Optimizing the Therapeutic Index of sdAb-Based Radiopharmaceuticals Using Pretargeting.

Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Methods: Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for 177Lu-TRT. Key pretargeting parameters (sdAb-injected mass and lag time) were optimized in healthy mice and U87MG (FAP+) xenografts. A TRT study in a pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (PDX) model was performed as a pilot study for sdAb-based pretargeting applications. Results: Modification of 4AH29 with trans-cyclooctene (TCO) moieties did not modify the sdAb pharmacokinetic profile. A 200-µg injected mass of 4AH29-TCO and an 8-h lag time for the injection of [177Lu]Lu-DOTA-PEG7-tetrazine resulted in the highest kidney therapeutic index (2.0 ± 0.4), which was 5-fold higher than that of [177Lu]Lu-DOTA-4AH29 (0.4 ± 0.1). FAP expression in the tumor microenvironment was validated in a PDAC PDX model with both immunohistochemistry and PET/CT imaging. Mice treated with the pretargeting high-activity approach (4AH29-TCO + [177Lu]Lu-DOTA-PEG7-tetrazine; 3 × 88 MBq, 1 injection per week for 3 wk) demonstrated prolonged survival compared with the vehicle control and conventionally treated ([177Lu]Lu-DOTA-4AH29; 3 × 37 MBq, 1 injection per week for 3 wk) mice. Mesangial expansion was reported in 7 of 10 mice in the conventional cohort, suggesting treatment-related kidney morphologic changes, but was not observed in the pretargeting cohort. Conclusion: This study validates pretargeting to mitigate sdAbs' kidney retention with no observation of morphologic changes on therapy regimen at early time points. Clinical translation of click-chemistry-based pre-TRT is warranted on the basis of its ability to alleviate toxicities related to biovectors' intrinsic pharmacokinetic profiles. The absence of representative animal models with extensive stroma and high FAP expression on cancer-associated fibroblasts led to a low mean tumor-absorbed dose even with high injected activity and consequently to modest survival benefit in this PDAC PDX.

Absorbed Dose-Response Relationship in Patients with Gastroenteropancreatic Neuroendocrine Tumors Treated with [177Lu]Lu-DOTATATE: One Step Closer to Personalized Medicine.

[177Lu]Lu-DOTATATE has been approved for progressive and inoperable gastroenteropancreatic neuroendocrine tumors (GEP-NETs) that overexpress somatostatin receptors. The absorbed doses by limiting organs and tumors can be quantified by serial postinfusion scintigraphy measurements of the γ-emissions from 177Lu. The objective of this work was to explore how postinfusion [177Lu]Lu-DOTATATE dosimetry could influence clinical management by predicting treatment efficacy (tumor shrinkage and survival) and toxicity. Methods: Patients with GEP-NETs treated with [177Lu]Lu-DOTATATE between 2016 and 2022 and who underwent dosimetry were included. Absorbed doses were calculated for healthy organs (liver, kidneys, bone marrow, and spleen) and tumors using PLANET Dose and the local energy deposition method based on serial posttreatment SPECT/CT. Up to 5 lesions per site were selected and measured on images collected at baseline and 3 mo after treatment end (measurement masked to the somatostatin receptor imaging uptake). For toxicity assessment, laboratory parameters were regularly monitored. Clinical data, including time to death or progression, were collected from the patients' health records. Correlations between absorbed doses by organs and toxicity and between absorbed doses by lesions and tumor volume variation were studied using regression models. Results: In total, 35 dosimetric studies were performed in patients with mostly grade 2 (77%) tumors and metastases in liver (89%), lymph nodes (77%), and bone (34%), and 146 lesions were analyzed: 1-9 lesions per patient, mostly liver metastases (65%) and lymph nodes (25%). The median total absorbed dose by tumors was 94.4 Gy. The absorbed doses by tumors significantly decreased between cycles. The absorbed dose by tumors was significantly associated with tumor volume variation (P < 0.001) 3 mo after treatment end, and it was a significant prognostic factor for survival. Toxicity analysis showed a correlation between the decrease of hematologic parameters such as lymphocytes or platelet concentrations and the absorbed doses by the spleen or bone marrow. The mean absorbed dose by the kidneys was not correlated with nephrotoxicity during the studied period. Conclusion: In patients treated with [177Lu]Lu-DOTATATE for GEP-NETs, tumor and healthy organ dosimetry can predict survival and toxicities, thus influencing clinical management.

Structural approach to charge density waves in low-dimensional systems: electronic instability and chemical bonding.

The charge density wave (CDW) instability, usually occurring in low-dimensional metals, has been a topic of interest for longtime. However, some very fundamental aspects of the mechanism remain unclear. Recently, a plethora of new CDW materials, a substantial fraction of which is two-dimensional or even three-dimensional, has been prepared and characterised as bulk and/or single-layers. As a result, the need for revisiting the primary mechanism of the instability, based on the electron-hole instability established more than 50 years ago for quasi-one-dimensional (quasi-1D) conductors, has clearly emerged. In this work, we consider a large number of CDW materials to revisit the main concepts used in understanding the CDW instability, and emphasise the key role of the momentum dependent electron-phonon coupling in linking electronic and structural degrees of freedom. We argue that for quasi-1D systems, earlier weak coupling theories work appropriately and the energy gain due to the CDW and the concomitant periodic lattice distortion (PLD) remains primarily due to a Fermi surface nesting mechanism. However, for materials with higher dimensionality, intermediate and strong coupling regimes are generally at work and the modification of the chemical bonding network by the PLD is at the heart of the instability. We emphasise the need for a microscopic approach blending condensed matter physics concepts and state-of-the-art first-principles calculations with quite fundamental chemical bonding ideas in understanding the CDW phenomenon in these materials.

Clinical and genetic features of patients suffering from CMT4J.

Mutations in the FIG4 gene have been identified in various diseases, including amyotrophic lateral sclerosis, Parkinson's disease, and Charcot-Marie-Tooth 4 J (CMT4J), with a wide range of phenotypic manifestations. We present eight cases of CMT4J patients carrying the p.Ile41Thr mutation of FIG4. The patients were categorized according to their phenotype. Six patients had a pure CMT; whereas, two patients had a CMT associated with parkinsonism. Three patients had an early onset and exhibited more severe forms of the disease. Three others experienced symptoms in their teenage years and had milder forms. Two patients had a late onset in adulthood. Four patients showed electrophysiological evidence of conduction blocks, typically associated with acquired neuropathies. Consequently, two of them received intravenous immunoglobulin treatment without a significant objective response. Interestingly, two heterozygous patients with the same mutations exhibited contrasting phenotypes, one having a severe early-onset form and the other experiencing a slow disease progression starting at the age of 49. Notably, although 7 out of 8 patients in this study were compound heterozygous for the p.Ile41Thr mutation, only one individual was found to be homozygous for this genetic variant and exhibited an early-onset, severe form of the disease. Additionally, one patient who developed the disease in his youth was also diagnosed with hereditary neuropathy with pressure palsies. Our findings provide insights into the CMT4J subtype by reporting on eight heterogeneous patient cases and highlight the potential for misdiagnosis when conduction blocks or asymmetrical nerve conduction study results are observed in patients with FIG4 mutations.

Gadolinium-Based Nanoparticles Sensitize Ovarian Peritoneal Carcinomatosis to Targeted Radionuclide Therapy.

Ovarian cancer (OC) is the most lethal gynecologic malignancy (5-y overall survival rate, 46%). OC is generally detected when it has already spread to the peritoneal cavity (peritoneal carcinomatosis). This study investigated whether gadolinium-based nanoparticles (Gd-NPs) increase the efficacy of targeted radionuclide therapy using [177Lu]Lu-DOTA-trastuzumab (an antibody against human epidermal growth factor receptor 2). Gd-NPs have radiosensitizing effects in conventional external-beam radiotherapy and have been tested in clinical phase II trials. Methods: First, the optimal activity of [177Lu]Lu-DOTA-trastuzumab (10, 5, or 2.5 MBq) combined or not with 10 mg of Gd-NPs (single injection) was investigated in athymic mice bearing intraperitoneal OC cell (human epidermal growth factor receptor 2-positive) tumor xenografts. Next, the therapeutic efficacy and toxicity of 5 MBq of [177Lu]Lu-DOTA-trastuzumab with Gd-NPs (3 administration regimens) were evaluated. NaCl, trastuzumab plus Gd-NPs, and [177Lu]Lu-DOTA-trastuzumab alone were used as controls. Biodistribution and dosimetry were determined, and Monte Carlo simulation of energy deposits was performed. Lastly, Gd-NPs' subcellular localization and uptake, and the cytotoxic effects of the combination, were investigated in 3 cancer cell lines to obtain insights into the involved mechanisms. Results: The optimal [177Lu]Lu-DOTA-trastuzumab activity when combined with Gd-NPs was 5 MBq. Moreover, compared with [177Lu]Lu-DOTA-trastuzumab alone, the strongest therapeutic efficacy (tumor mass reduction) was obtained with 2 injections of 5 mg of Gd-NPs/d (separated by 6 h) at 24 and 72 h after injection of 5 MBq of [177Lu]Lu-DOTA-trastuzumab. In vitro experiments showed that Gd-NPs colocalized with lysosomes and that their radiosensitizing effect was mediated by oxidative stress and inhibited by deferiprone, an iron chelator. Exposure of Gd-NPs to 177Lu increased the Auger electron yield but not the absorbed dose. Conclusion: Targeted radionuclide therapy can be combined with Gd-NPs to increase the therapeutic effect and reduce the injected activities. As Gd-NPs are already used in the clinic, this combination could be a new therapeutic approach for patients with ovarian peritoneal carcinomatosis.

Radiopharmaceuticals as combinatorial partners for immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of multiple cancer types. However, only a fraction of patients with cancer responds to ICIs employed as stand-alone therapeutics, calling for the development of safe and effective combinatorial regimens to extend the benefits of ICIs to a larger patient population. In addition to exhibiting a good safety and efficacy profile, targeted radionuclide therapy (TRT) with radiopharmaceuticals that specifically accumulate in the tumor microenvironment has been associated with promising immunostimulatory effects that (at least in preclinical cancer models) provide a robust platform for the development of TRT/ICI combinations. We discuss preclinical and clinical findings suggesting that TRT stands out as a promising partner for the development of safe and efficient combinatorial regimens involving ICIs.

Clonogenic assay to measure bystander cytotoxicity of targeted alpha-particle therapy.

Radiation therapy induces targeted effects in the cells that are irradiated and also non-targeted effects (i.e. bystander effects) in non-irradiated cells that are close to or at short distance (<∼1 mm) from irradiated cells. Bystander effects are mediated by intercellular communications and may result in cytotoxic and genotoxic modifications. Their occurrence and relative contribution to the irradiation outcome are influenced by several parameters among which the particle linear energy transfer seems to be prominent. Bystander effects were first observed after external radiation therapy, but have been described also following targeted radionuclide therapy. Therefore, we propose a method to investigate their occurrence in experimental conditions where cells are exposed to radiopharmaceuticals. In this approach, clonogenic cell death is the biological endpoint of the bystander effects caused by irradiation with alpha particles (a potent inducer of the bystander response).

Rapid communication: insights into the role of extracellular vesicles during Auger radioimmunotherapy.

PURPOSE: Non-targeted effects, including bystander and systemic effects, play a crucial role during Auger targeted radionuclide therapy. Here, we investigated whether small extracellular vesicles (sEVs) produced by irradiated cells could contribute to the bystander cytotoxic effects in vitro and also to therapeutic efficacy in vivo, after their injection in tumor xenografts. MATERIALS AND METHODS: B16F10 melanoma donor cells were exposed to radiolabeled antibodies (Auger radioimmunotherapy, RIT) for 48 h or to X-rays (donor cells). Then, donor cells were incubated with fresh medium for 2 h to prepare conditioned medium (CM) that was transferred onto recipient cells for bystander effect assessment, or used for sEVs enrichment. Resulting sEVs were incubated in vitro with recipient cells for determining bystander cytotoxicity, or injected in B16F10 melanoma tumors harbored by athymic and C57BL/6 mice. RESULTS: In vitro analysis of bystander cytotoxic effects showed that CM killed about 30-40% of melanoma cells. SEVs isolated from CM contributed to this effect. Moreover, the double-stranded DNA (dsDNA) content was increased in sEVs isolated from CM of exposed cells compared to control (not exposed), but the difference was significant only for the X-ray condition. These results were supported by immunodetection of cytosolic dsDNA in donor cells, a phenomenon that should precede dsDNA enrichment in sEVs. However, sEVs cytotoxicity could not be detected in vivo. Indeed, in athymic and in immunocompetent mice that received four intratumoral injections of sEVs (1/day), tumor growth was not delayed compared with untreated controls. Tumor growth was slightly (not significantly) delayed in immunocompetent mice treated with sEVs from X-ray-exposed cells, and significantly with sEVs purified from CM collected after 48 h of incubation. These results highlight the need to determine the optimal conditions, including radiation absorbed dose and sEVs collection time, to obtain the strongest cytotoxic effects. CONCLUSIONS: This study demonstrates that sEVs could play a role during Auger RIT through bystander effects in vitro. No systemic effects were observed in vivo, under our experimental conditions. However, X-rays experiments showed that sEVs collection time might be influencing the nature of sEVs, a parameter that should also be investigated during Auger RIT.

A Recanalized Umbilical Vein Hypermetabolic Thrombosis Mimicked an Hepatocellular Carcinoma Recurrence: A New Pitfall?

ABSTRACT: A transarterial left hepatic artery radioembolization involving 90Y microspheres was performed on a cirrhotic man with hypermetabolic 18F-FDG segment III hepatocellular carcinoma. During the 18F-FDG PET/CT follow-up, the disappearance of the hypermetabolic lesion was initially observed. Then, a focal segment III hypermetabolism reappeared mimicking a recurrence before disappearing without any treatment. Finally, the hepatic MRI demonstrated that the transitory segment III hypermetabolism matched a thrombus of the dilated recanalized umbilical vein.

Immunostimulatory effects of radioimmunotherapy.

Radiation therapy (RT) is known for its ability to kill cancer cells in an immunogenic manner. Recent preclinical data demonstrate that targeted alpha-particle therapy shares with RT the capacity to elicit immunostimulatory effects, standing out as a promising strategy to circumvent immune checkpoint inhibitor resistance in immunologically 'cold' tumors.

From the target cell theory to a more integrated view of radiobiology in Targeted radionuclide therapy: The Montpellier group's experience.

Targeted radionuclide therapy (TRT) is used to treat disseminated or metastatic tumours in which conventional external beam radiotherapy (EBRT) would have unacceptable side effects. Unlike EBRT, TRT delivers low doses at a continuous low dose rate. In EBRT, the effect increases progressively with the dose rate, and biological effects (tumour control and normal tissue damage) are related to the dose according to a sigmoid curve model. This model is part of the so-called quantitative radiobiology that is mostly based on the target cell theory, according to which cell death is due to (lethal) radiation hits to vital cellular targets. This model was developed for EBRT, but was adapted to low dose-rate situations by including a parameter that reflects the time needed to repair tissue damage. However, a growing body of evidence indicates that the model should take into account also the biological effects, which are due to intercellular communications (bystander effects) and amplify the effects of radiation, as well as the immune system. Moreover, extranuclear targets must be considered, although induced intracellular and intercellular signalling pathways may ultimately result in DNA damage. It is likely that bystander effects and immune response always contribute to the overall response to TRT at different levels, and that dose and dose rate are key parameters in controlling their real contribution. We hypothesize that the dose rate is the key determinant in the balance between the physical and DNA-centred response on one side, and the biological response that integrates all subcellular compartments and intercellular signalling pathways on the other side.

Therapeutic antibodies - natural and pathological barriers and strategies to overcome them.

Antibody-based therapeutics have become a major class of therapeutics with over 120 recombinant antibodies approved or under review in the EU or US. This therapeutic class has experienced a remarkable expansion with an expected acceleration in 2021-2022 due to the extraordinary global response to SARS-CoV2 pandemic and the public disclosure of over a hundred anti-SARS-CoV2 antibodies. Mainly delivered intravenously, alternative delivery routes have emerged to improve antibody therapeutic index and patient comfort. A major hurdle for antibody delivery and efficacy as well as the development of alternative administration routes, is to understand the different natural and pathological barriers that antibodies face as soon as they enter the body up to the moment they bind to their target antigen. In this review, we discuss the well-known and more under-investigated extracellular and cellular barriers faced by antibodies. We also discuss some of the strategies developed in the recent years to overcome these barriers and increase antibody delivery to its site of action. A better understanding of the biological barriers that antibodies have to face will allow the optimization of antibody delivery near its target. This opens the way to the development of improved therapy with less systemic side effects and increased patients' adherence to the treatment.

In Silico Investigation of the Biological Implications of Complex DNA Damage with Emphasis in Cancer Radiotherapy through a Systems Biology Approach.

Different types of DNA lesions forming in close vicinity, create clusters of damaged sites termed as "clustered/complex DNA damage" and they are considered to be a major challenge for DNA repair mechanisms resulting in significant repair delays and induction of genomic instability. Upon detection of DNA damage, the corresponding DNA damage response and repair (DDR/R) mechanisms are activated. The inability of cells to process clustered DNA lesions efficiently has a great impact on the normal function and survival of cells. If complex lesions are left unrepaired or misrepaired, they can lead to mutations and if persistent, they may lead to apoptotic cell death. In this in silico study, and through rigorous data mining, we have identified human genes that are activated upon complex DNA damage induction like in the case of ionizing radiation (IR) and beyond the standard DNA repair pathways, and are also involved in cancer pathways, by employing stringent bioinformatics and systems biology methodologies. Given that IR can cause repair resistant lesions within a short DNA segment (a few nm), thereby augmenting the hazardous and toxic effects of radiation, we also investigated the possible implication of the most biologically important of those genes in comorbid non-neoplastic diseases through network integration, as well as their potential for predicting survival in cancer patients.

Basic aspects of the charge density wave instability of transition metal trichalcogenides NbSe3and monoclinic-TaS3.

NbSe3and monoclinic-TaS3(m-TaS3) are quasi-1D metals containing three different types of chains and undergoing two different charge density wave Peierls transitions atTP1andTP2associated with type III and type I chains, respectively. The nature of these transitions is discussed on the basis of first-principles DFT calculation of their Fermi surface (FS) and electron-hole response function. Because of the stronger inter-chain interactions, the FS and electron-hole response function are considerably more complex for NbSe3thanm-TaS3; however a common scenario can be put forward to rationalize the results. The intra-chain inter-band nesting processes dominate the strongest response for both type I and type III chains of the two compounds. Two well-defined maxima of the electron-hole response for NbSe3are found with the (0a*, 0c*) and (1/2a*, 1/2c*) transverse components atTP1andTP2, respectively, whereas the second maximum is not observed form-TaS3atTP2. Analysis of the different inter-chain coupling mechanisms leads to the conclusion that FS nesting effects are only relevant to set the transversea*components in NbSe3. The strongest inter-chain Coulomb coupling mechanism must be taken into account for the transverse coupling alongc*in NbSe3and along botha*andc*form-TaS3. Phonon spectrum calculations reveal the formation of a giant 2kFKohn anomaly form-TaS3. All these results support a weak coupling scenario for the Peierls transition of transition metal trichalcogenides.

Revisiting the Radiobiology of Targeted Alpha Therapy.

Targeted alpha therapy (TAT) using alpha particle-emitting radionuclides is in the spotlight after the approval of 223RaCl2 for patients with metastatic castration-resistant prostate cancer and the development of several alpha emitter-based radiopharmaceuticals. It is acknowledged that alpha particles are highly cytotoxic because they produce complex DNA lesions. Hence, the nucleus is considered their critical target, and many studies did not report any effect in other subcellular compartments. Moreover, their physical features, including their range in tissues (<100 µm) and their linear energy transfer (50-230 keV/µm), are well-characterized. Theoretically, TAT is indicated for very small-volume, disseminated tumors (e.g., micrometastases, circulating tumor cells). Moreover, due to their high cytotoxicity, alpha particles should be preferred to beta particles and X-rays to overcome radiation resistance. However, clinical studies showed that TAT might be efficient also in quite large tumors, and biological effects have been observed also away from irradiated cells. These distant effects are called bystander effects when occurring at short distance (<1 mm), and systemic effects when occurring at much longer distance. Systemic effects implicate the immune system. These findings showed that cells can die without receiving any radiation dose, and that a more complex and integrated view of radiobiology is required. This includes the notion that the direct, bystander and systemic responses cannot be dissociated because DNA damage is intimately linked to bystander effects and immune response. Here, we provide a brief overview of the paradigms that need to be revisited.

Targeted Radionuclide Therapy Using Auger Electron Emitters: The Quest for the Right Vector and the Right Radionuclide.

Auger electron emitters (AEEs) are attractive tools in targeted radionuclide therapy to specifically irradiate tumour cells while sparing healthy tissues. However, because of their short range, AEEs need to be brought close to sensitive targets, particularly nuclear DNA, and to a lower extent, cell membrane. Therefore, radioimmunoconjugates (RIC) have been developed for specific tumour cell targeting and transportation to the nucleus. Herein, we assessed, in A-431CEA-luc and SK-OV-31B9 cancer cells that express low and high levels of HER2 receptors, two 111In-RIC consisting of the anti-HER2 antibody trastuzumab conjugated to NLS or TAT peptides for nuclear delivery. We found that NLS and TAT peptides improved the nuclear uptake of 111In-trastuzumab conjugates, but this effect was limited and non-specific. Moreover, it did not result in a drastic decrease of clonogenic survival. Indium-111 also contributed to non-specific cytotoxicity in vitro due to conversion electrons (30% of the cell killing). Comparison with [125I]I-UdR showed that the energy released in the cell nucleus by increasing the RIC's nuclear uptake or by choosing an AEE that releases more energy per decay should be 5 to 10 times higher to observe a significant therapeutic effect. Therefore, new Auger-based radiopharmaceuticals need to be developed.

Radiation-Induced Immunity and Toxicities: The Versatility of the cGAS-STING Pathway.

In the past decade, radiation therapy (RT) entered the era of personalized medicine, following the striking improvements in radiation delivery and treatment planning optimization, and in the understanding of the cancer response, including the immunological response. The next challenge is to identify the optimal radiation regimen(s) to induce a clinically relevant anti-tumor immunity response. Organs at risks and the tumor microenvironment (e.g. endothelial cells, macrophages and fibroblasts) often limit the radiation regimen effects due to adverse toxicities. Here, we reviewed how RT can modulate the immune response involved in the tumor control and side effects associated with inflammatory processes. Moreover, we discussed the versatile roles of tumor microenvironment components during RT, how the innate immune sensing of RT-induced genotoxicity, through the cGAS-STING pathway, might link the anti-tumor immune response, radiation-induced necrosis and radiation-induced fibrosis, and how a better understanding of the switch between favorable and deleterious events might help to define innovative approaches to increase RT benefits in patients with cancer.