Drug Carriers in Medicine and Biology - Monoclonal Antibodies

Volker Schellenberger at Genencor International discussed the use of mAb-enzyme fusion proteins for the activation of anticancer prodrugs within solid tumors. The major issues in this field are immunogenicity of foreign enzymes, achieving high tumor-to-blood ratios, and having prodrugs that are unstable, but that release active drugs with well-characterized dose-response effects. A version of -lactamase was described that was deimmunized according to the results of a T-cell stimulation assay. This enzyme was used to activate a prodrug of melphalan, a drug that is used in high-dose isolated limb perfusion for the treatment of melanoma. The fusion protein was optimized for thermal stability, resistance toward proteolysis, and selective binding under the slightly acidic conditions found within solid tumors. Clinical studies with the fusion protein/prodrug combination are planned.
Dr. David Scheinberg from the Memorial Sloan-Kettering discussed the use of mAbs for the delivery of short-lived -emitting isotopes to tumors. 225Ac was attached to several different mAbs through a functionalized DOTA chelator, and the resulting conjugates had very high radiochemical purities. This isotope is of interest, since it has a relatively long half-life (10 days) and sequentially decays through three other atoms, resulting in the release of four particles. The mAb-225Ac constructs effected high levels of cytotoxic activities in hematologic and solid tumors in an antigen-specific manner. Spheroid studies showed that the activities were centered around high concentrations of the conjugates, consistent with the short path length of the particles. One of the issues with 225Ac is that the daughter ions may not remain in the chelated form. When metal scavengers were used, kidney exposure was significantly reduced. The approach of using 225Ac in the targeted form is of interest, since the half-life may allow for significant levels of intratumoral penetration, and the dosimetry to tumors can be highly favorable. It is possible that one of the recombinant constructs described by Anna Wu would be ideally suited for 225Ac delivery.
Molecules that form supramolecular assemblies were prominently featured in the meeting. Timothy Deming from the University of California at Santa Barbara described self-assembly of block copolymers into hydrogels. New synthetic methods involving catalysts for the preparation of amino acid based block copolymers were described. The use of transition metal complexes as end groups to control the addition of each -amino acid N-carboxyanhydride (NCA) monomer to the growing polymer chain permitted control of the propagation reaction and resulted in copolymers of defined structure and narrow distribution of molecular weights. Diblock copolypeptide amphiphiles self-assembled at low concentrations to form the three-dimensional structure of hydrogels. The unique properties of these hydrogels depended on their structural parameters. Segments that were -helical formed gels more readily than -strands, which in turn were better than random coils. The porosity, fast response, and rapid rearrangement after stress of self-assembled hydrogels may prove useful in biomedical applications, particularly for drug delivery. Large molecules such as proteins and DNA could be loaded into the hydrogel structure to be released as the hydrogel responded to a physiological trigger.
Samuel Stupp from Northwestern University described amphiphiles that form three-dimensional fibrous networks upon addition to aqueous solutions. Specific peptide sequences were incorporated into the amphiphiles to impart features into the resulting nanofibers such as promotion of neuronal, pancreatic islet, and bone growth. Other amphiphiles were described that generate chemokine binding networks that inhibit angiogenesis or that support cell differentiation. The observation that rapid selective differentiation was linked to the amplification of bioactive epitope presentation to cells by nanofiber scaffolds will have a great impact on the design of numerous self-assembling systems. This is an emerging area of research with potentially a large number of medical applications.
M. Reza Ghadiri from the Scripps Institute described cyclic peptide sequences that spontaneously form nanotubes capable of penetrating through cell walls and membranes. The resulting nanotubes are able to kill bacterial cells within 5 min, whether they are growing or static. Depending on the peptide sequence employed, the resulting nanotubes can be specific for Gram-positive and Gram-negative bacteria, virally infected cells, and subsets of mammalian cells.
A number of talks concerned the use of polymers and polymer conjugates for therapy and imaging. The potential of vesicular carriers for drug delivery were described by Karen Wooley from Washington University, Kazunori Kataoka from the University of Tokyo, and Alexander Kabanov from the University of Nebraska. Dr. Wooley described supramolecular assemblies formed from dilute solutions of block copolymers. The resulting micelles assume a variety of shapes, depending on the hydrophilicities and amphiphilic characteristics of the building blocks. Such nanoparticles have been shell cross-linked and further modified with peptide sequences to promote binding to cell surfaces and/or transduction. Dr. Kataoka described nanocarriers consisting of block copolymers of poly(ethylene glycol) and poly(aspartic acid) and a dendrimer with a photosensitizer in its core. Enhanced photodynamic effects of these constructs, over cationic dendrimer photosensitizer and polyionic complex micelles, were clearly demonstrated. This approach is suitable for the delivery of photosensitizers of various structures.
Dr. Kabanov used Pluronic block copolymers as an example to demonstrate the effect of macromolecules and macromolecular therapeutics on gene expression profiles in treated cells. In particular the mechanism through which the Pluronic block copolymers sensitize multidrug resistant tumors to anthracycline antibiotics was presented. The mechanism of sensitization may involve disruption of mitochondrial activity and target cell ATP depletion. A relationship has been established between the extent of the cytotoxic activity of doxorubicin and the extent of ATP depletion by Pluronic block copolymer. Data from clinical trials of doxorubicin-containing Pluronic-based micelles were presented.
One of the most advanced polymer-drug conjugates in the clinic, polyglutamic acid-paclitaxel (Xyotax), was described by Jack Singer at Cell Therapeutics. This water soluble conjugate releases the anticancer drug paclitaxel through polymer degradation and ester bond hydrolysis. The advantages of Xyotax over paclitaxel appear to be higher intratumoral drug concentrations, lower systemic toxicity, and activity in settings where paclitaxel is inactive. Phase III clinical trials are in advanced stages, and the outcome should be available next year. If approved, this would represent the first covalent polymer-drug conjugate for cancer therapy and would provide validation for the whole concept.
The meeting included talks surrounding novel targeted constructs for drug delivery. Charles Wilson from the Archemix Corporation described aptamers, which are molecules composed of small oligonucleotides selected for binding through a reiterative PCR-based process. Aptamers to such proteins as thrombin and platelet derived growth factor, together with their pharmacokinetic parameters in unmodified or poly(ethylene glycol) modified forms, were described. Michael Flanagan from Sunesis Pharmaceuticals demonstrated that new small molecular weight drugs could be developed through a process known as tethering. Proteins were mutated at several residues near their binding sites with cysteines, and were then allowed to react with chemical cocktails containing agents with reactive disulfides. Molecules that bound were then tethered to other molecules that bound to nearby cysteines on the proteins, leading to new entities with high binding affinities. This resulted in the identification of new agents that inhibited such proteins as thymidylate synthase and interleukin 2. Peter Dervan from California Institute of Technology described minor groove binding distamycin derivatives that bound in a base-specific manner. Small molecular weight agents were generated to bind to HIF, a promoter for vascular endothelial growth factor, whose expression was down-regulated.
Drug carriers have had pronounced effects on imaging technologies. Jonathan Lindner from the University of Virginia described the diagnostic imaging and therapeutic applications of encapsulated microbubbles and other acoustically active microparticles. Application of these agents relies on surface modification with mAbs, peptides, or other ligands that facilitate binding to target cells involved in inflammation, angiogenesis, and atherogenesis. Ultrasonic irradiation of free or targeted microbubbles produces cavitation and other high-energy events such as microstreaming and ballistic shell dispersion that proved useful for imaging. Specific examples were discussed in which ultrasound destruction of payload-bearing microbubbles was used to amplify and site-localize gene and drug delivery. Other imaging technologies described in the meeting included quantum dots that were modified with biomolecules for cell recognition, described by Marcel Bruchez from the Quantum Dot Corporation. Zheng-Rong Lu from the University of Utah presented the design and synthesis of new macromolecular gadolinium complexes as MRI contrast agents. These new macromolecular structures, containing degradable disulfide bonds, permit control of the signal-to-background ratio, resulting in MRI blood pool contrast enhancement and increased sensitivity of tumor detection. Dr. Alexei Bogdanov from Massachusetts General Hospital described imaging technologies that allowed fo