STABILITY TESTING OF ANTI-VACCINE ANTIBODIES IN HUMAN SERUM

Background: To generate exhaustive data on the stability of human anti-vaccine or anti-drug antibodies, we designed a GLP study using anti-vaccine antibodies (AVA) samples collected from over 100 different subjects. Samples were selected from various time points after vaccination to cover the heterogeneity of the polyclonal human immune response. Samples were analyzed shortly after serum collection using specific ELISAs and re-analyzed after long-term storage or multiple cycles of freeze-thaw (FT). The general acceptance criteria for incurred sample reanalysis (ISR) for ligand binding assays (LBA) were applied, as well as some alternative stricter acceptance criteria promoted by various white papers. Results: Anti-vaccine antibodies are stable in undiluted serum for at least three cycles of freeze-thaw and at least 3.5 years storage at a nominal temperature of -80°C. Conclusion: The nature of the humoral immune response allows extending the conclusion of this study to all anti-vaccine and anti-drug antibodies, making it unnecessary to test long-term stability for each new biological therapeutics or vaccine program

STABILITY OF ANTI-IMMUNOTHERAPEUTIC ANTIBODIES IN FROZEN HUMAN SERUM SAMPLES

Background: To generate exhaustive data on the stability of human anti-vaccine or anti-drug antibodies, we designed a GLP study using anti-vaccine antibodies (AVA) samples collected from over 100 different subjects. Samples were selected from various time points after vaccination to cover the heterogeneity of the polyclonal human immune response. Samples were analyzed shortly after serum collection using specific ELISAs and re-analyzed after long-term storage or multiple cycles of freeze-thaw (FT). The general acceptance criteria for incurred sample reanalysis (ISR) for ligand binding assays (LBA) were applied, as well as some alternative stricter acceptance criteria promoted by various white papers. Results: Anti-vaccine antibodies are stable in undiluted serum for at least three cycles of freeze-thaw and 3.5 years storage at a nominal temperature of -80°C. Conclusion: The nature of the humoral immune response allows extending the conclusion of this study to all anti-vaccine and anti-drug antibodies, making it unnecessary to test long-term stability for each new biological therapeutics or vaccine program

Pillars article: the dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996. 86: 973-983

The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, spätzle regulatory gene cassette, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways

EBF Recommendation for stability testing of anti-drug antibodies; lessons learned from anti-vaccine antibody stability studies.

Long- and short-term stability testing of the analyte is one of the key parameters in bioanalytical method validation in support of pharmacokinetics. However, for immunogenicity testing the scientific rationale for long- and short-term stability testing on quality control (QC) samples most often spiked with polyclonal antibody raised in a different species should be questioned. Therefore, the European Bioanalysis Forum (EBF) formed a Topic Team (TT) to discuss the scientific rationale for stability testing of anti-drug antibodies (ADA). A review of EBF member companies’ experience on ADA stability and data from vaccine projects was the basis of this discussion. EBF recommends to perform short-term stability testing but not to perform long-term stability testing of ADAs in non-clinical and clinical studies

Nonclinical safety evaluation of scAAV8-RLBP1 (CPK850) for treatment of RLBP1 retinitis pigmentosa

Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of RLBP1 deficiency with a self-complementary AAV8 vector carrying the gene for human RLBP1 under control of a short RLBP1 promoter (CPK850)1. In this communication, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation. In Rlbp1-/- mice dosed at a range of CPK850 levels, a minimum efficacious dose of 3x107 vg in a volume of 1ul was observed. For safety assessment in these and Rlbp1+/+ mice, optical coherence tomography (OCT) and histopathological analysis indicated retinal thinning that appeared to be dose-dependent for both Rlbp1 genotypes with no qualitative difference noted between Rlbp1+/+ and Rlbp1-/- mice. In a non-human primate study, RLBP1 mRNA expression was detected and dose dependent intraocular inflammation and retinal thinning were observed. Inflammation resolved slowly over time, and did not appear to be exacerbated in the presence of anti-AAV8 antibodies. Biodistribution was evaluated in rats as well as from satellite animals in the non-human primate study. The vector was largely detected in ocular tissues as well as at low levels in the optic nerve, superior colliculus and lateral geniculate nucleus with limited distribution outside of these tissues. These data suggest that an initial subretinal dose of ~3x107 vg/uL CPK850 could safely be used in clinical trials

Nonclinical Safety Evaluation of scAAV8-RLBP1 for Treatment of RLBP1 Retinitis Pigmentosa

Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of RLBP1 deficiency with a self-complementary AAV8 vector carrying the gene for human RLBP1 under control of a short RLBP1 promoter (CPK850).1 In this article, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation. In Rlbp1−/− mice dosed at a range of CPK850 levels, a minimum efficacious dose of 3 × 107 vg in a volume of 1 μL was observed. For safety assessment in these and Rlbp1+/+ mice, optical coherence tomography (OCT) and histopathological analysis indicated retinal thinning that appeared to be dose-dependent for both Rlbp1 genotypes, with no qualitative difference noted between Rlbp1+/+ and Rlbp1−/− mice. In a non-human primate study, RLBP1 mRNA expression was detected and dose dependent intraocular inflammation and retinal thinning were observed. Inflammation resolved slowly over time and did not appear to be exacerbated in the presence of anti-AAV8 antibodies. Biodistribution was evaluated in rats and satellite animals in the non-human primate study. The vector was largely detected in ocular tissues and low levels in the optic nerve, superior colliculus, and lateral geniculate nucleus, with limited distribution outside of these tissues. These data suggest that an initial subretinal dose of ∼3 × 107 vg/μL CPK850 can safely be used in clinical trials

Proceedings of the 14th European immunogenicity platform open symposium on immunogenicity of biopharmaceuticals

ABSTRACTBiologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals and its one-day training course gives experts and newcomers across academia, industry, and regulatory agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we report the discussions that took place at the EIP’s 14th Symposium, held in April 2023. The topics covered included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assessment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated with new modalities, which were discussed in a dedicated session