Gut–liver MPS offer attractive opportunities to study absorption and metabolism processes for orally administered drugs, especially prodrugs. Modelling and simulation must be included from the outset for robust study design and parameter generation.

Microphysiological systems (MPS) are complex and more physiologically realistic cellular in vitro tools that aim to provide more relevant human in vitro data for quantitative prediction of clinical pharmacokinetics while...

Antibody-based immunotherapy is a promising strategy for targeting chemo-resistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell-surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated using CrossMab and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) in the context of human leukocyte antigen (HLA) A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary AML cells was mediated in ex vivo long-term co-cultures utilizing allogenic (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18) or autologous, patient-derived T cells (mean specific lysis: 54±12% after 11-14 days; ±SEM; n=8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 45.4±9.0% vs 70.8±8.3%; p=0.015; ±SEM; n=9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase I trial in patients with r/r AML (NCT04580121).

Since evidence is rising that extracellular matrix (ECM) fibers might serve as reservoirs for growth factors and cytokines, we investigated the interaction between fibronectin (FN) and interleukin-7 (IL-7), a cytokine of immunological significance and a target of several immunotherapies. By employing a FN fiber stretch assay and Förster resonance energy transfer (FRET) confocal microscopy, we found that stretching of FN fibers increased IL-7 binding. We localized the FN binding site on the CD loop of IL-7, since a synthetic CD loop peptide also bound stronger to stretched than to relaxed FN fibers. On the basis of a structural model, we propose that the CD loop can bind to FN, while IL-7 is bound to its cognate cell surface receptors. Sequence alignment with bacterial adhesins, which also bind the FN N-terminus, suggests that a conserved motif on the CD loop (TKSLEEN and the truncated SLEE in human and mouse IL-7, respectively) might bind to the second FN type I module (FnI) and that additional epitopes enhance the stretch-upregulated binding. FN fiber stretching might thus serve as a mechano-regulated mechanism to locally concentrate IL-7 in an ECM-bound state, thereby upregulating the potency of IL-7 signaling. A feedback model mechanism is proposed that could explain the well-known, but poorly understood, function of IL-7 in ECM homeostasis. Understanding how local IL-7 availability and signaling might be modulated by the tensional state of the ECM niche, which is adjusted by residing stroma cells, is highly relevant for basic science but also for advancing IL-7 based immunotherapies.

The superior clinical therapeutic effects and broad applications of bispecific T cell engagers (BiTEs) has transformed treatment strategies in oncology. While benefits of the novel immunotherapies are indisputable, the safety aspects require to be intensively investigated. Considering challenges of animal models for accessing efficacy and safety of biologics leading to failed clinical trials, there is unmet need for human disease relevant systems, which facilitate development of new clinical candidates. We developed a high-throughput, 384-well-format co-culturing platform for combined assessment of anti-tumor efficacy and liver safety of immunotherapeutics. As a model of liver we employed 3D spheroids composed of primary human hepatocytes and Kupffer cells with preserved metabolic and inflammatory functions. In parallel, the solid tumor model was aggerated of human cancer cell lines (HCT116-GFP) and primary cancer associated fibroblasts, mimicking the tumor microenvironment. Both models were cultured in automation-friendly wells pairwise interconnected with microfluidic channels. Gravity-driven tubeless flow ensured tissue-tissue interaction. To evaluate our system, we treated 3D spheroid models with runimotamab (HER2xCD3 BiTE) in the presence of peripheral blood mononuclear cells (PBMCs). Tumor viability and growth was assessed by fluorescence measurements, while liver toxicity and function were monitored by release of liver aminotransferases ALT/AST, albumin secretion and live confocal microscopy. Immune cell activation was assessed by a cytokine bead array. The treatment with runimotamab resulted in a significant decrease of fluorescence and size of tumor spheroids. At the same time, we observed an induced secretion of cytokines with a peak of expression of IL-2, TNFα after 24h and INFγ, IL-6 and IL-17A after 48h. Cytokine release was coupled with elevated levels of clinically relevant liver damage biomarkers ALT and AST with peak at 72h timepoint. This data suggests potential risk of cytokine release syndrome (CRS) followed by liver damage. Interestingly, redosing of the antibody was not followed by another release of liver enzymes, similarly to clinical observations. In summary, we have developed a human disease relevant, high-throughput platform for the evaluation of novel immunotherapies closely emulating clinical results. Automation-compatible with up to 192 co-culture conditions per plate it represents a powerful tool for clinical candidate development. Citation Format: Michal Rudnik, Ozlem Yavaş Grining, Simon Hutter, Frauke Greve, Daniela Ortiz Franyuti, Ramona Matheis, Ekaterina Breous-Nystrom, Olivier Frey. Combined investigation of anti-tumor efficacy and liver safety of bispecific T cell engagers in immune-competent high-throughput co-culturing platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2747.