Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma

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Dickerson, Adrienne L. Lehnert, Donald K. Hamlin, Kevin P. Labadie, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4456645/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Aug, 2024 Read the published version in EJNMMI Research → Version 1 posted 5 You are reading this latest preprint version Abstract Purpose: Glypican-3 (GPC3)-targeted radioisotope immuno-positron emission tomography (immunoPET) may lead to earlier and more accurate diagnosis of hepatocellular carcinoma (HCC), thus facilitating curative treatment, decreasing early recurrence, and enhancing patient survival. We previously demonstrated reliable HCC detection using a zirconium-89-labeled murine anti-GPC3 antibody (89Zr-αGPC3M) for immunoPET. This study evaluated the efficacy of the humanized antibody successor (αGPC3H) to further clinical translation of a GPC3-based theranostic for HCC. Methods:In vitro αGPC3 binding to HepG2 cells was assessed by flow cytometry. In vivo 89Zr-αGPC3H and 89Zr-αGPC3M tumor uptake was evaluated by PET/CT and biodistribution studies in an orthotopic xenograft mouse model of HCC. Results: αGPC3H maintained binding to GPC3 in vitro and 89Zr-αGPC3H immunoPET identified liver tumors in vivo. PET/CT and biodistribution analyses demonstrated high 89Zr-αGPC3H tumor uptake and tumor-to-liver ratios, with no difference between groups. Conclusion: Humanized αGPC3 successfully targeted GPC3 in vitro and in vivo. 89Zr-αGPC3H immunoPET had comparable tumor detection to 89Zr-αGPC3M, with highly specific tumor uptake, making it a promising strategy to improve HCC detection. hepatocellular carcinoma glypican-3 (GPC3) Zirconium-89 immuno-positron emission tomography (immunoPET) theranostics Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Hepatocellular carcinoma (HCC) is increasing in incidence worldwide and has become the fastest growing cause of cancer death in the United States, with a median survival of less than one year [ 1 – 3 ]. In order to improve survival with current treatments, HCC must be detected early when it is amenable to surgical resection or transplantation [3.4]. However, multiphase, computed tomography (CT) or magnetic resonance imaging frequently misses lesions less than 1 cm, resulting in diagnostic uncertainty, delayed diagnosis, and early recurrence following resection [ 5 , 7 ]. Innovative technology capable of detecting HCC with enhanced sensitivity and specificity is therefore imperative and pressing. Radioisotope theranostics, including immuno-positron emission tomography (immunoPET) and radioimmunotherapy (RIT), is an emerging field with the potential to transform HCC diagnosis and therapy [ 8 ]. While yttrium-90 microspheres and iodine-131-labeled lipiodol and metuximab are used in radioembolization therapy, there are currently no FDA approved theranostics for HCC. However, glypican-3 (GPC3)-targeted radioisotopes have shown promise in preclinical and early clinical studies [ 6 , 9 – 20 ]. GPC3 is a cell surface antigen expressed on up to 80% of HCCs but absent in liver parenchyma and benign lesions, making it an accessible and specific target for a theranostic approach [ 14 , 15 , 21 ]. GPC3-based imaging has the potential to facilitate earlier, definitive HCC diagnosis and subsequent RIT, thus improving patient survival [ 16 ]. Our group previously demonstrated that immunoPET using zirconium-89 ( 89 Zr)-labeled murine antibody targeting GPC3 ( 89 Zr-αGPC3 M ) reliably identified small HCCs in mice [ 6 , 10 , 11 ]. Natarajan et al. described the use of 89 Zr-labeled humanized αGPC3 for HCC detection in a patient-derived xenograft model [ 16 ]. We built on this important work by humanizing our radioimmunoconjugate (αGPC3 H ) and performing in vitro and novel in vivo comparisons to its murine predecessor. Here, we report that 89 Zr-αGPC3 H targets GPC3 comparably to 89 Zr-αGPC3 M , resulting in highly specific tumor uptake and successful HCC detection. Materials and Methods Creative Biolabs, Inc. (Shirley, NY) constructed αGPC3 H by engrafting of the parental murine antibody’s complementarity-determining region (CDR). Flow cytometry was used to evaluate in vitro binding of αGPC3 M , a chimeric intermediary (αGPC3 C ), αGPC3 H , and αGPC3-deferoxamine (DFO) to HepG2 cells. Orthotopic xenograft models of HCC were generated as previously described in athymic nude mice (Jackson Laboratories) [ 10 – 12 , 22 ]. Two weeks after HepG2 cell liver injection, bioluminescence imaging (BLI) was used to estimate tumor establishment. αGPC3 was conjugated with DFO and labeled with 89 Zr [ 10 ]. (For simplicity, 89 Zr-DFO-αGPC3 is written as 89 Zr-αGPC3.) Mice (n = 11 per group) were injected retro-orbitally with 8.1 to 10 megabecquerels (MBq) of 89 Zr-αGPC3 H or 89 Zr-αGPC3 M . Mice with tumors predicted using BLI (n = 6 per group) underwent PET/CT five days after 89 Zr-αGPC3 injection. Maximum activity concentration (MBq/mL) was measured in a 2D region of interest (ROI) to calculate tumor radioisotope uptake (percent injected dose per milliliter, %ID/mL), tumor-to-liver ratio, and tumor maximum standardized uptake value (SUV max ). Biodistribution studies using gamma counts were performed separately in non-tumor-bearing, non-imaged mice two days after injection and in PET-imaged mice after imaging completion to determine %ID/g for select organs and tumors. Livers from PET-imaged mice were processed for histopathology. Details provided in Supplementary Methods. Results Humanized aGPC3 and αGPC3-DFO maintains GPC3 binding in vitro Binding to HepG2 cell surface GPC3 by unconjugated αGPC3 M , αGPC3 C , and αGPC3 H was confirmed by flow cytometry (Fig. 1 a). Binding of DFO-conjugated and αGPC3 M to GPC3 was overall similar to the unconjugated antibody (Fig. 1 b). Binding of αGPC3 H and αGPC3 C to GPC3 was greater than αGPC3 M . Bioluminescence imaging predicts tumor establishment Tumors were identified with BLI (Fig. 2 ). Mice were assigned to 89 Zr-αGPC3 H and 89 Zr-αGPC3 M injection such that mean photon emission (photons/sec) in tumor-containing ROIs was similar between groups (Table 1 ). Table 1 Mouse-specific immunoPET and biodistribution data correlated with tumor size H1 H2 H3 H4 H5 H6 b M7 M8 M9 M10 M11 M12 b Biolum. (Photons/ Sec) 3.7x10 8 5.5x10 8 a 9.2x10 8 7x10 7 1.6x10 8 1.4x10 8 4.1x10 8 2.5x10 8 2.1x10 9 4.7x10 8 2x10 7 Rank c 5 3 2 9 7 8 6 10 1 4 11 Tumor Weight (g) 0.003 0.031 0.005 0.115 0.003 0.003 0.003 0.003 0.036 0.043 Rank 6–10 4 5 1 6–10 6–10 6–10 6–10 3 2 Tumor %ID/mL (PET) 24 131 67 245 21 15.5 14.9 24 142 111 Rank 6 3 5 1 8 9 10 6 2 4 Liver %ID/mL (PET) 12.5 7.9 9.6 7.3 8.1 8.9 6.7 7.0 5.4 5.9 5.6 4.7 Tumor: Liver 1.9 16.5 6.9 34 2.6 2.3 2.1 4.5 24 19.9 Rank 10 4 5 1 7 8 9 6 2 3 SUV max 9.2 47 23 104 9.1 6.0 5.6 9.0 55 45 Tumor %ID/g (Gamma counter) -426 d 100 982 109 85 16.8 58 458 156 56 Rank 10 5 1 4 6 9 7 2 3 8 Liver %ID/g (Gamma counter) 20 10.2 16.2 11.3 10.5 10.9 7.6 8.3 6.1 6.4 6.5 7.7 Tumor: Liver -21 d 9.8 61 9.6 8.1 2.2 7.0 75 24 8.8 Rank 10 4 2 5 7 9 8 1 3 6 a Not measured due to minimal apparent signal. b H6, M12: no tumor identified on PET/CT or histopathology (blank = not applicable). c Rank from largest (1) to smallest (10/11). d -426 included in Fig. 4 c calculations; -21 excluded from Fig. 4 d (see Supplementary Methods). αGPC3 H is amenable to 89 Zr radiolabeling The radiochemical purity of both 89 Zr-αGPC3 antibodies was > 98% and the specific activity was 0.14 GBq/mg. Details provided in Supplementary Methods. Humanized 89 Zr-aGPC3 immunoPET reliably identifies tumors Five of six mice injected with 89 Zr-αGPC3 H and 89 Zr-αGPC3 M , respectively, demonstrated discrete hepatic localizations of increased PET intensity consistent with tumors (H1-H5, M7-M11; Fig. 3 a). Mean bioluminescence of PET-identified tumors (Table 1 ) was equivalent between groups (4.8x10 8 vs 6.3x10 8 +/- 4.5x10 8 photon/sec, p = 0.75). Histopathology identified tumors in H1-H5 and M7-M11, but not in mice without PET-identified tumors (H6, M12) (Fig. 3 b). Tumor uptake (97 vs 61 +/- 50%ID/mL, p = 0.42) and tumor-to-liver ratio (12 vs 11+/- 7.6, p = 0.68) were not significantly different between groups, despite significantly increased liver uptake in the 89 Zr-αGPC3 H -injected mice (9.1 vs 6.1 +/- 1.0%ID/mL, p = 0.02) (Fig. 3 c-e). SUV max was equivalent between groups (39 vs 24 +/- 21, p = 0.51) (Fig. 3 f). No difference in tumor radioimmunoconjugate uptake on biodistribution studies In non-imaged mice, the liver had the highest %ID/g calculated from gamma counter measurements followed by the lungs and spleen, with no significant difference between 89 Zr-αGPC3 H and 89 Zr-αGPC3 M -injected mice (mean 10 vs 8.8 +/- 4.6%ID/g, p = 0.77) (Fig. 4 a). In PET-imaged mice, tumor uptake was 7-fold greater than other organs, with equivalent organ uptake (mean 7.1 vs 6.3 +/- 2.7%ID/g, p = 0.75), tumor uptake (170 vs 149 +/- 241%ID/g, p = 0.93), and tumor-to-liver ratio of %ID/g (22 vs 24 +/- 19, p = 0.94) (Fig. 4 b-d). Discussion Humanized αGPC3 specifically targeted GPC3 in vitro and in vivo , enabling HCC detection with immunoPET in an orthotopic xenograft mouse model. This proof-of-concept study builds on our prior research validating a murine radioimmunoconjugate for a theranostic approach to HCC, with potential to improve diagnosis, treatment, and survival [ 6 , 10 – 12 ]. Our results demonstrate that humanization of 89 Zr-αGPC3 did not alter the highly avid binding to GPC3 on HepG2 cells and liver tumor xenografts. First, flow cytometry established at least equivalent, if not greater, binding of αGPC3 H to GPC3 compared with αGPC3 M , with minimal change when conjugated with DFO. Next, quality assurance of 89 Zr labeling confirmed that 89 Zr-αGPC3 H maintained high purity and specific activity. The majority of our experiments focused on the novel in vivo comparison between 89 Zr-αGPC3 H and 89 Zr-αGPC3 M . Five of six tumors in each group were detected by immunoPET, with no difference between groups in mean IVIS bioluminescence. PET/CT data revealed no significant difference in mean tumor uptake and tumor-to-liver ratios (%ID/mL). Similarly, biodistribution analysis showed no difference in mean organ uptake, tumor uptake, and tumor-to-liver ratios (%ID/g). While finding comparability between 89 Zr-αGPC3 H and 89 Zr-αGPC3 M achieved the study’s primary goal, additional details are worth noting. First, tumor uptake varied based on tumor size, with higher uptake in larger tumors as previously demonstrated [ 6 ]. %ID/g (gamma counter) results were greater than %ID/mL (PET) due to limited PET/CT spatial resolution causing partial volume effect; hence, there could be a larger discrepancy between %ID/g and %ID/mL values in mice with smaller tumors (e.g., H3, M9) (Fig. 3 , Table 1 ). While further consideration of the clinical impact of partial volume effect is warranted, this finding does highlight the successful detection of small tumors with 89 Zr-αGPC3 immunoPET. Second, background liver uptake was greater in the 89 Zr-αGPC3 H group, which could imply Fc-mediated liver uptake of 89 Zr-αGPC3 H . However, our prior studies of mice injected with 89 Zr-αGPC3 M compared with non-GPC3-targeting and GPC3-blocked controls demonstrated similar background liver uptake [ 6 , 11 , 16 ]. Furthermore, the tumor-to-liver ratio by nature adjusts for such variables, with no difference between groups suggesting that tumor uptake was also proportionally higher in the humanized antibody group. In fact, tumor-to-liver ratios of 12 or greater indicate 89 Zr-αGPC3 H is highly specific for GPC3-expressing tumors [ 16 ]. Tumor presence was histopathologically confirmed in mice with PET-identified tumors, while no tumors were found on histologic analysis of livers without PET-identified tumors. A limitation here is that, while meticulous gross examination of the liver and histopathologic analysis of suspected tumors was performed, serial sectioning of the entire left hepatic lobe was not undertaken due to limited funding. Therefore, the discordance between BLI and PET for H6 and M12 is unresolved. Of note, the three-week interval between imaging modalities was longer than in previous studies and thus tumor involution may have occurred. Our study is similar to those from other groups in that it underscores the potential of human αGPC3 to detect HCC with immunoPET, however, there are key differences. Tumor-to-liver ratios by PET/CT and biodistribution analyses were notably higher than those reported by Natarajan et al . using a similar 89 Zr-labeled human αGPC3 IgG antibody and Fayn et al . using 89 Zr-labeled GPC3-targeting HN3 single-domain antibodies. In addition, there was a greater relative difference between tumor uptake and uptake in organs such as the heart, lungs, gastrointestinal tract, and kidneys on biodistribution analysis [ 16 , 17 ]. While different methods for model development and radioimmunoconjugate injection used may affect the results such that they are not directly comparable [ 16 , 17 ], it is possible that our humanized antibody has a higher specificity for GPC3-expressing tumors. Furthermore, it should be noted that tumor-to-liver ratios were measured five days after injection in this study compared with one to seven days after injection in the aforementioned studies, however our prior experiments with 89 Zr-αGPC3 M demonstrated high tumor-to-liver ratios calculated from four hours up to seven days after injection [ 6 , 11 ]. Finally, Carrasquillo et al . conducted a phase I clinical study of PET/CT in HCC patients using αGPC3 codrituzumab labeled with iodine-124 ( 124 I). While this valuable work underscores the clinical translatability of radiolabeled antibodies against GPC3, there was no tumor uptake in one patient and low tumor-to-liver ratios in several others [ 18 ]. The authors stated that 89 Zr could have been a reasonable alternative to 124 I, and our findings support further investigation of 89 Zr-αGPC3 immunoPET to overcome challenges encountered with other radioimmunoconjugates. We appreciate the rigorous and ongoing work by our colleagues in the field and believe that parallel approaches to developing GPC3-targeted radiolabeled imaging agents will be beneficial [ 14 – 20 , 23 , 24 ]. In conclusion, humanized αGPC3 successfully targeted GPC3 in vitro and in vivo . Compared with our previously validated murine antibody, 89 Zr-αGPC3 H immunoPET demonstrated comparable HCC detection with highly specific tumor uptake in an orthotopic xenograft mouse model, affirming the efficacy and clinical translatability of 89 Zr-αGPC3 H immunoPET for HCC detection [ 16 ]. Given our GPC3-targeted murine radioimmunoconjugates were previously validated for both immunoPET and cytotoxic RIT, immediate next steps include assessing treatment response using αGPC3 H -based RIT. This developing theranostic joins a growing field of other solid tumors, including colorectal, breast, prostate, renal cell cancers, non-Hodgkin’s lymphoma, and neuroendocrine tumors, and has the potential to transform HCC management [ 10 , 25 , 26 ]. Abbreviations hepatocellular carcinoma (HCC) computed tomography (CT) immuno-positron emission tomography (immunoPET) radioimmunotherapy (RIT ) glypican-3 (GPC3) zirconium-89 ( 89 Zr) murine antibody targeting GPC3 (αGPC3 M ) humanized αGPC3 (αGPC3 H ) chimeric intermediary (αGPC3 C ) deferoxamine (DFO) region of interest (ROI) maximum standardized uptake value (SUV max ) iodine-124 ( 124 I) Declarations Funding: This work was supported by the Wayne and Joan Kuni Foundation Imagination Grant and the NIH/NCI Cancer Center Support Grant P30 CA015704. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions : Lindsay K. Dickerson, Adrienne L. Lehner, Donald K. Hamlin, Kevin P. Labadie, Yawen Li, D. Scott Wilbur, Robert Miyaoka, and James O. Parkcontributed to the study conception and/or design. Material preparation, data collection, and analysis were performed by Lindsay K. Dickerson, Adrienne L. Lehnert, Donald K. Hamlin, Kristin E. Goodsell, Yongjun Liu, and James O. Park. The first draft of the manuscript was written by Lindsay K. Dickerson with editorial input from all authors. All authors read and approved the final manuscript. Data Availability : All data will be available after publication upon request to the corresponding author. Ethics Approval: This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Washington (Protocol #4304-02). This study was performed in accordance with the University of Washington Office of Animal Welfare guidelines for the humane use of animals and was carried out in compliance with the ARRIVE guidelines. Specifically, the minimum number of mice needed to achieve experimental goals were used. Cages were supplied with sufficient bedding, food, water, and enrichment throughout the experiment. Appropriate anesthetic/analgesic, ophthalmic lubricant, heating pads, and sterile surgical technique were used for survival surgical procedures to minimize animal distress. Mice were monitored closely during and immediately after surgery for adequate depth of anesthesia and full recovery, respectively; isotonic saline was administered subcutaneously for fluid support as needed. Mice were evaluated daily for the first week after surgery for signs of distress, weight loss, agitation, sedation, lameness, failure to eat/drink, poor or aberrant grooming, abnormal posture, wound, or skin ulcerations. A composite of general appearance, body condition score (BCS), body weight, and abdominal girth measurements were used to assess well-being. After the first week, mice were evaluated three times weekly for the remainder of the experiment. For bioluminescence imaging and PET/CT, mice were anesthetized and cared for as described in theSupplementary Methods. Both the research team and Veterinary Services were actively involved in evaluation and care of the mice throughout the experiment. At the completion of the experiment, mice were euthanized using CO 2 followed by cervical dislocation according to IACUC protocol. References Philips CA, Rajesh S, Nair DC, Ahamed R, Abduljaleel JK, Augustine P. Hepatocellular carcinoma in 2021: An exhaustive update. Cureus. 2021. https://doi.org/10.7759/cureus.19274 . Chen ZY, Chhatwal J. Changing epidemiology of hepatocellular carcinoma and role of surveillance. In: Hoshida Y, editor. Hepatocellular Carcinoma: Translational Precision Medicine Approaches. Totowa, NJ: Humana; 2019. Singal AG, Parikh ND, Rich NE, John BV, Pillai A. Hepatocellular carcinoma surveillance and staging. In: Hoshida Y, editor. Hepatocellular Carcinoma: Translational Precision Medicine Approaches. Totowa, NJ: Humana; 2019. Llovet JM, Kelley RK, Villanueva A, et al. Hepatocellular carcinoma. 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Supplementary Files ParkSupplementaryMethodsHumanizedGPC3.docx Cite Share Download PDF Status: Published Journal Publication published 22 Aug, 2024 Read the published version in EJNMMI Research → Version 1 posted Reviewers agreed at journal 10 Jun, 2024 Reviewers invited by journal 07 Jun, 2024 Editor invited by journal 07 Jun, 2024 Editor assigned by journal 06 Jun, 2024 First submitted to journal 30 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Goodsell","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Kristin","middleName":"E.","lastName":"Goodsell","suffix":""},{"id":311892291,"identity":"be677f85-f80d-4f18-81a1-6e5d2e2885f5","order_by":5,"name":"Yongjun Liu","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Yongjun","middleName":"","lastName":"Liu","suffix":""},{"id":311892292,"identity":"6031bca9-50cd-45bf-a9e4-8f4bdd8bc799","order_by":6,"name":"Yawen Li","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Li","suffix":""},{"id":311892293,"identity":"04b554f6-1a9d-44c7-b297-f9a377809cb2","order_by":7,"name":"D. Scott Wilbur","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"Scott","lastName":"Wilbur","suffix":""},{"id":311892294,"identity":"1840d852-fe24-40cc-80f2-88fc2ab7f27d","order_by":8,"name":"Robert Miyaoka","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Miyaoka","suffix":""},{"id":311892295,"identity":"4dffedda-1375-4770-8fd7-e7bff516a3e8","order_by":9,"name":"James O. Park","email":"","orcid":"https://orcid.org/0000-0001-6939-1255","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"O.","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2024-05-21 18:43:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4456645/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4456645/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13550-024-01134-1","type":"published","date":"2024-08-22T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59215421,"identity":"339db1fc-6168-4a15-8a70-93e770d9b6ca","added_by":"auto","created_at":"2024-06-27 18:56:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":129236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHumanized αGPC3 binds to GPC3-expressing HepG2 cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro. \u003c/strong\u003e\u003c/em\u003e(a) Flow cytometric mean fluorescence intensity (MFI) of αGPC3\u003csub\u003eM \u003c/sub\u003e(green), αGPC3\u003csub\u003eC \u003c/sub\u003e(aqua), and αGPC3\u003csub\u003eH \u003c/sub\u003e(blue) compared with controls (unlabeled HepG2 cells and FITC-labeled secondary antibody alone, gray). (b) MFI of deferoxamine (DFO)-conjugated αGPC3\u003csub\u003eM \u003c/sub\u003e(dark green) and αGPC3\u003csub\u003eH \u003c/sub\u003e(navy) compared with control (gray)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4456645/v1/6a1cca0d5aa1bf123e5bbbe6.png"},{"id":59215420,"identity":"de922cab-7118-4f1f-bbf8-8b92934dd1ac","added_by":"auto","created_at":"2024-06-27 18:56:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":692054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioluminescence imaging predicts tumor establishment. \u003c/strong\u003eRepresentative BLI image demonstrating predicted tumor establishment in three of the five mice shown. ROI = region of interest. (Position 1 to 5 from left to right; mean photon emission not calculated for positions two and four)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4456645/v1/d369381d862454243ada5e3f.png"},{"id":59215422,"identity":"631a7da7-0b1f-4b22-b1a7-8da3125ccfd5","added_by":"auto","created_at":"2024-06-27 18:56:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2810598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHumanized \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e89\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eZr-αGPC3 immunoPET reliably identifies tumors.\u003c/strong\u003e (a) Axial (top) and sagittal (bottom) PET/CT images of \u003csup\u003e89\u003c/sup\u003eZr-aGPC3\u003csub\u003eH\u003c/sub\u003e-\u003csub\u003e\u0026nbsp; \u003c/sub\u003eand \u003csup\u003e89\u003c/sup\u003eZr-aGPC3\u003csub\u003eM\u003c/sub\u003e-injected mice. (b) Select H\u0026amp;E-stained liver sections. Blue arrowheads denote tumors. Scale bars 100 μm. (c) Tumor radioisotope uptake (%ID/mL). (d) Liver and tumor %ID/mL by mouse. (e) Tumor-to-liver ratio of %ID/mL; circle denotes largest tumors. (f) Tumor maximum standardized uptake (SUV\u003csub\u003emax\u003c/sub\u003e). Each point or thin bar (d) denotes the value for each tumor-bearing mouse (n=5/group); large bars denote the mean with SEM\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4456645/v1/274e968a910ae70eecb1a8ce.png"},{"id":59215423,"identity":"10171f0b-3f05-4d5d-892a-ac3f7b81f1d8","added_by":"auto","created_at":"2024-06-27 18:56:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":154495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue biodistribution is not significantly different between humanized and murine \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e89\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eZr-αGPC3-injected mice.\u003c/strong\u003e (a-b) Mean tissue radioisotope uptake (%ID/g) in mice not imaged with PET (non-tumor-bearing) (n=5/group) (a) and imaged with PET (majority tumor-bearing) (n=6/group) (b), two and five days after injection, respectively. (c) Tumor uptake and (d) tumor-to-liver ratio (%ID/g) in PET-imaged mice. Each point denotes the value for each tumor-bearing mouse (humanized n=4 with negative value excluded (see\u003cem\u003e \u003c/em\u003eSupplementary Methods), murine n=5); bars denote mean with SEM when applicable\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4456645/v1/c430bf2f25f34a197ff3249e.png"},{"id":63300748,"identity":"c6935851-5a07-4cf5-a0e5-917cdce5e461","added_by":"auto","created_at":"2024-08-26 16:17:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4327122,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4456645/v1/5dcbd21d-78a0-42f8-b241-34a08cadfffb.pdf"},{"id":59215424,"identity":"631c87d9-7c46-4cfe-9f54-883d0e4dcbeb","added_by":"auto","created_at":"2024-06-27 18:56:50","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":37435,"visible":true,"origin":"","legend":"","description":"","filename":"ParkSupplementaryMethodsHumanizedGPC3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4456645/v1/cfafa068a2744308bbdce6db.docx"}],"financialInterests":"","formattedTitle":"Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is increasing in incidence worldwide and has become the fastest growing cause of cancer death in the United States, with a median survival of less than one year [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In order to improve survival with current treatments, HCC must be detected early when it is amenable to surgical resection or transplantation [3.4]. However, multiphase, computed tomography (CT) or magnetic resonance imaging frequently misses lesions less than 1 cm, resulting in diagnostic uncertainty, delayed diagnosis, and early recurrence following resection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Innovative technology capable of detecting HCC with enhanced sensitivity and specificity is therefore imperative and pressing.\u003c/p\u003e \u003cp\u003eRadioisotope theranostics, including immuno-positron emission tomography (immunoPET) and radioimmunotherapy (RIT), is an emerging field with the potential to transform HCC diagnosis and therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While yttrium-90 microspheres and iodine-131-labeled lipiodol and metuximab are used in radioembolization therapy, there are currently no FDA approved theranostics for HCC. However, glypican-3 (GPC3)-targeted radioisotopes have shown promise in preclinical and early clinical studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. GPC3 is a cell surface antigen expressed on up to 80% of HCCs but absent in liver parenchyma and benign lesions, making it an accessible and specific target for a theranostic approach [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. GPC3-based imaging has the potential to facilitate earlier, definitive HCC diagnosis and subsequent RIT, thus improving patient survival [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur group previously demonstrated that immunoPET using zirconium-89 (\u003csup\u003e89\u003c/sup\u003eZr)-labeled murine antibody targeting GPC3 (\u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e) reliably identified small HCCs in mice [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Natarajan et al. described the use of \u003csup\u003e89\u003c/sup\u003eZr-labeled humanized αGPC3 for HCC detection in a patient-derived xenograft model [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We built on this important work by humanizing our radioimmunoconjugate (αGPC3\u003csub\u003eH\u003c/sub\u003e) and performing \u003cem\u003ein vitro\u003c/em\u003e and novel \u003cem\u003ein vivo\u003c/em\u003e comparisons to its murine predecessor. Here, we report that \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e targets GPC3 comparably to \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e, resulting in highly specific tumor uptake and successful HCC detection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eCreative Biolabs, Inc. (Shirley, NY) constructed αGPC3\u003csub\u003eH\u003c/sub\u003e by engrafting of the parental murine antibody\u0026rsquo;s complementarity-determining region (CDR). Flow cytometry was used to evaluate \u003cem\u003ein vitro\u003c/em\u003e binding of αGPC3\u003csub\u003eM\u003c/sub\u003e, a chimeric intermediary (αGPC3\u003csub\u003eC\u003c/sub\u003e), αGPC3\u003csub\u003eH\u003c/sub\u003e, and αGPC3-deferoxamine (DFO) to HepG2 cells. Orthotopic xenograft models of HCC were generated as previously described in athymic nude mice (Jackson Laboratories) [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Two weeks after HepG2 cell liver injection, bioluminescence imaging (BLI) was used to estimate tumor establishment. αGPC3 was conjugated with DFO and labeled with \u003csup\u003e89\u003c/sup\u003eZr [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. (For simplicity, \u003csup\u003e89\u003c/sup\u003eZr-DFO-αGPC3 is written as \u003csup\u003e89\u003c/sup\u003eZr-αGPC3.) Mice (n\u0026thinsp;=\u0026thinsp;11 per group) were injected retro-orbitally with 8.1 to 10 megabecquerels (MBq) of \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e or \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e. Mice with tumors predicted using BLI (n\u0026thinsp;=\u0026thinsp;6 per group) underwent PET/CT five days after \u003csup\u003e89\u003c/sup\u003eZr-αGPC3 injection. Maximum activity concentration (MBq/mL) was measured in a 2D region of interest (ROI) to calculate tumor radioisotope uptake (percent injected dose per milliliter, %ID/mL), tumor-to-liver ratio, and tumor maximum standardized uptake value (SUV\u003csub\u003emax\u003c/sub\u003e). Biodistribution studies using gamma counts were performed separately in non-tumor-bearing, non-imaged mice two days after injection and in PET-imaged mice after imaging completion to determine %ID/g for select organs and tumors. Livers from PET-imaged mice were processed for histopathology. Details provided in Supplementary Methods.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHumanized aGPC3 and αGPC3-DFO maintains GPC3 binding in vitro\u003c/h2\u003e \u003cp\u003eBinding to HepG2 cell surface GPC3 by unconjugated αGPC3\u003csub\u003eM\u003c/sub\u003e, αGPC3\u003csub\u003eC\u003c/sub\u003e, and αGPC3\u003csub\u003eH\u003c/sub\u003e was confirmed by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Binding of DFO-conjugated and αGPC3\u003csub\u003eM\u003c/sub\u003e to GPC3 was overall similar to the unconjugated antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Binding of αGPC3\u003csub\u003eH\u003c/sub\u003e and αGPC3\u003csub\u003eC\u003c/sub\u003e to GPC3 was greater than αGPC3\u003csub\u003eM\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBioluminescence imaging predicts tumor establishment\u003c/h2\u003e \u003cp\u003eTumors were identified with BLI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Mice were assigned to \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e and \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e injection such that mean photon emission (photons/sec) in tumor-containing ROIs was similar between groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMouse-specific immunoPET and biodistribution data correlated with tumor size\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eH4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eH5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eH6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eM7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eM9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eM10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eM11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eM12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiolum.\u003c/p\u003e \u003cp\u003e(Photons/\u003c/p\u003e \u003cp\u003eSec)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.7x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.2x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.6x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.4x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.1x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.5x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.1x10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4.7x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e2x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRank\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor\u003c/p\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor %ID/mL (PET)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003cp\u003e%ID/mL\u003c/p\u003e \u003cp\u003e(PET)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor: Liver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e19.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSUV\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor\u003c/p\u003e \u003cp\u003e%ID/g\u003c/p\u003e \u003cp\u003e(Gamma counter)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-426\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e458\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver %ID/g\u003c/p\u003e \u003cp\u003e(Gamma counter)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor: Liver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-21\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003ea\u003c/sup\u003eNot measured due to minimal apparent signal.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003eb\u003c/sup\u003eH6, M12: no tumor identified on PET/CT or histopathology (blank\u0026thinsp;=\u0026thinsp;not applicable).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003ec\u003c/sup\u003eRank from largest (1) to smallest (10/11).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003ed\u003c/sup\u003e -426 included in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec calculations; -21 excluded from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed (see Supplementary Methods).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eαGPC3\u003csub\u003eH\u003c/sub\u003e is amenable to \u003csup\u003e89\u003c/sup\u003eZr radiolabeling\u003c/h2\u003e \u003cp\u003eThe radiochemical purity of both \u003csup\u003e89\u003c/sup\u003eZr-αGPC3 antibodies was \u0026gt;\u0026thinsp;98% and the specific activity was 0.14 GBq/mg. Details provided in Supplementary Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHumanized \u003csup\u003e89\u003c/sup\u003eZr-aGPC3 immunoPET reliably identifies tumors\u003c/h2\u003e \u003cp\u003eFive of six mice injected with \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e and \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e, respectively, demonstrated discrete hepatic localizations of increased PET intensity consistent with tumors (H1-H5, M7-M11; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Mean bioluminescence of PET-identified tumors (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was equivalent between groups (4.8x10\u003csup\u003e8\u003c/sup\u003e vs 6.3x10\u003csup\u003e8\u003c/sup\u003e +/- 4.5x10\u003csup\u003e8\u003c/sup\u003e photon/sec, p\u0026thinsp;=\u0026thinsp;0.75). Histopathology identified tumors in H1-H5 and M7-M11, but not in mice without PET-identified tumors (H6, M12) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Tumor uptake (97 vs 61 +/- 50%ID/mL, p\u0026thinsp;=\u0026thinsp;0.42) and tumor-to-liver ratio (12 vs 11+/- 7.6, p\u0026thinsp;=\u0026thinsp;0.68) were not significantly different between groups, despite significantly increased liver uptake in the \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e-injected mice (9.1 vs 6.1 +/- 1.0%ID/mL, p\u0026thinsp;=\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-e). SUV\u003csub\u003emax\u003c/sub\u003e was equivalent between groups (39 vs 24 +/- 21, p\u0026thinsp;=\u0026thinsp;0.51) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNo difference in tumor radioimmunoconjugate uptake on biodistribution studies\u003c/h2\u003e \u003cp\u003eIn non-imaged mice, the liver had the highest %ID/g calculated from gamma counter measurements followed by the lungs and spleen, with no significant difference between \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e and \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e-injected mice (mean 10 vs 8.8 +/- 4.6%ID/g, p\u0026thinsp;=\u0026thinsp;0.77) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In PET-imaged mice, tumor uptake was 7-fold greater than other organs, with equivalent organ uptake (mean 7.1 vs 6.3 +/- 2.7%ID/g, p\u0026thinsp;=\u0026thinsp;0.75), tumor uptake (170 vs 149 +/- 241%ID/g, p\u0026thinsp;=\u0026thinsp;0.93), and tumor-to-liver ratio of %ID/g (22 vs 24 +/- 19, p\u0026thinsp;=\u0026thinsp;0.94) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHumanized αGPC3 specifically targeted GPC3 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, enabling HCC detection with immunoPET in an orthotopic xenograft mouse model. This proof-of-concept study builds on our prior research validating a murine radioimmunoconjugate for a theranostic approach to HCC, with potential to improve diagnosis, treatment, and survival [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results demonstrate that humanization of \u003csup\u003e89\u003c/sup\u003eZr-αGPC3 did not alter the highly avid binding to GPC3 on HepG2 cells and liver tumor xenografts. First, flow cytometry established at least equivalent, if not greater, binding of αGPC3\u003csub\u003eH\u003c/sub\u003e to GPC3 compared with αGPC3\u003csub\u003eM\u003c/sub\u003e, with minimal change when conjugated with DFO. Next, quality assurance of \u003csup\u003e89\u003c/sup\u003eZr labeling confirmed that \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e maintained high purity and specific activity. The majority of our experiments focused on the novel \u003cem\u003ein vivo\u003c/em\u003e comparison between \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e and \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e. Five of six tumors in each group were detected by immunoPET, with no difference between groups in mean IVIS bioluminescence. PET/CT data revealed no significant difference in mean tumor uptake and tumor-to-liver ratios (%ID/mL). Similarly, biodistribution analysis showed no difference in mean organ uptake, tumor uptake, and tumor-to-liver ratios (%ID/g).\u003c/p\u003e \u003cp\u003eWhile finding comparability between \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e and \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e achieved the study\u0026rsquo;s primary goal, additional details are worth noting. First, tumor uptake varied based on tumor size, with higher uptake in larger tumors as previously demonstrated [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. %ID/g (gamma counter) results were greater than %ID/mL (PET) due to limited PET/CT spatial resolution causing partial volume effect; hence, there could be a larger discrepancy between %ID/g and %ID/mL values in mice with smaller tumors (e.g., H3, M9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While further consideration of the clinical impact of partial volume effect is warranted, this finding does highlight the successful detection of small tumors with \u003csup\u003e89\u003c/sup\u003eZr-αGPC3 immunoPET. Second, background liver uptake was greater in the \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e group, which could imply Fc-mediated liver uptake of \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e. However, our prior studies of mice injected with \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e compared with non-GPC3-targeting and GPC3-blocked controls demonstrated similar background liver uptake [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Furthermore, the tumor-to-liver ratio by nature adjusts for such variables, with no difference between groups suggesting that tumor uptake was also proportionally higher in the humanized antibody group. In fact, tumor-to-liver ratios of 12 or greater indicate \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e is highly specific for GPC3-expressing tumors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTumor presence was histopathologically confirmed in mice with PET-identified tumors, while no tumors were found on histologic analysis of livers without PET-identified tumors. A limitation here is that, while meticulous gross examination of the liver and histopathologic analysis of suspected tumors was performed, serial sectioning of the entire left hepatic lobe was not undertaken due to limited funding. Therefore, the discordance between BLI and PET for H6 and M12 is unresolved. Of note, the three-week interval between imaging modalities was longer than in previous studies and thus tumor involution may have occurred.\u003c/p\u003e \u003cp\u003eOur study is similar to those from other groups in that it underscores the potential of human αGPC3 to detect HCC with immunoPET, however, there are key differences. Tumor-to-liver ratios by PET/CT and biodistribution analyses were notably higher than those reported by Natarajan \u003cem\u003eet al\u003c/em\u003e. using a similar \u003csup\u003e89\u003c/sup\u003eZr-labeled human αGPC3 IgG antibody and Fayn \u003cem\u003eet al\u003c/em\u003e. using \u003csup\u003e89\u003c/sup\u003eZr-labeled GPC3-targeting HN3 single-domain antibodies. In addition, there was a greater relative difference between tumor uptake and uptake in organs such as the heart, lungs, gastrointestinal tract, and kidneys on biodistribution analysis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While different methods for model development and radioimmunoconjugate injection used may affect the results such that they are not directly comparable [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], it is possible that our humanized antibody has a higher specificity for GPC3-expressing tumors. Furthermore, it should be noted that tumor-to-liver ratios were measured five days after injection in this study compared with one to seven days after injection in the aforementioned studies, however our prior experiments with \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eM\u003c/sub\u003e demonstrated high tumor-to-liver ratios calculated from four hours up to seven days after injection [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Finally, Carrasquillo \u003cem\u003eet al\u003c/em\u003e. conducted a phase I clinical study of PET/CT in HCC patients using αGPC3 codrituzumab labeled with iodine-124 (\u003csup\u003e124\u003c/sup\u003eI). While this valuable work underscores the clinical translatability of radiolabeled antibodies against GPC3, there was no tumor uptake in one patient and low tumor-to-liver ratios in several others [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The authors stated that \u003csup\u003e89\u003c/sup\u003eZr could have been a reasonable alternative to \u003csup\u003e124\u003c/sup\u003eI, and our findings support further investigation of \u003csup\u003e89\u003c/sup\u003eZr-αGPC3 immunoPET to overcome challenges encountered with other radioimmunoconjugates. We appreciate the rigorous and ongoing work by our colleagues in the field and believe that parallel approaches to developing GPC3-targeted radiolabeled imaging agents will be beneficial [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, humanized αGPC3 successfully targeted GPC3 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Compared with our previously validated murine antibody, \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e immunoPET demonstrated comparable HCC detection with highly specific tumor uptake in an orthotopic xenograft mouse model, affirming the efficacy and clinical translatability of \u003csup\u003e89\u003c/sup\u003eZr-αGPC3\u003csub\u003eH\u003c/sub\u003e immunoPET for HCC detection [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Given our GPC3-targeted murine radioimmunoconjugates were previously validated for both immunoPET and cytotoxic RIT, immediate next steps include assessing treatment response using αGPC3\u003csub\u003eH\u003c/sub\u003e-based RIT. This developing theranostic joins a growing field of other solid tumors, including colorectal, breast, prostate, renal cell cancers, non-Hodgkin\u0026rsquo;s lymphoma, and neuroendocrine tumors, and has the potential to transform HCC management [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ehepatocellular carcinoma (HCC)\u003c/p\u003e\n\u003cp\u003ecomputed tomography (CT)\u003c/p\u003e\n\u003cp\u003eimmuno-positron emission tomography (immunoPET)\u003c/p\u003e\n\u003cp\u003eradioimmunotherapy (RIT\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eglypican-3 (GPC3)\u003c/p\u003e\n\u003cp\u003ezirconium-89 (\u003csup\u003e89\u003c/sup\u003eZr)\u003c/p\u003e\n\u003cp\u003emurine antibody targeting GPC3 (\u0026alpha;GPC3\u003csub\u003eM\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003ehumanized \u0026alpha;GPC3 (\u0026alpha;GPC3\u003csub\u003eH\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003echimeric intermediary (\u0026alpha;GPC3\u003csub\u003eC\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003edeferoxamine (DFO)\u003c/p\u003e\n\u003cp\u003eregion of interest (ROI)\u003c/p\u003e\n\u003cp\u003emaximum standardized uptake value (SUV\u003csub\u003emax\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003eiodine-124 (\u003csup\u003e124\u003c/sup\u003eI)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the Wayne and Joan Kuni Foundation Imagination Grant and the NIH/NCI Cancer Center Support Grant P30 CA015704.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Lindsay K. Dickerson, Adrienne L. Lehner, Donald K. Hamlin, Kevin P. Labadie, Yawen Li, D. Scott Wilbur, Robert Miyaoka, and James O. Parkcontributed to the study conception and/or design. Material preparation, data collection, and analysis were performed by Lindsay K. Dickerson, Adrienne L. Lehnert, Donald K. Hamlin, Kristin E. Goodsell, Yongjun Liu, and James O. Park. The first draft of the manuscript was written by Lindsay K. Dickerson with editorial input from all authors. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e: All data will be available after publication upon request to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u0026nbsp;\u003c/strong\u003eThis study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Washington (Protocol #4304-02). This study was performed in accordance with the University of Washington Office of Animal Welfare guidelines for the humane use of animals and was carried out in compliance with the ARRIVE guidelines. Specifically, the minimum number of mice needed to achieve experimental goals were used. Cages were supplied with sufficient bedding, food, water, and enrichment throughout the experiment. Appropriate anesthetic/analgesic, ophthalmic lubricant, heating pads, and sterile surgical technique were used for survival surgical procedures to minimize animal distress. Mice were monitored closely during and immediately after surgery for adequate depth of anesthesia and full recovery, respectively; isotonic saline was administered subcutaneously for fluid support as needed. Mice were evaluated daily for the first week after surgery for signs of distress, weight loss, agitation, sedation, lameness, failure to eat/drink, poor or aberrant grooming, abnormal posture, wound, or skin ulcerations. A composite of general appearance, body condition score (BCS), body weight, and abdominal girth measurements were used to assess well-being. After the first week, mice were evaluated three times weekly for the remainder of the experiment. For bioluminescence imaging and PET/CT, mice were anesthetized and cared for as described in theSupplementary Methods. Both the research team and Veterinary Services were actively involved in evaluation and care of the mice throughout the experiment. At the completion of the experiment, mice were euthanized using CO\u003csub\u003e2\u003c/sub\u003e followed by cervical dislocation according to IACUC protocol. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePhilips CA, Rajesh S, Nair DC, Ahamed R, Abduljaleel JK, Augustine P. Hepatocellular carcinoma in 2021: An exhaustive update. 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The theranostic promise for neuroendocrine tumors in the late 2010s \u0026ndash; where do we stand, where do we go? \u003cem\u003eTheranostics\u003c/em\u003e. 2018; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/thno.30357\u003c/span\u003e\u003cspan address=\"10.7150/thno.30357\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"hepatocellular carcinoma, glypican-3 (GPC3), Zirconium-89, immuno-positron emission tomography (immunoPET), theranostics","lastPublishedDoi":"10.21203/rs.3.rs-4456645/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4456645/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Purpose: Glypican-3 (GPC3)-targeted radioisotope immuno-positron emission tomography (immunoPET) may lead to earlier and more accurate diagnosis of hepatocellular carcinoma (HCC), thus facilitating curative treatment, decreasing early recurrence, and enhancing patient survival. We previously demonstrated reliable HCC detection using a zirconium-89-labeled murine anti-GPC3 antibody (89Zr-αGPC3M) for immunoPET. This study evaluated the efficacy of the humanized antibody successor (αGPC3H) to further clinical translation of a GPC3-based theranostic for HCC. Methods:In vitro αGPC3 binding to HepG2 cells was assessed by flow cytometry. In vivo 89Zr-αGPC3H and 89Zr-αGPC3M tumor uptake was evaluated by PET/CT and biodistribution studies in an orthotopic xenograft mouse model of HCC. Results: αGPC3H maintained binding to GPC3 in vitro and 89Zr-αGPC3H immunoPET identified liver tumors in vivo. PET/CT and biodistribution analyses demonstrated high 89Zr-αGPC3H tumor uptake and tumor-to-liver ratios, with no difference between groups. Conclusion: Humanized αGPC3 successfully targeted GPC3 in vitro and in vivo. 89Zr-αGPC3H immunoPET had comparable tumor detection to 89Zr-αGPC3M, with highly specific tumor uptake, making it a promising strategy to improve HCC detection.","manuscriptTitle":"Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 18:56:45","doi":"10.21203/rs.3.rs-4456645/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-06-10T09:14:23+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-07T17:19:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"EJNMMI Research","date":"2024-06-07T10:04:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-06T19:26:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Research","date":"2024-05-31T01:20:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70761aa8-efdb-4393-9eb1-025771e0661b","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:10:23+00:00","versionOfRecord":{"articleIdentity":"rs-4456645","link":"https://doi.org/10.1186/s13550-024-01134-1","journal":{"identity":"ejnmmi-research","isVorOnly":false,"title":"EJNMMI Research"},"publishedOn":"2024-08-22 15:58:08","publishedOnDateReadable":"August 22nd, 2024"},"versionCreatedAt":"2024-06-27 18:56:45","video":"","vorDoi":"10.1186/s13550-024-01134-1","vorDoiUrl":"https://doi.org/10.1186/s13550-024-01134-1","workflowStages":[]},"version":"v1","identity":"rs-4456645","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4456645","identity":"rs-4456645","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}