Conditional activation of an anti-IgM antibody-drug conjugate for precise B cell lymphoma targeting

Cancerous B cells are almost indistinguishable from their non-malignant counterparts regarding their surface antigen expression. Accordingly, the challenge to be faced consists in elimination of the malignant B cell population while maintaining a functional adaptive immune system. Here, we present an IgM-specific antibody-drug conjugate masked by fusion of the epitope-bearing IgM constant domain. Antibody masking impaired interaction with soluble pentameric as well as cell surface-expressed IgM molecules rendering the antibody cytotoxically inactive. Binding capacity of the anti-IgM antibody drug conjugate was restored upon conditional protease-mediated demasking which consequently enabled target-dependent antibody internalization and subsequent induction of apoptosis in malignant B cells. This easily adaptable approach potentially provides a novel mechanism of clonal B cell lymphoma eradication to the arsenal available for non-Hodgkin's lymphoma treatment

Generation of a symmetrical trispecific NK cell engager based on a two-in-one antibody

To construct a trispecific IgG-like antibody at least three different binding moieties need to be combined, which results in a complex architecture and challenging production of these molecules. Here we report for the first time the construction of trispecific natural killer cell engagers based on a previously reported two-in-one antibody combined with a novel anti-CD16a common light chain module identified by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibodies simultaneously target epidermal growth factor receptor (EGFR), programmed death-ligand 1 (PD-L1) and CD16a with two Fab fragments, resulting in specific cellular binding properties on EGFR/PD-L1 double positive tumor cells and a potent ADCC effect. This study paves the way for further development of multispecific therapeutic antibodies derived from avian immunization with desired target combinations, valencies, molecular symmetries and architectures

Untersuchungen zur Substrattoleranz von Sactisynthasen für die Generierung Thioether‐verbrückter Sactipeptide mit neuen Eigenschaften

Sactipeptide sind ribosomal synthetisierte Peptide, die eine einzigartige Verknüpfung von Schwefel und α‐Kohlenstoffen enthalten. Die Bildung von Thioetherbrücken wird in diesen Molekülen durch Sactisynthasen katalysiert. Diese spezielle Art der Verknüpfung verleiht Sactipeptiden eine erhöhte strukturelle, thermische und proteolytische Stabilität, was sie zu attraktiven Gerüsten für die Entwicklung neuer Biotherapeutika macht. In diesem Artikel berichten wir über eine Studie zur Substrattoleranz der Sactisynthase AlbA, die die Bildung von Thioetherbrücken im Sactipeptid Subtilosin A katalysiert. Wir haben eine Modifikationsstelle innerhalb dieses Sactipeptids identifiziert, die ein Peptid‐Engineering ohne Beeinträchtigung der Bildung von Thioetherbrücken ermöglicht. Eine Reihe von natürlichen und hybriden Sactipeptidkonstrukten wurde hergestellt, um die AlbA vermittelte Bildung von Thioetherbrücken zu untersuchen und diese massenspektrometrisch zu identifizieren. In einer Proof‐of‐Principle‐Studie haben wir Subtilosin A mit einer neue Funktion ausgestattet, ein Thioether‐verbrücktes Streptavidin‐bindendes Peptid generiert und damit die Tür für das funktionelle Engineering von Sactipeptiden weiter geöffnet

Bispecific killer cell engagers employing species cross-reactive NKG2D binders redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies

NKG2D is an activating receptor expressed by natural killer (NK) cells and other cytotoxic lymphocytes that plays a pivotal role in the elimination of neoplastic cells through recognition of different stress-induced cell surface ligands (NKG2DL). To employ this mechanism for cancer immunotherapy, we generated NKG2D-engaging bispecific antibodies that selectively redirect immune effector cells to cancer cells expressing the tumor-associated antigen ErbB2 (HER2). NKG2D-specific single chain fragment variable (scFv) antibodies cross-reactive toward the human and murine receptors were derived by consecutive immunization of chicken with the human and murine antigens, followed by stringent screening of a yeast surface display immune library. Four distinct species cross-reactive (sc) scFv domains were selected, and reformatted into a bispecific engager format by linking them via an IgG4 Fc domain to a second scFv fragment specific for ErbB2. The resulting molecules (termed scNKAB-ErbB2) were expressed as disulfide-linked homodimers, and demonstrated efficient binding to ErbB2-positive cancer cells as well as NKG2D-expressing primary human and murine lymphocytes, and NK-92 cells engineered with chimeric antigen receptors derived from human and murine NKG2D (termed hNKAR and mNKAR). Two of the scNKAB-ErbB2 molecules were found to compete with the natural NKG2D ligand MICA, while the other two engagers interacted with an epitope outside of the ligand binding site. Nevertheless, all four tested scNKAB-ErbB2 antibodies were similarly effective in redirecting the cytotoxic activity of primary human and murine lymphocytes as well as hNKAR-NK-92 and mNKAR-NK-92 cells to ErbB2-expressing targets, suggesting that further development of these species cross-reactive engager molecules for cancer immunotherapy is warranted

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether‐Bond‐Forming Sactisynthase

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α‐carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in‐depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA‐mediated formation of thioether bridges, which were identified mass‐spectrometrically. In a proof‐of‐principle study, we re‐engineered subtilosin A to a thioether‐bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides

Generation of a symmetrical trispecific NK cell engager based on a two-in-one antibody

To construct a trispecific IgG-like antibody at least three different binding moieties need to be combined, which results in a complex architecture and challenging production of these molecules. Here we report for the first time the construction of trispecific natural killer cell engagers based on a previously reported two-in-one antibody combined with a novel anti-CD16a common light chain module identified by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibodies simultaneously target epidermal growth factor receptor (EGFR), programmed death-ligand 1 (PD-L1) and CD16a with two Fab fragments, resulting in specific cellular binding properties on EGFR/PD-L1 double positive tumor cells and a potent ADCC effect. This study paves the way for further development of multispecific therapeutic antibodies derived from avian immunization with desired target combinations, valencies, molecular symmetries and architectures

Better safe than sorry: dual targeting antibodies for cancer immunotherapy

Antibody-based therapies are revolutionizing cancer treatment and experience a steady increase from preclinical and clinical pipelines to market share. While the clinical success of monoclonal antibodies is frequently limited by low response rates, treatment resistance and various other factors, multispecific antibodies open up new prospects by addressing tumor complexity as well as immune response actuation potently improving safety and efficacy. Novel antibody approaches involve simultaneous binding of two antigens on one cell implying increased specificity and reduced tumor escape for dual tumor-associated antigen targeting and enhanced and durable cytotoxic effects for dual immune cell-related antigen targeting. This article reviews antibody and cell-based therapeutics for oncology with intrinsic dual targeting of either tumor cells or immune cells. As revealed in various preclinical studies and clinical trials, dual targeting molecules are promising candidates constituting the next generation of antibody drugs for fighting cancer

DataSheet_1_Conditional activation of an anti-IgM antibody-drug conjugate for precise B cell lymphoma targeting.pdf

Cancerous B cells are almost indistinguishable from their non-malignant counterparts regarding their surface antigen expression. Accordingly, the challenge to be faced consists in elimination of the malignant B cell population while maintaining a functional adaptive immune system. Here, we present an IgM-specific antibody-drug conjugate masked by fusion of the epitope-bearing IgM constant domain. Antibody masking impaired interaction with soluble pentameric as well as cell surface-expressed IgM molecules rendering the antibody cytotoxically inactive. Binding capacity of the anti-IgM antibody drug conjugate was restored upon conditional protease-mediated demasking which consequently enabled target-dependent antibody internalization and subsequent induction of apoptosis in malignant B cells. This easily adaptable approach potentially provides a novel mechanism of clonal B cell lymphoma eradication to the arsenal available for non-Hodgkin's lymphoma treatment.</p

Generation of a symmetrical trispecific NK cell engager based on a two-in-one antibody

To construct a trispecific IgG-like antibody at least three different binding moieties need to be combined, which results in a complex architecture and challenging production of these molecules. Here we report for the first time the construction of trispecific natural killer cell engagers based on a previously reported two-in-one antibody combined with a novel anti-CD16a common light chain module identified by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibodies simultaneously target epidermal growth factor receptor (EGFR), programmed death-ligand 1 (PD-L1) and CD16a with two Fab fragments, resulting in specific cellular binding properties on EGFR/PD-L1 double positive tumor cells and a potent ADCC effect. This study paves the way for further development of multispecific therapeutic antibodies derived from avian immunization with desired target combinations, valencies, molecular symmetries and architectures

Filtering and storage working memory networks in younger and older age

INTRODUCTION: Working memory (WM) is a multi-component model that among others involves the two processes of filtering and storage. The first reflects the necessity to inhibit irrelevant information from entering memory, whereas the latter refers to the active maintenance of object representations in memory. In this study, we aimed at a) redefining the neuronal networks sustaining filtering and storage within visual working memory by avoiding shortcomings of prior studies, and b) assessing age-related changes in these networks. METHODS: We designed a new paradigm that strictly controlled for perceptual load by presenting the same number of stimuli in each of three conditions. We calculated fMRI contrasts between a baseline condition (low filter and low storage load) and conditions that posed high demands on filtering and storage, respectively, in large samples of younger (n = 40) and elder (n = 38) participants. RESULTS: Our approach of comparing contrasts between groups revealed more extensive filter and storage WM networks than previous studies. In the younger group, filtering involved the bilateral insulae, the right occipital cortex, the right brainstem, and the right cerebellum. In the elder group, filtering was associated with the bilateral insulae, right precuneus, and bilateral ventromedial prefrontal cortex. An extensive neuronal network was also found during storage of information in the bilateral posterior parietal cortex, the left ventromedial prefrontal cortex, and the right precuneus in the younger participants. In addition to these brain regions, elder participants recruited the bilateral ventral prefrontal cortex, the superior, middle and inferior and temporal cortex, the left cingulum and the bilateral parahippocampal cortex. CONCLUSIONS: In general, elder participants recruited more brain regions in comparison to younger participants to reach similar accuracy levels. Furthermore, in elder participants one brain region emerged in both contrasts, namely the left ventromedial prefrontal cortex. Hence, elder participants seem to routinely recruit this brain region in demanding tasks, irrespective of whether filtering or storing is challenged