Finn C. Wiberg

Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog.

Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 Å structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the α/β-hydrolase domain and an eight-bladed β-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.

Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes

Neutrophil‐derived heparin‐binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7‐residue N‐terminally extended HBP form (pro‐HBP). Deletion of the pro‐peptide‐encoding cDNA sequence resulted in correctly processed HBP at the N‐terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 ± 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C‐terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose‐dependent manner the lipopolysaccharide (LPS)‐induced cytokine release from human monocytes.

Expression and Characterization of a 70-kDa Fragment of the Insulin Receptor That Binds Insulin MINIMIZING LIGAND BINDING DOMAIN OF THE INSULIN RECEPTOR

In order to characterize regions of the insulin receptor that are essential for ligand binding and possibly identify a smaller insulin-binding fragment of the receptor, we have used site-directed mutagenesis to construct a series of insulin receptor deletion mutants. From 112 to 246 amino acids were deleted from the α-subunit region comprising amino acids 469–729. The receptor constructs were expressed as soluble insulin receptor IgG fusion proteins in baby hamster kidney cells and were characterized in binding assays by immunoblotting and chemical cross-linking with radiolabeled insulin. The shortest receptor fragment identified was a free monomeric α-subunit deleted of amino acids 469–703 and 718–729 (exon 11); the mass of this receptor fragment was found by mass spectrometry to be 70 kDa. This small insulin receptor fragment bound insulin with an affinity (K d ) of 4.4 nm, which is similar to what was found for the full-length ectodomain of the insulin receptor (5.0 nm). Cross-linking experiments confirmed that the 70-kDa receptor fragment specifically bound insulin. In summary we have minimized the insulin binding domain of the insulin receptor by identifying a 70-kDa fragment of the ectodomain that retains insulin binding affinity making this an interesting candidate for detailed structural analysis.

Specificity of Insulin and Insulin-like Growth Factor I Receptors Investigated using Chimeric Mini-Receptors ROLE OF C-TERMINAL OF RECEPTOR α SUBUNIT

We have investigated the role of the C-terminal of the α-subunit in the insulin receptor family by characterizing chimeric mini-receptor constructs comprising the first three domains (468 amino acids) of insulin receptor (IR) or insulin-like growth factor I receptor (IGFIR) combined with C-terminal domain from either insulin receptor (IR) (residues 704–719), IGFIR, or insulin receptor-related receptor (IRRR). The constructs were stably expressed in baby hamster kidney cells and purified, and binding affinities were determined for insulin, IGFI, and a single chain insulin/IGFI hybrid. The C-terminal domain of IRRR was found to abolish binding in IR and IGFIR context, whereas other constructs bound ligands. The two constructs with first three domains of the IR demonstrated low specificity for ligands, all affinities ranging from 3.0 to 15 nm. In contrast, the constructs with the first three domains of the IGFIR had high specificity, the affinity of the novel minimized IGFIR for IGFI was 1.5 nm,whereas the affinity for insulin was more than 3000 nm. When swapping the C-terminal domains in either receptor context only minor changes were observed in affinities (<3-fold), demonstrating that the carboxyl-terminal of IR and IGFIR α-subunits are interchangeable and suggesting that this domain is part of the common binding site.

Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use.

The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well‐characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost‐efficient cell banking and single‐batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled‐up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic‐, protein‐, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition. Biotechnol. Bioeng. 2011;108:2171–2181.

Crystallization and molecular replacement solution of human heparin binding protein

The highly glycosylated protein, human heparin binding protein, has been crystallized in the primitive orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 39.0, b = 66.2 and c = 101.4 A. Ethanol was used as precipitant and glycerol as additive. A full data set has been collected to 3.1 A and diffraction was observed to at least 2.3 A. A molecular replacement solution using human neutrophile elastase as a search model was obtained, showing one molecule per asymmetric unit. The crystal packing showed no bad contacts and the R factor was 44.8% after ten cycles of rigid-body refinement.