Corrigendum: Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

[This corrects the article DOI: 10.3389/fimmu.2024.1345473.].

Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

AMG 256 is a bi-specific, heteroimmunoglobulin molecule with an anti-PD-1 antibody domain and a single IL-21 mutein domain on the C-terminus. Nonclinical studies in cynomolgus monkeys revealed that AMG 256 administration led to the development of immunogenicity-mediated responses and indicated that the IL-21 mutein domain of AMG 256 could enhance the anti-drug antibody response directed toward the monoclonal antibody domain. Anti-AMG 256 IgE were also observed in cynomolgus monkeys. A first-in-human (FIH) study in patients with advanced solid tumors was designed with these risks in mind. AMG 256 elicited ADA in 28 of 33 subjects (84.8%). However, ADA responses were only robust and exposure-impacting at the 2 lowest doses. At mid to high doses, ADA responses remained low magnitude and all subjects maintained exposure, despite most subjects developing ADA. Limited drug-specific IgE were also observed during the FIH study. ADA responses were not associated with any type of adverse event. The AMG 256 program represents a unique case where nonclinical studies informed on the risk of immunogenicity in humans, due to the IL-21-driven nature of the response.

Anti-drug Antibody Sample Testing and Reporting Harmonization.

A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.

Comparison of Titer and Signal to Noise (S/N) for Determination of Anti-drug Antibody Magnitude Using Clinical Data from an Industry Consortium.

During biotherapeutic drug development, immunogenicity is evaluated by measuring anti-drug antibodies (ADAs). The presence and magnitude of ADA responses is assessed using a multi-tier workflow where samples are screened, confirmed, and titered. Recent reports suggest that the assay signal to noise ratio (S/N) obtained during the screening tier correlates well with titer. To determine whether S/N could more broadly replace titer, anonymized ADA data from a consortium of sponsors was collected and analyzed. Datasets from clinical programs with therapeutics of varying immunogenicity risk levels (low to high), common ADA assay platforms (ELISA and MSD) and formats (bridging, direct, solid-phase extraction with acid dissociation), and titration approaches (endpoint and interpolated) were included in the analysis. A statistically significant correlation between S/N and titer was observed in all datasets, with a strong correlation (Spearman's r > 0.8) in 11 out of 15 assays (73%). For assays with available data, conclusions regarding ADA impact on pharmacokinetics and pharmacodynamics were similar using S/N or titer. Subject ADA kinetic profiles were also comparable using the two measurements. Determination of antibody boosting in patients with pre-existing responses could be accomplished using similar approaches for titer and S/N. Investigation of factors that impacted the accuracy of ADA magnitude measurements revealed advantages and disadvantages to both approaches. In general, S/N had superior precision and ability to detect potentially low affinity/avidity responses compared to titer. This analysis indicates that S/N could serve as an equivalent and in some cases preferable alternative to titer for assessing ADA magnitude and evaluation of impact on clinical responses.

Immunogenicity of erenumab: A pooled analysis of six placebo-controlled trials with long-term extensions.

BACKGROUND: Immunogenicity of erenumab, a human anti-calcitonin gene-related peptide receptor monoclonal antibody developed for migraine prevention, has been evaluated throughout clinical development. METHODS: Integrated post hoc analysis assessing immunogenicity of erenumab across six clinical trials in patients with episodic and chronic migraine (N = 2985). Anti-erenumab antibody incidence and potential impact on pharmacokinetics, efficacy, and safety were evaluated in short-term (double-blind treatment phase 12-24 weeks) and long-term (double-blind treatment phase plus extensions of up to 5 years) analyses. RESULTS: Anti-erenumab binding antibody incidence was low with few patients developing neutralizing antibodies. Antibody responses were mostly transient with low magnitude. Binding antibodies developed as early as 2-4 weeks after the first dose; the majority developed within the first 6 months and very few after the first year. Serum concentrations of erenumab in antibody-positive patients were generally lower than, but within the range of, antibody-negative patients. There was no impact of anti-erenumab antibodies on erenumab efficacy or safety with no differences between antibody-positive and antibody-negative patients in change in monthly migraine days or adverse event rates. CONCLUSIONS: This pooled analysis showed that immunogenicity had no meaningful clinical impact on efficacy or safety of erenumab in patients with migraine.Clinical Trial Registration: ClinicalTrials.gov, NCT01952574; ClinicalTrials.gov, NCT02456740; Clinicaltrials.gov NCT02483585; Clinicaltrials.gov, NCT02174861; Clinicaltrials.gov, NCT02630459; Clinicaltrials.gov, NCT03812224.

Anti-drug Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.

Recommendations for the Assessment and Management of Pre-existing Drug-Reactive Antibodies During Biotherapeutic Development.

Anti-drug antibodies (ADA) pose a potential risk to patient safety and efficacy and are routinely monitored during clinical trials. Pre-existing drug-reactive antibodies are present in patients without prior drug exposure and are defined by their ability to bind to a component of the drug. These pre-existing drug-reactive antibodies are frequently observed and could represent an adaptive immune response of an individual who has been previously exposed to antigens with structural similarities to the biotherapeutic. Clinical consequences of these antibodies can vary from no impact to adverse effects on patient safety, exposure, and efficacy, and are highly dependent on biotherapeutic modality, disease indications, and patient demographics. This paper describes how the immunogenicity risk assessment of a biotherapeutic integrates the existence of pre-existing drug-reactive antibodies, and provides recommendations for risk-based strategies to evaluate treatment-emergent ADA responses.

Assay signal as an alternative to titer for assessment of magnitude of an antidrug antibody response.

BACKGROUND: Titer methods are commonly used to characterize the magnitude of an antidrug antibody response. Assay S/N is an appealing alternative, but the circumstances under which use of signal-to-noise (S/N) is appropriate have not been well defined. RESULTS: We validated both titer and S/N-based methods for several therapeutics. S/N correlated strongly with titer both in aggregate and when examined on a per subject basis. Analysis of impact of antibody magnitude on pharmacokinetics yielded the same result using either method. Each assay demonstrated excellent precision, good linearity, and adequate drug tolerance. CONCLUSION: Under these circumstances, assay S/N is a valid alternative to titer for assessment of the magnitude of an antidrug antibody response.

Strategic characterization of anti-drug antibody responses for the assessment of clinical relevance and impact.

All therapeutic proteins have the potential to induce anti-drug antibodies (ADA). Clinically relevant ADA can impact efficacy and/or safety of a biological therapeutic. Immunogenicity assessment strategy evaluates binding and neutralizing ADA, and the need for additional characterization (e.g., epitope, titer and so on) is determined using a risk-based approach. The choice of characterization assays depends on the type, application and immunogenicity of the therapeutic. ADA characterization can impact the interpretation of the risk profile of a given therapeutic, and offers insight into opportunities for risk mitigation and management. This article describes common ADA characterization methods. Strategic assessment and characterization of clinically relevant ADA are discussed, in order to support clinical options for safe and effective patient care and disease management.

Recommendations for the characterization of immunogenicity response to multiple domain biotherapeutics.

Many biotherapeutics currently in development have complex mechanisms of action and contain more than one domain, each with a specific role or function. Examples include antibody-drug conjugates (ADC), PEGylated, fusion proteins and bi-specific antibodies. As with any biotherapeutic molecule, a multi-domain biotherapeutic (MDB) can elicit immune responses resulting in the production of specific anti-drug antibodies (ADA) when administered to patients. As it is beneficial to align industry standards for evaluating immunogenicity of MDBs, this paper highlights pertinent immunogenicity risk factors and describes steps involved in the design of a testing strategy to detect and characterize binding (non-neutralizing and neutralizing, NAb) ADAs. In a common tier based approach, samples identified as ADA screen positive are confirmed for the binding specificity of the antibodies to the drug molecule via a confirmatory assay. The confirmation of specificity is generally considered as a critical step of the tier based approach in overall ADA response evaluation. Further characterization of domain specificity of polyclonal anti-MDB ADA response may be required based on the analysis of molecule specific risk factors. A risk based approach in evaluating the presence of NAbs for MDB is discussed in this article. Analysis of domain-specific neutralizing antibody reactivity should be based on the risk assessment as well as the information learned during binding ADA evaluation. Situations where additional characterization of NAb specificity is possible and justified are discussed. Case studies demonstrating applicability of the risk factor based approach are presented. In general, the presence of a domain with high immunogenicity risk or presence of a domain with high endogenous protein homology may result in an overall high immunogenicity risk level for the entire MDB and can benefit from domain specificity characterization of immune response. For low immunogenicity risk MDBs, domain specificity characterization could be re-considered at later clinical phases based on the need to explain specific clinical observations. Inclusion of domain specificity characterization in early phase clinical studies for MDBs with limited clinical immunogenicity experience may be considered to help understand its value in later clinical development. It is beneficial and is recommended to have a well-defined plan for the characterization of ADA domain specificity and data analysis prior to the initiation of sample testing. Overall, best practices for immunogenicity evaluation of complex MDBs are discussed.

Mutations in the ExbB cytoplasmic carboxy terminus prevent energy-dependent interaction between the TonB and ExbD periplasmic domains.

The TonB system of Gram-negative bacteria provides passage across the outer membrane (OM) diffusion barrier that otherwise limits access to large, scarce, or important nutrients. In Escherichia coli, the integral cytoplasmic membrane (CM) proteins TonB, ExbB, and ExbD couple the CM proton motive force (PMF) to active transport of iron-siderophore complexes and vitamin B(12) across the OM through high-affinity transporters. ExbB is an integral CM protein with three transmembrane domains. The majority of ExbB occupies the cytoplasm. Here, the importance of the cytoplasmic ExbB carboxy terminus (residues 195 to 244) was evaluated by cysteine scanning mutagenesis. D211C and some of the substitutions nearest the carboxy terminus spontaneously formed disulfide cross-links, even though the cytoplasm is a reducing environment. ExbB N196C and D211C substitutions were converted to Ala substitutions to stabilize them. Only N196A, D211A, A228C, and G244C substitutions significantly decreased ExbB activity. With the exception of ExbB(G244C), all of the substituted forms were dominant. Like wild-type ExbB, they all formed a formaldehyde cross-linked tetramer, as well as a tetramer cross-linked to an unidentified protein(s). In addition, they could be formaldehyde cross-linked to ExbD and TonB. Taken together, the data suggested that they assembled normally. Three of four ExbB mutants were defective in supporting both the PMF-dependent formaldehyde cross-link between the periplasmic domains of TonB and ExbD and the proteinase K-resistant conformation of TonB. Thus, mutations in a cytoplasmic region of ExbB prevented a periplasmic event and constituted evidence for signal transduction from cytoplasm to periplasm in the TonB system.

Cytoplasmic membrane protonmotive force energizes periplasmic interactions between ExbD and TonB.

The TonB system of Escherichia coli (TonB/ExbB/ExbD) transduces the protonmotive force (pmf) of the cytoplasmic membrane to drive active transport by high-affinity outer membrane transporters. In this study, chromosomally encoded ExbD formed formaldehyde-linked complexes with TonB, ExbB and itself (homodimers) in vivo. Pmf was required for detectable cross-linking between TonB-ExbD periplasmic domains. Consistent with that observation, the presence of inactivating transmembrane domain mutations ExbD(D25N) or TonB(H20A) also prevented efficient formaldehyde cross-linking between ExbD and TonB. A specific site of periplasmic interaction occurred between ExbD(A92C) and TonB(A150C) and required functional transmembrane domains in both proteins. Conversely, neither TonB, ExbB nor pmf were required for ExbD dimer formation. These data suggest two possible models where either dynamic complex formation occurred through transmembrane domains or the transmembrane domains of ExbD and TonB configure their respective periplasmic domains. Analysis of T7-tagged ExbD with anti-ExbD antibodies revealed that a T7 tag was responsible both for our previous failure to detect T7-ExbD-ExbB and T7-ExbD-TonB formaldehyde-linked complexes and for the concomitant artefactual appearance of T7-ExbD trimers.

Identifying the subproteome of kinetically stable proteins via diagonal 2D SDS/PAGE.

Most proteins are in equilibrium with partially and globally unfolded conformations. In contrast, kinetically stable proteins (KSPs) are trapped by an energy barrier in a specific state, unable to transiently sample other conformations. Among many potential roles, it appears that kinetic stability (KS) is a feature used by nature to allow proteins to maintain activity under harsh conditions and to preserve the structure of proteins that are prone to misfolding. The biological and pathological significance of KS remains poorly understood because of the lack of simple experimental methods to identify this property and its infrequent occurrence in proteins. Based on our previous correlation between KS and a protein's resistance to the denaturing detergent SDS, we show here the application of a diagonal 2D (D2D) SDS/PAGE assay to identify KSPs in complex mixtures. We applied this method to the lysate of Escherichia coli and upon proteomics analysis have identified 50 nonredundant proteins that were SDS-resistant (i.e., kinetically stable). Structural and functional analyses of a subset (44) of these proteins with known 3D structure revealed some potential structural and functional biases toward and against KS. This simple D2D SDS/PAGE assay will allow the widespread investigation of KS, including the proteomics-level identification of KSPs in different systems, potentially leading to a better understanding of the biological and pathological significance of this intriguing property of proteins.

Structural basis of protein kinetic stability: resistance to sodium dodecyl sulfate suggests a central role for rigidity and a bias toward beta-sheet structure.

The term kinetic stability is used to describe proteins that are trapped in a specific conformation because of an unusually high-unfolding barrier that results in very slow unfolding rates. Motivated by the observation that some proteins are resistant to sodium dodecyl sulfate (SDS)-induced denaturation, an attempt was made to determine whether this property is a result of kinetic stability. We studied many proteins, including a few kinetically stable proteins known to be resistant to SDS. The resistance to SDS-induced denaturation was investigated by comparing the migration on polyacrylamide gels of identical boiled and unboiled protein samples containing SDS. On the basis of the different migration of these samples, eight proteins emerged as being resistant to SDS. The kinetic stability of these proteins was confirmed by their slow unfolding rate upon incubation in guanidine hydrochloride. Further studies showed that these proteins were also extremely resistant to proteolysis by proteinase K, suggesting that a common mechanism may account for their resistance to SDS and proteolytic cleavage. Together, these observations suggest that a rigid protein structure may be the physical basis for kinetic stability and that resistance to SDS may serve as a simple assay for identifying proteins whose native conformations are kinetically trapped. Remarkably, most of the kinetically stable SDS-resistant proteins in this study are oligomeric beta-sheet proteins, suggesting a bias of these types of structures toward kinetic stability.