A High Threshold of Biotherapeutic Aggregate Numbers is Needed to Induce an Immunogenic Response In Vitro, In Vivo, and in the Clinic.

BACKGROUND AND PURPOSE: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses. METHODS AND RESULTS: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials. CONCLUSION: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

Evaluation of the reproducibility of responses to nasal allergen challenge and effects of inhaled nasal corticosteroids.

BACKGROUND: Similar immune responses in the nasal and bronchial mucosa implies that nasal allergen challenge (NAC) is a suitable early phase experimental model for drug development targeting allergic rhinitis (AR) and asthma. We assessed NAC reproducibility and the effects of intranasal corticosteroids (INCS) on symptoms, physiology, and inflammatory mediators. METHODS: 20 participants with mild atopic asthma and AR underwent three single blinded nasal challenges each separated by three weeks (NCT03431961). Cohort A (n = 10) underwent a control saline challenge, followed by two allergen challenges. Cohort B (n = 10) underwent a NAC with no treatment intervention, followed by NAC with 14 days pre-treatment with saline nasal spray (placebo), then NAC with 14 days pre-treatment with INCS (220 µg triamcinolone acetonide twice daily). Nasosorption, nasal lavage, blood samples, forced expiratory volume 1 (FEV1), total nasal symptom score (TNSS), peak nasal inspiratory flow (PNIF) were collected up to 24 h after NAC. Total and active tryptase were measured as early-phase allergy biomarkers (≤30 min) and IL-13 and eosinophil cell counts as late-phase allergy biomarkers (3-7 h) in serum and nasal samples. Period-period reproducibility was assessed by intraclass correlation coefficients (ICC), and sample size estimates were performed using effect sizes measured after INCS. RESULTS: NAC significantly induced acute increases in nasosorption tryptase and TNSS and reduced PNIF, and induced late increases in nasosorption IL-13 with sustained reductions in PNIF. Reproducibility across NACs varied for symptoms and biomarkers, with total tryptase 5 min post NAC having the highest reproducibility (ICC = 0.91). Treatment with INCS inhibited NAC-induced IL-13 while blunting changes in TNSS and PNIF. For a similar crossover study, 7 participants per treatment arm are needed to detect treatment effects comparable to INCS for TNSS. CONCLUSION: NAC-induced biomarkers and symptoms are reproducible and responsive to INCS. NAC is suitable for assessing pharmacodynamic activity and proof of mechanism for drugs targeting allergic inflammation.

Validation of low-volume sampling devices for pharmacokinetic analysis: technical and logistical challenges and solutions.

While low-volume sampling technologies offer numerous advantages over venipuncture, implementation in clinical trials poses technical and logistical challenges. Bioanalytical methods were validated for measuring the concentration of crenezumab and etrolizumab in dried blood samples collected using Mitra and Tasso-M20. The data generated demonstrate that the concentrations of crenezumab and etrolizumab in dried blood collected by either device could be determined using calibrators prepared in serum. Drug concentrations from dried blood were converted to serum concentrations using patient hematocrit levels. Contract Research Organization experience in sample handling and analysis allowed us to compare differences between various low-volume sampling technologies. This study evaluated challenges and presented potential solutions for use of different low-volume sampling technologies for pharmacokinetic analysis.

Challenges with development of a pharmacokinetics assay to measure a variably glycosylated fusion protein.

Aim: Development of recombinant fusion proteins as drugs poses unique challenges for bioanalysis. This paper describes a case study of a glycosylated fusion protein, where variable glycosylation, matrix interference and high sensitivity needs posed unique challenges. Results: Six different assay configurations, across four different platforms were evaluated for measurement of drug concentrations. Two platforms that achieved the assay requirements were Simoa HD-1 and immune-capture LC-MS/MS-based assay. Conclusion: Both, Simoa HD-1 and the mass spectrometry-based methods were able to detect total drug by providing the adequate matrix tolerance, required sensitivity and detection of all the various glycosylated fusion proteins to support clinical sample analysis. The mass spectrometry-based method was selected due to robustness and ease of method transfer.

Considerations for Soluble Protein Biomarker Blood Sample Matrix Selection.

Blood-based soluble protein biomarkers provide invaluable clinical information about patients and are used as diagnostic, prognostic, and pharmacodynamic markers. The most commonly used blood sample matrices are serum and different types of plasma. In drug development research, the impact of sample matrix selection on successful protein biomarker quantification is sometimes overlooked. The sample matrix for a specific analyte is often chosen based on prior experience or literature searches, without good understanding of the possible effects on analyte quantification. Using a data set of 32 different soluble protein markers measured in matched serum and plasma samples, we examined the differences between serum and plasma and discussed how platelet or immune cell activation can change the quantified concentration of the analyte. We have also reviewed the effect of anticoagulant on analyte quantification. Finally, we provide specific recommendations for biomarker sample matrix selection and propose a systematic and data-driven approach for sample matrix selection. This review is intended to raise awareness of the impact and considerations of sample matrix selection on biomarker quantification.

Bioanalytical challenges in development of ultrasensitive Home Brew assays: a case study using IL-13.

Aim: IL-13 is a biomarker of type 2 inflammation that plays a critical role in asthma. IL-13 is present in serum at subpicogram levels. Methods: Simoa HD-1 technology was evaluated for the detection and quantitation of IL-13 by using a commercially available IL-13 kit and compared with a Simoa HomeBrew (HB) IL-13 assay as well as Immunological Multi-Parameter Chip Technology (IMPACT), an internal Roche platform. Performance of the assays was evaluated based on preset criteria for sensitivity, standard curve and controls' accuracy and precision, reproducibility and parallelism of endogenous analyte in serum samples. Results: The Simoa platform offered high assay sensitivity for evaluation of IL-13. Conclusion: This paper discusses the challenges and considerations when evaluating kits and/or developing HomeBrew assays using ultrasensitive platforms.

Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated biotherapeutics.

We describe a novel human immunoglobulin G2 (IgG2 )-tolerant and immune-competent heterozygous mouse model (Xeno-het) developed by crossbreeding a human Ig-tolerized XenoMouse® with a C57BL/6J wild-type mouse. The Xeno-het mouse expresses both mouse and human immunoglobulin G (IgG) genes, resulting in B-cells expressing human and mouse IgG, and secretion of human and mouse Ig into serum. This model was utilized to evaluate the immunogenicity risk of aggregated and chemically modified human antibodies. The mice were tested for their ability to break tolerance to self-tolerant monomeric antibodies. Aggregates made by mechanical stirring elicited an anti-drug antibody (ADA) response, but did not induce a robust and long-term memory B and T-cell response. Chemically modified antibodies made by oxidation were only weak and transient inducers of an immune response, as measured by a lack of both an ADA response and a B-cell antigen-specific response. Aggregate size was an important characteristic, as specific-sized protein-coated beads were able to elicit an immune response. We propose the use of this model to identify risk factors such as aggregation during manufacturing at early development for an increased potential immunogenicity risk.

Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses.

Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133), can enhance the in vitro innate immune response of a population of naive human peripheral blood mononuclear cells. This response depended on the aggregate type, inherent immunogenicity of the monomer, and donor responsiveness, and required a high number of particles, well above that detected in marketed drug products, at least in this in vitro system. We propose a cytokine signature as a potential biomarker of the in vitro peripheral blood mononuclear cell response to aggregates. The cytokines include IL-1ß, IL-6, IL-10, MCP-1, MIP-1α, MIP-1ß, MMP-2, and TNF-α. IL-6 and IL-10 might have an immunosuppressive effect on the long term immune response. Aggregates made by stirring induced the highest response compared with aggregates made by other methods. Particle size in the 2-10 µm range and the retention of some folded structure were associated with an increased response. The mechanism of aggregate activation at the innate phase was found to occur through specific cell surface receptors (the toll-like receptors TLR-2 and TLR-4, FcγRs, and the complement system). The innate signal was shown to progress to an adaptive T-cell response characterized by T-cell proliferation and secretion of T-cell cytokines. Investigating the ability of aggregates to induce cytokine signatures as biomarkers of immune responses is essential for determining their risk of immunogenicity.

Identification and inhibition of drug target interference in immunogenicity assays.

A well-designed anti-drug antibody (ADA) immunoassay is critical for appropriately monitoring the immunogenicity profile of a therapeutic protein during its development. AMG 386 is a peptide-Fc fusion protein that inhibits angiogenesis by preventing the interaction of angiopoietins with the Tie2 receptor. In bridging immunoassays for ADA, interference by the drug target, present in the assay sample, can result in false positive antibody detection. We used a statistical design-of-experiments approach to identify angiopoietin interference in bridging immunoassays of anti-AMG 386 antibodies. We also demonstrated that a high-affinity monoclonal antibody, directed against an epitope on angiopoietin that competes with AMG 386 binding, could inhibit the angiopoietin interference while preserving the detection of ADA. This report describes the development and validation of methodologies for evaluating and addressing drug target interference in bioanalytical assays that involve interactions between drug, ADA, immune complexes, and drug target.

Assessment of immunogenicity of romiplostim in clinical studies with ITP subjects.

Romiplostim is an Fc-peptide fusion protein that activates intracellular transcriptional pathways via the thrombopoietin (TPO) receptor leading to increased platelet production. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. Recombinant protein therapeutics can be at a risk of development of an antibody response that can impact efficacy and safety. Hence, a strategy to detect potential antibody formation to the drug and to related endogenous molecules can be useful. The immunogenicity assessment strategy involved both the detection and characterization of binding and neutralizing antibodies. The method for detection was based on a surface plasmon resonance biosensor platform using the Biacore 3000. Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in a neutralization assay. Serum samples from 225 subjects with immune thrombocytopenic purpura (ITP) dosed with romiplostim and 45 ITP subjects dosed with placebo were tested for romiplostim and TPO antibodies. Prior to romiplostim treatment, 17 subjects (7%) tested romiplostim antibody positive and 12 subjects (5%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, 11% of the subjects exhibited binding antibodies against romiplostim and 5% of the subjects with ITP showed binding antibodies against TPO. The antibodies against romiplostim did not cross-react with TPO and vice versa. No cases of anti-TPO neutralizing antibodies were detected in romiplostim-treated subjects. The incidence of anti-romiplostim neutralizing antibodies to romiplostim was 0.4% (one subject); this subject tested negative at the time of follow-up 4 months later. No impact on platelet profiles were apparent in subjects that had antibodies to romiplostim to date. In summary, administration of romiplostim in ITP subjects resulted in the development of a binding antibody response against romiplostim and TPO ligand. One subject developed a neutralizing antibody response to romiplostim that impacted the platelet counts of this subject. No neutralizing antibodies to endogenous TPO were observed.