Immunogenicity assays are designed to determine the immune response to biotherapeutics such as protein, peptide, antibody, oligonucleotide or DNA/RNA-based drugs and its clinical impact. In a recent IQ Consortium industry white paper published in the journal Molecular Therapy, researchers provided a framework for bioanalytical strategies related to the immunogenicity assessment of recombinant adenovirus associated virus (rAAV)- based gene therapies.

The need of immunogenicity assays for gene therapies (rAAV-based therapies)

When considering biotherapeutics for diseases, one must understand the likelihood and/or severity of immune response for which a given compound may elicit in the host. For this reason, it is imperative that before treatment, an individual’s immune response profile must be well characterized. Immunogenicity is the ability of a molecule or substance to provoke an immune response and the magnitude of that immune response. rAAVs are small, non-enveloped, non-replicative viruses which evoke a mild immune response and are not known to cause diseases yet are used as vectors for gene therapy. As of September 20, 2022, there are 151 clinical trials which utilize AAVs for treatment according to ClinicalTrials.gov. However, one major issue that may impact the efficacy of such therapeutics is the immunity of the patient/host. Natural exposure to wild type (wt) AAVs results in the development of antibodies against those AAVs; an individual may also have antibodies through maternal inheritance (Fig. 1). These pre-existing antibodies can bind to therapeutic AAVs (which use the same or similar capsids as vectors in gene therapy (GT)) and form large immune complexes that can inhibit the binding of the GT to the target cells or signal the immune response to attack the GT rAAV.

Fig.1 : AAV immunogenicity in humans

Thus, for successful GT intervention, a prior assay of the patient’s immunity profile should be assessed. Then, in conjunction with several methods of immunity suppression/modulation, a therapeutic regimen could be initiated. Such assays as immunoassays, flow cytometry (e.g. MACS), qRT-PCR, immune complex-based activation assays, compliment cleavage assays, cell-based neutralization assays, FluoroSpot, ELIspot, enzyme inhibition assays, cell-based Nab assays, and tetramer staining provide various avenues by which to characterize the host immune response with a focus on capsid immunogenicity and transgene product immunogenicity. These will be defined in section 3. This is a summary of the findings of a recent review published in Molecular Therapy (Yang, et al., 2022).

Assays used in immunogenicity testing of AAV vectors

1. Capsid Immunogenicity Assays
   1. Innate
      1. Assessment of cytokine and chemokine secretion/expression
         1. Immunoassays such asenzyme-linked immunosorbent assay (ELISA) or Electrochemiluminescent immunoassay (ECLIA)
         2. Flow cytometry (magnetic-activated cell sorting or MACS)
         3. qRT-PCR (assessing the gene expression levels in a given sample)
      2. Complement factors and activation
         1. Immunoassays
         2. Immune complex-based activation assays – example: kits that detect soluble IgG immune complexes (FcgR activation; Chen et al., 2022)
         3. Complement cleavage assays
      3. Humoral 

1. Anti-capsid binding antibodies (total antibodies or TAb)
   1. Immunoassay (Bridging and sandwich assays)
2. Anti-capsid TI (neutralizing antibodies)
   1. Cell-based neutralization assay
3. Cellular 

1. T cell response to capsid by measuring secreted factors (e.g. IFN-γ)
   1. FluouroSpot
   2. ELISpot
2. T cell phenotypes responding to capsid antigen
   1. Flow cytometry
   2. Tetramer staining 

1. Transgene product immunogenicity assays
   1. Humoral
      1. Anti-transgene product binding
         1. Immunoassays
      2. NAbs
         1. Cell-based NAb assay
         2. Immunoassay
         3. Enzyme inhibition assay
      3. Cellular
         1. T cell response to transgene antigen by secreted factors (e.g. IFN-γ)
            1. FluoroSpot
            2. ELISpot
         2. T cell phenotypes responding to transgene product
            1. Flow cytometry
            2. Tetramer staining

Regulatory requirements (FDA/EU) for immunogenicity assays/tests

The following is an outline of the relevant regulatory guidance documents, from the US Food and Drug Administration (FDA), International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), and the European Medicines Agency (EMA), to consider for immunogenicity assessments during study design for rAAV GTs:

1. FDA
   1. Guidance for Human Somatic Cell Therapy and Gene Therapy, March 1998
   2. Pre-clinical Assessment of Investigational Cellular and Gene Therapy Products, November 2013
   3. Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene Therapy Products, June 2015
   4. Human Gene Therapy for Hemophilia, January 2020
   5. Human Gene Therapy for Retinal Disorders, January 2020
   6. Human Gene Therapy for Rare Diseases, January 2020
   7. Human Gene Therapy for Neurodegenerative Diseases (draft)
2. EMA
   1. Guideline on the Quality, Non-Clinical and Clinical Aspects of Gene Therapy Medicinal Products, March 2018
3. ICH

S12 Nonclinical biodistribution considerations for gene therapy products (draft)

Conclusion 

The study findings highlighted a deeper comprehension of the elements and mechanism(s) that are responsible for immunological reactions will be highly helpful in expediting and optimizing the development of rAAV-based GTs. This understanding begins with optimal vector design and extends to, but is not limited to, the control of the manufacturing process, patient populations that are well-characterized, evaluation and practicality of repeat dosage, and clinical risk reduction. By lowering adverse events and ensuring maximum efficacy for patients with unmet medical requirements, each of these aspects is anticipated to contribute to more successful GT development.

Further reading

1. Yang, Tong-yuan, et al. “Immunogenicity Assessment of AAV-Based Gene Therapies: An IQ Consortium Industry White Paper.” Molecular Therapy – Methods & Clinical Development, vol. 26, 2022, pp. 471–494., https://doi.org/10.1016/j.omtm.2022.07.018.

Chen, Haizhang et al. “Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel.” EMBO molecular medicine vol. 14,1 (2022): e14182. doi:10.15252/emmm.202114182