(586b) Targeting the Breast Cancer Microenvironment Using Interleukin-12 Conjugated Pamam Dendrimers

Cancerous tumors require a blood supply for the delivery of
nutrients and removal of wastes. This is accomplished by stimulating new blood
vessel formation as the tumor grows. These blood vessels can be an ideal target
for therapy for a variety of reasons. The tumor vasculature is accessible to
therapeutic agents. The blood vessel (endothelial) cells, unlike the cancer
cells, are genetically stable. This means that therapeutics targeted to the
endothelial cell markers will not become ineffective due to changing targets.

New and developing blood vessels express a transmembrane
protein, α_Vβ₃ integrin that established
quiescent (not growing) blood vessels do not. A nine amino acid peptide has
been identified that binds to this integrin and has been examined for targeting
delivery of therapeutic molecules to tumor neovasculature. This peptide has the
amino acid sequence Cysteine-Aspartic acid-Cysteine-Arginine?Glycine-Aspartic
acid-Cysteine-Phenylalanine-Cysteine and is referred to as RGD-4C. This peptide
can be used to target molecules that inhibit new blood vessel formation
(antiangiogenic) or destroy the existing vasculature.

Many types of nanoparticles have
been examined as drug delivery vehicles, including radial polymers called
dendrimers. Ethylenediamine-core polyamidoamine (PAMAM) dendrimers are dense
radial polymers that contain a central ethylenediamine core, with each tertiary
amine nitrogen carrying two branched amidoamine groups. For each generation,
two additional branches are added to the terminal amino (-NH2) groups. Thus,
each arm of the dendrimer is split into an additional two branches at each
tertiary amine. The terminal primary amines remain reactive for further
conjugation, while the internal tertiary amines are very stable. A generation 5
PAMAM dendrimer has a measured diameter of 5.4 nm with 128 terminal functional
amine groups available for conjugating a variety of therapeutic, targeting and
imaging molecules.

Interleukin-12 (IL-12) is a regulatory cytokine involved in
the initiation and regulation of the cellular immune response. IL-12 is
produced by a variety of cells including macrophages, monocytes, dendritic
cells, and B cells typically in response to a foreign immunostimulating
molecule. IL-12 causes the production of interferon-gamma (IFN-γ) and
tumor necrosis factor-alpha (TNF-α) from both natural killer (NK) cells
and helper T cells. IL-12, through its stimulation of IFN-γ production,
enhances the immune response to cancers in mice and protects against
metastases. However, it can be toxic when delivered systemically.

In previous research, targeting delivery of IL-12 to a tumor
site using a protein fusion with RGD-4C resulted in localization at the tumor
site, expression of IFN-γ, decreases in cytotoxicity, and increased
angiogenesis. Using PAMAM dendrimers as delivery vehicles for the targeting
molecule (RGD-4C) and the therapeutic IL-12 will allow controlled ratios of
moieties for optimal cellular internalization and stimulation of IFN-γ. We
have conjugated the RGD-4C to a generation 5 PAMAM dendrimer and a fluorescent
molecule for detection purposes. We will present binding of the loaded
dendrimer to α_Vβ₃ integrin positive and
negative cells, optimization of IL-12 and RGD-4C conjugation method, and the
cytotoxic effects of the dendrimer and conjugated dendrimer in vitro.