Adeno-associated virus (AAV) gene transfer is a promising treatment for many patients with life-threatening genetic diseases. However, host immune response to the vector seems to pose a challenge for the durability and safety of AAV-mediated gene therapy. Today, AAV immunogenicity impacts on trial design and patient eligibility and remains a focus of preclinical studies that attempt to minimize host immune response against therapeutic vectors.

Clinical programs investigating AAV-vectored gene therapies have adopted the use of immune modulatory therapies to prevent or treat inflammatory responses and preserve transgene expression. We evaluated the pharmacodynamic and immunomodulatory effects of seven different immunosuppressive agents administered prophylactically prior to gene therapy dose administration and continued for four weeks in C57BL/6 mice. This study demonstrates prophylactic immune modulation prior to AAV administration can improve liver-directed transgene expression in mice.

Evolving drug modalities present ever-increasing challenges to immunogenicity prediction and risk assessment. In this case study, a heteroimmunoglobulin antibody against TL1A and TNF alpha was observed to have a high rate of immunogenicity in a clinical study contrary to risk assessment from available predictive tools. Characterization of the immune response is described focusing on the bioanalytical data. Finally, formation of large complexes is proposed to account for the immunogenicity observed.

I will present data from several studies performed in the last two years. The results show that SARS-CoV-2 infection induces multi-specific and multi-functional adaptive responses which is durable over the 6-8 months period. Neutralizing antibodies are likely key to protect from infection, but substantial contributions of T cells likely at the level of protection from severe disease. Side-by-side longitudinal comparison of different vaccine platforms reveals durable B and T cell responses, and allow correlation with vaccine efficacy. T cell reactivity is largely preserved at the level of SARS-CoV-2 variants, including Omicron and Delta.

Immunogenicity can compromise the safety and/or efficacy of therapeutic protein products. Due to the serious conditions they treat and lack of alternatives many protein therapeutics are approved even if they elicit immune responses. In the clinic, in addition to poor patient outcomes, the social and economic costs associated with neutralizing antibodies are considerable. In this presentation, I survey the immunogenicity of approved therapeutic proteins, discuss strategies for clinical management of immunogenicity, and identify challenges associated with circumventing the immune responses to approved protein therapeutics.

• Strategies to prevent or mitigate immunogenicity of therapeutic proteins and application of these strategies to prevent/mitigate immunogenicity of novel modalities: Cell and gene therapies​
- For what clinical scenarios are each strategy (deimmunization, immune suppression, therapeutic drug monitoring, immune tolerance induction) appropriate
- Retreatment of patients with AAV vectored therapeutics is blocked by humoral immunity to capsid
- Preexisting immunity to key elements of gene therapies (AAV, Cas9 proteins) may preclude patients from entry into clinical trials

• Transplantation of gene-corrected Autologous Hematopoietic Stem Cells mediated by lentiviral vectors in patients with “inborn errors of metabolism”- strategies for implementation
  -  Can lentivirus integration be targeted to avoid integration into oncogenes leading to tumorigenicity? To optimize expression/production of the therapeutic
  - What level and duration of immune suppression will be required post-transplant to prevent rejection of gene-corrected Autologous HSC?

• Are animal models of gene transfer able to predict immune response in humans? While immune-related toxicities seem to be rare in the clinic, is there a possibility that animal models are predictive, but the number of animals used for the preclinical studies is too low? 
• The immunogenicity assay toolbox improves every day. Do we have the right assays today to detect anti-AAV or anti-transgene responses in animals? Can in vitro assays be used instead and which one?
• How can the preclinical data generated be used in the clinic?  What is the true value of the pre-clinical assays and how can this be proved?​

• The multiplicity of translational and post-translational mechanisms affecting the MHC-II restricted immunopeptidome   
•  How to quantitatively assess the MHC-II ligandome and why it's important to know epitope copy numbers  
•  MHC-II presented tolerogenic and immunogenic peptides  ​

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the CNS characterized by T cell and Ab responses to the water channel protein Aquaporin 4 (AQP4). We developed a robust NMO model in C57BL/6 mice characterized by AQP4-specific Th17 and Ab responses resulting in high disease incidence and severity. We demonstrate the ability of tolerance induced by AQP4-encapsulating PLGA nanoparticles to both prevent and treat ongoing disease.

Anti-drug antibodies (ADAs) and hypersensitivity responses limit the use of many biotherapeutics. Generate Biomedicines has developed a generalizable method for computationally designing resurfaced proteins to avoid binding by preexisting ADAs. This technique was applied to L-asparaginase (ASN) to produce functional variants with up to 58 surface mutations. Resurfaced ASNs exhibited reduced binding to mouse, rabbit, and human ADAs, and alleviated hypersensitivity in an in vivo mouse model.

Comparative in vitro analytical methods to characterize innate immune response modulating impurities could potentially provide a more robust understanding of immunogenicity risk for biosimilars and help streamline their development. This talk will discuss the risks posed by innate immune response modulating impurities, available assays, and data interpretation, as well as common pitfalls and remaining knowledge gaps.

The presence of protein aggregates in therapeutic products has been correlated with ADA production and it has been shown that aggregates could play the role of danger signals facilitating T cell-dependent immune responses. Aggregates could be recognized by professional antigen-presenting cells, such as dendritic cells (DC), inducing their maturation and the activation of CD4+ T cells and thus helping B cell maturation and production of high-affinity ADA.

In vitro immunogenicity tools are used during the development of biologics to select clones with decreased clinical immunogenicity risk. Here we will discuss ongoing efforts to benchmark in vitro T cell activation assays and the cross-industry work led by HESI and AAPS to develop common reference biologics, a critical first step toward assay harmonization.

I will discuss all the molecular components that regulate the selection of the MHC-II presented peptides and 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.

Biotherapeutics have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Therefore, predicting the risk for immunogenicity of biotherapeutic products at early stages is a crucial need. This presentation will focus on in silico and in vitro T cell assay studies to characterize the immunogenic potential of different biotherapeutic proteins and their correlation to the clinically observed outcome.