Immunogenicity assessment is an important issue for ensuring the safety and efficacy of therapeutic protein products. Although the reliability of the anti-drug antibody (ADA) assay is one of the key points, there are some difficulties in assessing its validity because the analytes are polyclonal antibodies with variable and unknown characteristics. To elucidate the points to consider for the ADA assay, a Japanese research group was established that discusses the issues raised on the immunogenicity assessment. In this review, we first introduce the current situation regarding the development and immunogenicity assessment of therapeutic protein products in Japan. We then present our current view and recommendations on the ADA assay by considering its unique features. Therapeutic proteins such as monoclonal antibodies are currently essential in the treatment of cancer, autoimmune disease and other diseases. Many therapeutic protein products intended to address unmet medical needs are being developed worldwide, including in Japan, and therefore, ensuring their safety and efficacy is of paramount importance. Since protein has its intrinsic feature of immunogenicity owing to its structure containing potential B-cell and T-cell epitopes, therapeutic proteins have the potential to induce ADA even if the protein has the same amino acid sequence as endogenous human proteins. Proteins can be recognized as antigens by B cells and are also incorporated and digested by antigen-presenting cells. This process leads to the production of T-cell epitope peptides that are presented on major histocompatibility (MHC) II molecules. Various patient-and product-specific factors are suggested to affect the process of ADA induction [1]. The emergence of ADA in patients can potentially lead to loss of efficacy and/or adverse events. Therefore, immunogenicity risk assessment and risk-mitigating strategies are required during the development of therapeutic protein products [2].The appropriateness of the ADA assay is a key issue in immunogenicity assessment. The ligand-binding assay (LBA) is often used for detecting ADA in biological samples. Unlike the assay used for drug concentration analysis, the ADA assay has unique features, for example: the actual characteristics of the analyte (human ADA) are variable and unknown; there is no real reference standard: the experimentally prepared positive control for ADA is used as a surrogate reference standard to evaluate and control the assay performance; and the evaluated validation parameters such as sensitivity and drug tolerance limit (DTL) vary depending on the characteristics, mainly the affinity of the used positive control. In addition, matrix components that interfere with the assay in the study samples may vary depending on the disease and/or the individual patients. Therefore, it is practically impossible to completely assess the validity of the ADA assay, and the assay is associated with certain risks of obtaining an inappropriate result. As mentioned in the European Medicines Agency (EMA...
Target cells for 3H-labeled 1 alpha, 25(OH)2 vitamin D3 [1,25(OH)2D3, vitamin D] and its analog 3H-labeled 22-oxa-1 alpha, 25(OH)2 vitamin D3 (OCT) have been identified during endochondral and intramembranous ossification in developing, undecalcified, unembedded bone, using thaw-mount autoradiography. Two-day-old neonatal rats were injected with [3H]1,25(OH)2D3 or [3H]OCT; after 2 h leg, spine, and head were frozen and sectioned. In the epiphyseal-metaphyseal region specific nuclear concentrations of [3H]1,25(OH)2D3 and [3H]OCT were observed in identical cell populations, being low in cells of the articular and resting zone, intermediate in the proliferating zone, and highest in hypertrophic chondrocytes and in osteoblasts and precursor cells. In the primary spongiosa intertrabecular spaces there were a large number of cells with nuclear labeling--probably osteoblasts and precursor cells. In contrast, in the secondary spongiosa intertrabecular spaces, apparent blood-forming cells were mostly unlabeled. Osteoblasts along bone spicules and compact bone in long bones, vertebrae, and head also showed strong nuclear labeling, as did cells of the periosteum. These data suggest that 1,25(OH)2D3 and OCT regulate development, differentiation, and activities of chondrocytes and osteoblasts, including differentiation of resting chondrocytes into proliferating and hypertrophic chondrocytes that involve "chondroclastic" enlargement of lacunae and "trans-differentiation" of surviving hypertrophic chondrocytes; differentiation of stroma cells into osteoblasts; and in periosteum and other regions of intramembranous ossification differentiation of precursor cells and osteoblasts. Nuclear receptor binding and their selective and hierarchical distribution during cell differentiation appear to correspond to multiple genomic effects toward growth, regeneration and repair. The findings indicate a physiological significance and therapeutic potential of 1,25(OH)2D3 and in particular of its less hypercalcemic analog OCT.
Because of the therapeutic potential of oxacalcitriol (OCT, 22-oxa-dihydroxyvitamin D3), in vivo studies were conducted in adult and neonatal rats to identify the nuclear receptor sites of action in different tissues of the skin. Results were compared with those for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and oestradiol from previous studies. Autoradiograms were prepared from the dorsal skin of adult rats and the skin of the leg and head regions of neonatal rats 1 or 2 h after the injection of 3H-OCT. Specific nuclear concentrations of radioactivity, eliminated by competition with unlabelled OCT or 1,25(OH)2D3, were found in cells of the epidermis, outer hair sheath, hair bulb and sebaceous glands, but were absent or low in most fibroblasts of the dermis and hypodermis. The strongest nuclear binding of OCT was conspicuous in outer hair sheaths, where it was 1.5 to 3.2 times higher than in keratinocytes of the epidermis. The distribution of nuclear receptors for OCT was similar to that for 1,25(OH)2D3 but in part dissimilar to that for oestradiol. Oestradiol binding was found in the epidermis and hair sheaths, and also predominantly in fibroblasts of the dermis and hair dermal papillae. The results suggest genomic regulatory effects of OCT, similar to the effects of vitamin D, on proliferation, differentiation and activity of keratinocytes, growth and maintenance of hair, and proliferation and secretion of sebaceous glands. This may be utilized therapeutically, since OCT has a lower calcaemic effect than 1,25(OH)2D3.
1,25-Dihydroxy-22-oxavitamin D(3) (22-oxacalcitriol, OCT), is a new synthetic analogue of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3), calcitriol), to be used in the treatment of secondary hyperparathyroidism. This study used receptor micro-autoradiography in the parathyroid gland to determine and compare the time-course of receptor binding between OCT and 1,25(OH)(2)D(3). Mice were injected with 4 microg/kg of [26-(3)H]OCT or [26,27-methyl-(3)H]1,25(OH)(2)D(3), and killed at 5, 15, 30 min, 1, 2, 4, 8, 12, and 24 h afterwards. Thyroid-parathyroid tissue was excised and autoradiograms were prepared. Under identical conditions of dose and adjusted specific radioactivity between [(3)H]OCT and [(3)H]1,25(OH)(2)D(3), the plasma concentration of [(3)H]OCT was much lower than that of [(3)H]1,25(OH)(2)D(3). In the parathyroid at all time points, chief cell nuclei were labelled with varying degrees while connective tissue cells remained unlabelled. Nuclear receptor binding of [(3)H]OCT appeared equal to or higher than that of [(3)H]1,25(OH)(2)D(3). Nuclear uptake of [(3)H]OCT was maximal at 15 min and higher than that of [(3)H]1,25(OH)(2)D(3), which was maximal at 1 h after injection. Low levels of nuclear retention of the two compounds were still similarly detectable at 12 h. The results indicate the high affinity of OCT to parathyroid cells, and suggest that OCT has a higher therapeutic potential than 1,25(OH)(2)D(3), especially under clinical conditions, at which OCT with its lower calcaemic effect would allow treatment with a dose several times higher than 1,25(OH)(2)D(3).
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