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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. 

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.

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.

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.

p53 is involved in DNA damage response and is an exciting target for radiosensitization in cancer. Targeted radionuclide therapy against somatostatin receptors with ¹⁷⁷Lu-DOTATATE is currently being explored as a treatment for neuroblastoma. The aim of this study was to investigate the novel p53-stabilizing peptide VIP116 in neuroblastoma, both as monotherapy and together with ¹⁷⁷Lu-DOTATATE. Five neuroblastoma cell lines, including two patient-derived xenograft (PDX) lines, were characterized in monolayer cultures. Four out of five were positive for ¹⁷⁷Lu-DOTATATE uptake. IC₅₀ values after VIP116 treatments correlated with p53 status, ranging between 2.8&ndash;238.2 &mu;M. IMR-32 and PDX lines LU-NB-1 and LU-NB-2 were then cultured as multicellular tumor spheroids and treated with ¹⁷⁷Lu-DOTATATE and/or VIP116. Spheroid growth was inhibited in all spheroid models for all treatment modalities. The most pronounced effects were observed for combination treatments, mediating synergistic effects in the IMR-32 model. VIP116 and combination treatment increased p53 levels with subsequent induction of p21, Bax and cleaved caspase 3. Combination treatment resulted in a 14-fold and 1.6-fold induction of MDM2 in LU-NB-2 and IMR-32 spheroids, respectively. This, together with differential MYCN signaling, may explain the varying degree of synergy. In conclusion, VIP116 inhibited neuroblastoma cell growth, potentiated ¹⁷⁷Lu-DOTATATE treatment and could, therefore, be a feasible treatment option for neuroblastoma.