Preclinical Efficacy of a PSMA-Targeted Actinium-225 Conjugate (225Ac-Macropa-Pelgifatamab): A Targeted Alpha Therapy for Prostate Cancer.

PURPOSE: Initially, prostate cancer responds to hormone therapy, but eventually resistance develops. Beta emitter-based prostate-specific membrane antigen (PSMA)-targeted radionuclide therapy is approved for the treatment of metastatic castration-resistant prostate cancer. Here we introduce a targeted alpha therapy (TAT) consisting of the PSMA antibody pelgifatamab covalently linked to a macropa chelator and labeled with actinium-225 and compare its efficacy and tolerability with other TATs. EXPERIMENTAL DESIGN: The in vitro characteristics and in vivo biodistribution, antitumor efficacy, and tolerability of 225Ac-macropa-pelgifatamab (225Ac-pelgi) and other TATs were investigated in cell line- and patient-derived prostate cancer xenograft models. The antitumor efficacy of 225Ac-pelgi was also investigated in combination with the androgen receptor inhibitor darolutamide. RESULTS: Actinium-225-labeling of 225Ac-pelgi was efficient already at room temperature. Potent in vitro cytotoxicity was seen in PSMA-expressing (LNCaP, MDA-PCa-2b, and C4-2) but not in PSMA-negative (PC-3 and DU-145) cell lines. High tumor accumulation was seen for both 225Ac-pelgi and 225Ac-DOTA-pelgi in the MDA-PCa-2b xenograft model. In the C4-2 xenograft model, 225Ac-pelgi showed enhanced antitumor efficacy with a T/Cvolume (treatment/control) ratio of 0.10 compared with 225Ac-DOTA-pelgi, 225Ac-DOTA-J591, and 227Th-HOPO-pelgifatamab (227Th-pelgi; all at 300 kBq/kg) with T/Cvolume ratios of 0.37, 0.39, and 0.33, respectively. 225Ac-pelgi was less myelosuppressive than 227Th-pelgi. 225Ac-pelgi showed dose-dependent treatment efficacy in the patient-derived KuCaP-1 model and strong combination potential with darolutamide in both cell line- (22Rv1) and patient-derived (ST1273) xenograft models. CONCLUSIONS: These results provide a strong rationale to investigate 225Ac-pelgi in patients with prostate cancer. A clinical phase I study has been initiated (NCT06052306).

Mono- and multimeric PSMA-targeting small molecule-thorium-227 conjugates for optimized efficacy and biodistribution in preclinical models.

PURPOSE: PSMA (prostate-specific membrane antigen) is highly expressed on prostate cancer (PrCa) cells and extensively used as a homing target for PrCa treatment. Most prominently, PSMA-targeting conjugate PSMA-617, carrying a DOTA chelator and labeled with therapeutic radionuclides like beta-emitting lutetium-177 or alpha-emitting actinium-225, has shown clinical activity in PrCa patients. We sought to develop PSMA-targeting small molecule (SMOL) conjugates that show high uptake in PSMA-expressing tumors and fast clearance, and can easily be labeled with the alpha emitter thorium-227 (half-life 18.7 days). METHODS: A novel linker motif with improved competition against 3H-PSMA-617 on PSMA-expressing LNCaP cells was identified. A 2,3-hydroxypyridinone chelator modified with carboxyl groups (carboxy-HOPO) with increased hydrophilicity and robust labeling with thorium-227 was developed and allowed the synthesis of mono-, di-, tri-, and tetrameric conjugates. The resulting monomeric and multimeric PSMA SMOL-TTCs (targeted thorium conjugate) were evaluated for cellular binding, internalization, and antiproliferative activity. The in vivo antitumor efficacy of the PSMA SMOL-TTCs was determined in ST1273 and KUCaP-1 PrCa models in mice, and their biodistribution was assessed in cynomolgus monkeys, minipigs, and mice. RESULTS: The monomeric and multimeric PSMA SMOL conjugates were readily labeled with thorium-227 at room temperature and possessed high stability and good binding, internalization, and antiproliferative activity in vitro. In vivo, the monomeric, dimeric, and trimeric PSMA SMOL-TTCs showed fast clearance, potent antitumor efficacy, and high uptake and retention in prostate tumors in mice. No major uptake or retention in other organs was observed beyond kidneys. Low uptake of free thorium-227 into bone confirmed high complex stability in vivo. Salivary gland uptake remained inconclusive as mini pigs were devalidated as a relevant model and imaging controls failed in cynomolgus monkeys. CONCLUSION: Monomeric and multimeric PSMA SMOL-TTCs show high tumor uptake and fast clearance in preclinical models and warrant further therapeutic exploration.

Efficacy of a HER2-Targeted Thorium-227 Conjugate in a HER2-Positive Breast Cancer Bone Metastasis Model.

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.

A Targeted Thorium-227 Conjugate Demonstrates Efficacy in Preclinical Models of Acquired Drug Resistance and Combination Potential with Chemotherapeutics and Antiangiogenic Therapies.

Targeted alpha therapies (TAT) are an innovative class of therapies for cancer treatment. The unique mode-of-action of TATs is the induction of deleterious DNA double-strand breaks. Difficult-to-treat cancers, such as gynecologic cancers upregulating the chemoresistance P-glycoprotein (p-gp) and overexpressing the membrane protein mesothelin (MSLN), are promising targets for TATs. Here, based on the previous encouraging findings with monotherapy, we investigated the efficacy of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC) both as monotherapy and in combination with chemotherapies and antiangiogenic compounds in ovarian and cervical cancer models expressing p-gp. MSLN-TTC monotherapy showed equal cytotoxicity in vitro in p-gp-positive and -negative cancer cells, while chemotherapeutics dramatically lost activity on p-gp-positive cancer cells. In vivo, MSLN-TTC exhibited dose-dependent tumor growth inhibition with treatment/control ratios of 0.03-0.44 in various xenograft models irrespective of p-gp expression status. Furthermore, MSLN-TTC was more efficacious in p-gp-expressing tumors than chemotherapeutics. In the MSLN-expressing ST206B ovarian cancer patient-derived xenograft model, MSLN-TTC accumulated specifically in the tumor, which combined with pegylated liposomal doxorubicin (Doxil), docetaxel, bevacizumab, or regorafenib treatment induced additive-to-synergistic antitumor efficacy and substantially increased response rates compared with respective monotherapies. The combination treatments were well tolerated and only transient decreases in white and red blood cells were observed. In summary, we demonstrate that MSLN-TTC treatment shows efficacy in p-gp-expressing models of chemoresistance and has combination potential with chemo- and antiangiogenic therapies.

Tumor growth inhibition and immune system activation following treatment with thorium-227 conjugates and PD-1 check-point inhibition in the MC-38 murine model.

Targeted thorium-227 conjugates comprise the combination of a monoclonal antibody with specificity for a tumor cell antigen and a 3,2-HOPO chelator enabling complexation of thorium-227 (Th-227). The radiolabeled conjugate functions as an effective delivery system of alpha-particle radiation to the surface of the tumor cell inducing difficult to repair complex DNA damage and cell death. In addition, the mechanism of action of targeted alpha therapy (TAT) appears to involve a significant component linked to stimulation of the immune system. We report herein evidence of immune activation and long-lasting immune protection of a TAT in a syngeneic model using the MC-38 murine cell line. Firstly, MC-38 cells were irradiated ex vivo with the thorium labeled antibody before subcutaneous implantation into mice. These mice were then rechallenged with MC-38 cells contra-laterally. In the group receiving irradiated cells, 9 out of 10 animals had no measurable tumor growth compared to aggressive tumor growth in the control group. Secondly, in an efficacy study, 500 kBq/kg of thorium labeled antibody alone or in combination with PD-1 checkpoint inhibitor gave statistically significant tumor growth inhibition compared to vehicle control. Animals with no measurable tumors were once again rechallenged contra-laterally with MC-38 cells. The re-growth of tumors was significantly delayed (approx. 60 days) in the treatment group compared to age-matched controls (approx. 30 days) in the monotherapy group. Interestingly, in the TAT/ PD-1 combination group no re-growth was observed demonstrating the potential of combining a TAT with checkpoint inhibition therapy. Finally, tumors were excised from treated mice and analyzed by flow cytometry and immunohistochemistry (IHC). Analysis revealed significant infiltration of CD8+ T-cells and mature dendritic cells compared to vehicle controls. Together these results indicated that an ongoing immune response from treatment with alpha radiation could be enhanced by check-point inhibition.

89Zr-3,2-HOPO-Mesothelin Antibody PET Imaging Reflects Tumor Uptake of Mesothelin-Targeted 227Th-Conjugate Therapy in Mice.

The mesothelin (MSLN)-targeted 227Th conjugate is a novel α-therapy developed to treat MSLN-overexpressing cancers. We radiolabeled the same antibody-chelator conjugate with 89Zr to evaluate whether PET imaging with 89Zr-MSLN matches 227Th-MSLN tumor uptake, biodistribution, and antitumor activity. Methods: Serial PET imaging with protein doses of 4, 20, or 40 µg of 89Zr-MSLN and 89Zr-control was performed up to 168 h after tracer injection in human tumor-bearing nude mice with high (HT29-MSLN) and low (BxPc3) MSLN expression. 89Zr-MSLN and 227Th-MSLN ex vivo tumor uptake and biodistribution were compared at 6 time points in HT29-MSLN and in medium-MSLN-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 an SUVmean of 2.2 ± 0.5 in HT29-MSLN tumors. Ex vivo tumor uptake was 10.6% ± 2.4% injected dose per gram 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. Pretreatment SUVmean was 2.2 ± 0.2 in HT29-MSLN tumors, which decreased in volume on 227Th-MSLN treatment. BxPc3 tumors showed an SUVmean of 1.2 ± 0.3 and remained similar in size after 227Th-MSLN treatment. Conclusion: 89Zr-MSLN PET imaging reflected MSLN expression and matched 227Th-MSLN tumor uptake and biodistribution. Our data support the clinical exploration of 89Zr-MSLN PET imaging together with 227Th-MSLN therapy, both using the same antibody-chelator conjugate.

Targeted thorium-227 conjugates as treatment options in oncology.

Targeted alpha therapy (TAT) is a promising approach for addressing unmet needs in oncology. Inherent properties make α-emitting radionuclides well suited to cancer therapy, including high linear energy transfer (LET), penetration range of 2-10 cell layers, induction of complex double-stranded DNA breaks, and immune-stimulatory effects. Several alpha radionuclides, including radium-223 (223Ra), actinium-225 (225Ac), and thorium-227 (227Th), have been investigated. Conjugation of tumor targeting modalities, such as antibodies and small molecules, with a chelator moiety and subsequent radiolabeling with α-emitters enables specific delivery of cytotoxic payloads to different tumor types. 223Ra dichloride, approved for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC) with bone-metastatic disease and no visceral metastasis, is the only approved and commercialized alpha therapy. However, 223Ra dichloride cannot currently be complexed to targeting moieties. In contrast to 223Ra, 227Th may be readily chelated, which allows radiolabeling of tumor targeting moieties to produce targeted thorium conjugates (TTCs), facilitating delivery to a broad range of tumors. TTCs have shown promise in pre-clinical studies across a range of tumor-cell expressing antigens. A clinical study in hematological malignancy targeting CD22 has demonstrated early signs of activity. Furthermore, pre-clinical studies show additive or synergistic effects when TTCs are combined with established anti-cancer therapies, for example androgen receptor inhibitors (ARI), DNA damage response inhibitors such as poly (ADP)-ribose polymerase inhibitors or ataxia telangiectasia and Rad3-related kinase inhibitors, as well as immune checkpoint inhibitors.

Immunostimulatory effects of targeted thorium-227 conjugates as single agent and in combination with anti-PD-L1 therapy.

BACKGROUND: Targeted 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 vitro and in vivo in monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice. METHODS: The 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 studied in vitro on human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studied in vivo as 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 vivo depletion 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. RESULTS: MSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6, CCL20, CXCL10, and stimulator of interferon genes (STING)-related genes) in vitro as 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) activation in vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, p<0.05) as a single agent in human MSLN-expressing MC38 tumor-bearing immunocompetent mice. Combining MSLN-TTC with anti-PD-L1 further enhanced the efficacy (T:C 0.08, p<0.001) as evidenced by the increased number of tumor-free surviving animals. MSLN-TTC monotherapy caused migration of CD103+ cDC1 DCs and infiltration of CD8+ T cells into tumors, which was enhanced on combination with anti-PD-L1. Intriguingly, CD8+ T-cell depletion decreased antitumor efficacy. CONCLUSIONS: These in vitro and in vivo data 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.

In Vitro and In Vivo Comparison of 3,2-HOPO Versus Deferoxamine-Based Chelation of Zirconium-89 to the Antimesothelin Antibody Anetumab.

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).

Advances in Precision Oncology: Targeted Thorium-227 Conjugates As a New Modality in Targeted Alpha Therapy.

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.