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.

Immunogenicity assessment of bispecific antibody-based immunotherapy in oncology.

With increasing numbers of bispecific antibodies (BsAbs) and multispecific products entering the clinic, recent data highlight immunogenicity as an emerging challenge in the development of such novel biologics. This review focuses on the immunogenicity risk assessment (IgRA) of BsAb-based immunotherapies for cancer, highlighting several risk factors that need to be considered. These include the novel scaffolds consisting of bioengineered sequences, the potentially synergistic immunomodulating mechanisms of action (MOAs) from different domains of the BsAb, as well as several other product-related and patient-related factors.

Immune Complex Formation Is Associated With Loss of Tolerance and an Antibody Response to Both Drug and Target.

AMG 966 is a bi-specific, heteroimmunoglobulin molecule that binds both tumor necrosis factor alpha (TNFα) and TNF-like ligand 1A (TL1A). In a first-in-human clinical study in healthy volunteers, AMG 966 elicited anti-drug antibodies (ADA) in 53 of 54 subjects (98.1%), despite a paucity of T cell epitopes observed in T cell assays.

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 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.

Immunogenicity Risk Assessment for Multi-specific Therapeutics.

The objective of this manuscript is to provide the reader with a hypothetical case study to present an immunogenicity risk assessment for a multi-specific therapeutic as part of Investigational New Drug (IND) application. In order to provide context for the bioanalytical strategies used to support the multi-specific therapeutic presented herein, the introduction focuses on known immunogenicity risk factors. The subsequent hypothetical case study applies these principles to a specific example HC-12, based loosely on anti-TNFα and anti-IL-17A bispecific molecules previously in development, structured as an example immunogenicity risk assessment for submission to health authorities.

Phase 1, single-dose study to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of etelcalcetide in pediatric patients with secondary hyperparathyroidism receiving hemodialysis.

Background: Data on the safety, efficacy of etelcalcetide in children with secondary hyperparathyroidism (sHPT) are limited.

Methods: This phase 1 study (NCT02833857) evaluated the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) of single-dose etelcalcetide (0.035 mg/kg intravenously) in pediatric hemodialysis patients (two cohorts; 1: 12-< 18 years; 2: 2-< 12 years).

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.

Clinical immunogenicity of the d-amino acid peptide therapeutic etelcalcetide: Method development challenges and anti-drug antibody clinical impact assessments.

The immunogenicity risk assessment and bioanalytical strategy for novel therapeutics should account for both unique biophysical properties and potential consequences of immunogenicity. When assessing the immunogenicity risk of etelcalcetide, a peptide agonist of the calcium-sensing receptor, we considered the potential that the d-amino acid 'backbone' and biotransformation of etelcalcetide could allow the drug to act as a hapten. As a consequence, we validated and implemented a surface plasmon resonance immunoassay platform with both etelcalcetide and etelcalcetide-'carrier' surfaces to detect anti-drug antibodies (ADA).

A Proposal to Redefine Clinical Immunogenicity Assessment.

With more than 100 therapeutic proteins (TP) approved since the first EMA guidance on immunogenicity in 2007, a vast amount of clinical experience with a variety of therapeutic proteins has been gained. This has provided data on anti-drug antibodies (ADA) and their observed clinical impact, or lack thereof. It has become evident that not all ADA responses are clinically relevant.

BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms.

Follicular helper T cells (Tfh cells) are required for T cell help to B cells, and BCL6 is the defining transcription factor of Tfh cells. However, the functions of BCL6 in Tfh cells have largely remained unclear. Here we defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells.

Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses.

The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs).

Development and optimization of neutralizing antibody assays to monitor clinical immunogenicity.

The development of an unwanted immune response to a protein therapeutic is a constant concern and necessitates careful monitoring of this class of drugs during clinical development. Neutralizing antibodies can have a significant impact on bioavailability, efficacy and safety of a protein therapeutic. Consequently, immunogenicity testing is required prior to obtaining regulatory approval and in some cases even after a product is marketed.

Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell differentiation.

Follicular helper CD4 T (Tfh) cells provide B cells with signals that are important for the generation of high-affinity Abs and immunological memory and, therefore, are critical for the protective immunity elicited by most human vaccines. Transcriptional regulators of human Tfh cell differentiation are poorly understood. In this article, we demonstrate that Bcl6 controls specific gene modules for human Tfh cell differentiation.

IL-23 modulated myelin-specific T cells induce EAE via an IFNγ driven, IL-17 independent pathway.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) mediated by myelin-reactive CD4(+) T cells. An unresolved issue that has important clinical implications concerns the cytokines produced by myelin-reactive T cells that determine their pathogenicity. Initially, IL-12 polarized, IFNγ producing Th1 cells were thought to be essential for the development of EAE.

EAE mediated by a non-IFN-γ/non-IL-17 pathway.

Previous studies have shown that EAE can be elicited by the adoptive transfer of either IFN-γ-producing (Th1) or IL-17-producing (Th17) myelin-specific CD4(+) T-cell lines. Paradoxically, mice deficient in either IFN-γ or IL-17 remain susceptible to EAE following immunization with myelin antigens in CFA. These observations raise questions about the redundancy of IFN-γ and IL-17 in autoimmune demyelinating disease mediated by a diverse, polyclonal population of autoreactive T cells.

GM-CSF-dependent, CD103+ dermal dendritic cells play a critical role in Th effector cell differentiation after subcutaneous immunization.

Dendritic cells (DCs) play an important role in CD4(+) T helper (Th) cell differentiation and in the initiation of both protective and pathogenic immunity. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a DC growth factor critical for the induction of experimental autoimmune encephalomyelitis (EAE) and other autoimmune diseases, yet its mechanism of action in vivo is not fully defined. We show that GM-CSF is directly required for the accumulation of radiosensitive dermal-derived langerin(+)CD103(+) DCs in the skin and peripheral lymph nodes under steady-state and inflammatory conditions.

IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition.

The interleukin (IL)-12p40 family of cytokines plays a critical role in the development of experimental autoimmune encephalomyelitis (EAE). However, the relative contributions of IL-12 and IL-23 to the pathogenic process remain to be elucidated. Here, we show that activation of uncommitted myelin-reactive T cells in the presence of either IL-12p70 or IL-23 confers encephalogenicity.

The Th17-ELR+ CXC chemokine pathway is essential for the development of central nervous system autoimmune disease.

The ELR(+) CXC chemokines CXCL1 and CXCL2 are up-regulated in the central nervous system (CNS) during multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). However, their functional significance and the pathways regulating their expression are largely unknown. We show that transfer of encephalitogenic CD4(+) Th17 cells is sufficient to induce CXCL1 and CXCL2 transcription in the spinal cords of naive, syngeneic recipients.

Th17 and Th1 responses directed against the immunizing epitope, as opposed to secondary epitopes, dominate the autoimmune repertoire during relapses of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease with similarities to multiple sclerosis (MS). It has been suggested that relapses of EAE and MS may be associated with, and even driven by, T cells specific for novel epitopes that are primed during the course of tissue destruction in the target organ or in secondary lymphoid tissues. We show, however, that IFNgamma and IL-17 responses against the immunizing epitope remain dominant through out the course of multiphasic EAE.