Generation and functional characterization of anti-human and anti-mouse IL-36R antagonist monoclonal antibodies.

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, ß and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.

Efficient Qualitative and Quantitative Determination of Antigen-induced Immune Responses.

To determine the effectiveness of immunization strategies used in therapeutic antibody or vaccine development, it is critical to assess the quality of immunization-induced polyclonal antibody responses. Here, we developed a workflow that uses sensitive methods to quantitatively and qualitatively assess immune responses against foreign antigens with regard to antibody binding affinity and epitope diversity. The application of such detailed assessments throughout an immunization campaign can significantly reduce the resources required to generate highly specific antibodies. Our workflow consists of the following two steps: 1) the use of surface plasmon resonance to quantify antigen-specific antibodies and evaluate their apparent binding affinities, and 2) the recovery of serum IgGs using an automated small scale purification system, followed by the determination of their epitope diversity using hydrogen deuterium exchange coupled with mass spectrometry. We showed that these methods were sensitive enough to detect antigen-specific IgGs in the nanogram/µl range and that they provided information for differentiating the antibody responses of the various immunized animals that could not be obtained by conventional methods. We also showed that this workflow can guide the selection of an animal that produces high affinity antibodies with a desired epitope coverage profile, resulting in the generation of potential therapeutic monoclonal antibody clones with desirable functional profiles. We postulate that this workflow will be an important tool in the development of effective vaccines to combat the highly sophisticated evasion mechanisms of pathogens.

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1.

The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77 °C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the CH2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the CH2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.