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There are many factors that can influence the pharmacokinetics (PK) of a mAb or Fc-fusion molecule with the primary determinant being FcRn-mediated recycling. Through Fab or Fc engineering, IgG-FcRn interaction can be used to generate a variety of therapeutic antibodies with significantly enhanced half-life or ability to remove unwanted antigen from circulation. Glycosylation of a mAb or Fc-fusion protein can have a significant impact on the PK of these molecules. mAb charge can be important and variants with pI values of 1-2 unit difference are likely to impact PK with lower pI values being favorable for a longer half-life. Most mAbs display target mediated drug disposition (TMDD), which can have significant consequences on the study designs of preclinical and clinical studies. The PK of mAb can also be influenced by anti-drug antibody (ADA) response and off-target binding, which require careful consideration during the discovery stage. mAbs are primarily absorbed through the lymphatics via convection and can be conveniently administered by the subcutaneous (sc) route in large doses/volumes with co-formulation of hyaluronidase. The human PK of a mAb can be reasonably estimated using cynomolgus monkey data and allometric scaling methods.

Background and PurposeThere 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 ResultsHighly 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.ConclusionThe 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.

Background: The development of an anti-drug antibody (ADA)-tolerant pharmacokinetic (PK) assay is important when the drug exposure is irrelevant to toxicity in the presence of ADA. We aimed to develop and validate an ADA-tolerant assay for an exatecan-based antibody–drug conjugate (ADC) in monkey plasma. Results: The assay tolerated 5.00 µg/mL of ADA at 12 µg/mL of ADC. Its accuracy and precision results satisfied the acceptance criteria. Furthermore, the assay was free from hook and matrix effects and exhibited good dilutional linearity. Additionally, the ADC in plasma samples was stable under different storage conditions. Method: An ADA-tolerant ADC assay was configured with an anti-payload antibody for capture, and a drug-target protein combined with a horseradish peroxidase (HRP)-labeled antibody against a drug-target-protein tag for detection. Samples were firstly acidified to dissociate drug and ADA complexes, and to convert the carboxylate form to the lactone form of exatecan molecules; then, the ADAs in the samples were removed with a naked antibody-coated microplate. The treated samples were further incubated with coated anti-payload antibody and captured ADC molecules were quantified by the detection reagent. The developed assay was optimized and validated against regulatory guidelines. Conclusions: The assay met both methodological and sample-related ADA tolerance requirements, and was applicable to a nonclinical study in cynomolgus monkeys.

Background Anti-drug antibodies (ADAs) can impact on the efficacy and safety of biologicals, today used to treat several chronic inflammatory conditions. Specific patient groups may be more prone to develop ADAs. Rituximab is routinely used for ANCA-associated vasculitis (AAV) and as off-label therapy for systemic lupus erythematosus (SLE), but data on occurrence and predisposing factors to ADAs in these diseases is limited. Objectives To elucidate the rate of occurrence, and risk factors for ADAs against rituximab in SLE and AAV. Methods ADAs were detected using a bridging electrochemiluminescent (ECL) immunoassay in sera from rituximab-naïve (AAV; n  = 41 and SLE; n  = 62) and rituximab-treated (AAV; n  = 22 and SLE; n  = 66) patients. Clinical data was retrieved from medical records. Disease activity was estimated by the SLE Disease Activity Index-2000 (SLEDAI-2 K) and the Birmingham Vasculitis Activity Score (BVAS). Results After first rituximab cycle, no AAV patients were ADA-positive compared to 37.8% of the SLE patients. Samples were obtained at a median (IQR) time of 5.5 (3.7–7.0) months (AAV), and 6.0 (5.0–7.0) months (SLE). ADA-positive SLE individuals were younger (34.0 (25.9–40.8) vs 44.3 (32.7–56.3) years, p  = 0.002) and with more active disease (SLEDAI-2 K 14.0 (10.0–18.5) vs. 8.0 (6.0–14), p  = 0.0017) and shorter disease duration (4.14 (1.18–10.08) vs 9.19 (5.71–16.93), p  = 0.0097) compared to ADA-negative SLE. ADAs primarily occurred in nephritis patients, were associated with anti-dsDNA positivity but were not influenced by concomitant use of corticosteroids, cyclophosphamide or previous treatments. Despite overall reduction of SLEDAI-2 K (12.0 (7.0–16) to 4.0 (2.0–6.7), p  < 0.0001), ADA-positive individuals still had higher SLEDAI-2 K (6.0 (4.0–9.0) vs 4.0 (2.0–6.0), p  = 0.004) and their B cell count at 6 months follow-up was higher (CD19 + % 4.0 (0.5–10.0) vs 0.5 (0.4–1.0), p  = 0.002). At retreatment, two ADA-positive SLE patients developed serum sickness (16.7%), and three had infusion reactions (25%) in contrast with one (5.2%) serum sickness in the ADA-negative group. Conclusions In contrast to AAV, ADAs were highly prevalent among rituximab-treated SLE patients already after the first course of treatment and were found to effect on both clinical and immunological responses. The high frequency in SLE may warrant implementations of ADA screening before retreatment and survey of immediate and late-onset infusion reactions.

Biotherapeutics are a rapidly growing portion of the total pharmaceutical market accounting for almost one-half of recent new drug approvals. A major portion of these approvals each year are monoclonal antibodies (mAbs). During development, non-clinical pharmacology and toxicology testing of mAbs differs from that done with chemical entities since these biotherapeutics are derived from a biological source and therefore the animal models must share the same epitopes (targets) as humans to elicit a pharmacological response. Mechanisms of toxicity of mAbs are both pharmacological and non-pharmacological in nature; however, standard in silico predictive toxicological methods used in research and development of chemical entities currently do not apply to these biotherapeutics. Challenges and potential opportunities exist for new methodologies to provide a more predictive program to assess and monitor potential adverse drug reactions of mAbs for specific patients before and during clinical trials and after market approval.

Biological drugs have revolutionized the treatment of many chronic diseases, starting with cancer. They normally consist of antibodies that are also effectively used to treat several autoimmune diseases, including psoriasis. These products, called biologics, work by selectively blocking the activity of certain targets, mainly cytokines, which play a crucial role in the pathogenic and inflammatory processes involved in a particular disease. Unfortunately, a reduction in response to these biological treatments may occur over time, and this phenomenon is often due to the development of antibodies against the therapeutic antibodies. The immune responses directed to these therapeutics range from transient anti-drug antibodies (ADA) formation, with no clinical significance, to the generation of high titers and persistence of ADA, causing loss of efficacy. Considering the costs associated with the use of biological drugs, there is growing interest in identifying biomarkers that can predict clinical response to personalize treatments.

The FDA has released new draft guidance to standardize how immunogenicity of protein therapeutics is described in product labels. A key aspect to this new guidance is that companies should describe anti-drug antibodies that have clinical significance in addition to reporting ADAs’ incidence. Factors to consider when determining clinical significance include if those antibodies have a significant effect on the drug’s pharmacokinetics, pharmacodynamics, efficacy, and/or safety. While in many instances, the humoral response to protein therapeutics does not have any clinical significance, there are cases where there is a clinically significant effect and it is important to communicate this information to physicians and patients. This new guidance also delineates where immunogenicity information should be listed in product labels which should provide consistency in how this information is listed. There are many factors that contribute to a therapeutic’s immunogenicity and determining clinical significance is both complex and challenging, requiring that companies perform thorough analyses with scientific rigor. The analysis that is now proposed to understand clinical significance of ADAs is a new concept and will require companies to develop a strategy for compliance. This manuscript sets forth some of the key considerations in answering this important question. One of the benefits that this new guidance will provide is a common approach for describing the immune response to therapeutics that will be located in a dedicated section of the label, providing valuable consistency across protein therapeutics. Section 12.6 in the Clinical Pharmacology portion of the label will contain the relevant immunogenicity information, which will make it much simpler to find immunogenicity information in product labels. This new guidance is currently being utilized for new protein therapeutics and companies are being requested to systematically revise the labels of previously approved drugs for compliance, although an absolute timeline for this has not been established as of this writing.

Chimeric antigen receptor-engineered T cell therapies (CAR-T) are becoming powerful immunotherapeutic tools for treating malignancies, especially hematological malignancies. Like other biological drugs, CAR-T cell products can trigger unwanted immune responses in patients receiving the treatment. This might lead to treatment failure or life-threatening consequences. This immunogenicity could also affect the CAR-T cells’ cellular kinetics and clinical responses. In this review, we summarize the immunogenicity of biologics and their effects on PK/PD profiles, safety, and efficacy. We also introduce the mechanisms of immunogenicity induced by CAR-T cells and clinical evidence of immunogenicity of the currently FDA-approved CAR-T cell products. Particularly, we summarize the currently available immunogenicity data from each CAR-T cell product’s clinical trials, immunogenicity assays, sample types, and preclinical efficacy models, which were retrieved from the FDA and EMA websites. We also discuss a preclinical model that is promising for evaluating CAR-T cell immunogenicity.

A multicenter molecular biomarker survey was conducted in Multiple Sclerosis (MS) centers across Central-Eastern European countries, encompassing a population of 107 million. Our aim was to provide a “snapshot” for future studies investigating the use of molecular biomarkers in MS. A self-report questionnaire was distributed via email to MS centers in seven Central-Eastern European countries (Croatia, Czech Republic, Poland, Romania, Serbia, Slovakia, and Slovenia) and to four reference centers (two in Austria, one in Germany, and one in Denmark), focusing on cerebrospinal fluid (CSF) analysis and molecular biomarkers in MS. Responding centers routinely request CSF oligoclonal band (OCB) testing in suspected MS cases, although no consensus exists on the number of CSF-restricted bands required to define OCB positivity, either within or between countries. More than half of the surveyed centers in the Czech Republic, Slovakia, Slovenia, and the reference centers request kappa free light chain (κFLC) testing in patients with suspected MS. Neurofilament light chain (NfL) is frequently used as a molecular biomarker for MS in Romania, Slovakia, and the reference centers. In summary, besides the use of CSF-specific OCB there is no consensus among the surveyed countries regarding the use of molecular biomarkers in MS.

Anakinra is an effective, well-tolerated, long-term anti-inflammatory treatment for cryopyrin-associated periodic syndromes (CAPS), yet evidence shows that it can induce the development of anti-drug antibodies (ADA). This analysis aims to determine ADA occurrence in CAPS patients and elucidate their effects on anakinra dosing and drug efficacy. A post hoc analysis was performed on data from a long-term safety and efficacy study in patients with severe CAPS. Patients were initiated on an anakinra dose of 1.0–2.4 mg/kg once daily subcutaneously, which was increased (in 0.5–1.0 mg/kg increments) to 2.0–5.0 mg/kg/day according to clinical need (median 3.1 mg/kg/day). ADA, serum amyloid A (SAA), and C-reactive protein (CRP) levels were measured at various time points, and pharmacokinetic (PK) parameters at 1 and 3 months. Efficacy was evaluated using a diary symptom sum score (DSSS), and SAA and CRP levels were evaluated as proxies of efficacy. Safety was evaluated by an analysis of adverse events (AEs). Anakinra dose levels were unrelated to ADA status. A high proportion of patients with at least one post-baseline assessment developed ADA (83%), the majority (79%) within 3 months. However, anakinra treatment markedly improved symptoms and was effective regardless of the presence of ADA; the annual rates of AEs were comparable between ADA-negative and ADA-positive patients. While ADA are likely to occur in CAPS patients treated with anakinra, our evidence shows that chronic daily subcutaneous treatment with anakinra is safe and effective regardless of the development and presence of ADA.