Introduction: Interleukin 13 receptor alpha 2 (IL13Ra2) is a cell surface receptor that is frequently expressed in some of the most aggressive solid malignancies such as glioblastoma (GBM) and melanoma. IL13Ra2 also has limited expression in healthy tissue making it an ideal target for radioimmunotherapy. So far, no human IL13Ra2 antibody is clinically available. Five novel human monoclonal antibodies (mAbs) which specifically target IL13Ra2 were developed at the Sanders Tri-TDI. Here we report initial in vitro and in vivo studies with these antibodies following radiolabeling with ⁸⁹Zr using a human GBM (U-87 MG) cell line expressing IL13Ra2. The most promising antibody (KLG-3) was then studied in a melanoma model with human A-375 cells.

Methods: Five IL13Ra2-targeting mAbs (KLG-1-5) were developed using hybridoma technology, conjugated with p-Bz-SCN-DFO and radiolabeled with ⁸⁹Zr. Immunoreactivity assays were conducted with both the human IL13Ra1 and IL13Ra2 antigens to assess target specificity. Cell uptake and saturation binding assays were performed with U-87 MG and A-375 cells to determine binding affinity. Antibody internalization assays were conducted over 48 h. ImmunoPET studies were conducted on nude mice bearing subcutaneous U-87 MG and A-375 xenografts including serial biodistribution, imaging and pharmacokinetics of ⁸⁹Zr-mAbs. ImmunoPET blocking studies were conducted with excess cold antibody to ensure target specificity. Mass dose optimization studies were conducted to obtain the highest tumor-to-normal tissue ratios. Monte Carlo radiation dose modelling was conducted to determine feasibility of radiotherapy with these antibodies.

Results/Discussion: Conjugation of all mAbs with DFO was successful with high yields and showed quantitative radiolabeling with ⁸⁹Zr. All mAbs showed excellent target engagement in immunoreactivity assays with IL13Ra2 (90-98%), and none showed significant binding to IL13Ra1. In in vitro studies, labeled mAbs exhibited uptake in U-87 MG and A-375 cells (2.5-10.3% and 6.4-16.4%, respectively) and pico- to low nanomolar binding affinity (0.27-1.63 nM). All ⁸⁹Zr-mAbs had comparable U-87 MG cell internalization between 32-52% over 48 hours; ⁸⁹Zr-KLG-3 internalized significantly faster and to a greater degree (92%) in A-375 cells. Serial PET imaging studies over a period of 6 days showed continuously increasing tumoral uptake with maximal uptake at 144 h, and minimal non-specific binding. Intense tumor uptake ([%ID/g]_max) was observed for all ⁸⁹Zr-mAbs (35-71%) with significantly lower uptake in the IgG control (22%) in the U-87 MG model (n = 3-4 per group). Tumor uptake of KLG-1-5 was saturable with 10-fold mass excess carrier mAb (29-62%) showing specific target engagement. Lead mAb KLG-3 performed similarly well in the A-375 model with high tumor-to-normal tissue ratios (tumor [%ID/g]_max/ tissue [%ID/g]_mean) achieved at 144 h post injection of 7.4, 14.6, and 41.6 for blood, kidney and muscle respectively. Monte Carlo dosimetry estimates from ex vivo biodistribution studies suggest that for ¹⁷⁷Lu-labeled (i.e., therapeutic) KLG-3, tumorical absorbed doses will be achieved (271 Gy/mCi) with tolerable radiation burden for at-risk organs.

Conclusion: We developed and characterized five novel human IL13Ra2-targeted antibodies. Preliminary in vitro and in vivo data confirmed the specificity for IL13Ra2. ⁸⁹Zr-ImmunoPET studies in a GBM mouse model showed intense tumor mAbs uptake and high contrast, with lead mAb KLG-3 demonstrating the highest tumor-to-normal tissue ratios. Specific, high tumor uptake was also observed in a human melanoma model with ⁸⁹Zr-KLG-3 immunoPET. These results along with encouraging therapeutic dosimetry estimates support further translational development of the anti-IL13Ra2 theranostic platform.

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Figure 1. Representative MIP and axial PET/CT images of nude mice bearing U-87 MG xenografts injected with 20 mg (3.7 MBq) of ⁸⁹Zr-labeled IL13Ra2 antibodies. Imaging conducted six days post radiotracer injection.

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