Lymphomas arising from NK or gd-T cells are very aggressive diseases and little is known regarding their pathogenesis. Here we report frequent activating mutations of STAT3 and STAT5B in NK/T-cell lymphomas (n ¼ 51), gd-T-cell lymphomas (n ¼ 43) and their cell lines (n ¼ 9) through next generation and/or Sanger sequencing. STAT5B N642H is particularly frequent in all forms of gd-T-cell lymphomas. STAT3 and STAT5B mutations are associated with increased phosphorylated protein and a growth advantage to transduced cell lines or normal NK cells. Growth-promoting activity of the mutants can be partially inhibited by a JAK1/2 inhibitor. Molecular modelling and surface plasmon resonance measurements of the N642H mutant indicate a marked increase in binding affinity of the phosphotyrosine-Y699 with the mutant histidine. This is associated with the prolonged persistence of the mutant phosphoSTAT5B and marked increase of binding to target sites. Our findings suggest that JAK-STAT pathway inhibition may represent a therapeutic strategy.

Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximab (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Finally, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent "linker" for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.protein engineering | molecular recognition | cancer M onoclonal antibodies (mAbs) are indispensable tools in research laboratories and have become a central component in modern medicine. Thousands of antibodies are routinely used in research to detect and/or label specific proteins in a variety of settings. In recent years, dozens of mAbs that block signaling pathways, sequester growth factors, and/or induce an immune response have been successfully implemented in the clinic to treat cancer and other diseases, with hundreds still in active development (1, 2). Antibodies are being reengineered to best capitalize on their extraordinary ligand specificity to add new functionalities for a broad range of applications, mostly for clinical use, such as antibody-drug conjugates (ADCs), antibodydirected enzyme prodrug therapies (ADEPT), immune system engagement [e.g., Fc modifications (3), chemokine fusion, bispecific T-cell engagers (4)] and disease imaging (e.g., immunoPET and pretargeted radionuclide imaging; refs. 5 and 6).Invariably, these engineering efforts have been achieved either through posttranslational chemical modifications or manipulation of the gene sequence (7-9). Many of the chemical modifications lead to undesirable consequences that are detrimental to therapeutic and imaging applications (10). For example, to create ADCs to target cytotoxins to disease sites with the mAb, the chemical conjugation of the toxin (typically involves lysines, reduced cysteines, or sugars on the mAb) produ...

Magnetically separable hydrogenation catalysts serve as an example of a multifunctional M13 bacteriophage nanocomposite made by (a) growing Rh nanoparticles lengthwise along the pVIII region via nonspecific electrostatic interactions and (b) anchoring Fe3O4 nanoparticles on the pIII tip via materials-specific interactions identified through phage display methods

Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell lines, we detected distinct differences in receptor density and nano-organization upon treatment with therapeutic agents. This new platform can improve molecular quantification and can be developed to study the local protein environment of intact cells.