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2022 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. No significant financial relationships to disclose.

2002 Background: The presence of cancer stem cells (CSCs) has been demonstrated for glioblastomas (GBMs) and medulloblastomas. To target brain tumor CSCs and not normal stem cells, it would be desirable to identify specific markers. CD133+ has been used to isolate CSCs, but is also present on neural and hematopoietic stem cells. A tumor specific alteration of the EGF receptor, EGFRvIII, is present in many solid tumors, making it ideal for targeted therapies. We asked if EGFRvIII is expressed in brain tumor CSCs. Methods: Freshly resected brain tumors were dissociated using a modified protocol to preserve cell surface makers. Cells were then directly propagated in defined serum free stem cell growth media or first sorted for CD133 and EGFRvIII using FACS and then grown in defined media. Western blots and/or RT-PCR was used to analyze cells for expression of EGFRvIII and stem cell markers. Results: Tumor spheres were isolated from all brain tumors attempted: 11 GBMs, 1 ependymoma, 2 medulloblastomas and 1 a...