Molecular engineering strategies and methods for the expression and purification of IgG1-based bispecific bivalent antibodies.

In recent years, bispecific antibodies (BisAbs) have emerged as novel pharmaceutical candidates owing to their ability to engage two disease mediators simultaneously, thus providing a possible alternative therapeutic approach in complex diseases such as cancer and inflammation. Here we provide an overview of the molecular design, recombinant expression in mammalian cells and purification of BisAbs based on full-length IgG-scFv formats. Practical considerations and strategies to optimize transient expression and purification are also discussed.

Human natural killer cell receptors: insights into their molecular function and structure.

NK cells express receptors characterized by opposite functions that finely regulate their activities. Among inhibitory receptors, some are specific for different groups of MHC class I alleles, while others are still orphan receptors. On the contrary, various activating receptors are involved in the triggering of NK-mediated natural cytotoxicity. In general, their engagement induces human NK cells to kill target cells that are either HLA class I-negative or -deficient. Thus, the process of NK cell triggering mediated by Natural Cytotoxicity Receptors can be mainly considered as a non MHC-restricted mechanism. Here, a brief description of the molecular nature of these receptors, as well as, of their 3D-structures and of the implications for ligand recognition, is given.

Structural basis of MHC class I recognition by natural killer cell receptors.

Natural killer (NK)-cell function is regulated by NK receptors that recognize MHC class I (MHC-I) molecules on target cells. Two structurally distinct families of NK receptors have been identified, the immunoglobulin-like family (killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LIRs)) and the C-type lectin-like family (Ly49, CD94/NKG2A, NKG2D, CD69). Recently, the three-dimensional structures of several NK receptors were determined, in free form or bound to MHC-I. These include those of unbound KIRs, NKG2D, CD69, LIR-1 and the CD94 subunit of the CD94/NKG2A heterodimer. Together, these structures define the basic molecular architecture of both the immunoglobulin-like and C-type lectin-like families of NK receptors. In addition, crystal structures have been reported for the complex between Ly49A and H-2Dd, and for KIR2DL2 bound to HLA-Cw3. The complex structures provide a framework for understanding MHC-I recognition by NK receptors from both families and reveal striking differences in the nature of this recognition, despite the receptors' functional similarity.

Crystal structure of a superantigen bound to the high-affinity, zinc-dependent site on MHC class II.

MHC class II molecules possess two binding sites for bacterial superantigens (SAGs): a low-affinity site on the alpha chain and a high-affinity, zinc-dependent site on the beta chain. Only the former has been defined crystallographically. We report the structure of streptococcal pyrogenic exotoxin C (SPE-C) complexed with HLA-DR2a (DRA*0101, DRB5*0101) bearing a self-peptide from myelin basic protein (MBP). SPE-C binds the beta chain through a zinc bridge that links the SAG and class II molecules. Surprisingly, SPE-C also makes extensive contacts with the MBP peptide, such that peptide accounts for one third of the surface area of the MHC molecule buried in the complex, similar to TCR-peptide/MHC complexes. Thus, SPE-C may optimize T cell responses by mimicking the peptide dependence of conventional antigen presentation and recognition.

Design of selective eglin inhibitors of HCV NS3 proteinase.

Hepatitis C virus (HCV) infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals, estimated to be 100-200 million worldwide. Unfortunately, immunotherapy or other effective treatments for HCV infection are not yet available, and interferon administration has limited efficacy. Different approaches to HCV therapy are being explored, and these include inhibition of the viral proteinase, helicase, and RNA-dependent RNA polymerase and development of a vaccine. Here we present the design of selective inhibitors with nanomolar potencies of HCV NS3 proteinase based on eglin c. These eglin c mutants were generated by reshaping the inhibitor active site-binding loop, and the results emphasize the role played by residues P5-P4' in enzyme recognition. In addition, alanine scanning experiments provide evidence that the N terminus of eglin c also contributes to NS3 binding. These eglin inhibitors offer a unique tool for accurately assessing the requirements for effective inhibition of the enzymatic activity of NS3 and at the same time can be considered lead compounds for the identification of other NS3 inhibitors in targeted design efforts.

Postoperative drains at the donor sites of iliac-crest bone grafts. A prospective, randomized study of morbidity at the donor site in patients who had a traumatic injury of the spine.

A prospective, randomized study was performed to assess the effectiveness of postoperative closed suction drainage. One hundred and twelve consecutive procedures involving autologous iliac-crest bone graft were performed, from December 29, 1992, to July 1, 1993, following a traumatic injury of the spine in 108 patients. Sixty of the sites from which the bone graft had been obtained were drained with a single large Hemovac device. The drains were maintained for two to five days postoperatively. The remaining fifty-two incisions were closed without a drainage device. All patients were evaluated clinically for problems with wound-healing. The incisions were considered to be healed when they had been asymptomatic for one year. Of eleven patients who had problems with wound-healing, six had been managed with a drain and five had not. The findings of this study do not support the routine use of drainage at the donor sites of iliac-crest bone grafts.

Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein.

The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases. The HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent complex, which participates in processing the viral polyprotein. This proteolytic activity is believed to be essential for virus proliferation and thus the NS3 protease is a prime target for developing anti-HCV pharmacological agents. Recent X-ray crystallography structural studies have revealed the nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chain polypeptide. To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of making a beta-turn, to the N-terminus of the NS3 protease domain. This engineered single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/ NS4A non-covalent complex. Moreover, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor. The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineering for further structural studies and in vitro selection of potential drug-resistant mutants. These are important steps towards developing effective anti-protease compounds.

Rational design and functional expression of a constitutively active single-chain NS4A-NS3 proteinase.

BACKGROUND: The proteinase domain of the hepatitis C virus NS3 protein is involved in the maturation of the viral polyprotein. A central hydrophobic domain of the NS4A protein is required as a cofactor for its proteolytic activity. The three-dimensional structure of the proteinase domain alone and complexed with an NS4A-derived peptide has been solved recently and revealed that the N terminus of the proteinase is in near proximity to the C terminus of the cofactor. To study the molecular basis of the enzyme activation by its cofactor and to overcome the difficulties of structural and functional investigation associated with a two-species complex, we rationally designed a link to bridge the two molecules in order to have a single polypeptide construct. RESULTS: The engineered construct led to the production of a stable, monomeric protein with proteolytic activity that is independent from the addition of a synthetic peptide representing the cofactor domain of the NS4A protein. The protein is active on both protein and synthetic peptide substrates. Spectroscopic and kinetic analysis of the recombinant NS4A-NS3 single-chain proteinase demonstrated features superimposable with the isolated NS3 proteinase domain complexed with the NS4A cofactor. CONCLUSIONS: We designed a very tight connection between the NS3 and NS4A polypeptide chains with the rationale that this would allow a more stable structure to be formed. The engineered single-chain enzyme was indistinguishable from the NS3 proteinase complexed with its NS4A cofactor in all enzymatic and physico-chemical properties investigated.

Characterization of engineered hepatitis C virus NS3 protease inhibitors affinity selected from human pancreatic secretory trypsin inhibitor and minibody repertoires.

Given the extent of hepatitis C virus (HCV) infection as a worldwide health problem and the lack of effective treatment, the development of anti-HCV drugs is an important and pressing objective. Previous studies have indicated that proteolytic events mediated by the NS3 protease of HCV are fundamental to the generation of an active viral replication apparatus, as unequivocably demonstrated for flaviviruses. As a result, the NS3 protease has become a major target for discovering anti-HCV drugs. To gain further insight into the biochemical and biophysical properties of the NS3 enzyme binding pocket(s) and to generate biological tools for developing antiviral strategies, we decided to engineer macromolecular ligands of the NS3 protease domain. Phage-displayed repertoires of minibodies ("minimized" antibody-like proteins) and human pancreatic secretory trypsin inhibitor were sampled by using the recombinant NS3 protease domain as a ligate molecule. Two protease inhibitors were identified and characterized biochemically. These inhibitors show marked specificity for the viral protease and potency in the micromolar range but display different mechanisms of inhibition. The implications for prospective development of low-molecular-weight inhibitors of this enzyme are discussed.

Affinity selection of a camelized V(H) domain antibody inhibitor of hepatitis C virus NS3 protease.

The HCV genome encodes, within the NS3 gene, a serine protease whose activity specifically cleaves the viral polyprotein precursor. Proteolytic processing of HCV polyprotein precursor by the viral NS3 proteinase is essential for virion maturation and designing specific inhibitors of this protease as possible anti-viral agents is a desirable and practical objective. With a view to studying both the function of HCV NS3 protease and to designing inhibitors of this enzyme, we directed our interest towards engineering macromolecular inhibitors of the viral protease catalytic activity. We describe here the affinity-selection and biochemical characterization of one inhibitor, cV(H)E2, a 'camelized' variable domain antibody fragment, isolated from a phage displayed synthetic repertoire, which is a potent and selective inhibitor of proteolysis by the NS3 enzyme. In addition to being useful as a biological probe to study the function of HCV protease, this inhibitor can serve as a potential pharmacophore model to design antivirals. Moreover, the results suggest a way of engineering improved human-derived small recognition units tailored for enzyme inhibition.