Peter Bringmann

Site-specific modification of ED-B-targeting antibody using intein-fusion technology

BackgroundA promising new approach in cancer therapy is the use of tumor specific antibodies coupled to cytotoxic agents. Currently these immunoconjugates are prepared by rather unspecific coupling chemistries, resulting in heterogeneous products. As the drug load is a key parameter for the antitumor activity, site-specific strategies are desired. Expressed protein ligation (EPL) and protein trans-splicing (PTS) are methods for the specific C-terminal modification of a target protein. Both include the expression as an intein fusion protein, followed by the exchange of the intein for a functionalized moiety.ResultsA full-length IgG specific for fibronectin ED-B was expressed as fusion protein with an intein (Mxe GyrA or Npu DnaE) attached to each heavy chain. In vitro protocols were established to site-specifically modify the antibodies in high yields by EPL or PTS, respectively. Although reducing conditions had to be employed during the process, the integrity or affinity of the antibody was not affected. The protocols were used to prepare immunoconjugates containing two biotin molecules per antibody, attached to the C-termini of the heavy chains.ConclusionFull-length antibodies can be efficiently and site-specifically modified at the C-termini of their heavy chains by intein-fusion technologies. The described protocols can be used to prepare immunoconjugates of high homogeneity and with a defined drug load of two. The attachment to the C-termini is expected to retain the affinity and effector functions of the antibodies.

Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.

Significance This paper reports the first structure of WT-human glucose transporter 1 (hGLUT1), to our knowledge, cocrystallized with inhibitors. The structures provide a template to develop therapeutic inhibitors applicable to cancers, because cancer cells become dependent on greatly increased glucose consumption. This dependence results in up-regulation of glucose transporter expression, especially hGLUT1. The bound inhibitors include the natural compound cytochalasin B and two of a series of previously undescribed organic compounds that bind in the submicromolar range. Our results emphasize that modulation of glucose import by hGLUTs should focus on making good interaction points for compounds and that the actual chemical backbone of the inhibitor is of less importance. Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1–4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

Blockade of placental growth factor reduces vaso-occlusive complications in murine models of sickle cell disease

Vaso-occlusive crisis (VOC) is the most common and debilitating complication of sickle cell disease (SCD); recurrent episodes cause organ damage and contribute to early mortality. Plasma placental growth factor (PlGF) levels are elevated in SCD and can further increase under hypoxic conditions in SCD mice. Treatment with a PlGF-neutralizing antibody (anti-PlGF Ab) in SCD mice reduced levels of monocyte chemoattractant protein-3, eotaxin, macrophage colony-stimulating factor, and plasminogen activator inhibitor-1 significantly, and of macrophage-derived chemokine and macrophage inflammatory protein-3β moderately; this may contribute to inhibition of leukocyte recruitment, activation, and thrombosis. In subsequent experiments, anti-PlGF Ab treatment significantly reduced plasma lactate dehydrogenase levels, indicating possible reduction in cellular destruction and/or hemolysis. Histopathology studies revealed decreased incidence and severity of congestion in the lungs and spleen with repeated anti-PlGF Ab treatment. Furthermore, anti-PlGF Ab significantly reduced vaso-occlusion events under hypoxic conditions in a modified dorsal skinfold chamber model in SCD mice. Therefore, elevated PlGF levels may contribute to recruitment and activation of leukocytes. This can subsequently lead to increased pathology of affected organs in addition to mediating acute hypoxia/reoxygenation-triggered vaso-occlusion under SCD conditions. Thus, targeting PlGF may offer a therapeutic approach to reduce acute VOC and possibly alleviate long-term vascular complications in patients with SCD.