Loading drugs into carriers such as liposomes can increase the therapeutic ratio by reducing drug concentrations in normal tissues and raising their concentrations in tumors. Although this strategy has proven advantageous in certain circumstances, many drugs are highly hydrophobic and nonionizable and cannot be loaded into liposomes through conventional means. We hypothesized that such drugs could be actively loaded into liposomes by encapsulating them into specially designed cyclodextrins. To test this hypothesis, two hydrophobic drugs that had failed phase II clinical trials because of excess toxicity at deliverable doses were evaluated. In both cases, the drugs could be remotely loaded into liposomes after their encapsulation (preloading) into cyclodextrins and administered to mice at higher doses and with greater efficacy than possible with the free drugs.T here is currently wide interest in the development of nanoparticles for drug delivery (1-7). This area of research is particularly relevant to cancer drugs, wherein the therapeutic ratio (dose required for effectiveness to dose causing toxicity) is often low. Nanoparticles carrying drugs can increase this therapeutic ratio over that achieved with the free drug through several mechanisms. In particular, drugs delivered by nanoparticles are thought to selectively enhance the concentration of the drugs in tumors as a result of the enhanced permeability and retention (EPR) effect (8-18). The enhanced permeability results from a leaky tumor vascular system, whereas the enhanced retention results from the disorganized lymphatic system that is characteristic of malignant tumors.Much current work in this field is devoted to designing novel materials for nanoparticle generation. This new generation of nanoparticles can carry drugs-particularly those that are insoluble in aqueous medium-that are difficult to incorporate into conventional nanoparticles such as liposomes. However, the older generation of nanoparticles has a major practical advantage in that they have been extensively tested in humans and approved by regulatory agencies such as the Food and Drug Administration in the United States and the European Medicines Agency in Europe. Unfortunately, many drugs cannot be easily or effectively loaded into liposomes, thereby compromising their general use.In general, liposomal drug loading is achieved by either passive or active methods (9,(19)(20)(21)(22). Passive loading involves dissolution of dried lipid films in aqueous solutions containing the drug of interest. This approach can only be used for water-soluble drugs, and the efficiency of loading is often low. In contrast, active loading can be extremely efficient, resulting in high intraliposomal concentrations and minimal wastage of precious chemotherapeutic agents (9,23,24). In active loading, drug internalization into preformed liposomes is typically driven by a transmembrane pH gradient. The pH outside the liposome allows some of the drug to exist in an unionized form, able to migrate across the lipid bi...

Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics.

The use of proteins and peptides for therapeutic applications are often compromised by low biological stability, high renal clearance, and non-optimal biodistribution. Chemical attachment of poly-(ethylene glycol) (PEGylation) is often considered the most effective way to improve these pharmacologic properties by increasing circulation half-life as well as reduced renal clearance, immunogenicity and protease mediated degradation. However, random conjugation results in heterogeneous derivatives with undefined composition and can substantially lower the bioactivity of the modified protein, leading to unpredictable in vivo behavior. Site-specific modification of proteins is therefore an attractive approach to circumvent the non-specificity resulting from random conjugation. We have developed a novel technique named PRINT (PRotect, INcise Tag) for N-terminal specific bioconjugation of proteins and peptides. Conceptually, PRINT can be performed on any protein that has any N-terminal tag for purification and a protease cleavage site following the tag. The recombinant protein is first treated with an excess of citraconic anhydride to reversibly block all reactive primary amine sites. Proteolytic cleavage then exposes only a single amine (the primary amine at the N-terminus) for desired bioconjugation by amine-reactive NHS ester chemistry. Lowering of reaction pH results in removal of the citraconates, leaving N-terminal specific mono PEGylated protein molecules. We used Tumor Necrosis Factor-α (TNF-α) as a model protein as it suffers from inherent instability and short biological half-life, and exhibits toxic side effects at therapeutic concentrations in both small animals and human patients. We demonstrate that PRINT results in a single product with exquisite selectivity and specificity in contrast to conventional reaction using the same NHS reagent, which was further confirmed by mass spectrometric analyses. Subsequent de-blocking generated an N-terminal protected TNF-α molecule with enhanced serum stability, superior pharmacokinetic properties, and reduced systemic toxicity. Importantly, N-terminal protection by PRINT did not affect the bioactivity of TNF-α. Existing site-selective bioconjugation approaches are either specific to amino acid tags or involve substantial non-trivial chemical or biotechnological manipulations to synthesize a desired bioconjugate. In contrast, PRINT employs ubiquitously used recombinant DNA techniques and easily acquired commercial reagents to generate exquisite N-terminal selective protection. We show that PRINT is a robust, reproducible and mild strategy which is able to target the α -amine and provide N-terminal specific protection to proteins or peptides that suffer from similar issues. We believe that this approach is strongly orthogonal to current methods and will be applicable to many biotherapeutics and bioprobes that are currently being designed to treat cancer. Citation Format: Surojit Sur, Yuan Qiao, Anja C. Fries, Robert N. O’Meally, Robert N. Cole, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. PRINT: A protein bioconjugation method with exquisite N-terminal specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1341.