Direct observations of the effect of fine sediment deposition on the vertical movement of Gammarus pulex (Amphipoda: Gammaridae) during substratum drying

Benthic macroinvertebrates inhabit the streambed sediments of temporary streams during drying events. Fine sediment (< 2 mm in diameter) deposition and clogging of interstitial pathways reduces the connectivity between benthic and subsurface habitats, potentially inhibiting macroinvertebrate vertical movements. Direct observations within subsurface sediments are, however, inherently difficult. As a result, confirmation of macroinvertebrate vertical movement, and the effect of fine sediment, is limited. We used laboratory mesocosms containing transparent gravel sized particles (10–15 mm) to facilitate the direct observation and tracking of vertical movements by Gammarus pulex in response to water level reduction and sedimentation. Seven sediment treatments comprised two fine sediment fractions (small: 0.125–0.5 mm, coarse sand: 0.5–1 mm) deposited onto the surface of the substrate, and a control treatment where no fine sediment was applied. We found that G. pulex moved into the subsurface gravel sediments in response to drying, but their ability to remain submerged during water level reduction was impeded by fine sediment deposition. In particular deposition of the coarser sand fraction clogged the sediment surface, limiting vertical movements. Our results highlight the potential effect of sedimentation on G. pulex resistance to drying events in streams

In Vivo Binding and Retention of CD4-Specific DARPin 57.2 in Macaques

The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost.To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4(+) cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4(+) cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4(+) cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4(+) cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation.We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology

Crossing borders to bind proteins—a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation

CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins—high physical stability, specificity and low production costs—with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development

Abstract P4-12-30: A bivalent Her2 targeting DARPin with high efficacy against Her2-low and Her2-positive tumors

Abstract The approval of Herceptin, Perjeta and Kadcyla has dramatically improved the outcome of the 15% of breast cancer patients with high expression of Her2 (Her2-positive, IHC3+). In contrast, up to 50% of women with breast cancer express low levels of Her2 (Her2-low, IHC+2 or +1) at diagnosis, and these patients do not derive benefit from approved Her2 targeted therapies. It is estimated that 5-10% of Her2-positive patients will also develop Her2-low metastases refractory to treatment. Therefore, new therapeutic strategies are needed to alleviate the high tumor burden of Her2-low patients. We generated a bivalent Designed Ankyrin Repeat Protein (DARPin) containing two anti-Her2 moieties that target unique non-overlapping epitopes on Her2 (biparatopic). The biparatopic DARPin exerts a strong pro-apoptotic response on a panel of breast and gastric cancer cell lines, which cannot be fully recapitulated by treatment with Herceptin, Perjeta or both combined. DARPin treatment of Her2-positive breast cancer BT474 cells induces apoptosis (IC50 &amp;lt; 1nM) in virtually all cells within 24 hours, whereas single or combined treatment with Herceptin and/or Perjeta reduces proliferation by 50% (IC50 = 2-4nM). The biparatopic DARPin inhibits as well the viability (via apoptosis) of Her2-low MDAMB175 cells with a 100-fold higher potency than Herceptin. Mechanistically, treatment with the anti-Her2 biparatopic DARPin inhibits both Her2 and Her3 signaling, which in turn results in the induction of apoptosis. Interestingly, cell treatment with the mix of the two individual anti-Her2 DARPin moieties does not recapitulate the apoptotic effect of the biparatopic molecule, indicating that both moieties must be linked for maximum efficacy. In vivo, the biparatopic DARPin shows efficient anti-tumor activity in established breast cancer Her2-positive xenografts and Her2-low PDX tumor models. DARPin treatment of Her2-positive BT474 xenograft in BALB/c mice results in rapid tumor regression (50% tumor shrinkage after one dose) that is sustained at least during one month after the last dose. The biparatopic DARPin strongly inhibits tumor growth as well in a Her2-low breast cancer PDX in NMRI mice, where it demonstrates superior efficacy compared to Herceptin (DARPin/Herceptin tumor volume ratio &amp;lt; 30%). In summary, biparatopic targeting of Her2 enables inhibition of both Her2 and Her3, and blocks a tumor driver and its resistance mechanism. As a result, the DARPin interferes with both the proliferation and survival of tumor cells and triggers durable anti-tumor responses in vivo. DARPin treatment of Her2 positive tumors in mice show equivalent efficacy to Herceptin with faster kinetics; an effect that is magnified in Her2-low PDX tumors where the DARPin provides superior tumor control to Herceptin. Altogether, our data demonstrate the potential for the biparatopic DARPin to surpass some of the limitations of approved Her2 targeted agents, and warrants clinical investigation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-30.</jats:p