Peter T. Jones

Quaternary structure changes in aspartate transcarbamylase studied by X-ray solution scattering: Signal transmission following effector binding☆

The result of binding the effectors ATP and CTP to aspartate transcarbamylase was studied by X-ray solution scattering. Binding of substrate analogues produces a substantial change in the solution scattering curve, allowing us to monitor the proportion of the different quaternary structure states present in solution. In the initial solution this ratio was made roughly unity by adding either carbamyl phosphate and succinate, or N-(phosphonacetyl)-L-aspartate (PALA). ATP or CTP were then added, and their effect on the proportion of the different quaternary structure states was followed. When using carbamyl phosphate and succinate (weakly bound), ATP or CTP had a clear effect, as observed previously by monitoring the sedimentation rate (Changeux et al., 1968). However, when PALA (strongly bound) was used, the effect of CTP was very much smaller, and that of ATP was undetectable. This result supports the explanation by Tauc et al. (1982), that nucleotides act mostly through changing the affinity of the active sites for substrate, and only to a small extent by directly modifying the quaternary structure equilibrium in the case of CTP.

Generation of a panel of related human scFv antibodies with high affinities for human CEA.

BACKGROUND A human single chain Fv (scFv) specific for human carcinoembryonic antigen (CEA) has been isolated from a 2.0 x 10(9) phage display library from unimmunised human donors. The dissociation constant of the scFv has been measured by surface plasmon resonance (SPR) and found to be 7.7 x 10(-9) M, with an off-rate component of 6.2 x 10(-3) s-1. In order to investigate directly whether increased affinity leads to improved targeting of CEA-positive tumours, this scFv has been affinity matured by both targeted mutagenesis of the CDRs of heavy and light chains, and by light chain shuffling. STUDY DESIGN A partial randomisation scheme, biased towards amino acids commonly found as somatic mutations of germline antibody sequences, was used for directed diversification of VH and VL CDR3s. Diversification of the entire VL region was also introduced by light chain shuffling of the parental anti-CEA scFv. Selection of the mutagenised repertoires was carried out to enrich for antibodies with a reduced koff. RESULTS Sequencing the selected clones identified a number of amino acid changes in the VH CDR3, one of which gave a four-fold reduction in koff. Stringent selection of the light chain shuffled library resulted in several clones with a two- to three-fold reduction in koff. It has been possible to combine the selected changes from both mutagenesis approaches by using the mutagenised heavy chain and a light chain derived by shuffling to give a human scFv with a dissociation constant for human CEA of 6.0 x 10(-10) M. CONCLUSION A panel of human anti-CEA scFvs has been generated with differing dissociation constants for antigen, which will allow the correlation between tumour targeting efficiency in relation to binding affinity to be assessed directly. The scFv panel will be valuable in the optimisation of human antibodies for immunotherapy.

Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the CH2 domains

The serum half-lives of a wild-type recombinant mouse monoclonal antibody of the IgG2b isotype and a mutant antibody differing from the wild-type antibody by a single amino acid substitution introduced into the CH2 domain, the replacement of Asn 297 by Ala to delete the conserved site of heavy chain glycosylation, were determined in the rat. The biological half-life of the aglycosyl Asn 297-Ala mutant recombinant antibody (4.8 days) was significantly shorter than that of the normally glycosylated wild-type antibody (7.4 days) by enzyme immunoassay. A similar difference between the biological half-lives of 125I-labelled aglycosyl and wild-type antibodies (2.9 and 4.0 days, respectively) was determined by gamma counting. Analysis of serum samples demonstrated that both recombinant antibodies were present in the circulation predominantly as intact monomeric IgG and revealed no differences that could account for the more rapid elimination of the aglycosyl antibody. The results of this investigation indicate that the carbohydrate residues contribute only in part to the survival of IgG in vivo and suggest that the diminished half-life of the aglycosyl antibody is due to increased catabolism in the extravascular tissues.

Multiple binding sites on the CH2 domain of IgG for mouse FcγR11

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (FcγR). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fcγ R. This domain was mapped for interaction with mouse Fcγ R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human FcγR1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237 → Ala, Asn 297 → Ala, and Glu 318 → Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse FcγR11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by FcγR11 of the P388D1 cell line.

Recombinant mouse monoclonal antibodies with single amino acid substitutions affecting C1q and high affinity Fc receptor binding have identical serum half-lives in the BALB/c mouse

The serum half-lives of three recombinant mouse monoclonal antibodies, differing radically in their ability to bind to Clq or FcRI but only minimally in structure, were determined in the BALB/c mouse following intravenous administration. The wild-type antibody, a chimaeric antibody comprising variable domains binding 3-iodo-4-hydroxy-5-nitrophenylacetate and constant domains of the mouse IgG2b isotype, was eliminated from the bloodstream with biphasic kinetics: alpha-phase, 0.5 days; beta-phase, 7.0 days. The alpha- and beta-phase half-lives of mutant recombinant antibodies with single amino acid substitutions, either Glu 235-Leu allowing binding to the mouse FcRI, or Lys 322-Ala reducing Clq binding 30-fold, were indistinguishable from those of the wild-type antibody demonstrating that the biological half-life of intact mouse IgG is independent of the ability to bind Clq or FcRI. The major implication of the present study is that IgG molecules which have been genetically engineered to eliminate interaction with other components of the immune system should retain the long half-life typical of natural antibodies.

Phage antibodies against an unstable hapten: oxygen sensitive reduced flavin

It is difficult to raise antibodies against haptens and antigens that are unstable under the physiological conditions of the serum. Here we have used a phage antibody library to isolate antibody fragments against oxygen sensitive reduced flavin, by selection of the phage under anaerobic and reducing conditions at pH 5 and a pre‐elution step with the oxidized flavin. The binding of the reduced hapten to one of the antibody fragments was characterised by time‐resolved polarised fluorescence, and shown to be highly specific for the reduced flavin.

Solution structure of a soluble fragment derived from a membrane protein by shotgun proteolysis

We have previously reported a phage display method for the identification of protein domains on a genome-wide scale (shotgun proteolysis). Here we present the solution structure of a fragment of the Escherichia coli membrane protein yrfF, as identified by shotgun proteolysis, and determined by NMR spectroscopy. Despite the absence of computational predictions, the fragment formed a well-defined beta-barrel structure, distantly falling within the OB-fold classification. Our results highlight the potential of high-throughput experimental approaches for the identification of protein domains for structural studies.