Development of antibody fragment-based radiotheranostic agents for cancer
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With the increasing clinical use of antigen-targeted therapies for cancer, there is the need for a non-invasive method of assessing target antigen expression prior to treatment. In this study, we report the use of copper-64 (64Cu) radiolabeled anti-EphA2 minibody for pre-treatment diagnosis of antigen expression via Positron Emission Tomography (PET). Minibodies (VH, VL and CH3) are advantageous for use as targeting molecules over intact antibodies (IgG) due to accelerated serum clearance properties while maintaining high uptake and uniform distribution within the tumor, allowing high tumor to background ratios at earlier time points relative to IgG. As EphA2, a tyrosine kinase receptor, is overexpressed in various cancer types with minimal expression in normal tissue, rapid quantification of EphA2 expression could be beneficial for patient stratification. Recombinantly produced anti-EphA2 minibody was evaluated for purity, stability, molecular weight, and binding ability. Following conjugation to the bifunctional chelator, NOTA-SCN, and radiolabeling with 64Cu, [ 64Cu]Cu-NOTA-anti-EphA2 minibody was administered to HT1080-fibrosarcoma tumor-bearing nude mice for in-vivo PET imaging and ex-vivo biodistribution at 4 and 24 hours post-injection (p.i.). The [64Cu]Cu-NOTA-anti-EphA2 minibody exhibited rapid tumor targeting with 25.53 ± 2.92 percent injected dose per gram (%ID/g) at 4 hours, and 22.13 ± 7.68 %ID/g at 24 hours p.i. (n=4). There was fast clearance from the blood, with high tumor to blood ratios. Tumor SUVmean values obtained from region of interest (ROI) quantification of the PET images were 1.13 ± 0.03 and 1.08 ± 0.06 at 4 and 24 hours respectively. These findings demonstrate that anti-EphA2 minibody is an excellent targeting molecule, and [ 64Cu]Cu-NOTA-anti-EphA2 minibody is a promising immunoPET agent with potential for use for other theranostic applications. Targeted radionuclide therapy is rapidly becoming an attractive cancer treatment modality with the recent approval of radiopharmaceuticals like Lutathera and Pluvicto for neuroendocrine tumors and castration-resistant prostate cancers respectively. Despite significant advances made towards their clinical use, there is variability in patient responses, in part attributed to the inter and intra-tumoral heterogeneity of target antigen expression. Furthermore, a fixed dosing regimen, irrespective of tumor burden and physiological properties limits effectiveness of targeted radionuclide therapies. Addressing these limitations to maximize efficacy could significantly enhance the prospects of using targeted radionuclide therapies for cancers such as gliomas, which are resistant to conventional treatment methods. The goal of this project is to test the utility of [64Cu]Cu-NOTA-anti-EphA2 minibody for assessing antigen expression in gliomas for patient stratification, and predicting biological effects of cognate therapeutic isotope, copper-67 (67Cu), to enable more accurate and personalized therapeutic dosing. There is evidence of variable EphA2 expression amongst glioma patients, making it an excellent target for these investigations. The more favorable pharmacokinetics of the 85 kDa minibody compared to whole IgG is leveraged for improved theranostic outcomes. As previously describe, [64Cu]Cu-NOTA-anti-EphA2 minibody was prepared via conjugation of NOTA-SCN to anti-EphA2 minibody and radiolabeling with 64Cu. Orthotopic glioma tumors were established in C57BL/6 mice via the intracranial inoculation of GL261 cells in the right brain hemisphere. About 10 to 14 days post-inoculation, tumor volumes were confirmed via Magnetic Resonance Imaging (MRI), and 11.1 MBq (300 µCi) of [64Cu]Cu-NOTA-anti-EphA2 minibody was administered per mouse for PET/CT imaging at various time points. Image analysis, PET/MRI co-registrations, Standard Uptake Value (SUV) quantifications and dosimetry analysis were performed using Imalytics and OLINDA/EXM. The [64Cu]Cu-NOTA-anti-EphA2 minibody conjugate exhibited high and rapid in-vivo tumor targeting. SUVmean values were 0.66 ± 0.13, 0.73 ± 0.16, 0.88 ± 0.17, 1.18 ± 0.29 and 2.08 ± 0.58 at 1, 4, 10, 24 and 48 hours p.i. respectively. Using voxel-level-based dosimetry analysis via Imalytics, cumulative tumor doses of 0.07 ± 0.02 Gy/MBq and 0.28 ± 0.08 Gy/MBq were computed for [64Cu]Cu-NOTA-anti-EphA2 minibody and its cognate therapeutic counterpart, [67Cu]Cu-NOTA-anti-EphA2 minibody respectively. Effective dose results obtained via Imalytics were consistent with results from OLINDA/EXM, which converts from mouse to human. These results allowed the prediction of dose delivery as well as putative biological effects and/or toxicity of [67Cu]Cu-NOTA-anti-EphA2 minibody on organs like kidneys and heart. In summary, [64Cu]Cu-NOTA-anti-EphA2 minibody is a promising immunoPET agent that could be utilized for cancer diagnosis, antigen expression quantification and dosimetry analysis to guide and inform therapeutic dosing with [67Cu]Cu-NOTA-anti-EphA2 minibody. The cornerstones of targeted radionuclide therapies and other antigen-targeted therapies are effective targeting molecules like antibodies and peptides which play a key role in the precise delivery of cytotoxic payloads to tumor sites by binding to specific tumor-associated antigens or other proteins within the tumor microenvironment. This study focuses on designing, assessing and optimizing the tumor-targeting ability and pharmacokinetics of a novel dual-targeting molecule: anti-EphA2-CD11b bispecific antibody (BsAb). The investigation is geared towards evaluating the potential therapeutic application of this BsAb, which simultaneously targets EphA2, a tumor-associated antigen and CD11b, a protein expressed by immune cells heavily infiltrating the tumor microenvironment. Recombinantly produced anti-EphA2-CD11b-BsAb was conjugated to a bifunctional chelator, NOTA-SCN and then radiolabeled with 64Cu. The [64Cu]Cu-NOTA-anti-EphA2-CD11b-BsAb radioimmunoconjugate was subsequently administered to HT1080-fibrosarcoma-bearing nude mice via tail vein injection. In-vivo PET/CT imaging and ex-vivo biodistribution analysis were performed to determine tumor uptake and pharmacokinetic localization. At 4, 24 and 48 hours p.i., %ID/g values of [64Cu]Cu-NOTA-anti-EphA2-CD11b-BsAb in HT1080 xenograft were 5.35 ± 2.24, 4.44 ± 1.90 and 4.10 ± 0.60 respectively. There was high uptake in the liver as well as CD11b expressing organs: spleen, bone (marrow) and lung. Binding in these CD11b-rich organs were significantly reduced by co-administering the dose with non-radiolabeled anti-CD11b antibody and anti-EphA2- CD11b-BsAb, with the concurrent increase in tumor uptakes compared to non-blocked mice (8.39 ± 1.37 %ID/g for blocked and 4.44 ± 1.90 %ID/g for non-blocked at 24 p.i., P = 0.0175). Further optimization studies showed that at lower molar activity (3.7 MBq/nmol, 100 µCi/nmol), there were significantly higher tumor accumulations and reduced uptakes in CD11b-expressing organs compared to higher molar activity (22.2 MBq/nmol, 600 µCi/nmol). Anti-EphA2-CD11b-BsAb is a functional targeting molecule and would require optimization through molar activity or blocking with non-radiolabeled antibody to maximize tumor targeting.
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Ph. D.