Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

IgRepertoireConstructor: a novel algorithm for antibody repertoire construction and immunoproteogenomics analysis.

UNLABELLED: The analysis of concentrations of circulating antibodies in serum (antibody repertoire) is a fundamental, yet poorly studied, problem in immunoinformatics. The two current approaches to the analysis of antibody repertoires [next generation sequencing (NGS) and mass spectrometry (MS)] present difficult computational challenges since antibodies are not directly encoded in the germline but are extensively diversified by somatic recombination and hypermutations. Therefore, the protein database required for the interpretation of spectra from circulating antibodies is custom for each individual. Although such a database can be constructed via NGS, the reads generated by NGS are error-prone and even a single nucleotide error precludes identification of a peptide by the standard proteomics tools. Here, we present the IgRepertoireConstructor algorithm that performs error-correction of immunosequencing reads and uses mass spectra to validate the constructed antibody repertoires. AVAILABILITY AND IMPLEMENTATION: IgRepertoireConstructor is open source and freely available as a C++ and Python program running on all Unix-compatible platforms. The source code is available from http://bioinf.spbau.ru/igtools. CONTACT: ppevzner@ucsd.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The cryptophycins as potent payloads for antibody drug conjugates.

The cryptophycins are a potent class of cytotoxic agents that were evaluated as antibody drug conjugate (ADC) payloads. Free cryptophycin analog 1 displayed cell activity an order of magnitude more potent than approved ADC payloads MMAE and DM1. This potency increase was also reflected in the activity of the cryptophycin ADCs, attached via a either cleavable or non-cleavable linker.

A secreted protein microarray platform for extracellular protein interaction discovery.

Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor-ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell-cell or cell-extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.

Structure and function of a new class of human prolactin antagonists.

Delta 41-52 hPRL (human prolactin with residues 41-52 removed) is a lead compound for a new class of hPRL antagonists. The deleted sequence contains residues that functionally couple sites 1 and 2, the two hormone surfaces that each bind receptors. Delta 41-52 hPRL retains 0.03% agonist activity in FDC-1 cell bioassays, a 3054-fold reduction in activity, and displays approximately 100-fold less agonist activity than G129R hPRL, an antagonist that reduces the binding of hPRL receptor at site 2 during the formation of the heterotrimeric hormone/receptor complex. Replacement of various numbers and types of residues into the gap created by the deletion of residues 41 through 52 created hPRLs with varying agonist activities, suggested that manipulation of the sequence connecting the C-terminal of helix 1 with the disulfide bond (cysteines 58 with 174) linking helices 1 and 4 modulates articulation of these helices and influences agonist activity. We have compared the antagonist activities of G129R and Delta 41-52 hPRLs to induce apoptosis in Jurkat cells, a human lymphoid cell line displaying an autocrine/paracrine hPRL/receptor system. Delta 41-52 hPRL induces apoptosis in a time and dose-dependent fashion. Under these same conditions G129R hPRL fails to induce apoptosis. We conclude Delta 41-52 hPRL is a lead compound of a new class of hPRL antagonists capable at low concentrations of inducing apoptosis in human cells expressing an autocrine/paracrine hPRL/receptor system.