In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Formatting antibody fragments to mediate specific therapeutic functions.

Monoclonal antibodies are increasingly being used as therapeutic agents in a wide range of indications, including oncology, inflammation and infectious disease. In most cases the basis of the therapeutic function is the high degree of specificity and affinity the antibody-based drug has for its target antigen. However, the mechanism of action (MOA), the way the drug takes advantage of this specificity to mediate a therapeutic effect, varies considerably from drug to drug. Three basic potential categories of MOAs exist: antagonists, agonists and specific delivery mechanisms to target an active function to a particular cell type. The latter functions include selective cell killing, based on Fc-mediated events, recruitment of effector cells, and drug or radioisotope delivery. The majority of these mechanisms are not necessarily optimally mediated by an IgG structure; clearly, in the case of antibody-dependent cellular cytotoxicity or complement-mediated lysis, Fc is required. However, Fab fragments (the fragment comprising one antigen-binding arm of the Y-shaped IgG molecule) can be formatted to mediate most mechanisms and have the advantage that valency and half-life can be controlled to simplify the drug and address only the mechanism required. Moreover, Fab fragments can be produced in microbial expression systems which address manufacturing issues such as scale of supply and cost of goods.

Prolonged in vivo residence times of antibody fragments associated with albumin.

Antibody fragments can be expressed at a high level in microbial systems, but they may have limited therapeutic value because they are rapidly eliminated from the body. We demonstrate here that site-specific conjugation or binding of bacterially derived Fab' to the long-lived protein serum albumin allows full retention of the antibody's binding characteristics while imparting the albumin's longevity in vivo. In rats the area under the curve for Fab' conjugated to rat serum albumin was 17-fold greater than for the control of Fab' conjugated to cysteine. Again, a bispecific F(ab')(2) with specificity for rat serum albumin showed an area under the curve about 8-fold greater than did a F(ab')(2) without specificity to albumin. Genetic fusions of scFv to albumin were similarly long-lived and could be expressed in yeast to provide the basis of a cost-effective production system.

Expression and characterization of truncated forms of humanized L243 IgG1. Architectural features can influence synthesis of its oligosaccharide chains and affect superoxide production triggered through human Fcgamma receptor I.

The properties of IgG and its subcomponents are being exploited to generate new therapeutics with selected biological activities. In this study, a series of truncated, humanized IgG1 antibodies was expressed in Chinese hamster ovary cells, to evaluate the contribution of structural components to glycosylation and function. The series includes L243 IgG1 (alpha-MHC Class II) lacking a CH3 domain pair (DeltaCH3-IgG1), single-chain Fv fusion proteins with Fc or a hinge-CH2 domain, Fc with/out a hinge, and a single CH2 domain. Glycosylation of IgG Fc is important for recognition by effector ligands such as Fcgamma receptors. HPLC analysis of released and pyridylaminated oligosaccharides indicates that intact IgG1 and scFvFc antibodies are galactosylated and sialylated to levels similar to those observed previously for normal human IgG1. The truncated forms express increased levels of digalactosylated (30-83%) or sialylated (9-21%) oligosaccharide chains with the highest levels observed for the single CH2 domain. These data show which architectural components influence IgG glycosylation processing and that the (CH3)2 pair is particularly influential. When MHC Class II bearing (JY) cells were sensitized with L243 DeltaCH3-IgG1, scFvFc, or scFvhCH2 they elicited superoxide production, from U937 cells, at levels of 35-45% relative to that obtained for intact L243 IgG1 (100%). Mild reduction and alkylation of the hinge disulphide bonds of scFvhCH2 greatly decreased its capacity to trigger superoxide production. Thus, the L243 scFvhCH2 homo-dimer constitutes the minimal truncated form that binds the MHC Class II antigen and triggers superoxide production through FcgammaRI.

Changes in microvessel endothelial cell gene expression in an in vitro human breast tumour endothelial cell model.

Selective targeting of tumour-endothelium has been proposed as a means of therapy. The successful exploitation of this approach will rely on the identification of suitable targets expressed specifically on the tumour-associated endothelium. In an attempt to identify novel tumour-endothelium associated targets we have used differential mRNA display to identify genes up-regulated in an in vitro breast tumour-endothelial cell culture model. Confluent monolayers of human mammary microvessel endothelial cells (HuMMEC) were incubated for 5 days with MDA-MB-231 breast adenocarcinoma cell-conditioned medium (TCM). mRNAs isolated from TCM-treated and control cells were amplified using 104 combinations of four 3(') anchored T(12)VN primers and 26 'random' 10mers by RT-PCR and the products examined on DNA sequencing gels. Seventy-four sequences were cloned and the differential expression of five genes was confirmed using dot-blots. These were identified as procollagen type-IV, Tie-2/Tek receptor tyrosine kinase, NADH dehydrogenase subunit-6, and ferritin heavy-chain, which were up-regulated, and insulin-like growth factor binding protein-5, which was down-regulated. Increased endothelial expression of basement membrane proteins and tyrosine kinase receptors is known to occur during angiogenesis. Our data support the use of this model for further in vitro investigation of tumour angiogenesis.

Equilibrium and pre-equilibrium fluorescence spectroscopic studies of the binding of a single-immunoglobulin-binding domain derived from protein G to the Fc fragment from human IgG1.

A single-immunoglobulin-binding protein based upon the C2 domain of Protein G from Streptococcus has been shown to bind tightly to the Fc fragment of IgG1. The binding interaction results in a decrease in the fluorescence intensity from the sole Trp residue (Trp-48) in this domain. This spectral change has been used to monitor the binding interactions between the two proteins using equilibrium and pre-equilibrium fluorescence spectroscopy. Comparison of the data from the two techniques suggests that a conformational change occurs after the initial formation of the complex. Mutagenesis studies have shown that the Trp residue is important for binding and that replacement by a Phe residue is important for binding and that replacement by a Phe residue leads to a 300-fold decrease in the affinity for Fc gamma 1. Determination of the rate constants kon and koff at different values of pH between 4.0 and 9.0 suggest that variations in Kd are mediated predominantly by changes in kon. Competition experiments between SpG1 and a single-IgG-binding domain from Protein A from Staphylococcus aureus have been used to determine the affinity of the latter for Fc gamma 1.

The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy.

The stability and unfolding of an immunoglobulin (Ig) G binding protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations within each of the three alpha-helical regions (alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.

The identification of a lysine residue reactive to pyridoxal-5-phosphate in the glycerol dehydrogenase from the thermophile Bacillus stearothermophilus.

The glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is inactivated by incubation with pyridoxal-5-phosphate (PALP). The complex formed between the two can be trapped by reduction with sodium borohydride to yield a protein with an absorbance band at 325 nm and a fluorescence emission band at 430 nm, typical of trapped pyridoxal-5-phosphate moieties. Total loss of catalytic activity of the enzyme is associated with the modification of approximately one equivalent of the reagent; the incorporation of the reagent and the loss of activity can be prevented by the additional presence of the oxidised or reduced coenzyme. Peptides derived from the labelled protein have been sequenced and have identified Lys-97 as the reactive residue. Site-directed mutagenesis had been used to replace Lys-97 by a His residue. This mutated enzyme has no catalytic activity and fluorescence spectroscopy studies suggest that it is unable to bind NADH.

pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of protein A from Staphylococcus aureus.

A fusion protein, consisting of the N-terminal 81 amino acids from an inactive bovine DNase I (Q38,E39-E38,Q39) and two sequential synthetic IgG-binding domains based upon domain B of Protein A from Staphylococcus aureus has been shown to bind to porcine IgG with a similar affinity and pH profile to Protein A. The same residue in each B domain (Tyr111 and Tyr169) has been mutated by cassette mutagenesis to Ser, Glu, His, Lys or Arg and the effect of the mutation on binding interactions with porcine IgG investigated. The evidence presented suggests that the interactions at the B domain are highly sensitive to the presence of a charged residue.

Synthesis and mutagenesis of an IgG-binding protein based upon protein A of Staphylococcus aureus.

A novel protein able to bind with high affinity to the Fc fragment of IgG from a variety of animals has been produced by a gene synthesis approach. The IgG binding is accomplished by the presence of a single or two consecutive domains based upon domain B from protein A of Staphylococcus aureus. The IgG-binding moiety is fused to a peptide containing 21, 53 or 81 amino acids derived from the N-terminus of bovine DNase I. The latter is present to guide the expression of the protein in Escherichia coli into an inclusion body. This facilitates the high expression and recovery of the IgG-binding domains. The binding activity of this fusion protein is very close to that of the native protein A. Site-directed mutagenesis of the fusion protein and subsequent identification of changed binding interactions is reported.

The effect of DnaA protein levels and the rate of initiation at oriC on transcription originating in the ftsQ and ftsA genes: in vivo experiments.

The DnaA protein of Escherichia coli, essential for initiation at oriC, binds at a defined sequence which occurs at the chromosomal origin, near plasmid replication origins and in the promoters of the dnaA and mioC genes. This sequence also occurs at many other sites on the E. coli chromosome including three sites within the essential cell division genes ftsQ and A. Using an fts-lac fusion phage, lambda JFL100, we show here that fts gene expression responds both to reduced and increased intracellular levels of DnaA protein in a manner consistent with the hypothesis that DnaA protein regulates fts gene expression. Experiments using dnaC and dnaB-ts strains, however, suggest that DnaA control of fts transcription may be indirect, at least in part, with fts responding to the rate of initiation at oriC as well as to changes in DnaA protein level per se. It differs in this respect from dnaA gene expression which is unaffected when initiation of replication is inhibited by DnaB or DnaC inactivation. Strains integratively suppressed with pKN500 behave anomalously; neither fts nor dnaA transcription is significantly increased when DnaA is inactivated in these strains.