Recombinant antibodies for cancer diagnosis and therapy.

Recombinant antibodies now represent over 30% of biopharmaceuticals in clinical trials, highlighted by the recent approvals for cancer immunotherapy from the FDA which has awoken the biotechnology industry. Sales of these antibodies are increasing very rapidly to a predicted US$ 3 billion per annum worldwide by 2002. Since the development of new therapeutic reagent into commercial product takes 10 years, the recent FDA-approved antibodies are based on early antibody designs which are now considered primitive. Emerging technologies have created a vast range of novel, recombinant, antibody-based reagents which specifically target clinical biomarkers of disease. In the past year, radiolabelling of antibodies has increased their potential for cancer imaging and targeting. Recombinant antibodies have also been reduced in size and rebuilt into multivalent molecules for higher affinity. In addition, antibodies have been fused with many molecules including toxins, enzymes and viruses for prodrug therapy, cancer treatment and gene delivery. Recombinant antibody technology has enabled clever manipulations in the construction of complex antibody library repertoires for the selection of high-affinity reagents against refractory targets. Although phage display remains the most extensively used method, this year high affinity reagents have been isolated using alternative display and selection systems such as ribosome display and yeast display confirming the emergence of new display methods. Furthermore, innovative affinity maturation strategies have been developed to obtain high affinity reagents. This review focuses on developments in the last 12 months and describes the latest developments in the design, production and clinical use of recombinant antibodies for cancer diagnosis and therapy.

Direct selection of EGF mutants displayed on filamentous phage using cells overexpressing EGF receptor.

Understanding receptor-ligand interactions, and the signal transduction pathways they activate, is of great interest for the discovery of novel antagonists and agonists. In this report we describe a rapid and efficient procedure to evaluate the importance of several different epidermal growth factor (EGF) residues for the binding and activation of its receptor (EGFR). We constructed an EGF mutant library randomized at positions 13, 15 and 16 and expressed them on filamentous phages. Phage display is a powerful system, allowing rapid isolation of binding mutants. Since many of the most pharmacologically interesting receptors cannot be produced in a soluble form, we developed a technique to rapidly select receptor-binding molecules directly on cells. A luciferase assay, simple to perform, was then used to test their biological transduction activity and to rapidly detect mutants of interest. Analysis of the resulting sequences revealed that the wild-type amino acids at positions 13, 15 and 16 are optimized for binding and activity. EGF mutants with agonist properties were also isolated and tolerated substitutions were identified.

A simple luciferase assay for signal transduction activity detection of epidermal growth factor displayed on phage.

Studies on receptor-ligand interactions are important for the design of agonists or antagonists of natural ligands. We developed a luciferase reporter assay to screen epidermal growth factor receptor (EGFR) binding molecules rapidly for their ability to stimulate or inhibit signal transduction. Human EGF displayed on fd filamentous phage presented an activity similar to soluble EGF when tested for binding to the EGFR, for induction of cell cycle progression or in the luciferase assay. Two libraries of human EGF variants displayed on phage were constructed in which the aspartic acid residue at position 46 or the arginine residue at position 41 were randomised. EGF mutants displayed on phage were screened in parallel for binding to the EGFR using an ELISA assay and for transducing activity using the luciferase assay. Regarding the 46 position, most of the mutants retained the ability to bind the EGFR and their transducing activity corresponded perfectly with their binding. For the more crucial 41 position, only the wild-type EGF was able to bind the EGFR. Our approach allowed a simple determination of crucial positions and paved the way for identification of agonists with altered transduction activity.

Specific recognition of a tetrahedral phosphonamidate transition state analogue group by a recombinant antibody Fab fragment.

In order to obtain antibodies able to catalyse a peptide synthesis, a naive combinatorial library of human Fab antibody fragments was screened with the phosphonamidate transition state analogue of the reaction. Several Fab fragments were able to bind the analogue. Competitive binding studies performed with molecules containing representative parts of the hapten showed that two Fabs were able to recognize specifically the tetrahedral phosphorus present in the hapten.

The polymorphism ApoB/4311 in patients with myocardial infarction and controls: the ECTIM Study.

The polymorphism affecting codon 4311 of the apolipoprotein B gene (ApoB/4311) was investigated in a large case-control study in two French and one Northern Irish geographically defined populations. Cases were recruited 3 to 9 months after a myocardial infarction (MI) and controls were randomly selected from the population. The polymorphism was assessed using allele-specific oligonucleotides (ASO). The genotype frequencies of the ApoB/4311 polymorphism did not differ in Northern Ireland and France and were in Hardy-Weinberg equilibrium in all groups; strong associations with three other polymorphisms of the ApoB gene (XbaI, EcoRI, VNTR(34 repeats)) were observed and it was possible to identify highly sensitive and specific markers of the ApoB/4311 rare variant. Homozygotes for the ApoB 4311 rare variant were slightly less frequent in cases than in controls: 22 (4.4%) and 35 (6.7%) respectively (population adjusted chi 2 = 3.3 P less than 0.07), especially in Belfast: 6 (3.1%) and 12 (7.6%), respectively (P less than 0.06). Several lipid and lipoprotein parameters were measured. Consistently among control groups, rare homozygotes had lower mean levels of ApoB (P less than 0.02), triglycerides (P less than 0.02), and lipoprotein particles containing ApoE and ApoB (LpE:B; P less than 0.001) and a higher mean level of lipoprotein particles containing ApoAI and not ApoAII (LpAI; P less than 0.02) than heterozygotes and frequent homozygotes combined. The strong association between the ApoB/4311 polymorphism and LpE:B was also observed in patients with MI. When present in the homozygous form, the ApoB/4311 Asn----Ser variant is associated with a lipoprotein profile that is apparently favourable.