An allosteric antibody to the leptin receptor reduces body weight and reverses the diabetic phenotype in the Lep(ob) /Lep(ob) mouse.

OBJECTIVE: Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half-life and low immunogenicity could be useful in the treatment of this condition. METHODS: Phage display technology coupled with flow cytometry and cell-based in vitro assays were employed to identify an allosteric agonist of the mouse LEPR. LEP-deficient Lep(ob) /Lep(ob) mice were used to compare in vivo effects of LEP to antibody administration. To evaluate hypothalamic effects of treatment, changes in mRNA levels of neuropeptide Y and proopiomelanocortin were measured. RESULTS: XPA.80.037 is a monoclonal antibody that demonstrates allosteric agonism of the mouse LEPR. Treatment of Lep(ob) /Lep(ob) mice with XPA.80.037 markedly reduced hyperphagia and body weight, normalized blood glucose and plasma insulin levels, and corrected dyslipidemia. These metabolic alterations correlated with changes in mRNA levels of neuropeptide Y and proopiomelanocortin, suggesting that XPA.80.037 had hypothalamic effects. CONCLUSIONS: Agonist allosteric monoclonal antibodies to the LEPR can correct metabolic effects associated with LEP deficiency in vivo and thereby have the potential to treat conditions of LEP deficiency.

Sequence plasticity in the antigen-binding site of a therapeutic anti-HER2 antibody.

We have examined the plasticity of the antigen-combining site of a high-affinity antibody. In phage-displayed Fab libraries, selected CDR positions and one FR position of the humanized anti-Her2 antibody hu4D5 were substituted with all 20 amino acids. Antigen-binding selections were used to enrich for high-affinity variants, and a large number of sequences were obtained prior to convergence of the selected pool to a small set of clones. As expected, sequence variability of the antigen-binding site is overall diminished compared to known IgG sequences; however, certain positions retain much higher variability than others. The sequence variability map of the hu4D5 binding site is compared with a map derived from previous alanine-scanning of the antibody. Affinities of soluble Fab fragments for antigen confirm that multiple variants were selected with high affinity for antigen, including one variant with a single point mutation that was about threefold improved in affinity compared to the parental hu4D5. Interestingly, this mutation is one of the most radical in terms of changing side-chain chemistry (Trp for Asp) and occurs at the most plastic site as calculated by the Wu-Kabat variability coefficient. Thus variability mapping yields information about the antibody-antigen interaction that is useful and complementary to that obtained by alanine scanning mutagenesis.

The Erbin PDZ domain binds with high affinity and specificity to the carboxyl termini of delta-catenin and ARVCF.

Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: delta-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or delta-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and delta-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or delta-catenin complexed with beta-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherin-catenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and delta-catenin. Finally, we showed that endogenous delta-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that delta-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.