Supplementary Table S2: Recipient - Donor Characteristics

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.

Background: Ex vivo gene-modified autologous cell products are increasingly explored to cure monogenic disorders (e.g. sickle cell disease, thalassemia). Efficient removal of bone marrow (BM)-resident hematopoietic stem/progenitor cells (HSPCs) is a requirement for successful engraftment of such cell products. To accomplish this, various conditioning regimens have been utilized, each with their own unique profile of target specificity and on- and off-target toxicities. Currently most widely used is busulfan but unwanted non-hematologic toxicities have provided the impetus to develop more specific approaches that spare normal tissues better. Monoclonal antibodies (mAbs) conjugated with either small molecule toxins or radionuclides to selectively target phenotypically distinct cells have gained attention for this purpose. Of particular interest as target is CD45, a glycoprotein expressed at very high copy number exclusively on almost all blood cells. CD45-targeted radioimmunotherapy (RIT) with the beta-emitter, iodine-131, has proven effective to augment conditioning before allogeneic hematopoietic cell transplantation (HCT). Here, we evaluated CD45-directed RIT using a the highly potent alpha emitter, astatine-211 (211At) as payload as sole conditioning agent before autologous HCT of ex vivo gene-edited HSPCs in a nonhuman primate (NHP) HCT and gene therapy model. Methods: A humanized version of the human/NHP cross-reactive CD45 mAb, BC8 (HuBC8), was conjugated with isothiocyanatophenethyl-ureido-closo-decaborate(2-) (HuBC8-B10), a boron cage molecule to enable subsequent labeling with 211At as done in our early phase clinical trials. Simultaneously, NHP CD34+ cells were mobilized with G-CSF/AMD3100, collected via leukapheresis, and cryopreserved after being gene modified ex vivo using adenine base editors to reactivate fetal hemoglobin (HBG) production as well as to delete CD33. 72 hours after administration of a single dose of 211At-labeled HuBC8-B10, gene-edited CD34+ cells were thawed and infused. Animals received 300 µCi/kg (n=2) or 400 µCi/kg (n=1) of 211At with 0.5 mg/kg of HuBC8-B10. All animals were monitored for toxicities, and blood count recovery as well as engraftment of gene-modified HSPCs and blood lineages which were assessed serially using flow cytometry and next generation sequencing. Results: The editing efficiency of CD33 and HBG in the infusion product ranged from 50-77% and 15-38%, respectively, with no measurable impact on cell viability or erythro-myeloid differentiation potential of edited cells in colony-forming cell assays. A total of 2-4x106 CD34+ cells/kg were infused into the animals and rapid recovery of neutrophils and platelets seen in between 6-8 and 10-13 days, respectively. Transient weight loss over the first 20-30 days was seen (n=3) and gastric ulcer treatment needed (n=1). An 211At dose of 300 µCi/kg led to incomplete myeloablation with neutrophils remaining above 400/µl, whereas full myeloablation was seen at a dose of 400 µCi/kg. Ablation of monocytes, lymphocytes, and platelets was seen in all animals, whereas CD45-negative erythrocytes were spared and the hemoglobin remaining stable throughout the study. Two animals were entirely transfusion-independent and the third animal receive a single platelet transfusion. Dose-dependent engraftment of gene-editing in the peripheral blood was seen with 20-40% CD33-negative cells and 5-10% HBF reactivation in the two animals receiving 211At at 300 µCi/kg as compared to 80% of CD33-negative cells and 20% HBF reactivation in the single animal receiving 211At at 400 µCi/kg. Full recovery of the BM stem cell compartment was confirmed at 3-month post-transplant by flow cytometry. Conclusion: CD45-targeted alpha emitter-based RIT with 211At enables stable engraftment of ex vivo gene-edited autologous stem cell products. Our studies identify 211At-CD45 RIT as a targeted alternative for myeloablative conditioning followed by autologous transplantation of gene-modified HSPCs. 211At-HuBC8-B10 is well tolerated with only minimal adverse reactions observed in NHPs. While 211At-CD45 RIT demonstrates selective depletion of CD45-positive white blood cells as well as HSPCs in the BM, CD45-negative erythrocytes are spared, likely contributing to the minimal supportive care needs needed following autografting.

AbstractPurpose: Hematopoietic cell transplantation (HCT) has curative potential for myeloid malignancies, though many patients cannot tolerate myeloablative conditioning with high-dose chemotherapy alone or with total-body irradiation (TBI). Here we report long-term outcomes from a phase I/II study using iodine-131 (131I)-anti-CD45 antibody BC8 combined with nonmyeloablative conditioning prior to HLA-haploidentical HCT in adults with high-risk relapsed/ refractory acute myeloid or lymphoid leukemia (AML or ALL), or myelodysplastic syndrome (MDS; ClinicalTrials.gov, NCT00589316). Patients and Methods: Patients received a tracer diagnostic dose before a therapeutic infusion of 131I­anti-CD45 to deliver escalating doses (12–26 Gy) to the dose-limiting organ. Patients subsequently received fludarabine, cyclophosphamide (CY), and 2 Gy TBI conditioning before haploidentical marrow HCT. GVHD prophylaxis was posttransplant CY plus tacrolimus and mycophenolate mofetil. Results: Twenty-five patients (20 with AML, 4 ALL and 1 high-risk MDS) were treated; 8 had ≥ 5% blasts by morphology (range 9%–20%), and 7 had previously failed HCT. All 25 patients achieved a morphologic remission 28 days after HCT, with only 2 patients showing minimal residual disease (0.002–1.8%) by flow cytometry. Median time to engraftment was 15 days for neutrophils and 23 days for platelets. Point estimates for overall survival and progression-free survival were 40% and 32% at 1 year, and 24% at 2 years, respectively. Point estimates of relapse and nonrelapse mortality at 1 year were 56% and 12%, respectively. Conclusions: 131I-anti-CD45 radioimmunotherapy prior to haploidentical HCT is feasible and can be curative in some patients, including those with disease, without additional toxicity.

Background: CD45-targeted radioimmunotherapy (RIT) has long been explored as augmentation of conditioning before hematopoietic cell transplantation (HCT). The CD45 monoclonal antibody (mAb) most exploited for this purpose is BC8. Validating this approach, BC8 labeled with iodine-131 (131I-apamistamab [Iomab-B]) followed by allogeneic HCT was recently shown to improve outcomes of older adults with relapsed/refractory AML relative to conventional care. However, as a murine mAb, BC8 has important limitations, including substantial infusion toxicities and development of human anti-mouse mAbs, which preclude BC8 redosing and future use of any other murine mab. To overcome these limitations, we humanized BC8 and tested its anti-tumor properties in vivo using the potent alpha-emitter, astatine-211 (211At), as payload. Methods and Results: BC8 was humanized by grafting its complementarity-determining regions (CDRs) into the human variable domain germline heavy and light chain sequences with highest homology to BC8's sequences. Two variants were generated, one containing murine CDRs only and the other additionally including 2 murine residues within the non-CDR human light chain variable region and 4 murine residues within the non-CDR human heavy chain variable region. In competitive ELISA assays, CDR-grafted BC8 with murine back mutations (“HuBC8”) showed only minimally reduced binding to human CD45 compared to chimeric BC8 (ChiBC8) or murine BC8, and flow cytometrically determined binding of HuBC8 to CD45+ leukemia cell lines was similar to that of ChiBC8. For comparative in vivo testing, BC8, ChiBC8, and HuBC8 were conjugated with isothiocyanatophenethyl-ureido-closo-decaborate(2-) (B10-NCS), a boron cage molecule used for subsequent labeling with 211At as done in our early phase clinical trials with 211At-based RIT. For assessment of CD45+ cell targeting, NOD-Rag1null IL2rɣnull/J (NRG) mice were injected with CD45+ MOLM-13 cells in the flank to generate human AML cell flank tumors, followed by a single infusion of BC8 or HuBC8 labeled with 10 µCi of 211At. Tissues were harvested at 24 hours for analysis on a gamma counter and demonstrated similar tumor cell accumulation of radiolabeled BC8 and HuBC8. In vivo efficacy studies with ChiBC8, HuBC8, and non-binding isotype control mAbs (13R4; all human IgG4 frameworks) were performed by injection of 0.2x106 luciferase-transduced MOLM-13 or ML-1 cells into tail veins of NRG mice to generate disseminated human AML. 2 days later, groups of 8 mice were treated with radiolabeled mAbs (40 µCi 211At/animal); one group did not receive any mAb. In both leukemia models, ChiBC8 and HuBC8 extended the survival of treated mice relative to 13R4 (P<0.0001), without significant difference between ChiBC8 and HuBC8 (for MOLM-13: median survival 39.5 days [13R4]) vs. 60 days [ChiBC8] vs. 59 days [HuBC8]; for ML-1: 73 days vs. 138 days vs. 114 days), demonstrating potent in vivo anti-tumor efficacy of 211At-NCS-HuBC8 RIT. Since Fc engineering to minimize Fc receptor interactions has been shown to improve RIT and its therapeutic ratio (i.e. tumor-to-normal cell targeting), we then compared ChiBC8/IgG1 with ChiBC8 using frameworks with reduced Fc binding properties (IgG4, IgG4PAA, IgG2m4, and IgG2σ). There were no significant differences in CD45+ cell targeting in NRG mice bearing MOLM-13 flank tumors. However, there were substantial differences in anti-leukemia efficacies between different antibody frameworks, with ChiBC8/IgG1 labeled with 20 µCi 211At leading to longest survival of NRG mice whereas 211At-NCS-ChiBC8 with IgG4 ProAlaAla, IgG2m4, or IgG2σ did not extend survival of mice beyond what was accomplished with 211At-NCS-13R4 (P<0.0001; median survival 28.5 days [13R4/IgG1]) vs. not reached [ChiBC8/IgG1] vs. 95.5 days [ChiBC8/IgG4] vs. 36 days [ChiBC8/IgG2m4] vs. 27.5 days [ChiBC8/IgG4PAA] vs. 25 days [ChiBC8/IgG2σ]). Increasing the specific activity further increased the anti-leukemia efficacy of 211At-NCS-HuBC8. Conclusions: 211At-NCS-HuBC8 shows similar in vivo CD45+ cell targeting properties and anti-leukemia efficacy as murine 211At-NCS-BC8. Our studies identify the antibody framework (with greatest efficacy so far observed with IgG1) and specific activity as critical factors for the efficacy of 211At-NCS-HuBC8. Together, these study support further development of HuBC8 as antibody for possible clinical RIT applications.

The α-emitter 211At deposits a high amount of energy within a few cell diameters, resulting in irreparable DNA double-strand breaks while minimizing off-target toxicity. We investigated the use of the 211At-labeled anti-CD45 monoclonal antibody (mAb) 211At-CD45-B10 as a nonmyeloablative conditioning regimen for dog-leukocyte-antigen-haploidentical hematopoietic cell transplantation. Methods: Seventeen healthy dogs were injected with either a 0.50 (n = 14) or 0.75 (n = 3) mg/kg dose of anti-CD45 mAb labeled with 211At (8.436-23.199 MBq [0.228-0.627 mCi/kg]) on day -3. Peripheral blood stem cells from dog-leukocyte-antigen-haploidentical donors were given on day 0. Peripheral blood chimerism was calculated by polymerase chain reaction assays, and blood clearance of the radioimmunoconjugate was studied using enzyme-linked immunosorbent assay and radioactivity measurements of serial blood samples. Results: All dogs achieved donor chimerism by day 28 (range, 27%-100%). The hematopoietic engraftment rate was 100%, though engraftment durability was variable. No difference in absorbed dose to blood was seen for the 2 mAb dosing levels studied. Neutropenia (0-29 cells/μL), lymphocytopenia (36-130 cells/μL), and thrombocytopenia (1.5-9 × 103/μL) with prompt recovery were observed. The main adverse nonhematologic event related to 211At-CD45-B10 was mild reversible transaminitis. Graft-versus-host disease was not seen. Twelve of the 17 dogs survived over 30 d, with donor chimerism ranging from 3% to 99%. Conclusion: The results suggest that nonmyeloablative conditioning with 211At-CD45-B10 could be used in haploidentical hematopoietic cell transplantation though with variable engraftment.

Figures including: Supplemental Figure 1- Dose finding pharmacokinetics. Supplemental Figure 2- Dose finding tissue biodistribution. Supplemental Figure 3- body weight over time in a therapy study. Supplemental Figure 4- blood counts over time in a therapy study. Supplemental Figure 5- fusion protein binding to Ramos and Granta cell lines

Supplementary Materials, Methods and relevant AA sequences

Background Early intrahepatic recurrence is common after surgical resection of hepatocellular carcinoma (HCC) and leads to increased morbidity and mortality. Insensitive and nonspecific diagnostic imaging contributes to EIR and results in missed treatment opportunities. In addition, novel modalities are needed to identify targets amenable for targeted molecular therapy. In this study, we evaluated a zirconium-89 radiolabeled glypican-3 (GPC3) targeting antibody conjugate ( 89 Zr-αGPC3) for use in positron emission tomography (PET) for detection of small, GPC3 + HCC in an orthotopic murine model. Athymic nu/J mice received hepG2, a GPC3 + human HCC cell line, into the hepatic subcapsular space. Tumor-bearing mice were imaged by PET/computerized tomography (CT) 4 days after tail vein injection of 89 Zr-αGPC3. Livers were then excised for the tumors to be identified, measured, bisected, and then serially sectioned at 500 μm increments. Sensitivity and specificity of PET/CT for 89 Zr-αGPC3-avid tumors were assessed using tumor confirmation on histologic sections as the gold standard. Results In tumor-bearing mice, 89 Zr-αGPC3 avidly accumulated in the tumor within four hours of injection with ongoing accumulation over time. There was minimal off-target deposition and rapid bloodstream clearance. Thirty-eight of 43 animals had an identifiable tumor on histologic analysis. 89 Zr-αGPC3 immuno-PET detected all 38 histologically confirmed tumors with a sensitivity of 100%, with the smallest tumor detected measuring 330 μm in diameter. Tumor-to-liver ratios of 89 Zr-αGPC3 uptake were high, creating excellent spatial resolution for ease of tumor detection on PET/CT. Two of five tumors that were observed on PET/CT were not identified on histologic analysis, yielding a specificity of 60%. Conclusions 89 Zr-αGPC3 avidly accumulated in GPC3 + tumors with minimal off-target sequestration. 89 Zr-αGPC3 immuno-PET yielded a sensitivity of 100% and detected sub-millimeter tumors. This technology may improve diagnostic sensitivity of small HCC and select GPC3 + tumors for targeted therapy. Human trials are warranted to assess its impact.

Table S1 presents mouse survival data for in vivo experiments

PDF - 156K, Supplementary Figure 1, demonstrating CD38 receptor surface stability. Supplementary Figure 2, demonstrating animal body weight following anti-CD38 pretargeted radioimmunotherapy. Supplemental methods describing fusion protein construction as well as endocytosis and trichloroacetic acid (TCA) precipitation assays.

Click to increase image sizeClick to decrease image size Disclosure statementAll authors declare no competing conflict of interest.Data availability statementFor original data and reagents, please contact the corresponding author (rwalter@fredhutch.org).Additional informationFundingResearch reported in this publication was supported by the National Institutes of Health/National Cancer Institute (NIH/NCI; R01-CA172582).

BACKGROUND: Unprecedented reductions in multiple myeloma (MM) tumor burden after treatment with B cell maturation antigen (BCMA) directed bispecific T-cell engagers, or chimeric antigen receptor modified T-cells, have translated into improved progression free and overall survival; however, virtually all patients are predicted to ultimately relapse. The mechanisms of resistance to BCMA targeting agents are currently being explored, but presumably reflect persistence of MM cell subpopulations that either lack target antigen or otherwise evade immune-mediated tumor cell killing. Antigen targeted delivery of radiation to tumor cells leverages a unique effector mechanism that capitalizes on the exquisite sensitivity of malignant plasma cells to radiation. The α-emitter astatine-211 ( 211At) has particular promise as it deposits a very large amount of energy (~100 keV/μm) within a few cell diameters (50-90 μm) resulting in irreparable double-stranded DNA breaks. The high energy cell killing mediated by 211At is agnostic to MM cell heterogeneity, facilitates bystander delivery of radiation to target antigen negative clones, and limits radiation exposure to normal cells. We hypothesized that 211At targeting BCMA may be uniquely suited to eliminate residual MM cells. METHODS: We conjugated a human IgG1 anti-BCMA mAb and an isotype matched nonbinding control mAb (ofatumumab), with the amine-reactive labeling agent B10-NCS to enable 211At radiolabeling. In biodistribution studies of 211At-BCMA-B10 and 211At-ofatumumab-B10 using NOD.Cg-Rag1 tm1MomIl2rg tm1Wjl/SzJ (NRG) mice bearing flank MM tumor cell xenografts (MM1R or NCI-H929; n=5 mice/group), we demonstrated tumor-specific uptake of BCMA-B10 relative to control mAb and identified the optimal dose of 211At-mAb-B10 (210μg) for assessing therapeutic efficacy. In subsequent therapy studies, NRG mice (n=7-10/group) received 0.3-0.4e6 NCI-H929 luc tumor cells by tail vein injection 6-7 days prior to administration of 210 µg of 211At-BCMA-B10 or 211At-ofatumumab-B10 or no therapy (non-treatment control). Animal body weight was monitored serially, and disease was tracked using in vivo bioluminescence imaging (IVIS Spectrum) performed at 8 or more timepoints over 150 days. RESULTS: Disease elimination was defined as survival with no measurable disease by IVIS at 150 days following treatment. All mice receiving 6 or 8 µCi of 211At-BCMA-B10 (n=36) were cured (100%) after a single infusion of 211At-BCMA-B10. Mice in the untreated control groups uniformly experienced exponential tumor growth. At the lowest 211At dose (6 µCi), all BCMA targeted mice survived for more than 150 days while no untreated control mice survived beyond day 60 (median duration of survival 49 days [range 42-60 days]). Mice receiving 211At-ofatumumab had expected attenuation in tumor growth relative to untreated controls (presumably a consequence of non-specific radiation exposure); with a median duration of survival of 85 days; no mice survived beyond 108 days (range 65-108 days) [Figure 1, Figure 2]. No significant toxicity was observed in the 211At-BCMA-B10 treated groups. Among mice receiving 8 μCi of 211At-BCMA-B10 median animal body weight on day 14 post-treatment was 106% of baseline (range 97%-109%). Among mice receiving 6 μCi of 211At-BCMA-B10 the median body weight on day 14 was 107% of baseline (range 100% to 111%) and on day 150 the median weight was 123% of baseline (range 116% to 126%). CONCLUSIONS: Tumor responses are encouraging with 100% of mice cured of MM after receiving low doses of BCMA targeted 211At. While we have previously demonstrated that 211At-CD38 could eliminate MM in a similar model system, in that setting fewer than 50% of mice bearing the same NCI-H929 Luc tumors, and treated with 8 μCi of 211At, survived to day 150 [O'Steen S. Blood, 2019]. In contrast, here we show that 100% of mice are cured after receiving 6 μCi of 211At-BCMA-B10. These findings support further evaluation of BCMA targeted 211At in other tumor model systems and in clinical trials aimed at eliminating minimal residual disease.

Targeted radiopharmaceutical therapy with alpha-particle emitters (αRPT) is advantageous in cancer treatment because the short range and high local energy deposition of alpha particles enable precise radiation delivery and efficient tumor cell killing. However, these properties create sub-organ dose deposition effects that are not easily characterized by direct gamma-ray imaging (PET or SPECT). We present a computational procedure to determine the spatial distribution of absorbed dose from alpha-emitting radionuclides in tissues using digital autoradiography activity images from an ionizing-radiation quantum imaging detector (iQID). Data from 211At-radioimmunotherapy studies for allogeneic hematopoietic cell transplantation in a canine model were used to develop these methods. Nine healthy canines were treated with 16.9–30.9 MBq 211At/mg monoclonal antibodies (mAb). Lymph node biopsies from early (2–5 h) and late (19–20 h) time points (16 total) were obtained, with 10–20 consecutive 12-µm cryosections extracted from each and imaged with an iQID device. iQID spatial activity images were registered within a 3D volume for dose-point-kernel convolution, producing dose-rate maps. The accumulated absorbed doses for high- and low-rate regions were 9 ± 4 Gy and 1.2 ± 0.8 Gy from separate dose-rate curves, respectively. We further assess uptake uniformity, co-registration with histological pathology, and requisite slice numbers to improve microscale characterization of absorbed dose inhomogeneities in αRPT.

The alpha particle-emitting radionuclide astatine-211 (211At) is of interest for targeted radiotherapy; however, low in vivo stability of many 211At-labeled cancer-targeting molecules has limited its potential. As an alternative labeling method, we evaluated whether a specific type of astatinated aryl compound that has the At atom in a higher oxidation state might be stable to in vivo deastatination. In the research effort, para-iodobenzoic acid methyl ester and dPEG4-amino acid methyl ester derivatives were prepared as HPLC standards. The corresponding para-stannylbenzoic acid derivatives were also prepared and labeled with 125I and 211At. Oxidization of the [125I]iodo- and [211At]astato-benzamidyl-dPEG4-acid methyl ester derivatives provided materials for in vivo evaluation. A biodistribution was conducted in mice with coinjected oxidized 125I- and 211At-labeled compounds. The oxidized radioiodinated derivative was stable to in vivo deiodination, but unfortunately the oxidized [211At]astatinated benzamide derivative was found to be unstable under the conditions of isolation by radio-HPLC (post animal injection). Another biodistribution study in mice evaluated the tissue concentrations of coinjected [211At]NaAtO3 and [125I]NaIO3. Comparison of the tissue concentrations of the isolated material from the oxidized [211At]benzamide derivative with those of [211At]astatate indicated the species obtained after isolation was likely [211At]astatate.

Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia and higher-risk myelodysplastic syndrome (MDS). Of increasing interest are alpha-particle-emitting radionuclides such as astatine-211 ( 211 At) as they deliver large amounts of radiation over just a few cell diameters, enabling efficient and selective target cell kill. Here, we developed 211 At-based RIT targeting CD123, an antigen widely displayed on acute leukemia and MDS cells including underlying neoplastic stem cells. We generated and characterized new murine monoclonal antibodies (mAbs) specific for human CD123 and selected four, all of which were internalized by CD123+ target cells, for further characterization. All mAbs could be conjugated to a boron cage, isothiocyanatophenethyl-ureido- closo -decaborate(2-) (B10), and labeled with 211 At. CD123+ cell targeting studies in immunodeficient mice demonstrated specific uptake of 211 At-labeled anti-CD123 mAbs in human CD123+ MOLM-13 cell tumors in the flank. In mice injected intravenously with MOLM-13 cells or a CD123 NULL MOLM-13 subline, a single dose of up to 40 µCi of 211 At delivered via anti-CD123 mAb decreased tumor burdens and substantially prolonged survival dose dependently in mice bearing CD123+ but not CD123– leukemia xenografts, demonstrating potent and target-specific in vivo anti-leukemia efficacy. These data support the further development of 211 At-CD123 RIT toward clinical application.

Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide, with limited therapeutic options for advanced disease. Targeted alpha-therapy is an emerging class of targeted cancer therapy in which alpha-particle-emitting radionuclides, such as Th-227, are delivered specifically to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and in vivo efficacy of a Th-227-labeled GPC3-targeting antibody conjugate (Th-227-octapa-alpha GPC3) for treatment of HCC in an orthotopic murine model. Methods: The chelator p-SCN-Bn-H(4)octapa-NCS (octapa) was conjugated to a GPC3-targeting antibody (alpha GPC3) for subsequent Th-227 radiolabeling (octapa-alpha GPC3). Conditions were varied to optimize radiolabeling of Th-227. In vitro stability was evaluated by measuring the percentage of protein-bound Th-227 by gamma-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2-Red-FLuc cells was developed. Biodistribution and blood clearance of Th-227-octapa-alpha GPC3 were evaluated in tumor-bearing mice. The efficacy of Th-227-octapa-alpha GPC3 was assessed in tumor-bearing animals with serial measurement of serum alpha-fetoprotein at 23 d after injection. Results: Octapa-conjugated alpha GPC3 provided up to 70% Th-227 labeling yield in 2 h at room temperature. In the presence of ascorbate, at least 97.8% of Th-227 was bound to alpha GPC3-octapa after 14 d in phosphate-buffered saline. In HepG2-Red-FLuc tumor-bearing mice, highly specific GPC3 targeting was observed, with significant Th-227-octapa-alpha GPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. Twenty-three days after treatment, a significant reduction in tumor burden was observed in mice receiving a 500 kBq/kg dose of Th-227-octapa-alpha GPC3 by tail-vein injection. No acute off-target toxicity was observed, and no animals died before termination of the study. Conclusion: Th-227-octapa-alpha GPC3 was observed to be stable in vitro; maintain high specificity for GPC3, with favorable biodistribution in vivo; and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC.

Abstract The purpose of this study is to develop a thorium-227 (227Th) antibody radioimmunoconjugate targeting glypican-3 (GPC3) and to test its therapeutic efficacy in a hepatocellular carcinoma (HCC) orthotopic xenograft model. GPC3 targeting antibody (αGPC3) was conjugated to bifunctional chelator p-SCN-Bn-H4octapa (octapa), and αGPC3-octapa binding affinity for GPC3 was evaluated by flow cytometry. 227Th radiolabeling of this conjugate was optimized, and in vitro stability of 227Th-αGPC3-octapa was evaluated in PBS with and without free radical scavenging agent over 14 days. For in vivo evaluation, an orthotopic xenograft model was generated by hepatic subcapsular injection of human HCC HepG2 cells. In vivo biodistribution was assessed in blood, tumor and organs at 1, 7 and 21 days after tail vein injection of 227Th-αGPC3-octapa (500 KBq/kg). To test therapeutic efficacy, tumor-bearing animals were injected with 227Th-αGPC3-octapa (250 kBq/kg or 500 kBq/kg) and compared to an irrelevant control antibody, 227Th-αBHV1-octapa (500 kBq/kg), and to a no-treatment control. Tumor burden was assessed with serial serum alpha-fetoprotein (AFP) measurements, a marker of tumor burden validated in this model. Toxicity to 227Th-αGPC3-octapa was measured by serum comprehensive metabolic panel obtained 21 days after injection. GPC3 binding affinity was highest after conjugation of αGPC3 with 10 equivalents of octapa. The protein recovery from the conjugation process was >85%, and mass spectral analysis indicated an average of 3.3 octapa moieties per molecule of αGPC3-octapa. After two weeks, >98% of αGPC3-octapa and αBHV1-octapa had 227Th bound in the presence of scavenging agent. αGPC3-octapa maintained high affinity for GPC3 as measured by flow cytometry, indicating the octapa conjugation reaction did not alter immunoreactivity of αGPC3. In vivo, 227Th-αGPC3-octapa accumulated in the tumor over time and cleared from normal tissues with a %ID/g of <5% by 21 days after injection. Significant antitumor activity was observed after treatment with 227Th-αGPC3-octapa (500 kBq/kg) with mean AFP level of 10,696 ± 19,754ng/mL compared to 308,175 ± 362,372ng/mL and 145,154 ± 166,780ng/mL in the 227Th-αBHV1-octapa and no-treatment control group, respectively. Reductions in serum AFP was observed in all but one tumor-bearing mouse after 500kBq/kg 227Th-αGPC3-octapa therapy. Organ-specific toxicity was not observed after treatment with 227Th-αGPC3-octapa (500 KBq/kg) compared to no-treatment control. In conclusion, we report the development of a GPC3 targeted thorium conjugate and demonstrate its in vivo therapeutic efficacy in a murine orthotopic xenograft model of hepatocellular carcinoma. Citation Format: Kevin P. Labadie, Donald K. Hamlin, Aimee Kenoyer, Sara K. Daniel, Alan F. Utria, Andrew D. Ludwig, Heidi L. Kenerson, Delphine L. Chen, Johnnie Orozco, Raymond S. Yeung, Lily Li, Chris Orvig, Yawen Li, D Scott Wilbur, James O. Park. Glypican-3 targeted thorium-227 alpha therapy reduces tumor burden in an orthotopic xenograft model of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 928.

In a canine model of presensitization using donor blood transfusions, 100% of historical control dogs receiving 9.2 Gy total body irradiation (TBI) conditioning before dog leukocyte antigen (DLA)-identical marrow grafts had graft rejection. In this presensitization model, we investigated whether the addition of monoclonal antibody (mAb)-based targeted radioimmunotherapy (RIT) with astatine-211 (At-211) to TBI could overcome graft rejection. At-211 is an alpha-particle-emitting isotope that has a short path length, very high energy, and a short t1/2 of 7.2 hours, which allowed targeting radiation to the T cells responsible for graft rejection. Normal canine recipients were given three preceding transfusions of unirradiated whole blood on days -24, -17, and -10 before transplant from their DLA-identical marrow donors. At-211-anti-CD45 mAb was administered on day -3, and TBI followed by marrow grafts on day 0. Six of the 7 dogs (86%) achieved sustained engraftment as assessed by 100% donor chimerism in mononuclear cells, granulocytes, and CD3(+) T cells. One dog receiving the lowest CD34(+) cell content (0.35 x 10(6) cells/kg) rejected the graft. There were no late rejections in dogs followed up to 1 year. Graft-versus-host disease was seen in one dog. At-211-anti-CD45 mAb in combination with TBI as conditioning was successful in abrogating graft rejection in 86% of dogs in this presensitization model. At-211-anti-CD45 mAb conditioning with TBI may serve as a novel promising strategy to overcome graft rejection in heavily transfused patients with red cell disorders. (C) 2021 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.