Search

The COVID-19 pandemic caused by SARS-CoV-2 led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options, which can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2 infected animal models, but also for (ii) clearance of virus infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform “UniCAR” is currently under early clinical investigation. Recently, first proof-of-concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to a high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off-switch of UniCAR T-cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T-cells for the therapy of AML.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic with millions of infections and deaths worldwide and devastating impact on global economy. Up to now, vaccines and monoclonal antibody (mAb) therapies lack to provide a long-lasting protection against rapidly evolving new emerging SARS-CoV-2 variants. Thus, novel therapeutic options are pressingly needed especially for immunocompromised patients and/or patients with high risk for developing a severe coronavirus disease 2019 (COVID-19). In that regard, we developed a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2). This ACE2 decoy potently binds to the SARS-CoV-2 receptor binding domain (RBD), neutralizes SARS-CoV-2 as well as the Delta and Omicron variant and protects hamsters from a SARS-CoV-2 infection. To additionally use this ACE2 decoy for elimination of virus infected cells, we equipped it with an epitope tag. Thus, it can be applied as adapter molecule in the modular platform technologies UniMAB and UniCAR, which already demonstrated great success in the setting of malignant diseases. As adapter molecule the ACE2 decoy is able to efficiently recruit either universal chimeric antigen receptor (UniCAR) modified T cells or, in combination with an anti-peptide epitope-anti-CD3 bispecific Ab of the UniMAB system, unmodified T cells to efficiently kill SARS-CoV-2 RBD expressing human cells. Taken together, the ACE2 decoy represents a very promising immunotherapeutic drug for both SARS-CoV-2 variant neutralization and infected cell killing via the UniMAB and UniCAR system and might, therefore, clearly improve the treatment of COVID-19 patients.

Chimeric antigen receptor (CAR) T-cells are undoubtedly a promising approach in cancer immunotherapy. Nevertheless, mild to severe toxicities are associated with this approach including on-target/off-tumor effects and cytokine release syndrome. Aiming for increased clinical safety, adaptor CAR technologies were developed which include the modular universal CAR (UniCAR) platform developed by our group. UniCAR T-cells are exclusively activated in the presence of a target module (TM), which establishes the cross-link between cancer cells and UniCAR T-cells. These TMs are highly versatile molecules that can be constructed not only by using antibody fragments but also e.g. peptides specifically targeting a receptor or molecule on the cell´s surface. Somatostatin receptor (SSTR) subtype 2 is highly expressed in a variety of malignancies and has therefore been studied as a marker and target for cancer diagnosis and treatment. Currently, SSTR2 agonists and antagonists, such as Tyr3-octreotate (TATE) and BASS or JR11, respectively, are particularly well established and clinically implemented mostly used for diagnostic nuclear medicine. Given this and the proven flexibility and efficacy of the UniCAR system, we hereby aimed to develop small peptide-derived TMs targeting SSTR2 that can be used for both immunotherapeutic and diagnostic approaches. For that, the abovementioned SSTR2 agonist and antagonists were chemically linked to the E5B9 peptide and equipped with the radiometal chelator NODAGA. These TMs were tested in vitro and in vivo, in which they have proven to specifically redirect UniCAR T-cells to human neuroendocrine and breast SSTR2-expressing cancer cells. Furthermore, the enrichment of these anti-SSTR2 peptide TMs at the tumor site was confirmed by positron emission tomography (PET) studies. We hereby designed novel small peptide-derived TMs that can be used for redirection of UniCAR T-cells to SSTR2-expressing cancer cells as well as for PET imaging, proving to b

CAR T-cell therapy achieved unparalleled clinical success rates for treatment of patients with hematological malignancies. However, progress and clinical translation towards solid tumors is slow and hampered by many factors e.g. increased complexity, high heterogeneity and an immunosuppressive tumor microenvironment. Thus, CAR T-cell therapy alone might not result in durable antitumor responses. Combinatorial approaches are promising strategies to improve CAR T-cell efficacy in solid tumor treatment. In this regard, we here aim to combine conventional cancer theranostics with CAR T-cell immunotherapy in one single approach. By using the well-established UniCAR system, a novel, multifunctional tool termed PSCA-IgG4 target module (TM) was developed for dual prostate cancer theranostics. It comprises a human PSCA-specific binding domain, the hinge and Fc-domain of human IgG4 molecules as well as the UniCAR epitope E5B9. As shown by in vitro assays with PSCA-positive and PSCA-negative prostate cancer cells, the novel TM redirected UniCAR T cells for efficient tumor cell lysis in a strictly antigen- and TM-dependent manner. After radiolabeling with copper-64 or actinium-225, the novel PSCA-IgG4 TM was successfully applied for diagnostic imaging and targeted radioimmunotherapy. The 64Cu-labeled PSCA-IgG4 TM showed maximal tumor accumulation with optimal tumor-to-background ratios after 1.5 days. Furthermore, targeted alpha-therapy with the 225Aclabeled TM significantly delayed the outgrowth of established tumors in mice. In summary, the here presented, novel PSCA-IgG4 TM is a promising candidate for dual theranostics of prostate cancer that may help to overcome present hurdles in solid tumor therapy. After radiolabeling it facilitates not only targeted alpha-therapy and diagnostic imaging of PSCA, but can be also repurposed as a TM for UniCAR T-cell immunotherapy.

Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic PET imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with 64Cu2+ the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with 225Ac3+ and applied for targeted alpha therapy. A single injection of the 225Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy.

Modification of autologous T cells with tumor-specific chimeric antigen receptors (CARs) has been shown to be an effective tool for treatment of hematologic malignancies. However, clinical translation towards solid tumors is still limited as demonstrated by the suboptimal performance of CAR T cells in first in-human studies. Combining advances in CAR-T cell therapy and cancer theranostics may provide a promising multimodal approach to increase therapeutic efficacy and achieve durable responses in cancer patients. Here we present the development and functional characterization of different peptide- or antibody constructs targeting tumor-associated antigens or different structures of the tumor microenvironment. Equipment of these molecules with the E5B9 peptide epitope, enabled their use in the well-established UniCAR system. Accordingly, the constructs were able to specifically activate UniCAR T cells for efficient killing of both hematological and solid tumors in vitro and in vivo. Upon conjugation of chelators like DOTAGA and NODAGA, the peptide- or antibody derivates were further successfully applied for cancer theranostics, using e.g. 68Ga, 64Cu as diagnostic radionuclides and e.g. 67Cu, 225Ac as therapeutic radionuclides. PET imaging in xenotransplanted mice has demonstrated high contrast tumor accumulation. Consistent with the stable tumor accumulation, 225Ac-labeled molecules demonstrated therapeutic efficacy in a xenograft mouse model. In summary, E5B9-tagged peptide- or antibody constructs open a new era of cancer immunotheranostics as they can be used multimodally for (i) UniCAR-T cell therapy, (ii) non-invasive diagnostic imaging and (iii) targeted radioimmunotherapy. Prospectively, they could thus bridge the gap between the fields of CAR-T cells and cancer theranostics.

Background Chimeric antigen receptor (CAR) T-cells are living drugs successfully applied for treatment of patients with hematological malignancies. However, clinical translation towards solid tumor therapy faces several challenges and does not result in durable responses. In order to improve CAR T-cells´ therapeutic efficiency, combinations with other treatment and imaging modalities are intensively investigated. Methods For prostate cancer theranostics we developed a novel, multifunctional IgG4-based antibody construct directed against the prostate stem cell antigen (PSCA). Due to equipment with the UniCAR-tag E5B9 it can be used as a target module (TM) for the well-established UniCAR T-cell approach [1]. Functionality of the PSCA-IgG4 antibody was investigated in vitro by conducting co-cultivation assays with UniCAR T-cells and PSCApos/PSCAneg prostate cancer cells. After radiolabeling with copper-64 or actinium-225, imaging and radioimmunotherapeutic potential of the PSCA-IgG4 antibody were studied using NMRI Foxn1 nu/nu mice. Results UniCAR T-cells were specifically activated upon cross-linkage with PSCApos tumor cells via the novel PSCA-IgG4 antibody, which resulted in the secretion of pro-inflammatory cytokines and an efficient tumor cell lysis with EC50 values in the picomolar range. 64Cu- or 225Ac-labeled PSCA-IgG4 antibodies were successfully applied for PET imaging and radioimmunotherapy of prostate tumors in experimental mice. Antibodies specifically enriched at the tumor side whereby maximal tumor accumulation and optimal tumor-to-background ratios were reached after 31 hours. Treatment of tumor-bearing mice with 225Ac-labeled PSCA-IgG4 antibody further resulted in significant lower tumor sizes compared to the control group after 40 days. Conclusions We here present a novel, multifunctional PSCA-IgG4 antibody construct for prostate cancer theranostics that facilitates not only UniCAR T-cell immunotherapy, but is also a suitable tool for targeted alpha-th

Ziel/Aim Although CAR T-cell therapy has demonstrated tremendous clinical efficacy particularly in hematological malignancies, the success in solid tumors is still limited. The combination of the immunotherapeutic (Uni)CAR T-cell therapy with target modules (TM) in solid tumors and radiotherapy could be an additional treatment option. Therefore, we developed a human prostate stem cell antigen (PSCA)-specific, IgG4-based TM radiolabeled with copper-64 (Cu-64) for imaging and with actinium-225 (Ac-225) for treatment. Methodik/Methods A novel human PSCA-specific IgG4-based TM was conjugated with DOTAGA and radiolabeled with (Cu-64) and (Ac-255). The imaging and therapy were studied in NMRI Foxn1 nu/nu mice with xenotransplanted PSCA-expressing PC-3 tumors. Metabolic changes of the tumors were visualized with 18F-PSMA (PET), the physical size of the tumors and the distance between the vessels in the tumors (US) were measured. Ergebnisse/Results The radiochemical yield for the Cu-64/Ac-255 TM was 96 % and 52 % respectively, the RCP of the products were g.t. 97 %. The xenotransplanted mice were intravenously injected with the Cu-64 labeled TM given as single dose. After fast distribution, the blood activity concentration decreased very slowly. At 31 h p.i. the activity was maximal in the tumors thereby reaching the optimal tumor to background ratios and only the liver was also visible with much lower activity. The Ac-225 treatment resulted in significant lower tumor size as compared with the control group after 40 days. Doubling time of tumor volume was increased from 15.0 days (control) to 38.6 days of the treatment group. The 18F-PSMA ligand uptake was decreased in the tumor periphery. In comparison to the tumor size the vessel density decreased to a lower extent. Schlussfolgerungen/Conclusions We here present a Cu-64/Ac-225 labeled TM that allows the combination of (Uni)CAR T-cell immunotherapy with imaging with the Cu-64 labeled TM version and radiotherapy with the Ac

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Liposomes containing copper and the copper ionophore neocuproine were prepared and characterized for in vitro and in vivo anticancer activity. Thermosensitive PEGylated liposomes were prepared with di_erent molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) in the presence of copper(II) ions. Optimal, temperature dependent drug release was obtained at 70:30 DPPC to HSPC weight ratio. Neocuproine (applied at 0.2 mol to 1 mol phospholipid) was encapsulated through a pH gradient while using unbuffered solution at pH 4.5 inside the liposomes, and 100 mM HEPES bu_er pH 7.8 outside the liposomes. Copper ions were present in excess, yielding 0.5 mM copper-(neocuproine)2 complex and 0.5 mM free copper. Pre-heating to 45 °C increased the toxicity of the heat-sensitive liposomes in short-term in vitro experiments, whereas at 72 h all investigated liposomes exhibited similar in vitro toxicity to the copper(II)-neocuproine complex (1:1 ratio). Thermosensitive liposomes were found to be more effective in reducing tumor growth in BALB/c mice engrafted with C26 cancer cells, regardless of the mild hyperthermic treatment. Copper uptake of the tumor was verified by PET/CT imaging following treatment with [64Cu]Cu-neocuproine liposomes. Taken together, our results demonstrate the feasibility of targeting a copper nanotoxin that was encapsulated in thermosensitive liposomes containing an excess of copper.

Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. In the UniCAR system, the safety switch is controlled via a target module (TM) which is characterized by a small-size and short half-life. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing. This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. For long-term UniCAR therapy, the continuous infusion of TMs may not be an optimal setting for the patients. Thus, in later stages of treatment, single infusions of TMs with an increased half-life might play an important role in long-term surveillance and eradication of residual tumor cells. Given this, we aimed to develop and characterize a novel TM with extended half-life targeting the tumor-associated carbohydrate sialyl-Tn (STn).

Purpose: The purpose of this study was to evaluate a set of widely used nuclear medicine imaging agents as possible methods to study the early effects of systemic inflammation on the living brain in a mouse model of sepsis-associated encephalopathy (SAE). The lipopolysaccharide (LPS)-induced murine systemic inflammation model was selected as a model of SAE. Procedures: C57BL/6 mice were used. A multimodal imaging protocol was carried out on each animal 4 h following the intravenous administration of LPS using the following tracers: [99mTc][2,2-dimethyl-3-[(3E)-3-oxidoiminobutan-2-yl]azanidylpropyl]-[(3E)-3-hydroxyiminobutan-2-yl]azanide ([99m Tc]HMPAO) and ethyl-7-[125I]iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]ben-zodiazepine-3-carboxylate ([125I]iomazenil) to measure brain perfusion and neuronal damage, respectively; 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) to measure cerebral glucose uptake. We assessed microglia activity on another group of mice using 2-[6-chloro-2-(4-[125I]iodo-phenyl)-imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-methyl-acetamide ([125I]CLINME). Radiotracer uptakes were measured in different brain regions and correlated. Microglia activity was also assessed using immunohistochemistry. Brain glutathione levels were measured to investigate oxidative stress. Results: Significantly reduced perfusion values and significantly enhanced [18F]FDG and [125I]CLINME uptake was measured in the LPS-treated group. Following perfusion compensation, enhanced [125I]iomazenil uptake was measured in the LPS-treated group’s hippocampus and cerebellum. In this group, both [18F]FDG and [125I]iomazenil uptake showed highly negative correlation to perfusion measured with ([99mTc]HMPAO uptake in all brain regions. No significant differences were detected in brain glutathione levels between the groups. The CD45 and P2Y12 double-labeling immunohistochemistry showed widespread microglia activation in the LPS-treated group. Conclusions: Our results suggest that [125I]CLINME and

Radiotherapy is one of the most frequently applied treatments in oncology. Tissue-absorbed ionizing radiation damages not only targeted cells but the surrounding cells too. The consequent long-term induced oxidative stress, irreversible tissue damage, or second malignancies draw attention to the urgent need of a follow-up medical method by which personalized treatment could be attained and the actually dose-limiting organ could be monitored in the clinical practice. We worked out a special hemisphere irradiation technique for mice which mimics the radiation exposure during radiotherapy. We followed up the changes of possible brain imaging biomarkers of side effects, such as cerebral blood flow, vascular endothelial function, and cellular metabolic processes for 60 days. BALB/c mice were divided into two groups (n=6 per group) based on the irradiation doses (5 and 20 Gy). After the irradiation procedure arterial spin labeling (ASL), diffusion-weighted imaging (DWI) in magnetic resonance modality and [18F]fluoro-deoxy-D-glucose positron emission tomography (FDG-PET) scans of the brain were obtained at several time points (3, 7, 30, and 60 days after the irradiation). Significant physiological changes were registered in the brain of animals following the irradiation by both applied doses. Elevated standard uptake values were detected all over the brain by FDG-PET studies 2 months after the irradiation. The apparent diffusion coefficients from DWI scans significantly decreased one month after the irradiation procedure, while ASL studies did not show any significant perfusion changes in the brain. Altogether, our sensitive multimodal imaging protocol seems to be an appropriate method for follow-up of the health status after radiation therapy. The presented approach makes possible parallel screening of healthy tissues and the effectiveness of tumor therapy without any additional radiation exposure.

Background: Lung diseases (resulting from air pollution) require a widely accessible method for risk estimation and arly diagnosis to ensure proper and responsive treatment. Radiomics-based fractal dimension analysis of X-ray computed tomography attenuation patterns in chest voxels of mice exposed to different air polluting agents was performed to model early stages of disease and establish differential diagnosis. Methods: To model different types of air pollution, BALBc/ByJ mouse groups were exposed to cigarette smoke combined with ozone, sulphur dioxide gas and a control group was established. Two weeks after exposure, the frequency distributions of image voxel attenuation data were evaluated. Specific cut-off ranges were defined to group voxels by attenuation. Cut-off ranges were binarized and their spatial pattern was associated with calculated fractal dimension, then abstracted by the fractal dimension – cut-off range mathematical function. Nonparametric Kruskal-Wallis (KW) and Mann–Whitney post hoc (MWph) tests were used. Results: Each cut-off range versus fractal dimension function plot was found to contain two distinctive Gaussian curves. The ratios of the Gaussian curve parameters are considerably significant and are statistically distinguishable within the three exposure groups. Conclusions: A new radiomics evaluation method was established based on analysis of the fractal dimension of chest X-ray computed tomography data segments. The specific attenuation patterns calculated utilizing our method may diagnose and monitor certain lung diseases, such as chronic obstructive pulmonary disease (COPD), asthma, tuberculosis or lung carcinomas.