Targeted delivery of IL-12 might turn this cytokine into a safer, more effective cancer therapeutic. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). The addition of the human IgG1 moiety resulted in a longer plasma half-life of NHS-IL12 than recombinant IL-12, and a selective targeting to murine tumors in vivo. Data from both in vitro assays using human PBMCs and in vivo primate studies showed that IFN-gamma production by immune cells is attenuated following treatment with the immunocytokine, suggesting an improved toxicity profile than seen with recombinant IL-12 alone. NHS-IL12 was superior to recombinant IL-12 when evaluated as an anti-tumor agent in three murine tumor models. Mechanistic studies utilizing immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays all indicated that the anti-tumor effects of NHS-IL12 were primarily CD8+ T cell-dependent and likely IL-12-mediated. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation and chemotherapy.

BackgroundInterleukin (IL)-12 is a pro-inflammatory cytokine that mediates T-helper type 1 responses and cytotoxic T-cell activation, contributing to its utility as anti-cancer agent. Systemic administration of IL-12 often results in unacceptable toxicity; therefore, strategies to direct delivery of IL-12 to tumors are under investigation. The objective of this study was to assist the preclinical development of NHS-IL12, an immunocytokine consisting of an antibody, which targets necrotic tumor regions, linked to IL-12. Specifically this study sought to evaluate the safety, serum pharmacokinetics, anti-tumor activity, and immune modulation of NHS-IL12 in dogs with naturally occurring cancers.Methodology/Principal FindingsA rapid dose-escalation study of NHS-IL12 administered subcutaneously to dogs with melanoma was conducted through the Comparative Oncology Trials Consortium (COTC). Eleven dogs were enrolled in four dose-escalation cohorts; thereafter, an additional seven dogs were treated at the defined tolerable dose of 0.8 mg/m2. The expanded cohort at this fixed dose (ten dogs in total) was accrued for further pharmacokinetics and pharmacodynamics assessment. NHS-IL12 levels, serum cytokine concentrations, and peripheral blood mononuclear cell characterization (post-treatment) and draining lymph node immune profiling, and tumor biopsies (pre- and post-treatment) were collected. Adverse events included thrombocytopenia, liver enzymopathies, fever, and vasculitis. Correlation between interferon (IFN)-γ induction, adverse events, and NHS-IL12 exposure (maximum concentration and area under the concentration-time curve) were dose-dependent. Serum IL-10 levels and intratumoral CD8+ populations increased after treatment. Partial responses, according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, were observed in two dogs treated with NHS-IL12 0.8 mg/m2 and 1.6 mg/m2.Conclusions/SignificanceNHS-IL12 was administered safely to dogs with melanoma and both immunologic and clinical activity was observed. This study successfully defined a narrow therapeutic window for systemic delivery of NHS-IL12 via the subcutaneous route. Results will inform the design and implementation of first-in-human clinical trials of NHS-IL12 in cancer patients.

The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This new protein engineered platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. Alternating sequences from human IgA and IgG in the SEED CH3 domains generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of AG and GA SEED CH3 domains. Using a clinically validated antibody (C225), we tested whether Fab derivatives constructed on the SEED platform retain desirable therapeutic antibody features such as in vitro and in vivo stability, favorable pharmacokinetics, ligand binding and effector functions including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In addition, we tested SEED with combinations of binder domains (scFv, VHH, Fab). Mono- and bivalent Fab-SEED fusions retain full binding affinity, have excellent biochemical and biophysical stability, and retain desirable antibody-like characteristics conferred by Fc domains. Furthermore, SEED is compatible with different combinations of Fab, scFv and VHH domains. Our assessment shows that the new SEED platform expands therapeutic applications of natural antibodies by generating heterodimeric Fc-analog proteins.

Objective To determine levels of objectively measured physical activity (PA) and the proportion of adults with multimorbidity that adheres to PA guidelines. Methods All studies, where PA was measured at baseline using an activity monitor in an adult (≥18 years) multimorbid (≥80% of the population had ≥2 chronic conditions) population. A systematic literature search was performed in Medline, EMBASE, CINAHL, CENTRAL, ClinicalTrials.gov, opengrey.eu and google.com from inception up until 18th of January 2022. Risk of bias was assessed with a modified version of the Quality Assessment Tool for Quantitative Studies. A random-effects meta-analyses was performed to estimate daily minutes of sedentary behavior (SB), light PA (LPA), moderate PA (MPA), moderate to vigorous PA (MVPA) and steps. Proportions adhering to PA guidelines was narratively synthesized. Certainty of evidence was determined using The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. Results Fifteen studies (2,172 participants) were included. The most frequent combination of conditions were type 2 diabetes and hypertension (six studies). Participants spent a daily average of 500.5 (95% CI: 407.1 to 593.9) minutes in SB, 325.6 (95% CI: 246.4 to 404.7 minutes in LPA and 32.7 (95% CI: 20.2 to 45.3) minutes in MVPA. The mean daily number of steps was 5,145 (95% CI: 4264 to 6026) for people in free-living conditions. The proportion adhering to PA guidelines ranged widely (7.4% to 43%). All studies were rated as at high risk of bias and the certainty of evidence was very low. Conclusions PA levels and adherence varied from low to above guideline recommended levels for adults with chronic conditions, depending on PA intensity. The very low certainty of evidence calls for high quality studies focusing on detailed descriptions of PA behavior in people with multimorbidity. PROSPERO registration number CRD42020172456.

Monitoring brain activity at home using electroencephalography (EEG) is an increasing trend for both medical and non-medical applications. Gel-based electrodes are not suitable due to the gel application requiring extensive preparation and cleaning support for the patient or user. Dry electrodes can be applied without prior preparation by the patient or user. We investigate and compare two dry electrode headbands for EEG acquisition: a novel hybrid dual-textile headband comprising multipin and multiwave electrodes and a neoprene-based headband comprising hydrogel and spidershaped electrodes. We compare the headbands and electrodes in terms of electrode-skin impedance, comfort, electrode offset potential and EEG signal quality. We did not observe considerable differences in the power spectral density of EEG recordings. However, the hydrogel electrodes showed considerably increased impedances and offset potentials, limiting their compatibility with many EEG amplifiers. The hydrogel and spider-shaped electrodes required increased adduction, resulting in a lower wearing comfort throughout the application time compared to the novel headband comprising multipin and multiwave electrodes.

Introduction: The separation of graft-versus-leukemia (GvL) effect from graft-versus-host-disease (GvHD) is a major objective after allogeneic stem cell transplantation. We recently described two types of endogenous HLA class II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, presentation of DM-sensitive antigens rely on co-expression of HLA-DO - the natural inhibitor of HLA-DM. Since the expression of HLA-DO is not upregulated by inflammatory cytokines and restricted to B-cells, dendritic cells and thymic epithelial cells, DM-sensitive antigens cannot be presented on non-hematopoietic tissues. Therefore, usage of CD4 T-cells directed against DM-sensitive antigens might allow separation of GvL from GvHD. However, it remains elusive whether immunogenicity and anti-tumorigenic potential of DM-sensitive and DM-resistant antigens have comparable properties in vivo. Methods: Therefore, we sought to create an in vivo system using a DM-sensitive and a DM-resistant variant of the same model antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM or HLA-DO, respectively) to allocate the two model antigens ovalbumin (OVA) and murine Y-chromosome antigen DBY to their category. Furthermore, we introduced one to three amino acid substitutions within the MHC II restricted T-cell epitopes of the two antigens and tested DM-sensitivity or DM-resistance by T-cell activation using proliferation and IFN-g secretion as read-out in vitro. Finally, we vaccinated B6 mice with the generated epitope variants and measured expansion, phenotype and reactivity of OVA- or DBY-specific CD4 T-cells in vivo. Results: By testing T-cell recognition of OVA or DBY on murine B-cell lines overexpressing H2-M and H2-O, respectively, we could show that OVA leads to a more potent T-cell activation in the presence of H2-O demonstrating its DM-sensitive character. In contrast the wildtype epitope of DBY does not rely on H2-O expression for strong T-cell activation and was therefore assessed as DM-resistant antigen. By introducing one to three amino acid substitutions within the T-cell epitope we could generate one further DM-sensitive variant of OVA but also two DM-resistant counterparts. Likewise, we designed both DM-resistant and DM-sensitive epitope variants of murine DBY. To assess T-cell receptor avidity to our epitope variants presented on natural antigen presenting cells, titration of DM-sensitive and DM-resistant variants of the same antigen on untreated splenocytes from OVA or DBY T-cell receptor transgenic mice, respectively, were performed. We observed comparable activation of the same T-cell clone activated by either variant of the epitope as measured by proliferation and IFN-g secretion. Furthermore, upon vaccination of B6 mice with either variant of the epitope we could measure comparable expansion, phenotype, and reactivity of OVA- and DBY-specific T-cells both invivo and ex vivo. Conclusion: We successfully generated DM-sensitive and DM-resistant variants of the same epitope for the two model antigens OVA and murine DBY. With this tool we could demonstrate that DM-sensitive antigens are not inferior to their DM-resistant counterpart. Therefore, targeting DM-sensitive antigens after allogenic stem cell transplantation might be an interesting tool to improve the GvL effect with only limited GvHD. Disclosures Bernhardt: DFG TRR221/project A1 (German Research Foundation): Research Funding.

BackgroundGraft-versus-host-disease (GvHD) is a major problem in allogeneic stem cell transplantation. We previously described two types of endogenous human leukocyte antigen (HLA)-II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, DM-sensitive antigens rely on the expression of HLA-DO-the natural inhibitor of HLA-DM. Since expression of HLA-DO is not upregulated by inflammatory cytokines, DM-sensitive antigens cannot be presented on non-hematopoietic tissues even under inflammatory conditions. Therefore, usage of CD4+ T cells directed against DM-sensitive antigens might allow induction of graft-versus-leukemia effect without GvHD. As DM-sensitivity is likely linked to low affinity peptides, it remains elusive whether DM-sensitive antigens are inferior in their immunogenicity.MethodsWe created an in vivo system using a DM-sensitive and a DM-resistant variant of the same antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM and HLA-DO) to assign the two model antigens ovalbumin (OVA) and DBY to their category. Further, we introduced mutations within the two T-cell epitopes and tested the effect on DM-sensitivity or DM-resistance. Furthermore, we vaccinated C57BL/6 mice with either variant of the epitope and measured expansion and reactivity of OVA-specific and DBY-specific CD4+ T cells.ResultsBy testing T-cell recognition of OVA and DBY on a murine B-cell line overexpressing H2-M and H2-O, respectively, we showed that OVA leads to a stronger T-cell activation in the presence of H2-O demonstrating its DM-sensitivity. In contrast, the DBY epitope does not rely on H2-O for T-cell activation indicating DM-resistance. By introducing mutations within the T-cell epitopes we could generate one further DM-sensitive variant of OVA and two DM-resistant counterparts. Likewise, we designed DM-resistant and DM-sensitive variants of DBY. On vaccination of C57BL/6 mice with either epitope variant we measured comparable expansion and reactivity of OVA-specific and DBY-specific T-cells both in vivo and ex vivo. By generating T-cell lines and clones of healthy human donors we showed that DM-sensitive antigens are targeted by the natural T-cell repertoire.ConclusionWe successfully generated DM-sensitive and DM-resistant variants for two model antigens. Thereby, we demonstrated that DM-sensitive antigens are not inferior to their DM-resistant counterpart and are therefore interesting tools for immunotherapy after allogeneic stem cell transplantation.