Identification of Peptide Mimotope Ligands for Natalizumab.

Mimotope peptides selected from combinatorial peptide libraries can be used as capture reagents for immunoassay detection of therapeutic monoclonal antibodies (mAbs). We report the use of phage display libraries to identify peptide ligands (VeritopesTM) that bind natalizumab, a therapeutic mAb indicated for use in multiple sclerosis. PKNPSKF is identified as a novel natalizumab-binding motif, and peptides containing this motif demonstrated utility as capture reagents in enzyme-linked immunosorbent assays (ELISAs). A peptide containing the identified motif was shown to be competitive with the natural ligand (α4-integrin) and a neutralizing anti-idiotype antibody for natalizumab binding, indicating that VeritopesTM act as surrogate ligands that bind the antigen binding site of natalizumab. Affinity maturation further confirmed the motif sequence and yielded peptides with greater apparent affinity by ELISA. VeritopesTM are promising assay reagents for therapeutic drug level monitoring.

A cocktail of thermally stable, chemically synthesized capture agents for the efficient detection of anti-gp41 antibodies from human sera.

We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60(o)C.

Iterative in situ click chemistry assembles a branched capture agent and allosteric inhibitor for Akt1.

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties.

A selective 15N-to-(1)H polarization transfer sequence for more sensitive detection of 15N-choline.

The sensitivity and information content of heteronuclear nuclear magnetic resonance is frequently optimized by transferring spin order of spectroscopic interest to the isotope of highest detection sensitivity prior to observation. This strategy is extended to 15N-choline using the scalar couplings to transfer polarization from 15N to choline's nine methyl 1H spins in high field. A theoretical analysis of a sequence using nonselective pulses shows that the optimal efficiency of this transfer is decreased by 62% as the result of competing 15N-(1)H couplings involving choline's four methylene protons. We have therefore incorporated a frequency-selective pulse to support evolution of only the 15N-methyl 1H coupling during the transfer period. This sequence provides a 52% sensitivity enhancement over the nonselective version in in vitro experiments on a sample of thermally polarized 15N-choline in D2O. Further, the 15N T1 of choline in D2O was measured to be 217+/-38 s, the 15N-methyl 1H coupling constant was found to be 0.817+/-0.001 Hz, and the larger of choline's two 15N-methylene 1H coupling constants was found to be 3.64+/-0.0 1Hz. Possible improvements and applications to in vivo experiments using long-lived hyperpolarized heteronuclear spin order are discussed.