Analysis of selected and designed chimeric D- and L-α-helix assemblies.

D-peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base.

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).

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.

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.

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.

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).

Darolutamide Potentiates the Antitumor Efficacy of a PSMA-targeted Thorium-227 Conjugate by a Dual Mode of Action in Prostate Cancer Models.

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.

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.

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.

Preclinical Efficacy of a PSMA-Targeted Thorium-227 Conjugate (PSMA-TTC), a Targeted Alpha Therapy for Prostate Cancer.

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).

Role of hydrophobic and electrostatic interactions in coiled coil stability and specificity.

We have screened two coiled coil-forming libraries in which core a and electrostatic e/g positions have been partially randomized. We observed the relative ability of these residues to confer coiled coil stability using a protein-fragment complementation assay. Our studies continue with the Jun/Fos activator protein-1 (AP-1) leucine zipper complex, as it provides a valid therapeutic target, while representing one of the more simplistic examples of quaternary structure. In mammalian cells, 28 possible dimeric interactions result from combinations of cJun, JunB, JunD, cFos, FosB, Fra1, and Fra2. Consequently, peptides designed to target particular oncogenic members must bind with high affinity and also be specific if they are to function as desired. We have therefore tested the ability of core and electrostatic interactions to confer stable and specific peptides. A previously selected peptide (FosW) formed the template for the core and electrostatic libraries. The winner from the core randomization (FosW(Core)) bound specifically to cJun relative to cFos, FosB, Fra1, Fra2, and the FosW(Core) homodimer, as verified by thermal melting analyses and growth competitions in the presence of either a negative control "mock" peptide or a competitor fusion peptide (cFos-FosB-Fra1-Fra2). In contrast, the winner from the electrostatic e/g randomization (FosW(e/g)) bound to all respective complexes with high stability, suggesting that the more significant energetic contributions made by core residues may be enough to generate specificity as a consequence of positive design.

Selection of peptides interfering with protein-protein interaction.

Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

Cellular and Genetic Determinants of the Sensitivity of Cancer to α-Particle Irradiation.

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.

Synergistic Effect of a HER2 Targeted Thorium-227 Conjugate in Combination with Olaparib in a BRCA2 Deficient Xenograft Model.

Targeted thorium-227 conjugates (TTCs) represent a novel class of therapeutic radiopharmaceuticals for the treatment of cancer. TTCs consist of the alpha particle emitter thorium-227 complexed to a 3,2-hydroxypyridinone chelator conjugated to a tumor-targeting monoclonal antibody. The high energy and short range of the alpha particles induce potent and selective anti-tumor activity driven by the induction of DNA damage in the target cell. Methods: The efficacy of human epidermal growth factor receptor 2 (HER2)-TTC was tested in combination in vitro and in vivo with the poly ADP ribose polymerase (PARP) inhibitor (PARPi), olaparib, in the human colorectal adenocarcinoma isogenic cell line pair DLD-1 and the knockout variant DLD-1 BRCA2 -/- Results: The in vitro combination effects were determined to be synergistic in DLD-1 BRCA2 -/- and additive in DLD-1 parental cell lines. Similarly, the in vivo efficacy of the combination was determined to be synergistic only in the DLD-1 BRCA2 -/- xenograft model, with statistically significant tumor growth inhibition at a single TTC dose of 120 kBq/kg body weight (bw) and 50 mg/kg bw olaparib (daily, i.p. for 4 weeks), demonstrating comparable tumor growth inhibition to a single TTC dose of 600 kBq/kg bw. Conclusions: This study supports the further investigation of DNA damage response inhibitors in combination with TTCs as a new strategy for the effective treatment of mutation-associated cancers.

Synergistic Effect of a Mesothelin-Targeted 227Th Conjugate in Combination with DNA Damage Response Inhibitors in Ovarian Cancer Xenograft Models.

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.

Preclinical Combination Studies of an FGFR2 Targeted Thorium-227 Conjugate and the ATR Inhibitor BAY 1895344.

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.

Mesothelin-Targeted Thorium-227 Conjugate (MSLN-TTC): Preclinical Evaluation of a New Targeted Alpha Therapy for Mesothelin-Positive Cancers.

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).

Anetumab ravtansine inhibits tumor growth and shows additive effect in combination with targeted agents and chemotherapy in mesothelin-expressing human ovarian cancer models.

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.

Targeted alpha therapy using a novel CD70 targeted thorium-227 conjugate in in vitro and in vivo models of renal cell carcinoma.

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).