PULSE SPR: A High Throughput Method to Evaluate the Domain Stability of Antibodies.

Downstream purification of therapeutic antibodies requires candidates to be stable under various stress conditions, such as low pH. Current approaches to assess the conformational or colloidal stability of biologics may require significant amounts of material, and the testing may occur over an extended period of time. Furthermore, typical methodologies for early stability testing often do not directly address low pH stability, but focus more on conditions suitable for long-term drug product storage. Here we report a high-throughput method that measures protonation-induced unfolding of ligand binding sites for stability evaluation by surface plasmon resonance or PULSE SPR. This method, which requires only several micrograms of sample, is highly sensitive to the structural integrity of ligand binding sites and correlates well with thermal and chemical conformational stability. Combined with ligands targeting different regions of antibodies, this method allows a comprehensive assessment of antibody domain stability. By applying PULSE SPR, we found that antibodies with different complementarity-determining regions (CDRs), frameworks, formats, and interchain disulfide bonds showed different stabilities under acidic conditions. Additionally, biologics that aggregated in solution also had poor low pH stability. Taken together, PULSE SPR is a high-throughput and low sample consumption method that can be adopted to evaluate antibody domain stability and aggregation at low pH, which are two important aspects of therapeutic biologics.

IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFß.

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFß. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFß signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFß at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFß-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.

Analysis of 5518 unique, productively rearranged human VH3-23*01 gene sequences reveals CDR-H3 length-dependent usage of the IGHD2 gene family.

Clear and accurate understanding of diversity in antibody complementarity-determining regions (CDRs) is critical for antibody discovery and engineering. Previous observations of antibody CDR-H3 diversity were based on analyzing available antibody sequences in the public databases. The results may not accurately reflect that of natural antibody repertoire due to erroneous species annotation and the presence of man-made CDR loop diversity in public antibody sequence databases. In this study, in a precisely controlled germline context, we explored the relationship between amino acid composition and CDR-H3 length using 5518 unique productively rearranged human VH3-23*01 gene sequences. CDR-H3 length-dependent usage of the Cys-Xn-Cys motif is reported here.