Assembly of Protein Building Blocks Using a Short Synthetic Peptide

Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces

Apoptosis assays with lymphoma cell lines: problems and pitfalls

Much attention has been focused on the manner in which tumour cells die after treatment with cytotoxic agents. The basic question is whether cells die via apoptosis or via direct damage from the toxic agent. Various assays have been used to make this distinction. However, we show herein that some of the widely used assays for apoptosis do not in fact distinguish between apoptosis and other forms of cell death. More specifically: (1) A sub-G1 DNA content, identified by propidium iodide staining, does not distinguish between apoptotic and necrotic cells; (2) loss of mitochondrial membrane potential does not distinguish between apoptotic and necrotic cells, unless combined with an assay for an intact cell membrane; (3) subcellular fragments that arise from dead cells or from apoptotic bodies can interfere with some assays for apoptosis such as annexin V staining, as they may be close to the size of intact cells, making it difficult to decide where to set the size threshold; (4) irradiated cells display a large increase in nonspecific Ab binding. This may be partly due to an increase in cell size, but, regardless of the cause, it can lead to a mistaken conclusion that there is an increase in a particular antigen if appropriate control reagents are not tested; and (5) experiments utilising Ab crosslinking have neglected the role of cell aggregation, which can cause multiple problems including death from mechanical stress when cells are handled. Consideration of these factors will improve our ability to determine the mode of cell death

Latency reversal plus natural killer cells diminish HIV reservoir in vivo.

HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir

Efficient elimination of chronic lymphocytic leukaemia B cells by autologous T cells with a bispecific anti-CD19/anti-CD3 single-chain antibody construct

Recently, we have shown that a novel recombinant bispecific single-chain antibody construct (bscCD19 x CD3), induces highly efficacious lymphoma-directed cytotoxicity mediated by unstimulated peripheral T lymphocytes. Functional analysis of bscCD19 x CD3 has so far been exclusively performed with human B lymphoma cell lines and T cells from healthy donors. Here we analysed the properties of bscCD19 x CD3 using primary B cells and autologous T cells from healthy volunteers or patients with B-cell chronic lymphocytic leukaemia (B-CLL). We show that bscCD19 x CD3 induces T-cell-mediated depletion of nonmalignant B cells in all four cases and depletion of primary lymphoma cells in 22 out of 25 cases. This effect could be observed at low effector-to-target (E:T) ratios and in the majority of cases without additional activation of autologous T cells by IL-2. Even in samples derived from patients heavily pretreated with different chemotherapy regimens, strong cytotoxic effects of bscCD19 x CD3 could be observed. The addition of bscCD19 x CD3 to patients' cells resulted in an upregulation of activation-specific cell surface antigens on autologous T cells and elevated levels of CD95 on lymphoma B cells. Although anti-CD95 antibody CH-11 failed to induce apoptosis in lymphoma cells, we provide evidence that B-CLL cell depletion by bscCD x CD3 is mediated at least in part by apoptosis via the caspase pathway

Mesoscale computational protocols for the design of highly cooperative bivalent macromolecules

The last decade has witnessed a swiftly increasing interest in the design and production of novel multivalent molecules as powerful alternatives for conventional antibodies in the fight against cancer and infectious diseases. However, while it is widely accepted that large-scale flexibility ($10-100$ nm) and free/constrained dynamics (100 ns $- \mu$s) control the activity of such novel molecules, computational strategies at the mesoscale still lag behind experiments in optimizing the design of crucial features, such as the binding cooperativity (a.k.a. avidity). In this study, we introduced different coarse-grained models of a polymer-linked, two-nanobody composite molecule, with the aim of laying down the physical bases of a thorough computational drug design protocol at the mesoscale. We show that the calculation of suitable potentials of mean force allows one to apprehend the nature, range and strength of the thermodynamic forces that govern the motion of free and wall-tethered molecules. Furthermore, we develop a simple computational strategy to quantify the encounter/dissociation dynamics between the free end of a wall-tethered molecule and the surface, at the roots of binding cooperativity. This procedure allows one to pinpoint the role of internal flexibility and weak non-specific interactions on the kinetic constants of the NB-wall encounter and dissociation. Finally, we quantify the role and weight of rare events, which are expected to play a major role in real-life situations, such as in the immune synapse, where the binding kinetics is likely dominated by fluctuations.Comment: Paper + supplementary Informatio