Igor Fisch

Selection of Human Single Chain Fv Antibody Fragments Binding and Inhibiting Helicobacter pylori Urease

Single chain Fv antibody fragments (scFv) binding to purified Helicobacter pylori urease were selected from a nonimmune human antibody repertoire displayed on filamentous phage. After three rounds of screening on solid phase urease, 44 clones were found to bind the enzyme and four distinct scFv were identified by sequencing their heavy and light chain variable region genes (VH and VL). Two of the selected human scFv (scFv B4 and scFv D9) inhibited the activity of H. pylori urease with inhibitory constants (Ki) of 7 and 2 μM, respectively. Their affinity (Kd) for H. pylori urease as determined by surface plasmon resonance ranged from 17 to 42 nM. Both scFv were able to bind to urease present on the surface of living H. pylori organisms as demonstrated by flow cytometry analysis. The binding sites of scFv B4 and D9 were mapped by the use of two random hexapeptide libraries (X6 and CX6C) displayed on filamentous bacteriophage. The selected peptide sequences were shown to inhibit scFv binding to H. pylori urease and thus could be used in a vaccination strategy as epitopes mimicking (mimotopes) the region of urease recognized by these human scFv antibody fragments.

Hyphenation of multi-dimensional chromatography and mass spectrometry for the at-line-analysis of the integrity of recombinant protein drugs.

A robust tool is proposed for the rapid at-line verification of the identity and integrity of (recombinant) proteins, namely the hyphenation of multidimensional chromatography and mass spectrometry (MS). A recombinant human antibody produced in Chinese hamster ovary cells is taken as pertinent example. The recombinant human antibody is first captured from the production environment by affinity chromatography (rProtein A, isolation/concentration of the target molecule) and automatically transferred to an enzyme reactor (immobilized trypsin column) for digestion, thereby yielding different peptides corresponding to the protein sequence. The peptides are then separated on a reversed-phase column before being analyzed and identified by MS. This step does not require a fine resolution since the mass spectrometer can identify a variety of substances at the same time. The results are then analyzed in silico with suitable bio-informatic tools. When the gene sequence of the protein product is known, proteolytic cleavages can be predicted and the exact mass and hence the amino acid sequence of each peptide can thereby be deduced. Fitting experimental data and reference peptide sequences then provides important information about the integrity of the protein and more particularly about its sequence. In our case, the integrity of 45% of the light and 75% of the heavy chain sequences of the antibody could be verified within minutes.

The SPP Biotech Module 1: Proteins for Medical Applications a Multidisciplinary Effort for the Development of a Recombinant Anti-RhD Antibody Preparation of Hemolytic Disease of the Newborn

A review with 0 refs. is presented regarding studies of a multidisciplinary team to establish the prophylactic principle and the manufg. process for anti-Rh D therapy based on recombinant antibody prepns. produced from Chinese hamster ovary cells. [on SciFinder (R)]

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display: CHO Expression of Human Recombinant Anti-RhD Antibodies

Replacement of the hyperimmune anti‐Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti‐RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high‐affinity anti‐RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin‐treated red blood cells enabled the isolation of two high‐affinity Fab‐expressing phage clones, LD‐6‐3 and LD‐6‐33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants DIII, DIVa, DIVb, DVa, DVI types I and II, DVII, Rh33 and DFR. Full‐length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth‐adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti‐RhD antibody adapted to pilot‐scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody‐dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti‐RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.