Targeting brain tumor stem cells using a bispecific antibody directed against CD133+ and EGFRvIII+.
Posted on: 05/14/2009
A. Wong, S. Mitra, P. Gupta; Stanford University Medical Center, Stanford, CA
Background: Using the marker CD133, cancer stem cells (CSCs) have been demonstrated for glioblastomas (GBMs) and medulloblastomas. However, CD133 is also present on normal neural stem cells. EGFRvIII is a tumor specific EGF receptor. We hypothesized that a recombinant bispecific antibody directed against CD133 and EGFRvIII would be a highly specific reagent. Methods: Single chain antibodies (scFv) were cloned for anti-EGFRvIII and anti-CD133 using existing hybridomas and published sequences. scFv were cloned into a bicistronic vector containing a human CH3 constant domain. The bispecific antibody (BsAb) contains the anti-CD133 (AC133) and anti-EGFRvIII single chain Fv; monospecific but bivalent reagents for anti-CD133 and anti-EGFRvIII were also constructed. Results: U87 cells were co-transfected with increasing amounts of CD133 and decreasing amounts of EGFRvIII cDNAs. The BsAb showed the highest binding for cells expressing both epitopes, whereas Di-EGFRvIII and Di-AC133 had the highest affinity for cells expressing high levels of individual antigens. When cells expressing both antigens were mixed with cells expressing high levels of either antigen alone, the BsAb only recognized CD133+/EGFRvIII+ cells. We then explored the efficiency of tumor cell killing. Using human CD16-expressing NK cells as the effectors at an effector:target ratio of 10:1 and an 83 nM antibody concentration, the BsAb induced 86% lysis of U87-EGFRvIII/CD133 cells, 42.5% in U87-vIII but only 27.3% in U87-CD133 cells. Di-EGFRvIII efficiently induced cytotoxicity in U87-EGFRvIII/CD133 and U87vIII cells but not in U87-CD133 cells. Di-CD133 was the least effective at inducing ADCC. Finally, we studied the ability of the BsAb to induce ADCC on tumor spheres and normal neurospheres. The BsAb showed at least 4X greater lysis on tumor spheres that were CD133+/EGFRvIII+ over normal neurospheres expressing CD133 alone at an E:T of 10:1. This was seen at concentrations as low as 0.83 nM. Conclusions: A recombinant bispecific antibody directed against CD133 and EGFRvIII is highly specific for cells that are positive for both antigens, but poorly targets cells expressing CD133 alone. It can potentially be used as a therapeutic agent for specifically targeting CD133+/EGFRvIII+ cancer stem cells.
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