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Background

Neuroblastoma is an aggressive paediatric malignancy with poor outcomes in high-risk patients despite multimodal therapy. [¹⁷⁷Lu]Lu-DOTATATE, targeting somatostatin receptor 2 (SSTR2), is under clinical investigation for neuroblastoma. Terbium-161 (¹⁶¹Tb) shares chemical characteristics and a similar half-life with lutetium-177 (¹⁷⁷Lu) but offers advantages through its higher emission of Auger and conversion electrons, which may enhance therapeutic efficacy especially for micrometastases and recurrent disease. This study investigates [¹⁶¹Tb]Tb-DOTATATE as a potential complement for treatment of SSTR2-expressing neuroblastoma.

Results

DOTATATE was successfully radiolabelled with ¹⁶¹Tb, achieving high radiochemical yield and stability. [¹⁶¹Tb]Tb-DOTATATE demonstrated clear SSTR2-specific binding in vitro, as well as biodistribution and tumour uptake comparable to [¹⁷⁷Lu]Lu-DOTATATE in vivo. Treatment with [¹⁶¹Tb]Tb-DOTATATE reduced tumour growth in an activity-dependent manner, and demonstrated stronger early tumour control compared to [¹⁷⁷Lu]Lu-DOTATATE at comparable administered activities (**p < 0.01 at day 9). Tumour doubling times were longest in the 6 MBq [¹⁶¹Tb]Tb-DOTATATE group (29 vs. 18 days in controls), while the 3 MBq [¹⁶¹Tb]Tb-DOTATATE group showed similar doubling times to 4 MBq [¹⁷⁷Lu]Lu-DOTATATE (21 vs. 22 days). This was also reflected in survival, where 6 MBq [¹⁶¹Tb]Tb-DOTATATE achieved the greatest effect (50% increase in median survival), followed by 4 MBq [¹⁶¹Tb]Tb-DOTATATE (36%), while 3 MBq [¹⁶¹Tb]Tb-DOTATATE and 4 MBq [¹⁷⁷Lu]Lu-DOTATATE showed more modest improvements (23%). All treatment regimens were well tolerated, and no signs of treatment-related toxicity were observed.

Conclusion

[¹⁶¹Tb]Tb-DOTATATE demonstrates favourable targeting properties and activity-dependent therapeutic efficacy in preclinical neuroblastoma models without detectable toxicity. These findings encourage us to explore its potential as a complement to [¹⁷⁷Lu]Lu-DOTATATE, particularly for targeting minimal residual disease and micrometastases, and warrant further preclinical and clinical evaluation.

Targeted radionuclide therapy is an emerging potent therapeutic strategy in oncology. The cell surface antigen CD44v6 is a potential pan-cancer target for radionuclide therapy. This study aimed to evaluate the therapeutic efficacy, biodistribution, dosimetry, and safety profile of AKIR001, an antibody targeting CD44v6 labeled with ¹⁷⁷Lu.

Methods: The biodistribution and preclinical dosimetry of [¹⁷⁷Lu]Lu-AKIR001 were calculated in the highly CD44v6-expressing A431 murine xenograft model, with subsequent extrapolation to predict human dosimetry. Therapeutic efficacy was evaluated across 3 xenograft models, 2 with high and 1 with moderate levels of CD44v6, using multiple dosing levels, fractionation regimens, and combinations with cisplatin. Preclinical toxicology was evaluated in a cross-reactive rabbit model and complemented by a PET imaging study using ⁶⁸Ga-labeled AKIR001 in a cynomolgus macaque.

Results: Biodistribution studies confirmed the high and selective tumor uptake of [¹⁷⁷Lu]Lu-AKIR001, resulting in favorable dosimetry predictions for clinical application. Therapeutic evaluations demonstrated significant dose-dependent efficacy in all tested xenograft models, with fractionated dosing (2 doses) resulting in complete tumor regression in 80% of the animals in a radioresistant xenograft model. Biodistribution in rabbits demonstrated low uptake in normal tissues, and a good-laboratory-practice study using an excessive dose of AKIR001 was well tolerated, with no signs of adverse effects. PET imaging in a cynomolgus macaque corroborated these findings.

Conclusion: Collectively, these data strongly support the therapeutic efficacy, safety, and dosimetry of [¹⁷⁷Lu]Lu-AKIR001, justifying its advancement into clinical trials. A phase 1 clinical trial of [¹⁷⁷Lu]Lu-AKIR001for CD44v6-positive solid cancers (NCT06639191) is currently recruiting patients.

Multiple myeloma (MM) is a hematological disease of the plasma cell that remains clinically challenging despite the development of novel therapies. Epigenetic alterations have been demonstrated to contribute to MM pathogenesis, yet comprehensive studies into the links between different epigenetic regulatory systems in myeloma progression and drug resistance, though clinically relevant, are largely lacking. G9a and the DNA methyltransferases (DNMTs) are epigenetic modifiers that exhibit increased activity in MM, correlating with poor prognosis. To investigate the partnership between G9a and DNMTs, we used a combinatorial treatment approach involving small-molecule inhibitors. In-depth molecular analysis of the histone H3 lysine dimethylation distribution, the DNA methylome and the transcriptome of MM revealed a silencing mechanism involving G9a and DNMTs that represses key tumor suppressor genes. Moreover, dual inhibition of G9a and DNMTs reduced cell viability in primary MM cells and induced apoptosis in MM cell lines. This was accompanied by increased expression of apoptosis-related genes and decreased protein levels of the MM-associated oncoproteins IRF4, XBP1, and MYC. To assess the translational relevance of our in vitro findings, we evaluated the combination therapy in an in vivo preclinical xenograft MM model. Specifically, we demonstrate that the G9a inhibitor A366 synergizes with the DNMTs inhibitor decitabine to promote a robust tumor regression in vivo. Together, these data provide new insights into the cooperative role of G9a and the DNMTs in regulating gene silencing in MM, and support dual epigenetic inhibition as a promising therapeutic strategy.

Anaplastic thyroid cancer (ATC) is a rare but severe form of thyroid cancer responsible for approximately 50% of thyroid cancer deaths. Consequently, the identification of innovative therapies remains crucial for the effective treatment of ATC. Molecular radiotherapy is a rapidly growing field within oncology, and the cell surface antigen CD44v6, which is overexpressed in several cancers, is a plausible target for molecular radiotherapy of ATC. IHC of 39 patient samples with ATC was evaluated for CD44v6 expression. Biodistribution and dosimetry of iodine-125 (¹²⁵I)–/lutetium-177 (¹⁷⁷Lu)–labeled UU-40, a CD44v6-specific antibody, followed by in vivo efficacy in two ATC xenograft models with varying target expression levels (ACT-1 and BHT-101), accompanied by single-photon emission computed tomography (SPECT) imaging, evaluated radiolabeled UU-40 for therapeutic efficiency in ATC xenografts. The IHC revealed CD44v6 immunoreactivity in 46% of patient samples with ATC. The biodistribution favored ¹⁷⁷Lu-labeled UU-40 over the ¹²⁵I-labeled antibody and confirmed the in vivo specificity of both radioconjugates. The in vivo efficacy and accompanied SPECT imaging of a moderate CD44v6-expressing xenograft model (BHT-101) verified the tumor specificity, as well as the target-specific effect of ¹⁷⁷Lu-labeled UU-40 on tumor growth and survival. A 100% complete response rate was demonstrated as a result of therapy using a single dose of 16 MBq ¹⁷⁷Lu-labeled UU-40 in a high CD44v6-expressing xenograft model (ACT-1), and SPECT imaging revealed excellent tumor uptake of the radioconjugate at 14 days after injection. This study verifies the expression of CD44v6 in ATC and strengthens the superiority and promise of ¹⁷⁷Lu-labeled UU-40 over ¹³¹I-labeled UU-40 for antibody-based molecular radiotherapy of CD44v6-positive ATC.

Background: Glioblastoma (GBM) is the deadliest form of brain cancer, impacting both adults and children, marked by exceptionally high morbidity and mortality rates, even with current standard treatments such as surgery, radiation therapy, and chemotherapy. Therefore, there is a pressing need for new therapeutic strategies to improve survival and reduce treatment side effects. In this study, we investigated the effect of HSP90 inhibition in combination with radiotherapy in established and patient-derived glioblastoma cell lines.  

Methods: Potential radiosensitizing effects of the HSP90 inhibitor Onalespib were studied in XTT and clonogenic survival assays as well as in tumor-mimicking multicellular spheroid models. Further, migration capacity and effects on protein expression were studied after exposure to Onalespib and radiation using Proximity Extension Assay analysis.  

Results: HSP90 inhibition with Onalespib synergistically enhanced the radiosensitivity of glioblastoma cells grown in 2D and 3D models, resulting in increased cell death, reduced migration capacity and activation of the apoptotic signaling pathway. The proteomic analysis of glioblastoma cells treated with Onalespib, radiation, and their combination revealed significant alterations in protein expression profiles, involved in growth signaling, immune modulation pathways and angiogenesis. Moreover, the combination treatment indicated potential for enhancing cell cycle arrest and apoptosis, suggesting promising antitumor effects.  

Conclusion: These findings demonstrate that HSP90 inhibition may be a promising strategy to enhance the efficacy of radiotherapy in the treatment of GBM, potent

Neuroblastoma is a pediatric cancer with a five-year survival rate of merely 50% for high-risk cases. The treatment regimen is aggressive, leading to extensive side effects that significantly impact patients’ quality of life.

Targeted radionuclide therapy (TRT) involves the systemic administration of cancer-specific radioconjugates. This thesis focuses on TRT against the somatostatin receptor 2 (SSTR2) and the antigen CD44v6, two targets that are overexpressed in neuroblastoma,  

Radiosensitization renders cells more sensitive to radiation, which can improve the therapeutic efficacy and potentially reduce the radiation dose required to achieve an antitumor effect. This thesis investigates radiosensitization through the stabilization of p53 and the inhibition of heat shock protein 90 (HSP90), two proteins involved in the cellular response to DNA damage.

In papers I and II, we investigated the combination of the SSTR2-targeting radioconjugate ¹⁷⁷Lu-DOTATATE with the p53-stabilizing peptide VIP116 for neuroblastoma treatment. The combination therapy demonstrated enhanced antitumor effects in both in vitro and in vivo studies using mice bearing human neuroblastoma xenografts. Notably, the untreated and monotreated controls showed no nephrotoxicity.

In paper III, we demonstrated that combining external beam radiotherapy with the HSP90-inhibitor Onalespib produced additive or synergistic effects in vitro across a panel of neuroblastoma cell lines. Additionally, mice bearing syngeneic neuroblastoma tumor xenografts treated with this combination exhibited significantly improved therapeutic efficacy compared to control groups.

In paper IV, we developed and characterized human anti-CD44v6 antibodies for molecular radiotherapy. This work identified a lead candidate, UU-40, which demonstrated high affinity, strong tumor uptake, and favorable in vivodistribution, making it a promising candidate for future use against CD44v6-expressing cancers.

In conclusion, this thesis demonstrates that radiosensitization enhances the antitumor effects of radiation therapy in preclinical models of neuroblastoma. It is our hope that these discoveries will enable more effective and less harmful treatments for children with neuroblastoma. This thesis also produced an anti-CD44v6 antibody that holds great potential for future use in targeted radionuclide therapy, paving the way for innovative treatments for CD44v6-expressing cancers, including neuroblastoma. 

Purpose

Molecular radiotherapy is a treatment modality that is highly suitable for targeting micrometastases and [¹⁷⁷Lu]Lu-DOTATATE is currently being explored as a potential novel treatment option for high-risk neuroblastoma. p53 is a key player in the proapoptotic signalling in response to radiation-induced DNA damage and is therefore a potential target for radiosensitisation.

Methods

This study investigated the use of the p53 stabilising peptide VIP116 and [¹⁷⁷Lu]Lu-DOTATATE, either alone or in combination, for treatment of neuroblastoma tumour xenografts in mice. Initially, the uptake of [¹⁷⁷Lu]Lu-DOTATATE in the tumours was confirmed, and the efficacy of VIP116 as a monotherapy was evaluated. Subsequently, mice with neuroblastoma tumour xenografts were treated with placebo, VIP116, [¹⁷⁷Lu]Lu-DOTATATE or a combination of both agents.

Results

The results demonstrated that monotherapy with either VIP116 or [¹⁷⁷Lu]Lu-DOTATATE significantly prolonged median survival compared to the placebo group (90 and 96.5 days vs. 50.5 days, respectively). Notably, the combination treatment further improved median survival to over 120 days. Furthermore, the combination group exhibited the highest percentage of complete remission, corresponding to a twofold increase compared to the placebo group. Importantly, none of the treatments induced significant nephrotoxicity. Additionally, the therapies affected various molecular targets involved in critical processes such as apoptosis, hypoxia and angiogenesis.

Conclusion

In conclusion, the combination of VIP116 and [¹⁷⁷Lu]Lu-DOTATATE presents a promising novel treatment approach for neuroblastoma. These findings hold potential to advance research efforts towards a potential cure for this vulnerable patient population.

Thyroid cancer is the most common endocrine malignancy, affecting nearly 600,000 new patients worldwide. Treatment with the BRAF inhibitor sorafenib partially prolongs progression-free survival in thyroid cancer patients, but fails to improve overall survival. This study examines enhancing sorafenib efficacy by combination therapy with the novel HSP90 inhibitor onalespib. In vitro efficacy of sorafenib and onalespib monotherapy as well as in combination was assessed in papillary (PTC) and anaplastic (ATC) thyroid cancer cells using cell viability and colony formation assays. Migration potential was studied in wound healing assays. The in vivo efficacy of sorafenib and onalespib therapy was evaluated in mice bearing BHT-101 xenografts. Sorafenib in combination with onalespib significantly inhibited PTC and ATC cell proliferation, decreased metabolic activity and cancer cell migration. In addition, the drug combination approach significantly inhibited tumor growth in the xenograft model and prolonged the median survival. Our results suggest that combination therapy with sorafenib and onalespib could be used as a new therapeutic approach in the treatment of thyroid cancer, significantly improving the results obtained with sorafenib as monotherapy. This approach has the potential to reduce treatment adaptation while at the same time providing therapeutic anti-cancer benefits such as reducing tumor growth and metastatic potential.

Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.

Children of mothers with prenatal depressive symptoms (PND) have a higher risk of behavioral problems; fetal programming through DNA methylation is a possible underlying mechanism. This study investigated DNA methylation in cord blood to identify possible "at birth" signatures that may indicate susceptibility to behavioral problems at 18 months of age. Cord blood was collected from 256 children of mothers who had self-reported on symptoms of depression during pregnancy and the behavior of their child at 18 months of age. Whole genome DNA methylation was assessed using Illumina MethylationEPIC assay. The mother and child pairs were categorized into four groups, based on both self-reported depressive symptoms, PND or Healthy control (HC), and scores from the Child Behavior checklist (high or low for internalizing, externalizing, and total scores). Adjustments were made for batch effects, cell-type, and clinical covariates. Differentially methylated sites were identified using Kruskal-Wallis test, and Benjamini-Hochberg adjusted p values < 0.05 were considered significant. The analysis was also stratified by sex of the child. Among boys, we observed higher and correlated DNA methylation of one CpG-site in the promoter region of TPP1 in the HC group, with high externalizing scores compared to HC with low externalizing scores. Boys in the PND group showed lower DNA methylation in NUDT15 among those with high, compared to low, internalizing scores; the DNA methylation levels of CpGs in this gene were positively correlated with the CBCL scores. Hence, the differentially methylated CpG sites could be of interest for resilience, regardless of maternal mental health during pregnancy. The findings are in a relatively healthy study cohort, thus limiting the possibility of detecting strong effects associated with behavioral difficulties. This is the first investigation of cord blood DNA methylation signs of fetal programming of PND on child behavior at 18 months of age and thus calls for independent replications.