Albumin has a serum half-life of 3 weeks in humans. This feature can be used to improve the pharmacokinetics of shorter-lived biologics. For instance, an albumin-binding domain (ABD) can be used to ...recruit albumin. A prerequisite for such design is that the ABD-albumin interaction does not interfere with pH-dependent binding of albumin to the human neonatal Fc receptor (FcRn), as FcRn acts as the principal regulator of the half-life of albumin. Thus, there is a need to know how ABDs act in the context of fusion partners and human FcRn. Here, we studied the binding and transport properties of human immunoglobulin A1 (IgA1), fused to a
protein G-derived engineered ABD, in
and
systems harboring human FcRn. IgA has great potential as a therapeutic protein, but its short half-life is a major drawback. We demonstrate that ABD-fused IgA1 binds human FcRn pH-dependently and is rescued from cellular degradation in a receptor-specific manner in the presence of albumin. This occurs when ABD is fused to either the light or the heavy chain. In human FcRn transgenic mice, IgA1-ABD in complex with human albumin, gave 4-6-fold extended half-life compared to unmodified IgA1, where the light chain fusion showed the longest half-life. When the heavy chain-fused protein was pre-incubated with an engineered human albumin with improved FcRn binding, cellular rescue and half-life was further enhanced. Our study reveals how an ABD, which does not interfere with albumin binding to human FcRn, may be used to extend the half-life of IgA.
ABD - Albumin binding domain, ADA - anti-drug-antibodies, ADCC - Antibody-dependent cellular cytotoxicity, ELISA - Enzyme-linked Immunosorbent assay, FcαRI - Fcα receptor, FcγR - Fcγ receptor, FcRn - The neonatal Fc receptor, GST - Glutathione S-transferase, HC - Heavy chain, HERA - Human endothelial cell-based recycling assay, Her2 - Human epidermal growth factor 2, HMEC - Human microvascular endothelial cells, IgG - Immunoglobulin G, IgA - Immunoglobulin A, LC - Light chain, QMP - E505Q/T527M/K573P, WT - Wild type.
The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of T cell epitopes. ...However, studies on viral escape from T cell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 T cell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of T cell immunity.
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•Among 123 eluted or predicted SARS-CoV-2 HLA-ligands, 29 are CD8 T cell epitopes•Epitope immunoprevalence in non-vaccinated convalescents ranges from 3% to 100%•Epitope immunoprevalence and immunodominance is strongly correlated•Mutations in VOC are not driven by T cell escape
Meyer et al. identify peptide sequences from SARS-CoV-2 that are commonly recognized by CD8 T cells in convalescents. They demonstrate that these sequences are conserved in VOC. Thus, the emergence of VOC is not driven by escape from T cell immunity.
Summary
Fc receptors (FcR) are expressed on immune cells and bind to the Fc tail of antibodies. This interaction is essential for FcR‐mediated signaling and triggering of cellular effector functions. ...FcR activation is tightly regulated to prevent immune responses by non‐antigen bound antibodies or in the absence of ‘danger signals’. FcR activity may be modulated at the plasma membrane via cross‐talk with integrins. In addition, cytokines at the site of infection/inflammation can increase FcR avidity, a process referred to as inside‐out signaling. This regulatory mechanism has been described for FcγRI (CD64), FcγRIIa (CD32a), and FcαRI (CD89) and is also well‐known for integrins. Key cellular events during inside‐out signaling are (de)phosphorylation, clustering, cytoskeleton rearrangements, and conformational changes. The latter can be studied with antibodies that specifically recognize epitopes exposed by the active (high affinity) or inactive (low affinity) state of the FcR. These antibodies are important tools to investigate the role of FcR activation in disease settings. Research on FcR has gained momentum with the rise of monoclonal antibodies (mAb) entering the clinic for the treatment of cancer and other diseases. The clinical outcome of mAb therapy may be improved by increasing FcR avidity by cytokine stimulation.
Current combination therapies elicit high response rates in B cell malignancies, often using CD20 antibodies as the backbone of therapy. However, many patients eventually relapse or develop ...progressive disease. Therefore, novel CD20 antibodies combining multiple effector mechanisms were generated. To study whether neutrophil-mediated destruction of B cell malignancies can be added to the arsenal of effector mechanisms, we chimerized a panel of five previously described murine CD20 antibodies to the human IgG1, IgA1 and IgA2 isotype. Of this panel, we assessed
in vitro
antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and direct cell death induction capacity and studied the efficacy in two different
in vivo
mouse models. IgA antibodies outperformed IgG1 antibodies in neutrophil-mediated killing
in vitro
, both against CD20-expressing cell lines and primary patient material. In these assays, we observed loss of CD19 with both IgA and IgG antibodies. Therefore, we established a novel method to improve the assessment of B-cell depletion by CD20 antibodies by including CD24 as a stable cell marker. Subsequently, we demonstrated that only IgA antibodies were able to reduce B cell numbers in this context. Additionally, IgA antibodies showed efficacy in both an intraperitoneal tumor model with EL4 cells expressing huCD20 and in an adoptive transfer model with huCD20-expressing B cells. Taken together, we show that IgA, like IgG, can induce ADCC and CDC, but additionally triggers neutrophils to kill (malignant) B cells. We conclude that antibodies of the IgA isotype offer an attractive repertoire of effector mechanisms for the treatment of CD20-expressing malignancies.
Summary
B‐cell depletion induced by anti‐cluster of differentiation 20 (CD20) monoclonal antibody (mAb) therapy of patients with lymphoma is expected to impair humoral responses to severe acute ...respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) vaccination, but effects on CD8 T‐cell responses are unknown. Here, we investigated humoral and CD8 T‐cell responses following two vaccinations in patients with lymphoma undergoing anti‐CD20‐mAb therapy as single agent or in combination with chemotherapy or other anti‐neoplastic agents during the last 9 months prior to inclusion, and in healthy age‐matched blood donors. Antibody measurements showed that seven of 110 patients had antibodies to the receptor‐binding domain of the SARS‐CoV‐2 Spike protein 3–6 weeks after the second dose of vaccination. Peripheral blood CD8 T‐cell responses against prevalent human leucocyte antigen (HLA) class I SARS‐CoV‐2 epitopes were determined by peptide‐HLA multimer analysis. Strong CD8 T‐cell responses were observed in samples from 20/29 patients (69%) and 12/16 (75%) controls, with similar median response magnitudes in the groups and some of the strongest responses observed in patients. We conclude that despite the absence of humoral immune responses in fully SARS‐CoV‐2‐vaccinated, anti‐CD20‐treated patients with lymphoma, their CD8 T‐cell responses reach similar frequencies and magnitudes as for controls. Patients with lymphoma on B‐cell depleting therapies are thus likely to benefit from current coronavirus disease 2019 (COVID‐19) vaccines, and development of vaccines aimed at eliciting T‐cell responses to non‐Spike epitopes might provide improved protection.
Abstract
Für eine nachhaltige Bemessung von Stahlbeton‐ und Spannbetonbrücken in den Grenzzuständen der Tragfähigkeit und Gebrauchstauglichkeit ist eine realitätsgetreue Abschätzung der ...Torsionssteifigkeit in Abhängigkeit von der Höhe der Beanspruchung notwendig. Unter anderem aufgrund des aktuell begrenzten Kenntnisstands sind normative Grundlagen für die Nachrechnung von Brückenbauwerken lediglich eingeschränkt vorhanden und für die Brückenneubemessung nicht vorgesehen. Der vorliegende Beitrag gibt eine Übersicht über das Torsionstragverhalten und insbesondere die Torsionssteifigkeit von Stahlbeton‐ und Spannbetonbauteilen mit Plattenbalkenquerschnitt. Der Fokus liegt hierbei auf der mit der Reduzierung der Torsionssteifigkeit einhergehenden Schnittgrößenumlagerung im Gesamtsystem, was u. a. zu geringeren Bemessungstorsionsmomenten und folglich zu einer kleineren erforderlichen Bewehrungsmenge für die Torsionsbeanspruchung führt. Dieses wird anschaulich anhand einer Parameterstudie mit dem Modell nach Bieger sowie am Beispiel einer Brückennachrechnung und der Bemessung eines Brückenneubaus in Spannbetonbauweise erläutert.
Abstract
Effects of torsional stiffness on the load‐bearing behaviour of concrete bridges with T‐beam cross section
For a sustainable design of reinforced and prestressed concrete bridges in the ultimate limit state and serviceability limit state, a realistic estimation of the torsional stiffness as a function of the load level is necessary. Due to the current limited number of studies, normative bases for the recalculation of bridge structures are only available to a limited extent and are not intended for the design of new bridge constructions. The present article gives an overview of the torsional load‐bearing behavior and, in particular, the torsional stiffness of reinforced and prestressed concrete structures with T‐beam cross sections. The focus is on the redistribution of internal forces in the overall system associated with the reduction of torsional stiffness, which leads, among other things, to lower design torsional moments and consequently to a smaller amount of reinforcement required for torsional loading. This is explained clearly using a parameter study with the Bieger model and the example of a bridge recalculation as well as the design of a new bridge in prestressed concrete construction.
Für eine nachhaltige Bemessung von Stahlbeton‐ und Spannbetonbrücken in den Grenzzuständen der Tragfähigkeit und Gebrauchstauglichkeit ist eine realitätsgetreue Abschätzung der Torsionssteifigkeit in ...Abhängigkeit von der Höhe der Beanspruchung notwendig. Unter anderem aufgrund des aktuell begrenzten Kenntnisstands sind normative Grundlagen für die Nachrechnung von Brückenbauwerken lediglich eingeschränkt vorhanden und für die Brückenneubemessung nicht vorgesehen. Der vorliegende Beitrag gibt eine Übersicht über das Torsionstragverhalten und insbesondere die Torsionssteifigkeit von Stahlbeton‐ und Spannbetonbauteilen mit Plattenbalkenquerschnitt. Der Fokus liegt hierbei auf der mit der Reduzierung der Torsionssteifigkeit einhergehenden Schnittgrößenumlagerung im Gesamtsystem, was u. a. zu geringeren Bemessungstorsionsmomenten und folglich zu einer kleineren erforderlichen Bewehrungsmenge für die Torsionsbeanspruchung führt. Dieses wird anschaulich anhand einer Parameterstudie mit dem Modell nach Bieger sowie am Beispiel einer Brückennachrechnung und der Bemessung eines Brückenneubaus in Spannbetonbauweise erläutert.
Effects of torsional stiffness on the load‐bearing behaviour of concrete bridges with T‐beam cross section
For a sustainable design of reinforced and prestressed concrete bridges in the ultimate limit state and serviceability limit state, a realistic estimation of the torsional stiffness as a function of the load level is necessary. Due to the current limited number of studies, normative bases for the recalculation of bridge structures are only available to a limited extent and are not intended for the design of new bridge constructions. The present article gives an overview of the torsional load‐bearing behavior and, in particular, the torsional stiffness of reinforced and prestressed concrete structures with T‐beam cross sections. The focus is on the redistribution of internal forces in the overall system associated with the reduction of torsional stiffness, which leads, among other things, to lower design torsional moments and consequently to a smaller amount of reinforcement required for torsional loading. This is explained clearly using a parameter study with the Bieger model and the example of a bridge recalculation as well as the design of a new bridge in prestressed concrete construction.
The complement system is a powerful tool of the innate immune system to eradicate pathogens. Both in vitro and in vivo evidence indicates that therapeutic anti-tumor monoclonal antibodies (mAbs) can ...activate the complement system by the classical pathway. However, the contribution of complement to the efficacy of mAbs is still debated, mainly due to the lack of convincing data in patients. A beneficial role for complement during mAb therapy is supported by the fact that cancer cells often upregulate complement-regulatory proteins (CRPs). Polymorphisms in various CRPs were previously associated with complement-mediated disorders.
In this review the role of complement in anti-tumor mAb therapy will be discussed with special emphasis on strategies aiming at modifying complement activity. In the future, clinical efficacy of mAbs with enhanced effector functions together with comprehensive analysis of polymorphisms in CRPs in mAb-treated patients will further clarify the role of complement in mAb therapy.
Summary
Based on their mechanisms‐of‐action, CD20 monoclonal antibodies (mAbs) are grouped into Type I complement‐dependent cytotoxicity (CDC) and antibody‐dependent cell‐mediated cytotoxicity (ADCC) ...and Type II programmed cell death (PCD) and ADCC mAbs. We generated 17 new hybridomas producing CD20 mAbs of different isotypes and determined unique heavy and light chain sequence pairs for 13 of them. We studied their epitope binding, binding kinetics and structural properties and investigated their predictive value for effector functions, i.e. PCD, CDC and ADCC. Peptide mapping and CD20 mutant screens revealed that 10 out of these 11 new mAbs have an overlapping epitope with the prototypic Type I mAb rituximab, albeit that distinct amino acids of the CD20 molecule contributed differently. Binding kinetics did not correlate with the striking differences in CDC activity among the mIgG2c mAbs. Interestingly, chimerization of mAb m1 resulted in a mAb displaying both Type I and II characteristics. PCD induction was lost upon introduction of a mutation in the framework of the heavy chain affecting the elbow angle, supporting that structural changes within this region can affect functional activities of CD20 mAbs. Together, these new CD20 mAbs provide further insights in the properties dictating the functional efficacy of CD20 mAbs.
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