Development and characterization of CD133 positive cancer stem cell targeted toxins for use in carcinoma therapy

Abstract

Cancer Stem cells (CSC) have been shown to play an important role in a number of carcinomas. Although representing a subpopulation of many cancers, CSC are extremely important because they are more drug resistant than the more differentiated cancer cells which make up the bulk of most solid tumors. High numbers of CSC is an indicator of poor clinical outcome and have been shown to be a cause in drug refractory relapse, which is the single most urgent problem in carcinoma therapy. CD133 is a cell surface receptor that has been identified as a CSC marker in breast, brain, colon, pancreatic, and recently in Head and Neck (HN) carcinoma. Our laboratory bioengineered a targeted toxin called dCD133KDEL consisting of the scFv portion of a novel anti-CD133 monoclonal antibody on the same molecule as truncated pseudomonas exotoxin. Binding of dCD133KDEL was demonstrated on a variety of carcinoma lines and we verified the ability of the anti-CD133 scFv to sort tumor initiating cells. Since enhanced tumor initiation is a hallmark of CSC, we demonstrated that dCD133KDEL was able to prevent tumor initiation. Importantly, even though CD133 was expressed only on a subpopulation of cells, dCD133KDELprevented cell proliferation in vitro and had powerful anti-cancer effects in vivo in xenograft mouse models of head and neck cancer. The therapeutic potential of dCD133KDEL was further investigated in xenograft models of human breast and ovarian cancer where it was effective when administered systemically as well. To further study therapeutic potential, we assessed the reactivity of this drug on normal human progenitor cells since CD133 is a known progenitor cell marker. dCD133KDEL did not kill normal human CD133+ stem cells at the same concentrations as it did for carcinoma cells, indicating a therapeutic window exists. Drug safety studies were performed in mice and the maximum tolerated dose of dCD133KDEL was established. Liver damage was shown to be the dose limiting toxicity. Since CD133 positive cells can develop from CD133 negative cell populations, a phenomenon known as stem cell plasticity, we developed a bispecific targeted toxin (dEpCAMCD133KDEL) capable of targeting both CD133 and the epithelial cell adhesion molecule, EpCAM, an overexpressed marker on most carcinomas. dEpCAMCD133KDEL potently inhibited cell proliferation of a number of carcinoma lines in vitro and was also effective at eliminating tumor spheroids, which have been shown to be enriched for CSCs. This bispecific agent was also effective at causing tumor regression in a model of HN cancer in vivo. Because of the preliminary effectiveness of dCD133KDEL and dEpCAMCD133KDEL in preclinical evaluation, these drugs warrant further development for possible use in carcinoma therapy. These two CSC targeted toxins could be extremely useful in situations where our current drugs are failing because of the progression of a critical drug resistant CSC population. Since, targeted toxins work synergistically with current chemotherapy, these two targeted toxins could be used as an adjunct to current therapy to target the CSC population specifically, while traditional chemotherapy and radiation can still be used to target the rapidly dividing bulk of the tumor. We believe that future success in cancer treatment must include approaches to target CSC as well as the majority of less differentiated cancer cells that comprise a tumor.