An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.

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Neurons regulate their excitability by adjusting their ion channel levels. Degeneracy - achieving equivalent outcomes (excitability) using different solutions (channel combinations) - facilitates this regulation by enabling a disruptive change in one channel to be offset by compensatory changes in other channels. But neurons must co-regulate many properties. Pleiotropy - the impact of one channel on more than one property - complicates regulation because a compensatory ion channel change that restores one property to its target value often disrupts other properties. How then does a neuron simultaneously regulate multiple properties? Here we demonstrate that of the many channel combinations producing the target value for one property (the single-output solution set), few combinations produce the target value for other properties. Combinations producing the target value for two or more properties (the multi-output solution set) correspond to the intersection between single-output solution sets. Properties can be effectively co-regulated only if the number of adjustable channels (nin) exceeds the number of regulated properties (nout). Ion channel correlations emerge during homeostatic regulation when the dimensionality of solution space (nin - nout) is low. Even if each property can be regulated to its target value when considered in isolation, regulation as a whole fails if single-output solution sets do not intersect. Our results also highlight that ion channels must be co-adjusted with different ratios to regulate different properties, which suggests that each error signal drives modulatory changes independently, despite those changes ultimately affecting the same ion channels.

We have purified the heme-regulated eukaryotic initiation factor 2a subunit (eIF-2ar) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2a phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)- Protein synthesis in intact reticulocytes and their lysates is dependent on the availability of heme (1-4). In heme deficiency, protein synthesis is inhibited at the level of initiation due to the activation ofa heme-regulated inhibitor (HRI), also called the heme-controlled repressor (refs. 3, 5, and 6; for review see refs. 7 and 8). HRI is a cAMP-independent protein kinase that specifically phosphorylates the a subunit (eIF-2a) of the eukaryotic initiation factor 2 (eIF-2) (9-12). Phosphorylation of eIF-2a in reticulocyte lysates results in the binding and sequestration of reversing factor RF, also designated as guanine nucleotide exchange factor or eIF-2B, in a RF-eIF-2(aP) complex; the unavailability ofRF, which is required for the exchange of GTP for GDP in the recycling of eIF-2 and in the formation of the eIF-2-Met-tRNAf'GTP ternary complex, results in the cessation of the initiation of protein synthesis (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).HRI is present in heme-supplemented reticulocyte lysates in an inactive form (proinhibitor) (22). It is activated in lysates by heme deficiency (5,6,22) and by treatment with the sulfhydryl reagent N-ethylmaleimide (22-25), oxidized glutathione (23-25), high partial pressure of 02 (26), heavy metal ions (27), or heat shock (28,29). Activation of HRI is accompanied by its phosphorylation (30-34). The hemereversible form of HRI has been purified (30), and the binding of hemin directly to purified heme-reversible HRI has been demonstrated (33). We have observed that binding of hemin to HRI promotes intersubunit disulfide bond formation (34). The inhibition of autophosphorylation of HRI by hemin is very similar to that produced by thiol oxidation by diamide. A similar dimer is observed when HRI is treated with 1,6-bis(maleimido)hexane, a double sulfhydryl cross-linking agent. The cross-linked HRI dimer has little autokinase or eIF-2a kinase activity; these effects of 1,6-bis(maleimido)hexane are similar to the effects of hemin on HRI. These studies suggest that intersubunit disulfide formation by hemin is a likely mechanism by which hemin prevents the activation and inhibits the activity of HRI (34).To elu...

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