tool to explain such complex biological systems. This review focuses on these regulatory loops and discusses their importance on systems modeling of the immune system.

Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies.

Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.

The Role of Aggregates of Therapeutic Protein Products in Immunogenicity: An Evaluation by Mathematical Modeling.

Therapeutic protein products (TPP) have been widely used to treat a variety of human diseases, including cancer, hemophilia, and autoimmune diseases. However, TPP can induce unwanted immune responses that can impact both drug efficacy and patient safety. The presence of aggregates is of particular concern as they have been implicated in inducing both T cell-independent and T cell-dependent immune responses. We used mathematical modeling to evaluate several mechanisms through which aggregates of TPP could contribute to the development of immunogenicity. Modeling interactions between aggregates and B cell receptors demonstrated that aggregates are unlikely to induce T cell-independent immune responses by cross-linking B cell receptors because the amount of signal transducing complex that can form under physiologically relevant conditions is limited. We systematically evaluate the role of aggregates in inducing T cell-dependent immune responses using a recently developed multiscale mechanistic mathematical model. Our analysis indicates that aggregates could contribute to T cell-dependent immune response by inducing high affinity epitopes which may not be present in the nonaggregated TPP and/or by enhancing danger signals to break tolerance. In summary, our computational analysis is suggestive of novel insights into the mechanisms underlying aggregate-induced immunogenicity, which could be used to develop mitigation strategies.

Therapeutic outcomes, assessments, risk factors and mitigation efforts of immunogenicity of therapeutic protein products.

Therapeutic protein products (TPPs) are of considerable value in the treatment of a variety of diseases, including cancer, hemophilia, and autoimmune diseases. The success of TPP mainly results from prolonged half-life, increased target specificity and decreased intrinsic toxicity compared with small molecule drugs. However, unwanted immune responses against TPP, such as generation of anti-drug antibody, can impact both drug efficacy and patient safety, which has led to requirements for increased monitoring in regulatory studies and clinical practice, termination of drug development, or even withdrawal of marketed products. We present an overview of current knowledge on immunogenicity of TPP and its impact on efficacy and safety. We also discuss methods for measurement and prediction of immunogenicity and review both product-related and patient-related risk factors that affect its development, and efforts that may be taken to mitigate it. Lastly, we discuss gaps in knowledge and technology and what is needed to fill these.

A review of quantitative modeling of B cell responses to antigenic challenge.

A key role of B cells in the mammalian immune response is the generation of antibodies that serve to protect the organism against antigenic challenges. The same process may also be detrimental in the context of autoimmunity. Several modeling approaches have been applied to this aspect of the immune response, from predicting potential epitopes to describing B cells progress through developmental models and simulating antibody production. Here we review some of the modeling techniques, and summarize models that describe different activation mechanisms for B cells, including T cell dependent and independent models. We focus on viral infection as a prototype system, and briefly describe case studies in other disease areas such as bacterial infection and oncology. We single out aspects of the B cell response for which there are current knowledge gaps. We outline areas in need of further research in modeling applications to ultimately produce a "B cell module" for a complete immune response model.

Recombinant human L-ficolin directly neutralizes hepatitis C virus entry.

L-ficolin is a soluble pattern recognition molecule expressed by the liver that contributes to innate immune defense against microorganisms. It is well described that binding of L-ficolin to specific pathogen-associated molecular patterns activates the lectin complement pathway, resulting in opsonization and lysis of pathogens. In this study, we demonstrated that in addition to this indirect effect, L-ficolin has a direct neutralizing effect against hepatitis C virus (HCV) entry. Specific, dose-dependent binding of recombinant L-ficolin to HCV glycoproteins E1 and E2 was observed. This interaction was inhibited by soluble L-ficolin ligands. Interaction of L-ficolin with E1 and E2 potently inhibited entry of retroviral pseudoparticles bearing these glycoproteins. L-ficolin also inhibited entry of cell-cultured HCV in a calcium-dependent manner. Neutralizing concentrations of L-ficolin were found to be circulating in the serum of HCV-infected individuals. This is the first description of direct neutralization of HCV entry by a ficolin and highlights a novel role for L-ficolin as a virus entry inhibitor.

The development of a fully-integrated immune response model (FIRM) simulator of the immune response through integration of multiple subset models.

BACKGROUND: The complexity and multiscale nature of the mammalian immune response provides an excellent test bed for the potential of mathematical modeling and simulation to facilitate mechanistic understanding. Historically, mathematical models of the immune response focused on subsets of the immune system and/or specific aspects of the response. Mathematical models have been developed for the humoral side of the immune response, or for the cellular side, or for cytokine kinetics, but rarely have they been proposed to encompass the overall system complexity. We propose here a framework for integration of subset models, based on a system biology approach. RESULTS: A dynamic simulator, the Fully-integrated Immune Response Model (FIRM), was built in a stepwise fashion by integrating published subset models and adding novel features. The approach used to build the model includes the formulation of the network of interacting species and the subsequent introduction of rate laws to describe each biological process. The resulting model represents a multi-organ structure, comprised of the target organ where the immune response takes place, circulating blood, lymphoid T, and lymphoid B tissue. The cell types accounted for include macrophages, a few T-cell lineages (cytotoxic, regulatory, helper 1, and helper 2), and B-cell activation to plasma cells. Four different cytokines were accounted for: IFN-γ, IL-4, IL-10 and IL-12. In addition, generic inflammatory signals are used to represent the kinetics of IL-1, IL-2, and TGF-ß. Cell recruitment, differentiation, replication, apoptosis and migration are described as appropriate for the different cell types. The model is a hybrid structure containing information from several mammalian species. The structure of the network was built to be physiologically and biochemically consistent. Rate laws for all the cellular fate processes, growth factor production rates and half-lives, together with antibody production rates and half-lives, are provided. The results demonstrate how this framework can be used to integrate mathematical models of the immune response from several published sources and describe qualitative predictions of global immune system response arising from the integrated, hybrid model. In addition, we show how the model can be expanded to include novel biological findings. Case studies were carried out to simulate TB infection, tumor rejection, response to a blood borne pathogen and the consequences of accounting for regulatory T-cells. CONCLUSIONS: The final result of this work is a postulated and increasingly comprehensive representation of the mammalian immune system, based on physiological knowledge and susceptible to further experimental testing and validation. We believe that the integrated nature of FIRM has the potential to simulate a range of responses under a variety of conditions, from modeling of immune responses after tuberculosis (TB) infection to tumor formation in tissues. FIRM also has the flexibility to be expanded to include both complex and novel immunological response features as our knowledge of the immune system advances.