To harness the immune system's cytotoxic capacity to fight solid tumors, we developed tetravalent, bifunctional antibodies that recognize EGFRvIII, the deletion variant III of EGFR, and either CD3 or ...CD16A on immune cells, thereby directing T cells or NK-cells to eliminate EGFRvIII+ cancer cells.Using phage display, we identified scFv antibodies that selectively bind to EGFRvIII. These highly EGFRvIII-specific scFv antibodies were substantially improved by affinity maturation achieving KDs in the 100 pM range or lower and used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties. Mono- and bivalent binding constants, specificity for EGFRvIII and CD3 or CD16A, cytotoxic activity, and target-dependent effector cell activation were characterized in a panel of in vitro assays. TandAbs exhibited exquisite specificity towards the EGFRvIII antigen in Western Blot, SPR, ELISA, and FACS assays of EGFRvIII+ cells. No binding was observed to recombinant EGFR or to EGFR+ cells. The TandAbs apparent affinities for EGFRvIII were up to 25-fold improved relative to the monovalently binding scFvs, resulting in a KD of 11 pM for the best TandAb.EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs with high affinity for EGFRvIII were similarly potent in killing assays, displaying cytotoxicity towards EGFRvIII+ F98 glioma, transfected CHO or human DKMG cells with EC50 in the range of 1 pM – 10 pM. No cytotoxicity was observed on EGFR+ cells or EGFRvIII-negative cells demonstrating the high selectivity of EGFRvIII TandAbs for the tumor-specific EGFRvIII. Importantly, in the absence of EGFRvIII+ target cells in vitro TandAbs did not elicit T- or NK-cell activation, as demonstrated by their lack of proliferation. Binding to EGFRvIII in different solid tumor types and its absence from healthy tissues was shown by immunohistochemistry using a high affinity EGFRvIII-binding bivalent Diabody.In summary, EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs provide an opportunity to develop cytotoxic antibodies that solely target cancer, sparing normal tissues and thereby reduce the side effects associated with EGFR therapy.
We investigated whether antibodies against intracellular tumor-associated antigens support tumor-specific immunity when administered together with a treatment that destroys the tumor. We propose that ...released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic cells. We cloned the first human monoclonal antibodies against the Cancer/Testis (CT) antigen, NY-ESO-1. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates cross-presentation of a NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells, and whether this results in the maturation of dendritic cells in vitro. We investigated the efficacy of 12D7 in combination with chemotherapy using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1. Human dendritic cells that were incubated with NY-ESO-1:12D7 immune complexes efficiently stimulated NY-ESO-1(157-165)/HLA-A2-specific human CD8+ T cells to produce interferon-γ, whereas NY-ESO-1 alone did not. Furthermore, the incubation of dendritic cells with NY-ESO-1:12D7 immune complexes resulted in the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil (5-FU) plus 12D7 was significantly more effective than chemotherapy alone. We propose systemic injection of monoclonal antibodies (mAbs) against tumor-associated antigens plus a treatment that promotes the local release of those antigens resulting in immune complex formation as a novel therapeutic modality for cancer.
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Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a ...medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG
1
-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with K
D
values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages in vitro. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status. In vivo, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2-4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status.
Abbreviations: ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype
Redirection of immune cells to efficiently eliminate tumor cells holds great promise. Natural killer cells (NK), macrophages, or T cells are specifically engaged with target cells expressing markers ...after infection or neoplastic transformation, resulting in their activation and subsequent killing of those targets. Multiple strategies to redirect immunity have been developed in the past two decades, but they have technical hurdles or cause undesirable side-effects, as exemplified by the T cell-based chimeric antigen receptor approaches (CAR-T therapies) or bispecific T cell engager platforms. Our first-in-class bispecific antibody redirecting innate immune cells to tumors (AFM13, a CD30/CD16A-specific innate immune cell engager) has shown signs of clinical efficacy in CD30-positive lymphomas and the potential to be safely administered, indicating a wider therapeutic window compared to T cell engaging therapies. AFM13 is the most advanced candidate from our fit-for-purpose redirected optimized cell killing (ROCK®) antibody platform, which comprises a plethora of CD16A-binding innate immune cell engagers with unique properties. Here, we discuss aspects of this modular platform, including the advantages of innate immune cell engagement over classical monoclonal antibodies and other engager concepts. We also present details on its potential to engineer a fit-for-purpose innate immune cell engager format that can be equipped with unique CD16A domains, modules that influence pharmacokinetic properties and molecular architectures that influence the activation of immune effectors, as well as tumor targeting. The ROCK® platform is aimed at the activation of innate immunity for the effective lysis of tumor cells and holds the promise of overcoming limitations of other approaches that redirect immune cells by widening the therapeutic window.
Adaptive memory recall requires a rapid and flexible switch from external perceptual reminders to internal mnemonic representations. However, owing to the limited temporal or spatial resolution of ...brain imaging modalities used in isolation, the hippocampal-cortical dynamics supporting this process remain unknown. We thus employed an object-scene cued recall paradigm across two studies, including intracranial electroencephalography (iEEG) and high-density scalp EEG. First, a sustained increase in hippocampal high gamma power (55 to 110 Hz) emerged 500 ms after cue onset and distinguished successful vs. unsuccessful recall. This increase in gamma power for successful recall was followed by a decrease in hippocampal alpha power (8 to 12 Hz). Intriguingly, the hippocampal gamma power increase marked the moment at which extrahippocampal activation patterns shifted from perceptual cue toward mnemonic target representations. In parallel, source-localized EEG alpha power revealed that the recall signal progresses from hippocampus to posterior parietal cortex and then to medial prefrontal cortex. Together, these results identify the hippocampus as the switchboard between perception and memory and elucidate the ensuing hippocampal-cortical dynamics supporting the recall process.
The implementation of agronomic activities, based on the use of biostimulants, is an important element of agroecological practices. Therefore, comprehensive research was carried on the use of ...biostimulants. A field experiment was performed in 2016-2018 with common bean of Mexican Black cultivar. In particular growing seasons, bean plants were treated with Kelpak SL (seaweed extracts) and Terra Sorb Complex (free amino acids) in the form of single and double spraying with two solutions concentrations. According to the obtained data, application of biostimulants increased the yield of bean. Better results were observed after the use of Kelpak SL. The application of preparations influenced nutritional and nutraceutical quality of bean seeds. Terra Sorb Complex caused the highest increase in proteins level. In the light of achieved data, biostimulants in similar level decreased the starch accumulation. The most promising results, in the context of nutraceutical value of bean, were obtained in the case of increasing level of fiber. A positive impact of biostimulants on the seeds antioxidant potential was noted, expressed by the increased synthesis of phenolics, flavonoid, anthocyanins and antioxidant activities. Results of this study, directly indicate economic benefits from the use of biostimulants, which are extremely important to the farmers.
To analyze the influence of a cilioretinal artery (CRA) on macular and peripapillary vessel density in healthy eyes as measured using optical coherence tomography angiography.
A total of 83 eyes of ...83 patients were included in this study. Optical coherence tomography angiography was performed using the RTVue XR Avanti with AngioVue (Optovue Inc). The macula was imaged with a 3 × 3-mm scan, whereas for the optic nerve head a 4.5 × 4.5-mm scan was taken. Optical coherence tomography angiography images of the optic nerve head were screened for the presence of a CRA.
In 31 eyes, a CRA was detected (37.3%). The vessel density in eyes with a CRA was significantly lower within the optic nerve head (P = 0.005) but higher in the peripapillary capillary network (P < 0.001) and (whole en face) macular superficial capillary plexus (P = 0.025), when compared with eyes with no CRA.
Our findings reveal that in eyes with a CRA, the vessel density in the peripapillary and macular superficial capillary plexus is increased, whereas the optic nerve head perfusion (as indicated by vessel density in the inside disk region) is decreased. This has to be considered when analyzing quantitative optical coherence tomography angiography parameters in scientific and clinical applications.
Purpose
The SALUS study aims to improve the healthcare situation for glaucoma patients in Germany. In order to detect diurnal intraocular pressure (IOP) fluctuations, inpatient monitoring of IOP in ...an eye hospital for a minimum of 24 h is the current standard. SALUS assesses the benefits of a new form of outpatient care, where IOP can be measured by the patients themselves at home using a self-tonometer. This approach should promote the patient’s health competence and empowerment within the healthcare system while reducing treatment costs.
Methods
The SALUS study is a randomized controlled, open non-inferiority trial, alongside an economic analysis, determining whether outpatient monitoring of IOP with self-tonometry is at least as effective as current standard care and would reduce treatment costs. Participants (
n
= 1980) will be recruited by local ophthalmologists in the area of Westphalia-Lippe, Germany, and randomized to receive 7-day outpatient or 24-h inpatient monitoring. Participants in both study arms will also receive 24-h blood pressure monitoring. Furthermore, patient data from both study groups will be collected in an electronic case file (ECF), accessible to practitioners, hospitals, and the study participants. The primary endpoint is the percentage of patients with IOP peaks, defined as levels 30% above the patient-specific target pressure. Data will also be collected during initial and final examinations, and at 3, 6, and 9 months after the initial examination.
Results
The study implementation and trial management are represented below.
Conclusion
SALUS is a pioneering prospective clinical trial focused on the care of glaucoma patients in Germany. If SALUS is successful, it could improve the healthcare situation and health literacy of the patients through the introduction of various telemedical components. Furthermore, the approach would almost certainly reduce the treatment costs of glaucoma care.
Trial registration
ClinicalTrials.gov ID:
NCT04698876,
registration date:
11/25/2020.
DRKS-ID:
DRKS00023676,
registration date:
11/26/2020.
Purpose To kinematically and biomechanically compare 4 different types of tibial tunnel management in single-stage anterior cruciate ligament (ACL) revision reconstruction with the control: primary ...ACL reconstruction using a robotic-based knee testing setup. Methods Porcine knees and flexor tendons were used. One hundred specimens were randomly assigned to 5 testing groups: (1) open tibial tunnel, (2) bone plug technique, (3) biodegradable interference screw, (4) dilatation technique, and (5) primary ACL reconstruction. A robotic/universal force-moment sensor testing system was used to simulate the KT-1000 (MEDmetric, San Diego, CA) and pivot-shift tests. Cyclic loading and load-to-failure testing were performed. Results Anterior tibial translation increased significantly with all of the techniques compared with the intact ACL ( P < .05). In the simulated KT-1000 test, groups 2 and 3 achieved results equal to those of primary ACL reconstruction ( P > .05). The open tunnel and dilated tunnel techniques showed significantly greater anterior tibial translation ( P < .05). The results of the simulated pivot-shift test were in accordance with those of the KT-1000 test. No significant differences could be observed regarding stiffness or maximum load to failure. However, elongation was significantly lower in the primary ACL reconstruction group compared with groups 1 and 3 ( P = .02 and P = .03, respectively). Conclusions Filling an incomplete and incorrect tibial tunnel with a press-fit bone plug or a biodegradable interference screw in a standardized laboratory situation provided initial biomechanical properties and knee stability comparable with those of primary ACL reconstruction. In contrast, the dilatation technique or leaving the malplaced tunnel open did not restore knee kinematics adequately in this model. Backup extracortical fixation should be considered because the load to failure depends on the extracortical fixation when an undersized interference screw is used for aperture fixation. Clinical Relevance Our biomechanical results could help orthopaedic surgeons to optimize the results of primary ACL revision with incomplete, incorrect tunnel placement.
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