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Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15–30% of breast cancers but has low expression in normal tissue, making it attractive for targeted alpha therapy (TAT). HER2-positive breast cancer typically metastasizes to bone, resulting in incurable disease and significant morbidity and mortality. Therefore, new strategies for HER2-targeting therapy are needed. Here, we present the preclinical in vitro and in vivo characterization of the HER2-targeted thorium-227 conjugate (HER2-TTC) TAT in various HER2-positive cancer models. In vitro, HER2-TTC showed potent cytotoxicity in various HER2-expressing cancer cell lines and increased DNA double strand break formation and the induction of cell cycle arrest in BT-474 cells. In vivo, HER2-TTC demonstrated dose-dependent antitumor efficacy in subcutaneous xenograft models. Notably, HER2-TTC also inhibited intratibial tumor growth and tumor-induced abnormal bone formation in an intratibial BT-474 mouse model that mimics breast cancer metastasized to bone. Furthermore, a match in HER2 expression levels between primary breast tumor and matched bone metastases samples from breast cancer patients was observed. These results demonstrate proof-of-concept for TAT in the treatment of patients with HER2-positive breast cancer, including cases where the tumor has metastasized to bone.

Isolated kidneys (A, B) and bone marrow (C, D) were co-stained with a CD33-specific antibody (A, C) and a CD45-specific antibody (B, D).

The clinical efficacy of the first approved alpha pharmaceutical, Xofigo (radium-223 dichloride, 223RaCl2), has stimulated significant interest in the development of new alpha-particle emitting drugs in oncology. Unlike radium-223 (223Ra), the parent radionuclide thorium-227 (227Th) is able to form highly stable chelator complexes and is therefore amenable to targeted radioimmunotherapy. We describe the preparation and use of a CD33-targeted thorium-227 conjugate (CD33-TTC), which binds to the sialic acid receptor CD33 for the treatment of acute myeloid leukemia (AML). A chelator was conjugated to the CD33-targeting antibody lintuzumab via amide bonds, enabling radiolabeling with the alpha-emitter 227Th. The CD33-TTC induced in vitro cytotoxicity on CD33-positive cells, independent of multiple drug resistance (MDR) phenotype. After exposure to CD33-TTC, cells accumulated DNA double-strand breaks and were arrested in the G2 phase of the cell cycle. In vivo, the CD33-TTC demonstrated antitumor activity in a subcutaneous xenograft mouse model using HL-60 cells at a single dose regimen. Dose-dependent significant survival benefit was further demonstrated in a disseminated mouse tumor model after single dose injection or administered as a fractionated dose. The data presented support the further development of the CD33-TTC as a novel alpha pharmaceutical for the treatment of AML. Mol Cancer Ther; 15(10); 2422–31. ©2016 AACR.

Blood samples from animals treated with vehicle and CD33-TTC, administered at the different dose levels, were taken at Day -1, Day 67 and Day 123. (A) White blood cell counts, expressed in 109/L. (B) Red blood cell counts, expressed in 1012/L.

Supplementary figure legends.

Thorium-227 is purified from actinium-227 (227Ac). Thorium-227 decays via its alpha- and beta-emitting daughters to stable nonradioactive 207Pb. The energy emitted at each decay step (in MeV) are summarized. When thorium-227 decays to radium-223, the 3,2-HOPO chelator is unable to retain the daughter nuclide radium-223 which will detach from the antibody-chelator conjugate.

Binding of CD33 antibody and CD33 antibody chelator-conjugate to CD33-positive HL-60 cells by FACS staining in comparison to an isotype control. Bound antibodies were detected using secondary PE-labeled anti-human antibody. Median fluorescence intensity (MFI) was recorded and plotted against antibody concentrations. EC50 values were determined by fitting data using Graph Pad software.

SCNA and survival after irradiation.

The file contains description of Supporting Methods.

Longer doubling times in cells most resistant to alpha-particle irradiation.

Purpose:Prostate-specific membrane antigen (PSMA) is an attractive target for radionuclide therapy of metastatic castration-resistant prostate cancer (mCRPC). PSMA-targeted alpha therapy (TAT) has shown early signs of activity in patients with prostate cancer refractory to beta radiation. We describe a novel, antibody-based TAT, the PSMA-targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) consisting of the alpha-particle emitter thorium-227 complexed by a 3,2-HOPO chelator covalently linked to a fully human PSMA-targeting antibody.Experimental Design:PSMA-TTC was characterized for affinity, mode of action, and cytotoxic activity in vitro. Biodistribution, pharmacokinetics, and antitumor efficacy were investigated in vivo using cell line and patient-derived xenograft (PDX) models of prostate cancer.Results:PSMA-TTC was selectively internalized into PSMA-positive cells and potently induced DNA damage, cell-cycle arrest, and apoptosis in vitro. Decrease in cell viability was observed dependent on the cellular PSMA expression levels. In vivo, PSMA-TTC showed strong antitumor efficacy with T/C values of 0.01 to 0.31 after a single injection at 300 to 500 kBq/kg in subcutaneous cell line and PDX models, including models resistant to standard-of-care drugs such as enzalutamide. Furthermore, inhibition of both cancer and cancer-induced abnormal bone growth was observed in a model mimicking prostate cancer metastasized to bone. Specific tumor uptake and efficacy were demonstrated using various PSMA-TTC doses and dosing schedules. Induction of DNA double-strand breaks was identified as a key mode of action for PSMA-TTC both in vitro and in vivo.Conclusions:The strong preclinical antitumor activity of PSMA-TTC supports its clinical evaluation, and a phase I trial is ongoing in mCRPC patients (NCT03724747).

The file contains Supporting Figures.

Figures and Figure Legends for Supplementary Figures 1, 2, 3, 4 and 5

Targeted α-particle–emitting radionuclides have great potential for the treatment of a broad range of cancers at different stages of progression. A platform that accurately measures cancer cellular sensitivity to α-particle irradiation could guide and accelerate clinical translation. Here, we performed high-content profiling of cellular survival following exposure to α-particles emitted from radium-223 (223Ra) using 28 genetically diverse human tumor cell lines. Significant variation in cellular sensitivity across tumor cells was observed. 223Ra was significantly more potent than sparsely ionizing irradiation, with a median relative biological effectiveness of 10.4 (IQR: 8.4–14.3). Cells that are the most resistant to γ radiation, such as Nrf2 gain-of-function mutant cells, were sensitive to α-particles. Combining these profiling results with genetic features, we identified several somatic copy-number alterations, gene mutations, and the basal expression of gene sets that correlated with radiation survival. Activating mutations in PIK3CA, a frequent event in cancer, decreased sensitivity to 223Ra. The identification of cellular and genetic determinants of sensitivity to 223Ra may guide the clinical incorporation of targeted α-particle emitters in the treatment of several cancer types.Significance:These findings address limitations in the preclinical guidance and prediction of radionuclide tumor sensitivity by identifying intrinsic cellular and genetic determinants of cancer cell survival following exposure to α-particle irradiation.See related commentary by Sgouros, p. 5479

The file contains Supporting Tables.

Nrf2 activation and alpha-particle irradiation.

Incubation time does not impact alpha-particle irradiation sensitivity on the basis of cellular doubling time.

Purpose:Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue.Experimental Design:The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer.Results:BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data.Conclusions:These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).

Cellular morphometry affects survival after alpha-particle irradiation.

CCLE cell lines profiled and radiation sensitivity data.

Gene mutations and survival after irradiation.

Supplementary Table 1

High-throughput profiling of cellular survival after gamma irradiation.

Supplementary Methods

ssGSEA and survival after irradiation.

Purpose: Androgen receptor (AR) inhibitors are well established in the treatment of castration-resistant prostate cancer and have recently shown efficacy also in castration-sensitive prostate cancer. Although most patients respond well to initial therapy, resistance eventually develops, and thus, more effective therapeutic approaches are needed. Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer and presents an attractive target for radionuclide therapy. Here, we evaluated the efficacy and explored the mode of action of the PSMA-targeted thorium-227 conjugate (PSMA-TTC) BAY 2315497, an antibody-based targeted alpha-therapy, in combination with the AR inhibitor darolutamide. Experimental Design: The in vitro and in vivo antitumor efficacy and mode of action of the combination treatment were investigated in preclinical cell line–derived and patient-derived prostate cancer xenograft models with different levels of PSMA expression. Results: Darolutamide induced the expression of PSMA in androgen-sensitive VCaP and LNCaP cells in vitro, and the efficacy of darolutamide in combination with PSMA-TTC was synergistic in these cells. In vivo, the combination treatment showed synergistic antitumor efficacy in the low PSMA-expressing VCaP and in the high PSMA-expressing ST1273 prostate cancer models, and enhanced efficacy in the enzalutamide-resistant KUCaP-1 model. The treatments were well tolerated. Mode-of-action studies revealed that darolutamide induced PSMA expression, resulting in higher tumor uptake of PSMA-TTC, and consequently, higher antitumor efficacy, and impaired PSMA-TTC–mediated induction of DNA damage repair genes, potentially contributing to increased DNA damage. Conclusions: These results provide a strong rationale to investigate PSMA-TTC in combination with AR inhibitors in patients with prostate cancer.

Introduction: [227Th]Th-3,2-HOPO-MSLN-mAb, a mesothelin (MSLN)-targeted thorium-227 therapeutic conjugate, is currently in phase I clinical trial; however, direct PET imaging using this conjugate is technically challenging. Thus, using the same MSLN antibody, we synthesized 3,2-HOPO and deferoxamine (DFO)-based zirconium-89 antibody conjugates, [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb, respectively, and compared them in vitro and in vivo. Methods: [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb were evaluated in vitro to determine binding affinity and immunoreactivity in HT29-MSLN and PDX (NCI-Meso16, NCI-Meso21) cells. For both the zirconium-89 conjugates, in vivo studies (biodistribution/imaging) were performed at days 1, 3, and 6, from which tissue uptake was determined. Results: Both the conjugates demonstrated a low nanomolar binding affinity for MSLN and >95% immunoreactivity. In all the three tumor types, biodistribution of [89Zr]Zr-DFO-MSLN-mAb resulted in higher tumor uptake(15.88-28-33%ID/g) at all time points compared with [89Zr]Zr-3,2-HOPO-MSLN-mAb(7–13.07%ID/g). [89Zr]Zr-3,2-HOPO-MSLN-mAb femur uptake was always higher than [89Zr]Zr-DFO-MSLN-mAb, and imaging results concurred with the biodistribution studies. Conclusions: Even though the conjugates exhibited a high binding affinity for MSLN, [89Zr]Zr-DFO-MSLN-mAb showed a higher tumor and lower femur uptake than [89Zr]Zr-3,2-HOPO-MSLN-mAb. Nevertheless, [89Zr]Zr-3,2-HOPO-MSLN-mAb could be used to study organ distribution and lesion uptake with the caveat of detecting MSLN-positive bone lesions. Clinical trial (NCT03507452).

Radium-223, a bone-specific alpha particle therapy, increased survival of patients with metastatic castration-resistant prostate cancer (mCRPC) and bone metastases (Parker et al., NEJM 2013) 177Lu-PSMA-617 is a small molecule radioligand that delivers beta-particle radiation to cancer cells expressing prostate-specific membrane antigen (PSMA). Despite improving overall survival of patients with heavily pretreated mCRPC (Sator et al., NEJM 2021), responses to 177Lu-PSMA-617 are often not durable and many patients develop progressive disease presumably due to microscopic osseous lesions. Compared to beta-particle radiation, alpha particles have a shorter range and higher linear energy transfer, making them more suitable for treating microscopic lesions. In this study, we evaluated preclinical antitumor efficacy and potential beneficial combination effects of radium-223 combined with 177Lu-PSMA-617 in the intratibial LNCaP xenograft model mimicking progressive prostate cancer micrometastases in bone. LNCaP prostate cancer cells were inoculated into the right tibiae of male NOD.scid mice. Six weeks after inoculation, the mice were randomized to treatment groups based on serum prostate-specific antigen (PSA) and treated with two injections (Q4Wx2, i.v.) of vehicle, radium-223 (330 kBq/kg), 177Lu-PSMA-617 (10 MBq) or their combination 177Lu-PSMA-617 was given 24 hours after radium-223. PSA and bone formation marker procollagen type I intact N-terminal propeptide (PINP) were measured from blood samples and tumor-induced abnormal bone growth was determined by radiography. Terminal blood samples were analyzed for hematologic status at sacrifice. The combination treatment of radium-223 and 177Lu-PSMA-617 demonstrated robust antitumor efficacy, as indicated by lower serum PSA (mean change relative to pretreatment levels: 63.9 % vs 631 % (vehicle control), p=0.026), decreased tumor-induced abnormal bone area in tumor-bearing tibiae (p In conclusion, these preclinical results show that the combination of radium-223 and 177Lu-PSMA-617 is well-tolerated and efficacious in inhibiting osseous tumor growth and improving response. The combination of radium-223 and 177Lu-PSMA-I&T is currently being investigated in patients with mCRPC in a phase 1/2 trial (AlphaBet, NCT05383079). Citation Format: Arne Scholz, Matias Knuuttila, Justyna Zdrojewska, Mari I. Suominen, Esa Alhoniemi, Christoph A. Schatz, Sabine Zitzmann-Kolbe, Sanna-Maria Käkönen, Urs B. Hagemann. Radium-223 demonstrates increased antitumor activity in combination with 177Lu-PSMA-617 in the intratibial LNCaP xenograft model of bone metastatic prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5043.

Patients with advanced prostate cancer have few treatment options. While androgen receptor (AR) inhibitors are the standard of care for the systemic treatment, patients often develop resistance despite an initial response. Thus, novel therapeutic approaches are required. Prostate-specific membrane antigen (PSMA; FOLH1) is highly expressed in prostate cancer with limited expression in normal tissues. Therefore, PSMA presents an attractive target for radionuclide therapy and PSMA-targeted therapies have already shown efficacy in the clinical setting. Here, we evaluated the efficacy of 225Ac-pelgifatamab, a PSMA-targeted actinium-225 conjugate, in combination with the AR inhibitor darolutamide in androgen-dependent and -independent prostate cancer models. AR inhibitors are known to upregulate PSMA expression, therefore providing a rationale for combining 225Ac-pelgifatamab with darolutamide and potentially resulting in an enhanced treatment effect. In vitro, darolutamide induced the expression of PSMA in androgen-dependent VCaP and androgen-independent 22Rv1 cells by more than 10-fold and 2-fold, respectively. The efficacy of darolutamide in combination with 225Ac-pelgifatamab was synergistic in these cells with combination indexes of 0.12 and 0.47, respectively. In the androgen-dependent, patient-derived ST1273 model, a single i.v. injection of 225Ac-pelgifatamab at 75 kBq/kg resulted in a treatment/control (T/C) ratio of 0.22 on day 30, while twice daily (BID) oral (p.o.) treatment with 100 mg/kg darolutamide showed a T/C ratio of 0.29. However, the combination of darolutamide and 225Ac-pelgifatamab resulted in enhanced antitumor efficacy with a T/C ratio of 0.008. Furthermore, 5/10 mice remained tumor-free 86 days after the 225Ac-pelgifatamab injection. In the androgen-independent cell line-derived 22Rv1 model, darolutamide (100 mg/kg, p.o., BID) monotherapy was not efficacious with a T/C of 0.84 on day 16 but a single i.v. dose of 225Ac-pelgifatamab at 150 kBq/kg resulted in a T/C ratio of 0.69. Remarkably, the combination of darolutamide and 225Ac-pelgifatamab resulted in further enhanced efficacy with a T/C ratio of 0.34. Flow cytometric analysis of isolated 22Rv1 tumors showed a 50-fold increase in PSMA expression upon darolutamide treatment compared with vehicle. Taken together, these results suggest that upregulation of PSMA expression contributes to the higher efficacy observed when darolutamide is combined with 225Ac-pelgifatamab. In conclusion, our results provide a proof of concept for investigating the combination of 225Ac-pelgifatamab with darolutamide in the clinical setting. Citation Format: Christoph A. Schatz, Bernard Haendler, Sabine Zitzmann-Kolbe, Aasmund Larsen, Hartwig Hennekes, Carsten H. Nielsen, Maria Z. Alfsen, Alan Cuthbertson, Stefanie Hammer, Arne Scholz, Urs B. Hagemann. PSMA-targeted actinium-225 conjugate (225Ac-pelgifatamab) potentiates the antitumor efficacy of darolutamide in androgen-dependent and -independent prostate cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5047.

BackgroundTargeted thorium-227 conjugates (TTCs) are an emerging class of targeted alpha therapies (TATs). Their unique mode of action (MoA) is the induction of difficult-to-repair clustered DNA double-strand breaks. However, thus far, their effects on the immune system are largely unknown. Here, we investigated the immunostimulatory effects of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC)in vitroandin vivoin monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice.MethodsThe murine cell line MC38 was transfected with the human gene encoding for MSLN (hMSLN) to enable binding of the non-cross-reactive MSLN-TTC. The immunostimulatory effects of MSLN-TTC were studiedin vitroon human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studiedin vivoas monotherapy or in combination with anti-PD-L1 in MC38-hMSLN tumor-bearing immunocompetent C57BL/6 mice. Experiments were supported by RNA sequencing, flow cytometry, immunohistochemistry, mesoscale, and TaqMan PCR analyses to study the underlying immunostimulatory effects.In vivodepletion of CD8+ T cells and studies with Rag2/Il2Rg double knockout C57BL/6 mice were conducted to investigate the importance of immune cells to the efficacy of MSLN-TTC.ResultsMSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6,CCL20,CXCL10, and stimulator of interferon genes (STING)-related genes)in vitroas determined by RNASeq analysis. The results, including phospho-STING activation, were confirmed on the protein level. Danger-associated molecular pattern molecules were upregulated in parallel, leading to dendritic cell (DC) activationin vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, pConclusionsThesein vitroandin vivodata on MSLN-TTC demonstrate that the MoA of TTCs involves activation of the immune system. The findings are of relevance for other targeted radiotherapies and may guide clinical combination strategies.

Purpose Fibroblast growth factor receptor 2 (FGFR2) has been previously reported to be overexpressed in several types of cancer, whereas the expression in normal tissue is considered to be moderate to low. Thus, FGFR2 is regarded as an attractive tumor antigen for targeted alpha therapy. This study reports the evaluation of an FGFR2-targeted thorium-227 conjugate (FGFR2-TTC, BAY 2304058) comprising an anti-FGFR2 antibody, a chelator moiety covalently conjugated to the antibody, and the alpha particle–emitting radionuclide thorium-227. FGFR2-TTC was assessed as a monotherapy and in combination with the DNA damage response inhibitor ATRi BAY 1895344. Methods and Materials The in vitro cytotoxicity and mechanism of action were evaluated by determining cell viability, the DNA damage response marker γH2A.X, and cell cycle analyses. The in vivo efficacy was determined using human tumor xenograft models in nude mice. Results In vitro mechanistic assays demonstrated upregulation of γH2A.X and induction of cell cycle arrest in several FGFR2-expressing cancer cell lines after treatment with FGFR2-TTC. In vivo, FGFR2-TTC significantly inhibited tumor growth at a dose of 500 kBq/kg in the xenograft models NCI-H716, SNU-16, and MFM-223. By combining FGFR2-TTC with the ATR inhibitor BAY 1895344, an increased potency was observed in vitro, as were elevated levels of γH2A.X and inhibition of FGFR2-TTC–mediated cell cycle arrest. In the MFM-223 tumor xenograft model, combination of the ATRi BAY 1895344 with FGFR2-TTC resulted in significant tumor growth inhibition at doses at which the single agents had no effect. Conclusions The data provide a mechanism-based rationale for combining the FGFR2-TTC with the ATRi BAY 1895344 as a new therapeutic approach for treatment of FGFR2-positive tumors from different cancer indications.

Targeted 227Th conjugates (TTCs) represent a new class of therapeutic radiopharmaceuticals for targeted α-therapy. They comprise the α-emitter 227Th complexed to a 3,2-hydroxypyridinone chelator conjugated to a tumor-targeting monoclonal antibody. The high energy and short range of the α-particles induce antitumor activity, driven by the induction of complex DNA double-strand breaks. We hypothesized that blocking the DNA damage response (DDR) pathway should further sensitize cancer cells by inhibiting DNA repair, thereby increasing the response to TTCs. Methods: This article reports the evaluation of the mesothelin (MSLN)-TTC conjugate (BAY 2287411) in combination with several DDR inhibitors, each of them blocking different DDR pathway enzymes. MSLN is a validated cancer target known to be overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancer, with low expression in normal tissue. In vitro cytotoxicity experiments were performed on cancer cell lines by combining the MSLN-TTC with inhibitors of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related (ATR), DNA-dependent protein kinase, and poly[adenosine diphosphate ribose] polymerase (PARP) 1/2. Further, we evaluated the antitumor efficacy of the MSLN-TTC in combination with DDR inhibitors in human ovarian cancer xenograft models. Results: Synergistic activity was observed in vitro for all tested inhibitors (inhibitors are denoted herein by the suffix “i”) when combined with MSLN-TTC. ATRi and PARPi appeared to induce the strongest increase in potency. Further, in vivo antitumor efficacy of the MSLN-TTC in combination with ATRi or PARPi was investigated in the OVCAR-3 and OVCAR-8 xenograft models in nude mice, demonstrating synergistic antitumor activity for the ATRi combination at doses demonstrated to be nonefficacious when administered as monotherapy. Conclusion: The presented data support the mechanism-based rationale for combining the MSLN-TTC with DDR inhibitors as new treatment strategies in MSLN-positive ovarian cancer.

Rationale: Mesothelin targeted thorium-227 conjugate (227Th-MSLN) is a novel targeted alpha therapy developed to treat mesothelin overexpressing cancers. We radiolabeled the same antibody-chelator conjugate with zirconium-89 (89Zr-MSLN) to evaluate if positron emission tomography (PET) imaging with 89Zr-MSLN matches with 227Th-MSLN tumor uptake, biodistribution, and antitumor activity.Experimental design: Serial PET imaging with protein doses of 4, 20, or 40 μg 89Zr-MSLN and 89Zr-control was performed up to 168 h post tracer injection (pi) in high (HT29-MSLN) and low (BxPc3) mesothelin expressing human tumor-bearing nude mice. 89Zr-MSLN and 227Th-MSLN ex vivo tumor uptake and biodistribution were compared at 6 time-points in HT29-MSLN and in medium mesothelin expressing (OVCAR-3) tumor-bearing mice. 89Zr-MSLN PET imaging was performed before 227Th-MSLN treatment in HT29-MSLN and BxPc3 tumor-bearing mice.Results: 89Zr-MSLN PET imaging showed mean standardized uptake value (SUVmean) in HT29-MSLN tumors of 2.2 ± 0.5. Ex vivo tumor uptake was 10.6% ± 2.4% injected dose per gram (%ID/g) at 168 h. 89Zr-MSLN tumor uptake was higher than uptake of 89Zr-control (P = 0.0043). 89Zr-MSLN and 227Th-MSLN showed comparable tumor uptake and biodistribution in OVCAR-3 and HT29-MSLN tumor-bearing mice. SUVmean was 1.8-fold higher in HT29-MSLN than in BxPc3 tumors, matching with stronger 227Th-MSLN antitumor activity.Conclusion: 89Zr-MSLN PET imaging reflected mesothelin expression and matched with 227Th-MSLN tumor uptake, biodistribution, and antitumor activity. Our data support the clinical exploration of 89Zr-MSLN PET imaging together with 227Th-MSLN therapy, both using the same antibody-chelator conjugate.

Targeted α therapy (TAT) offers the potential for the targeted delivery of potent α-particle-emitting radionuclides that emit high linear energy transfer radiation. This leads to a densely ionizing radiation track over a short path. Localized radiation induces cytotoxic, difficult-to-repair, clustered DNA double-strand breaks (DSBs). To date, radium-223 (223Ra) is the only TAT approved for the treatment of patients with metastatic castration-resistant prostate cancer. Thorium-227 (227Th), the progenitor nuclide of 223Ra, offers promise as a wider-ranging alternative due to the availability of efficient chelators, such as octadentate 3,2-hydroxypyridinone (3,2-HOPO). The 3,2-HOPO chelator can be readily conjugated to a range of targeting moieties, enabling the generation of new targeted thorium-227 conjugates (TTCs). This review provides a comprehensive overview of the advances in the preclinical development of TTCs for hematological cancers, including CD22-positive B cell cancers and CD33-positive leukemia, as well as for solid tumors overexpressing renal cell cancer antigen CD70, membrane-anchored glycoprotein mesothelin in mesothelioma, prostate-specific membrane antigen in prostate cancer, and fibroblast growth factor receptor 2. As the mechanism of action for TTCs is linked to the formation of DSBs, the authors also report data supporting combinations of TTCs with inhibitors of the DNA damage response pathways, including those of the ataxia telangiectasia and Rad3-related protein, and poly-ADP ribose polymerase. Finally, emerging evidence suggests that TTCs induce immunogenic cell death through the release of danger-associated molecular patterns. Based on encouraging preclinical data, clinical studies have been initiated to investigate the safety and tolerability of TTCs in patients with various cancers.

Despite the recent advances in the treatment of ovarian cancer, it remains an area of high unmet medical need. Epithelial ovarian cancer is associated with high levels of mesothelin expression, and therefore, mesothelin is an attractive candidate target for the treatment of this disease. Herein, we investigated the antitumor efficacy of the mesothelin-targeting antibody-drug conjugate (ADC) anetumab ravtansine as a novel treatment option for ovarian cancer in monotherapy and in combination with the antitumor agents pegylated liposomal doxorubicin (PLD), carboplatin, copanlisib and bevacizumab. Anetumab ravtansine showed potent antitumor activity as a monotherapy in ovarian cancer models with high mesothelin expression. No activity was seen in mesothelin-negative models. The combination of anetumab ravtansine with PLD showed additive anti-proliferative activity in vitro, which translated into improved therapeutic in vivo efficacy in ovarian cancer cell line- and patient-derived xenograft (PDX) models compared to either agents as a monotherapy. The combination of anetumab ravtansine with the PI3Kα/δ inhibitor copanlisib was additive in the OVCAR-3 and OVCAR-8 cell lines in vitro, showing increased apoptosis in response to the combination treatment. In vivo, the combination of anetumab ravtansine with copanlisib resulted in more potent antitumor activity than either of the treatments alone. Likewise, the combination of anetumab ravtansine with carboplatin or bevacizumab showed improved in vivo efficacy in the ST081 and OVCAR-3 models, respectively. All combinations were well-tolerated. Taken together, these data support the development of anetumab ravtansine for ovarian cancer treatment and highlight its suitability for combination therapy with PLD, carboplatin, copanlisib, or bevacizumab.

The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a novel targeted alpha therapy (TAT) approach which is currently under investigation in a FiH trial in patients with metastatic castration resistant prostate cancer (mCRPC; NCT03724747). It consists of the alpha emitter thorium-227 complexed to a 3,2-HOPO chelator conjugated to a PSMA targeting antibody delivering radiation to PSMA expressing tumor cells and their microenvironment resulting in the induction of potentially cytotoxic DNA double-strand breaks (DBS). Combining with the PARP inhibitor olaparib may act synergistically via inhibition of DDR pathways. Here we evaluated the efficacy of PSMA-TTC in combination with olaparib that demonstrated a positive outcome in patients with homologous recombination repair-mutated mCRPC. We explored potential synergies of the combination treatment in prostate cancer models in vitro and in vivo and studied the mode of action by protein and RNA analysis. In vitro, synergy of the combination of PSMA-TTC with olaparib with a combination index (CI) of 0.7 was observed in three cell lines, namely LNCaP, 22Rv1 and MDA-PCa2b. with the first two (LNCaP and 22Rv1) carrying mutations in BRCA2. Only moderate synergy with a CI of 0.8 was seen in BRCA2 wt VCaP cells. In vivo, dose dependent anti-tumor activity with single i.v. injections of PSMA-TTC at 150 and 300 kBq/kg was seen in the LNCaP prostate cancer model resulting in a tumor/control (T/C) ratio of 0.3 and 0.09 at day 28, respectively. In contrast, olaparib given at 50 mg/kg QD did not show activity at day 28. The 300 kBq/kg PSMA-TTC dose combined with olaparib showed the most pronounced anti-tumor activity (T/C=0.04) and significantly increased the number of animals with complete tumor regressions compared to high dose PSMA-TTC alone (6 CRs vs 2 CRs, p = 0.0397, Chi-square) on day 28. At the end of the consecutive surveillance period on day 117, 7/8 (87.5%) animals remained tumor-free (putatively cured) in the combination group vs 4/10 (40%) mice in the PMSA-TTC monotherapy group (Chi-square test, P = 0.0400). Additive or synergistic toxicological effects for the combination treatment were not seen based on body weight loss and fatal tox rate. At the 117-day time point both the 300 kBq/kg PSMA-TTC monotherapy and combination with olaparib group demonstrated 0.3% and 9.3% body weight gain indicating good tolerability. Changes in markers of DNA damage response following treatment will be presented. In summary, combination of PSMA-TTC with olaparib shows synergistic preclinical anti-tumor activity and support further clinical investigation of this combination treatment in prostate cancer patients. Citation Format: Christoph A. Schatz, Stefanie Hammer, Antje Wengner, Urs B. Hagemann, Dominik Mumberg, Arne Scholz. PSMA-Targeted Thorium Conjugate (BAY 2315497) and olaparib combination show synergistic anti-tumor activity in prostate cancer models [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 1393.

// Urs B. Hagemann 1 , Dessislava Mihaylova 1 , Steinar R. Uran 1 , Joergen Borrebaek 1 , Derek Grant 1 , Roger M. Bjerke 1 , Jenny Karlsson 1 and Alan S. Cuthbertson 1 1 Thorium Conjugate Research, Bayer AS, Oslo, Norway Correspondence to: Urs B. Hagemann, email: urs.hagemann@bayer.com Keywords: targeted alpha therapy (TAT), alpha particles, radioimmunotherapy, thorium-227, renal cell carcinoma Received: January 25, 2017 Accepted: March 13, 2017 Published: April 07, 2017 ABSTRACT The cell surface receptor CD70 has been previously reported as a promising target for B-cell lymphomas and several solid cancers including renal cell carcinoma. We describe herein the characterization and efficacy of a novel CD70 targeted thorium-227 conjugate (CD70-TTC) comprising the combination of the three components, a CD70 targeting antibody, a chelator moiety and the short-range, high-energy alpha-emitting radionuclide thorium-227 ( 227Th ). In vitro analysis demonstrated that the CD70-TTC retained binding affinity to its target and displayed potent and specific cytotoxicity compared to an isotype control-TTC. A biodistribution study in subcutaneous tumor-bearing nude mice using the human renal cell carcinoma cell line 786-O demonstrated significant uptake and retention with 122 ± 42% of the injected dose of 227Th per gram (% ID/g) remaining in the tumor seven days post dose administration compared to only 3% ID/g for the isotype control-TTC. Tumor accumulation correlated with a dose dependent and statistically significant inhibition in tumor growth compared to vehicle and isotype control-TTC groups at radioactivity doses as low as 50 kBq/kg. The CD70-TTC was well tolerated as evidenced by only modest changes in hematology and normal gain in body weight of the mice. To our knowledge, this is the first report describing molecular targeting of CD70 expressing tumors using a targeted alpha-therapy (TAT).

Purpose: Prostate-specific membrane antigen (PSMA) is an attractive target for radionuclide therapy of metastatic castration-resistant prostate cancer (mCRPC). PSMA-targeted alpha therapy (TAT) has shown early signs of activity in patients with prostate cancer refractory to beta radiation. We describe a novel, antibody-based TAT, the PSMA-targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) consisting of the alpha-particle emitter thorium-227 complexed by a 3,2-HOPO chelator covalently linked to a fully human PSMA-targeting antibody. Experimental Design: PSMA-TTC was characterized for affinity, mode of action, and cytotoxic activity in vitro. Biodistribution, pharmacokinetics, and antitumor efficacy were investigated in vivo using cell line and patient-derived xenograft (PDX) models of prostate cancer. Results: PSMA-TTC was selectively internalized into PSMA-positive cells and potently induced DNA damage, cell-cycle arrest, and apoptosis in vitro. Decrease in cell viability was observed dependent on the cellular PSMA expression levels. In vivo, PSMA-TTC showed strong antitumor efficacy with T/C values of 0.01 to 0.31 after a single injection at 300 to 500 kBq/kg in subcutaneous cell line and PDX models, including models resistant to standard-of-care drugs such as enzalutamide. Furthermore, inhibition of both cancer and cancer-induced abnormal bone growth was observed in a model mimicking prostate cancer metastasized to bone. Specific tumor uptake and efficacy were demonstrated using various PSMA-TTC doses and dosing schedules. Induction of DNA double-strand breaks was identified as a key mode of action for PSMA-TTC both in vitro and in vivo. Conclusions: The strong preclinical antitumor activity of PSMA-TTC supports its clinical evaluation, and a phase I trial is ongoing in mCRPC patients (NCT03724747).

Targeted α-particle–emitting radionuclides have great potential for the treatment of a broad range of cancers at different stages of progression. A platform that accurately measures cancer cellular sensitivity to α-particle irradiation could guide and accelerate clinical translation. Here, we performed high-content profiling of cellular survival following exposure to α-particles emitted from radium-223 (223Ra) using 28 genetically diverse human tumor cell lines. Significant variation in cellular sensitivity across tumor cells was observed. 223Ra was significantly more potent than sparsely ionizing irradiation, with a median relative biological effectiveness of 10.4 (IQR: 8.4–14.3). Cells that are the most resistant to γ radiation, such as Nrf2 gain-of-function mutant cells, were sensitive to α-particles. Combining these profiling results with genetic features, we identified several somatic copy-number alterations, gene mutations, and the basal expression of gene sets that correlated with radiation survival. Activating mutations in PIK3CA, a frequent event in cancer, decreased sensitivity to 223Ra. The identification of cellular and genetic determinants of sensitivity to 223Ra may guide the clinical incorporation of targeted α-particle emitters in the treatment of several cancer types. Significance: These findings address limitations in the preclinical guidance and prediction of radionuclide tumor sensitivity by identifying intrinsic cellular and genetic determinants of cancer cell survival following exposure to α-particle irradiation. See related commentary by Sgouros, p. 5479

The clinical efficacy of the first approved alpha pharmaceutical, Xofigo (radium-223 dichloride, 223RaCl2), has stimulated significant interest in the development of new alpha-particle emitting drugs in oncology. Unlike radium-223 (223Ra), the parent radionuclide thorium-227 (227Th) is able to form highly stable chelator complexes and is therefore amenable to targeted radioimmunotherapy. We describe the preparation and use of a CD33-targeted thorium-227 conjugate (CD33-TTC), which binds to the sialic acid receptor CD33 for the treatment of acute myeloid leukemia (AML). A chelator was conjugated to the CD33-targeting antibody lintuzumab via amide bonds, enabling radiolabeling with the alpha-emitter 227Th. The CD33-TTC induced in vitro cytotoxicity on CD33-positive cells, independent of multiple drug resistance (MDR) phenotype. After exposure to CD33-TTC, cells accumulated DNA double-strand breaks and were arrested in the G2 phase of the cell cycle. In vivo, the CD33-TTC demonstrated antitumor activity in a subcutaneous xenograft mouse model using HL-60 cells at a single dose regimen. Dose-dependent significant survival benefit was further demonstrated in a disseminated mouse tumor model after single dose injection or administered as a fractionated dose. The data presented support the further development of the CD33-TTC as a novel alpha pharmaceutical for the treatment of AML. Mol Cancer Ther; 15(10); 2422–31. ©2016 AACR.

Mesothelin-targeted thorium-227 conjugate (MSLN-TTC) is a targeted alpha therapeutic consisting of a MSLN-targeting antibody anetumab covalently attached to a 3,2-HOPO chelator complexed with the alpha emitter thorium-227. It has potent in vivo activity in cell and patient-derived xenograft (PDX) models in monotherapy and in combination with DNA damage repair inhibitors. We evaluated the antitumor efficacy of MSLN-TTC in a mesothelin overexpressing ovarian PDX model (ST206B) in monotherapy and in combination bevacizumab or the multikinase inhibitor regorafenib which both have anti-angiogenic activity. The biodistribution of MSLN-TTC was studied in parallel. Athymic mice were transplanted s.c. with ST206B-derived tumor fragments and treated with MSLN-TTC (250 kBq/kg, Q7Dx2, 0.43 mg/kg) alone or in combination with bevacizumab (1.25 or 5 mg/kg, i.p., Q5D; or 5 mg/kg, single dose) or regorafenib (7.5, 15, or 30 mg/kg, QD, p.o. for 28 days). The biodistribution of the respective treatment groups was analyzed using a germanium detector. For molecular analyses, tumors were stained for γ-H2AX, Ki67, CD31 and α-SMA. MSLN-TTC monotherapy showed high in vivo potency at 2 × 250 kBq/kg with a Treated/Control (T/C) ratio of 0.28 (n=5) on day 29. Monotherapy treatment with bevacizumab and regorafenib showed dose-dependent antitumor activity with T/C ratios ranging from 0.31 (regorafenib 30 mg/kg) to 0.33 (bevacizumab 5 mg/kg, single dose). The in vivo efficacy of MSLN-TTC was further enhanced by combination therapy with bevacizumab (T/C 0.21; n=5) or regorafenib (T/C 0.15; n=5). Combination of MSLN-TTC and regorafenib (30 mg/kg) resulted in more complete and partial responses (3/4 CRs; 1/4 PR) as compared to MSLN-TTC (2/3 PRs; 1/3 PD) at day 141. All treatments were well-tolerated based on body weight assessment; a reversible myelosuppression of white blood cells was observed for MSLN-TTC monotherapy and combination with bevacizumab or regorafenib did not worsen it. In the biodistribution study, tumor-specific accumulation of MSLN-TTC was observed over 336 h (14 days) with lower accumulation in the MSLN-TTC + bevacizumab group (1.25 mg/kg, Q5D, p Citation Format: Urs B. Hagemann, Christine Ellingsen, Claudia Kamfenkel, Dieter Zopf, Jesper Fonslet, Carsten Nielsen, Hartwig Hennekes, Alan S. Cuthbertson, Dominik Mumberg. MSLN-targeted thorium-227 conjugate demonstrates increased antitumor activity in combination with bevacizumab and regorafenib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5355.

Targeted thorium-227 conjugates (TTCs) represent a new class of targeted alpha therapy (TAT). TTCs consist of an antigen targeting moiety, which is covalently attached to a 3,2-HOPO chelator, enabling specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. TTCs have demonstrated potent preclinical in vivo activity in monotherapy and in combination with DNA damage inhibitors. In the present work, the immunostimulatory effects of the mesothelin (MSLN) targeted thorium-227 conjugate (MSLN-TTC) was studied in immunocompetent mice, bearing human MSLN-transfected MC38 cells, in monotherapy and in combination with the PD-L1 inhibitor (PD-L1i) atezolizumab. Surface expression of DAMPs (calreticulin, HMGB1, HSP70 and HSP90), immunoinhibitory markers (PD-L1 and CTLA-4) and immunostimulatory markers (IFNAR, B7H2 and ICOS-L) was analyzed on murine, human MSLN-transfected MC38 colorectal cancer cells (MC38-hMSLN cells) after a 3-day exposure to MSLN-TTC. In vivo, C57/Bl6 mice were inoculated s.c. with MC38-hMSLN cells and treated with a single i.v. dose of non-radiolabeled MSLN-conjugate (0.14 mg/kg), isotype control-TTC (250 kBq/kg; 0.14 mg/kg), MSLN-TTC (250 kBq/kg; 0.14 mg/kg), PD-L1i (1.5 mg/kg; Q3D4; i.p.) or MSLN-TTC/PD-L1i combination. Tumors, lymph nodes, spleen and plasma were analyzed for MSLN and molecular markers. Tumor-free survivors were re-challenged with MC38-hMSLN or vector cells 125 d post-treatment. In a parallel arm of the experiment, CD8+ T cells were depleted using YTS169.4 antibody. MSLN-TTC treatment resulted in a specific dose-dependent increase of IFNAR, PD-L1 and HMGB1 in MC38-hMSLN cells in vitro. MSLN-TTC (T/C 0.38; Day 22) and PD-L1i (T/C 0.0.42; Day 22) monotherapies showed specific antitumor activity (T/C of radiolabeled isotype control was 0.76, Day 22) and their combination was synergistic (T/C 0.08; Day 22) with increased numbers of tumor free survivors. All three treatments increased HMGB1 levels in plasma. Induction of DSBs was observed in MSLN-TTC (+/- PD-L1i) treated tumors. Re-challenge of tumor-free survivors treated with MSLN-TTC, PD-L1i or MSLN-TTC/PD-L1i resulted in rejection of MC38-hMSLN, but not vector-cells. Infiltration of CD8+ T cells into tumors and their increase in tumor-draining lymph nodes were observed in MSLN-TTC, PD-L1i and MSLN-TTC/PD-L1i-treated groups but not in the vehicle group. Depletion of CD8+ T cells decreased the efficacy of PD-L1i and to a lower extent, MSLN-TTC and MSLN-TTC/PD-L1i. These results suggest that the efficacy of MSLN-TTC may involve activation of CD8+ T cells. These findings support the hypothesis that upon binding to antigen-positive cancer cells, TTC treatment induces DNA double strand breaks, leading to apoptosis and potentially immunogenic cell death, resulting in activation of the adaptive immune system. Citation Format: Urs B. Hagemann, Pascale Lejeune, Lisa Bartnitzky, Kathleen Stadelmann, Veronique Cruciani, Roger M. Bjerke, Sandra Berndt, Claudia Kamfenkel, Manuela Brand, Manuela Steinbach, Stefan Stargard, Christoph A. Schatz, Jenny Karlsson, Hartwig Hennekes, Alan S. Cuthbertson, Dominik Mumberg. Targeted thorium-227 conjugates demonstrate synergistic activity in combination with PD-L1 inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2257.

Targeted 227Th conjugates (TTCs) represent a new class of therapeutic radiopharmaceuticals for targeted α-therapy. They comprise the α-emitter 227Th complexed to a 3,2-hydroxypyridinone chelator conjugated to a tumor-targeting monoclonal antibody. The high energy and short range of the α-particles induce antitumor activity, driven by the induction of complex DNA double-strand breaks. We hypothesized that blocking the DNA damage response (DDR) pathway should further sensitize cancer cells by inhibiting DNA repair, thereby increasing the response to TTCs. Methods: This article reports the evaluation of the mesothelin (MSLN)-TTC conjugate (BAY 2287411) in combination with several DDR inhibitors, each of them blocking different DDR pathway enzymes. MSLN is a validated cancer target known to be overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancer, with low expression in normal tissue. In vitro cytotoxicity experiments were performed on cancer cell lines by combining the MSLN-TTC with inhibitors of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related (ATR), DNA-dependent protein kinase, and poly[adenosine diphosphate ribose] polymerase (PARP) 1/2. Further, we evaluated the antitumor efficacy of the MSLN-TTC in combination with DDR inhibitors in human ovarian cancer xenograft models. Results: Synergistic activity was observed in vitro for all tested inhibitors (inhibitors are denoted herein by the suffix “i”) when combined with MSLN-TTC. ATRi and PARPi appeared to induce the strongest increase in potency. Further, in vivo antitumor efficacy of the MSLN-TTC in combination with ATRi or PARPi was investigated in the OVCAR-3 and OVCAR-8 xenograft models in nude mice, demonstrating synergistic antitumor activity for the ATRi combination at doses demonstrated to be nonefficacious when administered as monotherapy. Conclusion: The presented data support the mechanism-based rationale for combining the MSLN-TTC with DDR inhibitors as new treatment strategies in MSLN-positive ovarian cancer.

Purpose: Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue. Experimental Design: The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer. Results: BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data. Conclusions: These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).

Background The number of radiolabeled monoclonal antibodies (mAbs) used for medical imaging and cancer therapy is increasing. The required chemical modification for attaching a radioactive label and all associated treatment may lead to a damaged mAb subpopulation. This paper describes a novel method, concentration through kinetics (CTK), for rapid assessment of the concentration of immunoreactive mAb and the specific radioactivity, based on monitoring binding kinetics. Methods The interaction of radiolabeled mAb with either the antigen or a general mAb binder such as Protein A was monitored in real time using the instrument LigandTracer. As the curvature of the binding trace has a distinct shape based on the interaction kinetics and concentration of the functional mAb, the immunoreactive mAb concentration could be calculated through reverse kinetic fitting of the binding curves, using software developed for this project. The specific activity, describing the degree of radioactive labeling, was determined through the use of calibrated signal intensities. Results The performance of the CTK assay was evaluated on the basis of various mAb-based interaction systems and assay formats, and it was shown that the assay can provide accurate and repeatable results for immunoreactive concentration and specific activity, with both accuracy and relative SD values below 15%. Conclusion By applying reverse kinetics on real-time binding traces it is possible to estimate the functional concentration and specific activity of radiolabeled mAb. The CTK assay may in the future be included as a complement to current quality assessment methods of radiolabeled mAbs.