New agents for targeting of IL-13RA2 expressed in primary human and canine brain tumors.

Interleukin 13 receptor alpha 2 (IL-13RA2) is over-expressed in a vast majority of human patients with high-grade astrocytomas like glioblastoma. Spontaneous astrocytomas in dogs resemble human disease and have been proposed as translational model system for investigation of novel therapeutic strategies for brain tumors. We have generated reagents for both detection and therapeutic targeting of IL-13RA2 in human and canine brain tumors. Peptides from three different regions of IL-13RA2 with 100% sequence identity between human and canine receptors were used as immunogens for generation of monoclonal antibodies. Recombinant canine mutant IL-13 (canIL-13.E13K) and canIL-13.E13K based cytotoxin were also produced. The antibodies were examined for their immunoreactivities in western blots, immunohistochemistry, immunofluorescence and cell binding assays using human and canine tumor specimen sections, tissue lysates and established cell lines; the cytotoxin was tested for specific cell killing. Several isolated MAbs were immunoreactive to IL-13RA2 in western blots of cell and tissue lysates from glioblastomas from both human and canine patients. Human and canine astrocytomas and oligodendrogliomas were also positive for IL-13RA2 to various degrees. Interestingly, both human and canine meningiomas also exhibited strong reactivity. Normal human and canine brain samples were virtually negative for IL-13RA2 using the newly generated MAbs. MAb 1E10B9 uniquely worked on tissue specimens and western blots, bound live cells and was internalized in GBM cells over-expressing IL-13RA2. The canIL-13.E13K cytotoxin was very potent and specific in killing canine GBM cell lines. Thus, we have obtained several monoclonal antibodies against IL-13RA2 cross-reacting with human and canine receptors. In addition to GBM, other brain tumors, such as high grade oligodendrogliomas, meningiomas and canine choroid plexus papillomas, appear to express the receptor at high levels and thus may be appropriate candidates for IL-13RA2-targeted imaging/therapies. Canine spontaneous primary brain tumors represent an excellent translational model for human counterparts

IL-13Rα2 is a Glioma-Restricted Receptor for Interleukin-13

We have found that binding sites for interleukin-13. ( IL13) are overexpressed in a vast majority of high-grade astrocytomas. (HGAs). These binding sites for IL-13 are distinct from the physiological receptor in that it does not bind IL-4. We also demonstrated that IL-13 receptor alpha 2 protein chain. (IL-13Rα2), an IL-4-independent receptor for IL-13, is abundant among HGAs, but not in normal organs. To examine if IL-13Rα2 is the tumorassociated site for IL-13, we stably transfected normal Chinese hamster ovary. (CHO) cells and glioma G-26 cells to express either human. (h) or murine. (m) IL13Rα2. CHO-hlL-13Rα2(+) cells and G-26-hlmlL13Rα2(+) cells, not CHO and G-26 parental or mock -transfected cells, specifically bound IL-13 in an IL-4-independent manner. The IL-13Rα2(+) cells also became highly susceptible to the killing by an IL-13-based cytotoxic fusion protein. In loss of function studies, a HGA cell line, SNB-19, was transfected with antisense. (as) hIL-13Rα2, as-SNB-19-hIL-13Rα2(+) cells lost their natural affinity towards IL-13 and became resistant to IL-13-based cytotoxins. The fact, that IL13Rα2-positive cells bind IL-13 independent of IL-4, become susceptible to IL-13 cytotoxins, cells deprived of IL-13Rα2 receptor lose these features, demonstrates that IL-13Rα2 is the brain tumor-associated receptor for IL-13

Immunoreactive IL-13RA2 in human and canine brain tumor specimens/cells by purified MAb’s of Peptide 1

<p><b><i>A</i></b>, Human glioblastoma (G), <b><i>B</i></b>, oligodendroglioma (O), astrocytoma (A), normal brain (NB), and G14 human GBM tumor lysate; meningioma (M), <b><i>C</i></b>, tissue lysates immunoreactivity using Western blots. Canine astrocytoma, glioblastoma and normal brain, <b><i>D</i></b>; oligodendroglioma, gliosarcoma (GSO) and mixed astro-oligo (AO), <b><i>E</i></b>; and choroid plexus papilloma (CPP) and meningioma, <b><i>F</i></b> tissue lysates immunoreactivity using western blots. Western blot of cell lines and parent tumor tissue obtained from dogs with spontaneous GBM, <b><i>G</i></b>. Immunoprecipitation of IL-13RA2 from U-251 MG cell lysate using either MAb 2G12C3, MAb 2G12E2 or a polyclonal antibody (R&D Systems #AF146); the polyclonal antibody was used for the receptor detection after immunoprecipitation<b><i>, H</i></b>.</p

MAb 1E10B9 binds to live GBM cells.

<p>Flow cytometry on human U-251 MG cells, canine GBM G06-A cells, and human T98G cells using MAb 1E10B9, <b><i>A.</i></b> Internalization of MAb 1E10B9 by G48a human GBM cells, <b><i>B.</i></b> Internalization of MAb 1E10B9 by U-251, G06-A, and T98G cells after 4-hr incubation, <b><i>C</i></b>.</p

Quantitative TaqMan RT-PCR comparing expression of IL-13RA2 in canine primary brain tumors.

<p>Elevated expression, relative to normal canine brain cortex, is seen predominantly in high grade glial tumors, essentially mirroring protein expression determined by western blotting. Off scale values are marked with an asterisk and value. MEN – meningioma; AST – astrocytoma; GBM – glioblastoma multiforme; OLIGO – oligodendroglioma.</p