Increased complexity and protein engineering of biotherapeutics have increased the potential for human immunogenicity in patients. Comprehensive immunogenicity risk assessment strategies in combination with protein engineering can mitigate these risks resulting in safer and more efficacious therapeutics for patients. Machine learning tools can be used to mine in vitro data sets to develop high throughput computational tools to push comprehensive immunogenicity risk assessment earlier into the discovery process.

Lead sequence optimization consists of sequential steps addressing efficacy, safety, and developability. However, sequence optimization of one parameter leads to unwanted consequences on the other two resulting in suboptimal drug substances. We present a framework to simultaneously de-risk immunogenic and physico-chemical sequence liabilities while maintaining parental efficacy parameters. This framework employs open source in silico tools and is achieved by simultaneous humanization, de-immunization, and sequence optimization of antibody-based biotherapeutics.

The complexity of the immune system makes prediction of responses difficult, especially to biologic therapies. Risk factors and drivers of the immune response are somewhat known, though combining all the factors into a meaningful prediction of the clinical outcome of immunogenicity for a new biotherapeutic remains elusive. Here, I review the steps to build an Immunogenicity simulator, assess its performance, and identify challenges in accuracy and its broader application.

I will discuss all the variables which contribute qualitatively and quantitatively, to the composition of the MHC II ligandome. Altogether, ensuring that the immunopeptidome landscape is highly sensitive to any changes in the composition of the intra- and extracellular proteome for a comprehensive survey of the microenvironment for MHC II presentation to CD4 T cells.

An Integrated Summary of Immunogenicity (ISI) is a component of regulatory dossiers for biopharmaceutical product submissions, facilitating product review, approval, and labeling. A multifactorial approach is required, incorporating immunogenicity risk assessment, bioanalytical strategy, and evaluation of clinical impact of anti-drug antibodies and neutralizing antibodies on pharmacokinetics, clinically relevant biomarkers, efficacy and safety parameters. This presentation will discuss our experience developing ISIs, including overall strategy and case examples.

ADA responses vary widely not only between different drugs, but also between recipients. Interestingly, it is frequently observed that an ADA response may be transient. Here we discuss examples of such variable responses, the factors contributing to dampening the ADA response, and the clinical significance thereof.

The presence of anti-drug antibodies (ADA) is an important factor in contextualizing clinical study PK, PD, safety, and efficacy. Recent efforts in harmonizing ADA validation and incorporating immunogenicity information into drug product labeling underscore the need for consistency and scientific rigor to facilitate proper communication and understanding of ADA impact. To support this effort, a cross-industry group established harmonized recommendations and a report template for clearly summarizing the essential aspects of ADA clinical study testing and reporting. This talk will present the recommendations for ADA bioanalytical report elements such as the method, critical reagents, equipment, data analysis, and study results.

• Identify assay design parameters to ensure detection of relevant antibodies
• Planning assay implementation timelines using immunogenicity risk assessment
• Designing schedule of assessments within clinical trials
• Integrate multiple findings to assess clinical relevance
• Conducting analysis using immunogenicity status as an outcome, not a baseline characteristic
  

• In-silico and in-vitro tools: State-of-the-art 
• In-vitro/in-vivo correlation of immunogenicity: What do we know?
• Immunogenicity strategy: How do we select the right assay?
• Interpretation of results and decisions: De-immunization vs immunogenicity risk and impact on project timelines
• Examining the regulatory requirements
  

Determining the neutralizing activity of an anti-drug antibody response is critical to understanding the safety and efficacy profile of a therapeutic.  In order to achieve a sensitive assay, the concentrations of reagents in the assay system need to be carefully balanced; excess amounts of target or therapeutic can lead to inaccurate results.  We will discuss considerations for mitigating the influence of these interferents during assay development.

Interference from free drug in patient serum is a key challenge in assessment of neutralizing antibodies (NAb). PABAD (Precipitation, acid Dissociation and Biotin-drug as Assay Drug), our new approach for assaying NAbs improve the drug tolerance of NAb assay while being compatible with both acid-sensitive and stable NAbs. Minimal requirement of Biotin-drug, unnecessity for magnetic beads, are additional advantages of PABAD that reduce cost and time of NAb assessment.

Chimeric Antigen Receptor T (CAR T) cell therapies have emerged as a novel approach to treat cancers. In the recently approved CAR T therapies, the immune responses are observed but their impact and relevance need further evaluation. We have used a reverse translational approach to understand the nature of immune responses through use of patient samples to further characterize humoral and cellular immune responses and relate to clinical outcome of disease. The reverse translational findings could provide opportunities for better design of the next generation of CAR T modalities by optimizing the sequences as a potential immunogenicity risk mitigation.

Discussion of gene therapy immunogenicity has focused mainly on anti-AAV antibodies, particularly pre-existing responses. However, the transgene protein may also induce a humoral or cellular immune response. This presentation will discuss the approaches to assessing immunogenicity against the expressed transgene product.

Bispecific antibodies are a novel class of complex molecules. Despite great interest in these new modalities to increase efficacy for treatment of complex diseases, recent clinical data indicate unique development challenges with high immunogenic potential which could potentially cause the failure of clinical program. Here, we present the evaluation of preexisting reactivity, anti-drug antibodies, and domain specificity to understand the immunogenicity response to multiple domain biotherapeutics.