Advancing Surgical Arrhythmia Ablation: Novel Insights on 3D Printing Applications and Two Biocompatible Materials.

To date, studies assessing the safety profile of 3D printing materials for application in cardiac ablation are sparse. Our aim is to evaluate the safety and feasibility of two biocompatible 3D printing materials, investigating their potential use for intra-procedural guides to navigate surgical cardiac arrhythmia ablation. Herein, we 3D printed various prototypes in varying thicknesses (0.8 mm-3 mm) using a resin (MED625FLX) and a thermoplastic polyurethane elastomer (TPU95A). Geometrical testing was performed to assess the material properties pre- and post-sterilization. Furthermore, we investigated the thermal propagation behavior beneath the 3D printing materials during cryo-energy and radiofrequency ablation using an in vitro wet-lab setup. Moreover, electron microscopy and Raman spectroscopy were performed on biological tissue that had been exposed to the 3D printing materials to assess microparticle release. Post-sterilization assessments revealed that MED625FLX at thicknesses of 1 mm, 2.5 mm, and 3 mm, along with TPU95A at 1 mm and 2.5 mm, maintained geometrical integrity. Thermal analysis revealed that material type, energy source, and their factorial combination with distance from the energy source significantly influenced the temperatures beneath the 3D-printed material. Electron microscopy revealed traces of nitrogen and sulfur underneath the MED625FLX prints (1 mm, 2.5 mm) after cryo-ablation exposure. The other samples were uncontaminated. While Raman spectroscopy did not detect material release, further research is warranted to better understand these findings for application in clinical settings.

Targeted α-Therapy Using 225Ac Radiolabeled Single-Domain Antibodies Induces Antigen-Specific Immune Responses and Instills Immunomodulation Both Systemically and at the Tumor Microenvironment.

Targeted radionuclide therapy (TRT) using targeting moieties labeled with α-particle-emitting radionuclides (α-TRT) is an intensely investigated treatment approach as the short range of α-particles allows effective treatment of local lesions and micrometastases. However, profound assessment of the immunomodulatory effect of α-TRT is lacking in literature. Methods: Using flow cytometry of tumors, splenocyte restimulation, and multiplex analysis of blood serum, we studied immunologic responses ensuing from TRT with an antihuman CD20 single-domain antibody radiolabeled with 225Ac in a human CD20 and ovalbumin expressing B16-melanoma model. Results: Tumor growth was delayed with α-TRT and increased blood levels of various cytokines such as interferon-γ, C-C motif chemokine ligand 5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1. Peripheral antitumoral T-cell responses were detected on α-TRT. At the tumor site, α-TRT modulated the cold tumor microenvironment (TME) to a more hospitable and hot habitat for antitumoral immune cells, characterized by a decrease in protumoral alternatively activated macrophages and an increase in antitumoral macrophages and dendritic cells. We also showed that α-TRT increased the percentage of programmed death-ligand 1 (PD-L1)-positive (PD-L1pos) immune cells in the TME. To circumvent this immunosuppressive countermeasure we applied immune checkpoint blockade of the programmed cell death protein 1-PD-L1 axis. Combination of α-TRT with PD-L1 blockade potentiated the therapeutic effect, however, the combination aggravated adverse events. A long-term toxicity study revealed severe kidney damage ensuing from α-TRT. Conclusion: These data suggest that α-TRT alters the TME and induces systemic antitumoral immune responses, which explains why immune checkpoint blockade enhances the therapeutic effect of α-TRT. However, further optimization is warranted to avoid adverse events.

Radiotheranostic Agents in Hematological Malignancies.

Radioimmunotherapy (RIT) is a cancer treatment that combines radiation therapy with tumor-directed monoclonal antibodies (Abs). Although RIT had been introduced for the treatment of CD20 positive non-Hodgkin lymphoma decades ago, it never found a broad clinical application. In recent years, researchers have developed theranostic agents based on Ab fragments or small Ab mimetics such as peptides, affibodies or single-chain Abs with improved tumor-targeting capacities. Theranostics combine diagnostic and therapeutic capabilities into a single pharmaceutical agent; this dual application can be easily achieved after conjugation to radionuclides. The past decade has seen a trend to increased specificity, fastened pharmacokinetics, and personalized medicine. In this review, we discuss the different strategies introduced for the noninvasive detection and treatment of hematological malignancies by radiopharmaceuticals. We also discuss the future applications of these radiotheranostic agents.

Emerging applications of nanobodies in cancer therapy.

Cancer is a heterogeneous disease, requiring treatment tailored to the unique phenotype of the patient's tumor. Monoclonal antibodies (mAbs) and variants thereof have enabled targeted therapies to selectively target cancer cells. Cancer cell-specific mAbs have been used for image-guided surgery and targeted delivery of radionuclides or toxic agents, improving classical treatment strategies. Cancer cell-specific mAbs can further inhibit tumor cell growth or can stimulate immune-mediated destruction of cancer cells, a feature that has also been achieved through mAb-mediated manipulation of immune cells and pathways. Drawbacks of mAbs and their variants, together with the discovery of camelid heavy chain-only antibodies and the many advantageous features of their variable domains, referred to as VHHs, single domain antibodies or nanobodies (Nbs), resulted in the exploration of Nbs as an alternative targeting moiety. We therefore review the state-of-the-art as well as novel exploitation strategies of Nbs for targeted cancer therapy.

Targeted Radionuclide Therapy with Low and High-Dose Lutetium-177-Labeled Single Domain Antibodies Induces Distinct Immune Signatures in a Mouse Melanoma Model.

Targeted radionuclide therapy (TRT) using probes labeled with Lutetium-177 (177Lu) represents a new and growing type of cancer therapy. We studied immunologic changes in response to TRT with 177Lu labeled anti-human CD20 camelid single domain antibodies (sdAb) in a B16-melanoma model transfected to express human CD20, the target antigen, and ovalbumin, a surrogate tumor antigen. High-dose TRT induced melanoma cell death, calreticulin exposure, and ATP-release in vitro. Melanoma-bearing mice received fractionated low and high-dose TRT via tumor targeting anti-human CD20 sdAbs, as opposed to control sdAbs. Tumor growth was delayed with both doses. Low- and high-dose TRT increased IL10 serum levels. Low-dose TRT also decreased CCL5 serum levels. At the tumor, high-dose TRT induced a type I IFN gene signature, while low-dose TRT induced a proinflammatory gene signature. Low- and high-dose TRT increased the percentage of PD-L1pos and PD-L2pos myeloid cells in tumors with a marked increase in alternatively activated macrophages after high-dose TRT. The percentage of tumor-infiltrating T cells was not changed, yet a modest increase in ovalbumin-specific CD8pos T-cells was observed after low-dose TRT. Contradictory, low and high-dose TRT decreased CD4pos Th1 cells in addition to double negative T cells. In conclusion, these data suggest that low and high-dose TRT induce distinct immunologic changes, which might serve as an anchoring point for combination therapy.

CS1-specific single-domain antibodies labeled with Actinium-225 prolong survival and increase CD8+ T cells and PD-L1 expression in Multiple Myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by the presence of clonal plasma cells in the bone marrow niche. Despite significant therapeutic advances, MM remains incurable for the majority of patients. Targeted radionuclide therapy (TRNT) has emerged as a promising treatment option to eradicate residual cancer cells. In this study, we developed and characterized single-domain antibodies (sdAbs) against the MM-antigen CS1 and evaluated its therapeutic potential in MM using TRNT. We first validated CS1 as potential target for TRNT. CS1 is expressed in normal and malignant plasma cells in different disease stages including progression and relapse. It is expressed in dormant as well as proliferating MM cells, while low expression could be observed in environmental cells. Biodistribution studies demonstrated the specific uptake of anti-CS1 sdAbs in tissues of 5TMM cell infiltration including bone, spleen and liver. TRNT using anti-CS1 sdAbs labeled with actinium-225 significantly prolonged survival of syngeneic, immunocompetent 5T33MM mice. In addition, we observed an increase in CD8+ T-cells and more overall PD-L1 expression on immune and non-immune cells, implying an interferon gamma signature using actinium-225 labeled CS1-directed sdAbs. In this proof-of-principle study, we highlight, for the first time, the therapeutic potential and immunomodulating effects of anti-CS1 radionuclide therapy to target residual MM cells.

A non-internalised CD38-binding radiolabelled single-domain antibody fragment to monitor and treat multiple myeloma.

BACKGROUND: Antibody-based therapies targeting CD38 are currently used as single agents as well as in combination regimens for multiple myeloma, a malignant plasma cell disorder. In this study, we aimed to develop anti-CD38 single-domain antibodies (sdAbs) that can be used to trace CD38+ tumour cells and subsequently used for targeted radionuclide therapy. SdAbs are derived from Camelidae heavy-chain antibodies and have emerged as promising theranostic agents due to their favourable pharmacological properties. METHODS: Four different anti-CD38 sdAbs were produced, and their binding affinities and potential competition with the monoclonal antibody daratumumab were tested using biolayer interferometry. Their binding kinetics and potential cell internalisation were further studied after radiolabelling with the diagnostic radioisotope Indium-111. The resulting radiotracers were evaluated in vivo for their tumour-targeting potential and biodistribution through single-photon emission computed tomography (SPECT/CT) imaging and serial dissections. Finally, therapeutic efficacy of a lead anti-CD38 sdAb, radiolabelled with the therapeutic radioisotope Lutetium-177, was evaluated in a CD38+ MM xenograft model. RESULTS : We retained anti-CD38 sdAb #2F8 as lead based on its excellent affinity and superior stability, the absence of competition with daratumumab and the lack of receptor-mediated internalisation. When intravenously administered to tumour-xenografted mice, radiolabelled sdAb #2F8 revealed specific and sustained tumour retention with low accumulation in other tissues, except kidneys, resulting in high tumour-to-normal tissue ratios. In a therapeutic setting, myeloma-bearing mice received three consecutive intravenous administrations of a high (18.5 MBq) or a low radioactive dose (9.3 MBq) of 177Lu-DTPA-2F8 or an equal volume of vehicle solution. A dose-dependent tumour regression was observed, which translated into a prolonged median survival from 43 days for vehicle-treated mice, to 62 days (p = 0.027) in mice receiving the low and 65 days in mice receiving the high (p = 0.0007) radioactive dose regimen, respectively. CONCLUSIONS: These results highlight the theranostic potential of radiolabelled anti-CD38 sdAbs for the monitoring and treatment of multiple myeloma.

Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33.

Nanobodies (Nbs) are the smallest antigen-binding, single domain fragments derived from heavy-chain-only antibodies from Camelidae. Among the several advantages over conventional monoclonal antibodies, their small size (12-15 kDa) allows them to extravasate rapidly, to show improved tissue penetration, and to clear rapidly from blood, which are important characteristics for cancer imaging and targeted radiotherapy. Herein, we identified Nbs against CD33, a marker for acute myeloid leukemia (AML). A total of 12 Nbs were generated against recombinant CD33 protein, out of which six bound natively CD33 protein, expressed on the surface of acute myeloid leukemia THP-1 cells. The equilibrium dissociation constants (KD) of these six Nbs and CD33 range from 4 to 270 nM, and their melting temperature (Tm) varies between 52.67 and 67.80 °C. None of these Nbs showed leukemogenicity activity in vitro. The selected six candidates were radiolabeled with 99mTc, and their biodistribution was evaluated in THP-1-tumor-bearing mice. The imaging results demonstrated the fast tumor-targeting capacity of the Nbs in vivo. Among the anti-CD33 Nbs, Nb_7 showed the highest tumor uptake (2.53 ± 0.69 % injected activity per gram (IA/g), with low background signal, except in the kidneys and bladder. Overall, Nb_7 exhibits the best characteristics to be used as an anti-CD33 targeting vehicle for future diagnostic or therapeutic applications.

Stromal-targeting radioimmunotherapy mitigates the progression of therapy-resistant tumors.

Radioimmunotherapy (RIT) aims to deliver a high radiation dose to cancer cells, while minimizing the exposure of normal cells. Typically, monoclonal antibodies are used to target the radionuclides to cancer cell surface antigens. However, antibodies face limitations due to their poor tumor penetration and suboptimal pharmacokinetics, while the expression of their target on the cancer cell surface may be gradually lost. In addition, most antigens are expressed in a limited number of tumor types. To circumvent these problems, we developed a Nanobody (Nb)-based RIT against a prominent stromal cell (stromal-targeting radioimmunotherapy or STRIT) present in nearly all tumors, the tumor-associated macrophage (TAM). Macrophage Mannose Receptor (MMR) functions as a stable molecular target on TAM residing in hypoxic areas, further allowing the delivery of a high radiation dose to the more radioresistant hypoxic tumor regions. Since MMR expression is not restricted to TAM, we first optimized a strategy to block extra-tumoral MMR to prevent therapy-induced toxicity. A 100-fold molar excess of unlabeled bivalent Nb largely blocks extra-tumoral binding of 177Lu-labeled anti-MMR Nb and prevents toxicity, while still allowing the intra-tumoral binding of the monovalent Nb. Interestingly, three doses of 177Lu-labeled anti-MMR Nb resulted in a significantly retarded tumor growth, thereby outcompeting the effects of anti-PD1, anti-VEGFR2, doxorubicin and paclitaxel in the TS/A mammary carcinoma model. Together, these data propose anti-MMR STRIT as a valid new approach for cancer treatment.

Pharmacokinetics of radiolabeled dimeric sdAbs constructs targeting human CD20.

Single-domain antibody fragments (sdAbs) are the smallest functional antigen-binding fragments, derived from heavy chain-only camelid antibodies. When designed as radiolabeled monomeric probes for imaging and therapy of cancer, their fast and specific targeting results in high tumor-to-background ratios early after injection. However, their moderate absolute uptake into tumors might not always be sufficient to treat cancerous lesions. We have evaluated the pharmacokinetics of seven constructs derived from a CD20-targeting monomeric sdAb (αCD20). The constructs differed in affinity or avidity towards CD20 (dimeric αCD20-αCD20 and αCD20 fused to a non-targeting control sdAb, referred to as αCD20-ctrl) and blood half-lives (αCD20 fused to an albumin-targeting sdAb (αAlb) = αCD20-αAlb). The constructs were radiolabeled with 111In (imaging) and 177Lu (therapy) using the bifunctional chelator CHX-A"-DTPA and evaluated in vitro and in vivo. In mice, tumor uptake of 177Lu-DTPA-αCD20 decreased from 4.82 ±â€¯1.80 to 0.13 ±â€¯0.05% IA/g over 72 h. Due to its rapid blood clearance, tumor-to-blood (T/B) ratios of >100 were obtained within 24 h. Although in vitro internalization indicated that dimeric 177Lu-DTPA-αCD20-αCD20 was superior in terms of total cell-associated radioactivity, this was not confirmed in vivo. Blood clearance was slower and absolute tumor uptake became significantly higher for αCD20-αAlb. Blood levels of 177Lu-DTPA-αCD20-αAlb decreased from 68.30 ±â€¯10.53 to 3.58 ±â€¯0.66% IA/g over 120 h, while tumor uptake increased from 6.21 ±â€¯0.94 to 24.90 ±â€¯2.83% IA/g, resulting in lower T/B ratios. Taken together, these results indicate that the increased size of dimeric αCD20-αCD20 or the fusion of monomeric αCD20 to an albumin-targeting moiety (αAlb) counterbalance their improved tumor targeting capacity compared to monomeric αCD20.

Same-Day Imaging Using Small Proteins: Clinical Experience and Translational Prospects in Oncology.

Imaging of expression of therapeutic targets may enable stratification of patients for targeted treatments. The use of small radiolabeled probes based on the heavy-chain variable region of heavy-chain-only immunoglobulins or nonimmunoglobulin scaffolds permits rapid localization of radiotracers in tumors and rapid clearance from normal tissues. This makes high-contrast imaging possible on the day of injection. This mini review focuses on small proteins for radionuclide-based imaging that would allow same-day imaging, with the emphasis on clinical applications and promising preclinical developments within the field of oncology.

Evaluation of [99mTc]Radiolabeled Macrophage Mannose Receptor-Specific Nanobodies for Targeting of Atherosclerotic Lesions in Mice.

PURPOSE: Macrophage accumulation characterizes the development of atherosclerotic plaques, and the presence of certain macrophage subsets might be an indicator of plaque phenotype and (in)stability. The macrophage mannose receptor (MMR) is expressed on alternatively activated macrophages and found at sites of intraplaque hemorrhage and neovascularization. It has been proposed as target to identify vulnerable plaques. Therefore, we aimed to assess the feasibility of using anti-MMR nanobodies (Nbs) as molecular tracers for nuclear imaging in an animal model of atherosclerosis. PROCEDURE: Anti-MMR and control Nb, radiolabeled with Tc-99m, were injected in ApoE-/- and/or C57Bl/6 mice (n = 6). In vivo competition studies involving pre-injection of excess of unlabeled anti-MMR Nb (n = 3) and injection of anti-MMR Nb in MMR-/- mice (n = 3) were performed to demonstrate specificity. At 3 h p.i. radioactive uptake in organs, tissues and aorta segments were evaluated. Autoradiography and immunofluorescence were performed on aortic sections. RESULTS: Significantly higher uptake was observed in all aortic segments of ApoE-/- mice injected with anti-MMR Nb compared to control Nb (1.36 ± 0.67 vs 0.38 ± 0.13 percent of injected dose per gram (%ID/g), p ≤ 0.001). Surprisingly, high aortic uptake was also observed in C57Bl/6 mice (1.50 ± 0.43%ID/g, p ≥ 0.05 compared to ApoE-/-), while aortic uptake was reduced to background levels in the case of competition and in MMR-/- mice (0.46 ± 0.10 and 0.22 ± 0.06%ID/g, respectively; p ≤ 0.001). Therefore, expression of MMR along healthy aortas was suggested. Autoradiography showed no specific radioactive signal within atherosclerotic plaques, but rather localization of the signal along the aorta, correlating with MMR expression in perivascular tissue as demonstrated by immunofluorescence. CONCLUSIONS: No significant uptake of MMR-specific Nb could be observed in atherosclerotic lesions of ApoE-/- mice in this study. A specific perivascular signal causing a non-negligible background level was demonstrated. This observation should be considered when using MMR as a target in molecular imaging of atherosclerosis, as well as use of translational animal models with vulnerable plaques.

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma.

Anti-CD20 radioimmunotherapy is an effective approach for therapy of relapsed or refractory CD20pos lymphomas, but faces limitations due to poor tumor penetration and undesirable pharmacokinetics of full antibodies. Camelid single-domain Ab fragments (sdAb) might circumvent some of the limitations of radiolabeled full antibodies. In this study, a set of hCD20-targeting sdAbs was generated, and their capacity to bind hCD20 was evaluated in vitro and in vivo A lead sdAb, sdAb 9079, was selected on the basis of its specific tumor targeting and significant lower kidney accumulation compared with other sdAbs. SdAb 9079 was then radiolabeled with 68Ga and 177Lu for PET imaging and targeted therapy. The therapeutic potential of 177Lu-DTPA-sdAb was compared with that of 177Lu-DTPA-rituximab and unlabeled rituximab in mice bearing hCD20pos tumors. Radiolabeled with 68Ga, sdAb 9079 showed specific tumor uptake, with very low accumulation in nontarget organs, except kidneys. The tumor uptake of 177Lu-DTPA-sdAb 9079 after 1.5 h was 3.4 ± 1.3% ID/g, with T/B and T/M ratios of 13.3 ± 4.6 and 32.9 ± 15.6. Peak tumor accumulation of 177Lu-DTPA-rituximab was about 9 times higher, but concomitantly with high accumulation in nontarget organs and very low T/B and T/M ratios (0.8 ± 0.1 and 7.1 ± 2.4). Treatment of mice with 177Lu-DTPA-sdAb 9079 significantly prolonged median survival compared with control groups and was as effective as treatment with rituximab or its 177Lu-labeled variant. Taken together, sdAb 9079 displays promising features as a theranostic drug to treat CD20pos lymphomas. Mol Cancer Ther; 16(12); 2828-39. ©2017 AACR.

Preclinical evaluation of an mRNA HIV vaccine combining rationally selected antigenic sequences and adjuvant signals (HTI-TriMix).

BACKGROUND: The development of a prophylactic vaccine against HIV-1 has so far not been successful. Therefore, attention has shifted more and more toward the development of novel therapeutic vaccines. Here, we evaluated a new mRNA-based therapeutic vaccine against HIV-1-encoding activation signals (TriMix: CD40L + CD70 + caTLR4) combined with rationally selected antigenic sequences [HIVACAT T-cell immunogen (HTI)] sequence: comprises 16 joined fragments from Gag, Pol, Vif, and Nef). METHODS: For this purpose, peripheral blood mononuclear cells from HIV-1-infected individuals on cART, lymph node explants from noninfected humans, and splenocytes from immunized mice were collected and several immune functions were measured. RESULTS: Electroporation of immature monocyte-derived dendritic cells from HIV-infected patients with mRNA encoding HTI + TriMix potently activated dendritic cells which resulted in upregulation of maturation markers and cytokine production and T-cell stimulation, as evidenced by enhanced proliferation and cytokine secretion (IFN-γ). Responses were HIV specific and were predominantly targeted against the sequences included in HTI. These findings were confirmed in human lymph node explants exposed to HTI + TriMix mRNA. Intranodal immunizations with HTI mRNA in a mouse model increased antigen-specific cytotoxic T-lymphocyte responses. The addition of TriMix further enhanced cytotoxic responses. CONCLUSION: Our results suggest that uptake of mRNA, encoding strong activation signals and a potent HIV antigen, confers a T-cell stimulatory capacity to dendritic cells and enhances their ability to stimulate antigen-specific immunity. These findings may pave the way for therapeutic HIV vaccine strategies based on antigen-encoding RNA to specifically target antigen-presenting cells.

Targeted alpha therapy using short-lived alpha-particles and the promise of nanobodies as targeting vehicle.

INTRODUCTION: The combination of a targeted biomolecule that specifically defines the target and a radionuclide that delivers a cytotoxic payload offers a specific way to destroy cancer cells. Targeted radionuclide therapy (TRNT) aims to deliver cytotoxic radiation to cancer cells and causes minimal toxicity to surrounding healthy tissues. Recent advances using α-particle radiation emphasizes their potential to generate radiation in a highly localized and toxic manner because of their high level of ionization and short range in tissue. AREAS COVERED: We review the importance of targeted alpha therapy (TAT) and focus on nanobodies as potential beneficial vehicles. In recent years, nanobodies have been evaluated intensively as unique antigen-specific vehicles for molecular imaging and TRNT. EXPERT OPINION: We expect that the efficient targeting capacity and fast clearance of nanobodies offer a high potential for TAT. More particularly, we argue that the nanobodies' pharmacokinetic properties match perfectly with the interesting decay properties of the short-lived α-particle emitting radionuclides Astatine-211 and Bismuth-213 and offer an interesting treatment option particularly for micrometastatic cancer and residual disease.