Reena Varkey

A Novel Anti-PcrV Antibody Providing Enhanced Protection against Pseudomonas aeruginosa in Multiple Animal Infection Models

ABSTRACT Pseudomonas aeruginosa is a major cause of hospital-acquired infections, particularly in mechanically ventilated patients, and it is the leading cause of death in cystic fibrosis patients. A key virulence factor associated with disease severity is the P. aeruginosa type III secretion system (T3SS), which injects bacterial toxins directly into the cytoplasm of host cells. The PcrV protein, located at the tip of the T3SS injectisome complex, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In an effort to identify a highly potent and protective monoclonal antibody (MAb) that inhibits the T3SS, we generated and characterized a panel of novel anti-PcrV MAbs. Interestingly, some MAbs exhibiting potent inhibition of T3SS in vitro failed to provide protection in a mouse model of P. aeruginosa infection, suggesting that effective in vivo inhibition of T3SS with anti-PcrV MAbs is epitope dependent. V2L2MD, while not the most potent MAb as assessed by in vitro cytotoxicity inhibition assays, provided strong prophylactic protection in several murine infection models and a postinfection therapeutic model. V2L2MD mediated significantly (P < 0.0001) better in vivo protection than that provided by a comparator antibody, MAb166, a well-characterized anti-PcrV MAb and the progenitor of a clinical candidate, KB001-A. The results described here support further development of a V2L2MD-containing immunotherapeutic and may suggest even greater potential than was previously recognized for the prevention and treatment of P. aeruginosa infections in high-risk populations.

Development of a new ARCHITECT automated periostin immunoassay

BACKGROUND Periostin is being investigated as a potential biomarker for T-helper-2 (Th2)-driven asthma or eosinophilic inflammation and may help to identify patients more likely to benefit from interleukin-13-targeted treatments. We report the development and analytic performance of the investigational use only ARCHITECT Periostin Immunoassay, a new automated assay developed to detect serum periostin concentrations. METHODS We assessed assay performance in terms of precision, sensitivity, linearity, interference from classical immunoassay interferents and representatives of common asthma medications, specimen handling, and isoform reactivity. The assay was also used to assess the biological variability of serum periostin concentrations in samples from healthy volunteers and from subjects with uncontrolled asthma (the intended use population). RESULTS The percentage CVs for 5-day total precision, assessed using two instruments, was <6% across 2 controls and one serum-based panel. Limit of quantitation was 4ng/mL (dilution adjusted concentration), suiting the needs for this application. Dilution analysis yielded linear results and no endogenous sample or drug interferences were observed. All known periostin isoforms expressed in the mature human lung were detected by the assay. CONCLUSION Our studies provide support that the ARCHITECT Periostin Immunoassay is a reliable and robust test for measuring serum periostin concentrations.

A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

ABSTRACT Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool.

Preclinical assessment of an antibody–PBD conjugate that targets BCMA on multiple myeloma and myeloma progenitor cells

MP, Ries RE, et al. Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia. Oncotarget. 2016;7:43281–94. 12. Humbert O, Peterson CW, Norgaard ZK, Radtke S, Kiem HP. A nonhuman primate transplantation model to evaluate hematopoietic stem cell gene editing strategies for betahemoglobinopathies. Mol Ther Methods Clin Dev. 2018;8:75–86. 13. Correnti CE, Laszlo GS, de van der Schueren WJ, Godwin CD, Bandaranayake A, Busch MA, et al. Simultaneous multiple interaction T-cell engaging (SMITE) bispecific antibodies overcome bispecific T-cell engager (BiTE) resistance via CD28 costimulation. Leukemia. 2018;32:1239–43. 14. Haworth KG, Ironside C, Norgaard ZK, Obenza WM, Adair JE, Kiem HP. In vivo murine-matured human CD3(+ ) cells as a preclinical model for T cell-based immunotherapies. Mol Ther Methods Clin Dev. 2017;6:17–30. 15. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5.