Many macromolecular therapeutics such as peptides, proteins, antisense oligodeoxynucleotides (ASODN), and short interfering RNA (siRNA) are active only in the cytoplasm or nucleus of targeted cells. Endocytosis is the primary route for cellular uptake of these molecules, which results in their accumulation in the endosomal-lysosomal trafficking pathway and loss of therapeutic activity. In this article, we describe the synthesis and pH-dependent membrane-destabilizing activity of a new "smart" polymer family that can be utilized to enhance the intracellular delivery of therapeutic macromolecules through the endosomal membrane barrier into the cytoplasm of targeted cells. These polymers are propylamine, butylamine, and pentylamine derivatives of poly(styrene-alt-maleic anhydride) (PSMA) copolymers. The PSMA-alkylamide derivatives are hydrophilic and membrane-inactive at physiological pH; however, they become hydrophobic and membrane-disruptive in response to endosomal pH values as measured by their hemolytic activity. Results show that the pH-dependent membrane-destabilizing activity of PSMA derivatives can be controlled by varying the length of the alkylamine group, the degree of modification of the copolymer, and the molecular weight of the PSMA copolymer backbone. Butylamine and pentylamine derivatives of PSMA copolymers exhibited more than 80% hemolysis at endosomal pH values, which suggests their potential as a platform of "smart" polymeric carriers for enhanced cytoplasmic delivery of a variety of therapeutic macromolecules.

Gelonin-based immunotoxins vary widely in their cytotoxic. Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ϳ5 ؋ 10 6 toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.Immunotoxins are a promising approach to the targeted delivery of highly potent, cancer-specific, cytotoxic agents. Immunotoxins are frequently composed of a targeting moiety (derived from antibodies or other cell-binding proteins) either chemically conjugated or genetically fused to highly cytotoxic plant or bacterial protein toxins. Clinical success for immunotoxins has been mostly limited to hematological malignancies due to transport limitations in solid tumors (1). Such limitations have been extensively studied experimentally (2) and with several computational models (3, 4).The potency of a particular immunotoxin is dependent on the ability to deliver the toxin to the cytoplasm, which is commonly considered to be the rate-limiting step. For some native toxins such as ricin, intracellular delivery is achieved through lectin binding, followed by internalization and toxin release with membrane fusion or retrograde trafficking (5). Immunotoxins attempt to recreate this scenario by replacing the indiscriminate lectin binding with cancer-specific antigen binding as a means of targeting and internalization (6). Subsequent intracellular trafficking, release, and endosomal escape are often achieved using existing toxin characteristics, translocation domains, protease cleavage sites, disulfide bonds, and/or signaling peptides (7-10). However, the inclusion of toxins with domains facilitating cytoplasmic access can also lead to increased nonspecific toxicity in vivo (11,12).Gelonin is a plant toxin and classified as a type I ribosomeinactivating protein because it lacks any cell-binding or cytoplasmic delivery domains. Recombinant gelonin (rGel) 2 is an ϳ30-kDa N-glycosidase with activity similar to the ricin A chain but exhibiting better stability and lower immunogenicity (13,14). The use of rGel in tumor-targeted cytotoxic agents has been well studied (15, 16). Furthermore, rGel has been shown to be active without cleavage from the binding domain and without negative impact on the targeting agent's pharmacokinetics (...

Targeted endocytic uptake is a first step towards tissue-specific cytoplasmic macromolecular delivery; however inefficient escape from the endolysosomal compartment makes this generally impractical at present. We report here a targeted cytolysin approach that dramatically potentiates endosomal release of an independently-targeted potent gelonin immunotoxin. Fibronectin domains engineered for affinity to epidermal growth factor receptor or carcinoembryonic antigen were fused to the plant toxin gelonin or bacterial pore-forming cytolysins. These fusion proteins display synergistic activity in both antigen-specific cytotoxicity in vitro, enhancing potency by several orders of magnitude, and in tumor growth inhibition in vivo. In addition, the number of internalized gelonin molecules required to induce apoptosis is reduced from ∼5×106 to < 103. Targeted potentiation shows promise for enhancing cytoplasmic delivery of other macromolecular payloads, such as DNA, siRNA, and miRNA.

Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.

<p>PDF file- 129 KB, Supplementary Methods; Supplementary Figure S1: Potentiator binding and cytotoxicity; Supplementary Figure S2: Potentiator hemolytic activity; Supplementary Figure S3: Potentiation of gelonin immunotoxin cytotoxicity; Supplementary Figure S4: Potentiaion of internalized cytotoxicity and reduction of gelonin immunotoxin TN50; Supplementary Figure S5: Plasma clearance of immunotoxin and potentiators; Supplementary Table S1: Independent dose escalation of therapeutic proteins; Supplementary Table S2: Determination of the minimum delay between in vivo doses of synergistic agents.</p>

<div>Abstract<p>Targeted endocytic uptake is a first step toward tissue-specific cytoplasmic macromolecular delivery; however, inefficient escape from the endolysosomal compartment makes this generally impractical at present. We report here a targeted cytolysin approach that dramatically potentiates endosomal release of an independently targeted potent gelonin immunotoxin. Fibronectin domains engineered for affinity to EGF receptor or carcinoembryonic antigen were fused to the plant toxin gelonin or bacterial pore-forming cytolysins. These fusion proteins display synergistic activity in both antigen-specific cytotoxicity <i>in vitro</i>, enhancing potency by several orders of magnitude, and in tumor growth inhibition <i>in vivo</i>. In addition, the number of internalized gelonin molecules required to induce apoptosis is reduced from approximately 5 × 10⁶ to less than 10³. Targeted potentiation shows promise for enhancing cytoplasmic delivery of other macromolecular payloads such as DNA, siRNA, and miRNA. <i>Mol Cancer Ther; 12(9); 1774–82. ©2013 AACR</i>.</p></div>

<p>PDF file- 129 KB, Supplementary Methods; Supplementary Figure S1: Potentiator binding and cytotoxicity; Supplementary Figure S2: Potentiator hemolytic activity; Supplementary Figure S3: Potentiation of gelonin immunotoxin cytotoxicity; Supplementary Figure S4: Potentiaion of internalized cytotoxicity and reduction of gelonin immunotoxin TN50; Supplementary Figure S5: Plasma clearance of immunotoxin and potentiators; Supplementary Table S1: Independent dose escalation of therapeutic proteins; Supplementary Table S2: Determination of the minimum delay between in vivo doses of synergistic agents.</p>