Sequence-simplified coiled coil polypeptides were synthesized and their folding properties characterized in order to define the role of charged border residues at the coiled coil interface for the controlled formation of homodimer and heterodimer structures. Three peptides were designed to form parallel coiled coils with valine and leucine occupying the hydrophobic interface positions a and d, respectively, of the heptad repeat abcdefg. The polypeptide designated E/K42, with the heptad repeat sequence VSSLESK, contained glutamate and lysine in the interface border positions e and g, respectively, and was designed to form a coiled coil homodimer at neutral pH. Two other polypeptides, designated E/E35 and K/K35, have the heptad repeats VSSLESE and VSSLKSK, respectively. E/E35 contains only glutamic acid at both e and g positions; K/K35, only lysine, E/E35 and K/K35 were designed to form a stable coiled coil heterodimer when combined at neutral pH. All three polypeptides were prepared by solid-phase synthesis and purified by reverse-phase high-performance liquid chromatography followed by size-exclusion chromatography. E/K42 formed a stable dimeric coiled coil structure as determined by circular dichroism and size-exclusion chromatography. The alpha-helical content of E/K42 was highest at neutral pH and decreased at extremes of pH. The alpha-helical structure of E/K42 at micromolar concentrations had a Tm of 62-65 degrees C and exhibited a concentration dependence of thermal denaturation consistent with dimer formation. In contrast to results with E/K42, a mixture of E/E35 and K/K35, but neither alone, forms alpha-helix at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)
Prostate cancer is the most common cancer and the second leading cause of cancer deaths among males in most Western countries. Autologous cellular immunotherapy for the treatment of cancer seeks to induce tumorspecific immunity in the patient and is consequently dependent on a suitable target antigen and effective presentation of that antigen to the patient's immune system. Prostatic acid phosphatase (PAP) has been tested as a target antigen due to its high and apparently specific expression in the prostate. We used a variety of approaches to analyze PAP expression, including immunohistochemistry, in situ hybridization, and quantitative polymerase chain reaction. We complemented these laboratory-based techniques with an in silico analysis of reported PAP expression in human cDNA libraries. Our studies confirmed that, while PAP expression is not restricted to prostate tissues, its expression in other human tissues is approximately 1-2 orders of magnitude less than that observed in the prostate. The relative specificity of PAP expression in the prostate supports its use as a target of autologous cellular immunotherapy. The approach described here, involving the use of multiple correlates of tissue-specific expression, is warranted as a prerequisite in selecting any suitable target for immunotherapy.
The development of protein conjugate therapeutics requires control over the site of modification to allow for reproducible generation of a product with the desired potency, pharmacokinetic, and safety profile. Placement of a single nonnatural amino acid at the desired modification site of a recombinant protein, followed by a bioorthogonal reaction, can provide complete control. To this end, we describe the development of copper-catalyzed azide-alkyne cycloaddition (CuAAC, a click chemistry reaction) for site-specific PEGylation of interferon β-1b (IFNb) containing azidohomoalanine (Aha) at the N-terminus. Reaction conditions were optimized using various propargyl-activated PEGs, tris(benzyltriazolylmethyl)amine (TBTA), copper sulfate, and dithiothreitol (DTT) in the presence of SDS. The requirement for air in order to advance the redox potential of the reaction was investigated. The addition of unreactive PEG diol reduced the required molar ratio to 2:1 PEG-alkyne to IFNb. The resultant method produced high conversion of Aha-containing IFNb to the single desired product. PEG-IFNbs with 10, 20, 30, and 40 kDa linear or 40 kDa branched PEGs were produced with these methods and compared. Increasing PEG size yielded decreasing in vitro antiviral activities along with concomitant increases in elimination half-life, AUC, and bioavailability when administered in rats or monkeys. A Daudi tumor xenograft model provided comparative evaluation of these combined effects, wherein a 40 kDa branched PEG-IFNb was much more effective than conjugates with smaller PEGs or unPEGylated IFNb at preventing tumor growth in spite of dosing with fewer units and lesser frequency. The results demonstrate the capability of site-specific nonnatural amino acid incorporation to generate novel biomolecule conjugates with increased in vivo efficacy.
Soluble extracellular protein antigens are notoriously poor stimulators of CD8+ cytotoxic T-lymphocyte (CTL) responses, largely because these antigens have inefficient access to an endogenous cytosolic pathway of the major histocompatibility complex (MHC) class I-dependent antigen presentation. Here, we present a strategy that facilitates antigen penetration into the cytosol of antigen-presenting cells (APC) by addition to the antigen of charge-modifying peptide sequences. As a result of this intervention, the charge modification enhances antigen uptake into APC by counteracting the repulsive cell surface charge, and then endosomal membranes are disrupted with a subsequent release of antigen into the cytosol. This technology significantly improves MHC class I-dependent antigen presentation to CTL, enabling a more efficient generation of specific CTL immunity in vivo. The strategy described here has potential for use in developing efficient vaccines for antigen-specific immunotherapy of human malignancies.
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