Immunogenicity Assessment & Clinical Relevance

Peptide products and manufacturing processes are increasingly complex. Despite significant advances in the analytical characterization of complex peptide drug products, gaps still exist in our understanding of the impact of impurities on a product immunogenicity risk. Currently available regulatory guidance does not put forward impurity qualification thresholds informed by immunogenicity risk and recommends qualification methods that present methodological challenges. This talk will provide an overview of these challenges and propose strategies to handle them.

Peptide products, like therapeutic proteins, carry the risk of triggering immune responses that can modify relevant clinical outcomes. Their immunogenicity evaluation is intimately dependent on whether the peptide in question is developed as a new molecular entity or as a generic version of an approved drug. This presentation will discuss the preparation of a suitable immunogenicity assessment founded on prediction studies and clinical immunogenicity data rom approved peptide drug products.

There is limited regulatory guidance on acceptable levels of API-related impurities in drug substances during clinical testing. A phase-appropriate, risk-based approach to evaluate the immunogenicity potential of impurities from an adaptive immune system perspective is presented. This approach frames the risk of the impurity in the context of both the immunogenicity potential of the API and dose level while also advocating for a shift from relative percentage to mass basis for impurity threshold considerations.

Therapeutic peptides can elicit unwanted immune responses, potentially leading to safety implications and reduced patient benefits. The Immunogenicity Risk Assessment (IRA) identifies immunogenicity (IG) risk factors and establishes tailored mitigation and bioanalytical monitoring strategies. This presentation highlights the IRA's role in therapeutic peptide development and its integration into the ISI for marketing applications.

Immunological responses to gene therapies continue to challenge the field, despite remarkable therapeutic advances. These immune reactions threaten safety profiles, therapeutic efficacy, and sustained clinical benefits. Effective immunogenicity management remains crucial for realizing the full clinical potential of gene-based treatments. This talk will present the FDA's perspective on immunogenicity considerations in gene therapy development and approval processes.

• What is your company's experience with submitting ISI for peptides, and when did you start preparing ISI? 
• What do you include in ISI?
• What is the stage adequate assessment in each phase of development (from discovery to post market) 
• What are the impurity thresholds and limits for peptide therapeutics, including their impact on clinical immunogenicity and Agency recommendations?
• Without clear guidance, what should the industry prioritize regarding peptide-related impurities and immunogenicity risk, such as impurity thresholds, impurity classes, and prediction tools?
• What pre-clinical approaches are commonly used to assess immunogenicity risk in peptides, and what are their pros and cons? Is there a tool that more accurately predicts this risk?
• What is your clinical ADA assessment strategy for peptides, such as banking samples but only testing if triggered, addressing cross-reactivity with endogenous counterparts, and performing Nab testing?
  

• Common challenges with overly sensitive assays that lead to high ADA positive incidence without clinical impact
• Considerations for assay development to mitigate these common challenges 
• Utility of placebo sample S/N values to define biological variability in the population and provide context for ADA datasets
• Data analysis approaches to identify clinically relevant thresholds using S/N
• Reimagining ADA assays as biomarker assays focused on context-of-use to define appropriate assay performance criteria
  

* The views expressed by the participants are those of the individuals and not necessarily those of their respective employers.

• In your experience, what magnitude (concentration) of ADA has typically shown impact on clinical parameters (PK, safety, efficacy)?
• Can you provide some examples about molecule type, route of administration, etc. which resulted in clinically impactful immunogenicity?
• In your experience, have you seen an association between level of drug exposure and magnitude of ADA required to have clinical impact?
• What range of trough concentration do you typically see for your drugs, especially for monoclonal antibodies?
• What challenges have you encountered in developing ADA assays with 100 ng/mL sensitivity, and in the presence of high drug concentrations? What mitigation strategies have you employed for the challenges above?
• Do you think expectations need to be reassessed for development of highly sensitive clinical ADA assays (≤100 ng/mL ADA) in the presence of high drug concentrations?
  

AAV are potent vectors used for gene delivery in gene therapy products. The success of gene therapy has been hindered by pre-existing and post treatment immune response against the AAV. Our studies show that females exhibit substantially higher preexisting antibody titers and neutralizing activities against multiple AAV serotypes compared to males. Beyond humoral immunity, we identified specific patterns in innate immune activation following AAV exposure in human peripheral blood mononuclear cells, where females demonstrate enhanced and accelerated cytokines production. Our data underscores the importance of considering biological sex in the design, evaluation, and safety assessment of gene therapy strategies.

Using development of an anti-LNP assay to illustrate the concepts, strategies to address pre-existing antibodies will be discussed. While some strategies will be experimental, others will include different ways of approaching the analysis, including use of AI.

Innovative multiplexed cell-based assays were designed to detect neutralizing antibodies (NAbs) against efinopegdutide, a dual GLP-1R/GCGR agonist peptide drug. Multiple assay formats were rigorously evaluated to develop assays that detect NAbs against the drug and its endogenous peptides, which carry the highest immunogenicity risk. Our multiplexing approach significantly reduces resource use, accelerates validation, and we established two cut points to effectively address matrix interference.

A survey of domain specificity for ADA responses to numerous T cell engaging bispecific antibodies was conducted. Data showed that ADA were specific for either both domains or only the T cell binding domain, indicating that the immune cell-binding arm is more likely to elicit a response. In addition, the domain mapping showed that ADA responses can evolve, which can begin with specificity to the T cell binding arm and later mature to the other arm.

Insulin icodec is a new weekly basal insulin. A neutralizing antibody (NAb) assay assessed samples in insulin-naïve Type 2 diabetes patients. Of 270 anti-drug antibody (ADA) positive subjects, 13% tested NAb positive. Higher ADA titers correlated with NAb detection. ADA presence or NAb status did not impact the efficacy or safety of insulin icodec. In vitro NAb testing added limited value to correlation of titer with clinical parameters.

This presentation will discuss the immunogenicity strategy for a multidomain biotherapeutic that includes a masked cytokine moiety. It will focus on our experiences with developing preclinical and clinical assays, highlighting the challenges encountered during assay development and explaining how a fit-for-purpose assay was implemented to support first-in-human studies.

mRNA-based therapies provide promising platforms for protein replacement in rare diseases involving dysfunctional intracellular proteins. We developed a de novo anti-protein antibody (APA) assay for an orphan disease target, where producing native reference protein was challenging. Due to limited protein availability, we validated a titer-only assay combining screening and titer steps, targeting a 5% false-positive rate. This approach achieved high sensitivity (<50 ng/mL), selectivity, protein tolerance, and precision within ±2 dilutions of median titer. We demonstrate when a simplified titer-only format is more effective than the standard 3-tier workflow, addressing cross-reactivity and challenges impacting clinical outcomes in orphan diseases.