Chimeric antigen receptor (CAR)-T cell therapy has shown limited success in the treatment of solid tumors, reinforcing the need to elucidate the in vivo biodistribution of these engineered T cells. Here, we integrate the anti-DOTA huC825 reporter ("Thor") platform into newly developed human anti-interleukin-13 receptor α-2 (IL13Rα2)-single-cell fragment variable (scFv)-derived CAR-T cells and investigate its utility for mapping CAR-T cell distribution in a xenograft mouse model of melanoma. Methods: We engineered anti-IL13Rα2-scFv-derived CAR-T cells expressing huC825 (KLG3BBz-huC825), evaluated detection sensitivity, and monitored CAR-T cell biodistribution via weekly [86Y]Y-aminobenzyl-DOTA PET/CT and therapeutic efficacy. Results: KLG3BBz-huC825 T cells demonstrated potent antigen-specific cytotoxicity and cytokine release in vitro. The Thor radiohapten capture platform offered exquisite detection sensitivity of only 3,000 engineered T cells and enabled prolonged spatiotemporal assessment of CAR-T cell kinetics up to 7 wk after infusion, corroborated by histopathology. Treatment with KLG3BBz-huC825 resulted in an overall survival benefit. Conclusion: The Thor platform offers a versatile and highly sensitive approach to study the real-time kinetics of CAR-T cells in vivo.

DOTA-radiometal (DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) hapten complexes of β-emitters (177Lu or 90Y) and α-emitters (225Ac) can be selectively targeted to tumors using an antitumor/anti-DOTA bispecific antibody (BsAb) approach in pretargeted radioimmunotherapy (DOTA-PRIT). Clinical translation of DOTA-PRIT may be accelerated by the development of a 68Ga companion diagnostic for PET imaging. Here, we report a novel radiohapten, which uses a 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) chelator for 68Ga labeling while retaining 175Lu in a benzyl-DOTA moiety for high-affinity antibody recognition (NODAGA-Pr; Pr = Proteus). Radiolabeling of NODAGA-Pr with 68Ga showed high radiochemical yield and purity, and in vitro studies confirmed high radiostability and negligible serum-protein binding. In vivo, using a DOTA-PRIT system targeting the GPA33 antigen in a human colorectal cancer mouse model, [68Ga]Ga-NODAGA-Pr demonstrated efficient tumor uptake and high-contrast PET imaging. Additional studies using an established DOTA-hapten test system─human embryonic kidney 293T cells expressing a transmembrane-anchored anti-DOTA scFv huC825 further confirmed its in vivo PRIT performance. These findings support [68Ga]Ga-NODAGA-Pr as a promising agent for pretargeted immunoPET and for imaging engineered cells expressing a radiohapten capture reporter gene, with favorable pharmacokinetics and tumor targeting well matched to existing therapeutic DOTA-radiohaptens.

EGFR signaling, which requires ligand shedding by ADAM proteases, drives the progression of a variety of cancers, including breast, ovarian and lung. We previously reported the generation and characterization of a fully human, affinity-matured anti-ADAM17 monoclonal antibody, D8P1C1, which inhibits both the proliferation of an array of cancer cell lines in vitro as well as breast cancer growth in a mouse xenograft model. Here, we show that the mAb inhibits the shedding of EGFR ligands and EGFR phosphorylation in cancer cell lines, thus explaining its anti-tumor effects. In a xenograft model with a high-grade serous ovarian cancer (HGSOC) cell line, D8P1C1 showed only modest therapeutic effect, without any discernible toxicity. These results suggest that ovarian cancers are less susceptible than breast cancers to therapeutic targeting of ADAM17- or EGFR-dependent signaling. Radioimmuno PET imaging with 89Zr-DFO-D8P1C1 confirmed tumoral accumulation of the mAb in high-grade and non-high-grade serous ovarian tumor xenografts. Furthermore, we report the generation and preliminary characterization of a bispecific T cell engager derivative of D8P1C1 with improved anti-tumor efficacy in vitro.

The 203Pb (half-life [t1/2], 51.9 h) and 212Pb (t1/2 = 10.6 h) theranostic pair shows great potential for radiopharmaceutical therapy. We have developed a highly versatile pretargeted radioimmunotherapy (PRIT) platform that utilizes antitumor antigen/anti-DOTA bispecific antibodies (BsAbs) in combination with rapidly clearing, low-molecular-weight DOTA-radiohaptens (DOTA-PRIT). In this study, we tested the hypothesis that high-therapeutic index (TI) targeting of 212Pb (an in vivo generator of α-emitting progeny, specifically 212Bi (t1/2 = 1.01 h) and 212Po (t1/2 = 0.3 µs), and its imaging surrogate, 203Pb (γ-emitter, 279 keV), would be feasible with anti-glycoprotein A33 (GPA33) DOTA-PRIT for treatment of human colorectal cancer (CRC). Methods: For efficient and stable chelation of lead and high-affinity recognition by BsAbs, we synthesized a TCMC-based radiohapten precursor named TCMC-Proteus (TCMC-Pr). We prepared [212Pb]Pb-TCMC-Pr and confirmed its stability in vitro and recognition by BsAbs. Using the 203Pb analog, we conducted serial biodistribution and SPECT/CT imaging studies with a 3-step GPA33 DOTA-PRIT approach and extrapolated dosimetry for 212Pb. We then established anti-GPA33 212Pb-DOTA-PRIT in mouse xenografts of human CRC (GPA33-expressing SW1222). Results: TCMC-Pr was successfully radiolabeled with 203Pb/212Pb and verified stable in serum, and specific BsAb binding was confirmed. For GPA33-pretargeted [203Pb]Pb-TCMC-Pr, the blood, SW1222 tumor, and kidney uptakes at 24 h after injection were 0.27 ± 0.14 %IA/g, 8.04 ± 2.94 %IA/g, and 0.88 ± 0.14 %IA/g, respectively. 212Pb doses to tumor, blood, and kidneys were 12,723 mGy/MBq, 313 mGy/MBq (TI, 40.6), and 1,075 mGy/MBq (TI, 11.8), respectively. During 212Pb-DOTA-PRIT therapy studies, we established efficacy and identified a double-cycle treatment regimen (938 kBq + 938 kBq separated by 48 h; total, 1.88 MBq) that led to prolonged survival including 3 of 5 histologic cures at the study endpoint of 137 d. Mice in this treatment group exhibited normal bone marrows and moderate tubulointerstitial lesions, with overall preserved renal function. Conclusion: We have developed a safe, curative GPA33-targeted 212Pb-DOTA-PRIT treatment in a preclinical model of human CRC. This research underscores the potential of 203/212Pb-DOTA-PRIT in managing CRC and provides a road map for its modular application to other human tumor target antigens compatible with DOTA-PRIT.

Rationale: Combining targeted alpha and beta therapy may address challenges such as toxicity, treatment resistance, and tumor heterogeneity. We evaluated the feasibility and therapeutic effectiveness of a DOTA-PRIT approach using a 177Lu/225Ac radioisotope cocktail, directly compared with monotherapies targeting GPA33 in human colorectal cancer (CRC) xenografts in mice. Methods: A three-step pretargeting regimen was employed: an anti-GPA33/anti-DOTA bispecific antibody (BsAb), a dendrimeric clearing agent, and radioligands labeled with 177Lu, 225Ac, alone or in combination. Serial biodistribution studies in GPA33(+) SW1222 xenografts evaluated how co-injection of 177Lu and 225Ac radioligands affected tumor uptake and biodistribution. iQID digital autoradiography was used to visualize isotope distribution in tumor and kidney samples. Mice bearing SW1222 and LS174T xenografts received mono-or combination-therapy regimens delivering 37-38 Gy to tumors. Dose-escalation studies, histopathology, and qPCR analysis of DNA-damage and apoptosis-related genes were also performed. Results: Biodistribution and autoradiography confirmed that the 177Lu-and 225Ac-labeled ligand effectively bound to pretargeted GPA33(+) xenografts when co-administered. High therapeutic indices were maintained across treatment groups, with autoradiography showing general overlap of co-injected probes. Combination therapy demonstrated comparable efficacy to monotherapies. At 150 d post-treatment, no treatment group had reached median survival; 5/9 mice receiving the cocktail (62.9 MBq 177Lu + 18.5 kBq 225Ac) were alive, including two tumor-free. In comparison, 4/8 mice in the 177Lu group and 8/10 in the 225Ac group survived, with 3 and 5 tumor-free animals, respectively. Combination therapy was well tolerated, showing no significant adverse effects on body weight or blood cell counts compared to healthy controls. Combined administration was safe up to 62.9 MBq 177Lu + 37 kBq 225Ac, resulting in 10/10 histological cures. Conclusions: Our findings confirm the feasibility of a combined 177Lu and 225Ac DOTA-PRIT in mice with established SW1222 xenografts, demonstrating tolerability and effectiveness comparable to monotherapy at equivalent average absorbed tumor doses.

Androgen receptor (AR) signaling is the key driver of prostate cancer. Thus, standard-of-care treatments focus on androgen-deprivation therapy followed by AR signaling inhibitors. Even when initially responsive, patients eventually develop resistance to therapies targeting AR signaling, leading to disease progression. To study lesion-to-lesion AR occupancy, the radiofluorinated analog of the endogenous androgen [18F]16β-fluoro-5α-dihydrotestosterone ([18F]FDHT) has been investigated as a potential AR imaging agent. In the literature, [18F]FDHT demonstrates slow clearance from healthy tissue, poor plasma stability in vivo, and nonapplicability in mouse studies. To overcome these limitations, we explored nonsteroidal selective androgen receptor modulator (SARM)-based pharmacophores as potential PET tracers. Here, we describe the performance of a 18F-radiolabeled AR tracer, [18F]F-SARM3. Methods: [18F]F-SARM3 was synthesized via 1-step copper-catalyzed radiofluorination, and its AR affinity (half-maximal inhibitory concentration [IC50]) and biological activity (half-maximal effective concentration [EC50]) were studied in various prostate cancer cell lines. The tracer's stability in vitro was also evaluated. [18F]F-SARM3 was evaluated in LNCaP and 22Rv1 (AR-positive) tumor-bearing male mice to assess its AR specificity and clearance profile, and its performance was compared with that of [18F]FDHT. Results: [18F]F-SARM3 was synthesized with a radiochemical yield and purity of 2.7 ± 1.4% and 97.9 ± 2.3%, respectively. In in vitro cell-binding assays, [18F]F-SARM3 demonstrated high affinity for ARs, similar to that of dihydrotestosterone (IC50, 20.2 ± 14.6 nM vs. 9.6 ± 4.0 nM, respectively, in 22Rv1 cells). [18F]F-SARM3 functions as a partial AR agonist, with an EC50 of 15.0 ± 12.0 nM. Furthermore, [18F]F-SARM3 is highly stable in phosphate-buffered saline and mouse blood (98.5% ± 1.2% and 98.6% ± 1.5% intact, respectively). The tracer's in vivo specificity was confirmed with the observed uptake in tumors and prostate glands of castrated mice, where uptake was blockable. In vivo specificity was not observed with [18F]FDHT. Conclusion: We developed a first-in-class SARM-based AR tracer that displays high affinity and selectivity for AR in vitro and in vivo. This represents a suitable PET tracer to image AR status in rodent models and provides a strong rationale for clinical translation of [18F]F-SARM3 as a high-affinity AR agonist PET imaging agent.

Introduction Malignant appendiceal epithelial tumors (AC) are rare and biologically diverse, with limited treatment options. Most patients with stage IV disease suffer from fatal, morbid peritoneal metastasis. Alpha and beta radiopharmaceutical therapies (RPT) hold promise for many human cancers, especially if delivered with precision platforms such as pretargeted radioimmunotherapy (PRIT). The self-assembling and disassembling (SADA) platform enables strong tumor retention of the antibody complex while allowing rapid clearance of unbound fragments, expanding the therapeutic window for curative PRIT with minimal toxicity. Objectives We report the use of GPA33-SADA PRIT to achieve cures of peritoneal AC xenografts. Materials and Methods Heterogeneity of target (GPA33, HER2, and B7H3) expression was measured by immunohistochemistry (IHC) on 26 flash frozen surgical, and 4 patient-derived AC xenograft (PDX) samples. Therapeutic index (AUCtumor/AUCnormal tissue) and dosimetry were derived using 2-144 hour biodistribution after a single dose of α-PRIT or β- PRIT using the subcutaneous (sq) PDX model. In therapy studies, BRG mice with sq or intraperitoneal (IP) PDX were treated with either (1) α-PRIT using [225Ac]-Proteus ([225Ac]Pr) split into 2 doses 7 d apart, or (2) β-PRIT using [177Lu]DOTA split into 3 doses 7 d apart,4 delivered by anti-GPA33-SADA. Toxicities (body weight, CBC, serum chemistry, terminal tissue histology) were measured acutely (first 30 d) and long term (at 160 d or time of death). Efficacy was measured by survival and response by pathology at time of sacrifice. Results Consistency of expression with heterogeneity of intensity was confirmed by IHC for GPA33, HER2 and B7H3. GPA33 was selected as the target because it is a well-studied and clinically established antigen in colorectal cancer. In the sq model, therapeutic index based on dosimetry for blood and kidney was 9.2 and 10.0 for Lu-177 β-PRIT, and 18.6 and 12.8 respectively for Ac-225.In therapy studies using the sq PDX models, α-PRIT delivered by GPA33-SADA (2 × 74 kBq [225Ac]Pr) yielded an median survival (MS) of 70 d vs 21 d in antibody-only or isotope-only controls (p < 0.0001). β-PRIT (3 × 111 MBq [177Lu]DOTA) achieved an MS of 68 d vs 27 d in controls. In therapy using the IP PDX models, 8 of 10 mice treated with α-PRIT had a pathological complete response at 160 d (MS 160 d vs 42 d controls, p < 0.0001), while β-PRIT extended survival to 147 d vs 30 d in controls. No dose-limiting acute (<30 d) or late (>5.5 mo) toxicities were observed. Transient leukopenia and mild reversible weight loss was observed; histology showed only minimal tubular degeneration. Conclusion Use of GPA33-SADA to deliver α-PRIT and β-PRIT achieved complete responses and pathologically-verified cures past 160 d in IP AC models with minimal short-term or long-term toxicities. These findings support the exceptional therapeutic window of SADA-based RPT as a potential safe and effective option for a major unmet need in peritoneal metastasis of appendiceal orgin, a disease with no established standard of care. Funding Acknowledgements Enid A. Haupt Endowed Chair (NKC), MSK's Cycle for Survival's Equinox Innovation Award in Rare Cancers, CRC philanthropic funds (GMN).

Rationale: Pretargeted radioimmunotherapy (PRIT), which combines systemic antibody-based targeting with ionizing radiation, is promising for treating liver metastases in patients with colorectal cancer (CRC). Previously, we established a three-step DOTA-PRIT regimen to deliver DOTA radiometal payloads to CRC using an anti-tumor/anti-DOTA bispecific antibody (BsAb) targeting cell surface glycoprotein A33 (GPA33), a tumor antigen target expressed on over 95% of primary and metastatic CRC; a clearing agent; and a monovalent 177Lu radiohapten called [177Lu]Lu-ABD. More recently, we developed a bivalent 177Lu radiohapten called [177Lu]Lu-Gemini to enhance tumor uptake and radiohapten retention. Here, we aimed to compare the efficacy and safety of bivalent vs. monovalent three-step DOTA-PRIT regimens in orthotopic CRC liver metastasis models, to mimic a clinical path forward. Methods: We established two orthotopic CRC liver metastasis models by inoculating either SW1222-luc (GPA33high) or LoVo (GPA33low) human CRC cells in athymic nude mice under ultrasonographic guidance. Tumor targeting efficacy and dosimetry of the radiohaptens were compared using ex vivo biodistribution studies, SPECT/CT, and quantitative autoradiography. We also performed a DOTA-PRIT experiment to compare the efficacy and safety profiles of bivalent (single-cycle [177Lu]Lu-Gemini, 48 h pretargeting interval) vs. monovalent (multicycle [177Lu]Lu-ABD, 24 h pretargeting interval) three-step DOTA-PRIT regimens, each designed to deliver comparable total radiation doses to tumors (around 50 Gy). Results: Both radiohaptens demonstrated efficient SW1222-luc tumor targeting, with [177Lu]Lu-Gemini showing superior targeting and tumor activity retention compared with [177Lu]Lu-ABD. In LoVo tumors, [177Lu]Lu-Gemini showed superior targeting, while [177Lu]Lu-ABD showed negligible targeting. Dosimetry estimates revealed higher SW1222-luc tumor mean absorbed doses for [177Lu]Lu-Gemini (119.88 cGy/MBq, 48 h pretargeting interval) compared with [177Lu]Lu-ABD (32.88 cGy/MBq, 24 h pretargeting interval), with more favorable blood and kidney therapeutic indices (50 and 9 for [177Lu]Lu-Gemini, and 15 and 5 for [177Lu]Lu-ABD, respectively). In the DOTA-PRIT experiment, both monovalent (injected activity: 3 × 44.4 MBq, 133.2 MBq total) and bivalent (injected activity: 44.4 MBq total) radiohapten regimens increased the median survival of treated mice compared with controls: 71 days for [177Lu]Lu-ABD-treated mice, 81 days for [177Lu]Lu-Gemini-treated mice, and 18 days for controls, without a statistical difference between treatment groups. Treatments were well tolerated, without significant weight loss or hematologic changes. Radiation-induced injuries were not identified histologically in the kidneys or bone marrow of mice submitted for necropsy. Conclusions: Our study demonstrates the exceptional benefit of a multivalent radiohapten strategy when treating an advanced model of CRC liver metastasis. Three-step GPA33 DOTA-PRIT with 177Lu-Gemini demonstrated that multivalency 1) improves PRIT therapeutic indices for blood and kidney and 2) has the potential to greatly reduce the administered activity without compromising the efficiency of the PRIT platform in clinically relevant models of target-rich and target-poor metastatic CRC.

Malignant appendiceal epithelial tumors (AC) present a rare and diverse group of neoplasms.1 Treatment options are limited especially with peritoneal recurrence. Alpha and beta particle therapy using radioisotopes, especially when delivered by a pre-targeted radioimmunotherapy (PRIT) approach, can be effective with a wide therapeutic window. SADA-PRIT exploits an initial long plasma half-life of SADA tetramers for increased tumor uptake and when unbound, their disassembly to monomer with rapid renal clearance, thereby maximizing therapeutic indices across all critical normal tissues.2, 3 6-week old female BRG mice with subcutaneous (sq) or intraperitoneal (IP) patient derived AC xenograft (PDX) were treated with two doses of α-PRIT using [225Ac]-Proteus ([225Ac]Pr, or three doses of β- PRIT using [177Lu]DOTA, 4 all delivered by anti-GPA33-SADA. Isotope doses were chosen based on previous dosimetry and given one week apart.2 Toxicity, tumor response and survival were followed up to 180 days. Log rank Mantel Cox statistics was used for median survival (MS) comparison. Positive immunohistochemistry was confirmed in 26/26 AC surgical samples and 4/4 Patient derived xenografts (PDX). Similar IHC patterns were seen for B7H3 and HER2. Using GPA33-SADA in sq models α-PRIT (2 x 74 kBq 225Ac) produced an MS of 70 days, compared to 21 days in the antibody-only and [225Ac]Pr-only control group; β-PRIT (3 x 111 MBq) provided an MS of 68 days, compared to 27 days in the antibody-only and [177Lu]DOTA-only control groups. In the IP AC PDX model, all 10/10 mice in the α-PRIT group are healthy and tumor-free at 100+ days, compared to an MS of 42d in both antibody-only and [225Ac]Pr-only control groups (p <0.0001). Neither [225Ac]Pr or [177Lu]DOTA caused dose-limiting acute (<30 days) or long term (to 6 months) toxicities. Transient weight loss and leukopenia were reversible. Mild tubular degeneration was documented by histology at necropsy. Use of GPA33-SADA to deliver α-PRIT and β-PRIT induced major tumor responses and significantly prolonged survival in sq AC and ip AC tumor models. Minimal myeloid, renal, hepatic, or neural toxicities were seen. IHC results support future testing of cocktailing of SADAs (specific for GPA33, B7H3, and HER2) to overcome tumor heterogeneity while reducing on-target off-tumor side effects. Nicole Aguirre, Michelle Kim, Hong-Fen Guo, Karina Leung, Sebastian E. Carrasco, Irene Cheung, Michael B. Foote, Andrea Cercek, Sarah M. Cheal, Darren R. Veach, Steven M. Larson, Georgios Karagkounis, Garrett Nash, Nai-Kong Cheung. Pre-targeted radioimmunotherapy using self-assembling dis-assembling (SADA) bispecific antibodies for the treatment of appendiceal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2150.

Chimeric antigen receptor (CAR) T cell therapy is limited by antigen escape, poor solid tumor penetration, and an off-tumor toxicity profile. CAR T cell-derived exosomes have been shown to express the parental CAR construct, allowing tumor targeting but limited inherent anti-tumor effects alone. Further engineering CAR T cells to modify their exosome cargo, such as to bind anti-tumor drugs or radionuclides, may allow for the generation of novel targeted delivery vehicles for cancer therapy. We previously engineered humanized C825 (huC825), an anti-metal-DOTA-hapten single chain variable fragment (scfv), into multiple different constructs, one of which is the 4h11 CAR against MUC16/Cancer Antigen 125, developed by Brenjens lab at Memorial Sloan Kettering Cancer Center. We showed that huC825 allows for theranostic potential based on high-affinity binding to various chelated radionuclides, such as for alpha or beta particle-based radiotherapy or diagnostic imaging. We derived exosomes from 4h11 and huC825-4h11 CAR T cells, as well as untransduced T cells, and characterized them for CAR and huC825 expression using Western blot and radio thin layer chromatography (radio-TLC), respectively, as well as with direct stochastic optical reconstruction microscopy (dSTORM). We performed binding and cytotoxicity assays in vitro after radiolabeling and purifying the exosomes with size-exclusion chromatography (SEC), or using a pretargeted approach, with various chelated radiotracers. 4h11 and huC825-4h11 CAR T cell exosomes were reliably manufactured from parental CAR T cells with high transduction rates (80-95%). Western blot using CD81 and ALIX exosomal markers illustrates that 4h11 and huC825-4h11 CAR T cell exosomes and their parental CAR T cells are positive for the 4h11 CAR. dSTORM imaging shows huC825-4h11 exosomes are positive for both 4h11 and huC825. Radio-TLC illustrates that radionuclides chelated by Y-aminobenzyl-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, further referred to as aminobenzyl-DOTA (ABD), such as [161Tb]Tb-ABD (therapeutic metal) or [177Lu]Lu-ABD (diagnostic and therapeutic), exhibit significant binding to huC825-4h11 CAR T cell exosomes compared to controls. We optimized a method for radiolabeling huC825-4h11 exosomes and purifying the product with SEC using various DOTA-chelated radionuclides. In vitro radiotracer binding assays using SKOV3 human ovarian cancer cells overexpressing MUC16 illustrate that only huC825-4h11 CAR T cell-derived exosomes selectively bind both MUC16-expressing tumor cells and DOTA-chelated radionuclides. Our work illustrates the potential of huC825-4h11 CAR T cell-derived exosomes as delivery vehicles for radionuclides for imaging or targeted therapy. Their 50-160 nm size may allow better solid tumor penetration in vivo than current cellular treatment modalities. Future directions include in vivo imaging and radiotherapy studies. Sarah Qureshy, Leah Gajecki, Darren Veach, Sang Gyu Lee, Irina Matei, Lukas Carter, David Lyden, David Scheinberg, Simone Krebs. Utilizing CAR T cell-derived exosomes for radiotheranostic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 585.

Purpose: Curative therapies remain a significant unmet need for advanced human colorectal cancer (CRC). The aim of this study was to establish a 3-step 225Ac-DOTA pretargeted radioimmunotherapy (PRIT) system for human CRC, targeting two individual antigens: glycoprotein A33 (GPA33) and human epidermal growth factor receptor 2 (HER2). Methods: In vitro cellular uptake and internalization assays, as well as survival assays (colony forming) were performed in GPA33- and HER2-positive SW1222 human CRC cells. In vivo biodistribution and therapy studies were performed with two human CRC xenograft mouse models. Results: For both antigens, treatment with up to 74 kBq 225Ac-DOTA-PRIT in SW1222-tumored mice significantly enhanced overall survival in comparison to controls, including histologic cures. The uptake of 225Ac at the tumor correlated with antigen expression (antigen expression for GPA33:HER2 is 5:1). Cellular assays showed no significant differences in internalized fraction or nucleus-associated radioactivity between the two targets. GPA33 had a higher absorbed dose to the nucleus (1.3 Gy vs. 0.65 Gy for HER2), resulting in reduced clonogenic survival. A single cycle of either GPA33 or HER2 DOTA-PRIT (37 kBq; 193.31 Gy and 113.91 Gy (relative biological effectiveness [RBE] = 5), respectively) was equally effective. No chronic nephrotoxicity was seen at ≤ 20 Gy (RBE = 5). The efficacy of GPA33-directed 225Ac-DOTA-PRIT was also confirmed in a patient-derived xenograft model. Conclusion: In summary, 225Ac-DOTA-PRIT to GPA33 or HER2 was highly effective and safe in preclinical models of human CRC. Tumor response to treatment could not be predicted by nuclear absorbed dose alone, highlighting the importance of comprehensive micro- and macro-dosimetry.

Radioimmunotherapy using 225Ac, a highly cytotoxic α-particle emitter, has potential for treating advanced breast cancer, especially human epidermal growth factor receptor 2 (HER2)-positive cases. We use a pretargeted radioimmunotherapy (PRIT) approach consisting of a 3-step intravenous regimen (step 1: bispecific anti-HER2/anti-DOTA antibody; step 2: clearing agent; step 3: 225Ac-radiolabeled Proteus DOTA, or [225Ac]Ac-Pr). Our goal was to establish curative 225Ac-PRIT with high therapeutic indices. Methods: The impact of [225Ac]Ac-Pr specific activity was evaluated in the BT-474 breast xenograft model. We tested the effects of [225Ac]Ac-Pr dosing during PRIT on tumor-targeting efficiency and tissue biodistribution. Using a 225Ac-PRIT regimen consisting of a ratio of 1.19 nmol of bispecific antibody to 0.60-0.66 nmol of [225Ac]Ac-Pr, we evaluated therapy in the BT-474 model and a patient-derived xenograft model. BT-474-tumor-bearing mice were treated with 1 or 2 cycles of 225Ac-PRIT (37 kBq/cycle) separated by 1 wk. A dose escalation study was performed on the BT-474 model to establish an absorbed radiation dose of approximately 40 Gy (relative biological effectiveness [RBE], 5) as a nephrotoxic dose, as no such histologic findings were observed in prior studies at the 20.7-Gy (RBE, 5) renal dose level. Results: In the BT-474 model, 100% (20/20) achieved complete responses and histologic cure in 17 of 20 (85%) of the treated animals. One-cycle and 2-cycle treatments were equally effective. Treatments were well tolerated, with no chronic radiation toxicity documented during necropsy at 175 d. Dosimetry estimates (RBE, 5) per 37 kBq administered for tumors and kidneys were 210 and 3.5 Gy, respectively. In the patient-derived xenograft model, a single 225Ac-PRIT treatment led to 60% (3/5) complete response and prolonged survival (>93 d) versus no treatment (30 d; P = 0.0185). Lastly, a 225Ac-PRIT regimen was identified that induces severe chronic nephrotoxicity (41.4 Gy/592 kBq; RBE, 5). Conclusion: Safe and effective 225Ac-PRIT regimens were developed in 2 preclinical models of advanced HER2-positive human breast cancer with tumor cure without dose-limiting nephrotoxicity. This study establishes crucial preclinical dosimetry benchmarks for 225Ac-PRIT and provides a compelling rationale for its advancement into the clinic.

S1. Hierarchical clustering based on Euclidean distance of RNA-seq data demonstrates separation of EBRT-treated (B1-3) and untreated (C1-3) samples.S2. Multi-dimensional scaling plot of RNA-seq data demonstrates separation of EBRT-treated (B1-3) and untreated (C1-3) samples.

Peritoneal carcinomatosis (PC), characterized by the dissemination of metastatic tumor cells throughout the peritoneal cavity from several gastrointestinal and gynecological malignancies, has significantly compromised patient survival. The standard of care is cytoreductive surgery with or without intraperitoneal chemotherapy. However, surgical resection often leaves behind microscopic or clinically occult disease due to the complex anatomy of the peritoneum, where intraperitoneal chemotherapy and systemic chemotherapy have shown limited success. To improve the therapeutic outcome, targeted therapy using radionuclides such as alpha, beta, and Auger emitters delivered by antibodies is actively being investigated. While preclinical murine models of PC have shown the potential of radioimmunotherapy (RIT) using various radioisotopes across a wide spectrum of antigen targets and tumor diagnoses with acceptable toxicities, successful clinical trials are lacking. Here, we retrospectively summarize preclinical and clinical PC studies, consider their translational potential, and examine paths to development that maximize the clinical benefit of RIT in this context.

Interleukin-13 receptor α-2 (IL13Rα2) is a cell surface receptor frequently expressed in solid malignancies, such as glioblastoma and melanoma, with limited expression in healthy tissue, rendering it an ideal target for noninvasive and specific tumor delineation. In this study, we report the development of 5 novel IL13Rα2-targeted human monoclonal antibodies (mAbs) KLG-1-5; in subsequent in vitro and in vivo studies after radiolabeling with 89Zr, we evaluate their performance to identify a lead candidate. Methods: Five novel human anti-IL13Rα2 mAbs KLG-1-5 were developed and in vitro binding properties and target specificity assessed. In vivo 89Zr-immuno-PET using KLG-1-5 was conducted in a subcutaneous U-87 MG glioblastoma mouse model, and a mass dose titration study was conducted with lead candidate KLG-3. Ex vivo biodistribution results were used to derive prospective dosimetry of 177Lu-labeled KLG-3. Targeting with KLG-3 was also verified in an A-375 melanoma model using the optimized conditions determined in the U-87 MG xenograft model. Results: In vitro studies confirmed target specificity and pico- to low nanomolar binding affinity. Immuno-PET studies with KLG-1-5 in U-87 MG xenografts demonstrated continuously increasing tumoral uptake with maximal uptake at 144 h after tracer injection, clearance of the unbound tracer from the blood pool, and little uptake in any other normal tissues, leading to high-contrast images. KLG-3 provided the highest tumoral uptake and tumor-to-normal tissue ratios and was chosen as the lead candidate, and further dose optimization with this antibody led to tumoral uptake of 97 ± 6 maximum percent of injected dose per gram at 144 h after tracer injection. Ex vivo biodistribution-derived prospective dosimetry for 177Lu-labeled KLG-3 predicted a favorable therapeutic index, encouraging the development of IL13Rα2-targeted radioimmunotherapy. Of note, KLG-3 performed similarly well in a melanoma model, emphasizing the versatility of this antibody. Conclusion: Lead candidate anti-IL13Rα2 mAb KLG-3 validated highly specific target binding in human glioblastoma and melanoma models, resulting in high-contrast PET images with minimal accumulation in off-target healthy tissues. Prospective dosimetry of its 177Lu-labeled counterpart suggested therapeutic efficacy at relatively low injected activities, supporting further pursuit of KLG-3 in future translational radioimmunotherapy applications.

The efficacy of fluorescence-guided surgery in facilitating the real-time delineation of tumours depends on the optical contrast of tumour tissue over healthy tissue. Here we show that CJ215-a commercially available, renally cleared carbocyanine dye sensitive to apoptosis, and with an absorption and emission spectra suitable for near-infrared fluorescence imaging (wavelengths of 650-900 nm) and shortwave infrared (SWIR) fluorescence imaging (900-1,700 nm)-can facilitate fluorescence-guided tumour screening, tumour resection and the assessment of wound healing. In tumour models of either murine or human-derived breast, prostate and colon cancers and of fibrosarcoma, and in a model of intraperitoneal carcinomatosis, imaging of CJ215 with ambient light allowed for the delineation of nearly all tumours within 24 h after intravenous injection of the dye, which was minimally taken up by healthy organs. At later timepoints, CJ215 provided tumour-to-muscle contrast ratios up to 100 and tumour-to-liver contrast ratios up to 18. SWIR fluorescence imaging with the dye also allowed for quantifiable non-contact wound monitoring through commercial bandages. CJ215 may be compatible with existing and emerging clinical solutions. A commercial near-infrared dye that is sensitive to apoptosis and that provides high tumour-to-muscle and tumour-to-liver contrast ratios facilitates fluorescence-guided tumour screening, tumour resection and the assessment of wound healing.

Fluorescence guided surgery (FGS) facilitates real time tumor delineation and is being rapidly established clinically. FGS efficacy is tied to the utilized dye and provided tumor contrast over healthy tissue. Apoptosis, a cancer hallmark, is a desirable target for tumor delineation. Here, we preclinically in vitro and in vivo, validate an apoptosis sensitive commercial carbocyanine dye (CJ215), with absorption and emission spectra suitable for near infrared (NIR, 650-900nm) and shortwave infrared (SWIR, 900-1700nm) fluorescence imaging (NIRFI, SWIRFI). High contrast SWIRFI for solid tumor delineation is demonstrated in multiple murine and human models including breast, prostate, colon, fibrosarcoma and intraperitoneal colorectal metastasis. Organ necropsy and imaging highlighted renal clearance of CJ215. SWIRFI and CJ215 delineated all tumors under ambient lighting with a tumor-to-muscle ratio up to 100 and tumor-to-liver ratio up to 18, from 24 to 168 h post intravenous injection with minimal uptake in healthy organs. Additionally, SWIRFI and CJ215 achieved non-contact quantifiable wound monitoring through commercial bandages. CJ215 provides tumor screening, guided resection, and wound healing assessment compatible with existing and emerging clinical solutions.

ADAM17 sheds EGFR/erbB ligands and triggers oncogenic pathways that lead to the progression of solid tumors. We targeted the ADAM17 disintegrin and cysteine rich domain region (D+C) to generate a panel of single-chain antibody fragments (scFvs) that selectively bind to the D or C domains of ADAM17, but not of ADAM10 or ADAM19. From the panel, we selected one scFv, referred to as C12, based on its high binding affinity towards the target, and re-formatted it to a full IgG for further studies. High-resolution cryo-electron microscopy studies documented that the mAb binds to the ADAM17 C-domain that in ADAM proteases, notably ADAM10 and ADAM17, is known to impart substrate-specificity. The C12 mAb significantly inhibited EGFR phosphorylation in cancer cell lines by hindering the cleavage of EGFR ligands tethered to the cell surface. This inhibition provides a mechanism for potential anti-tumor effects, and indeed C12 diminished the viability of a variety of EGFR-expressing cancer cell lines. Cell-based ELISA studies revealed that C12 preferentially bound to activated ADAM17 present on tumor cells, as compared to the autoinhibited ADAM17 that is the predominant form on HEK293 and other non-tumor cells. C12 also exhibited tumor growth inhibition in an ovarian cancer xenograft mouse model. Consistent with its selective tumor cell binding in vitro, radioimmuno PET (positron emission tomography) imaging with 89Zr-DFO-C12 in mouse xenograft models confirmed tumoral accumulation of the C12 mAb.

Radiolabeled small-molecule DOTA-haptens can be combined with antitumor/anti-DOTA bispecific antibodies (BsAbs) for pretargeted radioimmunotherapy (PRIT). For optimized delivery of the theranostic γ- and β-emitting isotope