rADDs: Novel Disintegrins Targeting Breast Cancer

Institution: University of Southern California
Investigator(s): Stephen Swenson, Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12IB-0153 Award: $244,500
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2006)
An effective treatment strategy for breast cancer requires two elements: (1) accurate diagnosis of disease and a mechanism to follow efficacy of treatment and (2) a targeted treatment of the disease that stops progression. What is needed to bridge these two elements is a disease-imaging agent capable of limiting disease progression, while also evaluating extent of disease and efficacy of therapy. Breast cancer therapy is difficult because the disease is represented by individual cells that are very heterogeneous with respect to growth and metastasis potential. In addition, therapy must exploit molecular and biochemical differences between the cancer cells and the surrounding normal breast tissues.

Cells that make up breast cancer display surface proteins that might be efficiently used to target for drug delivery and be detected by specific imaging agents. We hypothesize that a fragment of natural human ADAM proteins (A Disintegrin and Metalloproteinase), will bind to breast cancer cells with a high specificity and limit both tumor and blood vessel growth. The ADAM proteins play regulatory roles in cell-cell interactions through proteolytic processing of the extracellular domains of cell surface proteins, such as growth factors, signaling molecules, and adhesion molecules (e.g. integrins). Among the integrins binding to disintegrin domains of ADAM proteins is av▀3, which has been the focus of our previous research.

In this project we propose to produce a recombinant ADAM-Derived Disintegrin (rADD), characterize its properties and evaluate its anti-tumor efficacy in mouse models of human breast cancer. We postulate that we will be able to identify a rADD that: (i) can be expressed with retention of function; and (ii) possesses a high degree of affinity for integrins av▀3, a5▀1 and av▀5, which are important in both tumor growth and tumor induced angiogenesis. Also, we will label the rADD proteins radioactively with a short-lived PET (positron emission tomography) imaging agent. The labeled rADDs will then allow visualization via high resolution PET imaging which then permits identification and evaluation of both primary and metastatic tumors. We will test whether treatment with the recombinant rADDs will limit tumor growth, spread, and inhibit blood vessel growth into the tumor. Finally, we will use the PET labeled form of the rADDs to follow and evaluate efficacy of treatments in mouse models of human breast cancer. The proposal is innovative because it utilizes a human-derived protein to diagnose, treat and evaluate therapeutic efficacy.

Final Report (2008)
Breast cancer (BC) remains one of the most prevalent forms of cancer in women worldwide. In an effort to control BC, new and novel diagnostic and therapeutic strategies must be developed. In order for tumors to be successful (growing and spreading) they contain an excess of cell surface receptors (integrins) involved in three processes critical for dissemination; cell adhesion, cell migration and invasion. Our project exploits this integrin upregulation, since we are developing recombinant proteins that will selectively bind to integrins on tumor cells blocking processes critical to tumor growth and spread. In previous work funded by the CBCRP we have found that a snake venom protein, called contorostatin (CN), was a specific integrin inhibitor (i.e., ôdisintigrinö) on tumor cells. However, to advance this topic closer to clinical application we need to produce larger quantities the active portions of CN and related integrin-inhibitory proteins. Until now this effort has been hampered by the structural properties of CN as a highly-folded, cysteine-rich protein.

The Specific Aims of this project and our successes to date include:

1) Production of recombinant disintegrins (rDDs) that will bind with high affinity to integrins: ?v?3, ?5?1, and ?v?5. We have developed a unique bacterial expression system for small disulfide-rich disintegrin and disintegrin like proteins. We have successfully used this system to produce proteins that are found in nature as ôstand aloneö disintegrins. With respect to expression of a fragment of natural human ADAM proteins (A Disintegrin and Metalloproteinase, which are not found in nature) we have had some difficulty. As these proteins exist only as multi-domain proteins (ADAM), when expressed as a stand alone protein the rDDs aggregate and lose activity. We have recently found a way around this problem and are currently attempting expression and purification.

2) Characterization rDDs in breast cancer cell lines for their effects on cell adhesion, migration and invasion on extracellular matrix proteins. With regard to the characterization we have had success in characterizing recombinant disintegrins that are found as stand alone disintegrins in nature. These proteins are fully functional in limiting processes critical to BC progression. We are confidant that once we overcome the expression problem with the rDDs they will display more potent activity.

3) Using mouse models with human breast cancer xenografts, we are evaluating rDDs for efficacy in limiting tumor progression and angiogenesis and as PET-based imaging agents in disease treatment and progression. Toward this aim we have successfully shown that recombinant disintegrins are effective in limiting tumor growth and progression. In addition, we have shown that recombinant disintegrin therapy stops blood vessel growth into an established tumor.

With continued research we believe that the rDDs will prove to be useful as both breast cancer therapeutic and diagnostic/imaging agents.