Immune response against AAV-based viral delivery gene therapy products has a strong potential to impact safety and efficacy of the treatment. Pre- and post-treatment innate and adaptive responses have been investigated to determine and understand correlation with observed clinical signals. The presentation will focus on current industry experience related to methodologies applied to determine anti-GTx immunity. Recommendations generated during industry-wide conversations will be reviewed and discussed.

AAV gene therapy products involve complex and extensive immunogenicity assessment as these products are multicomponent. Challenges of immunogenicity assessment specific to gene therapy are related to the immune responses against these individual components, various modes of action for neutralizing antibodies, and the extent of immunogenicity assessment needed at different stages of clinical development. Case studies for developing antibody assays and strategies to overcome assay development challenges will be presented.

Cell and Gene Therapy drug development has led to an expanded interest in cellular immunogenicity as part of regulated bioanalysis.  Flow cytometry and ELISpot are playing a much larger role in the characterization of the patient's immune response to these therapies and leveraging these tools leads to a number of new challenges around sample collection, storage and shipment logistics and complex multiparameter analyses which will be discussed in this talk.

Engineering therapeutic proteins for improved clinical outcomes is now routine. Introduction of neo-sequences can however elicit immune response to the engineered protein-therapeutic (immunogenicity), which is an important safety concern. The potential consequences of immunogenicity range from loss in quality life to a life-threatening situation. In my talk I will discuss emerging trends in protein engineering that can allow the design of proteins with improved efficacy and lower immunogenicity.

Treatment of patients with mAbs generates varying levels of ADA. To gain proper insight into the clinically-relevant effect of ADA development we have characterized the immune response in patients in the post-approval clinical setting for titers, temporal patterns, isotype (IgM, IgG1-4), anti-idiotype characterization, and ADA detection using different assays. Depending on the purpose, alternative methods could be better suited for the detection of ADA than the standard bridging assays.

What is the immunogenicity profile of the Port Delivery System with ranibizumab (PDS) and how does it compare with that of monthly intravitreal (ITV) ranibizumab 0.5 mg injections? The immunogenicity profile of intravitreal ranibizumab 0.5 mg is well established. Incidences of anti-drug antibodies (ADAs) are low, and within a relatively narrow range of values (1–9%), and ADAs do not appear to impact safety, PK, or treatment effects. We will describe the PDS clinical immunogenicity profile and the potential impact of ADAs on visual outcomes, PK, and safety. Bioanalytical methods used in those studies will also be described.

• What is the current industry experience related to methodologies to determine anti-GTx immunity? 
• Value and main challenges related to harmonization of anti-GTx assays
  
• What are the recommendations generated during industry-wide conversations?

• Immunogenicity risk assessment
• What is relevant – humoral or/and cellular immunogenicity 
• Bioanalytical strategy and methods​

• New techniques for improving drug tolerance/target interference
• Effect of drug tolerance mitigation strategies on cut point factors through reduced variability of naïve sample responses
• Target selection in the face of glycosylation

• Cost of developing assays and new techniques
• Collecting good data
• Detecting ADA 
• Feedback from the FDA

Cell-based assays (CBA) for NAb are notoriously challenging: drug and soluble targets can generate false-positive/negative results even at low concentrations. In a CD20xCD3 bispecific CBA, prior anti-CD20 therapy (e.g., rituximab) was also shown to interfere. This was mitigated by addition of blocking mAbs, but each anti-CD20 therapy requires specific blockers. This highlights challenges with target-based NAb assays, both from exogenous therapies and endogenous anti-target antibodies (e.g., infectious disease).

Immunogenicity assessment is a critical part in drug development of biotherapeutics as anti-drug antibodies may impact drug PK, efficacy, and safety. Pre-existing or cross-reactive antibodies in drug naïve subjects may elevate assay cut points leading to inaccurate reporting of immunogenicity. In this presentation, we will discuss a case study with high prevalent pre-existing antibodies and approaches applied to ADA and NAb methods to overcome this challenge conferring accurate assessment of immunogenicity.

T cells engineered to target patient tumors proved to be extremely powerful addition to anti-cancer armamentarium. Expansion and long-term survival of engineered T cells are crucial for cancer therapy success. Patient’s immune response, both humoral and cellular may adversely affect T cells persistence. Therefore, ADA and cellular immunogenicity assays are needed to understand patient immune response and its effect on cellular therapy success. This presentation will share learnings gathered from the literature and experience, on factors that should be considered when setting an immunogenicity bioanalytical strategy.

Multi-domain molecules bring in added complexities during the development and validation of ADA assays. In this presentation, we will walk through a case study for a bispecific T cell engager that required multiple optimizations to achieve desired drug tolerance while avoiding a low screening cut-point factor and realizing confirmation with individual domains of the drug.

Current regulatory guidance states that ADA assay cut points need to be verified for appropriateness in different populations. For a mAb therapeutic, assay responses from over two thousand samples from seven different cancer types were compared using accepted statistical methods. Study-specific cut points may be required in some diseases like immunology and inflammation; however, based on our analysis, in-study cut points were not required for each new oncology disease indication.

MHC-associated Peptide Proteomics (MAPPs) allows the sequences presented for immune surveillance after natural processing in antigen presenting cells to be precisely defined. This technique has been primarily applied to understanding the risk of immunogenicity with therapeutic antibodies. This presentation will present findings from therapeutic peptides and compare those results to those obtained with endogenous incretin peptides. The impact of non-natural amino acids and acylation will be discussed.