I. Development of a Targetable Peptide-based Nanosphere System for Cancer Therapy
The purpose of this project is to develop a targetable nanosphere delivery system for the diagnosis and treatment of cancer. These biodegradable nanospheres (or peptide micelles) will consist of micellar assembly containing a core hydrophobic polypeptide region (C) a hydrophilic polyethylene glycol shell (S) and a targeting peptide moiety (T) attached to the surface of the shell. The hydrophobic core allows these micelles to solubilize very hydrophobic compounds and to release them slowly to the targeted site. The exterior of the micelle is designed to be non-immunogenic, thereby reducing the problems of reduced half-life or unwanted immune responses. The average diameter of these nanospheres is typically between 20 and 40 nm, a size which should allow these molecular assemblies to avoid the problems of renal excretion while enhancing the probability that they will be taken up by the targeted cells or tissues.
Unlike other targeting/delivery systems (such as stealth liposomes or earlier forms of polypeptide micelles), the individual components of our polypeptide micelles can be constructed using standard solid phase peptide synthesis. This allows for precisely controlled preparation of the micelle monomers. It also facilitates the covalent addition of targeting peptide groups to the polyethylene glycol (PEG) chain. In this particular system, the targeting groups will be short peptides which have been shown to bind to cell-surface antigens or receptors. One of the advantages of introducing targeting peptides to the surface of these nanospheres is that, while the target affinity for a single peptide may be low, the incorporation of multiple peptides into a micelle is expected to substantially increase the overall avidity. In addition, the use of small targeting peptides, instead of large proteins, means that the problems of protein denaturation, protein conjugation, protein induced immunogenicity and limited shelf-life can be avoided. Furthermore, these peptide-targeted micelles can be prepared completely synthetically, thereby eliminating the difficulties and costs associated with biologically derived products.
To date we have succeeded in developing both solid-phase and liquid-phase synthetic methods to prepare a wide range of micelle components (monomers) with a variety of "custom" peptide cores and a range of PEG conjugates. We have also identified optimum PEG-to-peptide ratios to enhance the formation and stability of the micelles. Extensive electron microsopy studies along with NMR characterization has allowed us to determine the stability, size and rotational dynamics of several of the micelle constructs. We have also succeeded in loading a number of hydrophobic drugs or drug-like molecules (doxorubicin, pyrene, sudan black) into these micelle and have assessed their stability in solution and serum. We are now working on refining the synthetic aspects of ataching targeting moieties to these micelle monomers. In particular, peptides which have been previously identified to bind cancer-associated antigens will be coupled to the PEG-peptide monomers. Two potential targeting peptides are bombesin and a short tetrapeptide (EPPT) recently identified that binds to breast cancer antigens. Because these nanospheres are potential pharmaceutical products, their in vitro and in vivo stability will also be studied in detail. Depending on the results of these stability tests, modifications to the hydrophobic core may have to be made (i.e. the addition of stabilizing disulfide bridges). In all cases, the biodistribution of radiolabelled micelles will be determined in the appropriate murine tumor models.
II. Design and Structural Characterization of a Prostate Cancer Antibody with Selected Peptide Epitopes and Peptide Mimetopes
It is the aim of this particular research program to study the molecular basis for antibody recognition and specificity. In working towards this goal, we have developed a multi- disciplinary approach designed to facilitate the detailed characterization of an antibody- antigen system that is of considerable medical interest. Specifically, we are using a combination of genetic engineering, peptide chemistry and NMR spectroscopy to study the structure and activity of a single chain antibody fragment (scFv) which is known to bind strongly to prostate specific antigen (PSA) -- a common marker for prostate cancer. Since the early 1980's it has been known that high serum PSA levels are well correlated with the presence of prostate cancer. By using simple immunodiagnostic assays it is now possible to quantitate serum PSA levels in a matter of hours. However, current problems with many commercial tests point to a lack of understanding regarding how PSA interacts with PSA-specific antibodies. Given the widespread use of PSA screening and the importance of accurate quantitative results, it is clear that a more detailed understanding must be gained about both the antibodies and the antigen(s) that are involved in these immunoassays. We believe that by studying the structure and activity of selected PSA- specific antibody fragments with (and without) PSA, it should be possible to identify specific residues that are vital to the binding and stability of PSA/antibody complexes. This kind of detailed information could assist protein chemists in the design and synthesis of more selective PSA antibodies which, in turn, could lead to the development of improved diagnostic tests for PSA or the creation of new prostate cancer therapeutics.
Over the past two years my laboratory has directed much of its effort at studying one particular PSA-specific antibody, a murine IgG variant known as B80.3. This antibody has shown good promise as both an ex vivo and an in vivo diagnostic tool for detecting and imaging of prostate cancer and prostate metastases. Furthermore, we have isolated, cloned and sequenced both the heavy and light-chain genes for B80.3, thereby making this antibody ideal for genetic manipulation and engineering. Because we are interested in characterizing the detailed structure and dynamics of this large antibody and its relatively large antigen, we have been acutely aware of the size limitations associated with our chosen technique (NMR spectroscopy). To address this "size" issue we have chosen to adopt a reductionist approach to understanding PSA/antibody recognition. By using genetic engineering techniques to disassemble both the ligand (PSA) and the receptor (the antibody) into their smallest functional components, we have simplified the system sufficiently to make it tractable to NMR characterization. We have succeeded in purifying several hundred milligrams of the scFv protein and have begun preliminary NMR studies with a variety of peptide mimetopes and epitopes. We hare expecting to have 15N labelled material ready within two months to conduct more detailed assignment work and further structural studies.
In studying this very interesting system we are employing several novel or innovative experimental approaches. These include: (i) the use of new kinds of expression vectors and host organisms to permit optimal protein expression; (ii) the development of novel and inexpensive isotopic labeling protocols for selected host cells; (iii) the use of a peptide mimic (both a mimetope and an epitope) to simulate the complete 33 kD PSA antigen; and (iv) the use of individual domain assignments and previously published homologous immunoglobulin NMR assignments to predict and assign B80.3 scFv resonances.
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