Electrodermal lability and sensorimotor preparation: effects on reaction time, contingent negative variation, and heart rate.

Electrodermal lability is a trait-like measure of spontaneous sympathetic resting activity. In the present study, we addressed whether interindividual differences in this lability have an impact on the reaction time (RT) and on two physiological indicators of a goal-oriented sensorimotor preparation in a long-running, forewarned RT task (S1-S2 paradigm). The two indicators were the brain's contingent negative variation (CNV) and a heart rate deceleration (HRD). The interindividual differences were determined by counting spontaneous skin conductance fluctuations during a 5-min resting phase and dividing the subjects into two groups: individuals below (stable) and above (labile) the median of these fluctuations. In the task, labile individuals had a shorter RT compared with stable individuals and showed in the final phase of preparation in both physiological indicators the stronger response. Thus, lability-dependent effects in forewarned RT tasks cannot be explained by differences in stimulus-driven or passively controlled processes alone. Rather, goal-oriented, deliberately controlled processes that serve to adequately prepare for an imperative stimulus-the S2 in our paradigm-also must be considered to explain them. Labile individuals not only react faster than stable ones but also intentionally prepare themselves more appropriately for the imperative stimulus. A norepinephrine hypothesis focusing on the tonic activity of the locus coeruleus (LC) is proposed as an explanation for these and other lability-dependent effects. The frequency of spontaneous electrodermal fluctuations at rest may represent a peripheral, noninvasive, and easily measurable indicator of the baseline LC activity during wakefulness.

Quality of Life in Patients 20-31 Years After Heart Transplantation.

BACKGROUND: Survival rates after heart transplantation (HTx) have significantly improved over the last decades. There is a growing need to understand the long-term psychological and somatic outcomes, which constitute quality of life (QoL), for these long-term survivors. METHODS: The QoL of patients (N = 75) living 20-31 years (M = 24.9 years, SD = 2.3 years) after orthotopic HTx was evaluated. In a first step, a detailed overview of the patients' somatic condition was assessed. Secondly, patients were compared to 58 control subjects in terms of self-reported QoL (SF-36) and psychological domains (GBB-24; HADS). Finally, a cluster analysis was conducted to identify patterns within the patient-reported outcome measures (PROMs) and to relate them to somatic, psychosocial, and demographic variables. RESULTS: 95.7% of the HTx-patients were in NYHA functional class I or II, and only 15.2% had a reduced LVEF. Compared to controls, long-term HTx patients had significantly lower scores on the physical component summary (PCS) of QoL and on the GBB-24 but not in the mental component summary (MCS) of QoL, or anxiety and depression (HADS). Clustering revealed two distinct groups of patients characterized by high versus low functioning and different levels of social support. CONCLUSIONS: Long-term survivors have a good functional, cardiac, and mental status, but report a lower physical QoL and higher levels of subjective complaints. The importance of social support for HTx recipients is once again highlighted.

Development and validation of a questionnaire on bodily experience in VAD patients (BE-S).

BACKGROUND: Little is known about the disturbance in bodily experience (BE) following ventricular assist device (VAD) implantation. The level of disturbance in BE serves as an indicator of the status of the patients' adaptation process to the device. This process encompasses coping with both the more affective, psychological conflicts and the more cognitive, practical challenges of living with the VAD. To provide an economical screening tool for everyday clinical practice, we refined and validated a questionnaire on BE in VAD patients. METHODS: Seven specific items were derived from clinical experience and presented to 365 VAD patients (85% male; time since implantation: 3-36 months). The item structure was examined using factor analyses and probabilistic test theory. Discriminant validity and change sensitivity were determined in relation to associated psychometric instruments. RESULTS: Four items were found to constitute the unidimensional bodily experience scale (BE-S). Besides a high internal consistency of the scale (ω = 0.86), the RMSEA of >0.01 indicates a very good model fit. The BE-S has high convergent validity with related constructs (Hospital Anxiety and Depression Scale, Kansas City Cardiomyopathy Questionnaire). Change sensitivity analyses proved the BE-S alone to be significantly sensitive to the temporal dynamics of psychological adaptation processes following VAD implantation. CONCLUSION: The BE-S constitutes a valid and economical tool for clinical practice to assess patients' disturbance in BE after VAD implantation. It is a valuable tool for identifying patients with difficulties in adapting to the VAD. Subsequently, it enables early and focused therapeutic support for these patients at risk.

Co-modulation of TNFR1 and TNFR2 in an animal model of multiple sclerosis.

BACKGROUND: Tumour necrosis factor (TNF) is a pleiotropic cytokine and master regulator of the immune system. It acts through two receptors resulting in often opposing biological effects, which may explain the lack of therapeutic potential obtained so far in multiple sclerosis (MS) with non-receptor-specific anti-TNF therapeutics. Under neuroinflammatory conditions, such as MS, TNF receptor-1 (TNFR1) is believed to mediate the pro-inflammatory activities associated with TNF, whereas TNF receptor-2 (TNFR2) may instead induce anti-inflammatory effects as well as promote remyelination and neuroprotection. In this study, we have investigated the therapeutic potential of blocking TNFR1 whilst simultaneously stimulating TNFR2 in a mouse model of MS. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein (MOG35-55) in humanized TNFR1 knock-in mice. These were treated with a human-specific TNFR1-selective antagonistic antibody (H398) and a mouse-specific TNFR2 agonist (EHD2-sc-mTNFR2), both in combination and individually. Histopathological analysis of spinal cords was performed to investigate demyelination and inflammatory infiltration, as well as axonal and neuronal degeneration. Retinas were examined for any protective effects on retinal ganglion cell (RGC) degeneration and neuroprotective signalling pathways analysed by Western blotting. RESULTS: TNFR modulation successfully ameliorated symptoms of EAE and reduced demyelination, inflammatory infiltration and axonal degeneration. Furthermore, the combinatorial approach of blocking TNFR1 and stimulating TNFR2 signalling increased RGC survival and promoted the phosphorylation of Akt and NF-κB, both known to mediate neuroprotection. CONCLUSION: These results further support the potential of regulating the balance of TNFR signalling, through the co-modulation of TNFR1 and TNFR2 activity, as a novel therapeutic approach in treating inflammatory demyelinating disease.

Novelty detection and orienting: effects on skin conductance and heart rate.

In a repetition-change paradigm it was explored whether the skin conductance response (SCR) and the heart rate (HR) response similarly reflect involuntary and voluntary orienting. Seven orienting stimuli, consisting of six contextually novel stimuli and one novel change, were presented to 144 participants. In order to evoke voluntary orienting, the signal value of the contextually novel stimuli was manipulated by task instruction. Results suggest that the SCR is a manifestation of the involuntary orienting response (iOR). The HR, however, showed a non-uniform pattern of response and turned out to be susceptible to voluntary orienting. While it responded to the last orienting stimulus, the novel change, with a clear-cut deceleration, the response to the first orienting stimulus had a polyphase structure and was sensitive to repetition and signal value. The HR response is, thus, of limited value as an indicator of the iOR because of its striking susceptibility to voluntary orienting.

Behavioral and neurophysiological signatures of interoceptive enhancements following vagus nerve stimulation.

An accruing body of research has shown that interoception (the sensing of signals from the body's internal milieu) relies on both a direct route (afforded by the vagus nerve) and a secondary route (supported by somatosensory mechanisms). However, no study has causally tested the differential role of these pathways, let alone via direct stimulation. To bridge this gap, we tested whether multidimensional signatures of interoception are modulated by noninvasive vagus nerve stimulation (nVNS). Sixty-three participants were divided into an nVNS and a sham-stimulation group. Before and after stimulation, both groups performed a validated heartbeat detection (HBD) task including a genuinely interoceptive condition (monitoring one's own heartbeat) and a control exteroceptive condition (tracking an aurally presented heartbeat). Electroencephalographic signals were obtained during both conditions to examine modulations of the heartbeat-evoked potential (HEP). Moreover, before and after stimulation, participants were asked to complete a somatosensory heartbeat localization task. Results from the interoceptive condition revealed that, after treatment, only the nVNS group exhibited improved performance and greater HEP modulations. No behavioral differences were found for the exteroceptive control condition, which was nonetheless associated with significant HEP modulations. Finally, no between-group differences were observed regarding the localization of the heartbeat sensations or relevant cardiodynamic variables (heart rate and or heart rate variability). Taken together, these results constitute unprecedented evidence that the vagus nerve plays a direct role in neurovisceral integration during interoception. This finding can constrain mechanistic models of the domain while informing a promising transdiagnostic agenda for interoceptive impairments across neuropsychiatric conditions.

Building the bodily self-awareness: Evidence for the convergence between interoceptive and exteroceptive information in a multilevel kernel density analysis study.

Exteroceptive and interoceptive signals shape and sustain the bodily self-awareness. The existence of a set of brain areas, supporting the integration of information coming from the inside and the outside of the body in building the sense of bodily self-awareness has been postulated, yet the evidence remains limited, a matter of discussion never assessed quantitatively. With the aim of unrevealing where in the brain interoceptive and exteroceptive signals may converge, we performed a meta-analysis on imaging studies of the sense of body ownership, modulated by external visuotactile stimulation, and studies on interoception, which involves the self-awareness for internal bodily sensations. Using a multilevel kernel density analysis, we found that processing of stimuli of the two domains converges primarily in the supramarginal gyrus bilaterally. Furthermore, we found a right-lateralized set of areas, including the precentral and postcentral, and superior temporal gyri. We discuss these results and propose this set of areas as ideal candidates to match multiple body-related signals contributing to the creation of a multidimensional representation of the bodily self.

Fatigue in multiple sclerosis is associated with multimodal interoceptive abnormalities.

BACKGROUND: Fatigue ranks among the most common and disabling symptoms in multiple sclerosis (MS). Recent theoretical works have surmised that this trait might be related to alterations across interoceptive mechanisms. However, this hypothesis has not been empirically evaluated. OBJECTIVES: To determine whether fatigue in MS patients is associated with specific behavioral, structural, and functional disruptions of the interoceptive domain. METHODS: Fatigue levels were established via the Modified Fatigue Impact Scale. Interoception was evaluated through a robust measure indexed by the heartbeat detection task. Structural and functional connectivity properties of key interoceptive hubs were tested by magnetic resonance imaging (MRI) and resting-state functional MRI. Machine learning analyses were employed to perform pairwise classifications. RESULTS: Only patients with fatigue presented with decreased interoceptive accuracy alongside decreased gray matter volume and increased functional connectivity in core interoceptive regions, the insula, and the anterior cingulate cortex. Each of these alterations was positively associated with fatigue. Finally, machine-learning analysis with a combination of the above interoceptive indices (behavioral, structural, and functional) successfully discriminated (area under the curve > 90%) fatigued patients from both non-fatigued and healthy controls. CONCLUSION: This study offers unprecedented evidence suggesting that disruptions of neurocognitive markers subserving interoception may constitute a signature of fatigue in MS.

Neuroimaging meta-analyses reveal convergence of interoception, emotion, and social cognition across neurodegenerative diseases.

INTRODUCTION: Simultaneous interoceptive, emotional, and social cognition deficits are observed across neurodegenerative diseases. Indirect evidence suggests shared neurobiological bases underlying these impairments, termed the allostatic-interoceptive network (AIN). However, no study has yet explored the convergence of these deficits in neurodegenerative diseases or examined how structural and functional changes contribute to cross-domain impairments. METHODS: A PRISMA Activated Likelihood Estimate (ALE) metanalyses encompassed studies meeting inclusion criteria: interoception, emotion, or social cognition tasks; neurodegenerative diseases (behavioral variant frontotemporal dementia (bvFTD), primary progressive aphasias (PPAs) Alzheimer's disease (AD), Parkinson's Disease (PD), multiple sclerosis (MS)); and neuroimaging (structural: MRI voxel-based morphometry; functional: fMRI and FDG-PET). RESULTS: From 20,593 studies, 170 met inclusion criteria (58 interoception, 65 emotion, and 47 social cognition) involving 7032 participants (4963 patients and 2069 healthy controls). In all participants combined, conjunction analyses revealed AIN involvement of the insula, amygdala, orbitofrontal cortex, anterior cingulate, striatum, thalamus, and hippocampus across domains. In bvFTD this conjunction was replicated across domains, with further involvement of the temporal pole, temporal fusiform cortex, and angular gyrus. A convergence of interoception and emotion in the striatum, thalamus, and hippocampus in PD and the posterior insula in PPAs was also observed. In AD and MS, disruptions in the AIN were observed during interoception, but no convergence with emotion was identified. INTERPRETATION: Neurodegeneration induces dysfunctional AIN across atrophy, connectivity, and metabolism, more accentuated in bvFTD. Findings bolster the predictive coding theories of large-scale AIN, calling for more synergistic approaches to understanding interoception, emotion, and social cognition impairments in neurodegeneration.

Plasma half-life extension of small recombinant antibodies by fusion to immunoglobulin-binding domains.

Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBDs) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C terminus of a single-chain Fv and a bispecific single-chain diabody, respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extents for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpG(C3)) followed by domains B and D of SpA, suggesting that SpG(C3) is particularly useful to extend the plasma half-life of small proteins.

STING-driven activation of T cells: relevance for the adoptive cell therapy of cancer.

Adoptive cell therapy (ACT) can successfully treat hematopoietic cancers but lacks efficacy against solid tumors. This is due to insufficient T cell infiltration, high tumor heterogeneity, frequent antigen loss with subsequent tumor escape, and the immunosuppressive tumor microenvironment (TME). Alternative methods to boost the anticancer efficacy of adoptively transferred cells are actively pursued. Among adjuvants that are utilized to stimulate anticancer immune responses, ligands of the stimulator of interferon genes (STING) pathway have received increasing attention. STING activation can trigger dendritic cell (DC) activation and endogenous immune responses, thereby preventing tumor escape. Activation of the STING pathway in the context of ACT was accordingly associated with improved T cell trafficking and persistence in the TME combined with the reduced presence of immunosuppressive cells. Recent findings also suggest cell-intrinsic effects of STING ligands on T cells. Activation of the STING signaling pathway was in this regard shown to enhance effector functions of CD4+ and CD8+ T cells, suggesting that the STING signaling could be exploited to harness T cell anticancer functions. In this review, we will discuss how the STING signaling can be used to enhance the anticancer efficacy of ACT.

Disturbance in bodily experience following ventricular assist device implantation.

BACKGROUND: Disturbance in bodily experience (BE) is a potential adverse consequence of ventricular assist device (VAD) implantation. The concept BE encompasses all cognitive and affective processes related to the subjective experience of one's own body. METHODS: A cross-sectional, multicenter study was performed, involving 365 VAD patients (85% male; time postimplant: 3-36 months). Patients completed a BE questionnaire (BE-S, 5-point Likert scale), and the disturbance in BE was analyzed based on sex, time since implantation (in the first, second, or third years postimplant), and patient acuity (elective vs emergent implantation). Subsidiary, patients' gratitude was surveyed. RESULTS: Disturbance in BE was not particularly pronounced (mean = 2.69, standard deviation = 1.17). Eighty-five percent of patients expressed high levels of gratitude. Disturbance in BE decreased (p = 0.04), while gratitude increased (p = 0.02) with time since implantation. Female patients showed more disturbance in BE (p = 0.01) and less gratitude (p = 0.01) compared to male patients. Among patients who underwent emergency implantation, the decrease in disturbance occurred predominantly in the third year, exceeding the level observed in elective implanted patients (p = 0.03). CONCLUSIONS: Disturbance in BE following VAD implantation does generally not reach excessive levels and tends to decrease over time. Our data indicate more disturbance and less gratitude in female patients. In emergently implanted patients, disturbance in BE is prolonged. Screening for disturbance in BE is recommended during follow-up, especially for these at-risk groups, to ensure early and focused psychological support.

Feasibility and Safety of Personalized, Multi-Target, Adoptive Cell Therapy (IMA101): First-in-Human Clinical Trial in Patients with Advanced Metastatic Cancer.

IMA101 is an actively personalized, multi-targeted adoptive cell therapy (ACT), whereby autologous T cells are directed against multiple novel defined peptide-HLA (pHLA) cancer targets. HLA-A*02:01-positive patients with relapsed/refractory solid tumors expressing ≥1 of 8 predefined targets underwent leukapheresis. Endogenous T cells specific for up to 4 targets were primed and expanded in vitro. Patients received lymphodepletion (fludarabine, cyclophosphamide), followed by T-cell infusion and low-dose IL2 (Cohort 1). Patients in Cohort 2 received atezolizumab for up to 1 year (NCT02876510). Overall, 214 patients were screened, 15 received lymphodepletion (13 women, 2 men; median age, 44 years), and 14 were treated with T-cell products. IMA101 treatment was feasible and well tolerated. The most common adverse events were cytokine release syndrome (Grade 1, n = 6; Grade 2, n = 4) and expected cytopenias. No patient died during the first 100 days after T-cell therapy. No neurotoxicity was observed. No objective responses were noted. Prolonged disease stabilization was noted in three patients lasting for 13.7, 12.9, and 7.3 months. High frequencies of target-specific T cells (up to 78.7% of CD8+ cells) were detected in the blood of treated patients, persisted for >1 year, and were detectable in posttreatment tumor tissue. Individual T-cell receptors (TCR) contained in T-cell products exhibited broad variation in TCR avidity, with the majority being low avidity. High-avidity TCRs were identified in some patients' products. This study demonstrates the feasibility and tolerability of an actively personalized ACT directed to multiple defined pHLA cancer targets. Results warrant further evaluation of multi-target ACT approaches using potent high-avidity TCRs. See related Spotlight by Uslu and June, p. 865.

Time is body: Multimodal evidence of crosstalk between interoception and time estimation.

Theoretical approaches propose a blending between interoception and time estimation. Interoception might constitute a neurophysiological mechanism for encoding duration. However, no study has assessed the convergence between interoception and time estimation using behavioral, neurophysiological, and functional anatomy signatures. We examined the multimodal convergence between interoception and time estimation using a two-fold approach. In study 1, we developed a dual design combining interoception (measuring heartbeat detection - HBD, and heartbeat evoked potential - HEP) with a time estimation paradigm (TEP, estimation of duration of a 120 s interval). In study 2, we performed a conjoint metanalysis (Multi-level Kernel Density Analysis, MKDA) of neuroimaging, including reports of interoception and time estimation. Both studies provide convergent evidence of time estimation's significant involvement in behavioral, electrophysiological (enhanced HEP), and neuroimaging (overlapping cluster in the right insula and operculum) signatures of interoception. Convergent results from both studies offer direct support for a shared mechanism of interoception and time estimation.

Birch Pollen Induces Toll-Like Receptor 4-Dependent Dendritic Cell Activation Favoring T Cell Responses.

Seasonal exposure to birch pollen (BP) is a major cause of pollinosis. The specific role of Toll-like receptor 4 (TLR4) in BP-induced allergic inflammation and the identification of key factors in birch pollen extracts (BPE) initiating this process remain to be explored. This study aimed to examine (i) the importance of TLR4 for dendritic cell (DC) activation by BPE, (ii) the extent of the contribution of BPE-derived lipopolysaccharide (LPS) and other potential TLR4 adjuvant(s) in BPE, and (iii) the relevance of the TLR4-dependent activation of BPE-stimulated DCs in the initiation of an adaptive immune response. In vitro, activation of murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs by BPE or the equivalent LPS (nLPS) was analyzed by flow cytometry. Polymyxin B (PMB), a TLR4 antagonist and TLR4-deficient BMDCs were used to investigate the TLR4 signaling in DC activation. The immunostimulatory activity of BPE was compared to protein-/lipid-depleted BPE-fractions. In co-cultures of BPE-pulsed BMDCs and Bet v 1-specific hybridoma T cells, the influence of the TLR4-dependent DC activation on T cell activation was analyzed. In vivo immunization of IL-4 reporter mice was conducted to study BPE-induced Th2 polarization upon PMB pre-treatment. Murine and human DC activation induced by either BPE or nLPS was inhibited by the TLR4 antagonist or by PMB, and abrogated in TLR4-deficient BMDCs compared to wild-type BMDCs. The lipid-free but not the protein-free fraction showed a reduced capacity to activate the TLR4 signaling and murine DCs. In human DCs, nLPS only partially reproduced the BPE-induced activation intensity. BPE-primed BMDCs efficiently stimulated T cell activation, which was repressed by the TLR4 antagonist or PMB, and the addition of nLPS to Bet v 1 did not reproduce the effect of BPE. In vivo, immunization with BPE induced a significant Th2 polarization, whereas administration of BPE pre-incubated with PMB showed a decreased tendency. These findings suggest that TLR4 is a major pathway by which BPE triggers DC activation that is involved in the initiation of adaptive immune responses. Further characterization of these BP-derived TLR4 adjuvants could provide new candidates for therapeutic strategies targeting specific mechanisms in BP-induced allergic inflammation.

Anti-inflammatory treatment in MPN: targeting TNFR1 and TNFR2 in JAK2-V617F-induced disease.

Chronic nonresolving inflammatory syndrome is a major disease feature in myeloproliferative neoplasms (MPNs). Systemic inflammation promotes the growth of the JAK2-V617F+ hematopoietic stem cell clone and is associated with constitutive symptoms (eg, fever, cachexia, and fatigue). Therefore, it is being discussed whether anti-inflammatory therapy, in addition to the well-established JAK inhibitor therapy, may be beneficial in the control of constitutive symptoms. Moreover, effective control of the inflammatory microenvironment may contribute to prevent transformation into secondary myelofibrosis and acute leukemia. Given the pivotal role of tumor necrosis factor α (TNF-α) in MPN and the distinct roles of TNF-α receptor 1 (TNFR1) and TNFR2 in inflammation, we investigated the therapeutic effects of αTNFR1 and αTNFR2 antibody treatment in MPN-like disease using the JAK2+/VF knock-in mouse model. Peripheral blood counts, bone marrow/spleen histopathology, and inflammatory cytokine levels in serum were investigated. αTNFR2 antibody treatment decreased white blood cells and modulated the serum levels of several cytokines [CXCL2, CXCL5, interleukin-12(p40)], as well as of macrophage colony-stimulating factor, but they lacked efficacy to ameliorate hematocrit and splenomegaly. αTNFR1 antibody treatment resulted in the mild suppression of elevated hematocrit of -10.7% and attenuated splenomegaly (22% reduction in spleen weight). In conclusion, our studies show that TNFR1 and TNFR2 play different roles in the biology of JAK2-V617F-induced disease that may be of relevance in future therapeutic settings.

The TNFR1 Antagonist Atrosimab Is Therapeutic in Mouse Models of Acute and Chronic Inflammation.

Therapeutics that block tumor necrosis factor (TNF), and thus activation of TNF receptor 1 (TNFR1) and TNFR2, are clinically used to treat inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. However, TNFR1 and TNFR2 work antithetically to balance immune responses involved in inflammatory diseases. In particular, TNFR1 promotes inflammation and tissue degeneration, whereas TNFR2 contributes to immune modulation and tissue regeneration. We, therefore, have developed the monovalent antagonistic anti-TNFR1 antibody derivative Atrosimab to selectively block TNFR1 signaling, while leaving TNFR2 signaling unaffected. Here, we describe that Atrosimab is highly stable at different storage temperatures and demonstrate its therapeutic efficacy in mouse models of acute and chronic inflammation, including experimental arthritis, non-alcoholic steatohepatitis (NASH) and experimental autoimmune encephalomyelitis (EAE). Our data support the hypothesis that it is sufficient to block TNFR1 signaling, while leaving immune modulatory and regenerative responses via TNFR2 intact, to induce therapeutic effects. Collectively, we demonstrate the therapeutic potential of the human TNFR1 antagonist Atrosimab for treatment of chronic inflammatory diseases.

Inhibition of Tumor Cell Growth and Cancer Stem Cell Expansion by a Bispecific Antibody Targeting EGFR and HER3.

The frequent activation of HER3 signaling as a resistance mechanism to EGFR-targeted therapy has motivated the development of combination therapies that block more than one receptor tyrosine kinase. Here, we have developed a novel tetravalent, bispecific single-chain diabody-Fc fusion protein targeting EGFR and HER3 (also known as ErbB3) that integrates the antigen-binding sites of a humanized version of cetuximab as well as a recently developed anti-HER3 antibody, IgG 3-43. This bispecific antibody combines the binding and neutralizing properties of the parental antibodies, as observed in biochemical and in vitro two-dimensional and three-dimensional cell culture assays, and gave rise to long-lasting growth suppression in a subcutaneous xenograft head and neck tumor model. In triple-negative breast cancer (TNBC) cell lines, treatment with the bispecific antibody inhibited the proliferation and oncosphere formation efficiency driven by HER3 signaling. In an orthotopic MDA-MB-468 tumor model, this translated into antitumor effects superior to those obtained by the parental antibodies alone or in combination and was associated with a reduced number of cells with stem-like properties. These findings demonstrate that the bispecific antibody efficiently blocks not only TNBC proliferation, but also the survival and expansion of the cancer stem cell population, holding promise for further preclinical development.

Improved monovalent TNF receptor 1-selective inhibitor with novel heterodimerizing Fc.

The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the pro-inflammatory responses mediated by TNF while leaving regenerative and pro-survival signals transduced by TNFR2 untouched. We recently generated a monovalent anti-TNFR1 antibody fragment (Fab 13.7) as an efficient inhibitor of TNFR1. To improve the pharmacokinetic properties of Fab 13.7, the variable domains of the heavy and light chains were fused to the N-termini of newly generated heterodimerizing Fc chains. This novel Fc heterodimerization technology, designated "Fc-one/kappa" (Fc1κ) is based on interspersed constant Ig domains substituting the CH3 domains of a γ1 Fc. The interspersed immunoglobulin (Ig) domains originate from the per se heterodimerizing constant CH1 and CLκ domains and contain sequence stretches of an IgG1 CH3 domain, destined to enable interaction with the neonatal Fc receptor, and thus promote extended serum half-life. The resulting monovalent Fv-Fc1κ fusion protein (Atrosimab) retained strong binding to TNFR1 as determined by enzyme-linked immunosorbent assay and quartz crystal microbalance, and potently inhibited TNF-induced activation of TNFR1. Atrosimab lacks agonistic activity for TNFR1 on its own and in the presence of anti-human IgG antibodies and displays clearly improved pharmacokinetic properties.

Diabody-Ig: a novel platform for the generation of multivalent and multispecific antibody molecules.

Multivalent mono- or bispecific antibodies are of increasing interest for therapeutic applications, such as efficient receptor clustering and activation, or dual targeting approaches. Here, we present a novel platform for the generation of Ig-like molecules, designated diabody-Ig (Db-Ig). The antigen-binding site of Db-Ig is composed of a diabody in the VH-VL orientation stabilized by fusion to antibody-derived homo- or heterodimerization domains, e.g., CH1/CL or the heavy chain domain 2 of IgE (EHD2) or IgM (MHD2), further fused to an Fc region. In this study, we applied the Db-Ig format for the generation of tetravalent bispecific antibodies (2 + 2) directed against EGFR and HER3 and utilizing different dimerization domains. These Db-Ig antibodies retained the binding properties of the parental antibodies and demonstrated unhindered simultaneous binding of both antigens. The Db-Ig antibodies could be purified by a single affinity chromatography resulting in a homogenous preparation. Furthermore, the Db-Igs were highly stable in human plasma. Importantly, only one short peptide linker (5 aa) per chain is required to generate a Db-Ig molecule, reducing the potential risk of immunogenicity. The presence of a fully functional Fc resulted in IgG-like pharmacokinetic profiles of the Db-Ig molecules. Besides tetravalent bispecific molecules, this modular platform technology further allows for the generation of other multivalent molecules of varying specificity and valency, including mono-, bi-, tri- and tetra-specific molecules, and thus should be suitable for numerous applications.